Enhanced Cytochrome P450 IA1 Activity of Self-Assembled Rat Hepatocyte Spheroids

Abstract

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.

Enhanced Differentiation of Adult Bone Marrow-Derived Stem Cells to Liver Lineage in Aggregate Culture

“…The sustained functions are synchronous to structural and morphological characteristics of spheroids, including the existence of tight cell-cell contact, retention of cell-cell junctions, and polarization. 17,[20][21][22]46,47 Under TEM, cells in the differentiated aggregates appeared to be abundant in tight junctions at cell-cell boundaries. Bile-canaliculi-like openings possessing microvilli and delimited by tight junctions were also highly visible.…”

“…[20][21][22] A number of factors such as the microenvironment, cell-cell interactions, and cellular polarization may play a positive role in sustaining liverspecific functions in those hepatocyte spheroids. Thus, it is worth exploring whether such positive effects of 3D cultivation translate to the differentiation of stem cells to hepatocyte-like cells.…”

Liver endothelial cells promote LDL-R expression and the uptake of HCV-like particles in primary rat and human hepatocytes

“…16,17 Analysis of the hepatic synthetic function (albumin, urea) and of hepatic metabolism (cytochrome P450 activity) showed that these three methods can maintain a high level of liverspecific function for over 4 weeks in vitro. However, analysis of LDL uptake in cultured hepatocytes was limited to nonpolarized cell lines such as HepG2, possibly due to loss of LDL-R expression in primary cultured hepatocytes.…”

Sustained P450 expression and prodrug activation in bolus cyclophosphamide-treated cultured tumor cells. Impact of prodrug schedule on P450 gene-directed enzyme prodrug therapy

“…33 The mechanism for the increased P450 expression in spheroids is unknown, however, it is of interest that the hepatocytes in the core of the spheroid exhibit higher P450 activity than those at the surface. 34 Further support for the cell density-dependent expression of P450 2B6 reported here is provided by the observation that another human P450, P450 3A4, also undergoes cell density-dependent expression, in that case in human primary hepatocytes, where the constitutive and the inducible levels of P450 3A4 are both decreased at lower cell densities in a cell-cell contactdependent process.…”