The impact of high hydrostatic pressure (40 MPa and 60 MPa) on the apoptosis rates and functional activity of cryopreserved porcine mesenchymal stem cells

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The aim of the present study was to examine the influence of two varied high hydrostatic pressure (HHP) values on the apoptosis (assessing caspase-8, survivin, CAD, Bax, BclxL and BclxS) and functional activity (using cocultures with bovine embryos) of porcine mesenchymal stem cells (pBMSCs). pBMSCs were isolated from porcine bone marrow and cultured in vitro. Before cryopreservation and storage in liquid nitrogen, pBMSCs were subjected to HHP values of 40 MPa and 60 MPa for 1 h at 24°C. After thawing, the cells were analysed for caspase-8 activity and protein expression of survivin, CAD, Bax, BclxL and BclxS. To indirectly test the influence of HHP on the functional activity of pBMSCs, in vitro maturated bovine oocytes were fertilized in vitro, and the obtained embryos were cultured under 4 different conditions: 1. monoculture in SOF medium; 2. coculture with pBMSCs in SOF medium; 3. coculture with pBMSCs subjected to 40 MPa HHP in SOF medium and 4. coculture with pBMSCs subjected to 60 MPa HHP in SOF medium. The quality of the developed blastocysts was analysed by TUNEL assay. HHP did not induce apoptosis in pBMSCs, as no significant difference was noted in the expression of any of the analysed apoptosis- related proteins between pBMSCs subjected to HHP (40 MPa or 60 MPa) and control. The highest number of obtained blastocysts was observed when the embryos were cultured in SOF. A highly significant difference (P<0.005) was noted between embryos cultured in SOF and embryos cultured in the presence of pBMSCs subjected to 60 MPa HHP or untreated pBMSCs. A significant difference (P<0.05) was noted between embryos cultured in SOF and embryos cultured in the presence of pBMSCs subjected to 40 MPa HHP. In conclusion, HHP does not induce apoptosis in pBMSCs. The obtained results of the blastocysts cocultured in vitro with pBMSCs (HHP-treated and untreated cells) imply that coculture with pBMSCs has a negative impact on the developmental rates of blastocysts.

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