RAPID HEPATOCYTE SPHEROID FORMATION - OPTIMIZATION AND LONG-TERM FUNCTION IN PERFUSED MICROCAPSULES

Enhancement of cell-cell interactions and, hence, long-term function i n liver support systems can be effected by controlling the diameters o f hepatocyte aggregates or spheroids. In this study, primary rat hepat ocytes were induced to rapidly form spheroids using an intermittent se ttling and agitation protocol. The cells were seeded into albumin coat ed flasks at densities ranging from 80,000 to 520,000 cells/cm(2). Hep atocytes were resuspended for 15 sec at 20-min intervals by placing th e flasks on a timer controlled linear shaker. At time points ranging f rom 8 to 24 hr, hepatocyte aggregates were imaged via light microscopy . Mean spheroid diameter and shape factor were determined using comput er analysis of captured images. Spheroid diameter could be controlled within the range of 60 to 240 mu m. For long-term evaluation, spheroid s were microencapsulated and cultured for 21 days under perfusion cond itions. Encapsulated spheroids secreted albumin at rates comparable to collagen sandwich control cultures for at least 14 days, with peak ra tes (similar to 80 mu cm/day/10(6) cells) exhibited after culture medi um changes. The results show that controlled, high efficiency hepatocy te aggregation can be accomplished in as little as 8 hr, and that the encapsulated spheroids exhibit long-term in vitro function.