Cryopreservation is a well-known laboratory technique for storing cells as well as other biological material at temperatures close to liquid nitrogen (-196°C).
Cryopreservation is a well-known laboratory technique for storing cells as well as other biological material at temperatures close to liquid nitrogen (-196°C). It offers the researcher a standby in case developing cells are damaged due to contamination, and it helps to minimize the incidence of genetic drift by permitting early passage cells to be used when existing cell cultures have been developed for an extended period. This article addresses the best procedures for freezing and thawing cells to keep them viable.
Before freezing, check the health of the cells
Make use of a suitable freezing medium
Freeze cells at an adequate concentration
Cells should preferably be preserved at a low passage number when cellular properties have had a minimum chance to change due to prolonged passaging. Before actually freezing cells, perform a viability count with Trypan Blue or another live/dead stain, as well as cleanliness and mycoplasma testing to check for contamination.
The cryopreservation medium is typically made up of growth media, a cryoprotectant such as DMSO or glycerol, and a protein supply (usually serum). Although the cryoprotectant is required to minimize cellular stress during the freeze-thaw process, the serum can be omitted; in cases where serum is to be eliminated, the growth media element can be augmented with conditioned serum-free medium or 10% cell culture grade BSA.
Passaging the cells or renewing the growth media 1–2 days before freezing ensures that the cells are viable and in an active growth period. Adherent cells, for example, should be at roughly 70–80 percent confluency when harvested for freezing.
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