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.

Tissue engineering with three-dimensional biomaterials represents a promising approach for developing hepatic tissue to replace the function of a failing liver. Herein, we address cell seeding and distribution within porous alginate scaffolds, which represent a new type of porous biomaterial for tissue engineering. The hydrophilic nature of the alginate scaffold as well as its pore structure and interconnectivity enabled the efficient seeding of hepatocytes into the scaffolds, that is, 70-90% of the initial cells depending on the seeding method. Utilization of centrifugal force during seeding enhanced cell distribution in the porous scaffolds, consequently enabling the seeding of concentrated cell suspensions (>1 x 10(7) cells/mL). Cell density in scaffolds affected hepatocyte viability as judged by MTT assay. At a cell density of 0.28 x 10(6) cells/cm3 scaffold, the number of viable hepatocytes decreased to 33% of its initial value within 7 days, whereas at the denser cultures, 5.7 x 10(6) cells/cm3 scaffold and higher, the cells maintained higher viability while forming a network of connecting spheroids. In the high-density cellular constructs, hepatocellular functions such as albumin and urea secretion, and detoxification (cytochrome P-450 and phase II conjugating enzyme activities), remained high during the 7-day culture. Collectively, the results of the present study highlight the importance of cell density on the hepatocellular functions of three-dimensional hepatocyte constructs as well as the advantages of alginate matrices as scaffoldings.

Two isomers of methanocarba (MC) thymidine (T), one an effective antiherpes agent with the pseudosugar moiety locked in the North (N) hemisphere of the pseudorotational cycle (1a, N-MCT) and the other an inactive isomer locked in the antipodean South (S) conformation (1b, S-MCT) were used to determine whether kinases and polymerases discriminate between their substrates on the basis of sugar conformation. A combined solid-state and solution conformational analysis of both compounds, coupled with the direct measurement of mono-, di-, and triphosphate levels in control cells, cells infected with the Herpes simplex virus, or cells transfected with the corresponding viral kinase gene (HSV-tk), suggests that kinases prefer substrates that adopt the S sugar conformation. On the other hand, the cellular DNA polymerase(s) of a murine tumor cell line transfected with HSV-tk incorporated almost exclusively the triphosphate of the locked N conformer (N-MCTTP), notwithstanding the presence of higher triphosphate levels of the S-conformer (S-MCTTP).