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Separation and culture method of skin mesenchymal stem cells

A technology of skin stem cells and mesenchymal stem cells, which is applied in the field of separation and cultivation of skin mesenchymal stem cells, can solve the problems of long cycle, low recurrence rate, instability, etc., achieve low cost, clear ingredients, and reduce production costs.

Inactive Publication Date: 2017-03-22
WEIFANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The existing mesenchymal stem cell culture method has a long cycle, is unstable, and has a low reproducibility rate, and is suitable for the culture of skin mesenchymal stem cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] A method for isolating and culturing skin mesenchymal stem cells, comprising the following steps:

[0027] 1) Obtaining of skin tissue: The skin tissue is normal skin surgically excised in a professional hospital or beauty salon, divided into 3cm 2 Put each part of the sample into PBS solvent, add 2% antibiotics, and store at 4°C under sterile conditions;

[0028] 2) Digest the skin tissue: add 0.05% trypsin solution containing 0.02% EDTA to the PBS solvent, digest at 37°C for 20 minutes, and finally, add medium containing 10% FBS to terminate the digestion;

[0029] 3) Obtaining skin stem cells: repeatedly blowing and beating the tissue block to separate the cells, filtering with a 40 μm pore size filter to remove impurities, and finally obtaining skin stem cells;

[0030] 4) Mesenchymal stem cell culture: the basic medium is DMEM / F12, and add 90U / ml penicillin, 90mg / streptomycin, 2mmol / L glutamine, 4mmol / L iron citrate, 4mmol / L nicotinamide, 30ng / ml bFGF, 40ng / mlEG...

Embodiment 2

[0032] A method for isolating and culturing skin mesenchymal stem cells, comprising the following steps:

[0033] 1) Obtaining of skin tissue: The skin tissue is normal skin surgically removed by a professional hospital or beauty salon, divided into 2cm 2 Put each part of sample into PBS solvent, add 2% antibiotics, and store at 5°C under sterile conditions;

[0034] 2) Digest the skin tissue: add 0.05% trypsin solution containing 0.02% EDTA to the PBS solvent, digest at 37°C for 15 minutes, and finally, add medium containing 10% FBS to terminate the digestion;

[0035] 3) Obtaining skin stem cells: repeatedly blowing and beating the tissue block to separate the cells, filtering through a 60 μm pore size filter to remove impurities, and finally obtaining skin stem cells;

[0036] 4) Mesenchymal stem cell culture: the basic medium is DMEM / F12, and 100U / ml penicillin, 100mg / ml streptomycin, 2mmol / L glutamine, 2mmol / L ferric citrate, 2mmol / L nicotinamide, 20ng / ml bFGF, 40ng / mlE...

Embodiment 3

[0038] A method for isolating and culturing skin mesenchymal stem cells, comprising the following steps:

[0039] 1) Obtaining of skin tissue: The skin tissue is normal skin surgically excised in a professional hospital or beauty salon, divided into 3cm 2 Put each part of the sample into PBS solvent, add 2% antibiotics, and store at 4°C under sterile conditions;

[0040] 2) Digest the skin tissue: add 0.05% trypsin solution containing 0.02% EDTA to the PBS solvent, digest at 37°C for 20 minutes, and finally, add medium containing 10% FBS to terminate the digestion;

[0041] 3) Obtaining skin stem cells: repeatedly blowing and beating the tissue block to separate the cells, filtering with a 40 μm pore size filter to remove impurities, and finally obtaining skin stem cells;

[0042] 4) Mesenchymal stem cell culture: the basic medium is DMEM / F12, and 5% fetal bovine serum is added, 100U / ml penicillin, 100mg / ml streptomycin, 2mmol / L glutamine, 5mmol / L iron citrate , nicotinamide 5...

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Abstract

The invention discloses a separation and culture method of skin mesenchymal stem cells. The separation and culture method comprises the following steps that (1) skin tissues are obtained; (2) the skin tissues are digested, specifically, a 0.05% of trypsin solution containing 0.02% of EDTA is added in a PBS solvent, digestion is conducted for 10-20 min at the temperature of 37 DEG C, and finally, a culture medium containing 10% of FBS is added to finish digestion; (3) skin stem cells are obtained; and (4) the mesenchymal stem cells are cultured, specifically, a basal culture medium is DMEM / F12, and penicillin, streptomycin, glutamine, iron citrate, nicotinamide, bFGF (basal fibroblast growth factors), EGF (epidermal growth factors) and B27 (antigen) additives are added, so that a culture solution is formed. According to the separation and culture method, separation is easy, more cells with higher activity can be obtained, and the repetition rate is quite high.

Description

technical field [0001] The invention relates to the field of cell separation and culture, in particular to a method for separation and culture of skin mesenchymal stem cells. Background technique [0002] Stem cells are a kind of primitive cells with self-renewal ability and multi-lineage differentiation potential under suitable microenvironment. Stem cells can be divided into embryonic stem cells, pluripotent stem cells, and committed stem cells in various tissues such as hematopoietic stem cells, neural stem cells, etc., according to the sequence in which they appear during individual development. At present, there have been experimental reports on the use of stem cells to treat myocardial infarction, lupus erythematosus, rheumatoid arthritis, nerve damage, and muscle atrophy. [0003] Human stem cells are a kind of superpotent stem cell population with multilineage differentiation potential existing in human tissues, which can be induced to differentiate into various tis...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0668C12N2500/24C12N2500/30C12N2501/00C12N2501/11C12N2501/115C12N2509/00
Inventor 马晓君
Owner WEIFANG MEDICAL UNIV
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