Human bone marrow discontinuous density gradient centrifugation for mesenchymal stem cell production

Reagents and materials:
1. Complete medium: α-MEM: α-low-limit basal medium containing glutamine, no nucleotide or deoxynucleotide; addition: 20% additional L-glutamine 2mmol/L, hybridized by FBS Tumor purification, non-heat inactivation, penicillin 100 U/ml, streptomycin 100 μg/ml. Filtration sterilization. Store at 4 ° C for no more than 2 weeks;
2. Phosphate buffer without calcium or magnesium;
3. Hank's balanced salt solution without calcium or magnesium;
4. Ficoll-Paque lymphocyte separation solution;
5. Polypropylene centrifuge tube, 15ml and 50ml;
6. Plastic tissue culture dish, diameter 15cm;
7. Plastic micropipette tip, 10μl;
8. 0.85% trypan blue salt solution;
9. Microcentrifuge tube;
10. Low temperature bench top centrifuge with bucket type rotor

experimental method:
1. Remove the anticoagulation tube cover and transfer the bone marrow fluid to a 50 ml centrifuge tube. Add room temperature HBSS to ensure the volume reaches 25ml;
2. Take another 50 ml centrifuge tube, add 20 ml of Ficoll-Paque lymphocyte separation solution, and gently add 25 ml of the cell suspension to the upper Ficoll layer. The interface between HBSS and Ficoll cannot be destroyed;
3. Turn off the brake and centrifuge at room temperature for 1800g for 30min;
4. After centrifugation, collect the white cell layer at the interface of Ficoll and HBSS and transfer it to a new 50 ml centrifuge tube;
5. The volume of the collected cell suspension was adjusted to 3 times with HBSS, and centrifuged at 1000 g for 10 min at room temperature. Repeat washing;
6. Resuspend the cells with 30 ml of CMM preheated to 37 °C;
7. 10 μl of the cell suspension was added to 10 μl of trypan blue, and the survival rate of the cells was evaluated by a hemocytometer, and the survival rate was required to be higher than 80%;
8. Transfer 30 ml of the cell suspension to a 15 cm diameter tissue culture dish and incubate in a 5% CO2 incubator for at least 15 h;
9. Remove the culture dish from the incubator and aspirate the medium;
10. Add 20 ml of pre-warmed PBSA, rinse the monolayer cells, and discard;
11. Repeat the rinsing process 3 times;
12. Add 30ml of preheated fresh CMM;
13. Repeat this rinse and update medium every other day for 6 days;
14. After 6 days, the monolayer cells were observed under an inverted microscope, and the adherent, fibroblast-like MSCs clones were clearly visible in the culture dish. Sometimes there may be signs of hematopoietic cell contamination, but these cells can be removed during passage. When the culture reaches 50%-60% confluence, the expansion and cryopreservation of MSCs culture can be performed;

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