cryopreservation
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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

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Presentation Transcript
benefits of freezing cells
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
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
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
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
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
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 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