RESUMO
We have previously described the design and operation of a microfabricated bioreactor that supports perfused 3D culture of liver cells and facilitates evolution of tissue-like morphological structures. Here, we describe the functional viability of cells maintained in this microarray bioreactor and examine the influence of different seeding protocols on the evolution of structure and function in comparison with static culture. Primary rat hepatocytes were seeded into the perfusion reactors either as single-cell suspensions immediately after isolation or as spheroidal aggregates formed over a 2- to 3-day period. Initial studies in which cells were cultured for 7 days postisolation revealed significantly greater functional activity and morphological stability of cells that were preaggregated for up to 3 days before seeding in the reactor, compared with direct seeding of single cells. Total albumin secretion and urea genesis rates in single-cell reactor cultures declined significantly during this initial culture period while remaining constant in preaggregated reactor cultures. Longer term studies indicate that rates of albumin secretion and urea genesis are maintained at constant levels through 15 days postisolation. These metabolic rates are an order of magnitude higher than observed for the same preaggregated structures cultured statically with comparable medium ratio and exchange conditions. The metabolic function data are supported by light microscopy images showing viable tissue structures, and electron microscopy images that reveal tight junctions, glycogen storage, and bile canaliculi.