Enhanced Morphology and Function in Hepatocyte Spheroids: A Model of Tissue Self-Assembly

Abstract

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.

Engineering hepatocellular morphogenesis and function via ligand-presenting hydrogels with graded mechanical compliance

“…Hepatocellular morphogenesis and assembly have been well established to be critical to the functional performance of liver-derived cells in vitro (Hansen et al, 1998;Singhvi et al, 1994a;Torok et al, 2001;Yuasa et al, 1993). In this study, we offer a bidimensional approach to systematically obtain distinct cell morphogenetic and functional outcomes by manipulating both substrate mechanical compliance and substrate bioactivity (ligand concentration).…”

“…This paradigm has been commonly observed for hepatocytes in vitro, particularly for culture systems that utilize diverse biophysical attributes to elicit markedly variant patterns of hepatocellular morphogenesis (Dispersio et al, 1991;Hamamoto et al, 1998;Hamilton et al, 2001;Hansen et al, 1994;Sawamoto and Takahashi, 1997;Singhvi et al, 1994a). Notably, hepatocyte cultures, biophysically induced to be 3D in nature, exhibit a compacted, spheroidal morphology and typically express elevated levels of liver-specific functions indicative of a highly differentiated state (Hamada et al, 1997;Hansen et al, 1998). This is in direct contrast to the levels of functional activity reported for cultures that have adopted a more 2D morphology (Baker et al, 2001;Hamamoto et al, 1998).…”

A Three-Dimensional Gel Bioreactor for Assessment of Cardiomyocyte Induction in Skeletal Muscle–Derived Stem Cells

“…37 Cell aggregate culture has been shown to enhance CM gene expression patterns, 22 increase the synthesis and release of ECM components, 26 and accelerate CM differentiation efficiency of embryonic stem cells 25 and liver stem cells. 23 Similarly, Albrecht et al found that chondrocyte matrix biosynthesis was dependent on cell cluster size, rather than overall cell density.…”

Biochemical and functional changes of rat liver spheroids during spheroid formation and maintenance in culture: I. morphological maturation and kinetic changes of energy metabolism, albumin synthesis, and activities of some enzymes

“…During this process, cells undergo significant environmental changes. The cells in spheroids resume cell-cell contacts, and survive and maintain liver-specific functions much longer and superior to cells cultured as monolayers [Landry et al, 1985;Lin et al, 1995;Lazar et al, 1995a;Juillerat et al, 1997;Hansen et al, 1998;Tzanakakis et al, 2001]. Liver spheroid culture, reported as early as the 1980s [Landry et al, 1985], was mainly employed in artificial liver studies [Lazar et al, 1995b;Hu et al, 1997].…”

“…These include self-assembly [Peshwa et al, 1994;Hansen et al, 1998;Yamada et al, 2001], magnetic bar rotationmediated [Yagi et al, 1993], gyrotatory-mediated [Walker et al, 2000], spinner-mediated [Sakai et al, 1996], and foam pore-trapping [Iijima et al, 1998] methods, and entrapping in collagen gel [Hansen et al, 1998] or alginated beads [Khalil et al, 2001]. Whatever the culture method, the liver cells in preparing threedimensional spheroid cultures experience a common process: intact liver tissue !…”