An isolation and culture method and application for porcine endometrial mesenchymal stem cells

An isolation and culture method for porcine endometrial mesenchymal stem cell (EMSCs), wherein the specific process comprises the following steps:

• (1) Pre-treatment
• Firstly, in the sterile room, cutting the porcine uterus, using pre-cooled sterile phosphate-buffered saline (PBS) added with magnesium chloride and calcium chloride to immerse, and rinsing;
• Secondly, peeling a serosa layer off to separate the endometrial layer from the muscular layer;
• Finally, placing the fragmented intima layer in an incubator at 37° C. to be digested with a basal medium supplemented with a digestive enzyme for 2-3 hours, and blowing every 15 minutes to obtain a cell suspension;
• (2) Centrifugation
• Firstly, filtering the cell suspension through cell filters, further preventing digestion by the addition of the MSCs complete culture medium (MSCCM), washing with PBS, and centrifuging at 1000 rpm for 5 minutes;
• Secondly, treating the cells with red blood cell lysate twice and washing with PBS;
• Finally, inoculating the cells in the MSCCM, and incubating at 37.0° C. in a humidified atmosphere of 5% CO2, replacing the MSCCM every 2-3 days, and the cells are P0 generation cells;
• (3) Cultivation and passage
• Firstly, pre-heating the MSCCM, trypsin with a concentration of 0.25% and PBS in a water bath at 37° C. in advance, when the cell density reaches 60%-80%, aspirating the MSCCM, and washing with PBS;
• Secondly, adding trypsin for digestion, when the cells are shrunk and dispersed, adding immediately an equal amount of MSCCM to terminate the digestion, transferring the cells to an EP tube and centrifuging at 1000 rpm for 5 minutes;
• Finally, removing the supernatant, adding MSCCM to re-suspend the cells, taking ⅓ of them and adding them into a new culture dish, shaking gently and mixing well, placing in a CO2 incubator for cultivation, and the obtained cells are P1 generation cells;
• (4) Counting, viability detection and culture
• Taking 100 μL of P1 generation cells, detecting the cell activity and counting, adjusting the cell density, inoculating the cells into the MSCCM for cultivation, and the cells after subculture again being the P2 generation cells, and by analogy.