A kind of separation and culture method of guinea pig common carotid artery endothelial cells

Abstract

The invention discloses a guinea pig common carotid artery endothelial cell separation and culture method. The method comprises the following steps: (1) cutting off common carotid artery from a guinea pig under a sterile condition; (2) cutting the common carotid artery into long sections, putting the long sections into a PBS solution, peeling off the outer membrane of common carotid artery, cutting common carotid artery in the vertical direction, putting the inner membrane of common carotid artery downwards, paving common carotid artery on a container filled with digestive fluid to make the inner membrane of common carotid artery fully contact the digestive fluid, then sealing the container, and allowing the container to stand still in an environment with a temperature of 4 DEG C; (3) adding fetal calf serum into the container, collecting the fluid in the container, subjecting the collected fluid to centrifugation in a centrifuge, removing the supernate to obtain common carotid artery endothelial cells, putting the obtained common carotid artery endothelial cells in an EBM culture medium, when the logarithmic phase is reached, transferring the common carotid artery endothelial cells to an incubator to keep on culturing the cells, and finally carrying out subculture. The provided separation and culture method has the advantages that the living rate is high, the damage to cells is low, and furthermore, the method is economic, practical, and simple, is easy to perform, and is suitable for massive production.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for separating and culturing guinea pig common carotid artery endothelial cells. Background technique [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. Endothelial cells (ECs) isolated from common carotid arteries of guinea pigs are vascular endothelial cells widely used in experimental research. [0003] Endothelial cells have poor growth ability in vitro, and primary culture is not easy to succeed. In the past, mechanical separation and tissue block transplantation were mainly used. Cut the umbilical vein, scrape the endo...

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

In recent years, the separation of endothelial cells has been gradually replaced by enzymatic digestion. The cells obtained by this method are relatively pure and can better maintain their biological activity. However, trypsin has a strong ability to decompose the cell membrane and can destroy the integrity of the cells. , affect the viability of cells, and the endothelial cells obtained from digestion may contain a large number of fibroblasts, which will affect subsequent experiments. The collagenase digestion time is long, the cells are not fully dissociated and the damage is large, and the cells obtained are very few. Culture Poor cell viability

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