Mesenchymal stem cells (MSCs) serum-free basal culture medium is a basic culture medium designed by Tongteng Xinchuang specifically for human mesenchymal stem cells, including umbilical cord mesenchymal, bone marrow mesenchymal, adipose derived mesenchymal, and other stem cells. Mesenchymal stem cells require serum-free basal culture with the addition of a certain proportion of nutritional supplements;, After preparing the complete culture medium, use it. Suitable for use in the recovery, passage, and reactor expansion stages of MSCs. Mesenchymal stem cell nutritional supplement is a human platelet lysate (hPL), which is non heterologous and does not contain animal serum. It is mainly used as a substitute for animal serum in traditional culture media. Nutritional supplements contain abundant growth factors and cytokines, aimed at providing the necessary nutrients for cell growth and proliferation. Compared with traditional culture media that require the addition of animal serum, serum-free basic culture media for mesenchymal stem cells used in conjunction with nutritional supplements can maintain undifferentiated growth of mesenchymal stem cells, preserve cell morphological characteristics and normal chromosome karyotypes, ensure high proliferation rate and differentiation potential of mesenchymal stem cells, and greatly reduce batch differences, as well as the risk of contamination by various viruses, mycoplasma, etc. This set includes 2L (500mL × 4) of serum-free basic culture medium for mesenchymal stem cells and 100mL of mesenchymal stem cell nutritional supplements.
Complete preparation of culture medium
1. Before use, thaw the mesenchymal stem cell nutritional supplement in a 37 ℃ water bath.
2. Add 5% mesenchymal stem cell nutrient additive (i.e. 25ml nutrient additive added to 500ml basic medium) to serum-free basic medium for mesenchymal stem cells, and prepare it into a complete culture medium before use.
3. Please use the prepared complete culture medium immediately or store it in 2- Under the condition of 8 ℃, use it up within 2 weeks.
Cell revival
1. Quickly dissolve a tube of frozen cells in a 37 ℃ water bath. After melting, quickly transfer the cell cryopreservation tube to a biosafety cabinet;
2. Gently aspirate the cells from the cryovial and transfer them to 15 ° C; In sterile centrifuge tubes of ml;
3. Slowly add preheated serum-free complete culture medium for mesenchymal stem cells, while gently shaking the centrifuge tube to ensure mixing;
4. Centrifuge 100-200g at room temperature for 3 minutes, then discard the supernatant;
5. Add an appropriate volume of complete culture medium to resuspend cells, count, and adjust cell density;
6. Add an appropriate amount of preheated culture medium to the culture vessel, and then add cell suspension to ensure that the density of live cells inoculated in the culture vessel is about 5 × 103; Per cm2;
7. Incubate in an incubator under recommended conditions of 37 ℃; 5 %CO2 ；
8. One day after recovery, discard the culture supernatant and rinse the monolayer cells with DPBS without calcium and magnesium ions, then remove the rinse solution; Add a suitable volume of preheated complete culture medium.
Note: It is recommended to use 50% of the original complete culture medium before cryopreservation and 50% of the complete culture medium prepared from this product during resuscitation to reduce cell stress and maintain cell stability.
Passaging Cells
1. When the cell fusion degree reaches 70-80%, passage can be carried out;
2. Before use, please preheat the recombinant trypsin solution and mesenchymal stem cell serum-free complete culture medium at 37 ℃;
3. Remove the culture medium from the culture vessel; Wash monolayer cells with DPBS without calcium and magnesium ions, and then remove the rinse solution;
4. Add suitable preheated recombinant trypsin to each culture vessel to ensure that the liquid covers all culture surfaces. Cultivate for 1-2 minutes under recommended cell culture conditions;
5. Use an inverted microscope to observe the cell culture bottle, ensuring that the cells become round and wrinkled;
6. Then add suitable preheated complete culture medium to each culture vessel to ensure that it can completely cover the culture surface; Transfer the cell suspension to a sterile centrifuge tube (if you want to increase cell density, rinse the culture bottle again with a suitable volume of DPBS free of calcium and magnesium ions, and then collect the liquid into the centrifuge tube);
7. Centrifuge 100-200g at room temperature for 3 minutes, then discard the supernatant;
8. Add an appropriate volume of culture medium to resuspend cells and perform live cell counting;
9. Adopting 5 × 103 pieces/cm2; Spread the density of live cells and gently shake the culture bottle to ensure uniform cell distribution;
10. Incubate in an incubator under recommended conditions of 37 ℃; 5 %CO2 ；
11. Change the culture medium or passage every 2-3 days according to the cell growth status, and preheat the new culture medium at 37 ℃ before adding it.