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Bi W.-W.,Central Laboratory of Siping Center Hospital | Bi W.-W.,Jilin Tuohua Biotechnology Co. | He L.-R.,Jilin Tuohua Biotechnology Co. | Li C.,Jilin Tuohua Biotechnology Co. | Nie D.-Z.,Jilin Tuohua Biotechnology Co.
Chinese Journal of Tissue Engineering Research | Year: 2013

Background: Amnion mesenchymal-derived stem cells are obtained from the placenta with a placenta amniotic membrane of about 600 cm2. Objective: To establish a kind of simple isolation and culture method of mesenchymal stem cells derived from human amnion in vitro, and to explore their biological properties. Methods: Mesenchymal stem cells derived from human amnion were harvested by trypsin digestion combined with direct adherence. The morphology of human amnion-derived mesenchymal stem cells was observed. The passage 5 cells were collected to draw a cell growth curve. Surface markers and cell cycle of the passage 5 cells were determined using flow cytometry. Passage 4 cells were obtained for osteogenic and adipogenic induction. After 4 months of cryopreservation, the resuscitated passage 4 cells were counted to determine cell survival rate and draw the cell growth curve. Results And Conclusion: A few of cells creped at day after primary seeding. About after 15 days, 80%-90% cells fused in a spindle shape. After passage, the cells showed even morphology and arranged spirally. The latency period of the passaged cells was 48 hours and logarithmic growth phase was about 4 days. After logarithmic growth phase, the cells entered the platform period. The flow cytometry results showed negative expression of CD34, CD14, HLA-DR, CD19, CD45, but positive expression of CD73, CD105, CD90 on the surface of mesenchymal stem cells. The alizarin red and oil red O staining was positive and confirmed osteogenic and adipogenic capacity of human amnion-derived mesenchymal stem cells. Flow cytometry results showed that 28% cells were in S phase. After cryopreservation, the survival rate of resuscitated cells was up to 90%, and the resuscitated cells had the same growth characteristic with the non-cryopreserved cells. These results confirm a simple method to proliferate a great amount of human amnion-derived mesenchymal stem cells.

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