[Isolation and culture of adipose-derived mesenchymal stem cells from inbreed line miniature pig of Wuzhishan and their biological characteristics]

Abstract

To establish a method for isolation, culture and identification of adipose-derived mesenchymal stem cells (ASCs) from the inbreed line miniature pig of Wuzhishan (ILMW). Methods: A total of 100 g adipose tissues were obtained from subcutaneous tissues of neck in six-month old healthy ILMW (3 samples, male). ASCs from ILMW (ILMW-ASCs) were isolated from adipose tissues through 0.1% collagenase digestion. The cells at the 3rd, 5th, 8th, 13th passages were collected. Cell morphology, size, phenotype, cell cycle, and apoptosis were monitored. Cell differentiation was induced and cell proliferation curve was drawn. Results: The ILMW-ASCs, fibroblast-like or whirlpool-like, began the adherence at 36 h and entered a logarithmic phase in the 5th day. Eighty percent of them were fused in the 7th day. The average diameter and volume of ILMW-ASCs were (17.00±0.54) µm and (2.58±0.24)×10-9 L, respectively. The expressions of CD29, CD44 and CD90 were positive, and there was no significant difference between the different passages (all P>0.05). The expressions of CD45, CD8a and HLA-DR were increased with the increase in passages after the 3th passage (all P<0.05). The adipogenic induction of ILMW-ASCs was observed by positive oil red O staining, and the osteogenic induction of ILMW-ASCs was determined by positive alizarin red staining. Apoptosis and senescence occurred in the 13 passage of ILMW-ASCs, and the proportion of S phase of cell cycle was lower than that in lower passages (all P<0.05). Conclusion: ILMW-ASCs are one of the best choice for porcine ASCs, which might provide a source of candidate stem cells for therapy of large animal disease models and tissue or organ repairment.