- A potential approach to facilitate the performance of implanted hepatocytes is to enable their aggregation and re-expression of their differentiated function prior to implantation. Here we examined the behavior of freshly isolated rat adult hepatocytes seeded within a novel three-dimensional (3-D) scaffold based on alginate. The attractive features of this scaffold include a highly porous structure (sponge-like) with interconnecting pores, and pore sizes with diameters of 100–150 μm. Due to their hydrophilic nature, seeding hepatocytes onto the alginate sponges was efficient. DNA measurements showed that the total cell number within the sponges did not change over 2 weeks, indicating that hepatocytes do not proliferate under these culture conditions. Nearly all seeded cells maintained viability, according to the MTT assay. Within 24 h post-seeding, small clusters of viable cells, were seen scattered within the sponge. More than 90% of the seeded cells participated in the aggregation; the high efficiency is attributed to the non-adherent nature of alginate. The spheroids had smooth boundaries and by day 4 in culture reached an average diameter of 100 μm, which is at the same magnitude of the sponge pore size. The cells appeared to synthesize fibronectin which was deposited on the spheroids. No laminin or collagen type IV were detected in the deposit. The 3-D arrangement of hepatocytes within the alginate sponges promoted their functional expression; within a week the cells secreted the maximal albumin secretion rate of 60 μg albumin/106 cells/day. Urea secretion rate did not depend on cell aggregation and was similar to that obtained when hepatocytes were cultured on collagen type I coated dishes (100 μg/106 cells/day). Our studies show that alginate sponges can provide a conducive environment to facilitate the performance of cultured hepatocytes by enhancing their aggregation. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 344–353, 2000.