Freezing cells

Whether you have received frozen or growing cells, ECACC experts advise you to prepare your own frozen stock of the cell line as soon as possible after receipt. Use asceptic techniques to ensure that you do not contaminate your cells.

1. Harvest the cells in the log phase of growth. For adherent cells harvest as close to 85% confluency as possible.
2. Check whether there are any special requirements for freezing the cell line. We recommend freezing cells in a cryopreservation solution consisting of 90% serum with 10% dimethyl sulfoxide (DMSO).  For some cell types DMSO may not be suitable.  If DMSO is not suitable, an alternative, such as glycerol, is specified in the cell line data on the product detail pages.
3. Centrifuge the cells at 150 x g for five minutes to create a pellet. Re-suspend the pellet in the cryopreservation solution to give a final cell concentration of 3-5x10⁶ cells per ml for adherent cells and between 6-8 x10⁶ cells per ml. Pipette 1ml aliquots into the plastic vials to be used for storage (preferably internally threaded and with an O-ring though this is dependent on your inventory system).
4. Freeze the cells at a cooling rate of 1-3ºC per minute, ideally using a programmable rate-controlled freezer. When the temperature reaches at least -130ºC transfer the vials to a gas phase liquid nitrogen storage vessel.  If you do not have a programmable rate controlled freezer you can use a polystyrene box or a vessel designed for slow freezing of cultures (such as a Mr Frosty) in a -80ºC freezer for a maximum of 24 hours prior to transfer to gaseous phase liquid nitrogen.  Advisory note: Test cell viability by thawing one vial after short term storage in gas phase liquid nitrogen.

Resuscitation of frozen cells

Advisory note: Wear personal protective equipment including laboratory coat, protective face mask and gloves when handling the frozen vials. On very rare occasions vials may explode on warming due to the expansion of trapped residual liquid nitrogen (refer to Material Safety Data Sheet on our Technical Support page 

The following guidance aims to help you establish a culture successfully and minimise cell damage and contamination: 

1. Transport the vial in dry ice or in a liquid nitrogen transport vessel to maintain the vial at low temperature until you are ready to resuscitate the cells
2. Quickly transfer the vial to a 37ºC water bath^*for approximately 1 to 2 minutes until no more than two ice crystals remain. Rapid thawing in this manner is essential to minimise damage to the cells; DO NOT thaw vials in an incubator or your hand *Do not totally immerse the vial in the water bath because this may cause contamination of the cells
3.  Before opening it wipe the entire vial with a tissue soaked in 70% alcohol
4. Open the lid and pipette the whole content of the vial into a sterile tube (for example 15ml capacity). Then slowly add 5ml of an appropriate pre-warmed medium that has already been supplemented with the required constituents
5. Count the viable cells to ensure the correct seeding density. This can be done microscopically using a haemocytometer and trypan blue stain (the non-viable cells will be stained blue) although other methods are available. Click here for a method and a cell counting calculator
6. Check whether the cells are adherent or suspension cells (if you are unsure check the ‘growth mode’ in the product detail pages on our website). 

i) For adherent cells refer to the cell line data on our website for the recommended cell seeding density, i.e. viable cells/cm², then calculate the amount of medium required^† and flask size necessary to achieve this. 

† For adherent cells the following culture medium volume ranges (minimum – maximum) are recommended for flask sizes: 25 cm² flask 5-10ml; 75 cm² flask 25–35ml; 175 cm² flask 40-50ml.

Check the cell line specific data to determine whether the cells require a pre-centrifugation step; adherent cells do not normally require this. However, if the cells are to be used immediately (for example for a cell based assay) a pre-centrifugation step may be advisable to remove residual cryoprotectant. Centrifugation should be at 100 - 150 x g for 5 minutes then re-suspend the pellet in fresh medium using the appropriate volume to achieve the correct seeding density

ii) A pre-centrifugation step is recommended for suspension cells to remove cryoprotectant. Centrifuge at 100 - 150 x g for 5 minutes then re-suspend the pellet in fresh medium using the appropriate volume to achieve the correct seeding density i.e. viable cells/ml. We recommend seeding your suspension cells at a relatively high density of 5-7 x 10⁵cells/ml

7. Incubate the cells at the temperature and CO₂ level recommended on the product detail page. Use flasks with vented caps to allow gaseous exchange if you are using a CO₂ incubator and if CO₂ is required to grow the cells

Hybridoma cultures from frozen

When recovering hybridoma cultures from frozen it is not unusual for growth initially to be slower than expected and there may be an observed decrease in viability. Establishment of an actively proliferating culture may take up to 2 weeks. Following resuscitation, seed at 4-5x10⁵ cells/ml. Observe after 24 hours and monitor daily until the cell density has reached 8-9x10⁵ cells/ml before subculturing. Centrifuge at 100 – 150 x g for 5 minutes then re-suspend the cells in fresh medium rather than diluting them.  

Often hybridoma cultures can benefit from being re-suspended with media supplemented with 20% foetal bovine serum (FBS) in the early critical stage of culture establishment immediately post resuscitation.