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Corneal limbus stem cell, and culture method as well as application thereof

A technology of limbal stem cells and culture methods, applied in the direction of non-embryonic pluripotent stem cells, animal cells, nervous system cells, etc., can solve the problems of poor treatment effect of eye diseases, low purity and high degree of differentiation of limbal stem cells, and achieve The effect of increasing the number, improving the curative effect, and reducing the degree of differentiation

Inactive Publication Date: 2016-09-21
SHEN ZHEN ISTEM REGENERATIVE MEDICINE SCI TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the limbal stem cells cultured by the existing methods have low purity and high degree of differentiation, which leads to poor therapeutic effect on the above-mentioned eye diseases when the limbal stem cells are used for transplantation

Method used

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  • Corneal limbus stem cell, and culture method as well as application thereof
  • Corneal limbus stem cell, and culture method as well as application thereof
  • Corneal limbus stem cell, and culture method as well as application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] 1. Obtain limbal tissue:

[0050] The eyeballs of infants and children who died due to various reasons were collected within 24 hours after death without eye diseases;

[0051] Place the human eyeball on a clean workbench, operate under strict aseptic conditions, cut along the 1mm behind the gray-white junction of the corneal limbus, remove the anterior segment tissue including the corneal limbus, and carefully move the anterior segment tissue to another container Remove iris, lens and other accessory tissues under a dissecting microscope in a plate with culture medium, and then tear off the corneal endothelial layer and Descemet's membrane;

[0052] Then, cut limbal tissue about 2.0-2.5 mm wide along a 1 mm ring in the transparent cornea, and divide it into tissue blocks of 2 mm×2 mm×2 mm. Take care to avoid clamping the cultured tissue pieces as much as possible throughout the operation.

[0053] 2. Obtain proliferative limbal tissue:

[0054] The limbal tissue was...

Embodiment 2

[0059] The steps of obtaining limbal tissue and proliferative limbal tissue in Example 2 are the same as those in Example 1, except that:

[0060] 3. Cell culture:

[0061] Place proliferative limbal tissue in 20ml containing FBS and Ca 2+ In the MEM culture medium, wherein, fetal calf serum 2g, the concentration of calcium ion is 0.06mmol / ml;

[0062] To the containing FBS and Ca 2+ 300ng of recombinant bovine basic fibroblast growth factor (Zhuhai Dongda Biopharmaceutical Co., Ltd.) was added to the MEM medium, and cultured until the 15th day.

Embodiment 3

[0064] The steps of obtaining limbal tissue and proliferative limbal tissue in Example 3 are the same as those in Example 1, except that:

[0065] 3. Cell culture:

[0066] Place proliferative limbal tissue in 20ml containing FBS and Ca 2+ In the MEM culture medium, wherein, fetal calf serum 2g, the concentration of calcium ion is 0.09mmol / ml;

[0067] To the containing FBS and Ca 2+ 300ng of recombinant bovine basic fibroblast growth factor (Zhuhai Dongda Biopharmaceutical Co., Ltd.) was added to the MEM medium, and cultured until the 15th day.

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Abstract

The invention discloses a corneal limbus stem cell, and a culture method as well as application thereof. The culture method of the corneal limbus stem cell comprises the following steps: acquiring a proliferative corneal limbus tissue; adding the proliferative corneal limbus tissue into an MEM culture medium containing FBS and Ca<2+>, and culturing, wherein the concentration of the Ca<2+> in the MEM culture medium containing the FBS and the Ca<2+> is 0.01mmol / L to 0.09mmol / L. According to the culture method of the corneal limbus stem cell, disclosed by the invention, the differentiation of the corneal limbus stem cell can be inhibited, the generation of other cells caused by the differentiation of the corneal limbus stem cell is avoided, the purity of the corneal limbus stem cell obtained through culture is guaranteed, the degree of differentiation is lowered, and thus the effect of treating corresponding eye diseases through transplanting the corneal limbus stem cell is improved.

Description

technical field [0001] The invention relates to the technical field of stem cells, in particular to a limbal stem cell and its culture method and application. Background technique [0002] The cornea consists of the epithelium, Descemet's membrane (Bowman's membrane), stroma, Descemet's membrane (Decemet's membrane) and the endothelium. The corneal epithelium has multiple layers of cells, including cells in the basal layer that connect with Bowman's membrane. The corneal epithelium has the functions of protecting the eyeball, stabilizing the tear film, and maintaining the transparency of the cornea. It is a constantly self-renewing tissue. Moreover, the maintenance of the integrity of the corneal epithelium depends on the continuous shedding of superficial cells and the continuous proliferation of basal layer cells. It is achieved by replacing exfoliated cells, and the continuous horizontal centripetal movement and vertical upward movement of epithelial cells originate from...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074A61K35/30A61P27/02
CPCA61K35/30C12N5/0623
Inventor 曾宪卓鲁菲
Owner SHEN ZHEN ISTEM REGENERATIVE MEDICINE SCI TECH CO LTD
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