Enhancement of cell-cell interactions and, hence, long-term function in liver support systems can be effected by controlling the diameters of hepatocyte aggregates or spheroids. In this study, primary rat hepatocytes were induced to rapidly form spheroids using an intermittent settling and agitation protocol. The cells were seeded into albumin coated flasks at densities ranging from 80,000 to 520,000 cells/cm2. Hepatocytes were resuspended for 15 sec at 20-min intervals by placing the flasks on a timer controlled linear shaker. At time points ranging from 8 to 24 hr, hepatocyte aggregates were imaged via light microscopy. Mean spheroid diameter and shape factor were determined using computer analysis of captured images. Spheroid diameter could be controlled within the range of 60 to 240 microns. For long-term evaluation, spheroids were microencapsulated and cultured for 21 days under perfusion conditions. Encapsulated spheroids secreted albumin at rates comparable to collagen sandwich control cultures for at least 14 days, with peak rates (approximately 80 microns/day/10(6) cells) exhibited after culture medium changes. The results show that controlled, high efficiency hepatocyte aggregation can be accomplished in as little as 8 hr, and that the encapsulated spheroids exhibit long-term in vitro function.