Monolayer and Spheroid Culture of Human Liver Hepatocellular Carcinoma Cell Line Cells Demonstrate Distinct Global Gene Expression Patterns and Functional Phenotypes

Tanaka

Biological & Pharmaceutical Bulletin

et al. 2017

Key words albumin; CYP1A1; human hepatoblastoma HepG2 cell; size control; spheroid Three-dimensional multicellular spheroids possess greater in vivo tissue-like morphology and function than two-dimensional monolayered cells. Therefore, multicellular spheroids are considered useful in vitro systems for drug screening, including tests of drug metabolism and toxicity, [1][2][3][4][5][6] and for studying tumor metastasis. 7,8) Furthermore, such spheroids have attracted much attention for application in cell-based therapy [9][10][11] and as a building block for the construction of large tissues. 12,13) In a previous study, we demonstrated that spheroid formation greatly prolonged the survival of insulinsecreting NIT-1 cells after transplantation in mice. 9)Spheroid size is a key parameter to determine the functions and properties of spheroids. It is reported that oxygen concentration is decreased in the core of the spheroids of human hepatoblastoma HepG2 cells, hepatocytes, and tumor cells. [14][15][16][17] This affects the expression of various molecules, including hypoxia inducible factor (HIF)-1. In addition, paracellular or transcellular transport is required in order for the inner cells to take up extracellular materials and to release secretory products into the culture media. Despite their importance, details regarding the relationship between spheroid size and cellular functions remain to be elucidated. In particular, few studies have investigated the effect of spheroid sizes over 300 µm on their functions and properties.Investigating the effects of spheroid size requires the preparation of size-controlled, multicellular spheroids. Several techniques for spheroid formation have been reported, including the spinner flask, hanging drop, and liquid overlay methods. [18][19][20] However, a common problem with these techniques is the difficulty in controlling spheroid size. In contrast, microfabrication is suitable for the development of sizecontrolled microwells with narrow size distribution. 10,21,22) In a previous study, we demonstrated that polydimethylsiloxane (PDMS)-based microwells constructed using a microfabrication technique could be used to obtain spheroids with a very narrow size distribution. 23) We also found that coating the microwells with poly(N-isopropylacrylamide) (PNIPAAm), a thermoresponsive polymer, increased the quality of the spheroids, since the coating prevented the cells from adhering to the PDMS-based microwells. 23)In the present study, we developed PDMS-based microwells of different sizes in order to prepare variably sized multicellular spheroids. HepG2 cells were used for the study as they represent one of the most extensively used cell lines to evaluate drug metabolism and toxicity. 3,24,25) Production of various proteins is an index of liver function. Albumin is the most abundant serum protein produced by hepatocytes. 26)Another important liver function is the metabolism of various compounds, including drugs. CYP enzymes are the major enzymes involved in drug metabolism. CYP1A1 is ...

“…1,2,23,31) The extensive cell-cell interaction could explain the enhanced albumin secretion and EROD activity in HepG2 cells when formulated in multicellular spheroids.…”

Section: Discussionmentioning

confidence: 99%

Optimization of Albumin Secretion and Metabolic Activity of Cytochrome P450 1A1 of Human Hepatoblastoma HepG2 Cells in Multicellular Spheroids by Controlling Spheroid Size

Tanaka

Biological & Pharmaceutical Bulletin

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

“…3C). Hepatocytes in aggregate or spheroid culture have been shown to be able to maintain viability and metabolic functions for a longer period than those in monolayer cultures, possibly due to the better retainment of in vivo hepatic morphological characteristics (33)(34)(35)(36). To further study the hiPSC-HPC aggregates, immunofluorescent stainings were performed on E-cadherin, an epithelial marker that had been shown to protect primary hepatocyte from apoptosis (37), and intracellular albumin, a functional hepatic marker.…”

Section: In Vitro Structural Characterization Of 3d Hepatic Triculturmentioning

confidence: 99%

Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting

Zhu

et al. 2016

701

661

The functional maturation and preservation of hepatic cells derived from human induced pluripotent stem cells (hiPSCs) are essential to personalized in vitro drug screening and disease study. Major liver functions are tightly linked to the 3D assembly of hepatocytes, with the supporting cell types from both endodermal and mesodermal origins in a hexagonal lobule unit. Although there are many reports on functional 2D cell differentiation, few studies have demonstrated the in vitro maturation of hiPSC-derived hepatic progenitor cells (hiPSC-HPCs) in a 3D environment that depicts the physiologically relevant cell combination and microarchitecture. The application of rapid, digital 3D bioprinting to tissue engineering has allowed 3D patterning of multiple cell types in a predefined biomimetic manner. Here we present a 3D hydrogel-based triculture model that embeds hiPSC-HPCs with human umbilical vein endothelial cells and adiposederived stem cells in a microscale hexagonal architecture. In comparison with 2D monolayer culture and a 3D HPC-only model, our 3D triculture model shows both phenotypic and functional enhancements in the hiPSC-HPCs over weeks of in vitro culture. Specifically, we find improved morphological organization, higher liver-specific gene expression levels, increased metabolic product secretion, and enhanced cytochrome P450 induction. The application of bioprinting technology in tissue engineering enables the development of a 3D biomimetic liver model that recapitulates the native liver module architecture and could be used for various applications such as early drug screening and disease modeling.3D bioprinting | in vitro hepatic model | iPSC | tissue engineering | biomaterials T he liver plays a critical role in the synthesis of important proteins and the metabolism of xenobiotic; the failure of these functions is closely related to disease development and drug-induced toxicity (1). For these reasons, in vitro liver models have been extensively developed to serve as platforms for pathophysiological studies and as alternatives to animal models in drug screening and hepatotoxicity prediction (2-4). Human primary hepatocytes, considered one of the most mature liver cell sources, lose many liver-specific functions rapidly when cultured in vitro due to the great discrepancies between the native and culture environments (5, 6). In addition, the practical difficulties in obtaining liver biopsy samples from every patient further hinder their use in personalized liver models. Consequently, hepatocytes derived from human induced pluripotent stem cells (hiPSCs), with the potential to be patient specific and easily accessible, have been widely acknowledged as the most promising cell source for developing personalized human hepatic models (4, 7).Many groups have reported monolayer differentiation of hiPSCs into hepatocyte-like cells (HLCs) and their ability to metabolize drugs (7-9). Nevertheless, hiPSC-derived HLCs are still considered immature in terms of many liver-specific gene expressions, functions, and...

Tissue Engineering Part C: Methods

“…Our results are similar to a previous study in which the cell cycle and cell proliferation were found to be up-regulated in HMs in comparison to hepatoma cells cultured in a 3D spheroid culture. 6 Although hepatocytes cultured in spheroids experience an environment that differs from CS configurations, they have been shown to exhibit metabolic and synthesis profiles similar to CS cultures. 6 …”

Section: Genome-wide Transcriptional Profiles Of Primary Hepatocytesmentioning

confidence: 99%

“…6 Although hepatocytes cultured in spheroids experience an environment that differs from CS configurations, they have been shown to exhibit metabolic and synthesis profiles similar to CS cultures. 6 …”

Section: Genome-wide Transcriptional Profiles Of Primary Hepatocytesmentioning

confidence: 99%

“…Global gene expression of human hepatocellular carcinoma cells (HepG2) in monolayer and spheroidal cultures revealed up-regulated metabolic functions in spheroids but not in monolayer cultures. 6 Since these data were taken at a single time point, they did not reveal temporal variations. Another study that monitored temporal gene expression in HMs cultured over a 3-day period revealed the down-regulation of cytochrome-P450 expression.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A Comparative Study of Genome-Wide Transcriptional Profiles of Primary Hepatocytes in Collagen Sandwich and Monolayer Cultures

Kim

Lasher

Milford

et al. 2010

Two commonly used culture systems in hepatic tissue engineering are the collagen sandwich (CS) and monolayers of cells. In this study, genome-wide gene expression profiles of primary hepatocytes were measured over an 8-day period for each cell culture system using Affymetrix GeneChips and compared via gene set enrichment analysis to elicit biologically meaningful information at the level of gene sets. Our results demonstrate that gene expression in hepatocytes in CS cultures steadily and comprehensively diverges from that in monolayer cultures. Gene sets up-regulated in CS cultures include several associated with liver metabolic and synthesis functions, such as metabolism of lipids, amino acids, carbohydrates, and alcohol, and synthesis of bile acids. Monooxygenases such as Cytochrome-P450 enzymes do not show any change between the culture systems after 1 day, but exhibit significant up-regulation in CS cultures after 3 days in comparison to hepatocyte monolayers. These data provide insights into the up-and down-regulation of several liver-critical gene sets and their subsequent effects on liver-specific functions. These results provide a baseline for further explorations into the systems biology of engineered liver mimics.