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.
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:
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
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.
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