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Method for monoclonal antibody ZUB1 immunological magnetic bead sorting primary generation human marrow mesenchyme stem cell

An immunomagnetic bead sorting and bone marrow mesenchymal technology, applied in the direction of bone/connective tissue cells, animal cells, vertebrate cells, etc., can solve the complex composition of CD45-GlycophorinA-cell population and the difficulty of immunolabeling to be directly sorted. The ideal labeling of human bone marrow MSCs, the difficulty of practical application of MSCs isolation and purification, etc., achieve the effect of good growth activity and proliferation potential, improve the time-consuming, and shorten the culture time.

Inactive Publication Date: 2006-11-22
ZHEJIANG UNIV
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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 monoclonal antibody ZUB1 immunological magnetic bead sorting primary generation human marrow mesenchyme stem cell
  • Method for monoclonal antibody ZUB1 immunological magnetic bead sorting primary generation human marrow mesenchyme stem cell
  • Method for monoclonal antibody ZUB1 immunological magnetic bead sorting primary generation human marrow mesenchyme stem cell

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Effect test

Embodiment 1

[0022] Separation of primary human bone marrow MSCs by monoclonal antibody ZUB1 immunomagnetic bead method

[0023] Bone marrow was collected from a healthy donor, anticoagulated with heparin, and Ficoll-paque (specific gravity 1.077) density gradient centrifugation to obtain bone marrow mononuclear cells. Monoclonal antibody ZUB1 (antibody subtype IgG1) disclosed in patent application number 200510061034. Nuclear cells were incubated, and after centrifugation and washing, anti-mouse IgG magnetic bead secondary antibody (Miltenyi Biotec Inc.) was added to incubate. After centrifugation and washing, human bone marrow MSCs positive for ZUB1 antigen were isolated from bone marrow mononuclear cells by immunomagnetic bead sorting system. ZUB1 antigen-positive cells and ZUB1 antigen-negative cells were inoculated on culture dishes respectively, and placed in low-glucose DMEM medium containing 10% (V / V) fetal bovine serum at 37°C with a volume fraction of 5% CO 2 Culture in an incuba...

Embodiment 2

[0027] Analysis of the proliferation ability of positive cells obtained by forward immune sorting using ZUB1 as a marker

[0028] Take the ZUB1 antigen-positive human bone marrow MSCs first and fifth generation cells cultured and passaged 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 ZUB1 as a marker have good proliferative activit...

Embodiment 3

[0030] Phenotypic characteristics of positive cells obtained by forward immuno-sorting using ZUB1 as a marker

[0031] The ZUB1 antigen-positive human bone marrow MSCs of the first and fifth passages cultured and passaged in vitro were treated with CD14-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 ZUB1 was expanded to the fifth generation in vitro. The phenotypic characteristics of positive cells after forward immuno-sorting with ZUB1 are those of human bone marrow MSCs. There is no significant d...

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Abstract

The invention discloses a human replacement of bone marrow charging stem cell method of single-antibody-ZUB1 immune magnetic bead sorting, which is characterized by the following: separating single core cell from bone marrow suspension liquid; selecting single-antibody-ZUB1 as mark to produce human bone marrow charging stem cell of ZUB1 positive antigen; possessing excellent growing activity and breeding potential; inducing into fat cell, sclerotomal cell and nerve cell out of the body directionally; improving the separating and purifying efficiency for original discharging stem cell; fitting for cellular biology study and clinical detection.

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 ZUB1, 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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IPC IPC(8): C12N5/0775
Inventor 黄河来晓瑜沈建根
Owner ZHEJIANG UNIV