Electron microscopy of endothelial cells in culture: IV. Surface replicas (original) (raw)
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Culturing endothelial cells of microvascular origin
Journal of Tissue Culture Methods, 2000
There is considerable divergence of opinion on the best methods for the isolation and in vitro culture of microvascular endothelium. Reports have either only described the isolation without mentioning culture conditions or have not fully defined the cell population being cultured. Even at the close of the 20th Century, the isolation and in vitro culture of endothelial cells of microvasculature origin still proves to be technically difficult and many questions remain. These questions need to be addressed by improvements to current methods of isolation and culture of Endothelial cells. A number of more 'high-tech' approaches to this isolation are being explored currently. Use of a more definitive panel of antibodies for immunocytochemical characterization, should enable a more confident characterization of the endothelial cell preparations cultured in vitro. Cell adhesion molecules such as ELAM and VCAM can be used to assist in determining cell population purity.
SURFACE PROPERTIES OF PULMONARY ENDOTHELIAL CELLS
Annals of the New York Academy of Sciences, 1983
Our long-term goal is to help elucidate the means by which pulmonary endothelial cells participate in establishing the quality of systemic arterial blood. Our special interests are in the cellular and molecular means by which the cells actively process polypeptide hormones, prohormones and other excitatory substances so as to determine which pass on to distant target tissues and which do not.' There appears to be a previously unrecognized close coupling, at the level of the endothelial cell, between the processing of circulating hormones, for example, degradation of bradykinin,* and cellular responses to the hormones, for example, release of PG12,3 many of which are perhaps influenced by and influence lung performance as well as the performance of distant tissues. High levels of integration are implied. Nonetheless, it remains true that our understanding of pulmonary endothelium as a metabolically active and responsive tissue will depend in large measure on understanding the detailed topography of the endcthelial surface in terms of relationships between enzymes, binding sites, and extracellular material. Here we report a method for obtaining surface replicas of endothelial cells showing previously unrecognized structures and the distribution of two known surface enzymes.
Endothelial cell cultures as a tool in biomaterial research
Journal of materials science. Materials in medicine
Progress in biocompatibility and tissue engineering would today be inconceivable without the aid of in vitro techniques. Endothelial cell cultures represent a valuable tool not just in haemocompatibility testing, but also in the concept of designing hybrid organs. In the past endothelial cells (EC) have frequently been used in cytotoxicity testing of materials, especially polymers, used in blood-contacting implants, as well as for investigating seeding technologies for vascular prostheses. At present the exponential development both in theory and practice of cell and molecular biology of the endothelium offers great promise in the biomaterial field. Up until now this EC research field has mostly been non-biomaterial orientated. Nevertheless, the relevance for biomaterial research is apparent. Four aspects will be concisely reviewed under the headings inflammation, with special reference to cell adhesion molecules (CAMs) and cytokines, angiogenesis, focusing on the healing response, ...
A new, rapid and reproducible method to obtain high quality endothelium in vitro
Cytotechnology, 2013
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 endotheliummimicking experimental model convenient for multiple research goals.
Isolation and culture of endothelial cells from human bone marrow
British Journal of Haematology, 1994
Adhesive interactions between haemopoietic progenitor cells and bone marrow sinusoidal endothelium are potentially important in the homing of these cells back to the extravascular compartment of the marrow to reestablish haemopoiesis following stem cell transplantation. A simple method for the isolation and culture of human bone marrow endothelial cells is described using bone marrow aspirates obtained from patients undergoing bone marrow harvests for autologous or syngeneic bone marrow transplantation. The method is based on the selective binding of the lectin Ulex europaeus agglutinin-1 (LEA-1) to endothelial cells. Magnetic Dynabeads coupled with UEA-1 were incubated with single cell suspensions of bone marrow following red cell lysis, and bound cells were isolated with a magnet. The isolated cells demonstrated positive immunofluorescence staining for von Willebrand factor. Cells were plated onto tissue culture flasks coated with extracellular matrix derived from human umbilical vein endothelial cells in an endothelial serum-free medium together with 5% fetal calf serum for 24 h. Cells were then cultured in endothelial serum-free growth medium supplemented with 5% fetal calf serum, endothelial cell growth supplement and heparin. After 2-4 weeks in culture. two morphologically different cell populations can be identified. One has a polygonal spindle-shaped morphology with a rapid growth rate, the other a rounded morphology and a slow growth rate. Both populations have a vesiculated cytoplasm. Positive immunostaining of the cells was demonstrated with a number of endothelial cell markers including von Willebrand factor, and antibodies to ICAM-1, VCAM-1, E-selectin, CD31 and BMAl20. Weibel-Palade bodies were observed by electron microscopy. Culture of these ceIls will allow detailed in vitro studies of adhesion mechanisms in the homing of haemopoietic progenitor cells.
Journal of Microscopy, 1996
Rat liver sinusoidal endothelial cells (LEC) contain fenestrae, which are clustered in sieve plates. Fenestrae control the exchange of fluids, solutes and particles between the sinusoidal blood and the space of Disse, which at its back side is flanked by the microvillous surface of the parenchymal cells. The surface of LEC can optimally be imaged by scanning electron microscopy (SEM), and SEM images can be used to study dynamic changes in fenestrae by comparing fixed specimens subjected to different experimental conditions. Unfortunately, the SEM allows only investigation of fixed, dried and coated specimens. Recently, the use of atomic force microscopy (AFM) was introduced for analysing the cell surface, independent of complicated preparation techniques. We used the AFM for the investigation of cultured LEC surfaces and the study of morphological changes of fenestrae. SEM served as a conventional reference.