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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 responses1,2,3,4. 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.......

Representative Results

HLMEC isolation
The main problem during the isolation of HLMVECs is the presence of contaminating cells since the microscopic capillaries cannot be easily separated from the stromal tissue. Therefore, achieving the highest possible purity at the earliest stages of the isolation process is crucial in order to reduce the culture passages and, thus, the cell aging. Likewise, an optimal isolation protocol should give the highest possible yield of pure HLMVECs. To achieve these goals, a new procedure wa.......

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.......

Acknowledgements

This work was supported by funds from the Italian Ministry of the University and Research (ex 60% 2021 and 2022) to R. P. and by grants from the Italian Cystic Fibrosis Foundation (FFC#23/2014) and from the Italian Ministry of Health (L548/93) to M. R.

....

Materials

NameCompanyCatalog NumberComments
0.05% trypsin-EDTA 1XGIBCO25300-054Used to detach cells from the culture plates
Anti CD31 Antibody, clone WM59DakoM0823Used for CD-31 staining in immunocytochemistry. Dilution used: 1:50
Anti vWF AntibodyThermo Fisher ScientificMA5-14029Used for von Willebrand factor staining in immunocytochemistry. Working dilution: 1:100
Autoclavable surgical scissorsAnyUsed for chopping specimens
Cell strainers 40 µmCorning431750Used during the second filtration
Cell strainers 70 µmCorning431751Using during the first filtration
Collagenase, Type 2WorthingtonLS004177Type 2 Collagenase used for enzymatic digestion. Working concentration: 2 mg/mL
Conjugated anti CD31 AntibodyBD Biosciences555445Used for cell sorting (1:20 dilution)
Dulbecco′s Phosphate Buffered Saline (PBS) with  CaCl2 and MgCl2Sigma-AldrichD8662Used for cell washing before medium change
Dulbecco′s Phosphate Buffered Saline (PBS) without CaCl2 and MgCl2Sigma-AldrichD8537Used for washing surgical specimens and cells before trypsinization
Endothelial Cell Growth Medium MVPromoCellC-22020HLMVEC growth medium
FibronectinSigma-AldrichF0895Fibronectin from human plasma used for plate coating. Working concentration: 50 µg/mL
Gelatin from porcine skin, type ASigma-AldrichG2500Used for plate coating
Type A gelatinSigma-Aldrichg-2500Gelatin from porcine skin used for plate coating. Working concentration: 1.5%

References

  1. Muller, W. A. Leukocyte-endothelial-cell interactions in leukocyte transmigration and the inflammatory response. Trends in Immunology. 24 (6), 326-333 (2003).
  2. Sumpio, B. E., Timothy Riley, J., Dardik, A. Cells in focus: En....

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Endothelial CellsLungTissue SampleBlood VesselsPurificationIsolationMicrovascularMacrovascularCollagenaseCell StrainerCentrifugationCell Culture

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