Culture method for efficiently obtaining adipose mesenchymal stem cells

A technology of mesenchymal stem cells and culture methods, which is applied in the field of high-efficiency acquisition of adipose-derived mesenchymal stem cells, can solve problems such as lack of clear safety application specifications, influence on cell activity, and stem cell restrictions, and achieve multi-directional differentiation ability, high activity rate, and The effect of less cell damage

Inactive Publication Date: 2016-12-14
中卫华医(北京)生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In terms of research on adipose stem cells, there are no reports on the extraction, expansion, cell activity and biological functions of adipose stem cells derived from different parts of the same donor.
And most of the culture methods use a single enzyme digestion method, the cell yield is low, the digestion time is long, and the cell activity is affected, and most of them are culture methods with serum. In clinical transformation, adipose-derived mesenchymal stem cells are used as seed cells in clinical applications. There is a big bottleneck, and there are many problems to be solved: such as the residue of animal-derived components, the introduction of foreign viruses in the cell culture system, and there are many controversies in terms of cell number and active biological functions, and there is no clear safety application specification, which makes stem cells It is very limited in clinical application

Method used

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  • Culture method for efficiently obtaining adipose mesenchymal stem cells
  • Culture method for efficiently obtaining adipose mesenchymal stem cells
  • Culture method for efficiently obtaining adipose mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1. The culture method for efficiently obtaining adipose-derived mesenchymal stem cells of the present invention is the isolation, culture, and cryopreservation of primary human autologous adipose-derived mesenchymal stem cells. The steps are as follows:

[0052] (1) Extraction of adipose tissue: In a regular medical beauty salon, use special liposuction equipment to extract 20ml of human fat under aseptic conditions, immediately put it into a sterile ziplock bag, seal it, and put it into a special incubator for adipose tissue, 4 degrees Conditions within 8-24 hours, sent to the laboratory.

[0053] (2) Separation of adipose-derived mesenchymal stem cells: open the sample storage device under sterile conditions, wash the adipose tissue derived from liposuction with D.Hanks, wash away blood cells and a small amount of mixed fibrous connective tissue, and at the same time apply the Plate detection to ensure that the fat is not contaminated before the experiment....

Embodiment 1-2

[0056] Example 1-2, the culture method for efficiently obtaining adipose-derived mesenchymal stem cells of the present invention differs from the above-mentioned Example 1-1 only in that the enzymatic hydrolysis is stopped after digestion at 37°C for 30 minutes; by 1.2×10 4 / cm 2 The density was inoculated into the blood-free medium of the culture bottle; when the cells were cultured to 80% confluency, they were digested with trypLETM, and inoculated into new culture bottles at a seeding ratio of 1:4 for culture.

Embodiment 1-3

[0057] Example 1-3, the culture method for efficiently obtaining adipose-derived mesenchymal stem cells of the present invention differs from the above-mentioned Example 1-1 only in that the enzymatic hydrolysis is stopped after digestion at 37°C for 40 minutes; 4 / cm 2 The density was inoculated into the blood-free culture medium of the culture bottle; when the cells were cultured to 85% confluency, they were digested with trypLETM, and inoculated into a new culture bottle for culture according to the seeding ratio of 1:5.

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Abstract

The invention relates to a culture method for efficiently obtaining adipose mesenchymal stem cells, and solves the problem that the cell viability obtained in the prior art is low. The culture method comprises the following steps: separation of the adipose mesenchymal stem cells: washing D.Hanks with fat tissues, detecting to guarantee no pollution, adding a solution mixture of I-type collagenase and trypLETM digestive enzyme with several times of volume, and stopping enzymolysis after carrying out rotary vibration digestion for dozens of minutes at the temperature of 37 DEG C, centrifuging to remove a liquid supernatant, and filtering with screen cloth to obtain an adipose mesenchymal stem cell suspension; culture of the adipose mesenchymal stem cells: inoculating a culture bottle bloodless culture medium with the adipose mesenchymal stem cell suspension, carrying out primary cell culture, observing with an inverted phase contrast microscope in the culture period, and changing a fresh culture medium, when the cells are cultured to 80% to 90% fusion, extensively inoculating a new culture bottle with the cells for culture after digestion with trypLETM, and subculturing for several generations to obtain the purified mesenchymal stem cells. The culture method has the advantages that the digestion is more sufficient, the cell yield is higher, the mesenchymal stem cells are protected from being damaged, and the viability is high.

Description

technical field [0001] The invention relates to a method for obtaining adipose-derived mesenchymal stem cells, in particular to a culture method for efficiently obtaining adipose-derived mesenchymal stem cells. Background technique [0002] Adipose tissue exists in large quantities in the human body, is easy to obtain, and has a strong ability to proliferate. After losing it, it will have little impact on the recipient. There is no immune response to the recipient, and more and more people are actively requesting liposuction. Therefore, adipose-derived mesenchymal stem cells have very broad prospects as a new source of adult stem cells. Especially for cells derived from mesoderm, ADSC has a similar differentiation ability to bone marrow mesenchymal stem cells, and can differentiate into fat, bone, cartilage, nerve and other cells, and the isolation and culture of mesenchymal cells from adipose tissue is compared with that from bone marrow. It is easier to achieve separation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0667C12N2509/00C12N2509/10
Inventor 王晶李亚平谷广其邱丽媛赵进军何文丽
Owner 中卫华医(北京)生物科技有限公司
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