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Primary isolation method of skin mesenchymal stem cells

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

Active Publication Date: 2019-01-18
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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  • 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 isolation method of skin mesenchymal stem cells. The method comprises: after skin tissue is pretreated, digested with DispaseII disperse enzyme, washed, repeatedly blown to separate cells, filtered and centrifuged to obtain cell precipitates which are resuspended in Lonza complete medium and then inoculated into a culture dish for 60-90min; The unattached cell suspension was inoculated into fibronectin-coated culture plates, and the cells were passaged when the cell confluence reached 80% - 90%. A large numb of skin mesenchymal stem cells can be isolate by using that primary isolation method of the invention, and the cell membrane of the skin mesenchymal stem cells isolated and cultured is not damage, the cell culture is stable, and mycoplasma pollution is not introduced, the cell vigor is good, and the skin mesenchymal stem cells can be stably cultured in the follow-up.

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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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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