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A new simple method for isolation of microvascular endothelial cells avoiding both chemical and mechanical injuries
1Department of Pathophysiology, Second Military Medical University, Shanghai, People's Republic of China.
Abstract:
Our study indicates that when small pieces of lung or muscles of chest wall are cultured, erythrocytes and leukocytes (PMNs) leave the tissues first, followed by vascular endothelial cells (ECs). Fibroblasts and other mixed cells grow after 72 hr culture. The ECs can then be isolated avoiding mechanical and chemical injuries. The lung tissue is obtained from the peripheral surface and muscles from the chest. It is then cut into pieces and cultured with DMEM containing 20% fetal bovine serum. After 60 hr culture, the tissues are discarded. The flask contains only ECs and blood cells. Blood cells can be cleared out after the cells are subcultured once or twice. The primary cells and the subcultured cells cultured on gelatinized culture dish give the capillary-like structure. Cells cultured on untreated dishes have regular cobblestone morphology and junctional contacts. The isolated cells were not mesothelial cells because the cells did not react to antibody against cytokeratin 18, while mesothelial cells reacted strongly to the antibody. The cells can be isolated from the lung tissue without pleura. The primary microvascular ECs are also cultured on microcarriers (cytodex 3). Because both mechanical and proteolytic injuries are avoided, the cells may be more similar to cells in the in vivo state. There are no significant differences in PMN-endothelium adherence and monolayer responses to second messengers, platelet activating factor, and phospholipase A2 when pulmonary and muscular microvascular endothelial cells are compared.
Insights
Researchers developed a method to isolate vascular endothelial cells (ECs) from lung and chest muscles. This technique minimizes injury, yielding cells suitable for studying microvascular functions in vivo.
Area of Science:
- Cell Biology
- Vascular Biology
- Tissue Engineering
Background:
- Endothelial cells (ECs) are crucial for vascular function and integrity.
- Isolating pure microvascular ECs from tissues can be challenging due to mixed cell populations.
- Understanding EC behavior in vitro requires methods that preserve their in vivo characteristics.
Purpose of the Study:
- To establish a reliable method for isolating primary microvascular endothelial cells from lung and chest wall muscles.
- To characterize the isolated cells and compare their morphology and function.
- To assess the suitability of these cells for studying endothelial cell-specific responses.
Main Methods:
- Culturing small tissue explants of lung and chest wall in DMEM with 20% fetal bovine serum.
- Sequential removal of blood cells (erythrocytes, leukocytes) and fibroblasts.
- Subculturing to purify endothelial cells and culturing on gelatinized or untreated dishes.
- Characterization using antibody against cytokeratin 18 and culturing on microcarriers (Cytodex 3).
Main Results:
- Erythrocytes and leukocytes were the first to leave cultured tissues, followed by vascular endothelial cells.
- Fibroblasts appeared after 72 hours; ECs could be isolated before significant fibroblast growth.
- Cells cultured on gelatinized dishes formed capillary-like structures, while those on untreated dishes showed cobblestone morphology.
- Isolated cells were confirmed as non-mesothelial via antibody testing.
- Microvascular ECs cultured on microcarriers showed no significant differences in PMN-endothelium adherence or responses to inflammatory mediators compared to pulmonary and muscular ECs.
Conclusions:
- A method was developed to isolate primary microvascular endothelial cells from lung and chest wall tissues, minimizing mechanical and chemical damage.
- The isolated cells exhibit distinct morphologies and form capillary-like structures, indicating their endothelial origin and functionality.
- This isolation technique provides a valuable tool for studying microvascular endothelial cells in a state closer to their in vivo condition, enabling research on inflammation and vascular responses.