Technical analysis: rat endothelial progenitor cell culture method - Database & Sql Blog Articles

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Technical analysis: rat endothelial progenitor cell culture method cell description

Endothelial progenitor cells are a heterogeneous group of cells that originate primarily from complex precursor cells in the bone marrow and are present in the peripheral circulation at different stages of differentiation. Because it is a precursor cell that can differentiate into endothelial cells, it is named endothelial progenitor cells. In 1997, Asahara et al. successfully isolated endothelial progenitor cells from human peripheral blood by immunomagnetic beads for the first time using the cell surface marker CD34, and proved that it has the ability to form blood vessels in vivo, and then proved the source in 1999. Endothelial progenitor cells derived from bone marrow can participate in physiological and pathological angiogenesis after birth in an angiogenic manner. The successful discovery of endothelial progenitor cells has completely changed the understanding of angiogenesis. It can selectively homing to damaged or ischemic sites, participate in local angiogenesis and angiogenesis, thereby forming blood vessels, and at the same time promoting endothelial cells. Repair and so on. In recent years, studies have shown that endothelial progenitor cells have a very broad application prospects, and can be used for angiogenesis, endothelial regeneration, cardiovascular disease treatment, tumor treatment, ischemic treatment, tissue engineering, gene therapy, etc., therefore, simple and fast It is necessary to obtain endothelial progenitor cells reliably and in sufficient quantities.

Reagents and materials

Adult rat

serum

Ficoll

M199 basic medium

Experimental procedure

1. Rats were intraperitoneally injected with 0.5 ml of 3% C11H17O3N2Na anesthetized to death, immersed in a burning volume of 75% ethanol for 15 min and then removed.

2. Remove the femur and tibia under sterile conditions to remove the muscles attached to the bone surface.

3. Dip the obtained bone into a small burning PBS containing phosphate buffer solution, and cut off the metaphyseal ends of the bones with scissors to expose the marrow cavity.

4. Take a 10 ml disposable syringe and take 6~8 ml of M199 basal medium to rinse the marrow cavity several times until the bones are white and translucent, and collect the cell suspension.

5. Take 4 ml of rat lymphocyte separation solution into a 12 ml centrifuge tube and incline at 45 °C. Then slowly add the cell suspension to the lymphocyte separation solution to make the volume ratio 1:2. The interface between the two is clear and should not be mixed with each other.

6. Place the centrifuge tube in a centrifuge and centrifuge at a radius of 12 cm, 2000 r/min, and centrifuge for 20 min.

7. Slowly take out the centrifuge tube. You can see that the liquid in the centrifuge tube is divided into 4 layers, the lowermost layer is red blood cells and granulocytes, the middle layer is lymphocyte separation solution, the uppermost layer is plasma and M199 medium, and the middle layer is the most There is a thin layer of cloud between the upper layers.

8. Insert the pipette directly into the cloud layer and slowly pump it;

9. Transfer the extracted liquid into another centrifuge tube, then add 10 ml PBS buffer, blow evenly, centrifuge, centrifuge radius 12 cm, 1500r/min, centrifuge for 5 min; 10, aspirate the supernatant, take 10 Mol PBS buffer, evenly blown, centrifuged, centrifuged at 12 cm, 1500 r/min, centrifuged for 5 min.

11. Aspirate the supernatant, add 5 ml of M199 complete medium, blow the cell pellet evenly, inoculate it in a T25 cell culture flask, and incubate it in a CO2 incubator.

Cell passage and cryopreservation

1. The cells grow to log phase, and the cells reach 80-90% fusion.

2. Remove the cell culture medium and wash the cells twice with PBS.

3. Add 0.25% trypsin and digest at 37 °C. Under the microscope, the cells were retracted and rounded, and the medium was completely digested.

4. Centrifuge, resuspend the cells and re-inoculate in fresh medium.

5, the cells can not be frozen.
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