Cryopreserved Lymphocytes Research Articles

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

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.

Advantages of cryoperserved lymphocytes for sequential evaluation of human immune competence. I. Mitogen stimulation.

Human lymphocytes were harvested from normal volunteer donors and cryopreserved. Various concentrations of dimethyl sulfoxide and human serum in the cryoprotective media were evaluated to optimize the recovery and function of viable cells. Multiple samples were then drawn from volunteers over a number of days, processed individually, and used in mitogen stimulation assays. These cells were also cryopreserved, immediately thawed, and cultured simultaneously with the fresh cells. In all instances fresh and cryopreserved lymphocytes exhibited similar levels of stimulation by mitogens. Cryopreserved cells from these sequential bleedings were then recovered and cultured simultaneously in mitogen stimulation assays. The results obtained in these assays with cryopreserved cell had less day-to-day variagion than those with cells cultured individually. Coefficients of variance of individual cultures of mitogen stimulation assays were reduced from 26-59% to 5-17% by use of cryopreserved cells. The findings suggested that use of cryopreservation techniques decreases the variability of cellular immune assays and thus alows more accurate longitudinal study of the immune competence of patients.

Spontaneous rosette and rosette-inhibition tests on fresh and cryopreserved lymphocytes.

Peripheral blood lymphocytes from 13 healthy adults were compared in the fresh state and after controlled freezing in liquid nitrogen. The mean percentage of cells forming spontaneous rosettes with sheep red blood cells was 52% in the fresh samples and 57% in frozen-thawed material. These figures are not significantly different. Fresh and frozen cells proved equally sensitive to the action of an antilymphocyte globulin preparation in inhibiting rosette formation. It is concluded that valid results may be obtained through the use of frozen preserved lymphocytes in rosette and rosette-inhibition tests.