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A primary isolation method of skin mesenchymal stem cells

A separation method and technology of mesenchymal stem cells, applied in the field of primary separation of skin mesenchymal stem cells, can solve the problems of cell membrane damage, mycoplasma pollution, instability, etc., and achieve the effect of good cell viability and stable cell culture

Active Publication Date: 2021-05-25
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Trypsin belongs to the proteolytic enzymes. Using this method to disperse tissues and separate cells will seriously damage the cell membrane, making it unstable during cell culture and even introducing mycoplasma contamination.

Method used

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  • A primary isolation method of skin mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1, Primary Separation of Skin Mesenchymal Stem Cells

[0030] 1. Spread 2ml of 20μg / ml fibronectin FN on a 10cm culture plate, incubate at 37°C for 30-60min, and set aside;

[0031] 2. Take the skin tissue (30mm×5mm), wash it several times with normal saline containing 1× double-antibody, and then wash once with 75% alcohol, and finally store it in glucose solution containing double-antibody at 4°C. Return to the laboratory and operate within 24 hours;

[0032] 3. Cut the skin tissue into a 3-5mm rectangle, wash it with sodium chloride injection, then wash it in 75% ethanol, and finally wash it again with sodium chloride injection;

[0033] 4. Cut the rectangle into small tissue pieces, put in 2U / ml Dispase II for digestion at 37°C for 1 hour;

[0034] After 5.1h, take out the tissue block and wash it with sodium chloride injection;

[0035] 6. Repeatedly blow the tissue block to separate the cells, and pass through a 100μm disposable cell sieve to remove imp...

Embodiment 2

[0040] Example 2, Primary Separation of Skin Mesenchymal Stem Cells

[0041] 1. Spread 2ml of 10μg / ml fibronectin FN on a 10cm culture plate, incubate at 37°C for 30-60min, and set aside;

[0042] 2. Take the skin tissue (30mm×5mm), wash it several times with normal saline containing 1× double-antibody, and then wash once with 75% alcohol, and finally store it in glucose solution containing double-antibody at 4°C. Return to the laboratory and operate within 24 hours;

[0043] 3. Cut the skin tissue into a 3-5mm rectangle, wash it with sodium chloride injection, then wash it in 75% ethanol, and finally wash it again with sodium chloride injection;

[0044] 4. Cut the rectangle into small tissue pieces, put in 0.5U / ml Dispase II for digestion at 37°C for 1 hour;

[0045] After 5.1h, take out the tissue block and wash it with sodium chloride injection;

[0046] 6. Repeatedly blow the tissue block to separate the cells, and pass through a 100μm disposable cell sieve to remove i...

Embodiment 3

[0051] Example 3, Primary Separation of Skin Mesenchymal Stem Cells

[0052] 1. Spread 2ml of 50μg / ml fibronectin FN on a 10cm culture plate, incubate at 37°C for 30-60min, and set aside;

[0053] 2. Take the skin tissue (30mm×5mm), wash it several times with normal saline containing 1× double-antibody, and then wash once with 75% alcohol, and finally store it in glucose solution containing double-antibody at 4°C. Return to the laboratory and operate within 24 hours;

[0054] 3. Cut the skin tissue into a 3-5mm rectangle, wash it with sodium chloride injection, then wash it in 75% ethanol, and finally wash it again with sodium chloride injection;

[0055] 4. Cut the rectangle into small tissue pieces, put in 2.5U / ml Dispase II for digestion at 37°C for 1 hour;

[0056] After 5.1h, take out the tissue block and wash it with sodium chloride injection;

[0057] 6. Repeatedly blow the tissue block to separate the cells, and pass through a 100μm disposable cell sieve to remove i...

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Abstract

The invention belongs to the field of stem cells, and discloses a primary separation method of skin mesenchymal stem cells. The skin tissue is pretreated, digested with Dispase II, washed and blown repeatedly to separate the cells from the tissue block, filtered and centrifuged. The obtained cell pellet was resuspended with Lonza complete medium and inoculated in a culture dish for 60-90 minutes; the non-adhered cell suspension was inoculated into a fibronectin-coated culture plate and cultured until the cell confluency reached 80%~ Passage after 90%. The cell membrane of skin mesenchymal stem cells isolated and cultured by the primary separation method of the present invention will not be damaged, the cell culture is stable, and mycoplasma pollution will not be introduced, a large number of skin mesenchymal stem cells can be separated, and the cell viability is good, which can Subsequent stable cultivation.

Description

technical field [0001] The invention belongs to the field of stem cells, and in particular relates to a primary separation method 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. 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 tissue cells under specif...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0668C12N2509/00
Inventor 王一飞陈海佳葛啸虎张梦晨王小燕
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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