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Primary rat hepatocytes can self-assemble to form multicellular spheroids when plated onto Primaria petri dishes. Spheroids have been observed to exhibit enhanced liver-specific functions and differentiated ultrastructure compared to monolayer cultures on dry collagen. With confocal scanning laser microscopy, CYP1A1 activity was evaluated in situ by detecting resorufin. This highly fluorescent molecule is the P450-mediated product of 7-ethoxyresorufin O-dealkylation (EROD). Significantly higher P450 activity was observed in spheroids compared to monolayers on collagen upon induction with 50 microM beta-naphthoflavone (BNF), a CYP1A inducer. This was confirmed by measuring microsomal EROD activity. The distribution of CYP1A1 activity within spheroids was heterogeneous, with higher activity localized to the hepatocytes in the interior. During the process of spheroid formation, cells were initially seen to attach and spread out as a monolayer. This stage was associated with relatively low CYP1A1 activity. As cells formed multicellular structures and aggregated into spheroids, the level of CYP1A1 activity increased over time. At least a fivefold higher fluorescence intensity was observed in spheroids compared to that of monolayers maintained on collagen. The higher P450 activity within spheroids may be associated with their ability to maintain a greater degree of differentiation compared to monolayers. These studies demonstrate the potential of hepatocyte spheroids as a model system for investigating drug metabolism, tissue engineering, and tissue self-assembly.

Hepatocytes can self-assemble into spheroids, which express enhanced differentiated function and may provide an important tool in tissue engineering. This study examines cell junction components as hepatocytes form either monolayer or spheroid, and correlates morphological changes with cytochrome P-450 function. Rat hepatocytes were cultured on either high-density (1 fxg/cm 2 ) or low-density (1 ng/cm 2 ) fibronectin, which promotes monolayers or spheroids, respectively. Immunofluorescence demonstrates that the cell-cell adhesion receptor cadherin localizes to cell borders in monolayers and aggregates. In contrast, actin cytoskeleton differs dramatically between the two morphologies. Actin and /3-catenin, which links cadherins to actin, colocalize in aggregates to a greater extent than in monolayers. The activity of cytochrome P-450 was measured in situ by determining the level of the fluorescent compound, resorufin, a product of P-450-mediated O-dealkylation of 7-ethoxyresorufin. As cells aggregate into spheroids, resorufin increases, with spheroids demonstrating at least fivefold higher fluorescence than monolayers. Spheroids placed on Primaria plates in the presence of serum disassemble into monolayers, with simultaneous decrease in fluorescence as cells develop a flattened morphology. Thus, we report findings that demonstrate a correlation between in vivo-like morphology and specific cytochrome P-450 function.

For decades, parenteral nutrition (PN) has been a successful method for intravenous delivery of nutrition and remains an essential therapy for individuals with intolerance of enteral feedings or impaired gut function. Although the benefits of PN are evident, its use does not come without a significant risk of complications. For instance, parenteral nutrition-associated liver disease (PNALD)—a well-described cholestatic liver injury—and atrophic changes in the gut have both been described in patients receiving PN. Although several mechanisms for these changes have been postulated, data have revealed that the introduction of enteral nutrition may mitigate this injury. This observation has led to the hypothesis that gut-derived signals, originating in response to the presence of luminal contents, may contribute to a decrease in damage to the liver and gut. This review seeks to present the current knowledge regarding the modulation of what is known as the “gut–liver axis” and the gut-derived signals which play a role in PN-associated injury.

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Primary rat hepatocytes can self-assemble to form multicellular spheroids when plated onto Primaria petri dishes or suspended in stirred vessels. These spheroids exhibit prolonged viability, enhanced liver-specific functions and differentiated ultrastructure compared to monolayer cultures. Upon transfer to collagen coated surface, or upon the addition of fetal bovine serum (FBS) to the culture, these spheroids began to disassemble and spread on the surface. The dynamics of cytochrome P450 CYP1A1/2 activity in the course of spheroid disassembly was examined in situ by detection of the fluorescent product, resorufin, of ethoxyresorufin O-dealkylation. Optical sectioning of the disassembling spheroids by confocal microscopy demonstrated that hepatocytes that reverted to monolayer exhibited markedly lower CYP1A1/2 activity than those that remained in a multilayered structure. This occurred whether the disassembly was caused by incubation with FBS-containing medium or by cultivation on a collagen-coated surface. When spheroids were cultured on the surface of agar, the disassembly process was retarded even in the presence of FBS. However, even in those intact spheroids, the exposure to FBS markedly decreased CYP1A1/2 activity. The decreased CYP1A1/2 activity was correlated to a diminished smooth endoplasmic reticulum as seen in the transmission electron micrograph. The results clearly demonstrate that the disassembly of hepatocyte spheroids led to decreased CYP1A1/2 activity. Furthermore, FBS contained a factor that caused CYP1A1/2 to decrease even in intact spheroids.

Several investigators have demonstrated that freshly harvested hepatocytes self-assemble into three-dimensional, compacted, freely suspended aggregates known as spheroids (1-3). These aggregates have smooth, undulating surfaces and average approx 120 µm in diameter. Hepatocyte spheroids exhibit enhanced liver-specific activities and prolonged viability, compared to cells maintained as a monolayer (4,5). Extensive cell-cell contacts, tight junctions, and microvilli-lined channels that resemble bile canaliculi have been observed between hepatocytes in spheroids (6,7). Thus, these cells appear to mimic the morphology and ultrastructure of an in vivo liver lobule. Reorganization of hepatocytes into these three-dimensional structures is hypothesized to contribute to their enhanced liver-specific functions. Because of their enhanced function and tissue-like ultrastructure, hepatocyte spheroids show great promise for use in tissue-engineering applications and drug metabolism studies.