EFFICIENT ASSEMBLY OF RAT HEPATOCYTE SPHEROIDS FOR TISSUE ENGINEERINGAPPLICATIONS

Freshly harvested primary rat hepatocytes cultivated as multicellular aggregates, or spheroids, have been observed to exhibit enhanced liver -specific function and differentiated morphology compared to cells cul tured as monolayers. An efficient method of forming spheroids in spinn er vessels is described. Within 24 h after inoculation, greater than 8 0% of inoculated cells formed spheroids. This efficiency was significa ntly greater than that reported previously for formation in stationary petri dishes. With a high specific oxygen uptake rate of 2.0 x 10(-9) mmol O-2/cell/h, the oxygen supply is critical and should be monitore d for successful formation. Throughout a 6-day culture period, spheroi ds assembled in spinner cultures maintained a high viability and produ ced albumin and urea at constant rates. Transmission electron microsco py indicated extensive cell-cell contacts and tight junctions between cells within spheroids. Microvilli-lined bile canaliculus-like channel s were observed in the interior of spheroids and appeared to access th e exterior through pores at the outer surface. Spheroids from spinner cultures exhibited at least the level of liver-specific activity as we ll as similar morphology and ultrastructure compared to spheroids form ed in stationary petri dishes. Hepatocytes cultured as spheroids are p otentially useful three-dimensional cell systems for application in a bioartificial liver device and for studying xenobiotic drug metabolism . (C) 1996 John Wiley & Sons, Inc.