In a serum-free cell culture system, it is very important to cryopreserve cells also in a serum-free freeze medium and not in a serum containing medium. Using an ordinary serum containing freeze medium, the serum affects the cell culture system over a long period. This influence can only be removed by dilution during a large number of passages after seeding.
- Adherent Cells: Remove adherent cells with Trypsin/EDTA. Neutralize Trypsin with Trypsin inhibito, pellet cells by centrifuging at low acceleration (200×g) for 3-5 minutes and remove Trypsin inhibitor solution. Resuspend the cells in cold Cryo-SFM at a concentration of 1 - 4 million cells/ml. Freeze the cells gradually (I °C/minute) and store them in liquid nitrogen.
- Cells in Suspension: Pellet the cells by centrifuge at low acceleration (200×g) for 4 minutes. Remove the supernatant, gently resuspend the cells in Cryo-SFM at a concentration of 1-5 million cells/ml. Freeze the cells gradually (1 °C/minute) and store them in liquid nitrogen.
- Thawing: Gently shake the vial in warm water (37 °C) until approximately 90% of the freeze medium is just thawed. Remove the vial immediately and continue shaking until the whole contents is thawed. (Do not allow longer incubation of the vial at 37 °C. The viability of the cells is drastically diminished in the thawed freeze medium.) Wipe it with 70% ethanol and remove the cap being careful not to touch the interior threads with fingers. Resuspend the contents of the vial gently using a pipette and dispense the contents of the vial into the equilibrated culture vessels.
Provitro's Cryo-SFM is a new formulation for cryopreservation of animal and human cells containing no serum, but rather DMSO, methyl cellulose and other cryoprotectant ingredients. After cryopreservation and thawing, a very high percentage of viable cells is obtained. Cells which are cryopreserved in Cryo-SFM also show excellent attachment ability as well as growth performance after thawing.
