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D 56 132
Volume 14
September
2010
9-10
 A
erosol albedometer
[page 26]
 Ba
cteria – Fighter Against Cancer
[page 20]
 A
daptability and Ergonomy
[page 36]
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• Editorial
Regeneration and Cell-Cell Signaling
Dear Reader, try to see
your body as a social
organism formed by
several billion individuals (cells). The way
these cells organize,
control and maintain
their integrity as a
functioning body, is a
highly sophisticated
communication network, established by only a handful of signaling
molecules. This sounds as if the system should
be simple but it isn’t. Each of the handful of signaling molecules organize a large set of jobs. For
example, the family of secreted wnt-factors is
involved in the development of three dimensional body plans, cell movement during gastrulation, patterning of the central nervous system,
and they are also involved in the occurrence of
polyposis cancer of the colon. The key are multiple layers of signal modulations by hundreds of
interacting molecules and crosslinks between
different signaling pathways.
Already at a very early stage in the development of an embryo, no matter if it will become a
morphologically complex body like a human, or
a very simple one like a Hydra (cnidaria) polyp,
the fate of every newly born cell is controlled by
communication between the surrounding and
the new one.
Already beginning from the fourth cleavage,
a sea urchin embryo (belonging to a taxonomic
group closely related to humans) produces cells
with different fates. At this point some cells become “master” cells that secrete signals which
are detected by other cells. Through the signals
the “master” cell gives advice, and the other
cells do what they were told to do. Such “master” cells with the capacity to instruct other cells
are organized in signaling centers at defined regions in a developing embryo. Ethel Browne
Harvey proved this in 1909 by transplanting the
tip of a Hydra head to the body wall of a second
Hydra. The second Hydra developed a new (secondary) head at the closest point to the tip of
the first. Some factors secreted by the tip of the
head induced the cells of the body wall of the
second animal to form an ectopic head. One of
these “head inducing factors” was later identified to be wnt 3. But such “master” cells are
also widely distributed in each and every tissue
in the body in an adult organism, ready to advise the surrounding cells managing a regeneration process in the case of damage.
Regeneration is a process that shares many
aspects with embryonic development. It is also a
process in which the communication between
cells is crucial for their “correct” behavior. And
again some “master” cells control the behavior
of the cells which repair the injured area. The
signaling factors involved in regeneration are
the same like in normal development. In the regeneration process of Hydra after cutting off the
complete head shows the expression of wnt 3.
At the basis of the evolution, animals had
much more capabilities for regeneration. The
larger, higher evolved and more complex an
organism is; the smaller are its regeneration capacities, leading to the assumption that regeneration capacities are “purposely” down-regulated
in higher organisms, maybe to prevent communication problems between cells leading to cancer, or for other yet unknown reasons. Hydra can
be completely dissolved in single cells. If some of
these cells are remixed and brought together (by
mild centrifugation) the cell-clumps reorganize
themselves and built up new completely intact
animals (Hobmayer et. al. Nature 2000). In sea
urchins you can perform the same experiment
with embryos up to the mid blastula stage. Comparable experiments can also be performed with
a variety of cell types of tissues of higher animals
like chicken retinae or even human cancer cells.
In the case of higher organisms it is necessary
to add signaling substances and growth factors
to the dissoluted cells, which then somehow (at
least partial) mimic the signaling environment
of a developing organism leading to processes
closely related with the embryonic development
and regenerative processes. These substances
are also used by Prof. Augustinus Bader for his
research on wound healing and regeneration. At
least on a theoretical level it should be possible
to induce regeneration far above the normal
(strongly down-regulated)level in our bodies by
adding signaling molecules and growth factors
that are responsible for the instruction of cells in
normal development and regeneration.
Sincerely,
Dr. Arne Kusserow
Editor-in-Chief
Dissociated and reaggregated Hydra cells. Emerging heads of de novo patterned polyps are clearly visible. The expression domains of the wnt3-gene are visualized in blue (in situ hybridization. Image by
Bert Hobmayer/University of Innsbruck; Nature 2000).
Sea urchin embryo at 32 cell stage. In this stage
some cells at the vegetal pole are already “master” cells that organize the cell fate of the
other cells.
G.I.T. Laboratory Journal 9-10/2010 ▪ 3
RESEARCH & DEVELOPMENT
C ontents •
EVENTS
Regenerative Medicine Focus on Regenerative Medicine 5
The Past, the Present, and the Future
JIB 2010 7
Advances in Metabolic Profiling 2010 8
PA R T I C L E M E A S U R E M E N T
Mass Spec Europe 8
Aerosol Albedometer
Lab-on-a-Chip World Congress 12
10
R. M. Nerem, Georgia Institute of Technology, Atlanta, Georgia, USA
26
A Tool for Measuring Optical Scattering and Extinction of
Dispersed Aerosols
Prof. J. Thompson, Texas Tech University, USA
Munich to Become the Capital of Biotech
Dealmaking in November 12
The ELRIG.de Meeting 2010 13
LabAutomation2011 14
BIOPROCESSING
Lost in Translation
30
Very Early Process Development for Biopharmaceuticals
Dr. S. Hellwig, Fraunhofer IME, Germany
MAGAZINE
World Congress on Preventive and Regenerative
Medicine
MATERIAL SCIENCES
6
Hanover, Germany, October 5–7, 2010
Prof. Dr. A. Bader, WCRM, Germany
Simple Solutions
Quantification Study of Drug Delivery by
Nanocarriers
32
A Cell Mass Spectrometry Approach
9
W.-Ping Peng et al., National Dong Hwa University, Taiwan
Simplicity is the ultimate sophistication P. Praet, GIT VERLAG
S creenin g
News
18
APPLICATION NOTE
Reproducible Cell Assays
P harmace u tics & D r u g D iscover y
Bacteria – Fighters Against Cancer
22
Consistency of Cryopreservation is a Necessity
20
Rolf O. Ehrhardt, MD, PhD and Brian Schryver, BioCision, LLC, Larkspur, USA
Bacteria as Promising Tools for Cancer Therapy
P H A R M A C E U T I C S & D R UG D I S C O V E R Y
Dr. S. Weiß and Dr. S. Leschner, Helmholtz Centre for Infection Research, Germany
Enzyme-Amplified Array Sensing of Proteins 24
Identification of Low Protein Concentrations
T echnolo g y & I nstr u mentation
Prof. V.M. Rotello, University of Massachusetts Amherst, USA
COVER STORY
On-line Oxygen Monitoring in Cell Culture
Adaptability and Ergonomy
Effects of Mitochondrial Modulators on O2 Dynamics of
Mammalian Cells
36
Built by Your Needs
Lynn S.G. and LaPres J. J., Michigan State University, East Lansing, MI, USA
K. Ansmann, Olympus Europa, Hamburg, Germany
News
ADVERTORIALS
38
High Energy Efficiency and Safety
39
J. Feddern, Siemens Building Technologies Division, Zug, Switzerland
Dynamic Image Analysis Beats Laser Diffraction
Nanoparticles in Liquids
J. Westermann, Retsch Technology, Haan, Germany
Count, Size and Visualize
White Giant or White Dwarf?
42
A. Malloy, NanoSight Limited, Wiltshire, UK
Particle Size Distribution Measurements of TiO2
Dr. Markus Ortlieb, Shimadzu, Düsseldorf, Germany
Transfer of USP-based HPLC Methods for Pantoprazole
Sodium to UPLC
44
20-fold increase in productivity
A.H. Schmidt, Steiner & Co., Berlin, Germany
4 ▪ G.I.T. Laboratory Journal 9-10/2010
48
The Dow Europe Laboratory in Horgen
APPLICATION NOTES
Particle Size Distributions
46
P rod u cts
51, 53, 54
I nde x / I mprint
Inside back Cover
52
• Events
Focus on regenerative medicine
© Michael Radtke/Flickr.com
The 5th World Congress for Preventive and Regenerative Medicine (WRCM) from the 5th to the
7th of October, will be a part of the conference
program of Biotechnica for the first time in 2010.
More than 500 scientists and clinical experts
from all over the world will be discussing new
possibilities for treating hitherto incurable or intractable diseases using regenerative therapies.
The program reflects the broad range of scientific approaches in the field of regenerative
medicine. Due to the considerable synergies between the two fields, the link with preventive
medicine has been highlighted this year for the
first time as the second main theme. The other
main subject focuses range from tissue culture
to the use of regenerative medicine in clinics,
anti-ageing medicine, and stem cell research.
Stem cells are considered to be the main hope
for the treatment of illnesses that are difficult to
cure. Since legal regulations govern research
work in the field of regenerative medicine, ethical and legal issues are also on the agenda. The event will take place in the Convention
Centre at the Hanover exhibition centre. It is being organized by Professor Augustinus Bader
who holds the chair of cell technologies and applied stem cell technology at the University of
Leipzig. The congress is rounded off by a poster
exhibition and a special presentation on the
subject of preventive and regenerative medicine
in the exhibition hall.
Specialists such as Professor William
Haseltine and Professor Madjid Samii will contribute as keynote speakers. Other speakers are
the pioneer of tracheal transplantation, Professor Paolo Macchiarini, and Professor Aubrey de
Grey who will present his theories on the reversing of ageing processes. Professor Jörg Wiltfang
will report on clinical pioneering work in the
field of bone regeneration in oral and maxillofacial surgery. Numerous other scientists have
confirmed that they will be attending, including
international experts from the fields of medicine,
technology and biology, as well as experts from
the pharmaceutical industry.
www.messe.de
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Quality in your hands
Magazine •
World Congress on Preventive and
Regenerative Medicine
Hanover, Germany, October 5–7, 2010
Biotechnica strongly developed its scientific program during the past few
years. This year more than 10 different international congresses will take
place in Hanover during Biotechnica. G.I.T. Laboratory Journal talked to the
Initiator of the WCPRM, Prof. Dr. Augustinus Bader.
Prof. Dr. Augustinus Bader, Initiator of the WCPRM
G.I.T. Laboratory Journal: Prof. Bader, this
occasion marks the fifth World Congress
that has been initiated by you. And it is the
first time Hanover is hosting the event.
What were the reasons to choose Hanover
as the venue?
A. Bader: This year, the fifth World Congress for
Preventive and Regenerative Medicine (WCRPM)
is entering into a strategic alliance with Biotechnica. The decision to combine the two was driven by the fact that the topics complement each
other. Biotechnology has delivered significant
anticipatory services for the clinical feasibility of
regenerative and preventative approaches. However, especially the clinical sector is still not very
familiar with these potentials and, at this time,
the biotechnological corporate structures that
are already established have only been linked
with the potential users of these technologies in
very few cases.
Through interaction the connections between
biotechnology and medical uses are to be significantly intensified and expanded far beyond
to what they have been to date. Consequently,
the fifth World Congress also is distinctly different from the event that has typically taken place
in this discipline, which could be dedicated primarily to the aspect of fundamental research or
materials development in these segments.
The focus of the Congress is on prevention
and regeneration. Which are the concrete
core topics participants will be able to gain
insights into?
A. Bader: In the discipline of regenerative technologies the key topics depicted will be stem cell
therapy processes used to treat the tissue categories of bones, cartilage, liver, the nervous system and the skin as well as the muscles including the heart. Additional associated tissue areas
will be examined in open discussion forums and
ad-hoc presentations.
As far as the discipline of preventative medicine is concerned, topics will comprise sports
medicine aspects, hormone therapy and antioxidant treatments. We are planning to combine
these issues, which have already been established in prevention and age research, with
questions arising from traditional pharmacology
with regard to the prevention of the major diseases that affect large portions of the population – diabetes and cardiovascular diseases.
Sports medicine aspects will demonstrate the
holistic approach of the prevention and regeneration concept of this Congress. We are planning to present biotechnological procedures in
the form of cross sectional platforms in both –
analytics and diagnostics; but we will also offer
insights into the product development sector for
laboratory and clinical requirements. Numerous
companies will present their solutions.
Who would benefit the most from attending this Congress?
A. Bader: The Congress is particularly interesting for individuals who are eager to learn about
cross sectional functions and cross sectional
topics with the objective of boosting the innovation potential for their own scopes of application. Extending their horizons allows scientists
and medical experts, as well as the true practitioners – i.e. surgeons working in hospitals, colleagues working in preventative and sports
medicine to participate in biotechnological stem
cell therapy innovations.
Opinion drivers from the discipline of medicine as well as from biotechnological fundamental research and stem cell technology will be in
attendance. The Congress targets decision makers and young scientists, who will be offered a
pertinent forum for the presentation of their results.
In addition, the event will cover the relevant
regulatory aspects, which will be presented by
the EMA (European Medicine Agency) and rep-
resentatives of the Paul Ehrlich Institute. Consequently, the event is also particularly interesting
for industrial continued GMP and GLP technologies education applications. Pertinent practical
training results are also possible due to the fact
that cooperating exhibitors are engaged in this
subject matter.
How important is stem cell research within
the scope of the innovative results and approaches?
A. Bader: As far as its innovative force is concerned, stem cell research is one of the most
powerful innovation drivers in the disciplines of
medical research and application. Principled
fundamental improvements can be anticipated
in areas such as bone-cartilage regeneration,
skin and liver regeneration, but also as far as the
regeneration of the human nervous system is
concerned. Based on the large tissue system of
animal and human bodies partial solutions for
smaller tissue constructions will already be possible with greater precision in certain scopes of
application.
Aspects of rapid prototyping, as well as a
method called organ printing will be presented;
as will biotechnological procedures or stem cell
treatment methods in the body that trigger tissue regeneration from the tissue itself. The entire
bandwidth of stem cell research, covering everything from cell therapy based on the bionic principle, embryonic cell types, fetal cell systems to
traditional tissue engineering will be introduced.
Would you like to give potential visitors
some recommendations on presentations
and speakers they should definitely not
miss?
A. Bader: Numerous key note speakers who are
highly respected around the globe, for instance
Professor Aubrey de Grey, who is one of the pri-
• Magazine
mary pioneers in age research, will come to the
event. Prof. Dr. Machens of the Klinikum rechts
der Isar and of the Technical University Munich
will present a report on the treatment of burn
trauma patients and initial results from clinical
trials. Prof. Su, who hails from Taiwan, will talk
about technologies available for the preparation
of blood platelet concentrates. This is a technology that will certainly be compatible with numerous applications in a variety of tissue areas.
Professor Cimen Karasu will make a presentation on preventative medicine; specifically on
aspects related to the prevention of age related
diseases and oxidative stress. Professor Dr. Halle
will segue to the discipline of sports medicine.
Contact
Andreas Guntermann
World Federation/
World Virtual Institute of Preventive & Regenerative
Medicine (Pyramed)
Hannover, Germany
[email protected]
www.regmed.net
© trixnbooze/Flickr.de
JIB 2010
A major meeting place in laboratory medicine,
the International Biology Days (Journées Internationales de Biologie – JIB) will be held at the
CNIT, Paris la Défense in France from November
2nd to 5th, 2010 (the 2nd is only dedicated to the
congress).
In this time of reform of the profession, the
event becomes a guide to changes and offers
not only a paying session dedicated to the ISOEN 15 189 accreditation norm during the congress but also “Café Scientifique” slots that deal
with current and pertinent issues.
Revealing know-how, the JIB gathers the
suppliers of medical biology laboratories (automated systems, analyzers, reagents, data processing, financing, services…). 200 exhibitors
meet with more than 10,000 professional visitors (private and hospital biologists, technicians,
biomedical engineers, researchers…) and show
them their new products and services on an International, European or French sneak preview.
The BioMI pavilion, Molecular Biology Initiatives,
created in 2008, continues its development on a
dedicated area within the exhibition.
Creating bridges, the JIB event works in partnership with the Ensaama – Olivier de Serres
school in order to create the “Tomorrow’s medical laboratory” (welcome desk and sample
rooms). Applied art students participate in this
contest and present their own projects on a privileged place.
Finally, the scientific influence of medical biology grows on the congress (2-3-4-5 Nov. 2010)
with the auspices of the IFCC and the support of
the EFCC. The scientific committee offers a special theme “Health & Environment: Challenges
for Laboratory Medicine” to the attendees, in order to better understand the mechanisms that
identify direct links between environment and
human health. The 2010 program also contains
a technological innovations session and a roundtable on Nov. 4th entitled “Mapping the future of
European Laboratory Medicine for Young Scientists”.
Contact
www.jib-sdbio.fr
Events •
Advances in Metabolic Profiling 2010
Select Biosciences is proud to announce their 6th
annual Advances in Metabolic Profiling conference, which will take place from 9th–10th
November 2010 at the Sheraton Hotel, Florence,
Italy.
The conference has been designed to give
expert guidance on this sector by gathering
some of the most influential and experienced
players in the field from Europe, America and
across the globe. It will also provide the opportunity to network with other colleagues from all
over the world.
The agenda will include the following key
subject areas appropriate to the latest in metabolic profiling:
▪▪ Clinical Applications of Metabolomics
▪▪ Fluxomics and Pathway Discovery
▪▪ Drug Discovery and Development
▪▪ Plant Metabolomics
▪▪ Nutrigenomics and Metabolomics
▪▪ Microbial Metabolomics – Novel Technologies for Metabolomics
▪▪ Bioinformatics and Data Handling for Metabolomics
▪▪ Data Fusion – Bringing Together Different
Omic Technologies
To guarantee a high attendance at this exciting
event, the traditional low registration fees and
group booking discounts will be maintained. Colocated with Mass Spec Europe, European Biomarkers Summit and Advances in Protein Crystallography, full conference passes include
admission to all sessions and the exhibition, as
well as conference documentation.
The conferences division of Select Biosciences is focused on organizing specialist biomedical
meetings. Experts from both academia and commerce are invited to present timely information
from current research through to commercial
implementation of new technologies. These
events also provide a unique networking facility
and the opportunity to reach a highly targeted
scientific audience.
Contact
Kirit Shah
Conference Producer
[email protected]
Aaron Woodley
Exhibition Manager
[email protected]
www.MetabolicProfiling.com
Mass Spec Europe
Select Biosciences will hold its 2nd annual Mass
Spectrometry Europe conference, in Florence Italy on 9–10th November 2010, under the auspices
of the Italian Chemical Society. It will be a two
day conference, co-located with Advances in
Metabolic Profiling, European Biomarkers Summit and Proteomics Europe. It will be focused on
all areas of mass spectrometry, from fundamental to applied research in the areas of biology,
chemistry, physics and other related disciplines.
Mass spectrometry is considered an ideal
technology for image analysis due to its high
sensitivity and molecular specificity. It has allowed researchers to profile, identify, characterize and quantify low molecular-weight compounds easily and is unequivocally an essential
tool for research in the 21st century.
This conference aims to aid the advancement
of this unique technology by providing a world
class agenda of presentations from influential
figures within the industry. It will allow the sharing of information and techniques, related to
mass spectrometry, as well as giving the opportunity for delegates to network with others from
all over the world.
Confirmed Speakers include:
▪▪ John Vickerman, Professor, University of Manchester
▪▪ Gianluca Giorgi, President of the Mass Spec
Division, Italian Chemical
▪▪ Frank Sobott, Group Leader, University of Oxford
▪▪ Pedro Cutillas, Head of Analytical Signaling
Group, Barts and the Queen Mary Medical
School
▪▪ Colin Creaser, Professor, University of Loughborough
▪▪ Sabine Becker, Head of Trace and Ultra Trace
Analysis, Research Centre Juelich
▪▪ John Langley, Head of Mass Spectrometry,
University of Southampton
Contact
Aaron Woodley
[email protected]
www.MassSpecEurope.com
• Magazine
Simple Solutions
Simplicity is the Ultimate Sophistication
olution of modern medicine. It stands to reason
“How Good Feelings Can Make You Healthy” in
Science is not only about complex formulas, figthat experts from all over the world react with
the last paragraph of the article), which was
ures or theories. It’s mainly about the results, not
some kind of mistrust to this and make their
published just shortly. Clever marketing to climb
how to get there. Albert Einstein already knew:
opinion public.
up the bestseller rankings?
”When the solution is simple, God is answerThe result was a chivvy after Bader. But it is
But no matter who is responsible for the aring.” But simple solutions are not accepted by
obvious that he is not the one to blame. Werner
ticle in the “Süddeutsche Zeitung”, he definitely
everybody. If one finds such a “god-given” anBartens is the author of the above-named article
achieved his objective: Everybody talks about
swer, envy and animosity are often the conseand therefore responsible for the content. Bader
this issue.
quence. Prof. Augustinius Bader from the Universeems to be pushed in the role of a victim, while
sity of Leipzig still adheres to this idea of simBartens is getting publicity for his new Book
plicity and got to know what it means to do so.
Philipp Praet, GIT VERLAG
(There is a reference to his new Book about
With his new stem cell derived product he gets
ready to change the medical Industry
– As simple as possible.
What you need to produce it are
stem cells from your body, taken from
your blood or your bones, cytokines,
growth factors and some other factors. All mixed together to result in a
crème – called Sanamander, or in an
injectable solution. The crème can be
used in several wounds such as flash
For more than 40 years we have been producing „Quality built on
burns, big lacerations or even paraTradition” in our plant in Germany. Our product groups stand for
plegia. Just coat your, e.g. burned skin
detailed experience in every-day use, ongoing technical advances
with the crème or inject the solution
as well as toughest demands on material, functionality and design.
into the injured tissue and let your
Users in most varying research, standard and special labs profit
body do the remaining work. The clue
from the variety, precision and reliability of all GFL products.
about this product is simple: Tell your
A vested quality demand in accordance with international
standards is documented for all GFL laboratory products with the
body that the damage can be fixed
certification to DIN EN ISO 9001: 2008.
by its own self-healing power; and
A tight-knit web of agencies and distributors worldwide ensures
your body will exactly do so.
a local presence to customers. GFL Laboratory equipment is used
Sounds like the best medical inin more than 150 countries worldwide.
vention for decades, if only the story
Deep Freezers
is reliable. During the last months,
Water Baths
Bader’s new invention, especially
Shaking Water Baths
some articles concerning this topic,
Water Stills
aroused a huge wave of disgust. Bad Incubators
er was attacked from various sides
Shakers
that the results aren’t proved by clinical trials. A fact that Bader himself
never disclaimed. Clinical trials are
long-lasting and expensive issues.
However, in his own understanding,
Sanamander is not a “miracle cure”,
as some press releases named it, nature is the only miracle. “We just figured out, how the self-healing process of our body is working, that’s
it.”, Bader states. At this point there
is a very simple way to prove whether
the crème can support wound healing or not. Wait until clinical trials are
performed. Time will prove the concept, – or not.
But if Bader is such a humble man,
where does all the disgust come
from? The stumbling block is an article written by Werner Bartens and
published in the “Süddeutsche ZeiGFL Gesellschaft für Labortechnik mbH · Schulze-Delitzsch-Strasse 4 · 30938 Burgwedel / Germany
tung” in Germany. Bartens, not Bader
Phone +49 (0)5139 / 99 58 - 0 · Fax +49 (0)5139 / 99 58 21 · E-Mail: [email protected] · www.GFL.de
describes Baders research as the rev- GFL at the ANALYTICA in Munich / Germany, 23 -26 March 2010: Hall B1 / Booth 231/330
Quality
built on Tradition
Magazine •
Regenerative Medicine
The Past, the Present, and the Future
Regenerative medicine is a rapidly growing, multidisciplinary field that
seeks to replace, repair, and/or enhance biological function in tissues and organ that has been lost due to congenital abnormalities, injury, disease, or
aging. In this the goal is to harness the intrinsic biological ability of the human body. The basic concept of using a more biologic approach in the development of medical implants and related treatments goes back to the first
half of the 20th century; however, the modern era only began a quarter century ago.
Robert M. Nerem, Ph.D.
Professor Emeritus at
the Georgia Institute of
Technology in Atlanta,
Georgia, USA
Neuronal stem cells from the brain with migrating precursor cells. (Credits: University of Tuebingen,
ZRM/Institute of Anatomy)
The initial focus was on replacement tissues, i.e.
developing substitute tissues outside of the body
for implantation into the body, with skin substitutes being some of the first targets. Although in
the 1990s these had moved into commercial development, by the beginning of this decade
commercial activity had for the most part encountered financial difficulties. Even while commercialization in this area was going through its
“ups and downs,” the science was moving
ahead. In the 1990s stem cell technology began
to emerge and the focus on replacement evolved
to include repair and regeneration with the result is that today there are a variety of approaches that are being pursued. The biological complexity of many if not most of the tissues and
organs of interest demands a broad range of approaches. Furthermore, this complexity suggests
that repair and/or regeneration may be the more
advantageous strategy. In this it must be recognized that the functional characteristics of a cell
is orchestrated by a “symphony of signals.” This
“symphony” is made up of soluble molecules,
the substrate and extracellular matrix to which
the cell adheres or is surrounded, cell-cell contact, and the physical forces, i.e. the mechanical
environment in which a cell resides.
The interest in stem cells is because a major
issue is cell source, and there are a variety of
types of stem cells. It is the embryonic stem cell
(ESC) that has the ability to produce every cell
type in the human body. Derived from embryos
created through in vitro fertilization, for some
such cells raise ethical issues. Then there are the
various adult stem cells, with the most common
being those that are bone marrow-derived such
as mesenchymal stem cells (MSCs). A more recent and exciting advance was the demonstration that a somatic cell like a skin cell could be
reprogrammed to create what is called an induced pluripotent stem (iPS) cell. Seemingly like
ESCs, there is still much that we have to learn
about iPS cells, and it may well turn out that iPS
cells, though similar to ESCs, have their own distinct characteristics. Finally, there are a variety of
progenitor cells in the human body. These have
limited potency. One example is the endothelial
progenitor cell that circulates in blood, homing
in so as to repair vascular injury. Which of these
different types of stem/progenitor cells will be
important will very much depend on the particular tissue or organ and whether the approach is
one of replacement, repair, or even regeneration.
As exciting as the advances in the biology of
stem cells and progenitor cells has been, regenerative medicine is more than stem cells. This is
because for a strategy to be successful will require that one delivers the cells and/or the necessary signals, i.e. those that will orchestrate the
desired cell function, at the right place and at
the right time. This “symphony of signals” could
be provided by the cells employed in the particular strategy used; however, if we actually understood the signals required and how and when to
deliver them, in many cases the approach might
be acellular in nature.
What is now becoming intriguing to many in
this field; however, is the concept of regeneration. The regenerative processes observed in
species such as the newt and salamander have
been replaced in the human by processes of in-
• Magazine
flammation and scar tissue formation. For the
human, the extent to which we can jump start
the regenerative process and supply the appropriate ingredients will dictate our success in
achieving tissue and organ regeneration. For
now it seems that the regeneration of adult human tissue is in the realm of science fiction;
however, there is much that can be learned from
the developmental process and thus from developmental biology, even though the geometric
scale and the time scale will be very different in
the adult than the developing embryo.
It is clear, however, that no single approach
will solve all problems; rather, each tissue and
each pathologic condition is likely to require a
different approach to obtain optimal results.
Furthermore, if stem cells are to be used, whether they are ESCs, MSCs, iPS cells, or some other
type of cell, there will be a need for the development of processing systems that will allow for
the expansion of cells and their differentiation
with the quality control necessary for their use
in clinical therapies.
The clinical translation of regenerative medicine, however, will require more than scientific
discovery and the advancement of the relevant
technologies. Regulatory agencies must become
educated in non-traditional approaches and develop appropriate guidelines for safe and effective delivery of regenerative medicine strategies.
Third party payers must come on board quickly
to sustain promising approaches and reward regenerative medicine strategies that have the
potential to significantly affect health care. Although the industry appears to have turned the
corner, these regulatory and reimbursement issues will need to be addressed if this industry is
to thrive. Furthermore, only then can the therapies developed through regenerative medicine
be available in the widest possible way.
With the ever accelerating advances in the
science and technology, regenerative medicine
has the potential of truly living up to the promise of delivering therapies for diseases, injuries,
and disorders where currently patients have no
options. As we look to the future, what are some
of the advances that can be envisioned? To start
with, there will be in vitro models of tissues and
organs fabricated from human cells and used in
drug development and toxicity testing. There will
be blood cells derived from stem cells and expanded in vitro, thus reducing the need for blood
donors. One can also envision an insulin-secreting, glucose responsive bioartificial pancreas,
and this may not be that far away. For children
born with a congenital heart defect, there will
be a tissue engineered heart valve fabricated of
living cells and one that grows with the child as
the child grows. There also will be cell-based
therapies for the repair of the wall of the heart
following a heart attack. And finally, perhaps the
real “holy grail” is the repair, even regeneration,
of the central nervous systems.
Acknowledgement
The author is an Institute Professor Emeritus at
the Georgia Institute of Technology in Atlanta,
Georgia, U.S.A. He serves as the Director of the
Georgia Tech/Emory Center (GTEC) for Regenerative Medicine, a unique interinstitutional research center bridging from the basic biology to
the enabling engineering technologies to the
clinical application. The author also is a Distinguished Visiting Professor in the World Class
University Project at Chonbuk National University in Jeonju, Korea, and while there he authored this article.
Professor Nerem will also present the plenary
opening lecture of the BioStar 2010 – 4th Congress on Regenerative Biology and Medicine,
13.–15. 10. 2010 in Stuttgart, Germany.
Contact
Robert M. Nerem
Parker H. Petit Institute for Bioengineering and
Bioscience
Georgia Institute of Technology
Atlanta, Georgia USA
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Events •
Lab-on-a-Chip World Congress
er will be running “Microfabrication Technologies for Microfluidic
Devices”.
Also at this year’s meeting we
are pleased to announce that there
will be a free workshop which is
being run by Aushon. Full details
will be announced on the website
soon.
Select Biosciences is pleased to announce their 2nd annual Lab-on-aChip World Congress. This year’s
event is being held in the fabulous
location of La Jolla, San Diego, CA,
USA - Thursday 28th and Friday 29th
October 2010.
The congress is being co-located with Microarray World Congress,
Molecular Diagnostics World Con-
gress and Single Cell Analysis Summit. All delegates will have access
to all four meetings and the large
combined exhibition, ensuring a
very cost effective trip.
The conference will focus on
Point of care diagnostics, Microfabrication/Engineering and Life science applications, and we have secured some fantastic speakers,
along with a brilliant keynote
speaker; Professor Jon Cooper from
Glasgow University in Scotland.
The conference can also boast
two training courses, which will
take place prior to the main event,
on Thursday 27th October. Nicole
Pamme will be running “Principles
and Applications of Microfluidics in
the Life Sciences” and Holger Beck-
Contact
Aaron Woodley
Exhibition Manager
[email protected]
Sara Spencer
Conference Producer
[email protected]
www.selectbiosciences.com
Munich to Become the Capital of
Biotech Dealmaking in November
For the first time in ten years BIOEurope, Europe´s largest partnering
event for the life science industry,
returns to Munich. From November
15–17, 2010 Munich will be the
center of dealmaking activity for
the biotech industry.
Munich is home to one of the
largest and most vibrant biotech
clusters in Europe. Featuring 180
life science companies, the Munich
biocluster employs 8,800 people.
The success of the region can be
attributed to a rapidly developing
commercial biotechnology industry,
world-class scientific research institutions, excellent infrastructure,
efficient access to capital, and
highly qualified employees.
The BIO-Europe event was last
held in Munich in 2001, and in the
intervening years it has grown into
the premier biotechnology business exchange in Europe. Horst
Domdey, Managing Director, Bavarian Biotechnology Cluster, said he
is looking forward to welcoming
the event back to Munich. “We
were just a baby biocluster, not
even a cluster really, when this
event was last held in Munich in
2001. It has been a busy time for
us, launching successful companies, drugs that have been ap-
proved, as well as new development programs.”
The Munich life science industry
today is made up of 43 pharmaceutical companies and 136 biotechnology companies, 8 of which
have successfully been listed as
public companies. Among the biotechs, 118 are SMEs between them
employing 2,600 people largely
dedicated to developing therapeutics and diagnostics.
BIO-Europe is a conference that
is designed to get partnerships
started. Partnering today is a main
engine of industry growth. For big
pharma, partnering is the primary
strategy for filling pipelines with
new medicines.
At BIO-Europe, attendees use
partneringONE software to screen
through the thousands of partnering opportunities, and automatically generate a calendar of meetings. This is proving to be the most
effective way to meet with potential new partners.
www.ebdgroup.com/bioeurope
• events
The ELRIG.de meeting 2010
Titrette
®
Oral sessions will cover the topics: Design
and quality of compound libraries, Storage, Logistics and Software. The speakers are experts in
drug discovery and laboratory automation from
the most important pharmaceutical companies
like Mirek Jurzak/Ingo Kober (Merck-Serono),
Thorsten Naumann (Sanofi-Aventis), Alexander
Hillisch (Bayer Schering), Julien Grimont (Actelion), and Jerome Giovannoni (Novartis), from
academic research institutes like Edgar Specker
(FMP, Leibnitz-Institut für Molekulare Pharmakologie) and Heiko Zimmermann (Fraunhofer-Institut für Biomedizinische Technik IBMT), leading
experts from the laboratory business; Doris
Hafenbradl (Biofocus), Johannes Knob (Amgen),
Ferry De Vugt (LabServices), Martin Frey, Hamilton, consultants: Dirk Schwammkrug (Logica)
and contract research providers: Jean-Yves Ortholand (Edelris).
© Michael Mertens/Flickr.com
The ELRIG.de forum 2010, organized by ELRIG.
de and G.I.T. Laboratory Journal will be held in
the new venue “Darmstadtium” in the heart of
Darmstadt, the “City of Science”.
On November 25 in Darmstadt, Compound
Management will be the core topic of this year’s
meeting of the German division of the Laboratory Robotics Interest Group. During the past
decade compound management developed towards a distinct discipline in industrial drug discovery. Compound management is the science
of the storing and the management of very large
collections of chemically or biologically active
substances which are systematically tested for
their pharmaceutical activity with high throughput screening assays. Compound libraries and
their management therefore gained an enormous attention in the past years since the status
of the individual compounds is crucial for the effective identification of possible drugs.
In the past years compound management
changed since a growing number of small biological substances entered the scene. Instrumentation that was originally developed for the storage and management of small chemical
substances was adapted to the new compounds,
thereby creating new challenges.
www.elrig.de
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Events •
LabAutomation2011
Palm Springs Convention Center, Ca, USA, January 29–February 2, 2011
In January 2011, the LabAutomation Conference
and Exhibition will celebrate 15 years of bringing science, technology and industry together.
Throughout these years the laboratory automation community has experienced significant and
positive changes. On July 1, 2010, the community experienced one of its most significant changes yet when two organizations, the Society for
Biomolecular Sciences (SBS) and the Association
for Laboratory Automation (ALA), united as Sections under the newly formed Society for Laboratory Automation and Screening (SLAS), an inclusive worldwide organization dedicated to
advancing scientific research and discovery
through laboratory automation and screening
technology. LabAutomation2011 will be the perfect time to experience the positive impact that
the SBS-ALA merger will have on both members
and our professional community.
The LabAutomation Conference and Exhibition will continue to deliver a first-class educational program and bring forth several new exciting offerings and opportunities. Presented by
the Laboratory Automation Section of SLAS, the
event is a powerful platform for education, peer
networking, and strengthening the laboratory
automation community. It provides participants
with direct access to the world’s top 100 podium
presentations, short courses, numerous vendor
-specific workshops, over 400 exhibit booths
and many new product launches.
Each year the event plays host to a line-up of
influential, forward-thinking industry visionaries.
In 2011, SLAS is proud to welcome Chad Mirkin,
Ph.D., who in 2009 was asked by President
Obama to participate as a member of the President‘s Council of Advisors on Science and Technology; John Butler, Ph.D., a Fellow and Group
Leader of the National Institute of Standards
and Technology; and Daryl Lund, Ph.D., Editor-inChief of Journal of Food Science, Institute of
Food Technologists.
LabAutomation2011 also boasts an extensive awards program that recognizes individuals
who have contributed to laboratory automation
and technology advancement. Among those
awards are the Young Scientists Award, which
provides travel to and lodging at LabAutomation2011 to students whose winning poster has
been selected at another event, and the SLAS Innovation Award, which recognizes the work of
those unique and special podium presentations
at LabAutomation that are exceedingly innovative and contribute to the exploration of technologies in the laboratory.
In the spirit of innovation, each year at LabAutomation dozens of new products are
launched. To celebrate these breakthroughs and
acknowledge the best of what‘s new, a team of
experts will select up to three of the most promising new products launched on the exhibit floor
for the official SLAS New Product Award (NPA)
Designation.
LabAutomation2011 participants will benefit
greatly from the highest caliber of scientific presentations covering a variety of industries and
scientific disciplines focusing on the following
educational tracks: Detection and Separation,
Micro- and Nanotechnologies, High-Throughput
Technologies, Informatics, and Evolving Applications of Laboratory Automation, featuring Agriculture and Food.
The Conference and Exhibition is a five-day
event taking place January 29-February 2 at the
Palm Springs Convention Center, Palm Springs,
CA, USA. To help make the LabAutomation2011
experience affordable, participants may take advantage of the Smart-Savers Discount Program.
This innovative cost-savings program offers discounts on everything from registration to hotel
to travel, including: hotel rates as low as $139
per night, $200 off airfare, complimentary registration for academics and those unemployed,
and more. For more detals and register, visit
www.labautomation.org/LA11.
www.labautomation.org/LA11
The revolution begins
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QIAGEN revolutionizes sample preparation
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Screening
News •
+++ News +++News +++
Expanded Collaboration
Argenta, a service division of Galapagos, announced it has signed a contract extension with Genentech, a member of the Roche Group. The extended agreement expands the portfolio of projects which are in collaboration with Genentech and allows Genentech to place projects directly into
Galapagos’ other service division, BioFocus. The collaboration dates back
to 2005. The current agreement covers a number of drug discovery programs that utilize Argenta’s expertise in computer-aided drug design
(CADD), medicinal chemistry, and in vitro biology and screening to discover
new chemical entities acting against undisclosed drug. Under the announced amendment, BioFocus will provide Genentech with integrated
medicinal chemistry, in vitro biology and ADME services.
www.argentadiscovery.com
www.biofocus.com
Spin-Out
Preclinical Oncology Services Limited (PRECOS) announced its official
launch following its spin-out from the University of Nottingham. The newly
formed company combines the innovation of one of the Country’s leading
universities with the scientific background and expertise of multi-disciplinary scientists. This places the company in a position to deliver specialist
services, backed by significant industry expertise and cutting-edge technology, to help both pharmaceutical and biotechnology companies develop
new anti-cancer drugs.
www.precos.co.uk
Collaboration for the Development of Biomarkers
Ariana Pharma announced that it has started a collaboration with the US
Food and Drug Administration (FDA). Ariana Pharma is providing its KEM
Biomarker technology to help enable FDA reviewers to analyze pharmacogenomic data combined with patient characteristics for biomarker signatures submitted through the FDA’s Voluntary Exploratory Data Submission
(VXDS) program. This collaboration directly relates to the FDA’s desire to
develop better tools for the analysis of genomic data in the context of the
development of personalized medicine. This collaboration is intended to
help the FDA systematically identify potential genomic “fingerprints” and
develop recommendations for the analysis of genomic data prior to submission of biomarker signatures.
www.arianapharma.com
Monoclonal Antibodies
GlaxoSmithKline (GSK) and Lonza announced that they have entered into
a new agreement under which Lonza will support the ongoing development of GSK’s biopharmaceutical pipeline by supplying manufacturing capacity for five early stage monoclonal antibodies. Under the terms of the
agreement, Lonza will initially manufacture clinical trial batches of five
compounds currently in Phase 1 and 2 for GSK. The company will also provide access to flexible capacity to enable GSK to respond to future demand
dependent upon progression of molecules through late stage development
and commercial launch. All other details of the agreement remain confidential.
www.lonza.com
www.gsk.com
Protein Research
Protagen, a specialist for in vitro diagnostics and GMP-compliant protein
analysis announced a second round of finance totaling a volume of € 10.0
Mio. After closing a first round of € 3.7 Mio. in August 2009, the NRW.
BANK, Dusseldorf, participated as new investor and joined the consortium
of MIG Fonds, Munich, S-Capital Dortmund, S-Venture Capital Dortmund
and KfW, Bonn, to finalize the actual round. Also the existing investors
contributed significantly to the second closing. The capital will be used for
the targeted expansion of the business unit Diagnostics and the clinical
validation of proprietary diagnostic marker proteins.
www.protagen.de
JX-594 for the Treatment of Cancers
Jennerex, a private clinical-stage biotherapeutics company focused on the
development and commercialization of first-inclass targeted oncolytic
products for cancer, and Transgene, a bio-pharmaceutical company specialized in the development of immunotherapeutic products, announced that
they have entered into an exclusive partnership to develop and commercialize JX-594 for the treatment of solid tumors in Europe, the Commonwealth of Independent States (CIS) and the Middle East. The lead cancer
biotherapeutic product, has shown anticancer activity and a welltolerated
safety profile in Phase 1 and Phase 2 clinical trials. Objective tumor response has been demonstrated in a variety of cancers including liver, colon,
kidney, lung and melanoma.
www.transgene.fr
www.jennerex.com
Metastatic Breast Cancer
Immutep announced the publication of a clinical research paper showing
that its lead product, IMP321, given with first-line paclitaxel achieved clinical benefit in 90 % of metastatic breast carcinoma (MBC) patients. Correlations were observed with both the patients’ monocyte (i.e. the primary target cell for the drug) count before treatment and the degree of activation of
monocytes during treatment. The study was an open-label fixed-dose-escalation trial carried out in three cancer centers in the Paris region. IMP321
induced both a sustained increase in the number and activation of APC
(monocytes and dendritic cells) and an increase in the percentage of NK and
long-lived cytotoxic effector-memory CD8 T cells. Clinical benefit was observed for 90 % of patients with only 3 progressors at 6 months. Also, the
objective tumor response rate of 50 % compared favorably to the 25 % rate
reported in the historical control group.
www.immutep.com
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Pharmaceutics & Drug Discovery •
Bacteria - Fighters Against Cancer
Bacteria as Promising Tools for Cancer Therapy
200 years ago the observation was already described that tumors
sometimes shrink when cancer patients undergo bacterial infections. This is the basis for bacteria‑mediated cancer therapy – an
alternative therapeutic approach of growing interest. Several bacterial strains are able to colonize solid tumors after systemic administration; a few even induce tumor reduction. Apparently, the
virulence of bacteria is crucial for an anti‑tumor effect. Now the
major challenge is to tailor bacterial strains that combine safety
with therapeutic efficacy.
History
At the beginning of the 19th century,
Vaultier noticed that bacterial infections
of cancer patients were associated with
shrinkage of the tumors [1]. A first report
of intentional treatment following this
groundbreaking observation dates to the
year 1868. The German physician W. Busch
placed a woman with an inoperable sarcoma into a bed that had been occupied
before by a patient suffering from erysipelas – a Streptococcus pyogenes infection.
The woman successfully became infected
and her tumor shrank. Unfortunately, she
died of the infection nine days later [2].
The toxicity of bacteria could not be handled at that time. Therefore, similar attempts with different bacteria or bacterial
products – like the well known Coley’s
toxin – failed, despite some success regarding the anti-tumor effect.
Nowadays, the situation has dramatically changed. Knowledge about hostpathogen interactions, the genome information of bacteria as well as state of the
art molecular methods allow modulation
of bacteria for particular purposes. Hence,
the approach of using bacteria in tumor
therapy presently undergoes a renaissance and is under intensive investigation.
Different types of bacteria can
colonize tumors
Amongst the bacteria that are able to target and colonize tumors are obligate an-
Dr. Siegfried Weiß and Dr. Sara Leschner,
Helmholtz Centre for Infection Research,
Braunschweig, Germany
aerobics like Clostridia and Bifidobacteria
but also facultative anaerobics like E.coli
or Salmonella. As it is a characteristic of
most solid tumors to have regions of low
oxygen, it appears reasonable to use bacteria that can only grow under hypoxic
conditions. This would avoid adverse effects on healthy tissues. However, it is just
this specificity which limits the potential
of obligate anaerobic bacteria. They will
leave well-oxygenated tumor areas unharmed from which the cancer can regrow. In contrast, facultative anaerobic
bacteria should have the potential to colonize all areas of a solid tumor and thus
exhibit a stronger anti-tumor effect.
Entering the tumor
Although it could be shown for various
bacterial strains that they can colonize
tumors, it is still not absolutely clear how
they manage to enter the cancerous tissue. This is a crucial point as an efficient
anti-tumor effect demands sufficient colonization. Different scenarios of the entry process can be envisioned. One suggests an active mechanism where
bacteria are chemoattracted by substances of quiescent or necrotic tumor
cells [3]. Alternatively, we propose that
bacteria are passively flushed into the
tumor tissue through its leaky vasculature. Upon intravenous application of S.
Typhimurium to tumor-bearing mice, a
rapid and strong influx of blood into the
tumor can be observed (fig. 1). At the
place of this hemorrhage, a huge necrosis forms. Salmonella are colonizing this
part and the region bordering the viable
tumor rim. TNF-α was identified as one
mediator that plays a crucial role in this
process [4]. We consider this an impor-
• Pharmaceutics & Drug Discovery
Fig. 1 Early events after an intravenous Salmonella infection of
tumor bearing mice. Top: A subcutaneous CT26 colon carcinoma
tumor in an uninfected mouse appears light and H&E stainings of
paraffin sections show mostly viable (V) tumor tissue. Middle:
Shortly after an i.v. infection with Salmonella, the tumor turns
dark due to a strong erythrocyte infiltration peaking at 12 h post
infection (p.i.). Bottom: The hemorrhage is cleared from the tumor
by 24 h p.i. and in the core of the tumor a necrotic region (N) has
developed. (Adapted from Leschner et al. PLoS ONE 4(8): e6692.
doi:10.1371/journal.pone.0006692)
tant finding. Clinical studies
with an attenuated mutant
strain of S. Typhimurium exhibiting a diminished potential to induce TNF-α showed
only very poor tumor colonization in human cancer patients [5]. Thus, the challenging task is to generate mutant
strains that are virulent
enough to efficiently colonize
tumors and at the same time
attenuated enough to be
safely administered.
Controlled expression of
therapeutic molecules
Particular bacteria do have
anti-tumor effects when administered systemically. Apart
from this natural ability to
cause tumor shrinkage, they
can also be used as vectors to
deliver therapeutic molecules
directly into the tumor, thus
enhancing killing of the cancerous cells. These molecules
could be bacterial toxins, cytokines that activate an anti-
tumor immune response or
prodrug converting enzymes.
Independent of which protein
to be expressed, it is essential
that the mode of expression
can be controlled tightly to
prevent adverse effects on
healthy tissues. This could be
achieved by the use of special
promoters to drive expression,
like inducible promoters. An
example would be the E.coli
promoter PBAD that can be induced by the sugar L-arabinose (fig. 2). Administration of
this sugar to mice infected
with bacteria that encode the
therapeutic molecule under
control of PBAD expression can
be started at any defined time
point [6]. Other possibilities
are the use of in vivo inducible
promoters. For instance, we
have defined several promoters that respond to the special
physiological conditions of the
tumor tissue but are silent in
other organs. Therefore, the
expression of a therapeutic
molecule can be rendered tu-
Fig. 2 The inducible promoter PBAD allows to induce gene
expression in tumor colonizing bacteria by addition of the sugar
L-arabinose. Tumor bearing mice were infected with Salmonella
that carry the bioluminescence operon of Photorhabdus luminescens under the control of the inducible promoter PBAD. The inducer L-arabinose was administered 24 h p.i. to the mice resulting in
bioluminescence in the tumor, which can be visualized by an in
vivo imaging system. Pictures show mice 0.5 h, 5 h and 24 h after
L-arabinose application.
mor specific. Using these control elements, the expression
of the potentially toxic substances in healthy tissues
should be prevented leaving
them unharmed by the therapy.
Conclusion
Many different bacteria have
demonstrated their potential
to be used in cancer therapy
as they are targeting solid tumors. However, the demands
for an ideal anti-cancer bacterium include more than that. It
has to be safe and efficient at
the same time. A combination
that appeared to be unsolvable when this therapy had
been applied first more than
100 years ago. With today’s
knowledge and the possibilities of molecular genetics to
tailor bacteria to the very special purpose, a successful application of bacteria in cancer
therapy appears to be in
reach.
References
[1] Barbe S. et al.: J.Appl.Microbiol. 101:571-578 (2006)
[2] Pawelek J.M. et al.: Lancet
Oncol. 4:548-556 (2003)
[3] Kasinskas R.W. et al.: Biotechnol.Bioeng. 94:710-721
(2006)
[4] Leschner S. et al.: PLoS.One.
4:e6692 (2009)
[5] Toso J.F. et al.: J.Clin.Oncol.
20:142-152 (2002)
[6] Loessner H. et al.: Cell Microbiol. 9(6):1529-37 (2007)
Authors
Dr. Siegfried Weiß and Dr. Sara
Leschner, Helmholtz Centre for
Infection Research, Braunschweig, Germany
Contact
Dr. Sara Leschner
Molecular Immunology
Helmholtz Centre for Infection
Research
Braunschweig
Germany
[email protected]
www.helmholtz-hzi.de
Application note •
Reproducible Cell Assays
Consistency of Cryopreservation is a Necessity
Cell Cryopreservation is a critical component of cell culture work.
The cells which survive the thermodynamic journey from the warm
temperature of the incubator to the –196°C environment of the
liquid nitrogen storage tank are free from the influences of time.
This capability provides the cell culturist with a means of taking a
snapshot of the culture at a given time in its history.
The challenge of cryogenic storage is, in a
word, ice. Cells are approximately 70%
water, and when chilled to below the
freezing point, ice crystals will form in the
cell interior, lethally disrupting the intracellular structures. Cryogenic storage methods are successful only because the process includes a reduction of the
intracellular water content prior to freezing, and, with the added benefit of a cryoprotectant, is successful in sufficiently
limiting ice crystal growth. As the freezing
process initiates in the extracellular fluid
space, the forming ice crystals exclude
and concentrate the dissolved solutes.
The degree of dehydration of the cell is
a key parameter that is controlled by the
rate of temperature decrease. If the rate
of temperature reduction is too low, the
cells will become dehydrated beyond the
critical water content survival limit due to
prolonged exposure to the exterior concentrated salt solution. In addition, the
added time spent in the high salt concentration environment can have a deleterious effect on cell health through exposure
to inappropriate pH, toxic ion levels and
concentrated solute-induced cell surface
protein denaturation. Conversely, should
the rate of temperature reduction be too
great, the cell interior will supercool and
ice crystals will nucleate, initiating interior
ice crystal growth while the interior water
percentage is still dangerously high.
The two opposing boundary conditions
restrict the freezing rate associated with a
peak of cell viability to a narrow range.
The value for the optimal freezing rate
may vary with cell type and is dependent
upon both cell size and membrane permeability. Fortunately, for a large portion of
cultured mammalian cell types, in the
presence of common cryoprotectants such
as DMSO, the optimal freezing rate will
coincide with a value of -1°C/min, and any
means of reliably attaining this rate of
freezing will be beneficial in the cryopreservation process.
Control Crossways
There are two main avenues for achieving
a controlled rate of cell freezing. The first
and most expensive one involves the use
of microprocessor-controlled refrigeration
systems that can be programmed to follow a pre-determined profile of temperature reduction.
The second avenue leads to the use of
passive freezing units, which exploit the
consistent thermodynamic principles of
temperature differentials and thermal
conductivity. Starting with a reliable thermal sink such as a -80°C deep freezer or
dry ice locker (-78°C), cell vials can be encased in a device that will, through an ap-
propriate combination of
thermal capacity and insulation, provide a freezing
profile with the desired
temperature
reduction
rate.
Numerous other protocols for cell freezing include
steps such as wrapping the
vials in paper towels, cotton or tissue, or encasing
the vials in recycled styrene
foam tube racks.
A common acceptable
threshold for the success of
these freezing methods is that sufficient cells be recovered alive upon
thawing to repopulate a culture flask
within a reasonable timeframe, while dismissing the fact that such methods can
result in cell cultures populated by a sub-
Fig. 1: High post-thaw cell viability. HUVEC cells were resuspended in freezing medium at
a concentration of 2 x 106 cells per ml. 1 ml aliquots were portioned into 1.8 ml Corning
cryovials and frozen at -1°C per minute in either a Biocision CoolCell or in an alcohol
filled cell freezing unit. Five vials frozen by either method were rapidly thawed and resuspended in growth media. Live cell count were obtained by the trypan blue exclusion method
• Application note
archived samples. The negative impact
upon archived samples due to repeated
temperature cycling is avoided in diligent
laboratory practice by assigning a common and remote region of the freezer to
the cell freezing process. This practice,
however, imposes a secondary concern in
that busy laboratories can often require
the freezing of samples generated by multiple researchers, and the combined heat
from two or more alcohol freezing containers in the same location will significantly alter the temperature reduction
profile of all containers present.
Alcohol-filled freezing containers also
require that the alcohol be changed every
five uses as absorbed moisture and evaporation can alter the heat capacity of the
system and thereby cause variance in the
thermal profile. In addition to the cost, the
alcohol replenishment results in continuous generation of contaminated solvent
that must be removed through hazardous
waste streams. In daily practice, tracking
the number of use cycles requires vigilance and, as most alcohol freezing units
are laboratory community property, the
consistency in maintenance descends to
the performance level of the least diligent
lab member.
Likewise, mistaken replacement of the
alcohol with an alcohol other than the required isopropanol is a repeated error
made by researchers unmindful of the fact
that different alcohols have significantly
different heat capacities and that switching alcohols will alter the freezing profile.
CoolCell System
set of the original culture. The selective
influences imposed upon the frozen cell
sample can result in a wide variation in
cell function and, in the worst case, lead
to unrepresentative cell performance, assay results, biomarker behavior or cellbased diagnostic parameters.
Alcohol-Filled Systems
The alcohol-filled systems rely on a large
thermal mass and high heat transfer to
slow the sample cooling rate to approximately –1°C/min. These insulation-free
designs depend upon the thermal conduction limits of the alcohol and the heat
transfer limits of the air inside the freezer
to regulate the heat flow, in effect controlling temperature reduction by temporarily
overwhelming the heat removal capacity
of the freezing unit. The heat lost from the
alcohol (250 mL) is approximately 10 x
greater than the heat removal required for
sample freezing, placing a greatly amplified thermal burden on the refrigeration
system that has the potential to cause
temperature fluctuations in locally stored
A recent alternative to alcohol-filled freezing containers is found in the radiallysymmetric insulation solid-state core (SSC)
based design of the BioCision CoolCell
product, which takes advantage of the
combination of precision insulation geometry and small solid core thermal ballast
(eye catcher).
The core has a total heat capacity that
is approximately 7% of the alcohol-filled
container system. The total heat capacity
of a fully loaded unit is less than that of a
typical freezer box of samples, therefore
the freezing unit can be confidently placed
next to previously archived samples without imposing a damaging thermal fluctuation. As the physical positioning of the insulation and the heat capacities of the
insulation and solid core materials are unalterable, when placed into the constant
temperature environment of a typical regulated deep freezer, the contained samples will experience very consistent freezing profiles (fig. 1).
Moreover, the unit can be used repeatedly and indefinitely with no maintenance
Fig. 2: Highly reproducible freezing profiles. 1 ml of cell freezing media was
placed into12 Cryovials. A thermocouple
probe was introduced into one vial in an
axial orientation with the probe end at
the center of the liquid volume. All vials
were equilibrated to 20°C, then loaded
into a Biocision CoolCell, placed into a
-80°C freezer and internal vial temperature was recorded by a data logger at 10 s
intervals. After a 4 h freezing cycle, the vials were removed, thawed and equilibrated to 20°C. The repeatability of the temperature profiles is shown with 5
consecutive freezing cycles
beyond insuring that it is dry at the time
of sample loading. This simple and widely
used method allows researcher to perform
cell cryopreservation with repeatability
and uniformity in freezing rate and postthaw performance profiles (fig. 2).
Conclusion
In summary, cryopreservation of cultured
cells is a proven and essential process. The
preservation of samples of PBMCs, stem
cells, patient cells, cell lines and other investigative cell material is of compromised
value if a the method of cryopreservation
imposes variable and unpredictable influences on the constituents of the emerging
cell population. Precision-engineered insulation alcohol-free cell freezing containers such as CoolCell represent standardizable means of providing reproducible cell
freezing profiles (figures 1 and 2). These
devices can contribute greatly in assuring
that valuable experimental assets are not
only preserved, but provide consistent and
meaningful results.
References
[1] Schryver B., and Ehrhardt, R.: Surprisingly
Unscientific World of Preanalytical Sample
Handling: A Simple Method for Standardization.“ American Biotechnology Laboratory, January 2010.
Contact
Rolf O. Ehrhardt, MD, PhD
Brian Schryver
BioCision LLC
Larkspur, CA, USA
Tel.: 001/888/478-2221
[email protected]
www.biocision.com
Pharmaceutics and Drug Discovery •
Enzyme-Amplified Array Sensing
of Proteins
Identification of Low Protein Concentrations
Irregular protein concentration levels in biofluids provide an indicator for the early detection of cancer and other disease states,
making protein sensing an important biomedical goal. In recent
studies we developed a highly sensitive enzyme-nanoparticle sensor array that uses enzymatic amplification to detect and identify
proteins at very low concentrations in both buffer solution and
biofluids.
Vincent M. Rotello,
PhD, Professor, Department of Chemistry, University of Massachusetts Amherst
Chemical Nose Approach in
Sensing
Most currently used methods for protein
sensing are based on specific recognition
between an immobilized capture agent,
such as an antibody or a receptor, and the
target protein. Sensory processes such as
taste and smell, however, utilize “differential” binding events where the receptors
bind through selective interactions rather
than specific. In this “chemical nose”
strategy, a sensor array is created with different receptors that are then trained to
generate a response pattern. This strategy
has been applied to a variety of small
molecule analytes [1]. More recently, this
method has been applied to proteins, albeit with relatively low sensitivity (1-350
µM) [2,3].
In our research we have focused on
the creation of more sensitive sensors for
proteins. Using the “chemical nose“ approach, we have developed gold nanoparticle sensor arrays for protein sensing, including nanoparticle-fluorescent polymer
conjugates that could identify proteins in
buffer at 4-215 nM[4] and an analogous
GFP (green fluorescent protein) sensor for
proteins in human serum that could detect and identify changes as small as 500
nM [5].
Enzyme-Amplified Array Sensing
(EAAS)
While the methods mentioned above are
efficient biosensing systems, the sensitivity of these displacement assays is
limited by the emissivity of the fluorescent species used. To enhance sensitiv-
Fig. 1: Molecular structures of the nanoparticles, and a schematic of the sensors comprised of β-gal and cationic nanoparticles. (a) Nanoparticles binding to β-gal inhibit the enzyme activity; (b) Molecular structures of the cationic gold nanoparticles (NP1-NP6); (c)
Release of β-gal by the protein.
ity, we applied enzymatic amplification
to “chemical nose”-based sensing. Our
enzyme-amplified array sensing (EAAS)
system features three components: (a)
β-galactosidase (β-gal) as the amplifying
enzyme element; (b) cationic functionalized gold nanoparticles (~2 nm core
diameter) as the receptors/ β-gal inhibitors, and (c) 4-methylumbelliferyl-β-Dgalactopyranoside (MUG) as the fluoro-
• Pharmaceutics and Drug Discovery
Fig. 2: Detection of proteins in phosphate buffer. (a) Fluorescence
responses pattern ratio in phosphate buffer. Each value is an average of six parallel measurements. (b) Canonical score plot of the
first three factors of the response patterns.
genic substrate. The anionic enzyme β-gal
(pI =4.6, Mw = 465 kDa) was chosen
due to its stability under a wide range of
temperature, pH, and ionic strength conditions, while our cationic gold nanoparticles were used to generate differential
affinity required for sensing. In practice,
electrostatic binding of the nanoparticle
to the enzyme inhibits activity without denaturing the β-gal (fig. 1 (a)). Analyte proteins bind the nanoparticles to displace
the enzyme and restores its activity (fig.
1 (c)) [6].
We first tested the EAAS system in
phosphate buffer, using a sensor array
comprised of six different functionalized
nanoparticles (fig. 1 (b)) and β-gal. We
chose nine biomedically relevant proteins
having various size, surface charges, molecular weights, and isoelectric points to
validate the methodology. The individual
target proteins generated highly reproducible rates of fluorogenesis, developing
distinguishable patterns from the six
nanoparticles. The resulting data were analyzed through linear discriminant analysis and all nine proteins were readily iden-
Fig. 3: Detection of proteins in desalted human urine. (a) Fluorescence responses patterns ratio in phosphate buffer. Each value is
an average of six parallel measurements. (b) Canonical score plot
of the first three factors of the response patterns.
tified (fig. 2), with a limit of detection/
identification of 1 nM.
To demonstrate the applicability of this
methodology for detection in real-world
biofluids, we first focused on human urine
– a highly used clinical sample due to its
availability and ease of collection [7].
However, it is a challenging biofluid for
sensor design due to high overall protein
concentrations (>1.5 μM, 0.150 g/L). Despite this challenging matrix, we successfully obtained reproducible fluorescence
that allowed full discrimination of proteins spiked into the analyte solution at 1
nM concentration (fig. 3). In our future
studies, we are developing platforms for
the application of this methodology to
clinical settings.
Support from the NSF (DMI-531171,
CHE-0808945, and DGE 0504485) and
NIH (GM077173) is gratefully acknowledged.
References
[1] Goodey A. et al.: J. Am. Chem. Soc.123,
(11), 2559–2570 (2001)
[2] Baldini, L. et al.: J. Am. Chem. Soc. 126,
(18), 5656–5657 (2004)
[3] Wright, A. T. et al.: Angew. Chem. Int. Ed.
44, (39), 6375–6378 (2005)
[4] You, C-C. et al.: Nat. Nanotechnol. 2, (5),
318–323 (2007)
[5] De, M., et al.: Nat. Chem. 1, (6), 461–465
(2009)
[6] Miranda, O. R. et al.: J. Am. Chem. Soc. 132,
(14), 5285–5289 (2010)
[7] Thongboonkerd, V.: Mol. BioSyst. 4, (8),
810–815 (2008)
Authors
Xiaoning Li, BS, PhD student; Brian Creran, AB,
PhD student; Vincent M. Rotello, PhD, Professor, Department of Chemistry, University of
Massachusetts Amherst
Contact
Rotello Research Group
Department of Chemistry
University of Massachusetts at Amherst
Amherst, USA
[email protected]
www.umass.edu/rotellogroup
Particle Measurement •
Aerosol Albedometer
A Tool for Measuring Optical Scattering and Extinction of
Dispersed Aerosols
Atmospheric particulate matter (aerosols) decrease visibility and are believed to affect climate by scattering and absorbing solar radiation aloft.
Scattering by aerosols leads to an increase in planetary albedo (reflectivity)
while light absorption can lead to warming of the atmosphere. In this work,
an instrument to simultaneously measure aerosol scattering, extinction, and
albedo is summarized. It is believed this method may find use in both field
and laboratory studies of aerosol optics.
Background and Rationale
The attenuation of a beam of monochromatic
light through an aerosol cloud can be modeled
through the Beer-Lambert law relationship:
(1)
where bext is the extinction coefficient (here we
use Mm–1 units) and z is the path length. In turn,
the extinction coefficient is the sum of scattering and absorption coefficients, and single scatter albedo (ω) the ratio between the effects of
scattering and extinction:
(2)
(3)
The reduction in local visibility and net climate
effect of the aerosol depends on a number of
factors including aerosol scattering coefficient
(bscat), extinction coefficient (bext), and the single
scatter albedo (ω). Therefore, precisely measuring these variables on both lab generated aerosol mimics and genuine ambient aerosols is of
considerable interest.
Traditionally, scattering and extinction have
been measured separately through nephelometry and long-path length optical loss/extinction
measurements (transmissometry). A major step
forward occurred in 2001 when Smith and Atkinson applied cavity ring-down spectroscopy
(CRDS) to aerosol extinction measurements [1].
Soon thereafter additional groups reported aerosol extinction measurements based on CRDS
[2,3]. CRDS provides the requisite sensitivity for
the measurement within a portable, compact instrument package. Similarly, interest in integrating sphere (reciprocal) nephelometry by several
groups [4, 5] has led to improvements in device
performance. All nephelometers cannot collect
light over all angles equally. This leads to an an-
▲ Image of wildfire smoke being transported
southward from Quebec over the great lakes
and northeastern United States. The image was
acquired by the Moderate Resolution Imaging
Spectroradiometer (MODIS) on the TERRA satellite on July 7, 2002. Visual inspection reveals the
smoke plume appears very different from the
other haze / clouds. This difference may be due
to a difference in single scatter albedo. Image
courtesy of Jesse Allen / NASA Earth Observatory.
◀ Fig. 1: Illustration of the albedometer. Extinction coefficient is measured through determining τ, the CRDS cell time constant. Scatter coefficient is determined by measuring intensity of
scattered light on a second independent channel
through the scattering photomultiplier. The photograph illustrates the interior of one-half of the
sphere nephelometer. The transparent tube, diffuse reflectance material, interference filter, and
arc shaped light baffle are apparent.
• Particle Measurement
gular truncation. For commercial devices, the
truncation angle is often 5–15°. The main technical advantage of integrating sphere nephelometry is this angle can be reduced to < 5°. Additionally, scattered light is collected over a solid
angle of nearly 4π steradians – a condition that
can help improve limits of detection. The albedometer directly builds upon these approaches by combining the technical advantages of
CRDS with integrating sphere nephelometry.
Device Function
Figure 1 illustrates the experimental setup similar to that originally reported in our recent technical works [4,5]. Ambient air is drawn through
either the aerosol inlet or an air filter. The filter
can remove particles from the sample which
provides a spectroscopic blank. The sample is
then drawn through either the internal volume
of a sphere nephelometer itself or a transparent
tube placed within the sphere. The albedometer
employs CRDS with a frequency doubled Nd:YAG
laser to make extinction measurements at 532
nm. In CRDS, the rate of light attenuation is
measured as a short pulse of light circulates in
an optical resonator formed between two highly
reflective mirrors (R > 0.999). After the light is
introduced into the resonator, the beam is
switched off and the light intensity then exponentially decays in time (first order) with a time
constant τ. The time constant τ is the time required for the intensity to fall to 1 / e of its original value. Since mirror reflectivity is fixed, only
light absorption and scattering by the sample
(placed between the mirrors) leads to a change
in rate of optical loss and a corresponding
change in τ. The cavity time constant (τ) can
then be linked to sample extinction coefficient
through the equation shown in figure 1 if mirror
reflectivity (R), distance between mirrors (L), and
time required for light to make one “round-trip”
through the cell (tr) is known. The effects of both
particles and absorbing gases can lead to changes in τ. Experimentally, the effect of gases is
subtracted by using filtered air as the spectroscopic blank. Ring down times on the order of
25–35 µs are often encountered for our system,
offering detection limits for bext < 1 Mm–1. In
the setup, ambient pressure and temperature
are also monitored at the measurement cell outlet. This allows for corrections in Rayleigh scattering due to changes in air density to be accounted for. Sample relative humidity (RH) is
also recorded since aerosol optical properties
are known to change with RH.
Simultaneously, on a second channel the device measures light scattered from the reflecting
beam through use of an integrating sphere
nephelometer and second photomultiplier tube.
The interior of the 30.5 cm sphere we use is
coated with a Lambertian diffuse reflectance
material (also illustrated in fig. 1) with R > 0.95
for the visible region. The scatter channel signal
also exhibits an exponential decay in time. For
quantitative analysis, the scatter channel detector signal (ISCAT) is ratioed to the CRDS channel
detector signal (ICRDS) at all points along the
curve. Strawa et al. [6] have shown this ratio is
linearly proportional to scattering coefficient
(bscat) through a constant K’ that takes into account efficiency of light collection, detector electronic gains etc. The ISCAT / ICRDS ratio is then
averaged for many points and this measurement
related to scattering coefficient through calibration with gases of known Rayleigh scatter coefficient (often CO2 and R-134a is used). In the
first generation design, aerosol filled the sphere.
The second generation instrument contains the
aerosol within a transparent glass tube to reduce sample volume and instrument response
time. Differential reflection off the glass tube
can bias light collection efficiency as a function
of scatter angle.
Figure 2 illustrates photographs and measured CRDS, scatter channel waveforms and
ISCAT / ICRDS ratios for ambient aerosols sampled in Pasadena, CA. The photographs and data
were taken roughly 6 hours apart and clearly
show the change in aerosol loading that occurred over this time. For the figure on the left,
dry PM2.5 aerosol extinction coefficient was
measured to be 60–65 Mm–1. The much hazier
photograph on the right illustrates a period of
time when PM2.5 aerosol extinction coefficient
was approximately 160 Mm-1. Viewing the data
traces, one can clearly see the change in the rel-
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Particle Measurement •
Fig. 2: View towards the San Gabriel mountains from Pasadena, CA on June 8, 2010. Photos were taken about 6 hours apart. Insets show optical data collected with the albedometer at approximately the same time the photos were taken. The light green trace represents CRDS data (ICRDS) and the black trace
is scatter channel measurements (Iscat). An increase in Iscat / ICRDS ratio (red trace) is clearly observed as the haze increases. A decrease in cavity time constant (τ) is observed for the thicker haze. The mountains are at a distance of approx. 8 km.
ative size of the CRDS and scatter channel signals. The ISCAT / ICRDS ratio increases from 1.5
to 3.3 with increased aerosol loading. A decrease in ring-down time constant (τ) from 16.6
to 11.2 µs is also reflected in the data.
S ample Data & Potential Technical Advantages
There are several perceived advantages of this
measurement platform. First, both scattering
and extinction measurements are made on the
exact same sample volume, simultaneously. This
helps eliminate measurement uncertainty due to
several different instruments being presented
fundamentally different aerosol samples. Aerosol particles can sometimes be lost in tubing
that connects different instruments. Furthermore, large differences in measurement volumes
can lead to imprecision and / or errors when reporting albedo. Use of the integrating sphere
design reduces truncation angle to approximately 3° on average. This increases the fraction
of scattered light which is collected. Another
unique feature of the design is that the scattering measurement is made during the CRDS transient as the beam circulates between the mirrors. Since the effective “time-constant” of the
integrating sphere itself is only on the order of
nanoseconds, any stray laser light introduced
into the sphere during the initial pulse is quickly
attenuated/removed. The CRDS beam continues
to circulate for many microseconds, and since
the scattering measurement occurs during this
time period, only light scattered from the resonating beam is detected. This assures the measurement is free of the effects of laser light scattered off the walls or surfaces of the sphere.
Conclusion
The aerosol albedometer offers simultaneous,
ensemble measurements of optical scattering
and extinction on ambient aerosols. It is believed
this platform may be of utility in making ambient measurements at fixed sites or on-board aircraft. The high sensitivity and rapid time response of the instrument may allow better
understanding of the effect of aerosols on radiative transport in earth’s atmosphere. Future developments will focus on further reduction in
device size and angular truncation. Additional
measurement wavelengths can also be added.
This research has been funded in part by the National Science Foundation (USA) under grants
1004114 and 634872
References
[1] Smith J.D. and Atkinson D.B.: Analyst, 126, 1216
(2001)
[2] Thompson J.E. et al.: Atmospheric Aerosol Measurements by Cavity Ringdown Turbidimetry.
Aerosol Sci. and Tech., 37(3), 221–230 (2003)
[3] Thompson, J.E. et al.: Monitoring Atmospheric Extinction Through Cavity Ringdown Turbidity” Anal.
Chem., 74 (9), 1962–1967 (2002)
[4] Varma R. et al.: Optics Lett. 28, 1007–1009
(2003)
[5] Fukagawa S. et al.: Applied Optics 44, 3520–3526
(2005)
[6] Thompson J.E. et al.: Optics Express 16(3),
2191–2205 (2008)
[7] Dial K.D. et al.: Anal. Chem. ASAP, DOI:10.1021/
ac100617j.
[8] Strawa A.W. et al.: J. Atmos. Ocean. Technol. 20,
454–465 (2003)
Contact
Prof. Jonathan Thompson
Associate Professor
Department of Chemistry & Biochemistry
Texas Tech University, USA
[email protected]
DEGASSER® series 300, can be validated
Semba Octave™, SMBC system in lab scale
Aerosol Detektor NQAD™, new technologie: WCPC
your individual partner in the LC
• HPLC
• GPC
• SMBC
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ERC GmbH
Otto-Hahn-Straße 28-30 · D-85521 Riemerling
phone +49(89) 660 55 696 · fax +49(89) 608 24 826
[email protected] · www.erc-hplc.de
The best testimony to the innovative power of Merck, its reliability
and close understanding of local and global markets is the sheer
diversity of its products. The Merck portfolio currently encompasses
more than 15,000 chemicals and reagents, active ingredients,
test kits and analytical systems. Every day, new products join
the fold, the result of purposeful research projects, specifically
tailored to the needs of the customer. Naturally, each project
meets Merck’s own high standards in terms of ultimate quality and
reliability – which spells peace of mind for you and more time to
concentrate on your work.
www.merck-chemicals.com
Does Merck Chemicals have something for stress?
Yes, take your pick
from nearly 15,000 products.
Each designed to put you at ease –
with ultimate purity, safety, reliability,
and plenty of fresh prospects.
The Barcelona® Chair by Knoll, Inc.
That’s what’s in it for you. Merck Chemicals
Bioprocessing •
Lost in Translation
Very Early Process Development for Biopharmaceuticals
Today, scientists are searching for proteins to be used as biopharmaceuticals in
the most remote corners of the plant and animal kingdoms. Drug candidates
are cloned, trimmed and modified and heterologously expressed in a handful of
relatively well-known host organisms. If things go extremely well for a new API
(active pharmaceutical ingredient), it is expressed and milligrams can be purified, proof-of-principle can be shown, toxicity seems to be no issue and the
need for a more useful amount for further characterization, usually along with
first considerations of conducting a clinical trial, arises.
In about a decade of upscaling and process development for a plethora of different API candidates at the Fraunhofer IME’s department for
Integrated Production Platforms, we have noticed that at this point, many promising candidates and sanguine dreams (and business plans)
die silently. This article tries to highlight a few of
putative showstoppers in early process development for biopharmaceuticals and to point at the
difficulties that will most likely occur as early as
possible.
Quick Success Versus the Search for a
Sustainable Expression
System
The first pitfalls in process development occur at
the very beginning. Unfortunately, the earlier a
decision is made that is not far-sighted enough,
the more damage will be done in later process
stages. The work of years will be reset to the
starting point because something in the process
simply turns out to be not feasible. Often, in an
long and sometimes challenging process of generating an expression clone, decisions are made
that are aimed at short-term objectives but cannot be adhered to in later phases. In other words,
a quick success is – consciously or not – preferred to longterm sustainability. For example,
commercially available bacterial expression kits
that are optimized for high-expression levels are
used in low-cell-density cultivations on complex
media. The kits often feature modular solutions
such as support for rare codons by additional
plasmids, deletions and additions in the host
strain that increase folding efficiency or stability,
strong promotors that rely on specific genetic
elements and environments and sometimes protein tags for purification or even detection of the
foreign protein. These kits are designed to make
life easier, but many elements or modules can
cause severe problems when it comes to their
usability in large-scale applications or regulatory
compliance.
Dr. Stehpan Hellwig,
Head of manufacturing,
Franunhofer IMEanufacturo
The decision for an expression cassette or
the use of a certain expression kit that promises
quick success is not wrong in itself. The problem
is that early achievements seduce to built on
them and continue the easy way without asking
how practicable it will be later. As we will see,
things that make life easier by rendering careful
design of the gene of interest, the expression
cassette and the expression system unnecessary
in the beginning might cause major discomfort
later on.
The Challenge of Scaling-Up
One of the misunderstandings we’ve been faced
frequently is the assumption that a bioreactor is
basically a large shake-flask featuring a built-in
autoclave. Processes that lead to a “fat band” in
a shake-flask or a cell cultivation system do not
always maintain their specific productivities
when translated to a bioreactor. The term “specific productivity” describes the level of expression or the accumulation of a product per biomass or per cell. Expression kits are often
tailored to low-cell density cultivations in very
rich media. Under these conditions, optimal supply of nutrients or energy and accumulation of
inhibitory metabolites is usually not a problem.
In medium with high cell density fermentations,
this may become limiting and decrease the specific productivity. Also, complex media compounds such as peptones or additives such as
protease inhibitors can be unacceptable from
the regulatory or economical point of view. As a
result, actual productivities may be lower than
the calculated numbers. This can often be fixed
by intensive process development, but it needs
resources and time to get there.
Scaling-up a laboratory purification process
to pilot scale usually includes major changes in
the separation steps. Laboratory filtration steps
sometimes rely on syringe filters and chroma-
• Bioprocessing
tography in gravity-driven columns. Also, “perform all steps on ice” is not an unusual direction
in laboratory protocols, but neither very specific
nor easy to in larger scales. Chromatography
media suited for reasonable processing times
and flow rates can differ fundamentally in terms
of resolution and capacity from those used in
gravity-flow approaches. Filtration processes
such as ultra-filtration or diafiltration, when carried out in tangential flow devices can introduce
thermal load to a process that may impair product integrity and cooling down some 50 liters of
an intermediate in a carboy takes surprisingly
long compared to 50 ml in a falcon tube.
Assuming that a process aimed at the production of a clinical-grade API has been scaled
up successfully, analytic results generated by the
QC department are likely to hold some surprises.
The purity of early-development protein preparations is usually overestimated - sometimes
simply due to the detection limits of the method
used. All of a sudden, with adequate overloading, bands of unknown identity pop above and
below the familiar band. Also, the resolution of
up-to date chromatography controllers will more
often than not raise questions regarding the homogeneity of the main elution peak by unveiling
shoulders or a distinct asymmetry that inevitably
call for further investigation and explanation.
IP issues
Assuming once again that all technical obstacles
can be adequately addressed and circumnavigated, there are legal issues that are often ignored until there is is a product, a market or
some profit foreseeable. There’s always something more urgent or more interesting to take
care of than analyzing which bits of somebody
else’s intellectual property you have been using
somewhere along the road. At this point, circumvention of a technology that has worked
well for a protein is an extremely painful decision to be made. But it’s either that or negotiation of license fees. An easily obtained research
license may turn out to be a costly mortgage on
the future if not addressed in time.
Time is Cash
Time may be linear or not, but in biopharmaceutical process development it definitely has more
dimensions than “time-to-market”. It may take
half an hour to spin down 500 ml of cultivation
broth, but in a pharmaceutical process, you’ll
still end up with 2 days for the same process
step if you account for buffer preparation, run
time and cleaning-in-place. The same applies to
other process steps in a very similar fashion.
Cost of goods also make a remarkably non-linear impression on most researchers. A raw material or consumable order somewhere under
500 Euros or dollars passes through easily without raising any eyebrows and a scale-up factor
of 100 is easily asked for. In fact we have been
asked several times for a quote on the scale-up
of laboratory protocols where the cost of buffers
alone exceeded the customer’s budget expectations for the entire project.
But these are relatively simple technical issues. More important are problems that cost
time, no matter if there is enough money available. We’ve all heard the calculations of how
many dollars of earnings are lost in a month’s
delay of the launch date of a blockbuster. Such
calculations may be rigged a little to impress the
audience, but in fact, the further a product is
down the pipeline, the more expensive and timeconsuming it becomes to clean up unsolved
problems.
Conclusion
There are many points to consider in the early
process development of biopharmaceuticals and
even this very limited and superficial flashlight
on the most easily made mistakes in the translation of a product from research to development
may be disillusioning. The take-home-message is
to involve know-how on the technical, regulatory and legal implications of biopharmaceuticals
as early as possible. On the other side, the availability and accessibility of existing information
and know-how as well as sophisticated technologies to efficiently produce and thoroughly analyze the biopharmaceuticals was never as abundant as today. Maybe it’s time to go back to one
of the fundamental virtues of scientists – think
twice before you pipet.
Fraunhofer IME – Department for Integrated Production Platforms
Fraunhofer IME is one of 5 Institutes of the
Fraunhofer Life science alliance. Building on 20
years of experience of recombinant protein production with an emphasis on antibody-derived
products and plant biotechnology, about 150
scientists are working on contract research and
development projects focused on Pharmaceutical Product Development, Plant Biotechnology
and Applied Ecology at the IME and the closelyattached institute for Molecular Biotechnology
at the Aachen University. Dr. Stephan Hellwig is
co-heading the Department of Integrated Production Platforms at the IME. The department
does contract research in protein production
process development and holds a manufacturing authorization for the GMP-compliant production of API for clinical phase one trials. Dr.
Hellwig is Head of Manufacturing in the GMP
facility.
Contact
Dr. Stephan Hellwig
Fraunhofer IME
Aachen, Germany
Head of Department – Integrated Production Platform
[email protected]
Material Sciences •
Quantification Study of Drug Delivery
by Nanocarriers
A Cell Mass Spectrometry Approach
Delivery of peptides, proteins, antibodies, vaccines and gene-based drugs by nanocarriers can greatly reduce drug
resistance and achieve a therapeutic effect in humans or animals. To understand the drug delivery efficiency by
nanocarriers, quantitative measurement is essential. We developed a cell mass spectrometry methodology that can
quantify nanocarriers uptake into mammalian cells. The quantitative study will help examine the mechanisms of different types of sustained release formulations with nanocarriers including liposomes, drug loaded biodegradable microspheres, viral nanoparticles and drug polymer conjugates.
nanocarriers is of importance in examining the
efficiency of drug delivery. However, only a few
studies have explored the quantitative measurement of the cellular uptake of nanocarriers during cell endocytosis and exocytosis process.
Traditional Quantitation Approaches
Dr. Wen-Ping Peng, Department of Physics, National Dong Hwa University, Hualien, Taiwan
Dr. Alice L. Yu, Genomics Research Center,
Academia Sinica,
Taipei, Taiwan
Dr. Chung-Hsuan Chen,
Genomics Research
Center, Academia Sinica, Taipei, Taiwan
Nanocarriers Used in Drug Delivery
The use of nanocarriers in drug delivery can enhance the intracellular concentrations of drugs.
In general, nanocarriers bind specific receptors
and enter the cancer cells via receptor-mediated
endocytosis. With the help of nanocarriers, drug
molecules can easily bypass the recognition of
P-glycoprotein and thus reduce the drug resistance [1]. The concentration of nanocarriers uptake into cells is proportional to the drug concentration. Therefore, the quantification of
Inductively coupled plasma atomic emission
spectroscopy (ICP-AES) and inductively coupled
plasma mass spectrometry (ICP-MS) are two
major techniques for the quantification of internalized nanoparticle (NP) elemental composition
and nanoparticle uptake [2,3]. ICP-AES and ICPMS have the advantages of sub-ppb detection
limits, high precision and high accuracy but are
confined to detection of elemental species, such
as gold NPs [4]. Another technique is “mass barcode” in which the gold NPs are encoded with
different functional groups and the uptake of
multiple functionalized gold-NP cells is measured by using laser desorption/ionization mass
spectrometry (LDI-MS) [5]. It is not widely adopted because the encoding process is tedious
and time-consuming and the ionization efficiencies for various functional groups with different
sizes of gold nanoparticles are different, which
makes quantitative measurement somewhat uncertain.
Cell Mass Spectrometry Approach
We have employed cell mass spectrometry
(CMS) technique to accurately measure cellular
uptake of nanocarriers [6]. The CMS technique is
unique in its ability to detect elemental species
as well as other nanomaterials. It streamlined
sample preparation and a rapid detection procedure. CMS provides an elegant way of weighing
particles with diameters greater than a few micrometers, which cannot be achieved by commercial mass spectrometers as shown in figure
1a [7,8].
Figure 1b shows the four parts of CMS apparatus: a laser-induced acoustic desorption of microparticles ion source without a matrix, a lowfrequency quadrupole ion trap for the
measurement of ultra-large m/z values, a pressure-controlled corona discharge to enhance the
number of charges on a cell or microparticle,
and a compact, low-noise charge detector for
total-charge measurement.
We used the CMS to quantify the uptake of
NPs by living cells [6]. We first determined the
weight range of living cells, e.g. the mouse leukemic monocyte/macrophage cell line Raw264.7,
the human embryonic-carcinoma cell line
NTERA2, and the human cervical-cancer cell line
HeLa and plotted the weight distribution dia
gram as shown in figure 1c. Next, we determined the uptake of NPs by measuring the mass
difference for the target cells treated with and
without gold NPs using CMS. Serial measurement of NP uptake by the living cells treated
with gold NPs for various time intervals showed
that NP uptaken by all the living cells reached a
plateau after 15 hours. This proved the same for
gold NPs in sizes ranged from 30 nm to 250 nm
as shown in figure 1d.
With the unique features of CMS, we found
that the amount of gold NPs swallowed by each
type of the living cells was the same as that determined by the ICP-MS. CMS required much
fewer experimental steps as compared with the
ICP-MS. Moreover, the total acquisition time
spent by the CMS was 5 times shorter than ICPMS. More importantly, CMS could detect the exact amount of particles absorbed by each cell,
instead of an average uptake per cell. Thus, it
can provide detailed information regarding the
heterogeneity in the particle uptake among each
individual cell. The same experiments were carried out using nonmetal polystyrene NPs, and
the result showed a similar trend in the uptake
kinetics of gold NPs and of nonmetal polystyrene NPs. To the best of our knowledge, CMS is
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think forward
Ion Trap MS
Material Sciences •
a)
Fig. 2: Types of nanocarriers for drug delivery
b)
c)
Nanodiamond+Proteins
Gold NP+RNAs
d)
Fig. 1: (a) Range of size and mass of molecules
analyzed by mass spectrometry. (b) CMS apparatus, including a quadrupole ion trap, a pulsed
Nd:YAG laser, a He–Ne laser, a charge detector,
and a CCD camera. The Nd:YAG laser induces cell
desorption. The He–Ne laser illuminates the
trapped cells so that they can be detected by the
CCD camera. (c) Mass histogram analysis of NTERA2 cells. (d) Kinetics of the cellular uptake of
30 nm and 250 nm gold nanoparticles.
efficiency, cellular uptake and cytotoxicity of
nanocarriers of various size and surface properties.
Conclusion
the only device that can measure the uptake of
nonmetal NPs by cells. Therefore it can be used
for the measurement of drug uptake when drugs
are carried by NPs such as virus and liposome. In
contrast, ICP-MS cannot be used for measurement of the uptake of nonmetal NPs. Besides
gold NPs and polystyrene NPs, CMS may also be
useful for detecting the uptake of nanocarriers
made of viruses, nanodaimonds, liposomes, carbon nanotubes, biodegradable polymers and
micelles as shown in figure 2 [6].
Size Dependency and Surface Modifications of Nanocarriers
Size and surface characteristics of nanocarriers
determine if drugs can be effectively delivered
to targeted tumor issue [9,10]. Figure 3 depicts a
variety of targeted nanocarriers modified with
different functional groups or drugs. Our CMS
approach could help evaluate the drug targeting
So far, CMS technology offers the best approach
to evaluate the cellular uptake of therapeutic
nanocarriers for drug delivery in cancer cells. It is
a rapid and accurate method for determining
the quantity of gold NPs uptake into cells, and it
can be used to determine the number of NPs
taken up into each individual cell, whereas ICPMS provides only a mean uptake for all cells.
Furthermore, CMS can be used to measure the
cellular uptake not only of metal nanocarriers
but also of nonmetal nanocarriers. In clinical use,
CMS may help evaluate the drug delivery efficiency of nanocarriers of various sizes, shapes
and surface properties and thus facilitate the development of optimal nanocarriers with the
most efficient drug delivery capacity.
References
[1] Cho K. et al.: Clin Cancer Res 14, 1310–1316
(2008)
Liposome+Prugs
Virus+Antibodies
Biodegrade
Polymer+Drugs
Fig. 3: Using nanocarriers as the base platform, a
variety of tissue-specific ligands or other molecules may be attached on the particle surface.
[2] Chithrani B. D. et al.: Nano Lett 6, 662–668
(2006)
[3] Yang P. et al.: Bioconjugate Chem 16, 494–496
(2005)
[4] Marquis B. J. et al.: Analyst 134, 425–439 (2009)
[5] Zhu Z. -J. et al.: J Am Chem Soc 2008, 130,
14139–14143 (2008)
[6] Lin H.-C. et al.: Angew Chem Int Ed 49, 3460–3464
(2010)
[7] Peng, W.-P. et al.: Angew Chem Int Ed 119,
3865–3869 (2007)
[8] Peng, W.-P. et al.: Mass Spectrom Rev. 23,
443–465 (2004)
[9] Liu Y. et al.: Biomaterials 31, 330–338 (2010)
[10]Singh P. et al.: Drug Dev Res 67, 23–41 (2003)
Authors
Dr. Wen-Ping Peng, Department of Physics, National
Dong Hwa University, Hualien, Taiwan
Dr. Alice L. Yu, Dr. Chung-Hsuan Chen, Genomics
Research Center, Academia Sinica, Taipei, Taiwan
Contact
Dr. Wen-Ping Peng
Department of Physics
National Dong Hwa University
Hualien, Taiwan
[email protected]
Cover story •
Adaptability and Ergonomy
Built by Your Needs
As a core component to almost all clinical and research work, microscopy has a
continuing and growing role within modern science and medicine. For example,
even with the increasing prevalence of molecular tests, the microscopic view
provides the unique opportunity to observe the physiology of a disease, or process. As a result, the quality of the diagnosis, or the progression of a research
project, is reliant upon the resolution and clarity of the images produced by the
microscope and its related imaging components. Consequently, both clinical and
research laboratories require upright microscope systems which can effectively
meet any needs.
With a host of peerless features, the Olympus
BX3 range of clinical and research microscopes
establish a new standard in microscopy, enabling users to truly build a microscope system
for their requirements. With the ability to adapt
to any working environment, each user’s preferences are met by the new microscope concept. A
workspace can therefore be completely tailored
by each user to their own needs; from the positioning of hardware and controls through to
personalizable software tools.
Following on from the highly successful BX2
range, the BX3 portfolio consists of: the BX46
ergonomic screening microscope, the BX43 and
BX system microscopes for clinical and research
tasks with optional coded and automation modules, and the BX63 research system with advanced automation as standard.
Future Proof and Adaptable to Any
Workflow
These microscopes are ideal for covering all your
current requirements and protocols, as well as
any future advances. Their modular nature ensures that they can facilitate multiple capabilities, including excellent fluorescence imaging.
The full range of Olympus digital imaging cameras are available for the series, enabling every
imaging requirement to be met. Furthermore, a
series of motorization options, combined with
the labSens or cellSens software packages, ensure that the user can automate as many, or as
few, features as required. As a result, the optimal imaging system for every eventuality can be
created with ease at any point in time, making
your lab completely future-ready.
As the basis of this concept in adaptability, a
new level of flexibility allows this microscope to
be ambidextrous, where many of the features
are easily adaptable for left- or right-handed-
Instruments of the BX3 series
ness. Building on this, users can completely define their working environment: from the microscope and imaging components to software
workflows, GUIs and the positioning of the controls on the desktop. As such, the BX63 research
system provides multiple options for managing
the entire microscopy and imaging process. At
the forefront of this concept, the programmable
touch-panel (fig. 1) controls all components of
the imaging system, enabling users to change
the objective, mirror unit and observation method, as well as navigate and focus the sample. In
addition, the research microscope can be fitted
with a detachable remote control, providing the
effect of traditional mechanical knobs for focus
(Z) and stage position (X, Y).This also automates
many of the optical adjustments required for
changing between contrast methods, including
condenser position; aperture stop; field stop; polarizer; fluorescence shutter; mirror turret position; and contrast method selected by the user.
True Color LED
The BX3 range features the novel, true-color LED
illumination system, which has a unique wavelength profile, providing a color rendering index
matching that of a halogen bulb with a daylight
filter. This makes it ideal for high color clarity
across the entire range of brightfield stains, a
capability not offered with standard LED illumination systems. Furthermore, the true-color LED
• Cover story
scope is more flexible, adapting to the users individual posture and therefore resulting in less
musculo-skeletal stress on the user.
Advanced Automation
Fig. 1: Programmable touch-panel
Fluorescence Illumination
system is controlled by the Light Intensity Manager (LIM), which automatically adjusts the intensity to user-defined levels on each objective
change, thus removing the need for manual adjustment to increase the efficiency of each
screen. In addition, users also benefit from the
Olympus has introduced a new fluorescence illumination concept to both the research and
clinical systems based around the unique fly-eye
lens system (fig. 2), which provides homogeneity
for fluorescence illumination and ease of use.
Furthermore, the new 8 position filter turret not
only provides the capacity for even the most
comprehensive multi-color fluorescence studies,
but also offers fast, tool-free mirror cube exchange (fig. 3). The cubes themselves are designed to maximize S/N by capturing >99% of
stray light and using advanced multi-layer filters
with steep cut-offs for highly defined wavelength selection.
The BX43 and the BX53 enable users to build a
system that suits their needs, with a host of optional automated components, including nosepiece, mirror turret, universal condenser, ND filter wheel and stage, allowing it to provide
similar contrast management capabilities to the
BX63. As a fully-motorized system, the BX63 is
focused via the nosepiece, allowing the stage to
be fixed for increased stability. Furthermore, the
new ultrasonic Piezo-driven motorized stage
uses highly accurate encoders which provide a
continuous X, Y read-out, enabling the precise
navigation to previously set coordinates at high
speeds. Due to this motorization and X, Y readout, the stage can also be positioned by hand
for rapid, gross sample alignment.
Consultancy and Training
Consultancy and training are essential functions
in all applications within microscopy and Olympus is at the forefront of providing excellent efficiency in this area, offering customizable, dualobservation and multi-discussion units for
laboratory conferencing. In addition, if fitted
with a digital imaging camera and the labSens
or cellSens software, the BX3 microscopes can
essentially act as a netcam. Using standard TCP/
IP protocols the labSens ‘NetCam solution’ enables the transfer of live and stored images
throughout the network for teaching, mentoring
or supervision.
Conclusion
Fig. 2: Fly-eye lens system
excellent life-span (approximately 20,000 hours)
and minimal power consumption associated
with LEDs, providing exceptionally low running
costs and reduced downtime.
Optically Superior
As well as integrating the UIS2 optical components, the BX3 range has also been optically optimized to improve workflows. For example, the
new wide-range condenser accommodates magnifications from 2–100 x without requiring a
swing-top lens. This cuts out one of the common
and time-consuming steps of microscope screening, offering substantial efficiency improvements.
Fig. 3: Tool-free mirror cube exchange
Setting New Standards in Ergonomy
In addition to the established ‘ergo-tube’, the
BX3 clinical microscopes are available with the
world’s first tilting/telescopic/lifting observation
tube that provides adjustment in three dimensions: eyepiece tilt –3° to +27º, tube extension
(backwards/forwards) of 55 mm, and lifting of
45 mm. The BX46 also features an ultra-low
fixed low-torque stage with ergo-grip controls,
ensuring that the movements and force required
by the operator are minimized. As their arms can
remain on the desk at all times, even placing a
sample on the stage requires very little effort.
This further increases efficiency as the micro-
The BX3 clinical and research microscopes form
part of the broader range of highly versatile systems. Through the fully-customizable automation and motorization, in combination with the
advanced camera and imaging software options,
Olympus can create an imaging system tailor
made for your individual needs. The unique
modular concept allows users to configure their
preferred workspace – whether this is a conventional set-up, or an automated system operated
via the intuitive software and touch-screen controls. Olympus has therefore provided the ultimate level of flexibility.
Contact
Katja Ansmann
Olympus Europa
Marketing Communications Manager
[email protected]
www.microscopy.olympus.eu
Company News •
Newsflow
Safety Protocol
Wyatt Technology offers a series of free of charge e-learning webinars on various
aspects of protein and macromolecular characterization. Hosted by some of the company’s leading scientists, these educational platforms represent state-of-the-art in
light scattering technology. 6 of the seminars are designed to optimize the usage of
Wyatt instrumentation and three are focused on the scientific background of light
scattering, and absolute macromolecular characterization.
www.wyatt.com
The CANopen Safety protocol (CiA 304) developed by CAN in Automation (CiA), international users’ and manufacturers group, is now published as an European standard and available as EN 50325–5 from the European Committee for Electrotechnical
Standardization (CENELEC) and any National body. The CANopen Safety protocol is
an addition to the CANopen protocol standardized in EN 50325–4, also known as
CiA 301. CANopen Safety is designed to allow safety-related communication based
on CAN according to IEC/EN 61508. The German TÜV has approved the protocol for
use for systems requiring Safety Integrity Level 3 (SIL 3).
www.can-cia.org
Chemical Dispersants in Sea Water
Paperless Lab Solutions
The United States Environmental Protection Agency (EPA) has published two rapid
screening methods for chemical dispersants in sea waters using Waters Acquity
UPLC/Quattro Premier XE (UPLC/MS/MS). These analytical procedures were developed by Dr. Lawrence Zintek, National Organic Methods Development Expert, and Dr.
David Schroeder, US EPA Region 5 Chicago Regional Laboratory (CRL) in collaboration with Dr. Johnson Mathew, Region 6 Houston Laboratory. These two methods
specifically target the analysis of dipropylene glycol monobutyl ether (DPGBE), ethylene glycol monobutyl ether (EGBE), and dioctyl sulfosuccinate (DOSS) in sea water;
three compounds used in the Gulf of Mexico in response to the oil leak.
www.waters.com
Thermo Fisher Scientific, announced that it has extended its reach across Central
Europe by making Vialis the latest member of its Informatics Global Partner Alliance.
Vialis, based in Switzerland, will provide local services and support for the full range
of Thermo Scientific informatics solutions, including laboratory information management systems, chromatography data systems, electronic laboratory notebooks and
spectroscopy software, as well as comprehensive laboratory automation and integration solutions.
www.thermofisher.com
www.vialis.ch.
Free of Charge e-learning Seminars
3D Cell Culture
50 Years of Innovations
Bruker was founded 50 Years ago, on September 7, 1960, in the suburbs of Karlsruhe,
Germany. As a part of their 50th birthday, Bruker management team rang the Opening Bell at the NASDAQ Stock at the NASDAQ MarketSite in New York City’s Times
Square.
www.bruker.com
Worldwide Distribution Agreement
Dionex and EMD Millipore, the Life Science division of Merck KGaA of Germany, announced the signing of a worldwide distribution agreement for the EMD Millipore
ICW-3000 water purification system for Dionex ion chromatography (IC) systems using RFIC-EG technology.
Dionex is now able to sell this water purifier and consumables directly to customers
globally. The system was designed by EMD Millipore specifically as an ultrapure water source for Dionex Reagent-Free IC systems with the powerful and convenient
“Just Add Water” technology that eliminates the need for eluent preparation. The
purification system’s easy installation with simple plumbing and control by the Dionex IC system make it very convenient to use.
www.merck.de
www.dionex.com
www.milipore.com
reinnervate, a life sciences company driving the adoption of routine 3D cell culture,
announced the commercial launch of ec23 a proprietary small molecule designed to
mediate the controlled and reproducible differentiation of cells in culture. The molecule has multiple applications in academic and pharmaceutical research. ec23 is a
synthetic retinoid based on all-trans retinoic acid (ATRA), which is known for its ability to modulate cell function and neural development. ATRA, as with other naturally
occurring retinoids, is unstable and degrades readily when exposed to light (even
low intensity light), which can result in cell culture heterogeneity and lack of reproducibility. The key advantage of the new molecule is that it is a more potent inducer
of neurogenesis than ATRA and is also entirely stable.
www.reinnervate.com
• Application Note
Particle Size Distributions
Dynamic Image Analysis Beats Laser Diffraction in a Micron to Millimeter Range
Laser diffraction is the most frequently used measurement technique for the
analysis of particle size distributions in the range 1 micron to 1 mm in the context of quality control. Modern laser diffraction systems offer some convincing
advantages such as short measurement times, easy operation and reproducible
analysis results. However, they also have various disadvantages: Even if the instruments have been calibrated and validated, an absolute particle size measurement is not possible. Various round robin tests have shown that the analysis
results depend strongly on the type of instrument and even on the particular
model and software version.
Principle of Dynamic Image
Processing Measurements
The particles move with the help of gravity,
compressed air or dispersed in liquid through
the measuring field. A light source illuminates
them from one side while a camera takes
their picture from the other side. The software
evaluates the projections of the particles to
determine the size distribution of all particles
of the sample in a very short time. The maximum dynamic measuring range is substantially extended by using two aligned cameras.
A high resolution camera detects small particles in a small measuring field. A camera with
lower resolution but a wider measuring field
simultaneously detects the larger particles,
allowing for rapid measurement with good
statistics.
The laser diffraction method is further limited by
the unsatisfactory detection for small outlier
volume fractions (over-size and under-size) of
approximately 2–3%, as well as the poor resolution of particles in the range from a few hundred
microns to millimeters. Although laser diffraction
systems are able to detect particles > 10 nanometers, only very few measuring channels are
provided for particle sizes of approximately
1 mm. The resolution for these particles is rather
poor. Thus, it is not possible to precisely resolve
multimodal size distributions, as particles of a
few hundred microns size difference are classified in the same size class.
The complex, indirect measurement algorithm used by laser diffraction is like a “black
box” for many users. The selection of the optimum evaluation parameters requires some experience; for the correct interpretation of the results it is often necessary to have some previous
Jörg Westermann,
Retsch Technology
knowledge about the sample characteristics.
Wrong assumptions and parameters lead to reproducible but inaccurate measurement results.
Static laser light scattering is a rapid method,
easy to carry out but difficult to evaluate. The
ideal measurement method should directly detect the individual particle characteristics, for
example by taking an image of the particle and
calculating it directly.
Now, such a direct measurement technology
for fine powders > 1 micron is available with the
new Camsizer XT (see Infobox). It uses the measurement technology of the ISO 13322-2 Dynamic Image Analysis standard, and beats laser
diffraction with regards to resolution and detection limits by more than a factor 10.
Until recently, dynamic image analysis was
only established for the measurement of dry,
pourable powders and granules in the size range
above 30 µm. Thanks to an advanced computer
and camera technology finer particles can now
be displayed more sharply and evaluated in real
time. The evaluation speed achieves 275 pictures
per second, with up to a few 100 particles in
each image.
For the measuring range of 1 micron and
above the image analysis method now also offers convincing benefits: As the particle images
are taken directly with a camera of extremely
high resolution, their size and shape can be accurately determined, even over a few orders of
magnitude and consequently with a much higher resolution when compared with laser diffraction. The following application examples show
the superiority of dynamic image analysis.
Accurate Determination of Oversized
Particles
The laser light scattering method always detects
a particle collective, i.e. the scattering signal is
an average of many particles. Small amounts of
undersized or oversized particles only cause a
Application Note •
Fig. 1: Measurement of silicon carbide (abrasive) with a size distribution of
1–10 µm and a mean value of 5 µm (dry dispersion with compressed air).
minor change in the light scattering pattern and therefore cannot
be reliably detected with laser diffraction. Depending on the sample
material, a volume fraction of
2–3% is considered as the absolute detection limit. The image
analysis method, however, evaluates individual particles and detects, depending on the operation
mode, every single particle of the
sample. Only a few particles in the
sample are enough for reliable detection, even if these particles
amount to less than 0.01% of the
entire sample volume. This opens
up new perspectives for the characterization and ensures improved
quality of the production monitoring process.
Highly Precise Particle Size
Measurement
The laser diffraction method is
based on the assumption that all
particles are spherical. The real particle shape which deviates from the
spherical shape changes the light
scattering pattern; however, the
software cannot transfer these
changes to a particular distribution
of size and shape. Although it is not
possible to differentiate between
the length and width of a particle,
both parameters are included in the
calculation of the “particle size”.
As a result, the particle size distribution is often presented wider
than it actually is and with a poorer
resolution.
If dynamic image processing is
used for particle analysis, it is possible to determine the length,
width and equivalent diameter
separately (see fig. 3). Thus it is
possible to obtain various size distributions from one measurement,
depending on which size definition
is considered.
Figure 5 shows the deviations
between the actual particle shape
and the ideal spherical shape the
laser scattering method is based
on. Spheres have a b/l ratio of 1.0.
The majority of particles in the
above example have a b/l ratio of
< 0.9, i.e. they are clearly not
spherical. Measuring the particle
shape with digital image pro
cessing thus leads to a more detailed knowledge of the sample
quality.
With dynamic image processing,
just like with laser diffraction, the
particles need to pass the field of
view individually to ensure that
each particle is analyzed individually. Agglomerates or particles which
stick together give the impression
of larger particle sizes. That is why
both methods involve dispersion
with compressed air or, alternatively,
in liquid. The dispersion parameters
have to be adjustable in a way that
strong agglomerations can be separated without destroying the primary particles. Dynamic image processing provides information about the
effectiveness of the dispersion tool
as the particle projections are available as pictures at all times.
For particles smaller than 1 micron, laser diffraction remains unrivaled. Image analysis with visible
light encounters its physical limits
here: as soon as the particle size
comes down to the wave length of
Fig. 2: Comparison of two different samples with different fractions of
oversized particles. Sample 2 (red) contains 0.2 % more over-size at 20 µm.
It is impossible to detect such small differences with laser diffraction.
Fig. 3: Schematic representation of length (xL), width (xW) and equivalent diameter (xA).
Fig. 4: The digital image processing method determines the size distribution
with the help of the particle width (xW, red), the particle length (xL, green)
and the equivalent diameter (xA, blue). The orange curve represents the results of laser diffraction. The results of image processing are more detailed
with a better resolution. The accuracy of the image analysis results is confirmed impressively by sieve analysis and microscopy.
Fig. 5: Shape analysis with digital image processing. The graphic shows the
width-to-length ratio (w/l) of the sample represented in figure 4. 20 % of
the particles are twice as long as they are broad, approx. 1 % are three
times as long.
the light, it is no longer possible to
produce sharp pictures of them.
Summary
Dynamic image Processing is an
established method for size and
shape analysis of free flowing, dispersed particles with hundreds of
customers and applications all over
the world. With the help of the latest camera technology the method
is now available also for particle
sizes from 1 micron, to 3 mm, a
size range which was previously
covered exclusively by laser diffraction. The same advantages are now
available for fine powders as well
as for larger particles: reliable de-
tection of over-size, high resolution
and excellent reproducibility of the
particle size results, information
about particle shape, as well as
easy operation, short measuring
times, and an intuitive, simple measuring principle. Indirect methods
with limited accuracy, such as laser
diffraction but also complex optical
methods with unreliable statistics,
such as microscopy, become increasingly outdated.
Kontakt
Jörg Westermann
Retsch Technology GmbH
Haan, Germany
[email protected]
www.retsch.com
NEW
PARTICLE ANALYZER
CAMSIZER XT
Particle size and particle shape
analysis of fine powders
Q Digital image processing with
patented two-camera-system
(acc. to ISO 13322-2)
Q Wide dynamic measuring range
IURPǋPWRPP
Q Reliable detection of smallest
amounts of “undersize” and
“oversize”
Q Very short measurement time
of 1 – 3 minutes
Q Modular system X-Change for
dry and wet dispersion
Q Measurement results are 100%
compatible to sieve analysis if
required
www.retsch.com/camsizerxt
a : H a ll 9 ·
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Fax:
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+49 (0) 21 29 / 55 61 - 87
[email protected]
Ideal for:
– metal and ore powders
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powders / granules /
fine pellets
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– detergents and
enzymes
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– foodstuffs (pulverized
and granulated)
– plastic fibers / powders
– refractory products
– abrasives (mediumsize and small grit)
and many more
White Giant or White Dwarf?
Particle Size Distribution Measurements of TiO2
What have white tattoo ink, milk, toothpaste and lines on tennis courts in
common? In all cases TiO2 (titanium dioxide) is used as a white pigment. It is
the widest used white pigment due to its brightness and very high refractive
index of 2.7. TiO2 is a naturally occurring mineral. Approximately four million
tons of this pigment are consumed annually worldwide. In daily use, it can
be found nearly everywhere - in paints, coatings, plastics, papers, inks, medicines as well as in toothpaste. It is applied even in the food industry as E171,
e.g. for whitening of skimmed milk.
Applications
However, TiO2 has additional surprising features.
It is a photocatalyst under ultraviolet (UV) light
– and under visible light if it is doped with nitrogen ions or with metal oxides.
The photocatalyst TiO2 captures ultraviolet
light and forms activated oxygen from water or
oxygen in the air. This process is similar to photosynthesis, in which chlorophyll captures sun-
Dr. Markus Ortlieb, Product specialist Particle Size Analyzers,
Shimadzu Europa
light to turn water and carbon dioxide into oxygen and glucose. The activated oxygen formed is
strong enough to oxidize and decompose organic materials and smelling gas. It is even strong
enough to kill bacteria. Photocatalyst coating
technology therefore adds advanced functions
to building materials, for instance sterilizing, deodorizing and anti-fouling.
The Graetzel cell (a type of chemical solar
cell) uses the same effect. The cell is composed
of a porous layer of nanoparticles of titanium
dioxide, covered with a molecular dye that absorbs sunlight.
The pigment-coated titanium dioxide is
placed between two electrodes in an electrolyte
solution. Sunlight is absorbed by the pigmentcoated titanium dioxide causing an electron to
be injected into the conduction band of the
semiconductor TiO2. These electrons travel
through a wire from the anode to the cathode,
creating an electrical current. In this way, energy
from the sun is converted into electricity. Most
of the materials used to make this cell are lowcost, easy to manufacture and flexible, allowing
them to be integrated into a wide variety of objects and materials.
However, TiO2 is produced in varying particle
sizes, oil and water dispersible, and with varying
coatings always depending on the application
field and industry.
Methods
Measuring Small Particles – Laser Diffraction
One method commonly used to measure particle
size is laser diffraction (fig. 1). When light hits a
particle, the resulting shadow image is not
sharply defined due to the wave-like nature of
light. It rather shows a light intensity pattern depending on the wavelength and the particle size.
In order to simplify the analysis of this intensity
pattern, a laser is used as a light source. In this
way, only a single wavelength needs to be considered.
The relationship between measured light intensity pattern and particle size can be derived
from the so-called Mie theory. For sufficiently
large particle diameters, the Fraunhofer approximation, which can be derived from Mie theory,
can also be applied. Mie theory is able to describe the pattern of light and dark circles
around the scattering center,
whose width increases with decreasing particle size. Small particles generate a system of rings that
are widely spaced. Rings at smaller
distances originate from a large
particle.
Most laser diffraction particle
size analyzers on the market are
equipped with several light sources
and/or detectors. Using this approach, a broad measuring range
can be realized. Using such instruments is problematic since detectors or the light source have to be
switched if the entire measurement
range is to be covered. This causes
further problems - switching needs
time, recalibration is needed and
the quality of measurement data is
poor in the overlapping areas of
the detectors. In the following
measurements (fig. 1) a particle
size analyzer was used with just a
single light source, a single optical
system and a single measurement
theory of laser diffraction (Shimadzu SALD-7101). This setup features
a perfect seamless and wide measuring range. There are no points of
discontinuity over the entire measuring range.
Figure 1 shows an example of a
particle size distribution. This setup
uses laser diffraction as the measurement principle. This method is
perfectly suited for larger particles
starting from 10 nm up to a range
of a few millimeters.
Fig. 1: Particle Size Distribution of TiO2: Average of ten consecutive measurements including standard deviation. Used Setup: Shimadzu SALD 7101
with batch cell. Measurement range: 10 nm – 300 µm
Is TiO2 a White Giant or
White Dwarf?
Measuring Tiny Particles –
Induced Grating Method
With dynamic light scattering
(DLS), the conventional method for
measuring particles in the range of
a few nanometers, the problem is
that light scattered by particles decreases sharply for particle sizes of
less than 100 nm. Furthermore, in
the single nano region with particle
sizes of less than 10 nm, physical
restrictions make it difficult to detect scattered light. Measurement
of particle sizes then becomes difficult. The IG method uses diffracted light instead of scattered light,
and is free from these physical restrictions. Furthermore, it does not
require input of the refractive index
as a measurement condition. It allows simple measurement of nanoparticles with high sensitivity.
The IG method uses specific
electrodes dipped into the sample
solution. If an electrical field is ap-
plied to the electrodes, particles
are “trapped” between the free
spaces of the electrodes resulting
in a diffraction pattern. With dielectrophoresis switched off, the
particles diffuse back into solution
and the detected diffracted light
intensity decays. Based on the difference in diffusion velocities of
large (slow) and small (fast) particles, the primary diffracted light
will decrease at a faster or slower
rate depending on particle size.
This method enables stable
measurements with excellent reproducibilities, particularly in the
single nano range, as it is virtually
resistant to contamination and
even to the presence of small foreign particles. Special requirements
of ambient air quality as well as
sample filtration are therefore unnecessary. Measurements can also
be carried out without any problems in many different solvents
(fig. 2).
Figure 2 shows an example of a
particle size distribution measured
with Shimadzu’s IG-1000. This setup uses a new measurement method called Induced Grating, developed by Shimadzu. It is a dedicated
method for measuring nanoparticles. The measurement range starts
at 0.5 nm and goes up to 200 nm.
The critical range of measurements
less than 10 nm is thereby extended.
Both. TiO2 has a “giant” application field and is used as a white
pigment or as a photocatalyst. Depending on the application field,
the size range of the particles differs. Sometimes the particle diameter is in the range of nanometers,
and sometimes in the range of micrometers. Sophisticated and advanced tools help in determining
particle size distributions as accurately as possible.
Fig. 2: Particle Size Distribution of TiO2: Average of three consecutive measurements including standard deviation. Used Setup: Shimadzu IG-1000.
Measurement range: 0.5 nm – 200 nm. Measurement principle: Induced Grating.
Contact
Dr. Markus Ortlieb
Shimadzu Europa GmbH
Duisburg Germany
[email protected]
www.shimadzu.eu
Transfer of USP-based HPLC methods
for pantoprazole sodium to UPLC
20-fold increase in productivity
HPLC is a commonly used analytical
method for assaying and purity controlling of active pharmaceutical ingredients („API’s“) in the pharmaceutical industry. Method transfer to
the latest technologies can be timeconsuming and are therefore rarely
performed for the improvement of
validated methods. However, the
transfer of established methods to a
Pantoprazole sodium sesquihydrate
(5-(difluoromethoxy)-2-[(3,4-dimethoxypyridin2-yl)methylsulfinyl]-3H-benzoimidazole, sodium
salt, sesquihydrate)
UPLC (ultra performance liquid chromatography) system can be worth
the investment. In the reported case
such an investment was rewarded
with surprising savings in analysis
time, operational costs and improved resolution.
We demonstrate the successful method transfer
for the analysis of pantoprazole sodium from the
USP-recommended L1 column, run on a conventional HPLC system, to a sub 2 µm particle column
on a UPLC system. With some small optimization
changes, the final methodology reduced the an
alysis run time from 55 min with HPLC to just 3
min with UPLC, resulting in a 20-fold increase in
throughput and a remarkable reduction in solvent
consumption and waste disposal costs!
Pantoprazole sodium sesquihydrate is described in the USP and the European Pharmacopeia. The purity and assay testing for pantoprazole sodium is accomplished using high
performance liquid chromatography (HPLC) with
ultraviolet detection (UV) on L1-column in compliance with the USP 32—NF 27 monograph for
pantoprazole sodium.
Standard solutions of pantoprazol sodium and
related compounds A-F were prepared according
to the USP procedure. All solutions were protected from light by use of amber glass ware and
the autosampler’s sample compartments temperature was set to 4°C.
LC System: Alliance 2695XE with PDA-detector 2996 and
Empower 2 C/S-software (Waters)
Column: XTerra RP18, 3.9x 150 mm, 5 µm (Waters)
Mobile Phase A: 1.74 g/L dibasic potassium phosphate, adjusted with phosphoric acid (330 g/L) to a pH of 7.00 ±0.05
Mobile Phase B: Acetonitrile
Gradient: 0–40 min 20 => 80% B (linear)
40–45 min 80 => 20% B (linear)
45–55 min 20% B (re-equilibration)
Flow Rate: 1.0 mL/min
Injection Volume: 20 µL
Temperature: 40°C
Detection: 290 nm
Fig. 1: Original USP-method
Introduction
The active ingredient in Pantoprazole is Pantoprazole sodium (see eye catcher). It is commercially available as delayed-release 20 and 40 mg
tablets and is functioning as a proton pump inhibitor: The 20 mg tablets are used for the treatment of patients with conditions caused by gastric acid secretion (reflux disease) and associated
symptoms (heartburn, acid belches and pain on
swallowing). Indications for the 40 mg dosis are
the treatment of gastrointestinal diseases which
require a reduction in acid secretion (different
forms of ulcer).
Experimental
UPLC Conditions for Purity Testing –
Geometrically Scaled Gradient
LC System: Acquity UPLC system (Binary Solvent Manager,
Sample Manager, Column Manager) with Acquity UPLC PDA
eLambda-detector and Empower 2 C/S-software (Waters)
Column: Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7 µm (Waters)
Mobile Phase A: 1.74 g/L dibasic potassium phosphate, adjusted with phosphoric acid (330 g/L) to a pH of 7.00 ±0.05
Mobile Phase B: Acetonitrile
Gradient: 0–13 min 20 => 80% B (linear)
13–15 min 80 =>20% B (linear)
15–18 min 20% B (re-equilibration)
Temperature: 40°C
Detection: 290 nm
Fig. 2: Geometrically scaled method
ceptance criteria of the USP monograph. A typical
chromatogram is shown in figure 5.
Conclusion
UPLC Conditions for Purity Testing - Shortest Analysis
Time Gradient: 0.00–6.55 min 20 => 80% B (linear)
6.55–7 .37 min 80 => 20% B (linear)
7.37 – 9.01 min 20% B (re-equilibration)
Fig. 3: Shortest analysis time method
UPLC Conditions for Purity Testing – Further Optimized
Gradient: 0
.0–2.0 min 20 => 38% B (linear)
2.0–2.3 min 38 => 80% B (linear)
2.3–2.5 min 80 => 20% B (linear)
2.5–3.0 min 20% B (re-equilibration)
The USP methods for assay and purity of the active pharmaceutical ingredient pantoprazole sodium, were successfully transferred from the
USP-recommended L1 column on a conventional
HPLC system, to a small particle 1.7 µm column
on a UPLC system, while the system suitability
criteria of the USP monograph are still met.
With some small optimization changes the final methodology reduced the analysis run time
from 55 min with HPLC to just 3 min with UPLC
– that’s nearly a 20-fold increase in throughput
and reduction in solvent consumption and waste
disposal costs! Therefore, for our company, the
investment in the ultra-performance LC technology is rewarded with significant savings in analysis time and operational costs while at the
same time improving resolution.
Fig. 4: Further optimized method
Fig. 5: UPLChromatogram at 290 nm of a “real
sample” of pantoprazole API
HPLC Conditions for Purity Testing (fig. 1) (on
the basis of the synthetic route, this test is recommended by the USP monograph when impurities
C, D, E and F are potential related compounds)
The HPLC separation of pantoprazole and impurities is shown in figure 1. The USP requirements for system suitability – the resolution between pantoprazole related compound E and D+F
peaks is not less than 1.5 – are met. In addition,
the tailing factor is not more than 2 and the relative standard deviation for replicate injections of
the standard solution is not more than 5.0%.
The current HPLC-based runtime for assay
and purity is 55 min, with a retention time of 8.5
minutes for the active pharmaceutical ingredient
pantoprazole.
Method Transfer
To meet the USP L1 column requirements an Acquity UPLC BEH C18 column, 2.1 x 50 mm,
1.7 µm was chosen. The HPLC parameters (gradient, flow rate, injection volume) were scaled
down to UPLC by using the Waters UPLC Console Calculator (free download on www.waters.
com). The calculator allows transferring isocratic
or gradient LC methods thanks to fundamental
Contact
equations of chromatography. The program
Alexander H. Schmidt
takes into account the different system dwell
Head of QC and Lab manager
volumes, changes in column diameter and partiSteiner & Co., Deutsche Arzneimittelgesellschaft mbH
cle size and calculates the conditions of „Geo& Co. KG, Berlin, Germany
metrically scaled gradient“, „Maximum Peak
[email protected]
capacity“ or of „Shortest Analysis Time“.
www.steinerarznei-berlin.de
For UPLC Conditions
for Purity Testing - Geometrically Scaled Gradient see figure 2 and for
ProductFurtherat
the Shortest Analysis
ion at m
inform bra
nd.co
u
Time on Equal Peak Cawww.vacu
pacity see figure 3.
In comparison to the
HPLC method (runtime
55 min), the UPLC
method – calculated
with the shortest analysis time at equal peak
capacity – has a runtime of only 9 min, with
a retention time of 1.2
minutes for the active
pharmaceutical ingredient pantoprazole (Fig.
3).
Finally, the method
New vacuum pumps ME 1 and ME 1C
was further optimized
to a total runtime of 3
Filtration is probably the most common application
for vacuum in the laboratory. The new diaphragm
min (Fig. 4).
Tired of waiting for filtration?
Let us help you speed it up.
Application
This optimized UPLC
method was used to
check for impurities in
pantoprazole API, purchased from an Indian
manufacture. All impurities were within the ac-
pumps ME 1 and ME 1C offer a compact and high
performance solution. With their easy-to-use
functionality, they are perfect for both single and
multiple filtrations. The ME 1C also offers outstanding chemical resistance.
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On-line Oxygen Monitoring in
Cell Culture
Effects of Mitochondrial Modulators on O2 Dynamics of Mammalian Cells
Real-time measurements of oxygen and pH in mammalian cell cultures are important to understand the
metabolic dynamics in cell cultures. The observation of the time course of cellular oxygen consumption in
response to environmental conditions or toxicological insults can be substantial for successful culture of
mammalian cells. This type of monitoring could also be valuable for other applications such as tissue engineering and stem cell research.
Non-invasive Oxygen Detection
In the present work, oxygen levels in the media
of cells plated at 4 different cell densities and
varying oxygen tension (19% O2 (Fig. 2) and 7%
O2 (Fig. 3)) were continuously monitored. In addition, the O2 consumption rates in response to
mitochondrial modulators (Fig. 4) were also assessed. Mouse embryonic fibroblasts (MEFs)
were used as a representative mammalian cell
type, carbonyl cyanide m-chlorophenylhydrazone
(CCCP) was used as a metabolic uncoupler and
antimycin was used as an electron transport
chain inhibitor. The SDR SensorDish Reader was
applied for on-line measurement of DO and pH
in 24-well multidishes.
Effects of Cell Density
The first experiment was designed to investigate
the effects of cell density on the cellular oxygen
consumption rate and subsequent pO2 levels in
the media. MEF cells were plated at three separate concentrations (100,000; 33,000; and
10,000 cells/cm2) along with a “media only”
control. Measurements were made every 2 minutes for 10 hours at near atmospheric oxygen
levels (approx. 19% O2). The oxygen profile
(Fig. 2) clearly shows an effect of cell density on
the O2 consumption level of each treatment.
A lag period of < 1 hour is observed, presumably where the system is equilibrating to temperature and oxygen levels. After this period, all
the cell concentrations show a decrease in pO2
levels from the “media only” control wells. Finally, during the course of the incubation, the
deviation from the “media only” control wells is
proportional to the number of cells initially plated, implying that individual cellular respiration
is equivalent at all the cell densities tested.
The second experiment was designed to investigate the effects of low ambient oxygen levels on cell density-induced observations in the
first experiment. MEF cells were plated at three
separate concentrations (100,000; 33,000; and
Cell Culture in Phase Contrast with Fluorescence (DAPI),
friendly provided by Olympus
10,000 cells/cm2) along with a “media only”
control and then placed in a 7% O2 atmosphere
and monitored every 5 minutes for 10 hours. The
oxygen profile confirmed the effect of cell density on the O2 consumption level of each treatment (Fig. 3).
A short period of temperature equilibration
(< 1 hour) is observed, after which the residual
oxygen in the media is consumed by the cells in
a density dependent fashion. Interestingly, even
the “media only” is not fully equilibrated at the
new oxygen levels at 7% O2 after 10 hours.
However, the deviation from the “media only”
control wells by each treatment is proportional
to the number of cells plated.
Effects of Mitochondrial Modulators
The third experiment was designed to investigate the effects of specific mitochondrial modulators on the cellular oxygen consumption and
subsequent pO2 levels in the media. MEF cells
were plated at 33,000 cells / cm2 and allowed to
adhere to the culture dish for 4 hours. The culture media was then replaced with the treatment specific media, containing either 1 µL / mL
Fig. 1: SDR SensorDish Reader for on-line monitoring of dissolved oxygen
and pH in 24-well multidishes.
Fig. 2: Average pO2 levels in the culture media of MEF cells plated at differing densities (100,000; 33,000; 10,000 and 0 cell/cm2) and exposed to approx. 19% O2; n = 3 for each treatment.
dimethyl sulfoxide (DMSO, vehicle
control); 10 µM carbonyl cyanide
m-chlorophenylhydrazone (CCCP);
or 10 µM antimycin. Each well was
then topped with 1 mL of mineral
oil and the plate was incubated
under standard culture conditions
(5% CO2, 35% humidity; 37°C, and
19% O2 atmosphere) and monitored every 2 minutes for 10 hours.
The oxygen profile shows a pronounced effect of treatment on the
O2 consumption level of the cells
(Fig. 4).
Measurements from the 3 to 4
hour time points indicate that there
was little variation in the treatments prior to receiving the dosed
media. Within 30 minutes of dosing,
observable
differences
emerged in the oxygen consumption curves between the treatments. DMSO, the vehicle control,
caused little variation in the average O2 level in the MEF cells. A
steady state between oxygen consumption and oxygen ingress was
formed at about 17.5% O2. Antimycin, an inhibitor of Complex III
of the electron transport chain,
showed an observable decrease in
the oxygen consumption rate of
the cells, resulting in a steady state
higher than the one with DMSO.
Finally, treatment with CCCP, a mitochondrial uncoupler treatment
resulted in an observable increase
in cellular oxygen consumption.
Materials and Methods
The SDR SensorDish Reader (PreSens) (Fig. 1) was used for the noninvasive on-line monitoring of oxygen and pH in 24-well microtiter
plates.
Mouse embryonic fibroblast
(MEF) cells were grown in 24-well
multidishes with integrated oxygen
sensors (OxoDishes). Cells were
maintained in Dulbeco’s Modified
Eagle Medium (DMEM) supplemented with 10% heat inactivated
fetal bovine serum (FBS), 1% peni-
Fig. 3: Average pO2 levels in the culture media of MEF cells plated at differing densities (100,000; 33,000; 10,000 and 0 cells / cm2) and exposed to
7% O2; n = 3 for each treatment.
Fig. 4: Average pO2 levels in the culture media of MEF cells exposed to Antimycin, DMSO and CCCP at the 4 hour time point ; n = 4 for each treatment.
cillin (10,000 U/mL)/ streptomycin
(10,000 µg/mL), 1% L-glutamine
(200 mM), 1% non-essential amino
acids (10 mM) and 1% 1 M HEPES
(pH 7.8) under standard cell culture
conditions (5% CO2, 35% humidity
and 37°C). In all cases, cells were
grown in 1 mL of media/well in a
Coy O2 Controlled Glove Box. The
oxygen concentration in the culture
medium was monitored throughout the culture period at preset intervals using the SensorDish Reader software.
when exposed to 7% O2. Therefore,
preincubation of the medium at
the desired oxygen level is recommended. Finally, CCCP was observed to increase cellular oxygen
demand while antimycin was observed to decrease cellular oxygen
demand compared to the vehicle
control. The SDR SensorDish Reader
allows for constant and rapid
quantification of media oxygen
levels which can be used as a measure of cellular oxygen demand
and metabolic function.
Conclusion
We monitored media oxygen levels
in mammalian cell cultures and
showed a cell density dependent
oxygen consumption. The density
dependent differences observed at
19% O2 were also observed at 7%
O2, a more physiologically relevant
oxygen level. In addition, cell culture media with no cells took more
than 10 hours to reach equilibrium
Contact
Lynn S.G. and LaPres J. J.
Dept. of Biochemistry, Michigan State
University
East Lansing, MI, USA
Sarina Arain
PreSens Precision Sensing GmbH
Regensburg, Germany
[email protected]
www.presens.de
Advertorial •
ADVERTISEMENT
High Energy Efficiency and Safety
The Dow Europe Laboratory in Horgen
Dow Chemical Company is one of the world‘s largest chemical companies
headquartered in Midland (Michigan) in the United States. Dow is a worldwide leading R&D company for chemicals, plastics, and agricultural products.
Sustainable activities are a fundamental part of corporate strategy and Dow
sets an example at its own locations, including the European headquarters
located in Horgen, near Zurich in Switzerland.
The Siemens Building Technologies Division and
Dow Chemical have enjoyed a close cooperation
for many years, with many of Dow‘s R&D facilities using Siemens technologies within their
laboratory operations.
The Horgen facility, in addition to serving as
the European corporate headquarters, is an important R&D location extended continuously for
applied research and customer applications.
Safety of staff and the environment are key, but
energy efficiency at Horgen has also been a fundamental focus for a number of years. Thus, the
new lab equipment they employ must not only
meet existing standards but supersede them.
mate be achieved within the buildings by adhering to business and ecological criteria.
Siemens provides its laboratory solution as
an answer to this challenge: a solution offering
comprehensive building automation and control
solutions along with specific functionality for
the entire laboratory. The solution supports the
requirements set forth in the European or Swiss
standard EN14175 or SN EN14175 and is independent of the fume hood type. The solution
combines control competency and quality, satisfying the highest demands of laboratory buildings while being user-friendly, reliable, extendable and flexible.
Challenging Environment
Automation of Laboratory Fume Hoods
at DOW Chemical
A laboratory is a challenging work environment,
subject to stringent rules and regulations for
workplace safety. However, in addition to fulfilling the primary duty of protecting laboratory
staff from potential hazards, Dow‘s demanding
requirements for the technical plants in the new
laboratories also require that a comfortable cli-
The laboratory fume hood is an important protective piece of equipment in a laboratory environment, offering lab users a specially protected
area to carry out experiments and tests. Targeted
air flow or extraction ensures that neither contaminated air nor explosive concentrations exist
in the working environment, while preventing
uncontrolled return flow in the work area. To allow users to carry out experiments, the fume
hood normally features a glass front sash that
can be opened or closed, providing an effective
safety barrier. When the front sash is closed, lab
users are protected against contamination and
exposure to toxic fumes or gases.
To protect as well while the front sash is
open, the volume flow extracted must prevent
toxic gases from entering the room. Thus, fume
hoods with constant flow are designed to extract the volume flow constantly to prepare for a
possible opening of the front sash. This results in
greater energy demand as well as unpleasant
noise, particularly when the front sash is closed.
Dynamic adjustment of the volume flow to the
current position of the fume hood‘s front sash
thus represents a more efficient and comfortable
solution. Safety can be guaranteed at significantly lower volume flows when the front sash
is closed, i.e. at a minimum opening of ca. 15
mm. Dow Europe employs exactly this type of
technology.
• Advertorial
The fume hood controller, as part of the Siemens laboratory solution, uses specific sensors
to acquire the front sash position linear and direct, allowing calculation of the effective opening of the front sash. The setpoint for volume
flow is defined as per the opening of the front
sash. The fast-acting control system can prevent
toxic gases from entering the environment by
dynamically adjusting the extracted volume flow
when the front sash is opened. When closed, the
volume flow is lowered again.
Integrated Security
The fume hood controller is supplied as a typetested laboratory fume hood extraction control
unit with all components such as volume flow
controller and measuring elements, actuator,
and sensors.
Intelligence is integrated in an autonomous
automation station communicating via LON interface with room control and building automation and control. The fume hood controller constantly monitors the extracted volume flow and
triggers a local warning in the event of any deviation from a pre-determined level along with a
warning to the building automation and control
system. In addition, users are notified under SN
EN14175 when the front sash opening of 500
mm (still considered safe) is exceeded. The fume
hood controller is designed to identify and signal wire breaks at the periphery, thus signaling
interrupted communications.
The application of the fume hood controller
is based on type-tested functions customized locally via just a few parameters to meet individual project requirements:
▪▪ Manual fume hood extraction flow control
using a fume hood operator unit by Siemens.
Max 5 fume hood operating modes are available.
▪▪ Automatic fume hood extraction volume
flow in dependence on front sash or slide
window position
▪▪ Automatic calibration of pressure sensor
▪▪ Front sash position acquisition and alarming
as per SN EN14175
▪▪ Variable setpoint generation based on constant flow velocity, variable linear setpoint
generation
▪▪ Supports strategies for simultaneous control
and maintenance of minimum air exchange
rate via fume hoods
▪▪ Monitoring for wire breaks
▪▪ Switching to emergency operation: Fire,
emergency shutdown, purge, emergency underpressure, emergency overpressure
▪▪ Control of fume hood lighting via operator
unit or by room occupancy
▪▪ Control of supporting flow fan
▪▪ Integration of switchable fume hood equipment
▪▪ Control via presence sensors
▪▪ Operating mode change based on room occupancy and scheduler program
Siemens Total Building Solutions (TBS)
Concept
The laboratory solution from Siemens is based
on the DESIGO building automation and control
system and laboratory-specific applications. At
Dow Europe in Horgen, efficient operations and
fast alarm management/fault intervention is carried out via the management station, providing
information from the building automation and
control system on the state of all technical and
mechanical installations.
Thousands of data points for ventilation
plants, laboratory rooms and fume hoods, along
with hundreds of fire detectors, gas detectors,
and supervision of critical doors are presented
graphically on the management station in a user-friendly manner.
Summary
For many years, the management and R&D location Dow Chemical in Horgen has been setting
the standard for safety and energy efficiency
within one of the world‘s largest chemical companies.
The decision in favor of the Siemens laboratory solution sets yet another standard toward
dynamic control and monitoring of fume hoods,
precise room control and efficient plant operations. Seamless integration in the location-wide
building automation and control system is welcomed, especially by users, as it guarantees a
high degree of safety, fast alarm/fault intervention, and continued plant optimization.
The integrated solution approach by Siemens
allowed for implementing Dow Europe‘s requirements from laboratory fume hoods to laboratory
room control to primary plant control and comprehensive building automation and control system visualization from one supplier.
Despite comprehensive technical requirements, the project was implemented within a
short period of time and at a high standard of
quality thanks to type-tested individual functionality and standardized communications interfaces. Fast customization to special needs by
Dow Europe provided proof of the flexibility of
the Siemens lab solution.
Contact
Jens Feddern
Siemens Building Technologies Division, Zug,
Switzerland
[email protected]
www.siemens.com/lifesciences
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• Products
Particle Size Analysis System
Beckman Coulter has extended the range of its
LS 13 320 particle size
analyzer to allow highresolution, reproducible
measurement of samples
from .017 to 2000 µm.
The enhanced device also
adds Rosin-Rammler and
Folk & Ward Phi methods
to its standard analytical
capabilities. The instrument’s patented Tornado Dry Powder Dispersing System keeps sample particles
intact, allowing true measurement of the original samples. Reproducibility is
typically better than one percent. These features combine with the extended
size range to provide high-accuracy/high-resolution detection without the risk
of missing either the largest or the smallest particles in a sample.
The analyzer differs from other laser-based instruments by virtue of its
wide dynamic size range, number of detectors and sample measurement
options. Reverse Fourier optics incorporated in a patented fiber-optic spatial filter system optimize light scattering measurement. Polarization Intensity Differential Scattering (PIDS) technology, a Beckman Coulter exclusive,
furthers detection capabilities in pharmaceutical, plastics, food and beverage, and a variety of other applications.
Vacuum Pumps for Filtration and Solid
Phase Extraction
In March 2010 Vacuubrand
launched the diaphragm pumps
ME 1 and ME 1C. They complete
the range of compact vacuum
pumps for applications like filtration and sample preparation in
chemistry, microbiology, wastewater control and other analytical processes. The ultimate vacuum of 100
mbar obtains 90% of atmospheric pressure that is doing the work of forcing
the media through the filter. For aqueous filtration, the ME 1 is the optimal
choice; however, for more aggressive solvents, the ME 1C with its chemical resistance properties is the right solution. The new top mounted power switch
and the space saving design offers an easy-to-use functionality even with
gloves and requires minimal bench top space. An optional manual control valve
with dial gauge enables variable fine adjustment of the pumping speed (max.
0.7 m³/h). Both pumps allow an almost maintenance-free use. New designed
from the established technology of the three-stage model line MD 1 and MD 1C
they stand out due to a proven long diaphragm life.
Vacuubrand
www.vacuubrand.com
POCKET
MICROSCOPE
30 or 100 magnification
SPIRIG
handy and self-illuminating
Beckmann Coulter
www.beckmancoulter.com
www.spirig-30.com
a: H
Visit us at Biotechnic
all 9 · Booth B37
www.gitverlag.com
Advertorial •
Nanoparticles in Liquids
Count, Size and Visualize
With more and more manufacturing processes turning to the use of component materials having nanoscale dimensions, the need for characterization of nanoparticles becomes more critical. This applies from the fundamental understanding of how individual nanoparticles behave through processing to ensure reproducible performance of the finished product in use. This could apply to vaccine production, the development of drug delivery systems or to the
production of inks and pigments. In all cases, the ability to count and size particles is required. With a new technique,
nanoparticle tracking analysis, this is achieved in parallel with the real-time visualization of the particles.
Nanoparticle Tracking Analysis (NTA)
NTA is a light scattering method
for nanoparticle analysis. It is being
increasingly used for determining
nanoparticle size through simulta-
neously tracking and analyzing the
trajectories described by a number
of individual nanoparticles undergoing Brownian motion in a fluid.
The technique is centred on a
sample analysis module which
comprises a small metal housing
Fig. 1
containing a solid-state, singlemode laser diode (< 30 mW, 635
nm) configured to launch a finely
focused beam through the sample
of liquid containing a dilute suspension of nanoparticles placed directly above a specially designed
optical flat. The sample chamber is
approximately 250 μl in volume
and 500 μm deep and the sample
is introduced by syringe via a Luer
port. The sample is allowed to thermally equilibrate for 20 seconds
prior to analysis.
The beam is caused to refract at
the interface between the liquid
sample and the optical element
through which it is passed such
that it describes a path which is
close to parallel to the glass-sample interface, (Fig. 1)
Particles resident in the beam
(which is approximately 100 μm
wide by 25 μm deep), are visualized by a conventional optical microscope aligned normally to the
beam axis and which collects light
scattered from each and every particle in the field of view. A video of
typically 20-60 seconds duration is
taken (30 frames per second) of
the moving particles (Fig. 2a). The
video is analyzed by a proprietary
analysis program on a frame-byframe basis, each particle being
identified and located automatically and its movement tracked.
The thresholds for particle identification can be user adjusted, as can
the gain and shutter speed settings
of the camera, thus allowing the
user to optimize the image for a
particular sample type. The 8-bit
video sequence can be automatically or user-adjusted in terms of
image smoothing, background subtraction, setting of thresholds, removal of blurring etc. to allow particles of interest to be tracked
without interference from stray
flare or diffraction patterns which
can occasionally occur with nonoptimum sample types.
Particles diffusing into the scattering volume are identified and
followed for the duration of the
particle presence in the beam or
until they diffuse to within a certain distance of an adjacent particle at which point tracking is
• Advertorial
sults shown as a particle size distribution plot
(Fig. 2c).
Looking ahead
More than one hundred peer reviewed publications serve to illustrate the growing use of NTA
to provide quantitative data leading to practical
solutions for the characterization of nanoparticles. The ability to visualize individual particles
counting each one separately provides exceptional confidence in the data being reported.
ceased eliminating the possibility of analyzing
particle trajectories which cross behind each
other (Fig. 2b). Movements of individual particles are followed through the video sequence
and the mean squared displacement determined
for each particle. From these values, the diffusion coefficient and hence sphere-equivalent,
hydrodynamic radius can be determined using
the Stokes-Einstein equation and with the re-
Sensitive Chemiluminescent Detection of
Hydrogen Peroxide
Lumigen HyPerBlu, a novel chemiluminescent substrate from Beckman Coulter enables direct detection of hydrogen peroxide.
Combining a broad dynamic range
and bright, sustained chemiluminescence, the ready-to-use reagent
offers convenience for highthroughput screening laboratories.
Reaction of the substrate with hydrogen peroxide rapidly generates sustained
high-intensity luminescence for maximum sensitivity in solution assays. Direct
detection increases accuracy and simplifies data analysis. When coupled with
oxidases, the reagent also allows reliable, one-step, indirect detection of oxidases or their substrates. The reagent is provided as a single solution and does
not require mixing. It is stable for one year when stored in an amber bottle at
2–8°C.
Beckmann Coulter
www.beckmancoulter.com
Data Visualization
Merck Millipore, the Life Science division of Merck KGaA, introduced the first
software application for visualizing GPCR (G-protein coupled receptors), as well
as kinase activity. The new Data Analysis and Report Tool (DART), which can be
accessed through Merck Millipore’s Drug Discovery portal, creates an interactive map of target profiling assay results and enables drug researchers to make
faster, more informed decisions. The application projects each compound’s activity profile onto a map depicting clusters of target protein families. This map
provides scientists with graphical insight into cross-target interactions to help
drive structure-activity relationship (SAR) studies. Data limits, sizing, colors, and
target subclasses can be adjusted in
seconds, thereby turning numerical
data into a graphical display that can
highlight biologically significant conclusions.
Merck Millipore
www.millipore.com
Contact
Andrew Malloy
Head of Applications Science
NanoSight Limited, Minton Park
London Road, Amesbury, Wiltshire, UK
www.nanosight.com
Reporter Genes as Minicircle DNA
Plasmid Factory offers a number of
common reporter genes as minicircle.
It consists almost only of the “gene of
interest” – the reporter gene. Antibiotic resistance and the ori are removed. In order to compare the
Minicircle with the classical plasmid,
the company has developed the McBox. It is currently available as Luc (luciferase), GFP (Green Fluorescent Protein) and LacZ (ß-galactosidase). Custom
made minicircle DNA and minicircle products with S/MAR elements are also
available.
Plasmid Factory
web.plasmidfactory.com/en
Plate Washer
Tecan has developed the HydroSpeed plate washer, an advanced system for optimized washing of cells, beads and ELISAs in 96- and 384-well formats. It offers
full control over critical wash parameters via an intuitive touchscreen interface,
with drop-wise dispensing and tunable aspiration settings to help avoid loss of
material and maximize assay efficiency. The system’s AntiClogging function is
automatically rinsing and soaking the wash head when the system is idle between runs, and the instrument’s Easy X-change system allows rapid removal
and replacement of wash heads for intense ultrasonic cleaning. The washer uses
two magnets per well for
magnetic bead washing, offering fast bead settling and
high recovery rates, and can
also be equipped with a vacuum filtration module for
processing of non-magnetic
beads.
Tecan Trading
[email protected]
www.tecan.com/cell-protection
Products •
Optical Emission Spectrometer
SPE Concept
Spectro has presented its Spectro
Arcos 165 for the first time at Jaima
Expo. The optical emission spectrometer with inductively coupled
plasma (ICP-OES) records the elemental spectrum between 165 and
770 nanometer for every measurement; making it especially suited to
challenging tasks in environmental
analysis. The system rounds out the
ICP-OES analyzer product series between the flagship Spectro Arcos and the entry-level Spectro Genesis. The engineering of the system “is based completely on the high-end components utilized in our flagship Arcos system and achieves exactly the same detection
limits, the same precision and equally reproducible results,” explains Olaf
Schulz, the manufacturer’s Product Manager for ICP-OES spectrometers. The optical system is the only difference between the models: While the Arcos records
the entire spectrum starting at 130 nanometers for every measurement, the
trimmed down Arcos 165 measures the wavelength range beginning at 165
nanometers.
For sample preparation, cleaning and
concentration of neutral, acidic and basic analytes from various matrices (e.g.
urine, blood, tablets, food, water), Macherey-Nagel offers new Chromabond
HR-Xpert – a professional SPE concept.
Polymer-based RP- and mixed-mode ion
exchange phases fulfill the demands of
modern SPE phases. They provide an excellent enrichment of neutral, acidic and
basic compounds. The spherical support
polymer (PS/DVB) with optimized pore
structure and high surface facilitates
good reproducible, reliable and cost-efficient analysis with a broad spectrum of
applications. The divers mixed-mode phases Chromabond HR-XC, HR-XCW, HRXA and HR-XAW provide the option of more aggressive washing procedures for
matrix removal. Thus, cleaner samples and a protection of HPLC and GC instruments result.
Spectro Analytical Instruments
www.spectro.com
Semi-Preparative HPLC Pump
Cecil Instrument’s Adept HPLC
range has been enhanced with a
newly updated semi-preparative
pump. The pump expands and
streamlines the modularity of this
range. Each pump has a range of
0.01 to 50 ml/minute and may be
used isocratically or with others, to create a high pressure gradient. This makes
for smooth, easy and reproducible transitions in scaling up from analytical to
semi–preparative work. The use of semi-preparative pumps, with Autoquest autosamplers, column heater chillers, Wavequest UV/Visible ultra-fast scanning
detectors, and fraction collectors makes for complete automation in the longterm, fast and reliable collection of fractions. As with all the Cecil modular components, the semi-preparative pump may be used with third party systems.
Cecil Instruments
www.cecilinstruments.com
Vital Parameters Kept Under Surveillance
Presens Precision Sensing
has introduced the EOM-CO2mini. This electro-optical
module is a solution for original equipment manufacturers
(OEM) for customized monitoring devices. As for its small
footprint, it can easily be integrated and, assembled according to ISO
9001:2008, it enables a precise and non-invasive CO2 measurement. The instrument extends the manufacturer’s scope of supply for OEMs. Customers from
Scientific R & D as well as Biotech & Pharma can now monitor all parameters
which are essential for life: O2, pH and CO2.
PreSens Precision Sensing
www.presens.de
Machery-Nagel
www.mn-net.com
Absorbance Reading
BMG Labtech has introduced the
Spectrostar Nano. This ultra-fast,
full spectrum absorbance microplate reader with cuvette port is
suited for all absorbance assays. Its
rapid full spectrum analysis at a
resolution of 1 nm allows for absorbance assays never before possible on a microplate reader. Using
an ultrafast absorbance spectrometer, it can capture a full UV-Visible spectrum
from 220 nm to 1.000 nm in less than 1 sec/well and measure sample volumes
down to 2 µL. Use the built-in cuvette port for kinetic studies and quick experiments and measure all standard microplate formats up to 1536 wells. The microplate reader allows for single push button operation for basic commands, as
well as specific assay protocol set up. The most common absorbance assays
such as Elisas, DNA, RNA, Protein (Bradford, BCA, Lowry), cell growth, and beta-galactosidase can be performed with ease due to predefined protocols.
BMG Labtech
www.bmglabtech.com
Giving pH Electrodes a Treat
Metrohm has presented the “pHit
kit”. It contains all that is needed to
gently clean or regenerate pH electrodes: A step by step description of
the cleaning procedure; 50 mL cleaning solution; 50 mL 3 M KCl electrolyte
solution; 50 mL storage solution, and
two electrode vessels.
Metrohm
www.metrohm.com
• Company Index & Imprint
Agilent Technologies Applied Biosystems. Beckman Coulter 8
Out Back Cover
51, 53
Biocision 22
BioCision
7
Helmholtz- Zentrum für Infektionsforschung IME Fraunhofer Inst. f. Molekularbiologie u.
Angew.Ökologie 30
Kraeber 25
Macherey- Nagel 54
29
BMG Lab Technologies 54
Merck Brand Fabrik für Laborgeräte 13
Metrohm Bruker
33, 38
C- CIT 20
13
11
NanoSight 52
Cecil Instruments 54
National Dong Hua Univers. 32
CPC Colder. 31
Olympus Europa Holding David James Group 14
Phoenix MarCom 5, 27
Cover
22
Pittsburgh Conference on Analytical Chemistry 50
EBD 12
Plasmidfactory ERC 28
Presens 46, 54
9
Qiagen 15, 16, 19
GFL Ges. f. Labortechnik IMPRINT
Segment Sales Manager
Laboratory & Biotechnology
Published by
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Tel: +49 6151 8090 261 Fax: -144
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Select Biosciences 8, 12
Shimadzu Europa 35, 42
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Spectro Analytical Instruments 54
Spirig 51
Steiner Dt. Arzneimittel Gesellschaft 44
Tecan Group 53
Texas Tech University 26
Thermo Fisher Scientific 38
Univers. of Massachusetts 24
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Manuscripts should be addressed to the
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Reproduction, including excerpts, is permitted only with the permission of the
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The Path Is Clear
Introducing the 7500 Fast Dx Real-Time PCR Instrument
Forging a new path to clinical flexibility.
The new 7500 Fast Dx Real-Time PCR Instrument from Applied Biosystems
is a highly flexible, medium-throughput solution that may streamline your
assay development and validation efforts—allowing you to work directly on
an in vitro diagnostic platform. And the 7500 Fast Dx Real-Time PCR Instrument
is flexible and open enough to integrate seamlessly into your workflow from
single tube strips through 96-well plate setup.
• Available for in vitro diagnostic use*
– May help to minimize time and expense in obtaining assay clearance
• Open software design
– Full control over thermal cycling protocols
• 96-well block format
– Compatible with standard prep and setup instrumentation
It’s the perfect combination of speed, flexibility, and productivity—
the path is now clear.
*For In Vitro Diagnostic Use Only. The 7500 Fast Dx meets the requirements of the In Vitro Diagnostic Medical Devices Directive (98/79/
EC). The 7500 Fast Dx is for distribution and use in specific European countries only. Not for use in the USA.
©2010 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies
Corporation or their respective owners.
To learn more about the 7500 Fast Dx
Real-Time PCR Instrument, visit
www.appliedbiosystems.com/7500Dx

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