Human adipose derived stromal cell separation and culture method

A technique for separating and culturing mesenchymal stem cells, which is applied in the field of separating and culturing human adipose-derived mesenchymal stem cells, can solve problems such as cumbersome operation steps, pollution, and easy aging of cells, and achieve simplification of operation procedures, reduction of cytotoxicity, and reduction of pollution risks Effect

Inactive Publication Date: 2019-05-24
湖南南华生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional separation and cultivation method is to mix the fat with digestive enzymes and then put it into a shaker for digestion or add some active additives, which increases the cost of the separation process, causes mechanical damage to the cells, and the cumbersome operation steps also increase the risk of contamination; In addition, this separation method leads to risks such as poor quality of isolated cells, long culture period, and easy aging of cells; moreover, there is a risk of animal-derived contamination in the expansion culture with a culture system containing animal-derived components

Method used

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  • Human adipose derived stromal cell separation and culture method
  • Human adipose derived stromal cell separation and culture method
  • Human adipose derived stromal cell separation and culture method

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Embodiment 1

[0038] A method for isolating and culturing human adipose-derived mesenchymal stem cells, comprising step 1, collecting fat; step 2, removing impurities from fat; step 3, digesting with mixed enzymes; step 4, collecting and centrifuging; step 5, culturing and passage; step 6, digesting cryopreservation;

[0039] Wherein in above-mentioned step one, collecting fat comprises the following steps:

[0040] 1) Collect 2-100ml of human-derived fat, which can be abdominal fat mass, thigh fat mass, or fat suspension from liposuction, and put it into a sterile storage and transportation solution. The temperature for storage and transportation is 4~ 20°C, within 12 to 24 hours after collection, human-derived fat is transported to the laboratory for separation and processing;

[0041] 2) Soak the adipose tissue in 75% alcohol for 2-3 seconds, add 1-5 times the volume of normal saline to wash, transfer the fat to a centrifuge tube with hemostatic forceps after centrifugation, add 4-10 ti...

Embodiment 2

[0056] A method for isolating and culturing human adipose-derived mesenchymal stem cells, comprising the following steps:

[0057] 1) Collect 2-100ml of human-derived fat, which can be abdominal fat mass, thigh fat mass, or fat suspension from liposuction, and put it into a sterile storage and transportation solution. The temperature for storage and transportation is 4~ 20°C, within 12 to 24 hours after collection, human-derived fat is transported to the laboratory for separation and processing;

[0058] 2) Soak the adipose tissue in 75% alcohol for 2-3 seconds, add 1-5 times the volume of normal saline to wash, transfer the fat to a centrifuge tube with a hemostatic forceps after centrifugation, add 4-10 times the volume of normal saline, reverse the number Centrifuge once, repeat 2-3 times;

[0059] 3) Use liberase digestive enzyme to digest fat, digest it for 30 minutes, neutralize the digestion with medium containing 10% fetal bovine serum, and centrifuge;

[0060] 4) Se...

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Abstract

The invention discloses a human adipose derived stromal cell separation and culture method which includes the steps: firstly, fat collection; secondly, removing impurities in fat; thirdly, digestion by mixed enzyme; fourthly, collecting and centrifuging; fifthly, culture and passage; sixthly, digestion and freezing. According to the human adipose derived stromal cell separation and culture method,separation is implemented by the mixed enzyme, digestion time is short, acquired cells cannot be further purified by a lymphocyte separation medium and a filter screen, and liquid needs to be changedafter the cells fits with wall. According to the method, a separation process is low in cost, used reagents and materials are less in separation, operation processes are simplified, pollution risks in the operation process are reduced, animal source serums are omitted in the whole separation and culture process, cell toxicity is reduced, and a culture period is short by the aid of the mixed enzyme and a serum-free culture system.

Description

technical field [0001] The invention relates to the technical field of adipose-derived mesenchymal stem cells, in particular to a method for separating and culturing human adipose-derived mesenchymal stem cells. Background technique [0002] Adipose-derived mesenchymal stem cells, also known as adipose-derived stem cells, are a type of adult stem cells. Studies have shown that human adipose-derived, umbilical cord-derived, placenta-derived, and bone marrow-derived mesenchymal stem cells have self-renewal and multi-directional differentiation potentials, and can differentiate into osteocytes, chondrocytes, adipocytes, and epithelial cells under certain induction culture conditions. Cells, etc., and has an immunomodulatory effect. The ability of adipose-derived mesenchymal stem cells to differentiate into adipocytes is stronger than that of other tissue-derived mesenchymal stem cells. Adipose-derived mesenchymal stem cells have significant advantages in beauty and anti-aging....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 谭科芳姚青
Owner 湖南南华生物技术有限公司
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