Isolation and culture of primary microvascular endothelial cells in vitro

The application of microvascular endothelial growth factor and the development of immunomagnetic beads technology have made the cultivation and purification of microvascular endothelial cells relatively simple.

1. Brief introduction of microvascular endothelial cell culture

The microvascular endothelial cells of the main organs and tissues of the human body have been successfully cultured: skeletal muscle, heart, brain, stomach, retina, lung, skin, choroid, small intestine, fat, liver sinus, kidney, joint synovium, placenta, bone marrow, islet Microvascular endothelial cells of organ tissues such as the cornea and esophagus.

2. Isolation of microvascular endothelial cells

At present, there are mainly three methods for isolating endothelial cells, namely, enzymatic digestion, mechanical separation, and magnetic bead separation.

Enzymatic digestion: The advantage is that the cells are separated and the purity is high; the disadvantage is that the enzyme has a destructive effect on certain proteins. The digested tissue suspension requires serum to terminate the enzyme activity and the tissue suspension is passed through a 200 mesh metal screen to remove undigested tissue.

Mechanical separation method and magnetic bead separation method: the damage of the endothelial cells by the enzyme can be avoided, and the disadvantage is that other cells are more likely to be contaminated.

Magnetic Bead Separation: Endothelocytes are isolated from other cell populations using a specific medium (magnetic beads with endothelial-specific monoclonal antibodies on the surface). Although this method can separate microvascular endothelial cells that can be used for inflammation research, the amount of cells isolated is small.

Mechanical separation: for the separation of large vascular endothelial cells, this method avoids damage to endothelial cells by enzymes and other cells.

3. Purification of microvascular endothelial cells

(1) Purification method: mainly using nylon mesh or stainless steel mesh screen to filter cell suspension, physical scraping method, growth characteristic selection method, local digestion method, differential adhesion method and immunomagnetic beads method.

(2) Growth characteristic selection method: according to the growth characteristics of endothelial cells, that is, the adherent tissue block, after the culture for a period of time, except for the blood cells, the other cells have not left the tissue block and the microvascular endothelial cells swim first, then Immediately remove the tissue block, most of which are left with blood cells and endothelial cells. The blood cells are automatically eliminated after passage from 1st to 2nd generation, and the remaining is microvascular endothelial cells. This method is preferable because it avoids mechanical and chemical damage to cells and does not require special equipment.

(3) Local digestion method: When the boundary between different types of cell groups is obvious, a small amount of digestive enzyme may be added to the target cell area. After a period of time, the digestive juice is taken out together with the cells, and then the digestive enzyme is removed by centrifugation or terminated. After digestion, the cells were seeded in the wells of another culture plate for culture. This method, like physical scraping, requires a relatively large number of cells of interest and is relatively well defined by other cells.

Differential adhesion method: Endothelial cells adhere to the fibroblasts firmly, and the cells are digested with trypsin and monitored under an inverted microscope. When the fibroblasts shrink and become detached, the digestion is terminated immediately with the medium containing fetal bovine serum. The cell fluid was gently aspirated and most of the endothelial cells remained in the wells of the original culture plate. After several treatments, fibroblasts can be substantially removed.

4. Identification of microvascular endothelial cells

So far, microvascular endothelial cells of different organ tissues have not been found to have a common specific protein or marker.

Identification of microvascular endothelial cells:

(1) Comprehensive evaluation based on the origin of organs and tissues, in terms of morphology, phenotype, biochemistry and function.

(2) Microvascular endothelial cells generally grow in a single layer, with contact inhibition, and are typical paving stones. Weibel-Palade bodies can be seen under electron microscope.

(3) Surface expression of CD31, CD34, thrombomodulin, factor VIII and selectin, etc., cells can ingest low-density lipoprotein, produce prostaglandins, endocytosis, and bind to phytohemagglutinin to form a capillary-like network.

5. Training points of microvascular endothelial cells

According to the growth characteristics of microvascular endothelial cells, some special culture conditions are required, and these culture conditions may differ due to the origin of microvascular endothelial cells. The culture of microvascular endothelial cells is generally selected from basal medium, fetal bovine serum, double antibody, glutamine, and endothelial cell growth factor.