Experimental Materials:
1. a complete human cornea;
2. GMF-Saline G;
3. Neutral protease II; PriCellls primary cell isolation kit
4. L-type collagenase, 1mg/ml in DMEM/F12/GASP;
5. TrypLE Express or 0.25% trypsin formulated in GMF-Saline G;
6. DMEM/F12/GASP;
7. DEME/F12/2FB/GASP: DMEM/F12/GASP contains 2% FBS;
8. CMF/GASP;
9. 3.5cm plastic tissue culture dish or 6-well plate;
10. Scalpel or single-edged safety blade;
11. Cell strainer, 70 μm blood nylon filter;
12. Plastic blade, "Cell Lifter" or "Cell Scraper";
13. Curved iris shear, 11cm (4-3/8in);
14. Jeweler's é•Š, 10cm (4in);
15. Corneal scissors, 19mm blade, pointed;
16. Colibri sewing pliers, 0.1mm;
17. SDS sample buffer, using final concentration: 0.058 mol/L Tris, 5% glycerol, 1.67% SDS, 0.02% bromophenol blue;
18. PriCells primary cell special basal medium;
19. PriCells primary cell culture special additive;
experimental method:
1. Clean the cornea with GMF-Saline G for 3×5 min;
2. Cut off the residual sclera, conjunctiva and iris;
3. Add reagents from the PriCells Primary Cell Isolation Kit and shake overnight at 4 °C shaker;
4. The cornea to which the reagent was added was shaken at 4 ° C for 30 min;
5. Clean the cornea with DMEM/F12/GASP;
6. Carefully remove the epithelial and endothelial cells under a dissecting microscope;
7. Scrape the epithelial cell surface and endothelial cell surface of the basement membrane with a plastic scraper. Observed under the microscope to ensure complete removal of these cell layers;
8. Wash the corneal stroma once with fresh medium;
9. Cut the substrate into small pieces of about 2 mm with a scalpel or a safety blade or a fine surgical scissors;
10. The collagenase prepared with DMEM/F12/2FB/GASP was digested at 37 ° C for 3 h until most of the tissues disappeared;
11. Centrifuge at 400g for 10min, discard the supernatant;
12. Resuspend the cells with DMEM/F12/2FB/GASP, filter the digest with a 70 μm cell strainer, and centrifuge;
13. Repeat the above washing step 2 times, counting the cells after each pass;
14. The isolated mesenchymal cells were resuspended in MJM and inoculated to a plastic tissue culture dish at a density of 1 x 104/cm2;
15. Replace PriCells primary cell special basal medium + PriCells primary cell culture special additive every three days;
16. Passage digestion with trypsin when cells reach 90% confluence:
Veterinary Solution Disinfectant
Veterinary Solution Disinfectant,Povidone Iodine Disinfectant,Ivermectin Pour On Solution,Povidone Iodine Disinfectant Solution
Shandong Unovet Pharmaceutical Co.,Ltd. , https://www.unovetcn.com