Microscale Engineering of Tissue Models for Compound Screening

Microscale Engineering of Tissue (Liver)
Models for Compound Screening
Salman R. Khetani
Assistant Professor
Department of Mechanical Engineering
School of Biomedical Engineering
Isolated Primary Hepatocytes
Rapid Loss of Viability and Phenotypic Functions In Vitro
Albumin (µg/106 cells/hour)
0.35
0.30
0.25
0.20
0.15
Fresh Hepatocytes
0.10
0.05
Day
0.00
1
2
3
4
5
6
7
9
10
12
14
Albumin – marker of liver’s synthetic ability. Other
functions decline as well (i.e. CYP450)
2 weeks later
The Liver Microenvironment
Role of the Microenvironment in Modulating Hepatic Fates
Microscale Technologies For Tissue Engineering
Semiconductor-Driven Tools for Biological Applications
Khademhosseini A et al. PNAS 2006;103:2480-2487
Micropatterned Co-Cultures (MPCCs)
Role of Tissue Organization in Modulating Hepatic Functions
HUMAN Hepatocytes – Stromal Support Cells
Khetani and Bhatia, Nature Biotechnology, 26(1), p120-126, 2007
MPCC Functionality and Longevity
Mimicking Elements Of In Vivo Liver Physiology In Vitro
Albumin
Urea
 Other functions (i.e. CYP450 and conjugation enzymes, transporters)
show similar kinetics/stability
 Human and Monkey MPCCs – High levels of functions for ~1 month
 Rat MPCCs – High levels of functions for ~2 months
Khetani and Bhatia, Nature Biotechnology, 26(1), p120-126, 2007
T-Cadherin Induces Hepatocyte Functions
Cellular (CHO) and Purified (Recombinant) Protein Presentation
Khetani et al, FASEB J, 22(11), p3768-3775, 2008
Miniaturized MPCCs
HepatoPac™ by Hepregen Corporation (Medford, MA)
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Up to 96-well standard tissue culture plates
Compatible with fresh and cryopreserved
hepatocytes (human, rat, monkey)
Amenable to automated fluid handlers
Amenable to high content imaging
Prototype Development Funded by Deshpande Center at MIT
Drug Metabolism in MPCCs
Better Prediction of Drug Clearance & Metabolite Production
• Long-term continuous (up to 7 days) incubations allows for clearance
of low turnover compounds and detection of secondary metabolites
• Tight well-to-well variability (CV < 15%) allows rank ordering of
compounds based on intrinsic clearance
• With Pfizer (Groton, CT)
Wang W, Khetani S et al, Drug Metab Dispo, 38(10), p1900-1905, 2010
Detection of Drug-Induced Liver Injury in MPCCs
• 96-well format MPCCs
• Repeat dosing with drugs (45 drugs total) over 5 to 9 days
• Cellular functions assessed:
• Albumin
• Urea
• ATP
• Glutathione
• Hypotheses:
• Repeat dosing improves sensitivity without loss of specificity
• Functional assays are as sensitive as destructive toxicity endpoints
for rapid (and non-destructive) hazard identification
• Human cultures are more sensitive than rat ones
Detection of Drug-Induced Liver Injury in MPCCs
• Repeat dosing (2 vs. 4 doses) improves sensitivity
without compromising specificity (~90%)
• Human cultures are more sensitive than rat ones
(~70% vs. ~50%) for predicting human DILI hazards
• Albumin/urea are more sensitive than ATP and GSH
for rapid hazard (and non-destructive) identification
Kupffer Macrophages in MPCCs
Day 2
Day 10
Day 10
Hepatitis C (HCV)
A Global Epidemic

An estimated 170
million people
worldwide are infected

Leading cause of liver
transplants in U.S.

No vaccine available

Severe side effects of
current therapies
Replication of HCV in MPCCs for Several Weeks
Primary human hepatocyte in MPCCs are susceptible to HCV
Ploss/Khetani et al, PNAS, 107(7), p3141-3145, 2010
Type 2 Diabetes Mellitus (T2DM)
A Global Epidemic

~285 million people affected and rising due to obesity. T2DM
accounts for 90-95% of the cases

Difficulties maintaining glucose levels in the blood due to:


Reduced insulin output from pancreas AND

Insulin resistance in peripheral tissues
Complications include:


CVD, neuropathy, nephropathy, eye damage, foot damage,
hearing problems etc.
Glucose levels are maintained by the liver via storage (as
polymer glycogen) and de novo production (gluconeogenesis).
Gluconeogenesis (GNG) in MPCCs
Work of Mike Lehrer and Matt Davidson
Glycogen Production in MPCCs
5mM Glucose,
No Hormones
5mM Glucose +
Insulin
5mM Glucose +
Glucagon
Work of Mike Lehrer and Matt Davidson
Patient-derived Induced Pluripotent Stem Cells
• iPSCs have been generated (~5 years ago) by expression
of 4 transcription factors into somatic cells (i.e. skin)
• Human iPSCs can mimic human embryonic stem cells in
all aspects of pluripotency and differentiation.
• Great potential for personalized medicine
• Functions in iPSC-derived human hepatocytes (iPSCHHs) can be further improved (magnitude and
longevity) using microscale engineering approaches.
iPSCs-HHs in MPCCs: i-MPCCs
Cells from Cellular Dynamics International
•
•
•
•
i-MPH = micropatterned pure hepatocytes
i-MPCC = micropatterned co-cultures
CYP3A4-Glo from Promega: Contributions for CYP3A5 and CYP3A7?
Decline seen in sandwich cultures, which have ~5-10 fold lower activities
(on per cell basis) than MPCCs
Work of Dustin Berger, Matt Davidson
Toxicity Assessment in i-MPCCs
Cells from Cellular Dynamics International
• Diclofenac and
amiodarone are
hepatotoxins
• Aspirin and
propranolol are
relatively safe
compounds
Work of Matt Davidson, Brent Ware
Zonation in the Liver
• Zonation can lead to
zone-specific drug
toxicities in the liver
• Allows various
hepatocytes in the liver
to adapt to systemic
changes in the body
(i.e. fasting, feeding,
disease)
• Very few model
systems to study this in
vitro.
Modeling Oxygen In Vitro
Work of Dustin Berger
Culture on Softer (More Tissue-Like) Substrates
Chemo-mechanical Tuning of Polyelectrolyte Multilayers
Chen/Khetani et al, Biomaterials, 30, p1113-1120, 2009
Overall Vision for Microfabricated Tissue Models
Laboratory at CSU
Liver Disease
• Drugs
• Industrial chemicals
• Alcohol
• Viruses
• Nutrition
Engineering
• Microfabrication
• Polymers
• Computation/Modeling
• High-throughput
screening devices
• Multiplexed reporter
systems
Cell Sources: Human, Animal, Stem Cells
Why? Prevention, Diagnosis and Treatment of Liver Disease
Acknowledgments
Microfabricted Tissue Models Lab (CSU)
Academic Collaborators
Dustin Berger (PhD student)
Brent Ware (PhD student)
Christine Lin (Rotation PhD student)
Matthew Davidson (ME student)
Josh Pickrell (ME/SBME Undergraduate)
Alison Bailey (CBE/SBME undergraduate)
Hepregen Research Team
Charles Rice et al (Rockefeller)
Sangeeta Bhatia et al (MIT)
Mike Pagliassotti (CSU)
Adam Chicco (CSU)
Chuck Henry (Chemistry, CSU)
Hugo Rosen (CU-Boulder)
David Eddington (UIC)
Neil Kaplowitz (USC)
Simon Aoyama
Chitra Kanchagar
Stacy Krzyzewski
Amanda Moore
Julianne Shi
Jeannemarie Gaffney
Okey Ukairo
Industrial Collaborators
Scott Obach (Pfizer)
Yvonne Will (Pfizer)
Mike Aleo (Pfizer)
Funding
Pfizer Corporate Partnership
NIH Challenge Grant (PI: S.Khetani)
NSF SBIR Phase I and II (PI: S.Khetani)
FDA SBIR Phase I and II (PI: S.Khetani)
CSU Mech. Eng Start-up funds