Abstract

BackgroundThe in vitro culture of endothelial cells (ECs) is an indispensable tool for studying the role of the endothelium in physical and pathological conditions. Primary ECs, however, have a restricted proliferative lifespan which hampers their use in long-term studies. The need for standardized experimental conditions to obtain relevant and reproducible results has increased the demand for well-characterized, continuous EC lines that retain the phenotypic and functional characteristics of their non-transformed counterparts.ResultsPrimary feline ECs from aorta and vena cava were successfully immortalized through the successive introduction of simian virus 40 large T (SV40LT) antigen and the catalytic subunit of human telomerase (hTERT). In contrast to the parental ECs, the transformed cells were able to proliferate continuously in culture. Established cell lines exhibited several inherent endothelial properties, including typical cobblestone morphology, binding of endothelial cell-specific lectins and internalization of acetylated low-density lipoprotein. In addition, the immortalization did not affect the functional phenotype as demonstrated by their capacity to rapidly form cord-like structures on matrigel and to express cell adhesion molecules following cytokine stimulation.ConclusionThe ability to immortalize feline ECs, and the fact that these cells maintain the EC phenotype will enable a greater understanding of fundamental mechanisms of EC biology and endothelial-related diseases. Furthermore, the use of cell lines is an effective implementation of the 3-R principles formulated by Russel and Burch.

Highlights

  • The in vitro culture of endothelial cells (ECs) is an indispensable tool for studying the role of the endothelium in physical and pathological conditions

  • Cell morphology and growth characteristics Aorta At the completion of the primary isolation, aggregates of 20–50 aortic ECs could be seen floating in the medium

  • The frequency of clone formation approximate 5×10-5 and these clones of fast growing cells were further expanded before human telomerase reverse transcriptase (hTERT) transduction

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Summary

Introduction

The in vitro culture of endothelial cells (ECs) is an indispensable tool for studying the role of the endothelium in physical and pathological conditions. Endothelial cells line the inner surface of blood vessels. In this strategic position, they play a key role in a large number of important physiological processes, such as regulation of vascular tone and blood flow, fluid and solute exchange, haemostasis and coagulation, inflammatory responses and angiogenesis. In order to investigate the role of ECs in these events, it has been proven valuable to optimize techniques to isolate, culture and characterize these cells in vitro. When studying the biological characteristics of a disease, it is desirable to use ECs isolated from vessels of the appropriate size and functionality, that originate from the proper anatomical compartment

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