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Culture method of bone marrow derived endothelial progenitor cells

A technology of endothelial progenitor cells and culture methods, which is applied in the field of cell culture, can solve the problems of low culture efficiency, low passage times, and high culture costs, and achieve the effects of improving culture efficiency, solving complex operation processes, and improving viability

Active Publication Date: 2017-05-31
哈尔滨中科赛恩斯生物技术有限公司
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0012] In order to solve the technical problems of low passage times, high culture cost and low culture efficiency in the culture process of endothelial progenitor cells, the present invention provides a method for culturing bone marrow-derived endothelial progenitor cells. The adopted technical scheme is as follows:

Method used

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  • Culture method of bone marrow derived endothelial progenitor cells
  • Culture method of bone marrow derived endothelial progenitor cells
  • Culture method of bone marrow derived endothelial progenitor cells

Examples

Experimental program
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Effect test

Embodiment 1

[0047] 1. Configuration of complete medium

[0048] The complete medium is added in the serum medium EGM2: 10% (V / V) fetal bovine serum (FBS), 10ng / mL vascular endothelial growth factor VEGF, 0.4‰ (V / V) glucocorticoid, 1‰ (V / V) vitamin C, 1‰ (V / V) epidermal growth factor EGF, 5ng / mL basic fibroblast growth factor bFGF, 2ng / mL colony-stimulating factor (CSF), 1‰ (V / V) platelet source Sexual growth factor PDGF.

[0049] 2. Isolation and culture of mouse bone marrow mononuclear cells

[0050] The experimental animals were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.3 mL / 100 g). The mouse femur was taken under aseptic conditions, and the bone marrow was quickly and completely washed into a centrifuge tube with PBS, and the erythrocyte lysate was slowly added in a ratio of 1:5 after pipetting evenly. Bone marrow mononuclear cells were isolated after centrifugation at 1000rpm for 5min. After washing 3 times with PBS, mononuclear cells were separated into...

Embodiment 2

[0057] This example provides a method for culturing bone marrow-derived endothelial progenitor cells. The difference between this method and Example 1 is that the composition of the medium used is: EGM2+5%FBS+Hydrocortisone+Ascorbic Acid+EGF+VEGF+IGF +FGF. Specifically, 5% (v / v) fetal bovine serum FBS, 10ng / mL Hydrocortisone (glucocorticoid), 0.4‰ (V / V) Ascorbic Acid, 2‰ EGF, 2ng / mL were added to the EGM2 medium. mL of VEGF, 5ng / mL of IGF, 1‰ (V / V) of FGF. The obtained endothelial progenitor cells are cultured by this culture method for a total of 6 generations, and after 6 generations, the state of the cells is relatively poor, and the cells grow very slowly.

Embodiment 3

[0059] This example provides a method for culturing bone marrow-derived endothelial progenitor cells. The difference between this method and Example 1 is that no colony-stimulating factor CSF is added to the medium used. The endothelial progenitor cells are cultured by this culture method, and the obtained endothelial progenitor cells are passed down for 6 generations. After 6 generations, the cell state is poor, the speed of passage is slow, and there are many floating dead cells.

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Abstract

The invention discloses a culture method of bone marrow derived endothelial progenitor cells and belongs to the technical field of cell culture. The method comprises the steps of treating extracted bone marrow with a red blood cell lysis buffer, then, carrying out separating so as to obtain single karyocytes of the bone marrow, carrying out washing, then, inoculating a fibronectin precoated culture dish with the obtained single karyocytes, carrying out culture by using a complete culture medium, digesting the cells with trypsin after a period of time of culture, inoculating culture bottles with digested cells, and carrying out differential adherent culture and differential digestion culture. The used complete culture medium is prepared through adding fetal calf serum, a vascular endothelial growth factor, glucocorticoid, vitamin C, an epidermal growth factor, a basic fibroblast growth factor, a colony stimulating factor and a platelet-derived growth factor into a serum-free medium EGM2. The method has the characteristics that the operating steps are relatively simple, the spent time is short, the quantity of separation in one time is large, the number of transfer generations of the obtained cells is large, and the like.

Description

technical field [0001] The invention relates to a method for culturing bone marrow-derived endothelial progenitor cells, which belongs to the technical field of cell culture. Background technique [0002] Endothelial progenitor cells (EPCs) are the precursor cells of mature vascular endothelial cells, which were first proved by Asahara et al. in 1997 to have neovascular potential in the hematopoietic system. In a large number of previous studies, endothelial progenitor cells play an important role in cardiovascular and cerebrovascular diseases, peripheral vascular diseases, tumor angiogenesis, and wound healing, and can effectively promote angiogenesis in ischemic myocardium and improve limb ischemia. To repair the damaged vascular endothelium, more and more attention has been paid to the research on the isolation, purification, expansion, function and application of EPC. However, the number of endothelial progenitor cells in the body is very small, only 0.1% in the bone ma...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0692C12N2500/38C12N2500/90C12N2501/11C12N2501/115C12N2501/135C12N2501/165C12N2501/22C12N2501/39
Inventor 石佳宁陈思胡啸竹万娜肖阳
Owner 哈尔滨中科赛恩斯生物技术有限公司
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