Susceptibility of human T cells, T-cell subsets, and B cells to cryopreservation

We have studied the number of viable and functionally active T and B lymphocytes obtainable after cryopreservation to determine the best and most practical way to recover the maximal number of viable and functionally active cells. Assays were done on purified populations of human T and B cells recovered after cryopreservation. The results were compared to those obtained from similar types of cells fractionated from fresh and from cryopreserved mononuclear cells. The number of viable T cells recovered after cryopreservation was significantly lower than the number of viable T cells obtained from either fresh or cryopreserved mononuclear cells. The residual viable T cells recovered after cryopreservation showed significantly reduced blastogenic activity in response to pokeweed mitogen (PWM) stimulation. This occurred despite their normal blastogenic response to phytohemagglutinin and their normal ability to help B cells in the production of immunoglobulins following PWM stimulation. The reduction in the blastogenic responses of these T cells to PWM stimulation is attributed to the loss of a portion of the PWM responding subset of T cells. The loss in this subset of T cells was related to the exposure of cells to ammonium chloride prior to cryopreservation. The viability and functional abilities of B cells were not affected regardless of whether purification was done before or after cryopreservation. These findings indicate that extrinsic membrane damage to T cells induced prior to cryopreservation can affect the viability and responsiveness of a certain population of normal T cells. The damage can be minimized by reversing the sequence of T-cell isolation and freezing so that isolation of T cells is done after, rather than before, freezing. These results could be important in the study of T cells from patients with T-cell abnormalities, since the patients' cells could have an intrinsic membrane defect which would make them sensitive to freezing similar to that induced by extrinsic damage.