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Primary culture of normal retinal pigment epithelial cells
 Primary culture of normal retinal pigment epithelial cells
Experimental Materials:
1. Source of materials: human eyes, rabbit eyes or pig eyes;
2. Washing solution: 1×PBS containing no Ca 2+ and Mg 2+ , adding 100 IU/ml penicillin, 100 μg/ml streptomycin, pH 7.2;
3. Special materials: eye support and 7-0 nylon thread. A sterilized white rubber anti-mouth rubber plug is used as an eyeball holder;
4. Disinfectant: 200 IU/ml gentamicin saline;
5. Digestive juice: mixed digestive juice of 0.01% EDTA-0.125% trypsin;
6. Culture medium: DMEM medium containing 10% fetal bovine serum;
7. Culture vessels: culture flasks, 24-well culture plates or culture dishes;
experimental method:
1. Eyeballs were removed and immersed in 200 IU/ml gentamicin saline for 5 min. Cut the anterior segment of the eyeball 2 to 3 mm behind the serrated edge to remove the cornea, lens and vitreous. The retinal neuroepithelial layer was removed by separation. Making the posterior segment of the eyeball into an eye cup;
2. Place the eye cup on the eyeball holder (can be replaced by a disinfected saline bottle rubber stopper), and take the needle holder with 7-0 nylon thread in 4 symmetry directions, and fix the eye cup to the wall of the eyeball holder. On the edge
3. Using a capillary pipette, gently aspirate the retinal neuroepithelial layer from the eyecup. Rinse the eye cup twice with PBS solution and blot dry;
4. Add a mixed digest of 0.01% EDTA-0.125% trypsin and place in a sterile beaker. Digested in a 37 ° C incubator for 30 min;
5. Aspirate the digestive juice. Wash slightly with PBS solution. The serum culture solution is added to terminate the action of the digestive enzyme, and the inner wall of the eye cup is gently blown with a capillary pipette to cause the retinal pigment epithelial cells to fall off;
6. Collect the cell suspension in the eye cup and centrifuge (800r/min) for 8min;
7. Discard the supernatant and resuspend the pelleted retinal pigment epithelial cells with the culture medium. count;
8. Inoculate the culture flask at a density of 8 x 104 - 10 x 104 / ml;
9. Transfer to the incubator, culture according to the conventional method, and change the liquid twice a week;
10. The mechanical separation method can also be used directly: the retinal pigment epithelial cell layer is removed with a toothless sputum, and after rinsing with PBS, it is cut into 1.5 mm × 1.5 mm tissue pieces and directly inoculated into a 24-well culture plate. Carry out cultivation;