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Method for sorting primary human marrow mesenchy malstem cell by monoclonal antibody ZUB4 immunomagnetic bead

An immunomagnetic bead sorting and bone marrow mesenchymal technology, which is applied in the direction of bone/connective tissue cells, animal cells, vertebrate cells, etc., can solve the problem of complex components of CD45-GlycophorinA- cell populations and the difficulty of direct sorting by immunolabeling Ideal markers of human bone marrow MSCs, difficulties in the practical application of MSCs isolation and purification, etc., to achieve good growth activity and proliferation potential, improve time-consuming, and shorten the effect of culture time

Inactive Publication Date: 2009-10-28
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

The content of MSCs in bone marrow mononuclear cells is extremely low, and the content of MSCs in the sorted cells can be effectively increased by negative immuno-sorting. Some researchers used CD45 and Glycophorin A as markers to screen the CD45-Glycophorin A- cell population components Complex, still needs further purification
Therefore, the above-mentioned immune markers are difficult to be ideal markers for directly sorting human bone marrow MSCs
[0005] So far, researchers at home and abroad have been identifying human bone marrow MSCs through the combination of multiple surface molecules, combined with the characteristics of cell self-renewal and multilineage differentiation potential. The single specific surface molecule of MSCs is still being explored, which gives MSCs The practical application of separation and purification brings certain difficulties

Method used

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  • Method for sorting primary human marrow mesenchy malstem cell by monoclonal antibody ZUB4 immunomagnetic bead
  • Method for sorting primary human marrow mesenchy malstem cell by monoclonal antibody ZUB4 immunomagnetic bead
  • Method for sorting primary human marrow mesenchy malstem cell by monoclonal antibody ZUB4 immunomagnetic bead

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Separation of primary human bone marrow MSCs by immunomagnetic beads with monoclonal antibody ZUB4

[0023] Bone marrow was collected from a healthy donor, heparin anticoagulant, Ficoll-paque (specific gravity 1.077) density gradient centrifugation to obtain bone marrow mononuclear cells, and monoclonal antibody ZUB4 (antibody subtype IgG1) disclosed in patent application number 200510061036.1 was used to combine with bone marrow mononuclear cells Nucleated cells were incubated, centrifuged and washed, then incubated with anti-mouse IgG magnetic bead secondary antibody (Miltenyi Biotec Inc.), centrifuged and washed, and isolated from bone marrow mononuclear cells by immunomagnetic bead sorting system to obtain ZUB4 antigen-positive human bone marrow MSCs. ZUB4 antigen-positive cells and negative cells were inoculated on culture dishes respectively, and placed in low-sugar DMEM medium containing 10% (V / V) fetal bovine serum at 37°C with a volume fraction of 5% CO 2 Cultu...

Embodiment 2

[0027] Analysis of the proliferation ability of positive cells obtained by forward immune sorting using ZUB4 as a marker

[0028] Take the ZUB4-positive human bone marrow MSCs first and fifth generation cells cultured in vitro, and press 2×10 4 Cells / well density were seeded in 24-well culture plates, cultured for 1, 2, 3, 4, 5, 6, 7, and 8 days, and cell kinetics analysis was performed. The culture has the following common characteristics: After the cells are subcultured, the cells are completely attached to the wall in about 10 hours, and the cell shape becomes a spindle cell again; the incubation period of the subculture is about 24 to 36 hours; days; after the end of the logarithmic growth phase, it enters the plateau phase; there is no significant difference between the growth of the fifth generation and the first generation cells, see figure 2 . Positive cells obtained by forward immune sorting with ZUB4 as a marker have good proliferative activity and maintain a spin...

Embodiment 3

[0030] Phenotypic characteristics of positive cells obtained by forward immuno-sorting using ZUB4 as marker

[0031] The first and fifth passages of ZUB4-positive human bone marrow MSCs cultured and passaged in vitro were treated with CD 14-FITC, CD29-PE, CD34-PE, CD44-FITC, CD45-FITC, CD105-PE, CD166-PE, HLA-DR-FITC monoclonal antibody was used as a probe, and the expression of MSCs surface molecules was analyzed by flow cytometry. The results showed that CD29, CD44, CD166, and CD105 (SH2) positive markers showed a single peak, and the expressions of hematopoietic cell differentiation antigens CD14, CD34, CD45, and HLA-DR were all negative, see image 3 , image 3 The immunophenotype analysis of the positive cells obtained after forward immunosorting marked with ZUB4 was expanded to the fifth passage in vitro. The phenotypic characteristics of positive cells after forward immuno-sorting with ZUB4 are those of human bone marrow MSCs, and there is no significant difference in...

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Abstract

The invention provides the method of separating and choosing the dry cell in the marrow using the single resisting ZUB4 immune magnetoelectric bead. The medullary suspending liquid is separated to gain the single nuclear cell. Using the single clonal antibody ZUB4 for the sign, the full dry cell in the human marrow with the ZUB4 masculine antigen by the indirect immune magnetoelectric bead choosing system is gained. The full dry cell in the human body has the good developing active capability and the good proliferating potential. The ZUB4 antigen during the process of the cell bearing is steady and can be directionally induced and separated to the lipocyte, the bone cell, the nerve cell. The invention provides the perfect method of choosing and sublimating the full dry cell in the human marrow and advances the purity of the full dry cell between the original era and the efficiency of separating and culturing. The invention is propitious to the research and the extending in the clinic checking and the application of the biology characteristic of the full dry cell.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a new method for the separation, purification and in vitro culture of primary cells of human bone marrow mesenchymal stem cells. The method uses a newly prepared anti-human bone marrow mesenchymal stem cell monoclonal antibody ZUB4, through The method of immune sorting effectively separates and purifies mesenchymal stem cells from human bone marrow mononuclear cells, and cultures and expands them in vitro to induce multilineage differentiation. Background technique [0002] Human bone marrow mesenchymal stem cells (Mesenchymal stem cells, MSCs) are stem cells with high self-renewal and multi-lineage differentiation potential existing in the bone marrow, and are ideal seed cells for tissue engineering and regenerative medicine. There are a large number of basic and clinical research reports on bone marrow MSCs at home and abroad, which have broad prospects in the clinical applic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/08C12N5/0775
Inventor 黄河来晓瑜沈建根
Owner ZHEJIANG UNIV
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