No abstract

In this study, we investigated the applicability of C3A--a human hepatocyte cell line--as a predicting tool for drug metabolism by applying tissue-engineering methods. Cultivation of C3A cells within alginate scaffolds induced the formation of spheroids with enhanced drug metabolism activities compared to that of two-dimensional (2-D) monolayer cultures. The spheroid formation process was demonstrated via histology, immunohistochemistry, and transmission electron microscope (TEM) analyses. The C3A spheroids displayed multilayer cell morphology, characterized by a large number of tight junctions, polar cells, and bile canaliculi, similar to spheroids of primary hepatocytes. Spheroid formation was accompanied by a reduction in P-glycoprotein (Pgp) gene expression and C3A cell proliferation was limited mainly to cells on the spheroid outskirt. The 3-D constructs maintained a nearly constant cell number according to MTT assay. Drug metabolism by the two most important cytochrome p-450 (CYP) enzymes in human liver, CYP1A2 and CYP3A4, was tested using preferred drugs. With CYP1A2, 3-fold enhancement in activity per cell was seen for converting ethoxyresorufin to resorufin compared to C3A cell monolayers. The spheroids responded to the inducer beta-naphthoflavone and to the inhibitor furafylline of CYP1A2. Enhanced metabolizing activity of CYP3A4, measured by the amount 6beta-testosterone formed from testosterone, and that of the phase II enzyme glucuronosyltransferases (UGT) further indicated that the tissue-engineered C3A spheroids may provide an efficient experimental tool for predicting drug activities by these CYPs. Moreover, the maintenance of constant cell number, as well as the elevated hepatocellular functions and drug metabolism activities, suggest that the tissue-engineered C3A may be applicable in replacement therapies.

The present work explores cell cultivation in macroporous alginate scaffolds as a means to reproduce hepatocyte terminal differentiation in vitro. Newborn rat liver cell isolates, consisting of proliferating hepatocytes and progenitors, were seeded at high cell density of 125 x 10(6)/cm(3) within the scaffold and then cultivated for 6 wk in chemically defined medium. Within 3 days, the alginate-seeded cells expressed genes for mature liver enzymes, such as tryptophan oxygenase, secreted a high level of albumin, and performed phase I drug metabolism. The cells formed compacted spheroids, establishing homotypic and heterotypic cell-to-cell interactions. By 6 wk, the spheroids developed into organoids, with an external mature hepatocyte layer covered by a laminin layer encasing inner vimentin-positive cells within a laminin-rich matrix also containing collagen. The hepatocytes presented a distinct apical surface between adjacent cells and a basolateral surface with microvilli facing extracellular matrix deposits. By contrast, viable adherent cells within collagen scaffolds presenting the identical porous structure did not express adult liver enzymes or secrete albumin after 6 wk. This study thus illustrates the benefits of cell cultivation in macroporous alginate scaffolds as an effective promoter for the maturation of newborn liver cells into functional hepatic tissue, capable of maintaining prolonged hepatocellular functions.

Cultivation of primary adult hepatocytes creates a challenge because of their loss of hepatocellular functions if prevented from attaining polarized cell-cell and cell-matrix interactions. Thus, when hepatocytes are seeded in nonadhesive porous alginate scaffolds, they form 100-microm-diameter spheroids with enhanced cell-cell interactions. Using transmission electron microcopy (TEM), histology, and functional studies, we investigated the state of hepatocyte spheroids during in vitro cultivation. TEM of day 3 spheroids revealed multiple cell layers, with tight junctions between adjacent cells and microvillus-lined channels that resembled bile canaliculi, both structurally and functionally. When copper ions were added to the external medium, the spheroidal hepatocytes performed endocytosis and eventually secreted the heavy metal ions into the bile lumens. From day 8 on, however, there was a rapid decline in cell viability. Histology and TEM analysis of day 13 spheroids revealed a necrotic center, with one viable cell layer on the outskirts. The absence of DNA laddering and negative results in TUNEL assay indicated that apoptosis is not the main process leading to cell death. Cell necrosis may be a result of accumulated bile secretions in the compacted spheroids. Collectively, our results suggest that spheroids derived from adult hepatocytes may have limited utility in long-term applications.

By exploiting the salt-insensitive interaction of the cellulose-binding domain (CBD) of the Clostridium thermocellum cellulosome with cellulose, purification of CBD-fused versions of SecY and SecE, components of the translocation apparatus of the halophilic archaeon Haloferax volcanii, was undertaken. Following transformation of Haloferax volcanii cells with CBD-SecY- or -SecE-encoding plasmids, cellulose-based purification led to the capture of stably expressed, membrane-bound 68 and 25 kDa proteins, respectively. Both fusion proteins were recognized by antibodies raised against the CBD. Thus, CBD-cellulose interactions can be employed as a salt-insensitive affinity purification system for the capture of complexes containing the Haloferax volcanii translocation apparatus components SecY and SecE.