Cell lines are stored by cryopreservation. A cryoprotective agent (dimethyl sulfoxide, DMSO) is added to the culture which is frozen at a computer-controlled cooling rate of about 1 ̊C per minute until -60 ̊C. The freezing medium in which the cells are also stored is indicated for each individual cell line. In general, we use 7 volumes medium, 2 volumes fetal bovine serum, and 1 volume DMSO.
Frozen ampoules are stored in liquid nitrogen (LN2) at -196 ̊C. Two batches of ampoules are respectively prepared for storage as a seeding (master bank) and distribution stock (working bank). The seeding stock is used to replenish the distribution stock. At the DSMZ we normally freeze 20 ampoules for seeding stock and 25-50 ampoules for distribution. Cell viability in LN2 is maintained over a very long time (>10 years or more), provided, of course, that ampoules are continuously exposed to the gas or liquid phases.