Function of lymphocytes and macrophages after cryopreservation by procedures for pancreatic islets: Potential for reducing tissue immunogenicity

Abstract

The survival of tissue allografts can be extended by pretreating the tissue to remove the stimulatory leucocytes that populate the graft; with this in mind, we have recently begun to explore a cryobiological approach to modulating tissue immunogenicity by using the differential susceptibility of different cells to freezing injury. The sensitivity of leucocytes to fast cooling rates, which were used in procedures that have been reported to yield viable pancreatic islets of Langerhans, was examined. The loss of both cell numbers and the ability of peripheral blood lymphocytes to undergo blastogenic transformation in response to the mitogen concanavalin A after freezing and thawing was determined over a range of cell concentrations using the “curve-shift” method. Lymphocytes frozen at 1 °C/min by a control procedure that was designed to yield maximum survival of lymphocytes showed that although there was a decrease in the number of responding cells, there was no reduction in the ability of the recovered cells to undergo blastogenesis when compared with the response of nonfrozen cells. However, cooling at 1 °C/min in the experimental procedures resulted in both the loss of cells as well as a marked reduction in the ability of recovered cells to incorporate 125I-deoxyuridine into nucleic acid. Cells cooled at either 20 or 75 °C/min by any of the procedures showed total inability to respond to stimulation. Lysozyme is produced continuously by all types of macrophages in culture. The large net increase in total lysozyme content of macrophage cultures is therefore a useful measure of the viability of these accessory cells. Cooling at 1 °C/min by a control, optimized procedure yielded 91% survival of viable peritoneal exudate cells. Cooling at either 1 or 20 °C/min in the experimental procedures resulted in 72–75% survival of cells frozen by one method and 33% survival when frozen by an alternative procedure. Negligible recovery of viable cells was obtained after cooling at 75 °C/min. The preservation protocols employed in this study differ significantly in the variables known to influence the survival of the cells; these include the concentration of cryoprotectant (CPA), the length and temperature of exposure to CPA, the dilution regimen, and the optimum cooling rate for survival of pancreatic islets. This study therefore defines clearly those conditions most likely to effect a depletion of “passenger” lymphoid cells by freezing during the Cryopreservation of islets of Langerhans.