No abstract

Hepatocyte aggregation into spheroids attributes to their increased activity, but in the absence of a vascular network the cells in large spheroids experience mass transfer limitations. Thus, there is a need to define the spheroid size which enables maximal cell viability and productivity. We developed a combined theoretical and experimental approach to define this optimal spheroid size. Hepatocyte spheroids were formed in alginate scaffolds having a pore diameter of 100 microm, in rotating T-flasks or spinners, to yield a maximal size of 100, 200, and 600 microm, respectively. Cell viability was found to decrease with increasing spheroid size. A mathematical model was constructed to describe the relationship between spheroid size and cell viability via the oxygen mass balance equation. This enabled the prediction of oxygen distribution profiles and distribution of viable cells in spheroids with varying size. The model describes that no oxygen limitation will take place in spheroids up to 100 microm in diameter. Spheroid size affected the specific rate of albumin secretion as well; it reached a maximal level, i.e., 60 microg/million cells/day in 100-microm diameter spheroids. This behavior was depicted in an equation relating the specific albumin secretion rate to spheroid size. The calculated results fitted with the experimental data, predicting the need for a critical number of viable hepatocytes to gain a maximal albumin secretion. Taken together, the results on mass transport in spheroids and its effects on cell viability and productivity provide a useful tool for the design of 3D scaffolds with pore diameters of 100 microm.

No abstract

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.