A NEW METHOD FOR CONTINUAL QUANTITATION OF VIABLE CELLS ON ENDOTHELIALIZED POLYURETHANES

Many of the segmented polyurethanes currently used in cardiovascular p rostheses undergo either modification of their surface structure or ar e lined with a confluent monolayer of endothelial cells to improve the ir 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 asses sing the growth of endothelial cells on polyurethanes. Methods, such a s 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 solubilize rs with the various constituents (e.g., catalyzers, antioxidants) and/ or functional groups in the polyurethane formulations. In addressing t hese problems, we adapted a novel, highly reproducible fluorescent ass ay which is based on reduction by viable cells of an electrochemically sensitive compound, Alamar Blue. The bioreduced product is soluble an d stable in culture media and noncytotoxic. In addition, the assay is independent of the geometry or physicochemical properties of the polym eric 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.