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Method for separating and in vitro culturing stem cells

A technology of bone marrow mesenchymal stem cells and culture methods, which is applied in the new biological field of bone marrow mesenchymal stem cell isolation and in vitro expansion and culture, can solve problems such as difficulties and poor sorting effects, and improve the expansion efficiency and improve the expansion efficiency. The effect of increasing capacity and improving purification rate

Inactive Publication Date: 2003-09-03
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This kind of sorting requires a large number of mononuclear cells, so a large amount of bone marrow is needed for one sorting, otherwise the sorting effect is not good, which will have certain difficulties in the future clinical application

Method used

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  • Method for separating and in vitro culturing stem cells
  • Method for separating and in vitro culturing stem cells
  • Method for separating and in vitro culturing stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] A specific example taken by the method of the present invention is: after washing the bone marrow with an equal amount of calcium and magnesium-free PBS, suspending the cells with an equal amount of DMEM-LG culture fluid containing 10% FBS, and then slowly adding to a density of 1.073g / ml The mononuclear cell layer was collected by centrifugation on Percoll separation medium, washed 2-3 times with PBS, and washed with 5×10 6 / ml density inoculated in 10% FBS DMEM-LG medium for adherent culture.

[0031] 1. After the cells are layered in the culture flask, discard the culture medium and suspended cells. Adherent spindle cells were digested with Tryspin-EDTA and collected. Harvested cells were washed once with PBS and suspended in PBS. Then according to the instructions provided by MACS, CD14 was sorted from the suspension cells with the adsorption CD14 monoclonal antibody-magnetic bead separation system (MACS, Miltenyi Biotech Inc.) - Cells, and then use the adsorptio...

Embodiment 2

[0034] Separation and purification efficiency

[0035] Table 1 shows the proportions of different labeled cell surface antigens in cells at different stages of separation and purification determined by flow cytometry.

[0036] Table 1. Proportions (%) of different marker cell surface antigens in cells of different isolation and purification stages CD34 in isolation and purification stages + CD14 + CD105 + CD90 + Mononuclear cells 4.11 7.94 5.92 4.71 Primary adherent cells 0.35 8.19 54.51 42.63 CD14 - Cells 0.08 0.42 52.97 63.76 CD14 - CD105 + Cell 0.05 0.17 98.24 89.42

[0037] CD34 in primary isolated human bone marrow mononuclear cells + , CD14 + , CD105 + and CD90 + The average percentages of the cells were 4.11%, 7.94%, 5.29% and 4.71% respectively; after primary adherent culture into spindle cells, CD34+ Greatly reduced cells, CD105 + and CD90 + The proportion of cells increased, indicating that most of the primary adherent spindle cells were human bon...

Embodiment 3

[0039] Cell morphology at different culture stages

[0040] see figure 1 , 4 hours after primary cell inoculation, round cells with single nuclei can be seen attached to the wall, and cell colonies can be seen 2 days after inoculation. The formation of colonies can be seen. Each colony contains 20-50 cells, grows spindle-shaped, and is radial or concentric. Arranged in a circle. A large number of colonies appeared 4 days after inoculation and gradually layered on the bottom of the culture flask. It takes 14-16 days for cell expansion to form a monolayer in Teflen-75 flasks. But it can be seen from the figure that there are some suspended and adhered round cells. figure 1 Shown under an inverted microscope, the cells are spindle-shaped, 10×10 times.

[0041] see figure 2 , human bone marrow mesenchymal stem cells (CD34 - CD14 - CD105 + CD90 + ) under the culture system provided by the present invention (DMEM-LG: ginsenoside polysaccharide: 10% FBS) to carry out the fi...

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Abstract

A process for separating and external amplification culture of myeloid mesenchymal stem cells is disclosed. In order to effectively increase their external purifying rate and amplification power, after the adherent culture of primary cell is performed on the basis of separating single myeloid nuclea cell, the positive and negative immunoselections and integrated for purifying them and a culture system containing ginsenoside polyose is created for amplification culture to increase the external amplification and secondary culture capability.

Description

Technical field [0001] The invention belongs to a biotechnological method, and relates to a new biotechnological method for isolating bone marrow mesenchymal stem cells and expanding and culturing in vitro. This method can effectively purify bone marrow mesenchymal stem cells and improve their ability to expand in vitro. Background technique [0002] There is a kind of stem cells with multi-differentiation potential in the bone marrow stroma. Related studies have shown that this kind of stem cells can differentiate into osteoblasts, chondrocytes, adipocytes, myoblasts, nerve cells and cardiomyocytes, etc. This kind of stem cells is called bone marrow mesenchymal stem cells, which will be of great value in tissue engineering in medicine. [0003] Since the specific marker molecules of bone marrow mesenchymal stem cells have not been found yet, the isolation and purification of the stem cells is still an indirect method: the traditional method is the attachment method, that ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 王金福
Owner ZHEJIANG UNIV
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