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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Cryopreservation

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Cryopreservation

Cryopreservation


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


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