Expression of albumin and cytochrome P450 enzymes in HepG2 cells cultured with a nanotechnology-based culture plate with microfabricated scaffold

Abstract

-The Nanoculture plate (NCP) is a recently developed plate which essentially consists of a textured surface with specific characteristics that induce spheroid formation: microfabrications with a micro-square pattern on the culture surface. The NCP can be used to generate uniform adhesive spheroids of cancer cell lines using conventional techniques without the need of any animal compounds. In this study, we assessed the performance of human hepatoma cell line HepG2 cells cultured with an NCP to evaluate the effects of the NCP on their hepatocyte-specific functions. The NCP facilitated the formation of three-dimensional (3D) HepG2 cell architecture. HepG2 cells cultured with an NCP exhibited enhanced mRNA expression levels of albumin and cytochrome P450 (CYP) enzymes compared to those cultured with a two-dimensional (2D) conventional plate. The expression levels of two specific liver-enriched transcription factors, hepatocyte nuclear factor 4α (HNF4α) and CCAAT/enhancer binding protein α (C/EBPα), were higher in HepG2 cells grown with the NCP than those in HepG2 cells grown with conventional plates before albumin and CYP enzymes expression levels were increased. The inducibility of CYP1A2 and CYP3A4 mRNA following exposure to inducers in HepG2 cells cultured with an NCP was comparable to that in HepG2 cells cultured with conventional plates, while the expression levels of CYP1A2 and CYP3A4 mRNA following exposure to inducers were higher when using an NCP than when using conventional plates. These results suggest that the use of an NCP enhances the hepatocyte-specific functions of HepG2 cells, such as drug-metabolizing enzyme expression, making the NCP/ HepG2 system a useful tool for evaluating drug metabolism in vitro.

Influence of the pattern size of micropatterned scaffolds on cell morphology, proliferation, migration and F-actin expression

“…In cells cultured on 3D patterned scaffolds, the expression levels of adhesion signalling complexes, 43 focal adhesion kinase 13,43 and F-actin, 13,15,44 their proliferative, 8,13,20,22 cell aggregation [17][18][19][20] and metabolic 13,[19][20][21] capacities, and their differentiation potential, 9, 23 differ from those of cells cultured on flat surfaces.…”

Evaluation of Three-Dimensional Cultured HepG2 Cells in a Nano Culture Plate System: an In Vitro Human Model of Acetaminophen Hepatotoxicity

“…They also identified higher sensitivity to acetaminophen cytotoxicity in 3D-cultured HepG2 cells compared with conventional monolayer cultured HepG2 cells and suggested that this 3D-cultured HepG2 cell system may be useful as a human model of APAPinduced hepatotoxicity. However, the pathophysiological relevance of the mechanisms of APAP-induced cytotoxicity observed in 3D-cultured HepG2 cells remains a missing piece of the puzzle (16,17). Our current results of APAP-induced GSH depletion, mitochondrial dysfunction, and JNK pathway activation as well as cytoprotective action by NAC, CyA, and SP600125 observed in the NCP-HepG2 system, a 3D-cultured method, may explain mechanisms of cytotoxicity more accurately than conventionally cultured methods and be more useful in toxicological and pharmacological safety assessment.…”

“…These results suggest that 3D-cultured HepG2 cells may be a useful tool as an in vitro human model of APAP-induced hepatotoxicity. Other studies have reported on a new 3D-cultured HepG2 system using a nano culture plate (NCP) that shows higher expression of albumin and some CYP enzymes in NCP-cultured HepG2 cells compared with conventionally cultured cells (17). However, it remains to be demonstrated whether cell injury induced by APAP in 3D-cultured HepG2 cells in vitro was produced by corresponding human and rodent mechanisms of in vivo APAP-induced hepatotoxicity, and if antidotes such as NAC or other agents also show protective potential against APAP-induced hepatotoxicity in the 3D-cultivation system.…”

Comparative Study of the Effects of Antituberculosis Drugs and Antiretroviral Drugs on Cytochrome P450 3A4 and P-Glycoprotein

“…Their usage, however, is limited for several reasons: high cost, insufficient availability of donor organs, interdonor functional variability, and limited life span (32). Meanwhile, the well-established human hepatoblastoma, HepG2, can also be used for the same application and cytotoxicity testing because of its good proliferation, immortality, and stable viability; however, the intrinsic enzyme activities and the reactivity of CYPs, especially CYP3A4 and CYP2B6, to CYP modulators are entirely inadequate, even in the three-dimensional culture of HepG2 cells (33)(34)(35). Similarly, while immortalized human hepatic cell lines, i.e., Fa2N-4 cells and ADV-1 cells that are derived from BC2 cells, have been reported since 2000, the inherent characteristics of those lines restrict their routine application in the drug discovery setting (36,37).…”