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Preliminary study of human umbilical cord mesenchymal stem cells in the initial inoculum density of osteogenic differentiation main purpose of human umbilical cord mesenchymal stem cells in vitro separation, primary culture, amplification and purification, flow cytometry cell phenotypic identification, as well as osteogenic differentiation of human umbilical cord mesenchymal stem cells in the initial inoculum density affect other biological characteristics of the preliminary study, human umbilical cord mesenchymal stem cell tissue engineering research further animal experiments and clinical studies to provide a theoretical basis. Method (1) based Xiangya hospital operating room under sterile conditions Caesarean Obstetrics and Gynecology healthy pregnant women at term umbilical cord tissue samples of 15, the anatomy in the operating room under sterile conditions to remove the umbilical artery and vein, and the sterile PBS wash away hematocele placed in pre-prepared cell room spare medium sent to the central laboratory of Xiangya Hospital. (2) human umbilical cord mesenchymal stem cells in primary cell culture and identification: umbilical cord tissue is cut into pieces to 1mm3 tissue blocks, collagen type II protease digestion primary cultured cells prepared in a sterile console. After the primary cell culture success, flow cytometry to identify cell phenotype. (3) human umbilical cord mesenchymal stem cells passaged culture, and its growth curve plotted. Using trypan blue staining count living cells the number of primary cell cultures and identified after subcultured to 3rd generation, record the results, in time for the abscissa, the number of living cells for longitudinal coordinates of the cell growth curve. (4) different cell seeding density of human umbilical cord mesenchymal stem cells were cultured: 3rd generation cells were amplified in three different seeding density (1 × 105ml, 1 × 106/ml, 1 × 107/ml) were incubated for 18 days, after which each cell confluence, record the total number of each group of cells were harvested and incubation time growth curve. (5) the initial inoculation cell density of human umbilical cord mesenchymal stem cells into bone differentiation induced: 3rd generation cells to the aforementioned three seeding density packet inoculated into cell culture plate into bone induced differentiation experiments, the present study osteogenic induction culture medium containing 1.0 × 10-7mol / L dexamethasone, 50mg / L ascorbic acid, 50 mg / L of vitamin C, 1.0 × 10-3mol/Lβ- glycerophosphate DMEM / high glucose medium. Identification of the induced to differentiate into bone cells (1) alkaline phosphatase staining kit. (2) Akane red pigment staining. All the results were expressed as mean number ± standard deviation (X Guests s) that the analysis of the experimental data with SPSS.17.0 statistical software, and differences between groups in the homogeneity of variance test, the use of single-factor analysis of variance (ANOVA-LSD). Inspection level for a = 0.05, P lt; 0.05 was considered statistically significant. Adherent mononuclear cells cultured in vitro results (1) derived from human umbilical cord Wharton's jelly glue showed mesenchymal cells, mesenchymal cells showed a fibroblast-like morphology. (2) generation of 3,5,7 cell growth curve is basically the same. Growth common characteristics: within 24 hours after inoculation of the cells adherent subcultured incubation period is about 24-36 hours; subcultured in the logarithmic growth phase of approximately 3-5 days, active cell growth at this stage slowed quick, sustained 4 - 5d; 4-5 days after the end of the logarithmic growth phase, cell growth into the plateau. (3) expression of mesenchymal stem cell-associated antigens CD 105, CD44, do not express the hematopoietic stem cell antigen CD34, CD45, consistent with bone marrow-derived mesenchymal stem cells, just from different sources. (4) different initial inoculation density of human umbilical cord mesenchymal stem cells induced to osteogenic differentiation was significantly (p lt; 0.05), the initial seeding density of 1 × 106/ml cell group of human umbilical cord mesenchymal stem cells induced to differentiate into osteoblasts better than the other two density group. Conclusion umbilical cord sources of mesenchymal stem cells cultured in vitro amplification, and has a similar to bone marrow-derived mesenchymal stem cell biological morphology and antigen-table type, able to osteogenic induction of differentiation, and the most reasonable of into bone induced early initial inoculation density of approximately 1 × 106/ml.
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