Experimental Materials:
1. a complete human cornea;
2. GMF-Saline G;
3. Neutral protease II, 1.2 U/ml (diluted 2.4 U neutral protease II with DMEM/F12/GASP);
4. Trypsin / EDTA;
5. DMEM/F12/GASP;
6. DEME/F12/2FB/GASP: DMEM/F12/GASP contains 2% FBS;
7. CMF/GASP;
8. LSC medium;
9. Curved iris shear, 11cm (4-3/8in);
10. Jeweler's é•Š, 10cm (4in);
11. Corneal scissors, 19mm blade, pointed;
12. Colibri sewing pliers, 0.1mm;
experimental method:
1. Wash the cornea 3 × 5min;
2. Cut off the residual sclera, conjunctiva and iris;
3. Add 2 ml of neutral protease II, overnight at 4 ° C, slightly agitated;
4. Put the cornea added with neutral protease II into a shaker at 4 ° C for 30 min;
5. Clean the cornea with DMEM/F12/GASP;
6. Under the dissecting microscope, carefully remove the epithelial cells in the center of the cornea (when most of the central epithelial cells have been exfoliated) and remove the pigment epithelial cells from the limbus containing the Vogt barrier;
7. Digest the limbal epithelial cells with trypsin/EDTA at 37 ° C for 10 min;
8. Add an equal volume of DMEM/F12/2FB/GASP;
9. Centrifuge at 400g for 10min, discard the supernatant;
10. Wash once with DMEM/F12/2FB/GASP, centrifuge, and discard the supernatant;
11. Add LSC medium, scatter the cells and count with a hemocytometer;
12. Inoculate the cells at a density of 1 × 104 / cm 2 onto a tissue culture dish with a 3T3 feeder layer or a prepared human amniotic membrane;
13. Change the medium every three days;
14. Passage digestion with trypsin when cells reach 90% confluence:
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