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Method for isolating placental mesenchymal cells from tissue and culturing them into mesenchymal stem cells
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A technology of mesenchymal stem cells and cells, applied in the field of isolating stem cells
Active Publication Date: 2020-09-15
广东博雅干细胞科技有限公司
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[0083] 1. Pre-preparation of the mixed synthase digestion solution: pipette 22ml of HBSS (Hank's Balanced Salt Solution) containing calcium and magnesium ions, 0.4ml of Roche Liberase MNP-S enzyme (for example, purchased from Xibao Biology, article number: 5578582001), 0.7ml Put ml of DNA type I enzyme in a 50ml centrifuge tube, then add zincchloride (addition concentration is 0.2g / L, 0.25g / L, or 0.3g / L), mix well, and preheat at 37°C for more than 20min. The Hank's balanced salt solution consists of: 8.0g / L of NaCl, 0.4g / L of KCl, 0.1g / L of MgSO4 7H2O, 0.1g / L of MgCl2 6H2O, 0.06g / L of Na2HPO4 2H2O, 0.06g / L of KH2PO4, 1.0g / L of glucose, 0.14g / L of CaCl2, 0.35g / L of NaHCO3, 0.2g / L of phenol red, hydrochloric acid or sodiumhydroxide to adjust the pH to 7.4.
[0084] 2. Preparation of placenta lobule: take out the placenta from the collection bag and place it in a white porcelain plate, rinse with tissue cleaning soluti...
[0103] Take 2 tubes of frozen cells, thaw them quickly at 37°C, transfer the cells to a 15ml centrifuge tube, add 8ml of complete medium to recover by dripping; unless otherwise stated, the complete medium used in this article is DMEM-F12 culture containing 10% FBS base;
[0104] Then centrifuge at 1200rpm for 5min (acceleration 9, deceleration 7), remove the supernatant, add 5ml of complete medium to resuspend; each tube of cells is inoculated in a T75 culture flask, supplement the complete medium to 30ml, and place in a CO2 incubator ( Culture at 37°C, 5% CO2, saturated humidity); replace the medium with complete medium every 3-4 days, count according to the colony formation after 12 days of recovery, until the cell density is not less than 3000 cells / cm 2 Subsequent passages can be carried out;
[0105] 2. Cell subculture: Take the revived P0 generation cells and wash them with PBS, ...
Embodiment 3
[0119] Embodiment 3, biological characteristic identification of placental MSC
[0120] Refer to the granted patent The method of [0062] to [0089] of CN102676451A carries out the biological characteristic identification of placental mesenchymal stem cells, and the results show that the MSCs obtained by applying the method of the present invention have the ability to differentiate into osteoblasts, adipocytes, and chondrocytes. It is confirmed that the MSC obtained by the method of the present invention has stem cell characteristics.
[0121] For example, exemplarily, the induction differentiation test is performed on the P5 generation cells, and the results show that these cells have the ability to differentiate into osteoblasts, adipocytes, and chondrocytes. Typical micrographs of adipogenic, osteogenic, and chondrogenic differentiation are shown in Figure 6 .
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Abstract
The invention relates to a method for separating placental interstitial cells from tissue and cultivating placental interstitial cells into mesenchymal stem cells, and further relates to a mixed enzyme digestive fluid used for the method. Particularly, the method comprises the steps of treatment of placental lobules, mixed enzymedigestion and termination, primary cell collection, primary cellcryopreservation, cellrecovery and cell propagation. By using the method, the efficiency of separating the mesenchymal stem cells from placenta can be effectively improved. For example, obtained primary cells are a group of relatively pure mesenchymal cells (CD73 expression is greater than 60% and CD45 is not expressed), the quantity of cells obtained from each gram of tissue can reach 2.5*10<7> and the yield is stable; P1-P5 generations of cells of the mesenchymal stem cells obtained through subculture are mesenchymal stem cells, positive expression CD73, CD90 and CD105 is greater than 98%, and negative expression CD34, CD45, CD19, CD11b and HLA-DR is smaller than 2%; and the G2-stage cells of the P5 generation of cells are smaller than 1%, the multiplication capacity is high and the cells do not enter a division stage; and the cells have the ability of being differentiated into osteoblasts, lipoblasts and chondroblasts under the stimulation of a specific induction medium.
Description
technical field [0001] The present invention relates to a method for isolating stem cells from placenta, in particular to a method for isolating mesenchymal stem cells from placenta, and more particularly to a method for isolating mesenchymal stem cells from placenta tissue using the digestive enzyme composition of the unique formula of the present invention. A method for culturing stromal cells into mesenchymal stem cells. Using the method of the invention can effectively improve the efficiency of isolating mesenchymal stem cells from placenta. Background technique [0002] Mesenchymal stem cells (mesenchymal stem cells, MSCs) such as human mesenchymal stem cells were first isolated from bone marrow, a type of tissue stem cells derived from mesoderm with multi-lineage differentiation potential and self-renewal ability, in vivo and Under specific conditions in vitro, it has the ability to differentiate into various adult cells such as osteoblasts, chondrocytes, adipocytes, ...
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