HUVEC (human umbilical vein endothelial cell) separation, culture and subculture method

A technology of endothelial cells and separation methods, applied in the direction of artificial cell constructs, animal cells, vertebrate cells, etc., can solve the problems of mixed blood vessel wall cells, difficult to control strength, and small number of cells

Inactive Publication Date: 2012-12-19
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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Problems solved by technology

Cut the umbilical vein, scrape the endothelial cells with the back of a scalpel, or use a separation tube with a nylon mesh, but it is difficult to control the strength of this method. If the force is too

Method used

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Embodiment 1

[0013] Take the 20cm-long fresh umbilical cord of the newborn on the same day, put it in the sterile PBS solution containing double antibodies, and store it at 4°C; use a blunt needle to insert it into the umbilical cord vein, and rinse it with PBS solution until no blood is visible in the outflow fluid ;Clamp the lower end of the umbilical cord, add 15ml of collagenase type I (1mg / ml) to digest in a 37°C water bath for 15 minutes; Rinse the umbilical cord 3 times; centrifuge the collected solution at 2000r / min for 5 minutes; discard the supernatant, add 10ml M199 medium (containing phenol red, 20% newborn calf serum, 2mmol / L L-glutamine, 100U / ml penicillin , 100U / ml streptomycin, 100μg / ml vascular endothelial growth factor-heparin), blow off the cells with an elbow, transfer all the liquid into a gelatin-coated culture bottle, and store in CO 2 The incubator was incubated at 37°C. After 24 hours, discard the culture medium, wash it with PBS solution for 3 times, add 10ml of ...

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Abstract

The invention relates to an HUVEC (human umbilical vein endothelial cell) separation, culture and subculture method belonging to the technical field of biology. The method comprises the following steps: (1) separation of HUVEC: taking a fresh umbilical cord, cleaning with a sterile PBS (phosphate buffer solution) containing penicillin-streptomycin, adding collagenase to perform water bath digestion at 37 DEG C, centrifuging the digestion solution, then adding an M199 culture medium, transferring into a gelatin-coated culture bottle, and culturing in a CO2 incubator at 37 DEG C; (2) culture of HUVEC: after the HUVEC is cultured at 37 DEG C for 24 hours, pouring out the culture medium, cleaning with the PBS, adding a fresh M199 culture medium, and afterwards, changing the culture medium once every two days, wherein the HUVEC can be subcultured generally after being cultured for 5-7 days; and (3) subculture of HUVEC: pouring out the culture medium, cleaning with the PBS, adding the digestion solution to digest the cell, adding a DMEM (dulbecco's modified eagle medium) containing serum to terminate the reaction once the cell is rounded, centrifuging the digested cell, and adding a fresh culture medium, wherein the HUVEC subcultured for 2-3 generations is used for experiments. The separated HUVEC is economical and practical, is simple and easy to use, and is beneficial to obtaining required in-vitro experimental model cells.

Description

1. Technical field [0001] The invention relates to a method for separating, cultivating and passing down human umbilical vein endothelial cells HUVEC, and belongs to the field of biotechnology. 2. Background technology [0002] Vascular endothelial cells have a variety of physiological functions, can produce and secrete many biologically active substances, and play an important role in maintaining vasomotor and anticoagulation. The successful culture of vascular endothelial cells in vitro is the basis for studying the function of endothelial cells and their role in the occurrence and development of various diseases. Human umbilical vein endothelial cells (HUVEC) isolated from the free umbilical cord of the placental end of healthy puerpera are widely used in experimental research. [0003] Human umbilical vein endothelial cells have poor growth ability in vitro, and the primary culture is not easy to succeed. In the past, mechanical separation and tissue block transplantat...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 黄午阳李春阳闫征王兴娜王帆王健
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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