Culture medium used for culturing adipose mesenchymal stem cells, and applications thereof

A technology of basal medium and serum-free medium, which is applied in the direction of culture process, tissue culture, animal cells, etc. It can solve the problems of easy change of cell morphology, cell aging, difficulty in ensuring the consistency of medium batches, etc.

Inactive Publication Date: 2017-10-20
北京康爱瑞浩细胞技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, animal serum contains growth-promoting active ingredients and growth-inhibiting ingredients, and it may carry bacteria, viruses, and protein infectious diseases; in addition, the protein content in serum is high, and the components are complex. There are differences between batches in the application of FBS, which requires a lot of verification work
In view of this, some companies have developed a new gene

Method used

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  • Culture medium used for culturing adipose mesenchymal stem cells, and applications thereof
  • Culture medium used for culturing adipose mesenchymal stem cells, and applications thereof

Examples

Experimental program
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Example Embodiment

[0055]Example 1. Special culture medium for human adipose-derived mesenchymal stem cells

[0056] The special medium for human adipose-derived mesenchymal stem cells of the present invention is a-MEM medium as basic medium, a-MEM medium, recombinant human insulin, human serum albumin, transferrin, fibronectin, ascorbic acid , biotin, PDGF (platelet-derived growth factor), bFGF (basic fibroblast growth factor) and TGF-β (transforming growth factor-β) mixed culture medium. Among them, the concentration of recombinant human insulin in the special medium is 5 μg / ml; the concentration of human serum albumin in the special medium is 0.5 mg / ml, the concentration of transferrin in the special medium is 5 μg / ml, and the concentration of human serum albumin in the special medium is 5 μg / ml. The concentration of connexin in the special medium is 5ng / ml, the concentration of ascorbic acid in the special medium is 5μg / ml, the concentration of biotin in the special medium is 3μg / ml, and the...

Example Embodiment

[0057] Example 2. Primary isolation and culture of human adipose-derived mesenchymal stem cells and detection of proliferation ability

[0058] 1. Primary isolation of human adipose-derived mesenchymal stem cells

[0059] 1. Aseptically collect the liposuction fluid for liposuction, wash with PBS (GIBCO, catalog number C10010500BT) for several times, remove the drugs and blood cells used in liposuction, and obtain adipose tissue.

[0060] 2. Cut the adipose tissue obtained in step 1 into pieces, collect them in a 50ml centrifuge tube, add an equal volume of PBS, and shake vigorously. After shaking, let stand at room temperature until layers appear in the centrifuge tube, and collect the upper part.

[0061] 3. Wash the upper part collected in step 2 with PBS, add an equal volume of 0.1% collagenase type I (Sigma company, catalog number 9001-12-1) to digest at 37°C for 1 hour, centrifuge at 400g / min for 5min, suck up The upper layer of fat was mixed, and then filtered through ...

Example Embodiment

[0073] Example 3. Detection of biological characteristics of human adipose-derived mesenchymal stem cells

[0074] Take the P1 generation and P15 generation human adipose-derived mesenchymal stem cells obtained by culturing the medium-I in the second step of Example 2, and add 0.25% (v / v) Trypsin and 0.04% (v / v) Digest with EDTA, then collect cells, take 1×10 6 The cells were transferred to a flow tube, washed twice with PBS and then resuspended in PBS. The following labeled antibodies were added: CD90, CD45, HLA-DR and CD59, incubated at 4°C for 30 min, and washed twice with PBS. , and the labeled cells were analyzed by FACSCanto II flow cytometer of BD Company to detect the expression of surface markers CD90, CD45, HLA-DR and CD59. And no antibody was used as a negative control.

[0075] The result is as figure 2 shown ( figure 2 P1-control and P15-control in both refer to negative control). It can be seen from the figure: when the human adipose-derived mesenchymal s...

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Abstract

The invention discloses a culture medium used for culturing adipose mesenchymal stem cells, and applications thereof. The culture medium comprises insulin human, human serum albumin, transferring, fibronectin, ascorbic acid, biotin, PDGF, bFGF, and TGF-beta. It is confirmed by experiments that culturing with the culture medium is capable of obtaining a large amount of high quality mesenchymal stem cells, and the mesenchymal stem cells possess excellent stem cell characteristics, higher immunosuppression activity, and low immunogenicity, and are more suitable to be used for allogeneic cell therapy. The potential risk of clinical applications of exogenous animal serum is avoided, cell pollution rate is reduced, proliferation of human adipose mesenchymal stem cells is promoted, aging speed of human adipose mesenchymal stem cells in in-vitro culture is reduced. Requirements on large scale industrialized production of adipose mesenchymal stem cells needed by clinical therapy are satisfied, and problems such as unstable properties of different batches and high cost are solved at the same time.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a culture medium for culturing adipose-derived mesenchymal stem cells and an application thereof. Background technique [0002] Mesenchymal stem cells (mesenchymal stromal / stem cells, MSCs) are a kind of pluripotent stem cells derived from the mesoderm in the early stage of development, and are widely found in various body tissues such as "bone marrow", "fat", "umbilical cord" and "placenta". , "amnion", etc., and has self-renewal ability and multi-directional differentiation potential, as well as unique cytokine secretion function. Under different induction conditions, it can differentiate into bone, cartilage, fat, muscle, tendon, ligament, nerve, liver, cardiac muscle and other tissue cells, and it still has multi-directional differentiation potential after continuous subculture and freezing. Experimental studies have found that compared with bone marrow mesenchymal s...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0667C12N2500/25C12N2500/38C12N2500/90C12N2501/115C12N2501/135C12N2501/15C12N2501/998
Inventor 卢戌刘静维王跃刘雪松黄彩庭吴璇
Owner 北京康爱瑞浩细胞技术有限公司
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