Method for amplifying in vitro mesenchymal stem cells

A bone marrow mesenchymal and stem cell technology, applied in the field of cell expansion, can solve the problems of ineffective reduction of amputation rate, strong immunogenicity, insufficient tissue source immune rejection, etc., to solve tumorigenicity and sensitization problem, the effect of high amplification efficiency

Inactive Publication Date: 2009-04-22
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Diabetic foot is one of the most serious complications of diabetes. In the past, taking drugs was a common way to treat diabetic foot, but the effect of taking drugs is often not good, and can not effectively reduce the amputation rate; in recent years, stem cell transplantation has initially seen curative effect , but faced with two major problems of insufficient tissue source and immune rejection
Stem cell research has brought new hope to diabetic foot patients. Among them, adult bone marrow mesenchymal stem cells have the strongest multidirectional differentiation potential and are most suitable for treating diabetic foot, a mixed lesion including neurovascular diseases. The proliferating cells are often tumorigenic and highly immunogenic, which limit their clinical application

Method used

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  • Method for amplifying in vitro mesenchymal stem cells
  • Method for amplifying in vitro mesenchymal stem cells
  • Method for amplifying in vitro mesenchymal stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0025] (1) Extract 10ml of bone marrow with a 20ml sterile syringe filled with 2ml of normal saline containing 500u / ml heparin. Shake well and inject into a 50ml sterile centrifuge tube. After diluting with an equal amount of normal saline solution containing 20u / ml heparin, slowly drip along the tube wall and superimpose an equal volume of Ficoll human lymphocyte separation medium with a specific gravity of 1.077g / ml, centrifuge at 2000r / min for 30min, and use First suck out and discard the uppermost layer of lipid with a capillary pipette, then suck out about 20ml of 50% serum, add 80ml of DMEM to make a total of 100ml of medium containing 10% serum from the same subject as the bone marrow, filter, and freeze at -20°C (for the last 5 days of culture), then extend the capillary pipette into the mononuclear cell layer (the milky white cloudy buffy coat layer between the cell separation solution and the plasma), and gently suck out all the cells along the tube wall (the remainin...

Embodiment 2

[0031] (1) Observing the quantity and the shape of the cells obtained after the amplification of Example 1: figure 1 It is a shape diagram of cells obtained after amplification in Example 1. from figure 1 It can be seen that the cells are arranged in a spiral or parallel arrangement, and the cells are in the shape of a long spindle. It shows that the morphology of cells cultured by this method conforms to the morphological characteristics of human bone marrow mesenchymal stem cells.

[0032] (2) The characteristics of the surface antigens CD105+, CD44+, and CD34- of the cells obtained after amplification in Example 1 were detected by flow cytometry. figure 2 It is the surface antigen CD105+ characteristic map of the cells obtained after the amplification of the flow detection embodiment 1; image 3 It is the surface antigen CD44+ characteristic map of the cells obtained after the amplification of the flow detection embodiment 1; Figure 4 It is the surface antigen CD34- c...

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Abstract

The present invention relates to cell amplification, in particular to a method for amplifying in vitro bone marrow mesenchymal stem cells. The amplification method for in vitro bone marrow mesenchymal stem cells comprises the following steps:firstly taking bone marrow, and separating and collecting bone marrow mesenchymal stem cell; secondly uniformly and suspendedly mixing the collected cells, and then inoculating and culturing; thirdly, digesting and collecting the cells when the cells reach between 85 and 95 percent of fusion, and then subculturing; fourthly, after 16 days and cultivation of 3 generations, discarding the fetal calf serum culture medium; and fifthly, washing with PBS, and then adding the culture medium which contains 10 percent of serum from the same main body as the bone marrow for culture for 3 days; and then digesting by pancreatin and stopping the digestion by serum. Compared with the prior art, the method has a higher amplification efficiency, and the problems of oncogenesis and sensitization existed in the in-vitro culture of MSC are effectively solved.

Description

technical field [0001] The invention relates to cell expansion, in particular to an in vitro bone marrow mesenchymal stem cell expansion method. Background technique [0002] my country is the second country with a high incidence of diabetes after India. Diabetic foot is one of the most serious complications of diabetes. In the past, taking drugs was a common way to treat diabetic foot, but the effect of taking drugs is often not good, and cannot effectively reduce the amputation rate; in recent years, stem cell transplantation has initially seen curative effect on diabetic foot , but faced with two major problems of insufficient tissue sources and immune rejection. Stem cell research has brought new hope to diabetic foot patients. Among them, adult bone marrow mesenchymal stem cells have the strongest multidirectional differentiation potential and are most suitable for treating diabetic foot, a mixed lesion including neurovascular diseases. The proliferating cells are oft...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C12N5/08C12N5/0775
Inventor 陈兵陆德宾梁自文姜友昭李晓艳张忠辉
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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