Synthetic sandwich culture of 3D hepatocyte monolayer
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].
Section snippets
Materials
PET TE membranes with thickness of 9 μm, pore density of 3×107 pores/cm2 and pore diameter of 0.8 μm were purchased from Sterlitech (WA, USA). The galactose ligand, 1-O-(6′-aminohexyl)-d-galactopyranoside (AHG, M.W. 279) was synthesized previously [23], [24], [25]. GRGDS peptide was purchased from Peptides International (Kentucky, USA). Minusheet carriers were purchased from Minucells and Minutissue Vertriebs GmbH (Bad Abbach, Germany). Primary rabbit anti-E-Cadherin and anti-GAPDH antibody were
Fabrication and characterization of bioactive PET TE membranes to construct the synthetic sandwich culture
The synthetic sandwich culture was constructed by a PET-Gal as the bottom substratum and a PET TE membrane (GRGDS-modified or galactosylated or non-modified) as the top support. The entire sandwich construct was secured in the Minusheet Carriers (Fig. 1A).
The fabrication and characterization of the PET-Gal (bottom substratum) were described previously [26]. We fabricated here GRGDS-modified or galactosylated PET TE membranes (top support, Fig. 1B) based on the commercially available PET TE
Discussion
A novel synthetic sandwich culture was developed by overlaying a 3D hepatocyte monolayer formed on a PET-Gal with a GRGDS-modified PET TE membrane top support. This 3D hepatocyte monolayer has been characterized previously with improved cellular structure and polarities, enhanced cell–cell interactions, better differentiated functions compared to the hepatocyte monolayer on collagen-coated substratum. Due to the weak adhesive force obtained from the bottom galactosylated substratum as well as
Conclusions
We have established an ECM-free synthetic sandwich culture by maintaining a 3D hepatocyte monolayer between a GRGDS-modified PET TE membrane (top support) and a PET-Gal (bottom substratum). The 3D hepatocyte monolayer in the synthetic sandwich culture exhibited similar polarity formation, improved mass transfer, enhanced cell–cell interactions and higher differentiated functions compared with the hepatocytes in the conventional collagen sandwich culture. This synthetic sandwich culture can
Acknowledgments
We would like to thank Mr. Talha Arooz and Ms. Tse Kit Yan for the technical support. This work is supported in part by the Institute of Bioengineering and Nanotechnology, Biomedical Research Council, Agency for Science, Technology and Research (A*STAR) of Singapore (R185-001-045-305); Ministry of Education Grant R-185-000-135-112, National Medical Research Council Grant R-185-000-099-213 and Singapore-MIT Alliance Computational and Systems Biology Flagship Project funding to HYU. YND, RBH and
References (45)
- et al.
Effect of culture conditions on the expression and function of Bsep, Mrp2, and Mdr1a/b in sandwich-cultured rat hepatocytes
Biochem Pharmacol
(2006) - et al.
Advances in bioartificial liver devices
Hepatology
(2001) - et al.
Biotransformation and liver-specific functions of human hepatocytes in culture on RGD-immobilized plasma-processed membranes
Biomaterials
(2005) - et al.
Galactose-carrying polymers as extracellular matrices for liver tissue engineering
Biomaterials
(2006) - et al.
Specific binding of glucose-derivatized polymers to the asialoglycoprotein receptor of mouse primary hepatocytes
J Biol Chem
(2001) - et al.
Adhesion of chicken hepatocytes to polyacrylamide gels derivatized with N-acetylglucosamine
J Biol Chem
(1978) - et al.
Role of extracellular matrix composition and configuration in maintenance of hepatocyte polarity and function
Biomaterials
(1996) - et al.
Primary culture of rat hepatocytes in 96-well plates: effects of extracellular matrix configuration on cytochrome P450 enzyme activity and inducibility, and its application in in vitro cytotoxicity screening
Toxicol In Vitro
(2007) - et al.
Preparation of 6-aminohexyl d-aldopyranosides
Carbohydr Res
(1979) - et al.
3D hepatocyte monolayer on hybrid RGD/galactose substratum
Biomaterials
(2006)
Biological evaluation of RGD peptidomimetics, designed for the covalent derivatization of cell culture substrata, as potential promotors of cellular adhesion
Biomaterials
Preparation of isolated rat liver cells
Methods Cell Biol
Hepatic vectorial transport of xenobiotics
Chem Biol Interact
Recognition of complex oligosaccharides by the multi-subunit asialoglycoprotein receptor
Trends Biochem Sci
RGD peptides confer survival to hepatocytes via the beta1-integrin-ILK-pAkt pathway
J Hepatol
The selective modulation of endothelial cell mobility on RGD peptide containing surfaces by YIGSR peptides
Biomaterials
Hepatocyte adhesion, growth and differentiated function on RGD-containing proteins
Biomaterials
The differential cytotoxicity of methotrexate in rat hepatocyte monolayer and spheroid cultures
Toxicol In Vitro
Hepatocyte function and extracellular matrix geometry: long-term culture in a sandwich configuration
Faseb J
Adhesive interactions and the metabolic activity of hepatocytes
J Cell Sci Suppl
Effect of extracellular matrix topology on cell structure, function, and physiological responsiveness: hepatocytes cultured in a sandwich configuration
Faseb J
Primary hepatocyte culture: substratum requirements and production of matrix components
Fed Proc
Cited by (75)
Bioprinting of Biomimetic Tissue Models for Disease Modeling and Drug Screening
2022, 3D Bioprinting and Nanotechnology in Tissue Engineering and Regenerative MedicineFabrication and in vitro evaluation of a packed-bed bioreactor based on galactosylated poly(ethylene terephthalate) microfibrous scaffolds
2020, Biochemical Engineering JournalFabrication and evaluation of modified poly(ethylene terephthalate) microfibrous scaffolds for hepatocyte growth and functionality maintenance
2020, Materials Science and Engineering C3D bioprinting of functional tissue models for personalized drug screening and in vitro disease modeling
2018, Advanced Drug Delivery ReviewsCarbon nanotubes as biological transporters and tissue-engineering scaffolds
2018, Synthesis, Technology and Applications of Carbon Nanomaterials
- 1
These authors contribute equally to this work.