Thawing should be rapid (approximately 90 seconds). To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thaw a vial of cells cryopreserved in Serum-Free Cell Freezing Medium by gentle agitation in a 37☌ water bath.Warm the complete growth medium to 37☌ prior to use with the cells.Handling Procedure for Frozen Cells and Initiation of Cultures Once at -80☌, frozen cryovials should be transferred to the vapor phase of liquid nitrogen for long-term storage. The cells should not be left at -80☌ for more than 24 to 48 hours. Freeze the cells gradually at a rate of -1☌/min until the temperature reaches -70☌ to -80☌.Aliquot 1 mL of the cell suspension to each labeled cryovial.
Aspirate the medium and suspend the cell pellet in Serum-Free Cell Freezing Medium at a concentration 3 x 10 6 to 5 x 10 6 cells/mL.Decontaminate the vial by dipping in or spraying with 70% alcohol. Take the Serum-Free Cell Freezing Medium from storage and swirl to mix.Centrifuge the cells again at 125 x g for 5 to 10 minutes. Aspirate medium and (neutralized) dissociation solution (used with adherent cells) and resuspend the cell pellet in 2 to 8 mL fresh, pre-warmed, complete growth medium.After centrifugation, the cells should form a clean loose pellet.Do not over-centrifuge cells as this may cause cell damage.Centrifuge the cells at 125 x g for 5 to 10 minutes.Harvest the culture to prepare a cell suspension using your standard, cell-specific method.
If growing adherent cells under serum-free conditions, we recommend the use of ATCC ® 30-2101 Trypsin-EDTA Solution (1X) and ATCC ® 30-2104 Soybean Trypsin Inhibitor (50X Concentrate) to detach the cells. Handling procedure Cryopreservation of CellsĬryopreserve cells when cultures are actively growing.