Isolated culture method of human adipose-derived stem cells and construction method of stem cell bank

A human adipose-derived stem cell, isolation and culture technology, applied in animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the problem of low cell proliferation, unstable culture medium preservation, and inability to completely remove tissue blocks and miscellaneous cells, etc. question

Active Publication Date: 2014-03-26
GUIZHOU SHENQI PHARMA RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Chinese patent ZL201010120944.4 discloses a "method for obtaining human adipose adult stem cells and a method for constructing the stem cell bank", which facilitates the effective storage and use of adipose stem cell resources. In this patent, type I collagenase is used to digest adipose tissue , filter the digested product filter to remove undigested tissue pieces, resuspend the cells with DMEM complete culture medium after centrifugation, and finally inoculate into cell culture flasks for culture, change fresh culture medium after 4-5 days, digest and passage after the cells are full; China Patent application CN201210018269.3 has made further improvements to the existing cell separation method, using mixed collagenases of type I, type II and type IV for digestion. Although this method improves the digestion effect, it cannot completely remove the tissue In addition, when the cells are inoculated into culture flasks for culture, the culture medium used is not stable, the cell proliferation is low, and there is a certain degree of differentiation, resulting in the purity of the cells in the frozen stem cell bank still being affected

Method used

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  • Isolated culture method of human adipose-derived stem cells and construction method of stem cell bank
  • Isolated culture method of human adipose-derived stem cells and construction method of stem cell bank

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Embodiment 1

[0055] A method for isolating and culturing human adipose stem cells, comprising the steps of:

[0056] (1) Collection of human adipose tissue, which is discarded adipose tissue after abdominal fat resection or liposuction;

[0057] (2) Acquisition and separation of human adipose stem cells: take the collected adipose tissue, remove the blood vessels and fibers visible to the naked eye in the fat, and cut it into 1mm pieces 3 Large and small tissue pieces were washed with D-Hanks balanced salt solution with a pH value of 7.2-7.4 for several times until the eluate was clear, and residual blood was removed, and 0.2% (m / v) mixed collagenase ( The 1% mother liquor preparation method of this mixed collagenase is: add 0.7g type Ⅰ collagenase, 0.3g type Ⅳ collagenase) to 100ml D-Hanks balanced salt solution), shake and digest evenly at 37°C for 60 minutes, then place Centrifuge in a centrifuge at a speed of 1600 rpm for 60 minutes to remove the superficial fat, wash the underlying c...

Embodiment 2

[0061] A method for isolating and culturing human adipose stem cells, comprising the steps of:

[0062] (1) Collection of human adipose tissue, which is discarded adipose tissue after fat resection or liposuction of human thigh;

[0063] (2) Acquisition and isolation of human adipose stem cells: take the collected adipose tissue, remove the blood vessels and fibers visible to the naked eye in the fat, and cut it into 2mm 3 Large and small tissue pieces were washed with D-Hanks balanced salt solution with a pH value of 7.2-7.4 for several times until the eluate was clear, and residual blood was removed, and 0.3% (m / v) mixed collagenase ( The preparation method of the 1% mother liquor of this mixed collagenase is: add 0.7g type Ⅰ collagenase, 0.3g type Ⅳ collagenase) to 100ml D-Hanks balanced salt solution), shake and digest evenly at 37°C for 80 minutes, then place Centrifuge in a centrifuge at a speed of 1800 rpm for 4 minutes to remove the surface fat, wash the bottom cells ...

Embodiment 3

[0067] A method for isolating and culturing human adipose stem cells, comprising the following steps:

[0068] (1) Collection of human adipose tissue, which is discarded adipose tissue after human abdomen, thigh, or subcutaneous fat resection or liposuction;

[0069] (2) Acquisition and separation of human adipose stem cells: take the collected adipose tissue, remove the blood vessels and fibers visible to the naked eye in the fat, and cut it into 1mm pieces 3 Large and small tissue pieces were washed with D-Hanks balanced salt solution with a pH value of 7.2-7.4 for several times until the eluate was clear, and residual blood was removed, and 0.4% (m / v) collagenase ( The 1% mother liquor preparation method of this mixed collagenase is: add 0.7g type Ⅰ collagenase, 0.3g type Ⅳ collagenase) to 100ml D-Hanks balanced salt solution), shake and digest evenly at 37°C for 40 minutes, then place Centrifuge in a centrifuge at a speed of 1600 rpm for 10 minutes to remove the surface f...

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Abstract

The invention discloses an isolated culture method of human adipose-derived stem cells and a construction method of a stem cell bank. The isolated culture method comprises the following steps of: (1) collecting a human adipose tissue; (2) obtaining and separating the human adipose-derived stem cells; (3) culturing the stem cells; (4) detecting and cryopreserving; and (5) constructing the stem cell bank. According to the invention, the mixed collagenase prepared from D-Hanks balanced salt solution and containing type I and type VI collagenases is employed to digest the adipose tissue so that the tissue is digested more thoroughly, the number of parenchyma cells is obviously reduced and tissue blocks are removed; the MCDB-201 culture solution containing 10-15% of fetal calf serum and 10<3>-10<5>U / Ml LIF (Leukemia Inhibitory Factor) is used for culturing the isolated stem cells so that the cells proliferate quickly and are good in morphology; the differentiation of the stem cells can be effectively inhibited and the characteristics of the primary stem cells are ensured; and in the meantime, the human adipose-derived stem cells can be promoted to grow for a long time, and to keep the features such as self-renewal and multipotential differentiation; and moreover, the obtained stem cells are saved in the bank constructed so that better-quality seed cell source is guaranteed.

Description

technical field [0001] The invention relates to a method for separating and culturing human fat stem cells and a method for constructing a stem cell bank, belonging to the technical fields of medicine and biology. Background technique [0002] Stem cell research is one of the most striking hotspots in the fields of medicine and biology in recent years. Stem cells are a class of cells with the potential for self-renewal and differentiation. By studying their differentiation potential, expansion ability, differentiated cell function and whether the materials are safe and convenient to select the most suitable source of stem cells for tissue cell regeneration and repair. Human adipose-derived stem cells (ADSCs) are a kind of stem cells isolated from human adipose tissue in recent years with multipotential differentiation potential. Studies have found that adipose-derived stem cells can differentiate into osteoblasts, chondrocytes, cardiomyocytes, adipocytes, nerve cells, etc....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775G06F19/28
Inventor 张芝庭
Owner GUIZHOU SHENQI PHARMA RES INST
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