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Preparation for mobilizing mesenchymal stem cells and method for separating mesenchymal stem cells

A technology of mesenchymal stem cells and preparations, applied in the field of preparation and separation of mobilized mesenchymal stem cells, can solve the problems of limited cell sources, cumbersome culture steps, pollution, etc., and achieve the effect of high mobilization efficiency

Inactive Publication Date: 2013-06-12
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is feasible and easy to master, but it also has some disadvantages: limited source of cells, cumbersome culture steps, long time to expand to the required cell volume, non-humanization of culture medium additives, pollution problems, risk of tumorigenesis, etc.
[0007] Although the HIF-1 / SDF-1α-CXCR4 axis plays a key role in the mobilization of MSCs according to literature reports and our previous research results, there is still a lack of an effective MSCs mobilization preparation and a feasible method for MSCs mobilization.

Method used

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  • Preparation for mobilizing mesenchymal stem cells and method for separating mesenchymal stem cells
  • Preparation for mobilizing mesenchymal stem cells and method for separating mesenchymal stem cells
  • Preparation for mobilizing mesenchymal stem cells and method for separating mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] 1. Reagent preparation

[0038] (1) CoCl 2 : Weigh 400mg CoCl 2 Add 10ml of normal saline to the powder to make a 40mg / ml storage solution, filter it through a 0.22μm filter and store in -20°C. When animals were administered, the stock solution was diluted 10 times with physiological saline to adjust the concentration to 4 mg / ml.

[0039] (2) AMD3100: 5g of powder plus 5ml of normal saline to make a 1mg / ml solution, filtered through a 0.22μm filter, and stored at -20°C. Physiological saline was diluted 4 times to 1.25mg / ml for administration to mice.

[0040] 2. Grouping of animals

[0041] (1) To detect CoCl 2 and CoCl 2 Combined with the mobilization effect of AMD3100 on MSCs, the rats were divided into 4 groups with 5 rats in each group, which were as follows: ①Normal saline group (NS group): intraperitoneal injection of normal saline (with CoCl 2 same volume), a total of 7 days; ②CoCl 2 Group 7d: intraperitoneal injection of CoCl every day 2 10 mg / kg solut...

Embodiment 2

[0071] To detect CoCl 2 For the effective concentration of MSCs mobilization, we used the CFU-F method to study the daily intraperitoneal injection of CoCl in rats. 2 Solution 5mg / kg, 20mg / kg for 7 days, the number of CFU-Fs in the peripheral blood of rats, the results show that: CoCl 2 5mg / kg×7 days, CoCl 2 20mg / kg×7 days had a mobilization effect on rat MSCs, and the peripheral blood CFU-F increased compared with the normal saline control group, but the mobilization efficiency was lower than that of CoCl 2 10mg / kg×7 days group. The specific result is: CoCl 2 5mg / kg×7 days group vs. normal saline group (2.02±0.12 vs.1.27±0.08 CFU-Fs / ml, P2 20mg / kg×7 days group vs. normal saline group (1.68±0.08 vs.1.27±0.08 CFU-Fs / ml, P<0.05).

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Abstract

The invention provides a preparation for mobilizing mesenchymal stem cells and a method for separating the mesenchymal stem cells. The preparation for mobilizing the mesenchymal stem cells comprises CoCl2, wherein the CoCl2 is a hypoxia mimetic agent, and the dosage is in a range of 5-20mg / kg. The CoCl2 and AMD3100 are used in a combined manner. The dosage of the AMD3100 is 5mg / kg. According to the method for separating the mesenchymal stem cells by using the preparation, the preparation is used for actuating the mesenchymal stem cells to be mobilized, enter peripheral blood and then be separated. The method comprises the following separation steps of: sampling the periphery blood, separating mononuclear cells by using a lymphocyte separating medium, performing resuspension by using a DMEM (dulbeccos modified eagle medium) containing 20% of fetal bovine serum, inoculating to a culture flask, culturing for 7 days, and changing a culture solution, thus obtaining the mesenchymal stem cells of the peripheral blood on the 10th day. The mesenchymal stem cells of the peripheral blood highly express CD90, CD29 and CD44, do not express CD45 and CD34, and have the capacities of in vitro bone formation, fat formation and cartilage differentiation formation. The preparation is high in MSCs (mesenchymal stem cells) mobilization efficiency, and an effective preparation and an effective method are provided for tissue engineering and regenerative medicine.

Description

technical field [0001] The invention belongs to the field of biotechnology, and mainly relates to a preparation and separation method for mobilizing mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) are adult stem cells with strong proliferation ability and multilineage differentiation potential. Since MSCs can differentiate into bone, cartilage, fat, muscle, tendon, nerve and other tissues under certain conditions, MSCs have become very potential seed cells for tissue engineering and regenerative medicine. MSCs have broad clinical application prospects: treating bone, joint, and tendon defects and injuries; promoting regeneration of nerve cells, cardiomyocytes, and liver cells; supporting hematopoiesis and promoting hematopoietic stem cell implantation; preventing and treating graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation ); Immunomodulatory treatment of autoimmune disorders, etc. [0003] Bone marrow is t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 黄河刘丽珍余勤
Owner ZHEJIANG UNIV
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