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An endothelial progenitor cell and culture method technology, applied in the field of biological cell culture, can solve the problems of high EPC culture cost, difficulty in EPC separation and culture, etc., and achieve the effects of reducing culture cost, easy adherence, and fast growth rate.
Inactive Publication Date: 2015-03-04
贾瑞鹏
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[0004] The purpose of the present invention is to provide a kind of culture method of endothelial progenitor cell, to solve
Method used
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preparation example Construction
[0031] Step a. Preparation and storage of type I rat tail collagen;
[0032] Step b. type I rat tail collagen coating cell culture flask;
[0033] Step c. Separation of human peripheral blood mononuclear cells;
[0034] Step d. inoculate the above-mentioned isolated mononuclear cells in the cell culture flasks coated with type I rat tail collagen, add negative control group and positive control group to the experiment, and add DMEM medium in the three groups of culture flasks at 37 °C, 5%CO 2 , cultivated in an incubator with 95% humidity;
[0035] Step e. digestion and passage of EPC;
[0036] Step f. The cell flow cytometry analysis detection of EPC;
[0037] Step g. the immunofluorescence detection of EPC;
[0038] Step h. cell proliferation experiment, observe the proliferation ability of EPC;
[0039] Step i. Single cell cloning experiment to observe the proliferation and self-renewal ability of EPC;
[0040] Step j. FITC-UEA-1 extracellular adsorption and endocyto...
Embodiment
[0048] The preparation, purification, detection and coating of cell culture flasks of type I rat tail collagen include the following steps:
[0049] a) Wash the tail of the rat, soak it in 75% alcohol for 15 minutes, cut off the tail, remove the fur and cut it into small pieces, pull out the silver tail tendon, cut the tail tendon and soak it in normal saline, Add the tendon to 0.1% acetic acid at a ratio of 50ml, shake it to disperse the tendon in the acetic acid solution, and place it at 4°C for 72 hours;
[0050] b) Transfer the above-mentioned fully dissolved liquid into a sterile centrifuge tube, and centrifuge. Centrifugation conditions: 4°C, 4000rpm, 10min;
[0051] c) Take the above supernatant, aliquot and store at 4°C for future use;
[0052] d) using the Kjeldahl method to detect the content of collagen in the above supernatant;
[0053] e) Use the prepared type I rat tail collagen to coat the cell culture flask, and the coating concentration is 6-10ug / cm 2 . Ad...
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Abstract
The invention discloses an endothelial progenitor cell (EPC) culture method. The culture method comprises the following steps: inoculating human peripheral blood mononuclear cells in a cell culture bottle coated by I-type rat tail collagen, adding a DMEM culture medium in the cell culture bottle, putting the cell culture bottle in an incubator with the temperature of 37 DEG C, the CO2 content of 5 percent and the humidity of 95 percent for culturing; carrying out EPC digestion passage; then carrying out flow cytometric analysis on EPC; carrying out EPC immunofluorescence assay; carrying out a cell proliferative assay; carrying out a single cell clonogenic assay; carrying out FITC-UEA-1 extracellular adsorption and endocytosis Dil-acLDL experimental measurement; and carrying out an in-vitro tube formation experiment. The EPC culture method is simple, convenient and feasible. With the adoption of the EPC culture method, the cost can also be greatly lowered; the high-purity I-type rat tail collagen is prepared by the EPC culture method and used for culturing EPC by coating the cell culture bottle, and the culture medium which is low in cost is also adopted, so that the EPC is successfully cultured from human peripheral blood; through identification, the EPC cultured by the method disclosed by the invention has the phenotypic characteristics and the functions of the EPC cultured by the conventional method.
Description
technical field [0001] The invention belongs to the field of biological cell culture, in particular to a method for culturing endothelial progenitor cells. Background technique [0002] Endothelial progenitor cells (EPC) are a kind of precursor cells of endothelial cells, which have the potential to proliferate, migrate and differentiate into mature endothelial cells, and play an important role in the process of neovascularization in vivo. Asahara et al. first isolated and cultured EPCs from adult peripheral blood in 1997, which can be differentiated into endothelial cells in vitro and participate in the formation of new blood vessels in ischemic tissues. Subsequent studies further confirmed that these cells are a special cell subtype originating from the bone marrow. However, in healthy normal adults, the content of EPCs in bone marrow or peripheral blood is less, which makes the isolation and culture of EPCs more difficult. Usually, for the isolation and culture of EPC, ...
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