Synthetic sandwich culture of 3D hepatocyte monolayer

Abstract

The sandwich culture of hepatocytes, between double layers of extra-cellular matrix (ECM), is a well-established in vitro model for re-establishing hepatic polarity and maintaining differentiated functions. Applications of the ECM-based sandwich culture are limited by the mass transfer barriers induced by the top gelled ECM layer, complex molecular composition of ECM with batch-to-batch variation and uncontrollable coating of the ECM double layers. We have addressed these limitations of the ECM-based sandwich culture by developing an ‘ECM-free’ synthetic sandwich culture, which is constructed by sandwiching a 3D hepatocyte monolayer between a glycine-arginine-glycine-aspatic acid-serine (GRGDS)-modified polyethylene terephthalate (PET) track-etched membrane (top support) and a galactosylated PET film (bottom substratum). The bioactive top support and bottom substratum in the synthetic sandwich culture substituted for the functionalities of the ECM in the ECM-based sandwich culture with further improvement in mass transfer and optimal material properties. The 3D hepatocyte monolayer in the synthetic sandwich culture exhibited a similar process of hepatic polarity formation, better cell–cell interaction and improved differentiated functions over 14-day culture compared to the hepatocytes in collagen sandwich culture. The novel 3D hepatocyte monolayer sandwich culture using bioactive synthetic materials may readily replace the ECM-based sandwich culture for liver tissue engineering applications, such as drug metabolism/toxicity testing and hepatocyte-based bioreactors.

Introduction

In vivo, hepatocytes are organized into a polarized epithelium with distinct apical (bile canalicular) and basal (sinusoidal) domains [1]. The basal domain of the hepatocytes is in contact with a complex extracellular matrix (ECM) containing fibronectin, laminin, collagen I–V, and proteoglycans in the space of Disse [2]. The interactions of hepatocytes with the ECM environment are important for hepatic polarity and differentiated function maintenance [3]. In standard in vitro culture, primary hepatocytes cultured on substrates coated with ECM protein, such as collagen or fibronectin, typically exhibit spreading morphology with deteriorating differentiated functions and nearly no polarized structure [4]. This deteriorating process could be rescued by overlaying another ECM layer, such as collagen or basement membrane (Matrigel™), which mimics the ECM distribution in the space of Disse. Hepatocyte sandwich culture between double layers of ECM is an in vitro model with re-established hepatic polarity and stable differentiated functions [3], [5], [6]. The hepatocyte sandwich culture has been adopted in liver physiology studies [7], [8], drug metabolism/toxicity testing [9] and hepatocyte-based bioreactors [10], [11]. Further applications of the conventional ECM-based sandwich culture were hampered by the complex molecular compositions of the ECM with batch to batch variation [12], uncontrollable ECM coating, mass transfer barriers induced by the gelled ECM-coated top support (hindering the exchange of nutrients, xenobiotics or biochemical signals with the bulk culture medium), and shedding of the ECM coating from the top support during culture. In this study, we have addressed these limitations of the ECM-based sandwich culture by developing an ‘ECM-free’ synthetic sandwich culture, in which we replaced the natural ECM with bioactive polymeric materials to achieve improved mass transfer and stable differentiated functions.

A variety of synthetic substrata with bioactive components, such as cell adhesion peptides: Arg-Gly-Asp (RGD) [13], Tyr-Ile-Gly-Ser-Arg (YIGSR) [14], Gly-Phe-Hyp-Gly-Glu-Arg (GFOGER) [15] or sugar ligands: galactose [16], glucose [17], lactose [18], have been used for cell culture to replace natural ECM with well-controlled material properties and cellular responses. Previously, we have fabricated a galactosylated polyethylene terephthalate (PET-Gal) film for primary rat hepatocyte culture and identified a 3D hepatocyte monolayer formed on the PET-Gal [19]. The 3D hepatocyte monolayer exhibited 3D cellular structure and polarities, enhanced cell–cell interactions and differentiated functions compared to the 2D hepatocyte monolayer on collagen-coated substratum [19]. Here, we established a synthetic sandwich culture by overlaying the 3D hepatocyte monolayer on the PET-Gal (bottom substratum) with a porous PET track-etched (TE) membrane (top support). Since the biochemical compositions of ECM play essential roles in regulating hepatocyte morphology, polarity and differentiated functions in ECM-based sandwich culture [20], [21], [22], we investigated the influence of three different top support (galactosylated, GRGDS-modified or non-modified PET TE membrane) on the hepatocyte morphology, polarity and differentiated functions in the 3D hepatocyte monolayer of the synthetic sandwich culture. The synthetic sandwich culture with GRGDS-modified PET TE membrane (top support)/PET-Gal (bottom substratum) exhibited the optimal performances, in terms of stabilizing the 3D monolayer morphology, re-establishing hepatocyte polarity and maintaining other differentiated functions.

We compared this GRGDS-modified PET TE membrane/PET-Gal synthetic sandwich culture of 3D hepatocyte monolayer with the collagen sandwich hepatocyte culture. 3D hepatocyte monolayer in the synthetic sandwich culture exhibited similar dynamic process of polarity formation and biliary excretion, improved mass transfer, enhanced cell–cell interaction, differentiated functions compared with the hepatocytes in the collagen sandwich culture. This synthetic sandwich culture model can replace the ECM-based sandwich culture for relevant hepatocyte-based applications such as drug metabolism/toxicity testing and hepatocyte-based bioreactors [7], [8].