Routine technique of lymphocyte cryopreservation evaluated by in vitro tests of immune response

Abstract

A reliable technique for cryopreservation of lymphocytes which conserves mitogenic response and surface marker (E-Rosettes and immunofluorescence) capability is presented. Briefly, lymphocytes are separated on Ficoll-Hypaque according to the method of Boyüm, washed twice. Concentration is adjusted and the cells are then placed in an ice-bath at + 4°C in 7·5% Dimethyl sulfoxid (DMSO) and medium 199 supplemented with 20% AB serum. The cellular suspension is partitioned equally ( 1 ml containing 10 7 lymphocytes/vial) into freezing vials and placed in the chamber of a Planer R 201 Programmed Freezer with a cooling rate of 1°C/minute from + 4°C to − 60°C with rapid intense cooling between − 6°C and − 8°C to prevent surfusion and then a rapid gradient from − 60°C to − 160°C. Next, vials are transferred into a container of stored liquid nitrogen. At the moment of their employment, the cryopreserved lymphocytes are placed in a water-bath at + 40°C, then diluted 10 fold within 2 min in culture medium 199 with 20% AB serum. Lymphocytes are counted, cell viability is evaluated by the Blue trypan dye exclusion test after which cells are distributed for diverse immunological tests. The data which is presented shows no significant difference between tests involving fresh and cryopreserved lymphocytes.