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Comparative studies on in vitro reactivity of fresh and cryopreserved pig lymphocytes
E Koch1, M Larak, F Ellendorff
1Institut für Tierzucht und Tierverhalten (FAL), Federal Republic of Germany.
Insights
Cryopreservation significantly impairs pig peripheral blood mononuclear cells (PBMC) in vitro functions. Frozen-thawed PBMC show reduced intracellular calcium, proliferation, and cytotoxicity, indicating sensitivity to liquid nitrogen storage.
Area of Science:
- Immunology
- Cryobiology
- Veterinary Science
Background:
- Cryopreservation is essential for preserving biological samples, including immune cells.
- Understanding the impact of cryopreservation on specific immune cell functions is crucial for research and clinical applications.
- Pig lymphocytes are valuable models for studying human immune responses.
Purpose of the Study:
- To investigate the effects of cryopreservation on the in vitro reactivity of pig peripheral blood mononuclear cells (PBMC).
- To assess the impact of liquid nitrogen (LN2) storage on key lymphocyte functions, including calcium signaling, proliferation, and cytotoxicity.
Main Methods:
- Peripheral blood mononuclear cells (PBMC) were cryopreserved using controlled-rate freezing and stored in liquid nitrogen (LN2).
- Cell viability was assessed post-thaw using trypan blue exclusion.
- In vitro functions evaluated included intracellular calcium ([Ca2+]i) levels, phytohemagglutinin (PHA)- and alloantigen-induced proliferation (blastogenesis), and cell-mediated cytotoxicity.
Main Results:
- High cell recovery (74.7%) and viability (94.5%) were observed post-thaw.
- Cryopreserved PBMC exhibited significantly lower resting [Ca2+]i and a diminished [Ca2+]i response to mitogenic stimulation (PHA).
- Reduced [3H]thymidine incorporation was noted in frozen-thawed PBMC following PHA stimulation and in mixed lymphocyte cultures (MLC), with a severe deficiency in cytotoxic effector functions.
Conclusions:
- Pig PBMC are highly sensitive to cryopreservation and LN2 storage, impacting multiple immune functions.
- While cell viability remains high, functional parameters like calcium flux, proliferation, and cytotoxicity are significantly compromised.
- These findings highlight the need for careful consideration of cryopreservation protocols when assessing pig lymphocyte immune responses.
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.
