Method for obtaining a large number of adipose-derived mesenchymal stem cells from fat

A adipose stem cell and fat technology, which is applied in cell dissociation methods, animal cells, vertebrate cells, etc., can solve the problems of low cell viability, unsuitable for obtaining a large amount of adipose-derived mesenchymal stem cells, and low cell yield.

Active Publication Date: 2018-05-08
FIVE DIMENSION BY INCOSC HEALTH MANAGEMENT JIANGSU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0021] Although the prior art discloses some methods for culturing adipose-derived mesenchymal stem cells such as the above-mentioned ones, the inventors have found that these methods do not seem to be suitable for obt

Method used

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  • Method for obtaining a large number of adipose-derived mesenchymal stem cells from fat
  • Method for obtaining a large number of adipose-derived mesenchymal stem cells from fat

Examples

Experimental program
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Effect test

Embodiment 1

[0097] Example 1: Isolation and culture of adipose-derived mesenchymal stem cells

[0098] (1) Put the fat sample mixed with swelling fluid obtained by liposuction into a centrifuge tube, and centrifuge at 800rpm for 3min;

[0099] (2) Remove the top layer of fat, and pipette the suspended fat particles and the bottom cell sediment respectively, and set aside;

[0100] (31) Add mixed enzymes (0.05% collagenase type I+0.05% collagenase type II+0.03% neutral protease+0.03% DNase) with a volume ratio of 1:1 to the fat granules obtained in step (2), at 37 Centrifuge at 100rpm for 30min; after digestion, add an equal volume of complete medium to stop digestion, centrifuge at 1500rpm for 5min, discard the supernatant, and resuspend the cell pellet in PBS;

[0101] (32) Resuspend the bottom cell pellet obtained in step (2) with PBS, add an equal amount of erythrocyte lysate and let it stand for 10 minutes to lyse; after the erythrocytes are fully lysed, centrifuge at 1500 rpm for ...

Embodiment 2

[0109] Example 2: Isolation and culture of adipose-derived mesenchymal stem cells

[0110] (1) Put the fat sample mixed with swelling fluid obtained by liposuction into a centrifuge tube, and centrifuge at 500rpm for 5min;

[0111](2) Remove the top layer of fat, and pipette the suspended fat particles and the bottom cell sediment respectively, and set aside;

[0112] (31) Add mixed enzyme (0.05% collagenase type I+0.05% collagenase type II+0.03% neutral protease+0.03% DNase) with volume ratio 1:1.2 in the fat granule gained in step (2), at 37 Centrifuge at 100rpm for 20min; after digestion, add an equal volume of complete medium to stop digestion, centrifuge at 2000rpm for 3min, discard the supernatant, and resuspend the cell pellet in PBS;

[0113] (32) Resuspend the bottom cell pellet obtained in step (2) with PBS, add an equal amount of erythrocyte lysate and let it stand for 5 minutes to lyse; after the erythrocytes are fully lysed, centrifuge at 2000 rpm for 3 minutes...

Embodiment 3

[0120] Example 3: Isolation and culture of adipose-derived mesenchymal stem cells

[0121] (1) Put the fat sample mixed with swelling fluid obtained by liposuction into a centrifuge tube, and centrifuge at 1000rpm for 2min;

[0122] (2) Remove the top layer of fat, and pipette the suspended fat particles and the bottom cell sediment respectively, and set aside;

[0123] (31) Add mixed enzymes (0.05% collagenase type I+0.05% collagenase type II+0.03% neutral protease+0.03% DNase) with a volume ratio of 1:0.8 to the fat granules obtained in step (2), at 37 Centrifuge at 100rpm for 40 minutes; after digestion, add an equal volume of complete medium to stop digestion, centrifuge at 1000 for 8 minutes, discard the supernatant, and resuspend the cell pellet in PBS;

[0124] (32) Resuspend the bottom cell pellet obtained in step (2) with PBS, add an equal amount of erythrocyte lysate and let it stand for 15 minutes to lyse; after the erythrocytes are fully lysed, centrifuge at 100...

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Abstract

The invention relates to a method for obtaining a large number of adipose-derived mesenchymal stem cells from fat. On one hand, the invention relates to a method for isolating and culturing the adipose-derived mesenchymal stem cells. The method comprises the steps that fat samples doped with swelling fluid and obtained by liposuction are placed in a centrifuge tube, and centrifugation is conducted; the uppermost layer of fat is removed, and suspended fat particles and underlying cells are transferred and precipitated respectively for later use; mixed liquid of digestive enzyme is added in theobtained fat particles, shaking digestion is conducted, the digestion is terminated, and resuspension is conducted on cell precipitate; red blood cell lysis buffer is added to underlying cell precipitate, the underlying cell precipitate is allowed to stand, and splitting decomposition, centrifugation and resuspension of the cell precipitate are conducted; cell suspension is combined, and adipose-derived mesenchymal stem cells which serve as primary dipose-derived mesenchymal stem cells are isolated; culturing and reproduction are further conducted on the primary dipose-derived mesenchymal stemcells. The invention further relates to a adipose-derived mesenchymal stem cell preparation prepared by the method, the mixed liquid of the digestive enzyme for isolating the adipose-derived mesenchymal stem cells, and a culture medium for culturing the adipose-derived mesenchymal stem cells. Excellent technical effects as described in a description are shown in all aspects of the method for obtaining a large number of adipose-derived mesenchymal stem cells from the fat.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for obtaining adipose-derived mesenchymal stem cells, in particular to a method for culturing autologous adipose-derived mesenchymal stem cells. The present invention also relates to the medium used in the above-mentioned culture of autologous adipose-derived mesenchymal stem cells. When the culture medium of the present invention is used to culture the autologous adipose-derived mesenchymal stem cells, the excellent technical effect of the present invention can be exhibited. The obtained autologous adipose-derived mesenchymal stem cells can be used for autologous application. Background technique [0002] Mesenchymal stem cells (MCS) are hot spots of adult stem cells with multi-directional differentiation potential. It is a cell population with bone formation ability and hematopoietic function found in most adult organs. It can be extracted from bone marrow, umbilical cord b...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0667C12N2509/00
Inventor 王芳梅俊
Owner FIVE DIMENSION BY INCOSC HEALTH MANAGEMENT JIANGSU
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