Efficient combinatorial approach to isolating rat pulmonary endothelial cell phenotypes

Abstract

Endothelium from discrete vascular segments within the lung displays phenotypic and functional heterogeneity. However, isolation of segment specific endothelial cells (ECs) is a time and cost intensive process. Thus the aim of this current work was to develop an efficient method for obtaining ECs from rat pulmonary artery (PAECs), pulmonary vein (PVECs) and pulmonary microvasculature (PMVECs). Following surgical removal, vessels were digested using type II collagenase and trypsin. The enzymatically obtained cells were plated in D‐valine/heparin endothelial selective growth media on standard culture dishes. Non‐ECs resistant to the D‐valine media were removed by scraping. Lectin panning and differential cell binding were utilized to remove any remaining contaminating cells. Cell cultures were characterized using an EC marker panel: platelet endothelial cell adhesion molecule 1 (PECAM1), endothelial nitric oxide synthase (eNOS), von Willebrand factor (vWF), vascular endothelial cadherin (VE‐cadherin), and acetylated LDL uptake. Specific lectin binding was used to confirm the segmental origin of the ECs. PAECs bound Helix pomatia and Sambucus nigra, while PMVECs bound Griffonia simplicifolia. This combinatorial approach to isolating rat lung endothelial cells consistently yields multiple cell lines from individual animals. Supported by 5PO1HL066299.