Method for separating adipose-derived stem cells from fat and preparation in use

A technology of mesenchymal stem cells and fat, applied in the field of isolation and culture of autologous adipose-derived mesenchymal stem cells, can solve the problems of inapplicable acquisition of a large number of adipose-derived mesenchymal stem cells, low cell yield, and low cell viability

Active Publication Date: 2018-03-30
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 obtaining a large amount of adipose-derived mesenchymal stem cells from fat. For example, these methods are used in Low cell yield and / or low cell viability when harvesting adipose-derived mesenchymal stem cells from fat

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  • Method for separating adipose-derived stem cells from fat and preparation in use
  • Method for separating adipose-derived stem cells from fat and preparation in use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Example 1: Isolation and culture of adipose-derived mesenchymal stem cells

[0112] (1) Put the fat sample mixed with swelling fluid obtained by liposuction into a centrifuge tube, and centrifuge at 800rpm for 3min;

[0113](2) Remove the top layer of fat, and pipette the suspended fat particles and the bottom cell sediment respectively, and set aside;

[0114] (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 to the fat granule gained 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;

[0115] (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 5 minutes...

Embodiment 2

[0123] Example 2: Isolation and culture of adipose-derived mesenchymal stem cells

[0124] (1) Put the fat sample mixed with swelling fluid obtained by liposuction into a centrifuge tube, and centrifuge at 500rpm for 5min;

[0125] (2) Remove the top layer of fat, and pipette the suspended fat particles and the bottom cell sediment respectively, and set aside;

[0126] (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;

[0127] (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 minute...

Embodiment 3

[0134] Example 3: Isolation and culture of adipose-derived mesenchymal stem cells

[0135] (1) Put the fat sample mixed with swelling fluid obtained by liposuction into a centrifuge tube, and centrifuge at 1000rpm for 2min;

[0136] (2) Remove the top layer of fat, and pipette the suspended fat particles and the bottom cell sediment respectively, and set aside;

[0137] (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;

[0138] (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 discloses a method for separating adipose-derived stem cells from fat and a preparation in use. On one hand, the method for separating the adipose-derived stem cells from fat comprises the steps of conducting liposuction to obtain a fat sample doped with inflation liquid to be subjected to centrifugation; removing the uppermost-layer fat, and removing suspension fat particles and bottom-layer cell sediment respectively; adding the fat particles into digestive enzyme mixed liquid, conducting vibration digestion, stopping the digestion, conducting centrifugation, and re-suspendingcell sediment; re-suspending the cell sediment at the bottom layer, adding erythrocyte pyrolysis liquid to conduct pyrolysis, conducting centrifugal, and re-suspending the cell sediment; merging to obtain cell suspension, filtering, and separating out the adipose-derived stem cells serving as primary passage. The invention further relates to a method for separating and cultivating the adipose-derived stem cells. The prepared adipose-derived stem cell preparations are used for separating the digestive enzyme mixed liquid of the adipose-derived stem cells, and are used for cultivating a culturemedium of the adipose-derived stem cells. The method for separating the adipose-derived stem cells from fat has various advantages as shown in the description.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for isolating adipose mesenchymal stem cells from fat, in particular to a method for isolating and culturing autologous adipose mesenchymal stem cells. The present invention also relates to the culture medium and related test solution used in the above-mentioned culture of autologous adipose-derived mesenchymal stem cells. When the method of the present invention is used to isolate and culture the adipose-derived mesenchymal stem cells, the excellent technical effect of the present invention can be presented. 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0667C12N2501/105C12N2501/11C12N2501/115C12N2509/00C12N2501/15
Inventor 王芳梅俊
Owner FIVE DIMENSION BY INCOSC HEALTH MANAGEMENT JIANGSU
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