Method for culturing mesenchymal stem cell in vitro

A technology of mesenchymal stem cells and in vitro culture, applied in the field of cell engineering, can solve the problems of insufficient cell viability for clinical and scientific research, slow cell growth, high cost, etc., and achieve the effect of high viability cell source, high cell viability, and simple configuration

Inactive Publication Date: 2013-06-12
THE SECOND AFFILIATED HOSPITAL ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cell expansion cycle of this culture method is long, and in addition to the α-MEM basal medium, relatively expensive components such as horse serum need to be added to the culture medium, which is costly, has many componen

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  • Method for culturing mesenchymal stem cell in vitro
  • Method for culturing mesenchymal stem cell in vitro
  • Method for culturing mesenchymal stem cell in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Preparation of mesenchymal stem cells using the method of the present invention

[0030] Primary culture:

[0031] Using 1.073g / cm 3 Mononuclear cells were isolated from bone marrow with Percoll separation medium by gradient density centrifugation, and the mononuclear cells were inoculated into α-MEM medium containing 10% fetal bovine serum by volume at 37°C and 5% CO 2Adhesive culture was carried out under concentration conditions, and the primary mesenchymal stem cells were obtained until the adherent cells appeared and showed the shape of fibroblasts.

[0032] α-MEM culture medium: anhydrous calcium chloride 200mg / L, potassium chloride 400mg / L, anhydrous magnesium sulfate 97.67mg / L, sodium chloride 6800mg / L, sodium dihydrogen phosphate monohydrate 140mg / L, L -Alanine 25mg / L, L-arginine hydrochloride 126.98mg / L, L-asparagine 50mg / L, L-aspartic acid 30mg / L, L-cysteine ​​hydrochloride 100mg / L, L - Cystine hydrochloride 31.28mg / L, L-glutamic acid 75mg / L, L-...

Embodiment 2

[0035] Example 2: Identification of mesenchymal stem cells of the present invention

[0036] 1. Immunophenotyping

[0037] To prepare primary mesenchymal stem cells, take 1×10 4 More than one cells were added to different EP tubes, and PE or FITC-labeled anti-CD14, CD34, CD45, CD73, CD90, CD105 monoclonal antibodies were added to each tube and detected by flow cytometry, the results are shown in figure 1 . Depend on figure 1 It can be seen that the mesenchymal stem cells cultured in the present invention have no or low expression of CD14, CD34, and CD45, and high expression of CD73, CD90, and CD105, which meet the reference standards for mesenchymal stem cells proposed by the International Society for Cell Therapy.

[0038] 2. Morphological characteristics

[0039] The primary mesenchymal stem cells and the third generation mesenchymal stem cells cultured in the present invention were observed under a microscope, and the results are shown in figure 2 ,Depend on figure ...

Embodiment 3

[0045] Example 3: Colony Cloning Test of Mesenchymal Stem Cells

[0046] The mesenchymal stem cells cultured by the method described in the present invention and the Dexter-LTC culture system were subjected to colony clone hematoxylin staining (the culture environment was consistent), and statistical and significant difference analysis was performed (≥50 cells were counted as a colony), The results are shown in Table 1 and Figure 4 .

[0047] Table 1 Count of mesenchymal stem cell colony clone test

[0048] group

Number of colonies

The average number of cells in a colony

Dexter-LTC culture system

8.0±1.0

59.2±8.2

The method of the invention

11.3±1.53*

66.5±14.4*

[0049] Note: * means there is a significant difference compared with the Dexter-LTC system (P<0.05)

[0050] It can be seen from Table 1 that the number of mesenchymal stem cell colonies cultured according to the method of the present invention and the average...

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Abstract

The invention relates to the field of cell engineering, and discloses a method for culturing mesenchymal stem cell in vitro. The method comprises the following steps: separating from marrow, umbilical cord blood or umbilical cord through a gradient density centrifugation by using 1.073g/cm<3> of Percoll separation medium to obtain a mononuclear cell, inoculating the mononuclear cell to alpha-MEM nutrient solution containing fetal calf serum with volume percentage of 10%, and performing adherent culture under 5% of CO2 concentration at 37 DEG C until the anchorage-dependent cell is appeared and is in fibroblast to obtain the mesenchymal stem cell. The culture method provided by the invention is low in cost and simple for configuration without adding the components such as equine serum, I-inositol, I-glutamine, mercaptoethanl and the like, the cell activity is higher, the required period of cell expansion is shortened; and meanwhile, the method is superior to the existing Dexter-LTC culture system, and is capable of providing high-activity cell source for the corresponding therapy and research.

Description

technical field [0001] The invention relates to the field of cell engineering, and specifically provides a method for culturing mesenchymal stem cells in vitro. Background technique [0002] Mesenchymal stem cells (MSCs) were first discovered in bone marrow. With the continuous development of stem cell technology, people found that umbilical cord blood and umbilical cord are also rich in mesenchymal stem cells. Mesenchymal stem cells are pluripotent stem cells with self-renewal and multi-directional differentiation potential. They have the advantages of secreting hematopoietic growth factors, low immunogenicity, and easy transfection and expression of foreign genes. Under specific induction conditions in vivo or in vitro, It can be differentiated into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium, etc. It still has multi-directional differentiation potential after continuous subculture and cryopreservat...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 李忠俊叶兴德邓小军冉茜相丽欣
Owner THE SECOND AFFILIATED HOSPITAL ARMY MEDICAL UNIV
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