[Separation culture and biological characteristics analysis for bone marrow-derived mesenchymal stem cells from inbreed line miniature pig of Wuzhishan]

Abstract

Objective: To establish a method to isolate, culture and identify bone marrow-derived mesenchymal stem cells (BM-MSCs) from inbreed line miniature pig of Wuzhishan (ILMW) in vitro, to compare the biological characteristics of BM-MSCs derived from different pigs, and to supply BM-MSCs for investigating the repair mechanisms of renal injury in ILMW aft er unilateral ureteral obstruction.

Methods: Four or 10-months old ILMW (n=4 per group) were selected and 5 mL of bone marrow fluid was extracted at 1 cm position from iliac wing edge. Mononuclear cells were isolated by density gradient method, then were cultured in the complete medium containing 3 different kinds of fetal bovine serum (FBS) (10% FBS, 12% FBS or 15% FBS). The cells of Passage 1, Passage 3, Passage 5 or Passage 11 were collected to examine biological characteristics including morphology, phenotype, differentiation ability, growth curve and cell cycle.

Results: The BM-MSCs were attached to the culture dishes, which were fibroblast-like or whirlpoollike. Primary cultured cells began the adherence at 18 h and entered a logarithmic phase in the 6th day. Eighty percent of them were fused in the 9th day. There were no obvious anomalies in the subcultured cells. The expressions in cell surface antigens of CD29, CD44 and CD90 were positive, while the expressions of CD34 and CD45 were negative. There was no statistically significant difference between cells from different generations (all P>0.05). Under condition of osteogenic induction, alizarin red staining was positive at the 18th day, and alcian blue staining was positive at the 21th day. Cell cycle examination showed that the rate of G0/G1 was about 81.45%.

Conclusion: BM-MSCs of ILMW has advantages of earlier adherent time, more active proliferation, shorter cell subculture cycle, and stable biological characteristics after subculture, which is one of the best kinds of BM-MSCs coming from swine in mainland of China.