| Objective:To discuss the feasibility of improved method to isolate and purify SD rat bone marrow mesenchymal stem cells (BMSCs). Improved method is evaluated to confirm its effect.The purpose is providing high purity of BMSCs for further experimental study.Methods:(1) The femur and tibia of SD rat are isolated under sterile conditions, remove the muscles and fascia attached on the bones, cut the middle of the femur and tibia after washing with phosphate buffered saline (PBS), wash the bone marrow cavity with 5ml Low glucose DMEM medium repeatedly. The flushing fluid is filtered through 200 mesh stainless steel sieve and centrifugated with 2000rpm for 5 min, discard the supernatant and the fat layer, then adjust into single cell suspension after adding complete culture medium. Cells are seeded in four 25cm2 cell culture flask with 106/ml and placed in the cell incubator with 5% carbon dioxide and 37℃, then randomly divided into A and B groups,2 bottles in each group. (2) The first exchange of medium is after 48h, and the medium is changed every 3 days after the first exchange; (3) When the cell grow up to 80% to 90% confluence, add lml 0.25% trypsin to digest for 2～3min, then add 5ml Low glucose DMEM complete cells culture medium containing 12% fetal bovine serum (FBS), carefully adjust into single cell suspension and passaged with the ratio of 1:2; (4) When the 1st generation cells grow up to 80% to 90% confluence, cells of A group are digested into single cell suspension by lml 0.25% trypsin after washing with PBS, then slowly flow into the centrifuge tube with Percoll isolation medium (density 1.073) of the same volume, centrifuge with 2000rpm for 20 min, carefully draw the middle layer of white flocculent into another centrifuge tube, wash the cells with complete cells culture medium completely, adjust the cell density to 105/ml and inoculated 5ml into 25cm2 cell culture flask, continue to incubate in the cell incubator; cells of B group are digested into single cell suspension by 1ml 0.25% trypsin after washing with PBS, adjust the cell density to 105/ml,then directly fill 5ml into 25cm2 cell culture flask (without isolation by Percoll isolation medium), continue to incubate in the cell incubator; (5) The morphology changes and proliferation of cells are observed under microscope every day, and draw the growth curve of 4th,9th generation cells in two groups by the MTT method; (6) Immunohistochemistry identify the cell phenotype of 3rd generation BMSCs in A, B group; (7) Flow cytometer analyzes the CD34, CD44, CD45, CD90 positive cells percentage of 3rd generation cells in A, B group; (8) Cell cycle of 3rd generation BMSCs in A, B group is analyzed by flow cytometer; (9)The osteogenic potential of BMSCs acquired from two culture methods are identified by culturing with osteogenic differentiation culture medium.Results:(1) The growth curve of 4th and 9th generation BMSCs in two groups show that the proliferation ability of cells acquired from two culture methods have no significant difference (P>0.05); (2) Immunohistochemistry show that CD29, CD44, CD90, CD 106 of BMSCs in A group is almostly positive expression, the cell membrane and cytoplasm are brown, while CD34, CD45 is negative expression, the membrane and cytoplasm are not colored; CD29, CD44, CD90, CD 106 of 3th generation BMSCs in B group is positive expression, most of the cells CD34, CD45 is negative expression, a small positive expression, this means that the purity of BMSCs in A group is higher than B group; (3) Flow cytometry analyzes the expression of surface antigen sign of 3rd generation BMSCs in A, B group, CD44, CD90 is perfectly expressed in A group, CD45 and CD34 is almost not expressed. The CD44, CD90 positive expression rate of cells in A group is significantly higher than B group (P<0.05), while the CD34, CD45 positive expression rate is significantly lower than B group (P<0.05), this means that the purity of BMSCs in A group is higher than B group; (4) Cell cycle of 3rd generation BMSCs in A, B group shows that most of the cells are in the quiescent period, only a small number of cells in active proliferation stage, this is in line with characteristics of stem cell; (5) The 3rd generation BMSCs of A, B groups are cultured for 3 weeks by osteogenic differentiation culture medium, a lot of dark red nodules of calcium are seen in two groups after alizarin red staining, the rat bone marrow mesenchymal stem cells isolated and purified by both methods have good potential to osteogenic differentiation, they have no significant difference.Conclusions:(1) This research find a new method to isolate and purify SD rat bone marrow mesenchymal stem cells, successfully isolate a large number of bone marrow mesenchymal stem cells of high purity from single rat, and solve the homologous problem、comparability and rejection issues of SD rat bone marrow mesenchymal stem cells research. In addition, this research save a large number of bone marrow and stem cell resources; (2) The improved method is simple, fast and not affect the cell’s proliferation ability; (3) Bone marrow mesenchymal stem cells isolated and purified by improved method have good morphology and biological stability, have powerful proliferation and excellent osteogenic differentiation potential; (4) Effect of improved method to isolate and purify bone marrow mesenchymal stem cells of SD rat is better than the traditional bone marrow adherent method. |
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