Non-random cell killing in cryopreservation: implications for performance of the battery of leukocyte tests (BLT), I. Toxic and immunotoxic effects.

To eliminate between-tests error in longitudinal human studies, for specimen sharing, convenient scheduling, etc., it is necessary for us to freeze leukocytes as well as non-transformed, continuous T lymphocyte (CTL) lines. Two commonly used cryopreservation methods were compared in terms of efficacy. Isolated peripheral blood mononuclear cells and CTLs were each aliquoted into three sets of vials. Two sets each were frozen in a 1:1 mixture of 15% DMSO in Mixed Medium (MM) and 20% FBS in MM using a commonly employed styrofoam freezer insert method for liquid nitrogen refrigerators and a programmed freezer (temperature falls at an optimal rate), respectively. The remaining set was held in MM with 20% FBS at 20 degrees C during the 2-h freezing process. The cells were thawed and/or washed and assayed for viability and T helper (Th)/T suppressor (Ts) ratio. It is clear that inadequate freezing (via the styrofoam method) non-randomly damages cells of T cell subpopulations, Th being more sensitive than Ts. Further, it is shown that inadequate cryopreservation can confound results from a number of assessment methods owing to morphological and functional damages. The battery of leukocyte tests (BLT) under development in this laboratory, is designed to detect toxic, immunotoxic and genotoxic effects of in vivo mutagen exposure on human blood. It is concluded that minimization of non-random cell losses (as quantitated on the basis of morphology) and preservation of related regulatory cell function is essential if one would assess the in vivo and in vitro states of heterogeneous cells. Further, it is suggested that freezing methods should be used only after verification that selective damage to subpopulations is not occurring among cells that otherwise might wrongly be assumed to be intact.