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High-efficiency preparation method of human skin tissue pluripotent stem cells

A pluripotent stem cell and skin technology, applied in the field of cell engineering, can solve the problems of low cell yield and low differentiation efficiency, and achieve the effect of strong stem cell activity, high differentiation efficiency, and maintaining stem cell properties.

Inactive Publication Date: 2017-06-23
安徽安龙基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve the defects of low cell yield and low differentiation efficiency in the prior art, and provide a high-efficiency preparation method of human skin tissue pluripotent stem cells to solve the above problems

Method used

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preparation example Construction

[0034] The invention discloses a highly efficient preparation method of human skin tissue pluripotent stem cells, the specific steps are:

[0035] 1. Coated bottle preparation:

[0036] (1) Dissolving human type IV collagen and diluting it to 0.1g / L with PBS to make a coating solution;

[0037] (2) Take 5ml of the coating solution and add it to the culture bottle, lay it flat, and put it in a refrigerator at 4°C overnight;

[0038] (3), discard the supernatant, and place it in an ultra-clean workbench to dry;

[0039] (4), the coated bottle is stored in a 4°C refrigerator for later use;

[0040] 2. Preparation of skin stem cells:

[0041] (5) Take cheek or chin skin tissue and remove fat tissue;

[0042] (6), cut the skin into small pieces of 0.5mm×0.5mm, rinse with PBS three times;

[0043] (7), the tissue block is placed in a sterile plate, with the dermis facing down, and digested overnight at 4°C with a digestion solution containing 1U / ml neutral protease II;

[0044...

Embodiment 1

[0059] The invention discloses a highly efficient preparation method of human skin tissue pluripotent stem cells, the specific steps are:

[0060] 1. Coated bottle preparation:

[0061] (1) Dissolving human type IV collagen and diluting it to 0.1g / L with PBS to make a coating solution;

[0062] (2) Take 5ml of the coating solution and add it to the culture bottle, lay it flat, and put it in a refrigerator at 4°C overnight;

[0063] (3), discard the supernatant, and place it in an ultra-clean workbench to dry;

[0064] (4), the coated bottle is stored in a 4°C refrigerator for later use;

[0065] 2. Preparation of skin stem cells:

[0066] (5), take cheek tissue, remove fat tissue;

[0067] (6), cut the skin into small pieces of 0.5mm×0.5mm, rinse with PBS three times;

[0068] (7), the tissue block is placed in a sterile plate, with the dermis facing down, and digested overnight at 4°C with a digestion solution containing 1U / ml neutral protease II;

[0069] (8), use opht...

Embodiment 2

[0084] The invention discloses a highly efficient preparation method of human skin tissue pluripotent stem cells, the specific steps are:

[0085] 1. Coated bottle preparation:

[0086] (1) Dissolving human type IV collagen and diluting it to 0.1g / L with PBS to make a coating solution;

[0087] (2) Take 5ml of the coating solution and add it to the culture bottle, lay it flat, and put it in a refrigerator at 4°C overnight;

[0088] (3), discard the supernatant, and place it in an ultra-clean workbench to dry;

[0089] (4), the coated bottle is stored in a 4°C refrigerator for later use;

[0090] 2. Preparation of skin stem cells:

[0091] (5), remove the skin tissue of the chin, and remove the fatty tissue;

[0092] (6), cut the skin into small pieces of 0.5mm×0.5mm, rinse with PBS three times;

[0093] (7), the tissue block is placed in a sterile plate, with the dermis facing down, and digested overnight at 4°C with a digestion solution containing 1U / ml neutral protease ...

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Abstract

The invention discloses a high-efficiency preparation method of human skin tissue pluripotent stem cells. The method comprises the following steps: preparation of coating bottle: putting a coating bottle in a 4-DEG C refrigerator, and storing for later use; and preparation of skin stem cells: taking a cheek or chin skin tissue, removing the fat tissue, putting the tissue block in a sterile plate, digesting over night, digesting corium layer small blocks, filtering the cell suspension by a filter, keeping the adherent cells, culturing in an incubator, repeatedly beating upon the bottle bottom by using a suction tube until the cells shed, carrying out bottle subculture on the cells according to the ratio of 1:3, and continuing culturing by 2-5 generations. The method has the advantages of high cell yield, high differentiation efficiency and the like.

Description

technical field [0001] The invention relates to the field of cell engineering, in particular to a highly efficient preparation method of human skin tissue pluripotent stem cells. Background technique [0002] Pluripotent stem cells derived from adult tissues are an important source in the field of tissue engineering and regenerative medicine. They are easy to obtain and have broad medical application prospects. Although embryonic pluripotent stem cells have great potential in the field of regenerative medicine, their clinical application is limited due to the ethical issues of xenogeneic cell transplantation. Induced pluripotent stem cells (iPS) are another potential source, but iPS cells are potentially oncogenic, making their clinical application difficult. [0003] Epidermal stem cells are skin tissue-specific stem cells with potential pluripotency. It is derived from the ectoderm of the embryo, has relatively primitive differentiation ability, has the characteristics o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074
CPCC12N5/0625C12N2509/00
Inventor 韦玉军陆宝石李航吴远航苏军
Owner 安徽安龙基因科技有限公司
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