Enhancing the Drug Metabolism Activities of C3A— A Human Hepatocyte Cell Line—By Tissue Engineering Within Alginate Scaffolds

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.

Application of Three-Dimensional Culture Conditions to Human Embryonic Stem Cell-Derived Definitive Endoderm Cells Enhances Hepatocyte Differentiation and Functionality

“…22,23 Interestingly, we have also shown that hESC-DE cells behave differently from HepG2 cells when cultured in an Algimatrix 3D scaffold. Firstly, hESC-DE cells required the treatment of ROCKi to survive in the 3D culture after dissociation, whereas HepG2 cells do not require ROCKi to survive or to form spheroids.…”

Hepatocyte-Like Cells Derived from Human Amniotic Epithelial Cells Can Be Encapsulated Without Loss of Viability or Function In Vitro

“…Indeed, CYP3A4 activity in the encapsulated HLC was close to that of encapsulated HepG2 cells. Previous studies reported elevated CYP3A4 activity in hepatocytes grown on matrices or encapsulated to create a three-dimensional environment [45][46][47], and that the increase in CYP3A4 activity could be due to increased cell to cell contact, additional extracellular matrix and recovery of cell polarity within in the microcapsule [44,48]. Thus, encapsulated HLC may also be suitable as an in vitro drug screening tool.…”

Hemoglobin Regulates the Metabolic, Synthetic, Detoxification, and Biotransformation Functions of Hepatoma Cells Cultured in a Hollow Fiber Bioreactor

“…34 Therefore, it is important to conserve the ability of hepatocytes to synthesize albumin in a HF-based BLAD. The reported albumin synthesis rates for human hepatoma/immortalized hepatocytes (HepG2/C3A/HepLL) ranged from 0.0625 to 0.42 mg/h/10 6 cells [35][36][37][38] ; primary human hepatocytes ranged from 0.002 to 0.006 mg/h/10 6 cells 39 ; primary porcine hepatocytes ranged from 0.05 to 6.6 mg/h/10 6 cells 30,[40][41][42] ; and primary rat hepatocytes ranged from 0.2 to 0.4 mg/h/10 6 cells. 43 The capacity for albumin synthesis in our experimental HF bioreactors with C3A cells (0.4-0.8 mg/h/10 6 cells) is comparable or even higher than most of the aforementioned primary cells and cell lines derived from various sources.…”