The influence of different freezing procedures and different cryoprotective agents on the immunological capacity of frozen-stored lymphocytes.

The influence of a series of factors on the frozen storage of lymphocytes was investigated. The cells were frozen using different freezing programmes, using the cryoprotectants dimethyl sulphoxide and glycerol in different concentrations in the freezing medium, and with variations in the period of exposure of cells to cryoprotectants before freezing and after thawing. Cell viability was evaluated by quantitative measurements of the cell-mediated immune response after stimulation with phytohemagglutinin, pokeweed mitogen, concanavalin A, and allogenic cells in mixed lymphocyte cultures. The factors investigated were found to have an important effect on the immune response, so that careful investigation and exact specification of the freezing system are necessary before frozen cells are used in blast-transformation tests. The best freezing programme had a duration of approximately 40 min with a smooth progression through the temperature range where phase transition takes place. The optimum dimethyl sulphoxide concentration in this programme was 8--10%. Dimethyl sulphoxide had no toxic effect on the cells, and no equilibration period was necessary prior to freezing. An equilibration period of 15 min with 10% glycerol was even better than the optimum programme with dimethyl sulphoxide.