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Method for separating human adipose-derived stem cells from human adipose tissues

A human adipose stem cell and adipose stem cell technology, which is applied in the field of cell engineering, can solve the problems of low purity and activity of human adipose stem cell products, unclean separation, low purity, etc., and achieves favorable differentiation induction, reduced content of heterocytic cells, and good activity. Effect

Active Publication Date: 2015-02-18
深圳市赛欧细胞技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The present invention aims at the problems of unclean separation and low purity in the existing human adipose stem cell separation technology, which affect the differentiation induction of human adipose stem cells, resulting in low yield, purity and activity of human adipose stem cell products, by optimizing the steps of cleaning human adipose tissue and collagenase nitration step to provide a method for effectively isolating human adipose stem cells from human adipose tissue, so that the growth state of the passaged cells is good, the yield of human adipose stem cells is high, and the process cost is low

Method used

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  • Method for separating human adipose-derived stem cells from human adipose tissues
  • Method for separating human adipose-derived stem cells from human adipose tissues
  • Method for separating human adipose-derived stem cells from human adipose tissues

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Embodiment 1

[0034] This embodiment provides a method for isolating human adipose stem cells from human adipose tissue, and culturing the isolated human adipose stem cells. details as follows:

[0035] (1) Isolation of human adipose stem cells from human adipose tissue

[0036] Washing: 15 mL of human adipose tissue was taken, and the human adipose tissue was repeatedly washed three times with normal saline to remove blood in the human adipose tissue.

[0037] Collagenase digestion: put the washed human adipose tissue into a 50mL centrifuge tube, then add an equal volume of type Ⅰ collagenase solution to the centrifuge tube, and mix well. Put it in a constant temperature shaker, and digest at a centrifugation rate of 150r / min for 1h at 37°C. The liquid in the centrifuge tube is divided into three layers, the upper layer is the yellow oily fat cell layer, the middle layer is the fat tissue layer, and the lower layer is the liquid containing mononuclear cells.

[0038] Collection: suck ou...

Embodiment 2

[0053] This embodiment provides a method for isolating human adipose stem cells from human adipose tissue, and culturing the isolated human adipose stem cells. The specific operation is basically the same as that in Example 1, except that the centrifugation rate (150r / min) during collagenase digestion in step (1) is changed to 100r / min. Other operations and parameters are completely consistent with those of Example 1.

[0054] During the above culture process, the human adipose stem cells of P0, P1, and P2 generations were continuously observed.

[0055] After P0 generation human adipose stem cells were cultured for 48 hours, observed under an inverted microscope, no cells adhered to the wall, and after 72 hours, a very small amount of cells adhered to the wall, and a large number of miscellaneous cells were suspended in the serum-free medium. After a series of medium replacement, digestion and passage, the number of miscellaneous cells suspended in the serum-free medium of P...

Embodiment 3

[0057] This embodiment provides a method for isolating human adipose stem cells from human adipose tissue, and culturing the isolated human adipose stem cells. The specific operation is basically the same as that in Example 1, except that the centrifugation time (1 h) during collagenase digestion in step (1) is changed to 0.5 h. Other operations and parameters are completely consistent with those of Example 1.

[0058] During the above culture process, the human adipose stem cells of P0, P1, and P2 generations were continuously observed.

[0059] After P0 generation human adipose stem cells were cultured for 48 hours, observed under an inverted microscope, no cells adhered to the wall, and after 96 hours, a very small amount of cells adhered to the wall, and a large number of miscellaneous cells were suspended in the serum-free medium. After a series of medium replacement, digestion and passage, the number of miscellaneous cells suspended in the serum-free medium of P1 genera...

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Abstract

The invention relates to the field of cell engineering and particularly relates to a method for separating human adipose-derived stem cells from human adipose tissues. According to the method, by adding a small amount of low-molecular-weight heparin calcium into normal saline, a mixed solution is obtained and used as a cleaning fluid to clean the human adipose tissues, residual impurities such as red blood cells in the human adipose tissues can be well removed and thus human adipose-derived stem cells with relatively higher purity can be separated, the content of the parenchyma cell is significantly reduced, the differentiation induction of the human adipose-derived stem cells is facilitated, the consumption amounts of collagenase I, trypsase and a serum-free culture medium can be reduced and the cost of the process is also decreased. In addition, by centrifuging and digesting the human adipose tissues for 1 hour at a speed of 150r / min, the human adipose-derived stem cells with significantly better activity can be obtained.

Description

technical field [0001] The invention relates to the technical field of cell engineering, in particular to a method for isolating human adipose stem cells from human adipose tissue. Background technique [0002] In 2001, cell biologists used liposuction to isolate multidirectional stem cells from the aspirated human fat suspension for the first time, and found that they could differentiate into fat, bone, cartilage, muscle, vascular endothelium, Liver, pancreas, nerve and other cells; the adult stem cells with differentiation potential found in human adipose tissue are called Human Adipose Derived Mesnchymal Stem Cells (hADSCs). Compared with other stem cells, hADSCs have the following five significant advantages: (1) Abundant sources, easy to obtain materials. (2) It is easy to expand and not easy to age. Experiments have proved that hADSCs grow like fibroblasts after passage, and the cell growth rate does not slow down after multiple passages, that is, they can continue t...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 胡士庶张涤平崔兴日陈贤光
Owner 深圳市赛欧细胞技术有限公司
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