a. Harvest cells as usual (and wash once with complete medium).
b. Resuspend cells in complete medium and determine cell count/viability. Keep cells on ice.
c. Centrifuge and resuspend in ice-cold freezing medium: 90% calf serum/10% DMSO, at 106 – 107 cells/ml.
Do use Recovery™ Cell Culture Freezing Medium!
d. Transfer 1 ml aliquots to freezer vials on ice.
f. Place in the -70°C freezer overnight. Note: Cells should be exposed to freezing medium for as little time as possible prior to freezing
g. Next day, transfer to liquid nitrogen (DON’T FORGET) and log in the “Liquid Nitrogen Freezer Log” Book.
Option: Achieve cryopreservation in either an automated or manually controlled rate freezing apparatus following standard procedures (approximately 1°C decrease per minute). Transfer frozen cells to liquid nitrogen, storage at -125° to -200°C is recommended.
Cryogenic preservatives – DMSO, glycerol, Recovery™ Cell Culture Freezing Medium
Prevent ice crystal formation within cells that would rupture membranes and kill them
Since ice crystals still form outside the cells, would get dessication of the cell as water would leave cytoplasm to equilibrate inside and out. DMSO also can bind water in the cytoplasm, preventing it from leaving