Effect of High Hydrostatic Pressure Applied Before Cryopreservation on the Survival Rate and Quality of Porcine Mesenchymal Stem Cells After Thawing

ABSTRACT

The aim of the present study was to examine the effects of varied high hydrostatic pressure (HHP) values on survival rate, proliferation rate, cell multipotency (transcript expression of SOX2, C-MYC, and REX1) and apoptosis (expression of phosphatidylserine (PS), SURVIVIN at the RNA level and BAX at the protein level) of porcine mesenchymal stem cells (MSCs). MSCs were isolated from porcine bone marrow and cultured in vitro. Before cryopreservation and storage in liquid nitrogen, MSCs were subjected to HHP at the varied pressures of 20, 30, 40, 50, or 60 MPa for 1 h at 24°C. Immediately after thawing and after 8 days of in vitro culture, cells were subjected to trypan blue staining, cell counting, real-time Polymerase Chain Reaction (PCR), western blotting, and fluorescence microscopy. BAX protein expression was only estimated immediately after HHP to exclusively examine the impact of HHP on apoptosis of MSCs. The viability of MSC subjected to 40, 50, and 60 MPa and estimated immediately after thawing increased significantly (P < 0.001 for 60 MPa and P < 0.05 for 40 and 50 MPa) in comparison to control. The proliferation rate of MSCs subjected to 40 MPa HHP was significantly higher than in the control group (P < 0.02) after 8 days of in vitro culture. After 8 days of in vitro culture, no significant differences were noted in the survival rates, PS exposure, or levels of SOX2, C-MYC, REX1, and SURVIVIN gene expression in all analyzed groups compared to control. In conclusion: 40–60 MPa HHP has a positive impact by improving cell viability in short term. 20–60 MPa HHP does not induce nor decrease apoptosis in MSCs. Fortunately, HHP does not induce differentiation of MSC. Our results calls for further analysis using HHP values higher than 60 MPa.

KEYWORDS:

• Apoptosis
• cryopreservation
• HHP
• MSCs
• pig

Additional information

Funding

This project was supported by funding from the Ministry of Science and Higher Education, Project no. 11-006.1. This research was also funded by the Polish National Science Centre resources allocated on the basis of decision no. DEC-2014/15/B/NZ9/04288.