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Isolation and in vitro expansion method of human vein endothelial cells

An endothelial cell and vein technology, applied in the field of bioengineering, can solve the problems of low cell positive rate, high cost, inconvenient operation, etc., and achieve the effects of broad application prospects, short time-consuming and simple operation.

Active Publication Date: 2020-10-02
BEIJING JING MENG STEM CELL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The positive rate of cells in the first two methods is relatively low, and the cost of the third method is relatively high. At present, the fourth method is the main method, but this method needs to inject enzyme solution into the umbilical cord vein, and needs to maintain the digestion temperature, etc., and the operation is difficult. Convenience, it also requires that the sample must be more than 20 cm in length, too short will lead to too few collected cells, in addition, other pollutants are often brought in during the operation
[0004] During the culture of endothelial cells, the current culture methods add more animal-derived components such as fetal bovine serum and horse-bovine serum to meet the nutritional needs of endothelial cells during the growth process, but there are foreign proteins and unknown sources of pollution, etc. question
[0005] For example, in the Chinese invention patent application with the application number 200810222274.X, the publication date of January 28, 2009, and the invention title "A Vascular Endothelial Cell and Its Preparation Method and Application", in the later stage of the endothelial cell culture process, add A large amount of heterologous serum such as horse serum and fetal bovine serum has safety risks for the use of late-stage endothelial cells

Method used

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  • Isolation and in vitro expansion method of human vein endothelial cells
  • Isolation and in vitro expansion method of human vein endothelial cells
  • Isolation and in vitro expansion method of human vein endothelial cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1, separation of human vein endothelial cells

[0061] This embodiment uses veins from human umbilical cord to realize the separation of human vein endothelial cells, including the following specific implementation process. It can be understood that the following operations are for the purpose of clearly disclosing the continuous process and not limiting the present invention:

[0062] 1) Take the fresh umbilical cord of a normal delivery baby in a sterile biological safety cabinet (within 12 hours, if the time is too long, the endothelial cells will die due to ex vivo, and it is difficult to survive), wash the umbilical cord twice with 250mL normal saline, and squeeze the umbilical cord with tweezers Tissue, remove the residual blood in the umbilical cord, and then use tweezers and a scalpel to peel off the umbilical cord amniotic membrane and jelly, and pick about 10cm of the human umbilical cord vein;

[0063] 2) Clamp both ends of the human umbilical cord...

Embodiment 2

[0067] Example 2, In Vitro Expansion of Human Vein Endothelial Cells

[0068] In this example, human vein endothelial cells were expanded in vitro, including the following specific operations: using platelet lysate (purchased from UltraGRO) containing 10% (5-15%, V / V), 10 ng / mL (8- VEGF (endothelial cell growth factor, purchased from peprotech), 2 units of heparin sodium (purchased from Wanbang Biochemical Medicine) and IMDM medium (purchased from gibco) (or DMEM / F12, α-MEM and other Cell culture medium) to resuspend human vein endothelial cells in expansion medium, at 37°C, 5% CO 2 , cultured under saturated humidity, induce adherence for 24 hours (12-36 hours are acceptable), change the medium in full, remove non-adherent cells, add fresh expansion medium, change the medium once every other day, and wait until the degree of cell fusion reaches When 90% (80-90% is acceptable), digest and passage with 0.05% (0.05-1% acceptable, M / V) trypsin (purchased from sigma) to obtain hu...

Embodiment 3

[0070] Embodiment 3, identification of human vein endothelial cells

[0071] 1. Growth characteristics and morphological characteristics of human vein endothelial cells

[0072] Observe the human vein endothelial cells (the human vein endothelial cells isolated in Example 1 are expanded) induced in Example 2 to adhere to the wall for 2 days with a microscope, as figure 1 As shown, there are scattered adherent cells in the shape of paving stones under the microscope. After the full amount of medium is changed and cultured for 3-4 days, the formation of monoclonal cells can be seen ( figure 1 A), when the cells are cultured until about 90% of the cell confluence ( figure 1 B), the cells are in the shape of typical paving stones, with fast proliferation and relatively uniform morphology, which can be used for the identification of cell biological characteristics.

[0073] 2. Identification of surface markers of human vein endothelial cells by flow cytometry

[0074]The third g...

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Abstract

The invention relates to a method for obtaining human vein endothelial cells from human umbilical cord veins. The method comprises separation of the human vein endothelial cells and in vitro amplification of the human vein endothelial cells, and the separation of the human vein endothelial cells comprises: immobilizing umbilical vein outer cells with alcohol, digesting umbilical vein endothelial cells with collagenase, and collecting many pure umbilical vein endothelial cells; and platelet lysate free of animal source and serum components is used in the in vitro amplification process of the human vein endothelial cells , so potential threat of animalic nonhomology components and serum to the cell application in the cell culture process is avoided. The human vein endothelial cells obtained through using the method can be used for researching newborn blood vessel mechanism and screening antitumor newborn blood vessel medicines.

Description

technical field [0001] The invention belongs to the separation and expansion of cells in the technical field of bioengineering, in particular to a method for obtaining human vein endothelial cells, including the separation and expansion in vitro. Background technique [0002] Endothelial cells have many biological functions, and can be used in the remodeling of blood vessels in atherosclerosis, reducing vascular damage, and preventing cardiovascular diseases; alleviating the ischemic symptoms of lower limbs in patients with diabetes and preventing non-traumatic amputation; in addition, endothelial cells Positive reports have been reported in drug research targeting angiogenesis, tissue regeneration, and wound healing. [0003] The isolation methods of umbilical vein endothelial cells include: mechanical scraping, tissue block transplantation, magnetic bead sorting after tissue digestion, and collagenase grouting. The positive rate of cells in the first two methods is relati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071
CPCC12N5/0602C12N2500/84C12N2501/165C12N2509/00
Inventor 李志刚丁炜王煜骏刘洁杜颖王颖颖杨海莲吴兰兰阮志吕岩白光伟聂晨飞曾照民马瑞
Owner BEIJING JING MENG STEM CELL TECH
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