title: Comparative studies on in vitro reactivity of fresh and cryopreserved pig lymphocytes.
abstract: 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.
authors.affiliation: Institut für Tierzucht und Tierverhalten (FAL), Federal Republic of Germany.
authors.forename: E
authors.initials: E
authors.lastname: Koch
authors.forename: M
authors.initials: M
authors.lastname: Larak
authors.forename: F
authors.initials: F
authors.lastname: Ellendorff
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002470:Cell Survival; D015925:Cryopreservation; D003602:Cytotoxicity, Immunologic; D005260:Female; D066298:In Vitro Techniques; D008213:Lymphocyte Activation; D007959:Lymphocyte Culture Test, Mixed; D008214:Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D008297:Male; D011247:Pregnancy; D013552:Swine
chemical_list: D002118:Calcium