A New Method for Continual Quantitation of Viable Cells on Endothelialized Polyurethanes

Many of the segmented polyurethanes currently used in cardiovascular prostheses undergo either modification of their surface structure or are lined with a confluent monolayer of endothelial cells to improve their hemocompatibility. During the establishment of an endothelial cell lining on these biopolymers it is necessary to continually monitor the number of viable cells that are covering the substrate. Yet, not all of the conventional cell enumeration techniques are suitable for assessing the growth of endothelial cells on polyurethanes. Methods, such as direct cell counting, dye uptake, or DNA or protein staining require either a transparent scaffold or lead to termination of the culturing process prior to measurement. In addition, some of the spectroscopic assays are often hampered by interaction of the dyes and/or solubilizers with the various constituents (e.g., catalyzers, antioxidants) and/or functional groups in the polyurethane formulations. In addressing these problems, we adapted a novel, highly reproducible fluorescent assay which is based on reduction by viable cells of an electrochemically sensitive compound, Alamar Blue. The bioreduced product is soluble and stable in culture media and noncytotoxic. In addition, the assay is independent of the geometry or physicochemical properties of the polymeric surfaces. In the present study we focus on the implementation of this assay to monitoring attachment and growth of various endothelial cell types on segmented polyurethanes.