Method for sorting primary human marrow mesenchymal steam cell by monoclonal antibody ZUC3 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 the effects of maintaining biological characteristics, improving time-consuming, and shortening culture time
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Embodiment 1
[0021] Separation of primary human bone marrow MSCs by monoclonal antibody ZUC3 immunomagnetic bead method
[0022] Bone marrow was collected from a healthy donor, anticoagulated with heparin, and Ficoll-paque (specific gravity 1.077) density gradient centrifugation was used to obtain bone marrow mononuclear cells. Nuclear cells were incubated, and after centrifugation and washing, anti-mouse IgM magnetic bead secondary antibody (Miltenyi Biotec Inc.) was added to incubate. After centrifugation and washing, human bone marrow MSCs positive for ZUC3 antigen were isolated from bone marrow mononuclear cells by immunomagnetic bead sorting system. ZUC3 antigen-positive cells and ZUC3 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 incubator, and then change the medium every 3 days.
[0023] After 3 days of culture, the cells in...
Embodiment 2
[0026] Analysis of the proliferation ability of positive cells obtained by forward immune sorting using ZUC3 as a marker
[0027] Take the ZUC3 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 ZUC3 as a marker have good proliferative activit...
Embodiment 3
[0029] Phenotypic characteristics of positive cells obtained by forward immuno-sorting using ZUC3 as a marker
[0030] The ZUC3 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) markers appeared positive single peaks, 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 ZUC3 was expanded to the fifth passage in vitro. The phenotypic characteristics of positive cells after forward immuno-sorting with ZUC3 are those of human bone marrow MSCs, and there is no significant...
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