- Hypoxianet

Making Choices
How to select the equipment to produce the O2 environment you need
Oxygen 2011 Training School Jan 10, 2011
Making Choices Outline
• Basic Incubation Chamber Types & Primary Considerations
• InVivo Applications
Cabinets
Glove Boxes
• InVitro Applications
Tri Gas Incubators
Compact Static Chambers
Cabinets
Glove Boxes
• Translation of Gaseous O2 to Cells
Measurement
New Developments
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Basic Incubation Chamber Types
• Tri‐Gas Incubators
• Compact Static Chambers •
Airtight Flush Boxes that have no controllers
•
Place in glove boxes or incubators/ovens for temp control
• Cabinets
• Glove Boxes
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Major Glove Box & Cabinet Suppliers
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Billups‐Rothenberg (compact static chambers only)
Biospherix (glove boxes and unsealed cabinets)
Coy Laboratory Products (full line)
Don Whitley (glove boxes only)
Plas‐Labs (glove boxes only)
Ruskinn (glove boxes only)
NOTE: Standard
incubator suppliers
provide tri-gas
incubators
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Primary Considerations 1. Temperature
2. CO2 3. Humidity/Evaporation/ Condensation
4. O2 Level
• Cycling (ramp/soak) or Steady
• Dissolved vs. Gaseous
• Various Levels Simultaneously
• Step Change (e.g. Ischemia)
5. Continuity of 1‐4 throughout the protocol
6. Duration
7. Other Environmental
Factors
• Waste Removal
• Pressure
• Size or Number of Samples
Address all of these in any equipment selection
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InVivo or InVitro?
InVivo:
InVitro: Tissue Culture in…
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Flies
Rats
Mice
Rabbits
Zebra Fish
Worms
Multi‐well plates (can be 3D)
Flask
Perfusion Chambers
Dishes (can be 3D)
Microfluidics
NOTE: Equipment cross‐over rarely successful due to procedural & contamination issues.
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InVivo Applications
Two Primary Options: Cabinets or Glove Boxes
Key Deciding Factors
Cabinets
Glove Boxes
Manipulation Possible During Exposure
No
Yes
Number of Animals
Few
Few to Many
O2 Cycling
Quick
Slow
Duration of Exposure
Short
Long
Size of the Animals
Limited
Any Size
Economics
€
€€
NOTE: Both typically use ambient temperature passively
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InVivo Applications
Cabinets
• Unsealed with O2 control (may be insufficient to remove waste)
• Sealed with O2 control (lower gas usage)
• Waste build‐up (CO2, urea, humidity, etc.)
o Will be negligible w/short duration or small numbers of animals
o Space available beneath the cage shelf for holding small amounts of waste removal substances (soda lime, charcoal, and desiccant)
o Can also use capsules for waste removal (automatic or manual)
• Best suited for quick O2 cycling if use small size
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InVivo Applications
Glove Boxes
• Temperature control (completely vinyl construction preferred if using ambient temp)
• CO2 removal/control
o
Combination of duration, animal type, & glove box size determines how managed
o
Cartridges ‐ manual or automatic (redundancy to allow change out)
• Humidity removal/control (with chillers can be related to temp control)
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InVivo Applications
Glove Boxes (con’t)
• Duration (long when airlock/transfer chamber used to exchange supplies)
• Door/Entry for cage change‐out before exposure
• Size of cages and procedures dictate size of door/entry, air lock, & glove box
• Vinyl glove box —more ergonomic than rigid gloveless units
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InVitro Applications
Four Primary Options
1.
2.
Tri‐Gas Incubators
Compact Static Chambers • Airtight Flush Boxes that have no controllers • Place in glove boxes or incubators/ovens for temp control
3.
4.
Cabinets
Glove Boxes
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InVitro Applications
Tri‐Gas Incubator
• Control/Maintain O2, CO2, temperature, & humidity
• Continuity cannot be maintained if the door is opened
• Gas usage higher than sealed chambers
• Manipulations not possible during stable incubation
• Cannot cycle between multiple
O2 levels rapidly
• Hard to control at low O2 levels
(gas consumption high
due to being unsealed)
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Compact Chambers & Cabinets
Common Characteristics
• Place in CO2 incubators, ovens, or glove boxes for temp control
• Use mixed gases for CO2 inclusion
• Include open water source to minimize sample evaporation if critical
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InVitro Applications
Compact Static Chambers (uncontrolled O2 level)
• Sealed flush boxes have no O2 controller
• Procedural Notes specific to Static Chambers
o
Unable to compensate for leaks, plastic outgassing or diffusion, gas consumption, etc.
o
Must remove pressure generated if heated after gaseous flush
o
Limited size & ability to feed cords and tubes into chamber
o
Static environment so mixing depends on convection and/or diffusion
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InVitro Applications
Cabinet With Controls
Advances relative to static chamber
• Active O2 control
• CO2 can be controlled independently or mixed into the N2 tank (most common)
• Ideal for cycling or step change
• Can be “sealed” to minimize gas usage
• Feed‐thrus for adding extra tubing
and cords
• Dynamic air mixing enables
uniformity and control
• Unlike glove boxes, these do not
allow for sample manipulation
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InVitro Applications
Glove Boxes (sometimes called workstations)
Like adding gloves and airlock/transfer chamber to your incubator—
provides continuity throughout procedures
• Long duration incubation under experimental conditions possible
• Temperature control (typically entire glove box is heated)
o
Non‐insulated o
If possible, place control probes near any internal incubation site
o
Use reference temperature sensor to monitor temperature near the
plates
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Glove Boxes: CO2 Control
Independent CO2 Control
• Separate or Integrated as part of O2 Controller • pH of media maintained using bicarbonate‐CO2 buffering system (5% CO2 typical)
• Enables use of sophisticated media to set CO2 levels at physiologic levels (non 5%) while maintaining pH
• Facilitates hypercapnia studies
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Glove Boxes: Humidification Considerations
•
Humidification Requirements depend upon duration and sample volumes: longer durations + lower volumes need higher humidity to slow evaporation
•
Whole Glove Box Humidification limits ability to install equipment in glove box
•
Degree of Humidification limited by temperature of non‐insulated walls
o
Condensation can increase potential of contamination and prevent
proper function of some sensors if liquid covers gas exchange membrane
•
Humidified Incubation Box within Glove Boxes allows for humidification of samples and humidity control in the remainder of the glove box •
Non‐condensing Environment allows electronic equipment to be used inside of a glove box
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Glove Boxes as Customized Workstations
Customize Glove Box to enclose equipment, thus complete procedures are done under controlled conditions
• Eliminates potential of effects due to changes in the environmental temp, O2, pH, humidity, etc.
• Customization may include size changes for glove box, doors, or airlock; addition of feed thrus for tubes or cords; access ports, microscope view ports; connecting glove boxes, etc.
• Need non‐condensing environment for most electronics
• Consider heat generation by equipment
• Select vendor with customization capabilities
o
Some with only modular design flexibility have limitations
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Glove Boxes: Customization Examples
Flow Cytometer in Hypoxic Glove Box
http://www.youtube.com/watch?v=Qhx-2DZACY
Microscope for Live Cell Imaging in Hypoxic Glove Box
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Glove Boxes: Environmental Factors & Options
Options
Environmental Considerations
•
HEPA Filtration of interior atmosphere
• Favorable ergonomics of flexible vinyl for entire chamber
•
UV Lights for “sterilization”
• Limited durability of vinyl glove ports when not allowed to flex as an entire chamber
o
Limits materials of construction
•
H2O2 Decontamination of system
•
Diaphragm tops prevent glove fight‐back upon entry and minor vacuum upon exit (potential for O2 ingress) by adjusting to volume changes •
Sliding shelf in air lock eliminates need to reach at odd angles into air lock
• Lab space requirements for glove box and attached air lock
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Glove Boxes: O2 Control
• Cost‐effective sensor technology limits accuracy to +/‐0.5% with precise calibration (account for dilution of affects of humidity in air or use calibrated gases)
• Anoxic (ppm O2) w/catalyst & hydrogen gas mix or N2 purge
• Volume too large to cycle or perform fast step change but can place separate cabinets in glove box to cycle or for simultaneous multiple O2 levels
• Control using dissolved O2 levels is difficult due to long diffusion times, heat of evaporation, etc.
• Gloveless units allow barehanded access but will have O2 ingress
o
Vacuum/purge sleeves available to minimize O2 ingress, but the trade‐off is longer access times to the glove box interior
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Translation of Gaseous O2 to the Cells
Diffusion Rate may not Keep up with Respiration
O2 level in culture media at mouse embryonic fibroblasts
plated at differing densities (100k, 33k, & 10k cells/cm2)
Gaseous O2 is controlled and must diffuse through media to reach the cells
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pO2 [% oxygen]
Cross‐section of well
in multi‐well plate
16
14
12
Even at 19% O2, diffusion unable to
keep up with O2 loss in media due to
respiration.
100
33
10
10
0
8
0
= O2 molecule
= adherent cells
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2
3
4
5
6
7
8
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Time (hours)
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100
pO2 [% oxygen]
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33
14
10
12
0
10
To obtain or maintain desired O2
levels in media, need to consider cell
density and media pre-equilibration.
8
6
4
NOTE: Remember to pre‐equilibrate media with enough time, with low depth to surface area ratio or mixing, by adding heat labile proteins last, & accounting for plastic acting as O2 reservoir
2
0
0
1
2
3
4
5
6
Time (hours)
7
8
9
10
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Pericellular & Intracellular Measurement
Sensor Dish Reader – continuous monitor of media O2 level, observe relative consumption rates, & compare treatment affects Optode Sensor are “printed” on the floor each well
Data from a single well
MicroSensors
– can measure O2 at discrete locations more information later in the session
Luxcel Biosciences Sensors
– pericellular, intracellular, and organellar microscopic phosphorescent oxygen probes
more information later in the session
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Gas Permeable Plates – testing ongoing
Cells grown on gas permeable membranes to
avoid/minimize diffusion effects through media
cell
Semi-permeable membrane
O2 and CO2
Membrane replaces bottom of microwell plate
Two parallel membranes with media between them
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Summary: Use Application to Screen Chamber Types
Application
• Temperature
• CO2 3. Humidity/Evaporation/
Condensation
4. O2 Level
5. Continuity of 1‐4 thru protocol
6. Duration
7. Other Environmental
Factors
O2 Chamber Types
• Tri‐Gas Incubators
• Compact Static Chambers • Cabinets
• Glove Boxes
and consider verifying biomemetics of invitro systems
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Questions?
Karen Studer‐Rabeler
VP, Business Development
[email protected]
Brian Coy
Director of Sales and Marketing
[email protected]
(734) 475‐2200
Coy Laboratory Products, founded in 1969, initially provided flexible vinyl anaerobic chambers for microbiology research. In the early 1990’s we provided our first hypoxic glove box and controllers. Today all manufacturing and design is done in our facility in Grass Lake, MI enabling us to provide economical and
effective designs for both standard and custom equipment.