Cryopreservation of mammalian cell lines video protocol

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 (3:22 minutes).

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Materials and Reagents

  • 1–2 mL cryovials
  • CoolCell® freezing unit (BioCision)
  • Freezing media

Cryopreservation

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

  2. 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. Resuspend in cell culture media and transfer into a 50 mL Falcon tube.

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

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

  5. Centrifuge for 5 min at 1,000 rpm at room temperature.

  6. Prepare freezing media (Table 1).


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


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

  8. Add freezing media to the required cell density. For mammalian cells this is usually 1,000,000/mL of freezing media. Cells should not be at room temperature in freezing media for more than 10 min.

  9. Aliquot 1 mL into cryovials and secure the lids.

  10. Transfer the cryovials into a CoolCell (at room temperature) and put into a -80°C freezer. The CoolCell will ensure that the temperature decreases steadily by 1°C/minute.

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


Culture TypeFreezing mediaNotes
Cells cultured in FBS-containing media90% FBS + 10% DMSOMix well and warm to 37°C before use.
Cells cultured in serum-free media90% 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 freezing90% FBS + 10% glycerolMix well and warm to 37°C before use.

Table 1. Different types of freezing media for mammalian cell lines

Thawing frozen cell lines

  1. Remove cryovial from liquid nitrogen storage and place in a 37°C water bath until only about 80% defrosted (do not thaw at room temperature). This should take no longer than 1 minute.
  2. Using a pipette transfer the contents of the vial into a 15 mL Falcon tube containing about 10 mL of pre-warmed culture media.
  3. Centrifuge at 500–1,000 rpm for 5 min, discard supernatant and resuspend in the appropriate amount of cell culture media.
  4. Transfer cells into a culture vessel and transfer into a 37°C incubator.

FAQs

Why can't I let cells thaw at room temperature?

​Cells should generally be thawed as quickly as possible, above room temperature. This is because 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.

​​How long can I store cryopreserved cells?

​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​.

Found this protocol useful? View our counting cells protocol​​​.