The following article originally appeared on February 19, 2020 in Biocompare here.
Cryopreservation is an established laboratory technique used to store cells and other biological material at a temperature close to that of liquid nitrogen (−196°C). It provides researchers with a backup should growing cells be lost due to contamination and helps to minimize the occurrence of genetic drift by allowing early passage cells to be brought into use when current cultures have been grown for an extended time. This article discusses best practices for freezing and thawing cells to maintain a high viability.
- Check cell health before freezing
Cells should ideally be frozen at a low passage number, when cellular characteristics have had little time to change as a result of extended passaging. Before freezing cells, it is important to perform a viability count using Trypan Blue or another live/dead stain, and to check for contamination via sterility evaluation and mycoplasma testing.
- Freeze cells during logarithmic growth and at an appropriate concentration
Passaging cells or refreshing the growth media 1–2 days before freezing will ensure the cells are healthy and in an active phase of growth. For instance, adherent cells will ideally be at around 70–80% confluency upon harvest for freezing. The concentration at which cells are frozen may vary between cultures but it is typically in the region of 1 x 106–5 x 106 cells/mL in freezing media; freezing cells at too low or too high of a density can impact viability and should be avoided.
- Use a suitable freezing media
Cryopreservation media typically comprises the growth media, a cryoprotectant such as DMSO or glycerol, and a source of protein (usually serum). Although the cryoprotectant is essential to prevent cellular stress during the freeze-thaw process, it is possible to omit the serum; in situations where serum is to be avoided, the growth media component can be supplemented with conditioned serum-free media or 10% cell culture grade BSA.
- Begin the freezing process as soon as possible
To maintain viability, the freezing process should begin as soon as the freezing medium has been introduced to the cells. Placing the cryovials on wet ice before transferring them to the freezing container can expedite this.
- Freeze cells slowly
Freezing cells slowly is essential to prevent intracellular ice formation and can be achieved using a freezing container that provides a freezing rate of 1°C/minute. This should be placed at -80°C for a minimum of 4 hours but ideally overnight once the cell-containing cryovials have been added. Isopropanol-free systems avoid the cost and inconvenience of regular alcohol replacements and provide more uniform freezing to preserve cell viability.
- Check frozen cell stocks before transfer to liquid nitrogen
Once cells have been frozen at -80°C, it can be helpful to sacrifice one of the vials to confirm the stocks are viable and not contaminated before transferring the remainder to liquid nitrogen. However, leaving cells at -80°C for long periods can affect cell health, so transfer to liquid nitrogen should be performed as soon as possible after checks are completed.
- Store cells in the vapor phase of liquid nitrogen
Cells should be stored in the vapor phase of liquid nitrogen to prevent liquid from entering the tubes. Not only can this result in contamination, but it can also lead to vials failing as the liquid expands upon thawing.
- Ensure cells remain frozen prior to thawing for use
To prevent cellular viability from being compromised, cryopreserved cells should never be transferred from liquid nitrogen to the laboratory on wet ice. Dry ice or a liquid nitrogen container must always be used, especially if the cells are to be transported over a considerable distance or time.
- Thaw cells quickly
Once the cells have been removed from liquid nitrogen storage, the cryovial should be placed in a 37°C water bath until the contents are just thawed. The cells should then immediately be transferred to a large volume of pre-warmed media, with more sensitive cell types (stem cells, primary cells) being added dropwise to preserve viability. Although some cells may be pelleted by centrifugation before being gently resuspended with a pipette and added to a cell culture flask containing pre-warmed growth media, for other cell types it is preferable (more gentle) to transfer the cells to a flask straight after thawing and to perform a media change the next day to remove any residual cryoprotectant.
- Confirm that recently thawed cells are healthy
A quick visual inspection can provide an early indication of cell health, for instance confirming that adherent cells have attached to the flask. Any observations can be supported with a viability check upon initial passaging.
Corning offers a wide range of cryopreservation products designed to maintain cell health and viability. More information can be found here.