Rat bone marrow collection for the production of MSCs
Rat bone marrow collection for the production of MSCs
Reagents and materials:
1. Complete medium (CCM): α-MEM: α-low-limit basal medium containing glutamine, no nucleotide or deoxynucleotide; addition: 20% additional L-glutamine 2mmol/L, Purified by FBS hybridoma, non-heat inactivated, penicillin 100 U/ml, streptomycin 100 μg/ml. Filtration sterilization. Store at 4 ° C for no more than 2 weeks;
2. Gauze;
3. PBSA containing 100 U/ml penicillin, 100 μg/ml streptomycin and 0.2 μg/ml amphotericin B;
4. Hank's balanced salt solution (HBSS) without magnesium and calcium;
5. Plastic Petri dish, 15cm diameter;
6. A scalpel with #10 and #15 blades;
7. Surgical scissors for 1 and 2 foot blades;
8. Scorpion;
9. Hemostasis;
10. 15cm blade rongeur;
11. Syringe with Luer connector, 20ml;
12. Hypodermic needles, 23 and 18;
13. Microcentrifuge tube, 1.5ml and 0.5ml;
14. Polypropylene centrifuge tube, 15ml and 50ml;
15. Adult Lewis rats;
16. CO 2 rodent euthanasia chamber;
17. Eppendorf 5417C microcentrifuge or equivalent;
experimental method:
1. CO2 asphyxiation to kill animals, do not use drugs;
2. Wipe the hind limbs with alcohol and dissect the hind limbs without damaging the femur. Separating the femur from the limb;
3. Remove the muscles and tendons from the femur. Wipe off the remaining tissue on the bone with a sterile gauze;
4. Wipe the cleaned bone with alcohol and place it in a 50ml plastic tube;
5. Immerse the bone with an ice bath CCM containing an antifungal drug until the bone marrow extraction is ready. The time from bone separation to bone marrow extraction is as short as possible;
6. Transfer the centrifuge tube containing the femur to the microbiological safety cabinet and place the bone into a 15cm petri dish;
7. Extract the bone marrow as follows;
(1) Cut the ends of the bone with a rongeur to expose the internal bone marrow;
(2) Inhalation of 5 ml of CCM containing antifungal drugs into a syringe containing a 23G needle, and flushing each femur into a 50 ml centrifuge tube;
(3) Aspirate the washed CCM, repeatedly flush the femur 3 times, and transfer it to a 15 ml centrifuge tube;
(4) Centrifugation at 1000g, resuspending the bone marrow pellet with 1ml of CCM containing antifungal drugs;
(5) 1 ml of bone marrow cell suspension was added to 4 ml of fresh PBSA containing antibiotics and antifungal drugs in a 15 ml centrifuge tube. 5 ml of cell suspension obtained from each femur can be mixed in the same centrifuge tube;
8. Centrifuge the bone marrow cells at 1000g for 10min at 10°C for 10min, and wash the cells 3 times with 10ml PBSA containing antibiotics and antifungal drugs;
9. Finally, resuspend the bone marrow cells in 5 ml CMM and place on ice for later use;
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