Acquisition method of adipose-derived stem cells

A technique for obtaining fat stem cells, which is applied to animal cells, vertebrate cells, bone/connective tissue cells, etc., and can solve the problems of insufficient cell purity and insufficient thorough digestion of cells by type I collagenase

Active Publication Date: 2012-05-09
中源协和生物细胞存储服务(天津)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] During the process of cell extraction and culture, we found that there are some problems in the traditional cell separation method, such as the incomplete digestion of cells with type I collagenase, and there will be many undigested tissue pieces; when cells are inoculated into culture flasks, P0 There are also more tissue pieces and a large number of miscellaneous cells in the cultured cells of the P2 and P1 passages, resulting in insufficient cell purity during the cryopreservation of the P2~P3 passages

Method used

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  • Acquisition method of adipose-derived stem cells
  • Acquisition method of adipose-derived stem cells
  • Acquisition method of adipose-derived stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment -1

[0034] (1) Take 50mL of adipose tissue, wash with D-Hank's balanced salt solution with a pH value of 7.2-7.4, and repeatedly centrifuge to remove excess aqueous solution and blood;

[0035] Add 0.4% (m / v) collagenase I equal to the volume of the adipose tissue to digest, and digest evenly under the temperature condition of 37oC for 60 minutes, then place it in a centrifuge and centrifuge at a speed of 2000 rpm for 5 minutes. Remove superficial fat. The bottom cells were blown repeatedly with D-Hank's balanced salt solution with a pH value of 7.2-7.4, and cleaned; the cleaned liquid was filtered through a 100-mesh sieve to remove undigested tissue; when the tissue was filtered, it could be seen that there were many incomplete lumps presence of digestive tissue;

[0036] The filtrate was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded to obtain SVF cells.

[0037] (2) Add the obtained SVF cells to 10 mL of high-glucose DMEM culture medium containing 10...

Embodiment -2

[0042] (1) Take 50mL of adipose tissue, wash with D-Hank's balanced salt solution with a pH value of 7.2-7.4, and repeatedly centrifuge to remove excess aqueous solution and blood;

[0043] Add 0.3% (m / v) mixed collagenase equal to the volume of the extracted adipose tissue for digestion, shake and digest evenly at 37oC for 60 minutes, then place in a centrifuge and centrifuge at 2000 rpm for 5 minutes. Remove superficial fat. The bottom cells were blown repeatedly with D-Hank's balanced salt solution with a pH value of 7.2-7.4, and cleaned; the cleaned liquid was filtered through a 100-mesh sieve to remove undigested tissue; when the tissue was filtered, it could be seen that there were many incomplete lumps presence of digestive tissue;

[0044] The filtrate was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded to obtain SVF cells.

[0045] (2) Add the obtained SVF cells to 10 mL of high-glucose DMEM culture medium containing 10% fetal bovine serum, ...

Embodiment -3

[0050] (1) Take 50mL of adipose tissue, wash with D-Hank's balanced salt solution with a pH value of 7.2-7.4, and repeatedly centrifuge to remove excess aqueous solution and blood;

[0051] Add 0.4% (m / v) mixed collagenase equal to the volume of the adipose tissue obtained for digestion, and digest evenly at a temperature of 37oC for 60 minutes, then place it in a centrifuge and centrifuge at a speed of 2000 rpm for 5 minutes. Remove superficial fat. The bottom cells were blown repeatedly with D-Hank's balanced salt solution with a pH value of 7.2-7.4, and cleaned; the cleaned liquid was filtered through a 100-mesh sieve to remove undigested tissues; incompletely digested tissue blocks were visible during tissue filtration. Significantly decreased in Case-1 and -2;

[0052] The filtrate was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded to obtain SVF cells.

[0053] (2) Add the obtained SVF cells to 10 mL of high-glucose DMEM culture medium containi...

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Abstract

The invention relates to an acquisition method of adipose-derived stem cells. The method comprises the following specific steps of: digesting adipose tissues by use of mixed collagenase; culturing adipose-derived stem cells obtained through digestion in a culture solution; carrying out solution replacement and transfer of culture; and detecting to obtain the qualified adipose-derived stem cells. According to the invention, the problems that in the traditional cell separation method, cells can not be thoroughly digested by collagenase and the cell purity is low can be solved; and a separation method of adipose-derived stem cells is improved and optimized, so that tissues can be digested more thoroughly, and the purity during a culture process is higher, thus a more excellent seed cell source is ensured.

Description

technical field [0001] The invention relates to a method for obtaining 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. Studies have found that adipose-derived stem cells can differentiate into osteoblasts, chondrocytes, cardiomyocytes, adipocytes, nerve cells, etc. under specific conditions, and have low immunogenicity and immunoregulatory functions. Transplantation of allogeneic ADSCs will not It causes a strong immune response and later rejection, which provides favorable conditions for allogeneic transplantation of ADSCs. In addition, ADSCs have a wide range of sources and can be obtained from any human body through liposuction or fat resection. It is safe and painless, and it is stable in vitro culture, fast in expansion, and not easy to age. Therefore, ADSCs have become a new hotspot in current research, and have great a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 刘广洋刘拥军朱德琳聂艳波王斌
Owner 中源协和生物细胞存储服务(天津)有限公司
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