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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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  • 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 generation of serum-free medium. However, the existing commercially available serum-free medium has unstable chemical components, it is difficult to ensure the consistency of medium batches, the cell proliferation rate is not high, and Expensive and other issues
In addition, after the mesenchymal stem cells have been cultured for 5 to 6 passages, the cell morphology is prone to change, and some cells will appear aging.

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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Effect test

Embodiment 1

[0055]Embodiment 1, the special culture medium of human adipose-derived mesenchymal stem cells

[0056] The special medium for human adipose-derived mesenchymal stem cells of the present invention is based on a-MEM medium, and 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, fiber 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 concentration of PDGF in the special medium is 5ng / ml, the concentra...

Embodiment 2

[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 fat extract from the liposuction operation, wash it several times with PBS (GIBCO, catalog number C10010500BT), remove the drugs and blood cells used in the liposuction operation, and obtain adipose tissue.

[0060] 2. Cut up the adipose tissue obtained in step 1, collect it in a 50ml centrifuge tube, add an equal volume of PBS to shake vigorously, leave it at room temperature after shaking until layers appear in the centrifuge tube, and collect the upper layer.

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

Embodiment 3

[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 in the medium-I culture in step 2 of Example 2, and add 0.25% (v / v) Trypsin and 0.04% (v / v) EDTA for digestion, and then collect the cells, take 1 × 10 6 Transfer cells into a flow tube, wash twice with PBS, then resuspend with PBS, add the following labeled antibodies: CD90, CD45, HLA-DR and CD59, incubate at 4°C for 30 min, and wash twice with PBS , the labeled cells were analyzed with BD's FACSCanto II flow cytometer to detect the expression of surface markers CD90, CD45, HLA-DR and CD59. And no antibody was used as negative control.

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

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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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