Placenta source filling dry cell and production thereof
A stromal stem cell and stem cell technology, applied in the field of placenta-derived mesenchymal stem cells and their preparation, can solve the problems of lack of large-scale separation, purification and expansion methods of placenta-derived mesenchymal stem cells, and achieve low immunogenicity, easy Amplification, high yield effect
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Embodiment 1
[0015] Example 1 Perfusion method to obtain placenta-derived mesenchymal stem cells
[0016] Immediately after delivery, the placenta is placed in a sterile tray or container, injected with balanced salt solution containing anticoagulants and antibiotics via the umbilical artery, exsanguinated, and perfused. The anticoagulant can be heparin at 1~100u / mL, and the antibiotic can be penicillin and streptomycin at 100u / mL. Collect the perfusate, centrifuge to obtain the cell components, then resuspend the cell components, use percoll with a density of 1.073g / mL, centrifuge at a centrifugal force of 500g for 30 minutes, harvest the interfacial cell layer, wash with PBS and inoculate culture bottles or culture plates, or directly for other purposes. It is also possible to use D7-FIB or NGFR-labeled immunomagnetic beads to separate mesenchymal stem cells with certain special markers.
Embodiment 2
[0017] Example 2 Obtaining Placenta-derived Mesenchymal Stem Cells by Enzyme Digestion
[0018] Immediately after delivery, the placenta is placed in a sterile tray or container, injected with balanced salt solution containing anticoagulants and antibiotics via the umbilical artery, exsanguinated, and perfused. The anticoagulant can be heparin at 1~100u / mL, and the antibiotic can be penicillin and streptomycin at 100u / mL. After the initial perfusion, the placental leaflets were cut aseptically, and the placental tissue was digested with trypsin or collagenase to obtain a cell suspension, and then the cell suspension was passed through a cell sieve to remove tissue fragments and blood cells, and the obtained cells were resuspended at a density of 1.073 g / mL percoll, centrifuge at 500g for 30 minutes, harvest the interfacial cell layer, wash with PBS and inoculate culture flasks or culture plates, or directly use for other purposes. It is also possible to use D7-FIB or NGFR-lab...
Embodiment 3
[0019] Example 3 Identification of placenta-derived mesenchymal stem cells
[0020] According to literature reports, the method of inducing the differentiation of bone marrow mesenchymal stem cells in vitro induces placenta-derived mesenchymal stem cells to differentiate into osteoblasts, chondrocytes or adipocytes. Flow cytometry was used to detect the phenotypes of placenta-derived mesenchymal stem cells, such as CD29, CD34, CD44, CD45, CD105, CD166, HLA-DR, etc. At the same time, the cell cycle is detected to determine whether it is in G 0 -G 1 The proportion of cells in phase.
[0021] The results showed that the placenta-derived mesenchymal stem cells induced in vitro could differentiate into osteoblasts, chondrocytes, and adipocytes. Flow cytometry showed that the cells were positive for CD29, CD44, CD105, and CD166, and negative for CD34, CD45, and HLA-DR. The cells in G0-G1 phase accounted for more than 95%.
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