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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

Inactive Publication Date: 2020-08-21
BEIJING YULONG SHENGSHI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This invention requires the trophoblasts to be placed in a container with two culture chambers, and the culture process is cumbersome

Method used

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Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention provides a limbal epithelial stem cell isolated culture method, the limbal epithelial stem cells obtained by the isolated culture method have higher cell activity and stronger cell proliferation ability, and the culture method is simple, effective and low in rejection reaction. Wherein the number of cells subjected to primary culture is 9.5-105, the positive expression rate of a surface marker is detected by flow cytometry, and a result is negative expression of a surface symbolic antibody of an antibody AE5 corneal fibroblast, negative expression of CX43 corneal stem cells, positive expression of a PCNA antibody and positive expression of a BrdU antibody.

Description

technical field [0001] The invention belongs to the technical field of cell separation and culture, and in particular relates to a method for separation and culture of limbal epithelial stem cells. Background technique [0002] Limbal stem cells refer to the corneal limbus, which is the junction of the cornea, conjunctiva and sclera, located in the basal layer of the limbal epithelial cells. The main function is to renew and repair the corneal epithelium. [0003] The cultivation of limbal stem cells is based on the in-depth understanding of limbal stem cells and a major innovation of traditional methods, and has a good development prospect. [0004] A series of problems such as the differentiation and physiological and biochemical changes of the cultured corneal stem cells, how to choose a safe and reliable carrier, how the cultured stem cells will be better, and how to reduce the occurrence of rejection after allogeneic transplantation all need to be solved urgently. [0...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797
CPCC12N5/0623C12N2509/00
Inventor 张晓南吴芳春侍晓云谷涌泉张斌霍文卓
Owner BEIJING YULONG SHENGSHI BIOTECH CO LTD
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