Limbal epithelial stem cell isolated culture method
A technology for the separation and cultivation of limbal stem cells, which is applied in the field of separation and cultivation of limbal epithelial stem cells, can solve the problems of cumbersome culture process, achieve mild and fast digestion, simple and effective rejection, and low incidence of fibroblast contamination
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Embodiment 1
[0042] (1) The cornea was obtained and cleaned under sterile conditions; the corneal cleaning solution was CMF-Saline G, and it was cleaned for 3✕5min.
[0043] The eyeballs of infants and children who died due to various reasons were collected within 24 hours after death without any eye diseases. The human eyeballs were placed on a clean workbench and operated under strict aseptic conditions. Cut at 1 mm, remove the anterior segment tissue including the corneal limbus, carefully transfer the anterior segment tissue to another plate filled with culture medium, remove iris, lens and other accessory tissues under a dissecting microscope, and then tear off the cornea Cortex and Descemet's membrane; then, cut limbal tissue about 2.0-2.5 mm wide along the 1 mm ring in the transparent cornea, and divide it into 2 mm × 2 mm × 2 mm tissue blocks. Take care to avoid clamping the cultured tissue pieces as much as possible throughout the operation.
[0044] (2) Subtract the remaining sc...
Embodiment 2
[0067] (1) The cornea is obtained and cleaned under sterile conditions;
[0068] (2) Subtract the remaining sclera, conjunctiva and iris;
[0069] (3) Add dispase II to digest at 4°C overnight, stir slightly, dispase II
[0070] The digestion is gentle and fast, and the complete corneal epithelial layer can be obtained, and the incidence of fibroblast contamination is low. Add 2ml of dispase II;
[0071] (4) Put the cornea added with dispase II on a shaker at 4°C for 30 minutes.
[0072] (5) Wash the cornea with DMEM / F-12 / GASP;
[0073] (6) Under a dissecting microscope, carefully remove the epithelial cells in the center of the cornea and remove the pigment epithelial cells containing the Vogt palisade area in the corneal limbus. Most of the central epithelial cells have been peeled off during the removal process;
[0074] (7) Digest the limbal epithelial cell sheet with trypsin / EDTA for 3 minutes;
[0075] (8) Add an equal volume of DMEM / F-12 / 2FB / GASP;
[0076] (9) Cent...
Embodiment 3
[0084] (1) The cornea is obtained and cleaned under sterile conditions;
[0085] (2) Subtract the remaining sclera, conjunctiva and iris;
[0086] (3) Add dispase II to digest at 4°C overnight, stir slightly, dispase II
[0087] The digestion is gentle and fast, and the complete corneal epithelial layer can be obtained, and the incidence of fibroblast contamination is low. Add 2ml of dispase II;
[0088] (4) Put the cornea added with dispase II on a shaker at 4°C for 30 minutes.
[0089] (5) Wash the cornea with DMEM / F-12 / GASP;
[0090] (6) Under a dissecting microscope, carefully remove the epithelial cells in the center of the cornea and remove the pigment epithelial cells containing the Vogt palisade area in the corneal limbus. Most of the central epithelial cells have been peeled off during the removal process;
[0091] (7) Digest the limbal epithelial cell sheet with trypsin / EDTA for 3 minutes;
[0092] (8) Add an equal volume of DMEM / F-12 / 2FB / GASP;
[0093] (9) Cent...
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