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Method for isolated culture of adipose-derived stem cells

An adipose stem cell, separation and culture technology, applied in cell dissociation methods, cell culture active agents, tissue culture, etc., can solve the problems of low cell viability and low total number of adipose stem cells, and achieve the effect of large number and strong vitality

Active Publication Date: 2018-11-02
吉林省太阳鸟再生医学工程有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the total number of adipose stem cells obtained by this method is small, and the cell viability is low

Method used

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  • Method for isolated culture of adipose-derived stem cells
  • Method for isolated culture of adipose-derived stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0039] The isolation and culture method of adipose-derived stem cells in this embodiment is as follows:

[0040] 1. Let the adipose tissue containing interstitial fluid stand still, and after the adipose tissue and the liquid are naturally stratified, suck up the liquid below with a 10mL pipette; use a 50mL syringe to absorb an equal volume of PBS and add it to the adipose tissue, cover the bottle cap and shake the fat repeatedly tissue, let it stand for 5 minutes; use a 25mL pipette to discard the solution under the adipose tissue (repeat once).

[0041] 2. Transfer the adipose tissue to a 50mL centrifuge tube with a disposable pipette; add an equal volume of type I collagenase and type III collagenase mixture (0.08% and 0.02% by mass respectively), and turn the bottle upside down after sealing Shake to mix well. Transfer to a constant temperature air bath shaker at 37°C, and digest at 200R for 50min.

[0042] 3. Add 1mL FBS to each tube, cap the bottle and shake well, cent...

Embodiment 2

[0047] The isolation and culture method of adipose-derived stem cells in this embodiment is as follows:

[0048] 1. Let the adipose tissue containing interstitial fluid stand still, and after the adipose tissue and the liquid are naturally stratified, suck up the liquid below with a 10mL pipette; use a 50mL syringe to absorb an equal volume of PBS and add it to the adipose tissue, cover the bottle cap and shake the fat repeatedly tissue, let it stand for 5 minutes; use a 25mL pipette to discard the solution under the adipose tissue (repeat once).

[0049]2. Transfer the adipose tissue to a 50mL centrifuge tube with a disposable pipette; add an equal volume of type I collagenase and type III collagenase mixture (0.09% and 0.01% by mass respectively), and turn the bottle upside down after sealing Shake to mix well. Transfer to a constant temperature air bath shaker at 37°C, and digest at 100R for 30min.

[0050] 3. Add 1mL FBS to each tube, cap the bottle and shake well, centr...

Embodiment 3

[0055] The isolation and culture method of adipose-derived stem cells in this embodiment is as follows:

[0056] 1. Let the adipose tissue containing interstitial fluid stand still, and after the adipose tissue and the liquid are naturally stratified, suck up the liquid below with a 10mL pipette; use a 50mL syringe to absorb an equal volume of PBS and add it to the adipose tissue, cover the bottle cap and shake the fat repeatedly tissue, let it stand for 5 minutes; use a 25mL pipette to discard the solution under the adipose tissue (repeat once).

[0057] 2. Transfer the adipose tissue to a 50mL centrifuge tube with a disposable pipette; add an equal volume of type I collagenase and type III collagenase mixture (0.07% and 0.03% by mass respectively), and turn the bottle upside down after sealing Shake to mix well. Transfer to a constant temperature air bath shaker at 37°C, and digest at 250R for 60min.

[0058] 3. Add 1mL FBS to each tube, cap the bottle and shake well, cent...

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Abstract

The invention relates to the technical field of cell culture, in particular to a method for isolated culture of adipose-derived stem cells. The isolated culture method comprises the following steps: enabling adipose tissues containing tissue fluid to stand, layering the adipose tissues, collecting upper adipose tissues, and washing the adipose tissues with a PBS solution; digesting the washed adipose tissues with a solution containing type I collagenase and type III collagenase; after digestion, adding FBS, shaking well, carrying out centrifugation, and discarding supernatant to obtain adipose-derived stem cells; inoculating the adipose-derived stem cells into a high-sugar DMEM medium containing FBS for cell culture; and after culturing for 40-50 h, replacing the DMEM medium containing EGF, bFGF and FBS to continue culturing. According to the method provided by the invention, type I collagenase and type III collagenase are adopted to digest adipose tissues, so that the adipose tissuescan be fully digested while cell vitality is maintained to ensure that adipose-derived stem cells can be dissociated, the number of obtained adipose-derived stem cells is large, and the vitality is strong.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for separating and culturing fat stem cells. Background technique [0002] Adipose-derived stem cells (ADSCs) are a kind of stem cells isolated from adipose tissue in recent years with multipotential differentiation potential. Due to defects such as immune rejection and inflammatory response, traditional fat transplantation methods are difficult to obtain satisfactory curative effect. According to statistics, after autologous adipose tissue is transplanted to the defect site, usually 40% to 60% of it will be absorbed. Using stem cells in the patient's own adipose tissue to construct adipose tissue with complete biological structure and functional engineering will undoubtedly be the best solution to this problem. How to achieve the differentiation from stem cells to adipocytes is an unavoidable problem in the construction of engineered adipose tissue. As early as ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0667C12N2501/11C12N2501/115C12N2509/00
Inventor 李首一石晓川
Owner 吉林省太阳鸟再生医学工程有限责任公司
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