Methods for storing the cells in liquid nitrogen

Harvest cells and count as for passaging. 1x 10⁷ cells in 1 ml are required for each 1 ml cryovial, in 10% DMSO.

For example:
If 1ml cell suspension obtained from harvest contains 50 x 10⁶ cells/ml.:
For each cryovials, you need 100µl DMSO + 200µl cell suspension + 700µl CSM. Aliquot the cells and medium to the cryovials, only adding the 100µl DMSO at the last moment, and freeze in vapour over liquid N₂ immediately . This level of DMSO is extremely toxic and the cells will die if left exposed to it at room temperature for any length of time. After two hours, transfer the frozen vials to the liquid nitrogen freezer.

Thawing cells

Prepare a 5cm culture dish with 9ml complete medium. Set the water bath to 25 deg.C. Remove the cryovial from the freezer, thaw and transfer cells to culture dish as quickly as possible . The cells are now in 1% DMSO, tolerable but still slightly toxic. Incubate overnight, next day carefully remove all the medium without disturbing the cells and replace with 5ml fresh complete medium. Thereafter passage as usual.