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Method for separating and purifying mouse lung microvascular endothelial cell

An endothelial cell, separation and purification technology is applied in the field of separation and extraction of high-purity microvascular endothelial cells, which can solve the problems of difficult purification by manual operation, inability to determine the deblocking time, and difficulty in avoiding the pollution of pulmonary microvascular endothelial cells.

Inactive Publication Date: 2015-02-25
BEIJING UNIV OF AGRI
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Problems solved by technology

[0004] Currently, the in vitro culture of pulmonary microvascular endothelial cells mainly adopts enzyme digestion method and tissue patch method. Enzyme digestion method is easy to obtain a large number of microvascular endothelial cells in a short period of time, but non-microvascular endothelial cells will be uniformly mixed in it, and it is difficult to purify by simple manual operation; The patch method also cannot determine the best deblocking time due to differences in tissue samples and processing methods, and it is difficult to avoid contamination by the swimming of pulmonary microvascular endothelial cells

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  • Method for separating and purifying mouse lung microvascular endothelial cell

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Embodiment 1

[0017] 1. Main experimental materials.

[0018] (1) Experimental animals: 7-day-old SD rats.

[0019] (2) Hank's solution: purchased from GIBCO Company, product number 14170-112.

[0020] (3) 0.01 M PBS: Take 8.0 g of NaCl, Na 2 HPO 4 2.9 g, KH 2 PO 4 0.2 g and 0.2 g KCl, dissolved in 1000 mL ultrapure water, sterilized by 0.22 μm filter, and stored in a refrigerator at 4°C.

[0021] (4) Digestive solution: prepared with 0.01 M PBS, containing 0.2% collagenase (purchased from Worthington, Cat. No. LS004176) and 0.1% neutral protease (purchased from Sigma, Cat. No. D4693-1G), sterilized by filtration with a 0.22 μm sterile filter.

[0022] (5) Complete medium: DMEM basal medium (purchased from GIBCO, product number 11960-044), containing 20% ​​fetal bovine serum (purchased from GIBCO, product number 10099-141), 2 mM L-glutamine (purchased from GIBCO Company, product number 25030-081), 1% double antibody solution (100 IU / ml penicillin and 100 μg / ml streptomycin, purc...

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Abstract

A method for separating and purifying mouse lung microvascular endothelial cell is characterized by comprising using an enzyme to perform digestive treatment on subpleural lung marginal tissue to obtain a single-cell suspension, and performing CD31 immunomagnetic bead sorting to obtain the target cell. Concretely, the method comprises getting subpleural lung marginal tissue of a large suckling mouse, firstly using a mixed solution of collagenase II and neutral protease for digestion to prepare the single-cell suspension, then performing incubation combination with CD31 immunomagnetic bead, then utilizing a magnetic bead sorting purification system to screen CD31+ cell, and removing magnetic beads combined with CD31+ cell by using a magnetic-bead release solution, so as to obtain high-purity mouse lung microvascular endothelial cell. The advantages comprise that the high-purity mouse lung microvascular endothelial cell can be obtained from mouse lung tissue through separation extraction by using enzyme digestion to prepare the single-cell suspension and performing CD31 immunomagnetic bead sorting.

Description

technical field [0001] The invention relates to a method for separating and extracting high-purity microvascular endothelial cells from rat lung tissue. Background technique [0002] Microvascular endothelial cells are located on the inner surface of microvessels, and are the necessary barriers for nutrients, pathogenic factors, cytokines and drugs to enter the extravascular tissues from the blood circulation system. The key regulatory points of basic pathological changes such as immunity, tumorigenesis, etc. The in vitro culture of microvascular endothelial cells provides an important way to deeply understand their biological characteristics, and is of great significance to the research and development of ways to protect and treat diseases caused by microvascular endothelial cell dysfunction and drug diseases. [0003] The phenotype and function of microvascular endothelial cells in different tissues and organs are significantly heterogeneous, and the study of the physiolo...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 张涛姜代勋穆祥
Owner BEIJING UNIV OF AGRI
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