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

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...

AI Technical Summary

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 light, the number of cells obtained is very small; if the force is too heavy, it is easy to mix Other blood vessel wall cells such as fibroblasts, etc., and this method is easy to damage endothelial cells