How are fibroblast cultures frozen for cryogenic storage?
• 0.53mM EDTA in HBSS • 0.04% trypsin/0.53 mM EDTA in HBSS • Fibroblast Growth medium • Fibroblast Freeze medium (growth medium with 10% glycerol or 5% DMSO) Procedure • If cells are to be frozen in 10% glycerol, complete freeze medium may be kept at room temperature until used. Freeze medium prepared with DMSO should be kept refrigerated until used. • Each flask that is to be pooled for the freeze (freeze pool) should be examined microscopically for contamination and any unusual growth pattern. One flask should be maintained as a “backup” flask until the viability of the freeze can be checked. • Aspirate the growth medium from each flask. Add EDTA to each flask without dislodging cells and incubate at room temperature for 10 minutes. If cells begin to round or the edges of the cell sheet constrict, remove EDTA immediately. • Replace the EDTA solution with the EDTA/trypsin. Incubate the flasks at 37C for 4 to 7 minutes. Examine the flasks microscopically to make sure the cells begin to
