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Limbus corneae stem cell serum-free culture medium

A serum-free medium, limbal stem cell technology, applied in non-embryonic pluripotent stem cells, tissue culture, animal cells, etc.

Inactive Publication Date: 2008-02-13
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, there is no serum-free medium suitable for long-term expansion and culture of undifferentiated limbal stem cells at home and abroad.

Method used

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  • Limbus corneae stem cell serum-free culture medium
  • Limbus corneae stem cell serum-free culture medium
  • Limbus corneae stem cell serum-free culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[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...

Embodiment 2

[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.

Embodiment 3

[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.

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Abstract

The invention discloses a limbal stem cell serum-free medium, consisting of the following raw materials (calculated by weight percent): DMEM / F12 60ml-90ml, BSA 0.5g-4g, EGF 1Mug-4Mug, insulin 0.1mg-1mg, hydrogen 20ug-100ug, cholera toxin 1ug-10ug, transferrin 0.1mg-1mg, selenious acid 0.1ug-1ug, GCLCM 10ml-40ml, penicillin 5*103IU-2*104IU and streptomycin 5*103IU-2*104IU. The invention adopts the method that the culture medium is added with fibroblast conditioned medium, and the nutritive factors are supplemented with the hormone and cytokine combination, fibroblast conditioned medium and BSA. As a result, the nutritive needs for long-time in-vitro expansion and cultivation of the limbal stem cell serum-free medium are satisfied, and the medium is serum-free and suitable for commercial production.

Description

technical field [0001] The invention relates to the field of in vitro culture medium for stem cells, in particular to a serum-free culture medium for limbal stem cells and an application thereof. Background technique [0002] Cell culture is an important link in the production of genetically engineered products. Because the traditional serum-containing cell culture will bring many unfavorable factors to production and scientific research, since the 1950s, foreign countries have begun to develop serum-free medium. At the end of the 1980s and the beginning of the 1990s, serum-free media with commercial application value were launched one after another. One of the main characteristics of serum-free media is that the applicable cell lineage is narrow, and it is necessary to explore new formulas or optimal conditions for specific cell lines. . Limbal stem cells are the source of corneal cell proliferation and renewal. Studying the proliferation and differentiation of limbal ste...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C12N5/074
Inventor 弥胜利窦忠英
Owner NORTHWEST A & F UNIV
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