Cryopreservation

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

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

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 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 Some Repositories Prefer Glass 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