Co-culture of feeder cells to expand primary CD34+ cells in vitro
Reagents and materials:
1. MSCs medium;
2. 6-well culture plate;
3. Mitomycin C, 100 μg/ml;
4. Co-culture medium: IMDM added 10% FBS, 100 U/ml penicillin and 100 μl/ml streptomycin;
5. Cytokines (stem cell factor, IL-6, Flt-3 ligand, thrombopoietin);
experimental method:
1. Umbilical cord blood-derived mesenchymal stem cells (CB-MSCs) were used as feeder cells, and CB-MSCs were resuspended in MSCs medium at a cell concentration of 5×10 4 cells/ml;
2. Inoculate CB-MNCs into 6-well plates;
3. Change the fresh medium half-time twice a week;
4. When CB-MNCs reach more than 90% confluence, treat with 10μg/ml mitomycin C, 37 ° C for 2.5h;
5. Wash CB-MNCs twice with serum-free IMDM;
6. Resuspend 1×104/ml CD34+ cells in cytokine-containing co-culture medium (100 ng/ml stem cell factor, 100 ng/ml IL-6, 50 ng/ml Flt-3 ligand, 10 ng/ml thrombopoietin) );
7. Inoculate CD34+ cells onto feeder cells of CB-MSCs;
8. Replace 1/4 medium twice a week;
9. After 2 weeks, collect unattached cells;
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