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Human autologous adipose mesenchymal stem cell serum-free culture method

A serum-free culture and mesenchymal stem cell technology, applied in the field of serum-free culture of human autologous adipose-derived mesenchymal stem cells, can solve the problems of no clear safety application specifications, residual animal-derived components, and limited cell yield, so as to promote adherence and proliferation, high reproducibility, and high cell yield

Inactive Publication Date: 2020-02-28
赛瑞诺(北京)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In terms of current research on adipose stem cells, most of the primary isolation methods are one-step enzymatic hydrolysis, and the cell yield is limited; most of the culture methods are serum culture methods, and adipose-derived mesenchymal stem cells are used as seed cells in clinical transformation. 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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  • Human autologous adipose mesenchymal stem cell serum-free culture method
  • Human autologous adipose mesenchymal stem cell serum-free culture method
  • Human autologous adipose mesenchymal stem cell serum-free culture method

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Embodiment 1

[0037] This embodiment provides a serum-free culture method for human autologous adipose-derived mesenchymal stem cells, which specifically includes the following steps:

[0038] (1) Digestion solution: 0.1% type I collagenase, 1.5% bovine serum albumin and 0.5mg / mL deoxyribonuclease I;

[0039] Configure serum-free medium: α-MEM medium contains 2% platelet lysate, 10ng / ml vascular endothelial growth factor, 10ng / ml recombinant basic fibroblast growth factor, 5ng / ml platelet-derived growth factor dimer and 2mmol / L L-glutamine;

[0040] (2) Wash 50ml of adipose tissue twice with Duchenne's phosphate buffer solution, centrifuge at 1500rpm for 12min, then perform sterility test, and take aseptic adipose tissue for later use;

[0041] (3) Digest the sterile adipose tissue at 36°C for 45 minutes using a digestion solution twice the volume of the sterile adipose tissue, centrifuge at 1400 rpm for 10 minutes after stopping digestion, and obtain cell pellets and undigested adipose ti...

Embodiment 2

[0047] This embodiment provides a serum-free culture method for human autologous adipose-derived mesenchymal stem cells, which specifically includes the following steps:

[0048] (1) Digestion solution: 0.2% type I collagenase, 2.5% bovine serum albumin and 0.5mg / mL deoxyribonuclease I;

[0049] Configure serum-free medium: α-MEM medium contains 2% platelet lysate, 10ng / ml vascular endothelial growth factor, 10ng / ml recombinant basic fibroblast growth factor, 5ng / ml platelet-derived growth factor dimer and 2mmol / L L-glutamine;

[0050] (2) 50ml of adipose tissue was washed 3 times with Duchenne's phosphate buffer, centrifuged at 1800rpm for 8min, then sterility test was carried out, and sterile adipose tissue was taken for later use;

[0051] (3) Digest the sterile adipose tissue at 38°C for 35 minutes using a digestion solution whose volume is three times that of the sterile adipose tissue, centrifuge at a speed of 1600 rpm for 6 minutes after stopping digestion, and obtain ...

Embodiment 3

[0057] This embodiment provides a serum-free culture method for human autologous adipose-derived mesenchymal stem cells, which specifically includes the following steps:

[0058] (1) Digestion solution: 0.15% type I collagenase, 2% bovine serum albumin and 0.5 mg / mL deoxyribonuclease I;

[0059] Configure serum-free medium: α-MEM medium contains 2% platelet lysate, 10ng / ml vascular endothelial growth factor, 10ng / ml recombinant basic fibroblast growth factor, 5ng / ml platelet-derived growth factor dimer and 2mmol / L L-glutamine;

[0060] (2) Wash 50 ml of adipose tissue three times with Duchenne's phosphate buffer solution, centrifuge at 1650 rpm for 10 min, then perform sterility test, and take aseptic adipose tissue for later use;

[0061] (3) Digest the sterile adipose tissue at 37°C for 40 minutes using a digestion solution whose volume is 3 times that of the sterile adipose tissue, centrifuge at a speed of 1500 rpm for 8 minutes after stopping digestion, and obtain cell pe...

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Abstract

The invention relates to a human autologous adipose mesenchymal stem cell serum-free culture method which particularly includes the steps: selecting sterile adipose tissues and performing first digestion to obtain first cell suspension; performing second digestion on undigested adipose tissues to obtain second cell suspension; combining the first cell suspension and the second cell suspension andthen performing culture, passage and identification to obtain human autologous adipose mesenchymal stem cells. According to the adipose mesenchymal stem cell culture method, primary cell separation, multiplication culture and cryopreservation systems are serum-free culture media, and the risk of animal derived ingredient addition is completely avoided. The method is a two-step enzymic digestion method, by the aid of the specific proportion of digestive enzyme and the adipose tissues, the adipose tissues sufficiently contact with enzyme solution and can be sufficiently digested, the cells cannot be damaged, primary cells are high in yield, good in activity, high in repeatability and good in safety, and the method meets clinical application standards.

Description

technical field [0001] The invention belongs to the technical field of stem cell culture, and in particular relates to a serum-free culture method for human autologous adipose-derived mesenchymal stem cells. Background technique [0002] Since Zuk et al. successfully isolated stem cells from adipose tissue in 2001 and named them adipose-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells have received great attention. Zuk et al. found that they can be isolated from adipose tissue obtained from liposuction Developed a kind of programmed lipoaspirate (PLA) cells, which exhibit stable growth characteristics in vitro culture, and can differentiate into various cell directions in a specific environment, have the characteristics of stem cells, and are similar to bone marrow Compared with mesenchymal stem cells, adipose stem cells have convenient sources, mainly from human abdomen, thighs and arms, etc. They are huge in number, safe and easy to obtain, and a larg...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0667C12N2500/32C12N2500/80C12N2500/90C12N2501/115C12N2501/135C12N2501/165
Inventor 李哲浩
Owner 赛瑞诺(北京)生物科技有限公司
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