Cryopreservation of mammalian cell lines video protocol

Learn about cryopreservation of mammalian cell lines for long term storage in liquid nitrogen.

Cryopreservation is a method whereby cells are frozen, maintaining their viability, until they are defrosted months or years later. Cells are cryopreserved to minimize genetic change and avoid loss through contamination. It is best to cryopreserve cells when they are at their optimal rate of growth.

Materials and Reagents

Stage 1 - Cryopreservation

Materials required

Culture type
Freezing media
Notes
Cells cultured in FBS-containing media
90% FBS + 10% DMSO
Mix well and warm to 37°C before use
Cells cultured in serum-free media
90% conditioned media  + 10% DMSO

Use the supernatant from the centrifuge step (step 7)

Mix well and warm to 37°C before use

Cells that require glycerol for freezing
90% FBS + 10% glycerol
Mix well and warm to 37°C before use

Steps

Label cryovials with the date, name of researcher, cell number, passage number and cell type (and any other useful information, for example genetic modifications).

If cells are adherent, remove the cell culture media, wash in PBS, add enough trypsin to cover the cells and incubate for approximately 2 min in a 37°C incubator.

If cells are in suspension, just transfer the desired volume directly into a 50 mL Falcon tube.

Count cells using a hemocytometer to determine their viability. Cell viability should be at least 75% for cryopreservation.

Centrifuge for 5 minutes at 300 x g at room temperature.

Prepare freezing media (see Table 1).

Please note, DMSO is not suitable for all cell types, therefore glycerol can be used as an alternative.

Remove the supernatant (keep this; it is needed for the freezing media, see Table 1) and loosen the pellet gently.

Add freezing media to the required cell density.

Aliquot 1 mL into cryovials and secure the lids.

Transfer the cryovials into a CoolCell at room temperature.

The CoolCell will ensure that the temperature decreases steadily by 1°C/minute.

After approximately 24 h, remove the cryovials from the CoolCell and transfer into liquid nitrogen for long-term storage.

Cells frozen according to the protocols can be kept for several years in liquid nitrogen. Ideally, frozen cells should not be stored at -80 ºC for long periods of time (up to one week) and should be transferred into liquid nitrogen whenever possible. Stocking cells at -80ºC for long periods can compromise cell viability​.

Stage 2 - Thawing frozen cell lines

Steps

Remove cryovial from liquid nitrogen storage.

Cells should generally be thawed as quickly as possible, above room temperature as a slow thawing process can damage cells.

The freezing media contains essential cryoprotection agents, such as dimethyl sulfoxide (DMSO), to prevent the formation of intracellular and extracellular ice crystals that could damage the cell membrane and components. DMSO does have the drawback of being cytotoxic, and therefore cells should be thawed at a speed faster than is possible at room temperature in order to facilitate the quick dilution and removal of DMSO from the immediate environment of the cells.

The slow freezing process (-1ºC per minute) also helps prevent the formation of intracellular ice crystals by allowing sufficient efflux of water before freezing. Thawing the cells quickly serves the further purpose of preventing any crystals that have formed during the freezing process from damaging the cell membrane or components.

Using a pipette transfer the contents of the vial into a 15 mL Falcon tube containing about 10 mL of pre-warmed culture media.

Centrifuge at 300 x g for 5 minutes, discard supernatant and resuspend in the appropriate amount of cell culture media.

Transfer cells into a culture vessel.