Normal corneal endothelial cell culture

Normal corneal endothelial cell culture

Experimental Materials:

1. Corneal material: can be taken from the human eye, rabbit eyes or bulls eye, the human eye material is best from the fetus;

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 equipment: iris recovery device;

4. Disinfectant: 1:5000 liters of mercury solution and 100 IU/ml of gentamicin physiological saline;

5. Digestive juice: 0.25% trypsin-0.01% EDTA mixed solution;

6. Culture solution: The base solution was prepared by adding HEPES 6.0 g/L, NaHCO 3 2.2 g/L and L-glutamine 0.06 g/L from DMEM medium . The application solution is supplemented with 15% fetal bovine serum, penicillin 100 IU/ml, streptomycin 100 μg/ml in the base solution, and the pH is adjusted to 7.0-7.2 with 5% NaHCO 3 ;

experimental method:

1. Cut corneal endothelial tissue: When corneal epithelial cells are cultured with human eyes, it is best to take the cornea from the fetus. Because adult corneal cells are cultured in vitro, they are not as viable as fetal corneal cells;

1) 1 eyeball was taken, and the mercury-salt solution and the gentamicin physiological saline disinfectant were alternately washed twice to wash away the residual blood on the surface of the eyeball and perform preliminary disinfection treatment;

2) Under sterile conditions, a small circular incision is made 1 mm inside the limbus;

3) using an iris restorer to insert the posterior elastic membrane from the incision between the elastic membrane and the corneal stroma (parenchymal layer), and bluntly separating the entire corneal parenchyma;

4) After the corneal epithelial layer is completely separated, the whole endothelium layer tissue is circularly cut at a distance of 1 mm from the inner side of the limbus;

2. Inoculation and cultivation;

1) Place the obtained endothelial layer tissue in a Petri dish and add 2-3 drops of bovine serum;

2) Cut it into tissue pieces of about 1.5 mm × 1.5 mm with a corneal scissors;

3) inoculation of the tissue block endothelium face down in a culture flask with a toothless tweezers;

4) Let stand for 20-60 minutes in the incubator, and then add enough culture solution after the plant block is firmly adhered;

5) Cultivate according to conventional methods. Change the liquid twice a week;