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Method applicable to serum-free culture of mesenchymal stem cells

A technology of serum-free culture and serum-free medium, which is applied in the biological field, can solve the problems of heavy workload, incomplete FN, and high price, and achieve good self-renewal dryness, maintain self-renewal dryness, and promote proliferating effect

Inactive Publication Date: 2014-06-25
FUZHOU GENERAL HOSPITAL OF NANJING MILITARY COMMAND P L A
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of the existing solutions are: 1) The need to pre-coat the petri dish is heavy, and it is not suitable for large-scale cultivation
2) Need to use special protein such as fibronectin FN, which is expensive, about 1 million RMB per gram
3) The recombined FN is only a fragment and does not fully have the functions of all FN
Therefore, the serum-free medium based on this is expensive and inconvenient to use, making its popularity very low

Method used

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  • Method applicable to serum-free culture of mesenchymal stem cells
  • Method applicable to serum-free culture of mesenchymal stem cells
  • Method applicable to serum-free culture of mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Stimulating serum-free cultured bone marrow mesenchymal stem cells to secrete endogenous fibronectin

[0032] Steps:

[0033] 1) Bone marrow mesenchymal stem cells were divided into 5×10 4 Cells were seeded into 6-well plates.

[0034] 2) Add 2ml of medium 1 (control group, serum-free basal medium αMEM) and medium 2 (thrombin group, add thrombin 4 IU / ml to serum-free basal medium) respectively. 37°C, CO 2 The concentration was 5% for 24 h in a cell culture incubator.

[0035] 3) Collect the culture supernatant to measure the amount of fibronectin FN secreted by mesenchymal stem cells by ELISA method.

[0036] 4) The cells were collected to extract total cellular RNA, and real-time quantitative PCR was used to detect the expression of fibronectin mRNA in mesenchymal stem cells.

[0037] Results: After thrombin stimulation, the expression of fibronectin mRNA and the secretion of endogenous fibronectin in mesenchymal stem cells increased more than 6 times....

Embodiment 2

[0038] Example 2: Adhesion effect of serum-free cultured mesenchymal stem cells

[0039] Steps:

[0040] 1) Culture mesenchymal stem cells in medium 1 and (control group, serum-free basal medium αMEM) medium 2 (thrombin group, add thrombin 4 IU / ml to serum-free basal medium), 2×10 4 Cells were planted in 96-well plates and allowed to attach to the wall for 1 hour.

[0041] 2) Discard the medium and unattached cells.

[0042] 3) Wash 3 times with PBS to fully wash the unattached cells.

[0043] 4) Add MTT and incubate for 3-4h, discard MTT.

[0044] 5) Observe the number of cell adherence under a microscope.

[0045] 6) Add DMSO to dissolve adherent cells.

[0046] 7) Measure the absorbance value of the cells at a wavelength of 490nm to determine the cell adhesion.

[0047] Results: The number of adherent mesenchymal stem cells stimulated by thrombin group increased significantly in 1 h in serum-free culture. Such as image 3 As shown, the control group in the figure i...

Embodiment 3

[0048] Embodiment 3: The effect of thrombin group on the proliferation of mesenchymal stem cells in serum-free medium

[0049] Steps:

[0050] 1) Fully resuspend and mix the mesenchymal stem cells, and press 2×10 per well 3 The number of cells was planted in a 96-well plate, and medium 1 (control group, serum-free basal medium αMEM) and medium 2 (thrombin group, the concentration of thrombin added in the serum-free basal medium were divided into 0.25 and 0.5 respectively) , 1, 2, 4, 8, 10 IU / mL, 7 concentrations).

[0051] 2) 37°C, CO 2The concentration was 5% in the cell culture incubator for 3 days.

[0052] 3) Add CCK-8 and incubate for 3h.

[0053] 4) Measure the absorbance value of the cells at a wavelength of 450nm to determine the cell proliferation.

[0054] Results: This method can significantly stimulate the proliferation of mesenchymal stem cells in serum-free medium, and has a concentration-dependent effect, see Figure 4 .

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Abstract

The invention discloses a method applicable to serum-free culture of mesenchymal stem cells. The method comprises a step of adding a stimulant drug thrombin into a base culture medium to stimulate mesenchymal stem cells to secrete endogenous fibronectin. The method disclosed by the invention is a method which is simple and convenient, low in cost and high in safety, effectively solves an adherence problem of the serum-free cultured mesenchymal stem cells, and also obviously promotes the mesenchymal stem cells to propagate in a serum-free culture medium at the same time. The method is applicable to serum-free culturing and propagating of clinical research-level mesenchymal stem cells.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a serum-free culture method for mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) are a group of adult stem cells with high self-renewal ability and differentiation potential. It has attracted much attention because of its advantages of immune regulation, cytokine secretion, and convenient sampling, and has become an ideal seed cell for cell therapy. The research on mesenchymal stem cells has attracted more and more attention and has shown more and more broad application prospects. It has extremely important application value in the fields of cell therapy and tissue engineering. After the hematopoietic stem cell, it is the most researched and mature adult stem cell in clinical application. It was first reported that MSCs were used in clinical trials in 1995, and now the cultured MSCs have been widely used in clinical trials, suc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 陈津马予洁郭子宽谭建明
Owner FUZHOU GENERAL HOSPITAL OF NANJING MILITARY COMMAND P L A
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