A new, rapid and reproducible method to obtain high quality endothelium in vitro

Abstract

Human umbilical vein endothelial cells (HUVECs) cultured in vitro are a commonly used experimental system. When properly differentiated they acquire the so-called cobblestone phenotype; thereby mimicking an endothelium in vivo that can be used to shed light on multiple endothelial-related processes. In the present paper we report a simple, flexible, fast and reproducible method for an efficient isolation of viable HUVECs. The isolation is performed by sequential short trypsinization steps at room temperature. As umbilical cords are often damaged during labor, it is noteworthy that this new method can be applied even to short pieces of cord with success. In addition, we describe how to culture HUVECs as valid cobblestone cells in vitro on different types of extracellular matrix (basement membrane matrix, fibronectin and gelatin). We also show how to recognize mature cobblestone HUVECs by ordinary phase contrast microscopy. Our HUVEC model is validated as a system that retains important features inherent to the human umbilical vein endothelium in vivo. Phase contrast microscopy, immuno-fluorescence and electron microscopy reveal a tight cobblestone monolayer. Therein cells show Weibel-Palade bodies, caveolae and junctional complexes (comparable to the in vivo situation, as also shown in this study) and can internalize human low density lipoprotein. Isolation and culture of HUVECs as reported in this paper will result in an endothelium-mimicking experimental model convenient for multiple research goals.Electronic supplementary materialThe online version of this article (doi:10.1007/s10616-012-9459-9) contains supplementary material, which is available to authorized users.

Analysis of Vascular Mechanical Characteristics after Coronary Degradable Stent Implantation

“…e silicone chamber of the implanted cells was placed in a 37°C, 5% CO 2 environment, and the chamber was removed and the growth state of ECs are still lacking experimental support [21]. Due to individual di erences in in vivo experiments, it is still di cult to directly measure the stress on living intravascular cells [22]. erefore, this paper uses a self-developed in vitro culture device to deeply explore how circumferential stress (strain) a ects the growth state of ECs.…”

Multi‐Material Tissue Engineering Scaffold with Hierarchical Pore Architecture

“…On culture day 5, the endothelial cells were seen lining the flow channels and had utilized most of the available space under all conditions tested (Figure 3C–H). Co-staining with actin and CD-31, an endothelial cell marker, [28] demonstrated cell retention and cobblestone morphology on the open microchannels, [29] while a fluorescent cell-tracking dye demonstrated cell retention in the closed, perfused microchannels. While secondary micropores did not provide a clear benefit to the vascular cells in the device configuration tested herein, future device configurations are envisioned as stacked modular units comprising heart cell layers paired with dedicated, permeable microchannel layers, [8b] and in this envisioned configuration the secondary pores in the microchannels are expected to provide advantages of enabling transport between the individual modules and also reducing polymer volume fraction.…”

Enhanced assay of endothelial exocytosis using extracellular matrix components

“…Other studies have shown that confluency affects endothelial biology. Electron microscopy of confluent “cobblestone” appearing HUVEC cultures revealed myriad hallmarks of endothelial cells [53]. Confluent cells also express more endothelial nitric oxide synthase (eNOS or NOS3) than non-confluent cells [54, 55].…”