Cryopreservation
Benefits Of Freezing Cells
• Benefits Of Freezing A Validated Stock Of Cells
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Genotypic drift
Senescence leading to extinction of cell line
Transformation to tumor related properties
Contamination
Distribution to others
Saving reagents, time
Equipment failure such as incubator
Cross-contamination by other cell lines
Theoretical Background Of Cell Freezing
• Optimal Cell Freezing Is Characterized By
– Maximum number of viable cells upon thawing
– Minimum intracellular crystal formation
– Minimum formation of foci of high solute concentration
• Optimal Freezing Is Accomplished By
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Cooling slowly so water escapes
Cooling fast enough to avoid crystal formation
Use hydrophylic cryoprotectant
Storing at lowest possible temperatures
• minimize negative effect of solute foci on proteins
– Thaw rapidly
• minimize crystal growth and solute gradients
Cell Concentration And Freezing Medium
• High Cell Concentration Seems To Enhance Survival
– Possibly due to “leakiness” effect from cryogenic damage
– Centrifugation is avoided since dilution of cryoprotectant is high
when reseeding
• Ex. 1 mL of 1x107 cells diluted to 20 mL volume giving a 5x105 cells/mL. If
cryoprotectant was 10% it will become 0.5%
• Toxicity is unlikely at 0.5%
• Residual cryoprotectant can be removed as soon as cells start growing
• Freezing Medium
– DMSO, Glycerol
– DMSO used at 5-15%, 10% is common
– DMSO should be stored in glass or polypropylene
• Can dissolve rubber and some plastics leading to impurities
– Many laboratories increase FBS concentration to 40, 50 or 90%
Cooling Rate
• Optimal Cooling Rate: 1°C/minute
– Compromise between fast freezing minimizing crystal
formation and slow allowing for extracellular water
migration
• Cooling Curve Is Affected By
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Ambient temperature
Insulation
Specific heat of ampoule contents, volume of ampoule
latent heat absorption during freezing
Insulation During Freezing
• Use 2 Polystyrene Foam Boxes To Store Vials
– This set up provides insulation for 1°C/minute cooling
• Place In A –80°C Freezer
• Transfer To Liquid Nitrogen After 24 Hrs Or
-150°C Freezer
• If At Kean Just Leave It In The -80 °C
• Liquid Nitrogen Is Widely Used To Store Cells Long
Term
– -196 °C
– Several types of liquid nitrogen cryofreezers are available
Ampoules
• Use polypropylene cryovials
– Resistant to cracking
• Some Repositories Prefer Glass
– Better properties for long term storage
• Labeling Is Very Important
– Stored cells can outlive you! Proper labeling is essential
– In your label include
• Cell type, date and cell number
• Use an alcohol resistant marker
Thawing Stored Ampoules
• Thawing Of Cells Should Be Rapid
– Water bath @ 37°C
– Reason: minimize crystal formation
• Spray Vial With Alcohol To Avoid Contamination
• Dilution Should Be Done Slowly
– DMSO will cause severe osmotic damage if done fast
• Most Cells Do Not Require Centrifugation
• Some Do Require Centrifugation
– Ex. suspension growing cells