Method for primary culture of human umbilical vessel smooth muscle cells

Abstract

The invention relates to a method for primary culture of human umbilical vessel smooth muscle cells. The method comprises the following culture steps of: 1) cleaning blood on human umbilical cord, separating umbilical vein, and transferring to a sterile culture vessel; 2) cutting the blood vessel along the vertical axis direction, and removing endothelial cells while maintaining the internal membrane surface of the blood vessel upwards; 3) cutting the blood vessel into 5mm*5mm blocks, sucking and transferring to a culture bottle and uniformly paving on a bottle bottom; 4) inverting the culture bottle, and adding a DMEM (Eagle's minimal essential medium) culture liquid containing 20% fetal bovine serum; 5) placing the culture bottle in a 37 DEG C incubator containing 5% CO2, after the tissue blocks cling to the wall 4-6 hours later, carefully overturning the culture bottle, thus immersing the tissue blocks in the culture liquid, and standing and culturing; and 6) exchanging the liquid once every 3 days, and carrying out morphological and immunocytochemical identification on the cells when 3-5 generations of cells are generated.

Description

technical field [0001] The invention relates to the field of human cell culture in medicine, in particular to a method for primary culture of human umbilical vessel smooth muscle cells. Background technique [0002] Vascular smooth muscle cells (VSMCs) are the main cell components that constitute the media of blood vessels, and their contraction and relaxation, proliferation, migration and calcification are closely related to the occurrence of cardiovascular diseases. The culture of human umbilical vascular smooth muscle cells in vitro is the basis and important means for studying the pathogenesis and prevention of cardiovascular diseases. [0003] The in vitro culture of arterial vascular smooth muscle cells began in the 1970s, and was successfully cultured by Ross and Chamley using the tissue patch method; vascular smooth muscle cells cultured in vitro can be obtained from arteries in different parts of the human body, such as thoracic aorta and abdominal aorta , pulmonar...

AI Technical Summary

Problems solved by technology

However, this culture method has the following disadvantages for culturing human umbilical vein smooth muscle cells: 1. Care should be taken when tearing off the adventitia of the artery: since there is no obvious boundary between the adventitia and media of the umbilical vein, how to distinguish the adventitia during the operation? Membrane and media; how to check that the adventitia has been completely removed, and how to ensure that the smooth muscle of the media is not damaged in the process of tearing off the adventitia, etc., currently there is no uniform standard, which brings great difficulties to the experiment

2. After cutting the tissue piece into 1mm×1mm×1mm, when using a straw to move it into the culture bottle, the tissue piece is very small (only the size of a grain of yellow rice) and it is easy to be sucked into the straw and cause cell loss; 3 .Since the tissue is only about 1mm3, it is difficult to spread the tissue blocks evenly on the bottom of the bottle with a straw, and the spacing is 0.5-1cm. Often, 3-5 tissue blocks are gathered together; 4 . It takes a long time (about 2 hours on average) to complete the above-mentioned main operations in the traditional tissue block adherence method for culturing vascular smooth muscle cells. During this period, the Hanks solution or D-hanks solution must be replaced continuously. What is more important is that the umbilical vessels are long. Exposure to the outside for a long time can easily cause cell hypoxia and necrosis, reducing the survival rate of cell culture

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