Cryopreservation. Benefits Of Freezing Cells Benefits Of Freezing A Validated Stock Of Cells –Genotypic drift –Senescence leading to extinction of cell.

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1 Cryopreservation

2 Benefits Of Freezing Cells Benefits Of Freezing A Validated Stock Of Cells –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

3 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 –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

4 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 1x10 7 cells diluted to 20 mL volume giving a 5x10 5 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% Cell Concentration And Freezing Medium

5 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 –Ambient temperature –Insulation –Specific heat of ampoule contents, volume of ampoule – latent heat absorption during freezing Cooling Rate

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7 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

8 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 Ampoules

9 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 Thawing Stored Ampoules