Intensive promotion of spheroid formation by soluble factors in a hepatocyte-conditioned medium

We developed a hybrid artificial liver and a drug metabolism simulator usin g polyurethane foam (PUF) in which primary hepatocytes spontaneously form f unctional spheroids. Gel filtration liquid chromatography analysis of a hep atocyte-conditioned medium during spheroid formation showed that some subst ances secreted by primary rat hepatocytes accumulated advantageously inside the pores of PUF compared with outside. Similar substances were detected i n a hepatocyte-conditioned medium from a positively-charged surface by conc entrating the substances using an ultrafiltration membrane of a molecular w eight-cutoff of 50 kD. These substances were shown to act as soluble factor s on freshly isolated primary rat hepatocytes to promote spontaneous and ra pid spheroid formation, depending on their concentration by preventing them from initially attaching and spreading on a positively-charged surface. In particular, using 50-fold concentrated substances, about 80% of total hepa tocytes formed the floating spheroids within 72 h of culture. The resulting spheroids had a diameter distribution mainly ranging from 40 to 70 mu m an d expressed high-level liver-specific functions compared with a conventiona l monolayer.