Survival of tissue culture cells frozen by a two-step procedure to -196 degrees C. I. Holding temperature and time.

A two-step freezing procedure has been examined in order to separate some of the causes of damage following freezing and thawing. Different holding temperatures and times have been studied during the freezing of Chinese hamster tissue culture cells in dimethyl sulphoxide (5%, $\text{v}\text{v}$). Damage following rapid cooling to, time at, and thawing from different holding temperatures was found to increase at lower holding temperatures and at longer times. Damage on subsequent cooling from the holding temperature to ?196 °C and thawing was found to diminish at lower holding temperatures and longer times. The net result was that optimal survival from ?196 °C was obtained after 10 min at ?25 °C. Protection against the second step of cooling to ?196 °C was acquired at the holding temperature itself and was absent at ?15 °C without freezing.It seems that this technique will allow the different phases of freezing injury to be separated. These phases may include thermal shock to the holding temperature, hypertonic damage at the holding temperature and dilution shock on thawing from ?196 °C.