Microvascular and Macrovascular Endothelial Cell Isolation and Purification from Lung-Derived Samples

Summary

Although challenging, the isolation of pulmonary endothelial cells is essential for studies on lung inflammation. The present protocol describes a procedure for the high-yield, high-purity isolation of macrovascular and microvascular endothelial cells.

Abstract

The availability of cells isolated from healthy and diseased tissues and organs represents a key element for personalized medicine approaches. Although biobanks can provide a wide collection of primary and immortalized cells for biomedical research, these do not cover all experimental needs, particularly those related to specific diseases or genotypes. Vascular endothelial cells (ECs) are key components of the immune inflammatory reaction and, thus, play a central role in the pathogenesis of a variety of disorders. Notably, ECs from different sites display different biochemical and functional properties, making the availability of specific EC types (i.e., macrovascular, microvascular, arterial, and venous) essential for designing reliable experiments. Here, simple procedures to obtain high-yield, virtually pure human macrovascular and microvascular endothelial cells from the pulmonary artery and lung parenchyma are illustrated in detail. This methodology can be easily reproduced at a relatively low cost by any laboratory to achieve independence from commercial sources and obtain EC phenotypes/genotypes that are not yet available.

Introduction

The vascular endothelium lines the inner surface of the blood vessels. It plays key roles in regulating blood coagulation, vascular tone, and immune-inflammatory responses^1,2,3,4. Although the culture of endothelial cells (ECs) isolated from human specimens is essential for research purposes, it must be remarked that the ECs from different blood vessels (arteries, veins, capillaries) have specific functions. These cannot be fully recapitulated by human umbilical vein endothelial cells (HUVEC), which are easily available and widely used in st....

Protocol

This study was approved, and the protocol followed the guidelines of the human research ethics committee of the University of Chieti-Pescara (#237_2018bis). Figure 1 illustrates the isolation of endothelial cells from segments (1-3 cm long) of pulmonary parenchyma or pulmonary artery from deidentified human subjects (with written consent) undergoing thoracic surgery for various reasons, such as pneumothorax or lobectomy. In this latter case, the surgeons also collected a pulmonary artery seg.......

Discussion

The multiple roles played by vascular endothelial cells in human pathophysiology make these cells an indispensable tool for in vitro pathogenetic and pharmacological studies. Since ECs from different vascular sites/organs display peculiar features and functions, the availability of healthy and diseased ECs from the organ of interest would be ideal for research purposes. For instance, HLMVECs are essential for studies on lung inflammation; therefore, a methodology for the high-yield, high-purity isolation of thes.......

Materials

Name	Company	Catalog Number	Comments
0.05% trypsin-EDTA 1X	GIBCO	25300-054	Used to detach cells from the culture plates
Anti CD31 Antibody, clone WM59	Dako	M0823	Used for CD-31 staining in immunocytochemistry. Dilution used: 1:50
Anti vWF Antibody	Thermo Fisher Scientific	MA5-14029	Used for von Willebrand factor staining in immunocytochemistry. Working dilution: 1:100
Autoclavable surgical scissors	Any		Used for chopping specimens
Cell strainers 40 µm	Corning	431750	Used during the second filtration
Cell strainers 70 µm	Corning	431751	Using during the first filtration
Collagenase, Type 2	Worthington	LS004177	Type 2 Collagenase used for enzymatic digestion. Working concentration: 2 mg/mL
Conjugated anti CD31 Antibody	BD Biosciences	555445	Used for cell sorting (1:20 dilution)
Dulbecco′s Phosphate Buffered Saline (PBS) with  CaCl2 and MgCl2	Sigma-Aldrich	D8662	Used for cell washing before medium change
Dulbecco′s Phosphate Buffered Saline (PBS) without CaCl2 and MgCl2	Sigma-Aldrich	D8537	Used for washing surgical specimens and cells before trypsinization
Endothelial Cell Growth Medium MV	PromoCell	C-22020	HLMVEC growth medium
Fibronectin	Sigma-Aldrich	F0895	Fibronectin from human plasma used for plate coating. Working concentration: 50 µg/mL
Gelatin from porcine skin, type A	Sigma-Aldrich	G2500	Used for plate coating
Type A gelatin	Sigma-Aldrich	g-2500	Gelatin from porcine skin used for plate coating. Working concentration: 1.5%