Limbal stem cells and preparation method thereof
A technology of limbal stem cells and limbal stem cells, which is applied in the field of preparation of limbal stem cells, can solve the problems of single storage method, disordered structure, and no fixed ratio, etc., and achieve high purity of screening cells, slow renewal cycle, and high screening efficiency Effect
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[0066] One embodiment of the present invention provides a method for preparing limbal stem cells, comprising the following steps:
[0067] Obtain corneal tissue and pretreat the corneal tissue to be used;
[0068] Adding a cell cryopreservation solution containing a cell inducer to the pretreated corneal tissue;
[0069] Freezing corneal tissue with cell freezing solution;
[0070] The cell-inducing agent is a reagent that can induce local inflammation or stress response of cells in the corneal tissue, but does not directly cause cell death.
[0071] In the above method, the cells are screened and preserved by adding a cryopreservation solution containing a cell-inducing agent to the corneal tissue, and the added cell-inducing agent can induce local inflammation or apoptosis of the cells in the corneal tissue, and the low-temperature Freezing conditions can induce apoptosis or necrosis of adult cells in corneal tissue, while maintaining and preserving the activity of limbal ...
Embodiment 1
[0088] Example 1: Preparation of Rabbit Limbal Stem Cells
[0089] (1) Obtain corneal tissue and pretreat the corneal tissue to be used:
[0090] The eyeballs removed from New Zealand rabbits were washed 3 times with phosphate buffered saline (PBS) added with 1% penicillin-streptomycin solution, and the excess tissue around the outside of the eyeballs was removed with tissue scissors. 5mm of sclera and cut off the rest, remove the contents at the same time, scrape off the iris and other parts, and then wash 3 times with PBS.
[0091] (2) Add cell cryopreservation solution containing cell inducer to the pretreated corneal tissue:
[0092] First prepare the cell culture medium KCM for culturing limbal stem cells in vitro: high-glucose DMEM+10% fetal bovine serum+1% glutamine+1% non-essential amino acids+1% penicillin-streptomycin solution+10ng / ml epidermal growth factor (EGF)+5μg / ml transferrin+5μg / ml insulin, and add 4μg / ml prednisolone acetate and 20ng / ml staurosporine (STS)...
Embodiment 2
[0101] Example 2: Preparation of human limbal stem cells
[0102] (1) Obtain corneal tissue and pretreat the corneal tissue to be used:
[0103] The human eyes were washed with Kolabito eye drops for 5 to 6 times, and then washed with Ⅲ Aner Iodine and normal saline with gentamicin respectively, and the eyeballs were removed with tissue scissors to remove the excess tissue around the outside of the eyeballs. Keep 1-5mm of the sclera along the limbus and cut off the rest, remove the contents, scrape off the iris and other parts, and then clean the cornea with phosphate buffered saline (PBS) with 1% penicillin-streptomycin solution Wash 3 times.
[0104] (2) Add cell cryopreservation solution containing cell inducer to the pretreated corneal tissue:
[0105] First prepare the cell culture medium KCM for culturing limbal stem cells in vitro: high-glucose DMEM+10% fetal bovine serum+1% glutamine+1% non-essential amino acids+1% penicillin-streptomycin solution+10ng / ml epidermal g...
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