Selective destruction of leucocytes by freezing as a potential means of modulating tissue immunogenicity: membrane integrity of lymphocytes and macrophages.

It is now known, when a tissue allograft is transplanted, that antigen recognition alone is not sufficient for lymphocyte activation in the host. "Passenger" leucocytes (antigen-presenting cells) present in the donor tissue are now recognized as a major immunogenic stimulus. Removal of these contaminating leucocytes, using a variety of procedures, has enabled the immunogenicity of allografts to be reduced, thus enhancing the survival of tissue allografts. This initial study explores the possibility of using a cryobiological approach to modulating the immunogenicity of tissues by virtue of the well-recognized differential susceptibility of different cell types to freezing injury. The investigation was prompted by demonstrations that pancreatic islets can secrete insulin in response to a graded glucose challenge after cryopreservation using relatively fast cooling rates which would be expected to be suboptimal for leucocyte survival. Batches of rat peripheral blood lymphocytes, or peritoneal exudate cells (macrophages) were cooled at 0.3, 1, 5, 20, 75, or 200 degrees C/min using three different cryopreservation protocols reported to yield viable pancreatic islets. Cell survival was evaluated in terms of the numbers of cells recovered after freezing as well as a fluorometric viability assay which assessed the membrane integrity of cells. Optimum survival of both lymphocytes and macrophages after freezing and thawing was found at cooling rates in the range of 0.3 to 5 degrees C/min. A significant number (10-40%) of these lymphoid cells survived freezing at 20 degrees C/min and only after cooling at rates greater than 75 degrees C/min was survival reduced to a negligible level.