How to Cryopreserve Human Primary Cells

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Cryopreservation of Human Primary Cells (Target Cells)

Because cells are a valuable resource and replacement is expensive and time consuming, freezing and preserving extra fresh cells or cells that are not needed immediately for processing for long-term storage is vitally important.

Please note that freezing extra fresh cells after thaw may decrease viability and lead to poorer cell recovery after thaw.

Materials Needed

  • Sterile conical centrifuge tubes, 15mL or 50mL
  • Cold CryoStor® CS10 or 10% DMSO freezing medium
  • Washing buffer:
    • PBS without calcium or magnesium
    • 0.5% Human Serum Albumin (HSA)
    • 2mM EDTA
  • 70% ethanol
  • Sterile 1.2mL or 1.8mL cryogenic vials
  • Labels compatible for use in freezing and liquid nitrogen
  • Brooks Life Sciences CoolRack®, or other method to cool cryogenic vials
  • Brooks Life Sciences CoolCell® freezing container, or other control-rate freezing container

Notes

  • Always wear personal protective equipment and use universal precaution when working with human-derived biological materials and liquid nitrogen.

The protocol describes a general procedure for freezing cells. For cell-specific freezing protocols, please contact our scientists at (877) 944-4362.

Protocol

  1. Count the cells that will be cryopreserved.
  2. Calculate the amount of vials for cryopreservation [total cell count from step 1 ÷ size of preferred vial (i.e. 10M, 25M cells) = amount of vials].
  3. Print or write labels with the appropriate vial and donor information on each cryogenic vial.
  4. Place cryogenic vials in cryogenic vial holder, such as the CoolRack®, and place in -20°C freezer for at least 15 minutes.
  5. Spray bottle of CryoStor® CS10 / freezing medium with 70% alcohol and chill on ice.
  6. Spin down the cells in 50mL conical tubes at 400 x g for 10 min at 4°C.
  7. Discard the supernatant, loosen the cell pellet by gently tapping the tube, spray tube with 70% alcohol, and chill on ice.
  8. Remove the cryogenic vials from the -20°C freezer, spray with 70% alcohol, and transfer to the biosafety cabinet.
  9. Resuspend the cell pellet with the appropriate amount of CryoStor® CS10 / freezing medium.
  10. Mix the suspension well by gently pipetting up and down to ensure an even concentration solution of the cells and CryoStor® CS10 / freezing medium.
  11. Aliquot the cell solution into each cryogenic vial.
  12. Place the cryogenic vials into designated freezing containers.
  13. Transfer the freezing container to a -80°C freezer overnight.
  14. Transfer the cryogenic vials to vapor phase of liquid nitrogen tank.

Tips and Tricks

  1. Prefreezing the vials helps to reduce the shock to the cells for freezing.
  2. The concentration of CryoStor® CS10 is 0.5-10×106 cells/ mL of CryoStor. This is an optimal concentration for adding CryoStor to protect the cells.
  3. Storing at -80°C overnight is gentler than directly storing in liquid nitrogen and the rate of 1°C/minute is used to prevent the formation of ice crystals that would shear the cells and destroy them.
  4. Vapor phase of liquid nitrogen is gentler for the cells than liquid phase. Liquid phase can potentially introduce liquid contaminants to the cells.

For Technical Support, dial 877.944.4362 or email bioresearchproducts@hemacare.com.