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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. Source


Bi W.-w.,Central Laboratory of Siping Center Hospital | Li C.,Central Laboratory of Siping Center Hospital | Song G.-z.,Central Laboratory of Siping Center Hospital
Journal of Clinical Rehabilitative Tissue Engineering Research | Year: 2011

BACKGROUND: Adipose-derived mesenchymal stem cells (MSCs) are taken from fat tissue obtained by liposuction aspirates, which can be repeatedly drawn, and there are adequate sources of raw materials. OBJECTIVE: To establish the isolation and culture method of MSCs derived from human adipose in vitro, and to explore their biological properties. METHODS: Separation by collagen enzyme digestion was used to obtain MSCs from human adipose that were cultured in vitro until the cells reached 80% confluence and passaged. The P3, P7, P10 generation cells were collected to observe and analyze the cell growth curve. The surface markers of the P5 generation cells were detected by using flow cytometry. The P4 cells were induced into osteoblasts in vitro. The cryopreserved passage cells were recovered respectively in 2 and 6 months to detect cell survival rate after resuscitation. RESULTS AND CONCLUSION: Primary cells adhered to the wall at 3 days, then started the swift growth and formed the colony after 6 days, and reached 80%-90% fusions at 11 days, showing the desmoid shape. After passage, the cells maintained desmoid shape. The passage cells growth curves had the common characteristics: latent period was 24-48 hours, logarithm multiplication period was 3-4 days, and the cells entered the platform period at 5-6 days after the logarithm multiplication period. The flow cytometry examination showed that the expressions of CD34, CD14, HLA-DR were negative, CD44, CD105, CD13 were positive, and HLA-ABC were weakly positive on the surface of MSCs. After the recovery, the cell survival percentage reached above 90%; compared with the uncryopreserved passage cells, the cryopreserved passage cells had the same growth characteristics. Source

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