Cryoprotection of murine lymphocyte subpopulations using a microprocessor-controlled cooling system

Single-cell suspensions of splenic lymphocytes from 5- to 6-month-old C57BL/6 mice were cryopreserved using cooling rates ranging from ?0.25 to ?10.0 °C/min with the microprocessor-controlled cooling system developed in our laboratory. The cells (30 × 10⁶ cells/ml) were suspended in RPMI 1640 containing 10% FCS and 10% DMSO, and a total volume of 1.75 ml was frozen. Fluorescein-diacetate staining identified viable cells in unfrozen controls and frozen-thawed suspensions. Functional capacity was assessed in vitro by the incorporation of ³H]thymidine by dividing cells activated with graded concentrations of the T-lymphocyte mitogens, PHA-P and Con A, and the B-lymphocyte mitogen, LPS. High percentages of viable cells were recovered after cooling at rates ranging from ?0.5 to ?5.0 °C/min, as compared with those of unfrozen control suspensions. Incorporation of ³H]thymidine by T and B cells reached similar levels after cooling at rates ranging from ?0.25 to ?5.0 °C/min, and the percentage incorporation of ³H]thymidine as compared with that of unfrozen cells was 80–100%, except for T lymphocytes activated with PHA-P after cooling at ?5.0 °C/min. The relative response of cell suspensions to T- and B-cell mitogens, the relative mitogenic index, was unchanged from that of unfrozen controls in suspensions cooled at all rates including two (?0.25 and ?10.0 °C/min), which permitted recovery of only 55% of unfrozen cells. The importance of the constant cooling rates and rapid compensation for heat released at the phase change using the microprocessor-controlled system and of precise determinations of cellular viability and function are discussed and related to the apparent protection conferred on subpopulations of murine lymphocytes using cooling rates ranging from ? 0.25 to ?10.0 °C/min.