Dawn M. Wankowski

GTSF-2: A NEW, VERSATILE CELL CULTURE MEDIUM FOR DIVERSE NORMAL AND TRANSFORMED MAMMALIAN CELLS

SummaryThe aim of this study was to test the versatility of a new basal cell culture medium, GTSF-2. In addition to traditional growth-factors, GTSF-2 contains a blend of three sugars (glucose, galactose, and fructose) at their physiological levels. For these studies, we isolated normal endothelial cells from human, bovine, and rat large blood vessels and microvessels. In addition, GTSF-2 was also tested as a replacement for high-glucose-containing medium for PC12 pheochromocytoma cells and for other, transformed cell lines. The cell growth characteristics were assessed with a novel cell viability and proliferation assay, which is based on the bioreduction of the fluorescent dye, Alamar Blue. After appropriate calibration, the Alamar Blue assay was found to be equivalent to established cell proliferation assays. Alamar Blue offers the advantage that cell proliferation can be measured in the same wells over an extended period of time. For some of the cell types (e.g., endothelial cells isolated from the bovine aorta, the rat adrenal medulla, or the transformed cells), proliferation in unmodified GTSF-2 was equivalent to that in the original culture media. For others cell types (e.g., human umbilical vein endothelial cells and PC12 cells), GTSF-2 proved to be a superior growth medium, when supplemented with simple additives, such as endothelial cell growth supplement (bFGF) or horse serum. Our results suggest that GTSF-2 is a versatile basal medium that will be useful for studying organ-specific differentiation in heterotypic coculture studies.

In Vitro Testing of Endothelial Cell Monolayers Under Dynamic Conditions Inside a Beating Ventricular Prosthesis

Thromboembolic complications remain a major problem associated with the long-term clinical use of cardiac prostheses. A promising approach toward resolving this predicament is lining the blood contacting surfaces with a functional monolayer of endothelial cells (EC). In developing an endothelialized cardiac prosthesis, the authors in the past focused on establishing a confluent EC monolayer on the luminal surface of ventricular blood sacs. In this study, the authors concentrated on exposing the post confluent monolayers to the dynamic conditions inside a beating ventricle. The cells, derived from either bovine aortae or jugular veins, were grown to post confluence inside fully assembled ventricles on fibronectin or plasma cryoprecipitate coated, textured surfaces. After 11 days of culturing under static conditions, the endothelialized ventricles were connected to a mock loop that was run for 6 and 24 hr at 60 bpm and mean flow rate of 3.2 L/min. The status of the monolayer was evaluated by Alamar Blue assay before and after each run, and the extent of surface coverage was determined visually using bright field microscopic study after cell staining with KMnO4 and toluidine blue. In addition, morphometric information on cells/polyurethane surface was obtained with a scanning electron microscope. After 6 hr of pumping, cell staining revealed signs of moderate cell loss in fibronectin coated blood sacs, whereas in cryoprecipitate coated bladders the signs of denudation were marginal. In seven ventricles operated for 24 hr, Alamar Blue measurements indicated 35 +/- 16% of cell loss from monolayers established on fibronectin coating, but only 4.8 +/- 6.25% on cryoprecipitate. Thus, the current study demonstrates the feasibility of maintaining an intact endothelial surface in a beating ventricular prosthesis and indicates that the integrity of the endothelial lining is dependent upon a proper choice of surface macrostructure and protein coating.

Durability of Endothelial Cell Monolayers Inside a Beating Cardiac Prosthesis

Thromboembolic complications associated with the use of cardiac prostheses might be alleviated by lining the blood-contacting surfaces of these devices with a functional monolayer of endothelial cells. In the current study, we tested our hypothesis that precoating textured surfaces of artificial ventricles with various plasma proteins could enhance the resistance of endothelial cell monolayers to hemodynamic forces generated within an in vitro mock circulatory loop system. Bovine jugular vein endothelial cells were grown to confluence on the luminal surface of artificial ventricles constructed of textured, medical grade polyurethane (Biospan), which had been precoated with either fibronectin or plasma cryoprecipitate. Following 7 days of culturing under static conditions, the endothelialized ventricles were connected to a mock loop system, and exposed to pulsatile flow for 6 and 24h (60bpm, 3.21/min mean flow rate, 150mmHg ejection pressure). Retention of endothelial cells was evaluated by Alamar Blue assay before and after each run. Monolayer integrity and additional morphometric parameters were also assessed by direct visualization, employing various light and electron microscopic techniques. In ventricles which had been precoated with fibronectin, Alamar Blue assay indicated cellular retention to be 77% ± 4% and 72% ± 5% of static controls, after 6 and 24h, respectively. In marked contrast, cryoprecipitate-coated ventricles retained over 90% of their endothelial cell lining through 24 h of exposure to physiological hemodynamic conditions. These findings were confirmed by visual inspection. Our study demonstrates the feasibility of maintaining an intact endothelial surface in a beating ventricular prosthesis, and that the durability of the cell layer is highly dependent upon the selection of biomaterial surface topography and protein coating.

Factitious Angiogenesis III: How to Successfully Endothelialize Artificial Cardiovascular Bioprostheses by Employing Natural Angiogenic Mechanisms

In this series of NATO ASI meetings on angiogenesis, we introduced the concept of “factitious angiogenesis” to describe a novel approach in tissue engineering aimed at the generation of permanent, hemocompatible blood conduits (31,32). We define as blood conduits a variety of cardiovascular prostheses, such as artificial vascular grafts, ventricular assist devices and total artificial hearts, and skeletal muscle ventricles. Without belaboring the profound technical and surgical problems associated with their manufacture, implantation, and/or long term use, all of these cardiovascular prostheses share a major, common obstacle: the inadequate hemocompatibility of their blood-contacting surf aces, which are made of various types of biopolymers (23,30,52). We hypothesized that the hemocompatibility in these novel blood conduits can be significantly improved by lining their blood-contacting surfaces with a non-thrombogenic monolayer of autologous endothelial cells (ECs).

Successful endothelialization of cardiovascular prostheses

Hemocompatibility of the blood-contacting surface of artificial cardiac prostheses remains a major challenge for their long-term clinical use. We hypothesized that the thrombogenicity of segmented polyurethanes (PUS) used for creating the blood sacs can be reduced by lining the luminal surface with autologous endothelial cells (EC). Over the past years we have optimized our approach by 1. choosing an appropriate (detoxified) biomaterial (Biospan@), 2. generating a controlled, roughened PU surface and 3. precoating the luminal surface with an autologous protein complex (APC) made from plasma cryoprecipitate, 4. using autologous EC isolated from adipose tissue or the jugular vein, 5. designing a seeding device with 3-D rotation, which allows for the effective, uniform endothelialization of fully assembled cardiac prostheses,and 6. developing techniques for non-destructive monitoring of the extent of EC coverage. In vitro studies confirm the importance of exposing the EC lining the blood sacs to hemodynamic forces (flow and cyclic strain) prior to implantation for establishing a nonthrombogenic, shear-resistant EC monolayer. We recently tested fully endothelialized Ventricular Assist Devices (VADs) in vitro in a mock-loop circulation (3.2 l/min flow at 1 Hz) for 6 and 24 hours. At 6 hours, no denudation was observed. After 24 hours the integrity of the EC-monolayer on fibronectin-coated PUS was partially (<300/o) impaired. By contrast, virtually no denudation was observed when the cells were seeded on Biospan@ precoated with bovine cryoprecipitate. Similar results were obtained in our first er vivo tests in which fully endothelialized VADs were connected to an extracorporeal aorta-to aorta bypass in a calf model. Our results provide the first evidence for sucecssful in vitro and w vivo testing of endothelialized VADs and stress the importance of the composition of the extracellular matrix for maintaining a durable EC monolayer.