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
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.
- Make use of a suitable freezing medium
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.
- Freeze cells at an adequate concentration
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.
- Freeze your cells as early as possible
To keep the cells alive, the freezing process should commence as early as the freezing media is applied. This can be ramped up by placing the cryovials on wet ice before moving them to the freezing container.
- Use appropriate freezing media
The cryopreservation medium is typically made up of growth media, a cryoprotectant such as DMSO or glycerol, and a protein supply (usually serum). Even though cryoprotectant is required to minimize cellular stress during the thawing process, the serum can be omitted.
- Keep cells in the liquid nitrogen vapour phase
To avoid liquid from reaching the tubes, cells should be preserved in the vapour phase of liquid nitrogen. Not only can this cause infection, but it can also cause vials to fail as the liquid expands on freezing.
- Before thawing cells for usage, be sure they are still frozen
To avoid compromising cellular viability, cryopreserved cells should never be carried from liquid nitrogen to the lab on wet ice. Dry ice or a liquid nitrogen container should always be provided, especially if the cells are being transported over a long distance or over a long period of time.
- Quickly thaw frozen cells
After removing the cells from liquid nitrogen storage, place the cryovial in a 37°C water bath till the contents are thawed. To sustain viability, the cells should be promptly transported to a large volume of pre-warmed media, with more vulnerable cell types (stem cells, primary cells) added dropwise.
- Check if the cells are healthy
A fast thorough check can provide an early sign of cell health, such as identifying the presence of adhering cells on the flask. During the initial passaging, any findings can be supported by a viability check.
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