Limbus corneae stem cell serum-free culture medium

[0017] The making of embodiment 1 fibroblast conditioned medium

[0018] The key of the present invention is the production of fibroblast conditioned medium, which will now be described by taking the production of serum-free medium for limbal stem cells of Guanzhong dairy goats as an example.

[0019] Preparation of conditioned medium for fibroblasts: Under an ophthalmic operating microscope, aseptically cut the Guanzhong dairy goat corneal epithelial tissue (about 200 μm deep), cut the corneal epithelial tissue into small pieces of 5 mm × 5 mm, and stick the epithelial side up At the bottom of the dish, 3-4 pieces per dish were added with medium (DMEM / F12+2%BSA+10ng / mlEGF+10ug / ml insulin+penicillin and streptomycin each 100IU / ml) for cultivation. When the fibroblasts covered the plate, the tissue pieces were removed, and the fibroblasts were digested and subcultured at 1×10 5 / ml, inoculated in a 50mm plate, cultured for 48 hours, collected the culture medium, centrifuged (4...

[0020] Embodiment 2 culture medium formula,

[0021] The content of various components in each 100ml serum-free medium is: DMEM / F1260ml, BSA 0.5g, EGF1μg, insulin 100ug, hydrocortisone 20ug, cholera toxin 1ug, transferrin 100ug, sodium selenite 100ng, GCLCM 40ml , Penicillin 2×10 4 IU, streptomycin 2×10 4 IU.

[0022] Embodiment 3 culture medium formula

[0023] The contents of various components in each 100ml serum-free medium are: DMEM / F1270ml, BSA 1g, EGF1.5μg, insulin 300ug, hydrocortisone 40ug, cholera toxin 1ug, transferrin 300ug, sodium selenite 300ng, GCLCM 30ml, penicillin 1×10 4 IU, streptomycin 1×10 4 IU.