Comparative studies on in vitro reactivity of fresh and cryopreserved pig lymphocytes

The effect of cryopreservation on in vitro reactivity of pig lymphocytes was studied. Peripheral blood mononuclear cells (PBMC) were frozen by controlled-rate freezing and stored in liquid nitrogen (LN2) between 4 and 36 days. Following thawing 74.7 +/- 2.6% of cells were recovered of which 94.5 +/- 0.9% were viable as determined by trypan blue exclusion. Functional parameters measured included the concentration of free intracellular Ca2+ ([Ca2+]i) in resting and mitogen-stimulated PBMC, mitogen and alloantigen-induced blastogenesis, as well as cell-mediated cytotoxicity. Irrespective of storage time and cell donor, [Ca2+]i in frozen-thawed PBMC (67.7 +/- 4.3 nM) was significantly lower (P less than 0.001) when compared to fresh cells (96.2 +/- 4.5 nM). In addition, cryopreserved PBMC only weakly responded with an increase of [Ca2+]i after stimulation by various concentrations of phytohemagglutinin (PHA). Following activation by PHA (2 micrograms/ml) for 4 days fresh lymphocytes (84,047 +/- 5475 cpm) incorporated significantly more (P less than 0.005) [3H]thymidine than frozen PBMC (66,001 +/- 4117 cpm). A similar difference in proliferation rates (P less than 0.05) between fresh (10,046 +/- 1915 cpm) and frozen-thawed PBMC (5852 +/- 1304 cpm) was observed in one-way mixed lymphocyte cultures (MLC), while the spontaneous incorporation of radiolabel was unchanged in frozen stored cells. By using MLC-derived cytotoxic effector cells (E) and [3H]thymidine-labeled concanavalin A blasts as targets (T), cryopreserved PBMC displayed a severe deficiency of cytotoxic effector functions at all tested E:T ratios. These results indicate that pig PBMC are very sensitive to LN2 storage although some immunological functions are more affected by cryopreservation than others.