A new controlled-rate cooling apparatus for freezing hematopoietic cells for storage at -196 degrees C

An apparatus has been constructed to cool biological material at a controlled rate. The material to be frozen is placed in glass ampuls which are immersed in an aluminum bath containing ethyl alcohol and the bath is placed inside a freezer cabinet. Liquid nitrogen is pumped intermittently into the cabinet by means of a single-speed electric pump. The rate of cooling is controlled by a device that varies the interval between successive pumping cycles. The temperature fall is monitored by thermocouples placed inside selected glass ampuls and recorded as a plot on moving graph paper.This simple instrument is capable of cooling at an accurately controlled rate over the range of 0 to 7 °C/min. We chose for our studies a cooling rate of 1 °C/min which we could maintain with an accuracy of ±0.1 °C. Temperature fluctuations were, however, observed at the freezing plateau and varied considerably in magnitude and temperature at onset even for the same material cooled under the same conditions. Mouse bone marrow cells frozen by our technique and stored for various periods of time may, on reconstitution, form colonies in vivo and in vitro identical in morphology and number to those from unfrozen control cells. Our results suggest that expensive and intricate devices may not be necessary to obtain optimal recovery of viable cells after storage in liquid nitrogen. The apparatus is now in regular use for the storage of human bone marrow cells intended for use in treatment of patients with leukemia refractory to conventional measures.