Molecular mechanisms underlying the enhanced functions of three-dimensional hepatocyte aggregates

doi:10.1016/j.biomaterials.2013.11.063

Biomaterials

Volume 35, Issue 7, February 2014, Pages 2162-2171

https://doi.org/10.1016/j.biomaterials.2013.11.063 Get rights and content

Abstract

Three-dimensional (3D) culture of hepatocytes leads to improved and prolonged synthetic and metabolic functions, but the underlying molecular mechanisms are unknown. In order to investigate the role of 3D cell–cell interactions in maintaining hepatocyte differentiated functions ex vivo, primary mouse hepatocytes were cultured either as monolayers on tissue culture dishes (TCD) or as 3D aggregates in rotating wall vessel (RWV) bioreactors. Global gene expression analyses revealed that genes upregulated in 3D culture were distinct from those upregulated during liver development and liver regeneration. Instead, they represented a diverse array of hepatocyte-specific functional genes with significant over-representation of hepatocyte nuclear factor 4α (Hnf4a) binding sites in their promoters. Expression of Hnf4a and many of its downstream target genes were significantly increased in RWV cultures as compared to TCD. Conversely, there was concomitant suppression of mesenchymal and cytoskeletal genes in RWV cultures that were induced in TCDs. These findings illustrate the importance of 3D cell–cell interactions in maintaining fundamental molecular pathways of hepatocyte function and serve as a basis for rational design of biomaterials that aim to optimize hepatocyte functions ex vivo for biomedical applications.

Introduction

Three-dimensional (3D) cell culture is critical for understanding the function of cells in a physiologically relevant context and for tissue engineering complex solid organs like the liver [1]. 3D culture of hepatocytes has been shown to maintain the differentiated functions of hepatic cell lines [2] and primary cells [3], [4] as well as promote differentiation of stem cells toward the hepatic lineage [5], [6], [7], [8]. Sustained differentiated functions of hepatocytes in 3D culture could be harnessed for drug toxicity screening [9], [10] or to improve the efficacy of extracorporeal liver-assist devices [11]. In addition, 3D aggregation may facilitate transplantation of hepatocytes kept in culture, which would be particularly useful for hepatocytes derived from pluripotent stem cells [12], [13].

While differentiated functions are improved in hepatocytes cultured either on 3D scaffolds [14], [15], [16], [17] or as self-aggregated 3D spheroids [2], [3], [4], little is known about the underlying molecular mechanisms that lead to those improved functions in 3D culture. Using the rotating wall vessel (RWV) system, we previously showed that one important factor for sustaining differentiated functions of a human hepatocellular carcinoma cell line is sustained 3D cell–cell interactions [2]. RWVs are rotational bioreactors with a gas permeable membrane that, when in operation, are completely filled with culture media so that it functionally rotates as a solid-body around a horizontal axis. This creates a suspension culture environment with minimal fluid shear stress and turbulence, allowing cells to co-localize and self-aggregate with 3D spatial freedom [18]. Studies have shown that hepatocyte spheroids cultured under these conditions could develop into larger sizes with less hypoxic limitations to the inner core compared to stationary culture [19]. Human hepatocytes and non-parenchymal cells co-cultured within RWVs demonstrated formation of complex 3D micro-architectures, including microvilli, bile canaliculi, and sinusoidal fenestrations [20], [21].

Importantly, as tissue engineering strategies advance and the definitions of biomaterials evolve, self-aggregated hepatocyte spheroids developed within bioreactors may be considered biomaterials in their own right [22]. Self-aggregated cellular spheroids are the building blocks for organ printing, a bottom up synthesis approach to tissue engineering [23]. Therefore, understanding the molecular mechanisms that regulate hepatocyte functions within spheroids is critical for the optimization of organ function within a printed liver. Moreover, hepatocyte spheroids generated within RWVs allow investigation of the biological effects of 3D cell–cell interactions without the influence of contacting scaffolds, surfaces, or carriers. In this study, we performed global gene expression and promoter region analyses on primary mouse hepatocytes cultured on collagen-coated tissue culture dishes (TCDs) or RWVs to determine the underlying molecular mechanisms important for maintaining hepatocyte-specific functions in 3D culture.

Section snippets

Mice

Primary hepatocytes were isolated from male C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME). All mice were handled in accordance with the “Guide for the Care and Use of Laboratory Animals” and approved by the Institutional Animal Care and Use Committee.

Hepatocyte isolation and culture

Mouse primary hepatocytes were isolated by the two-step perfusion technique using Liver Perfusion and Liver Digest Media (Life Technologies, Pleasanton, CA), followed by separation using a 50% Percoll (GE Healthcare Life Sciences, Pittsburgh,

Morphology and function of primary mouse hepatocytes cultured within RWVs and on TCDs

Primary mouse hepatocytes cultured within RWVs self-organized into 3D aggregates that were 100 to 200 μm in their longest cross-sectional length by 24 h of culture (Fig. 1A). After 3 days in RWVs, hepatocyte cell shape remained round in densely-packed aggregates ranging from 200 to 300 μm in size. In contrast, hepatocytes on TCDs formed a monolayer of characteristic bi-nucleated cells within 24 h of culture (Fig. 1A). After 3 days in TCDs, cultured cells acquired irregular and pleomorphic

Discussion

In this study, we used global gene expression profiling and promoter region analyses to identify molecular differences between primary mouse hepatocytes cultured as monolayers on TCDs and as 3D aggregates generated in RWVs. Our results indicated that improved synthetic and metabolic functions in hepatocyte 3D aggregates reflected the upregulated expression of a diverse array of hepatocyte-specific functional genes compared to monolayer cultures. The pattern of differential gene expression was

Conclusions

Using global gene expression and promoter region analyses, we demonstrated that the master transcriptional regulator Hnf4a has a major role in maintaining differentiated hepatocyte functions in 3D culture. Concomitantly, expression of mesenchymal and cytoskeletal genes induced in monolayer cultures were suppressed in 3D aggregates. These findings provide a basis for rational design of biomaterials that aim to optimize hepatocyte-specific functions for in vitro and in vivo applications.

Acknowledgments

This work was supported by the American College of Surgeons Resident Research Scholarship, the UCSF Liver Center Pilot/Feasibility Grant P30-DK026743, National Institutes of Health grant K08-DK093708 (to TTC), and in part by UH3-AG037628 (to MHF). The authors thank Drs. Hobart W. Harris and Holger Willenbring for critical review of the manuscript and Ms. Pamela Derish for manuscript editing.