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A method for isolating and culturing human adipose-derived stem cells and a method for constructing a stem cell bank

A technology for the isolation and cultivation of human adipose stem cells, which is applied to animal cells, vertebrate cells, bone/connective tissue cells, etc., and can solve problems such as low cell proliferation, inability to completely remove miscellaneous cells in tissue blocks, and influence on cell purity

Active Publication Date: 2016-05-18
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

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  • A method for isolating and culturing human adipose-derived stem cells and a method for constructing a stem cell bank
  • A method for isolating and culturing human adipose-derived stem cells and a method for constructing a stem cell bank

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Embodiment 1

[0055] A method for separating and culturing human adipose stem cells includes the following steps:

[0056] (1) Collect human adipose tissue, which is adipose tissue discarded after human abdominal liposuction or liposuction;

[0057] (2) Obtain and separate human adipose stem cells: take the collected adipose tissue, remove the visible blood vessels and fibers in the fat, and cut it into 1mm 3 Large and small tissue pieces, wash several times with D-Hanks balanced salt solution with a pH value of 7.2-7.4 until the eluate is clear and remove residual blood. Add 0.2% (m / v) mixed collagenase equal to the volume of adipose tissue ( The 1% mother liquor of the mixed collagenase is prepared as follows: 100ml D-Hanks balanced salt solution is added 0.7g type I collagenase and 0.3 g type IV collagenase), shake and digest for 60 minutes at 37°C, then centrifuge Centrifuge at 1600 rpm for 60 minutes in the machine to remove the surface fat. The bottom cells are repeatedly washed with D-Han...

Embodiment 2

[0061] A method for separating and culturing human adipose stem cells includes the following steps:

[0062] (1) Collect human adipose tissue, which is the discarded adipose tissue after liposuction or liposuction;

[0063] (2) Obtain and separate human adipose stem cells: take the collected adipose tissue, remove the visible blood vessels and fibers in the fat, and cut them into 2mm 3 Large and small tissue pieces, wash several times with D-Hanks balanced salt solution with a pH value of 7.2-7.4 until the eluate is clear and remove residual blood. Add 0.3% (m / v) mixed collagenase equal to the volume of adipose tissue ( The 1% mother liquor of the mixed collagenase is prepared as follows: 100ml D-Hanks balanced salt solution is added 0.7g type I collagenase, 0.3g type IV collagenase), shake and digest for 80 minutes at 37°C, then centrifuge Centrifuge at 1800 rpm in the machine for 4 minutes to remove the surface fat. The bottom cells are repeatedly washed with D-Hanks balanced sal...

Embodiment 3

[0067] A method for separating and culturing human adipose stem cells includes the following steps:

[0068] (1) Collect human adipose tissue, which is the adipose tissue discarded after liposuction or liposuction in the abdomen, thigh, or subcutaneous liposuction;

[0069] (2) Obtain and separate human adipose stem cells: take the collected adipose tissue, remove the visible blood vessels and fibers in the fat, and cut it into 1mm 3 Large and small tissue pieces, wash several times with D-Hanks balanced salt solution with a pH value of 7.2-7.4 until the eluate is clear and remove residual blood. Add 0.4% (m / v) mixed collagenase equal to the volume of adipose tissue ( The 1% mother liquor of the mixed collagenase is prepared as follows: 100ml D-Hanks balanced salt solution is added 0.7g type I collagenase, 0.3g type IV collagenase), shake and digest at 37°C for 40 minutes, then centrifuge Centrifuge in the machine at 1600 rpm for 10 minutes to remove the surface fat. The bottom cel...

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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 cultivating human adipose 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 noticeable hotspots in the fields of medicine and biology in recent years. Stem cells are a type of cells with self-renewal and differentiation potential. By studying their differentiation potential, expansion ability, cell function after differentiation, and whether it is safe and convenient to obtain materials, select the most suitable source of stem cells for tissue cell regeneration and repair. Human adipose-derived stem cells (adipose-derivedstemcells, ADSCs) are a kind of stem cells with multidirectional differentiation potential isolated from human adipose tissue in recent years. Studies have found that adipose-derived stem cells can differentiate into osteoblasts, chondrocytes, cardiomyo...

Claims

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Application Information

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