Preparation method for simultaneously culturing stromal stem cells and endothelial progenitor cells from marrow at one time and application of stromal stem cells and endothelial progenitor cells
A technology of endothelial progenitor cells and stem cells, applied in bone/connective tissue cells, animal cells, extracellular fluid diseases, etc., can solve problems such as time-consuming, complicated methods, and high costs
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Embodiment 1
[0030] Simultaneous culture of stromal stem cells and endothelial progenitor cells in bone marrow
[0031] 20 mL of bone marrow from normal donors (who signed an informed consent form) were collected, separated by Ficoll density gradient centrifugation, washed three times with 1×PBS (pH 7.4), and the obtained mononuclear cells were counted. 20mL bone marrow to obtain about 40×106 cells mononuclear cells.
[0032] Half of mononuclear cells (approximately 20×10 6 cells) were resuspended in α-MEM complete medium (10% (v / v) fetal bovine serum, 20ng / mL basic fibroblast growth factor), added to 25cm 2 In a culture flask, 5 mL per bottle, the cell concentration is 2×10 6 cells / mL in 5% CO 2 Start the culture at 37°C.
[0033] The other half of mononuclear cells (about 20×10 6 cells) were resuspended in EGM-2 complete medium (5% (v / v) fetal bovine serum, 50ng / mL epidermal growth factor, 50ng / mL vascular endothelial growth factor, 50ng / mL human insulin-like growth cytokine, 1ug / mL...
Embodiment 2
[0036] Flow Cytometry Detection of Stromal Stem Cells and Endothelial Progenitor Cells Obtained from One-time Simultaneous Culture in Bone Marrow
[0037] Cultured to the last day (fifth generation), respectively take a bottle of grown stromal stem cells and endothelial progenitor cells and digest them with 0.025% (m / v) trypsin, stop the digestion solution with 0.5mL calf serum, and then at 1000rpm, Centrifuge at 4°C for 5 minutes, resuspend the cells in 1mL of α-MEM and EGM-2 medium respectively, take 20ul of the cell solution and dilute 20 times with 1×PBS (pH7.4), add 1 times the volume of trypan blue solution to the diluent , mix well and add to the cell counting plate, observe and count under an inverted microscope, the blue stained cells are dead cells, and the unstained cells are live cells.
[0038] Take trypan blue staining and count 3×10 6 Cells and stromal stem cells were divided into three groups for flow cytometry detection. The first group was added to centrifug...
Embodiment 3
[0043] This example illustrates the application of stromal stem cells combined with endothelial progenitor cells obtained through simultaneous culture in bone marrow of the present invention in an animal model of ischemic stroke.
[0044] The stromal stem cells and endothelial progenitor cell cultures obtained in Examples 1 and 2 were respectively taken as the fifth generation. After the cells were digested with 0.025% (m / v) trypsin and terminated with 0.5 mL of calf serum, they were centrifuged at 800 rpm for 5 min. Afterwards, the cell pellet was collected, washed 3 times with 0.9% sodium chloride solution, and then resuspended to 2×10 6 Each / mL concentration, prepare 2×10 6 The injection of stromal stem cells and endothelial progenitor cells at a concentration of 1 / mL was stored at 4°C for later use.
[0045] The rat model of ischemic stroke purchased from Capital Medical University was randomly divided into four groups A, B, C and D, with 10 rats in each group. Group A wa...
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