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Primary isolation culture method of adipose-derived stem cells

A technology for the separation and cultivation of adipose stem cells, which is applied in the direction of animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the problems of unsatisfactory separation and culture methods of ADSCs, slow proliferation speed, and cytotoxicity, and achieve favorable scale Chemical production and preparation, stable enzymatic hydrolysis conditions, and high cell viability

Active Publication Date: 2015-04-29
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, mechanical methods are still used to remove blood vessels, single enzyme digestion, lysing red blood cells with lysate, and flow cytometry to sort stem cells, etc., to obtain a relatively pure cell population of ADSCs as much as possible; add penicillin-streptomycin during culture Antibiotics such as antibiotics, although they have a certain antibacterial effect, they also cause varying degrees of toxicity to cells.
The whole process of isolation and culture of adipose stem cells is complicated, time-consuming and labor-intensive, and the production cost doubles
In addition, after actual operation, it was found that the effect was not very ideal. Due to the use of more chemical reagents, ADSCs had low vigor and slow proliferation after isolation; and the cell shape varied greatly.
Use low-concentration, single enzyme solution to enzymatically hydrolyze adipose tissue. Incomplete enzymatic hydrolysis leads to inability to fully release adipose stem cells
Overall, methods for isolating and culturing ADSCs are unsatisfactory

Method used

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  • Primary isolation culture method of adipose-derived stem cells
  • Primary isolation culture method of adipose-derived stem cells
  • Primary isolation culture method of adipose-derived stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] 1) Let the adipose tissue containing interstitial fluid stand still, and after the adipose tissue and the liquid are naturally separated, suck up the liquid below with a 10mL pipette; use a 50mL syringe to draw an equal volume of PBS and add it to the adipose tissue, cover the bottle cap and shake the adipose tissue repeatedly tissue, let it stand for 5 minutes; use a 25mL pipette to discard the solution under the adipose tissue (repeat once).

[0028] 2) Transfer the adipose tissue to a 50mL centrifuge tube with a disposable pipette; add an equal volume of type I collagenase and trypsin mixture (0.25% and 0.05% by mass), and shake the bottle upside down after sealing. Mix well. Transfer to a constant temperature air bath shaker at 37°C, digest at 200R for 50min

[0029] 3) Add 1 mL of FBS to each tube, cap the bottle and shake well, centrifuge at 1500rpm / min for 5min; discard the supernatant after centrifugation.

[0030] 4) According to the ratio of fat volume: 10% ...

Embodiment 2

[0034] 1) Let the adipose tissue containing interstitial fluid stand still, and after the adipose tissue and the liquid are naturally separated, suck up the liquid below with a 10mL pipette; use a 50mL syringe to draw an equal volume of PBS and add it to the adipose tissue, cover the bottle cap and shake the adipose tissue repeatedly tissue, let it stand for 5 minutes; use a 25mL pipette to discard the solution under the adipose tissue (repeat once).

[0035] 2) Transfer the adipose tissue to a 50mL centrifuge tube with a disposable pipette; add an equal volume of type I collagenase and trypsin mixture (0.05% and 0.1% by mass), and shake the bottle upside down after sealing. Mix well. Transfer to a constant temperature air bath shaker at 37°C, digest at 100R for 30min

[0036] 3) Add 1 mL of FBS to each tube, cap the bottle and shake well, centrifuge at 1000rpm / min for 3min; discard the supernatant after centrifugation.

[0037] 4) According to the ratio of fat volume: 10% F...

Embodiment 3

[0041] 1) Let the adipose tissue containing interstitial fluid stand still, and after the adipose tissue and the liquid are naturally separated, suck up the liquid below with a 10mL pipette; use a 50mL syringe to draw an equal volume of PBS and add it to the adipose tissue, cover the bottle cap and shake the adipose tissue repeatedly tissue, let it stand for 5 minutes; use a 25mL pipette to discard the solution under the adipose tissue (repeat once).

[0042] 2) Transfer the adipose tissue to a 50mL centrifuge tube with a disposable pipette; add an equal volume of type I collagenase and trypsin mixture (0.3% and 0.01% by mass), and shake the bottle upside down after sealing. Mix well. Transfer to a constant temperature air bath shaker at 37°C, digest at 250R for 60min

[0043] 3) Add 1 mL of FBS to each tube, cap the bottle and shake well, centrifuge at 1500rpm / min for 10min; discard the supernatant after centrifugation.

[0044] 4) According to the ratio of fat volume: 10% ...

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Abstract

The invention discloses a primary isolation culture method of adipose-derived stem cells. The primary isolation culture method comprises the following steps: (1) washing adipose tissues; (2) adding a mixed liquid of type-I collagenase and trypsase into the adipose tissues, wherein the volume of the mixed liquid is equal to the volumes of the adipose tissues; (3) adding FBS, covering a bottle cap, sealing, uniformly shaking, carrying out centrifugation, and discarding supernate after the centrifugation; (4) carrying out resuspension precipitation by virtue of a DMEM containing the FBS, uniformly blow-hitting to form cell suspension, and inoculating the cell suspension to a culture medium, wherein the volume ratio of precipitates to the DMEM containing the FBS is 1 to 1; (5) culturing the inoculated culture medium; and (6) transferring the cultured culturing medium into a cell incubator, and continuing to culture the culturing medium. Enzymolysis conditions explored in the primary isolation culture method are stable and efficient, the complete enzymolysis of the adipose tissues is achieved, and cells of each component are furthest released; on the premise that the activity of ADSCs is not influenced, the quantity of the ADSCs acquired in unit adipose volume is relatively stable.

Description

technical field [0001] The invention relates to a method for isolating and culturing stem cells, in particular to a method for primary isolating and culturing adipose stem cells. Background technique [0002] Human adipose-derived stem cells (Adipose-Derived Stem Cells, ADSCs) are a kind of stem cells isolated from human adipose tissue in recent years with multipotential differentiation potential. ADSCs can not only differentiate into adipose, cartilage, cardiomyocytes and other cells, but also secrete a variety of angiogenesis-promoting factors, apoptosis factors and other cytokines with biological activity. In addition, ADSCs also have the advantages of easy extraction, large amount of acquisition, and less pain in donor extraction. It has been widely used in the fields of tissue engineering and regenerative medicine. [0003] The in vitro isolation and culture of human adipose-derived stem cells has always been a difficult problem. In the methods of the prior art, impr...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 王一飞陈海佳葛啸虎卢瑞珊马岩岩王小燕
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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