Three-dimensional culture of hepatocytes on porcine liver tissue-derived extracellular matrix
Introduction
The liver is the only internal organ capable of natural regeneration of large portions of lost tissue. As little as one third of a liver can regenerate itself in vivo [1]. However, liver cells placed in culture rapidly lose their in vivo phenotypic characteristics and functional abilities. This situation has limited the ability to study regenerative properties and basic functions of liver cells in vitro. As shown previously, it is difficult to maintain liver-specific function of primary hepatocytes in culture for more than one week without an adequate supportive microenvironment including substrates, e.g. extracellular matrix (ECM) coatings or feeder layers [2]. The lack of a suitable culture platform has restricted drug discovery, toxicology, cancer, and tissue regeneration studies for liver cells. Despite decades of research, current culture products are not suitable for the expansion and maintenance of the highly specialized functions of hepatocytes. While some generic products and systems are available, they do not meet the specific culture requirements of primary liver cells. Thus, there is a need for cell culture systems that mimic the in vivo characteristics of hepatocytes.
Elements that influence human hepatocyte cultures include cell-ECM interactions, soluble growth factors and cytokines, physical factors (e.g. stress and strain) [3], [4], and cell–cell communications [5]. Importantly, cell-ECM interactions play a fundamental role in hepatocyte growth [6], [7], liver organ development [8], [9], tissue regeneration [6], [10], [11], wound healing [10], [12], [13] and malignancy [8], [14]. The liver ECM contains proteins and carbohydrates that provide support and anchorage for cells, segregate tissues, and regulate intercellular communication. Commercially available tissue extracts enriched in matrix (e.g. Matrigel, collagen-I, extracts from amnions) have been used successfully as culture substrata for many years [15]. However, they are not tissue-specific [16], [17]. We recently reported that tissue-derived ECM could be used successfully as a 2D substrate for the cell type that originated from that tissue [18]. Additionally, a 3D co-culture system using a porous ECM scaffold combined with dynamic culture conditions promoted the formation of a multilayered urothelium and infiltration of smooth muscle cells into the matrix. This construct could be used to engineer urological tissue for bladder or urethral tissue reconstruction [19].
Under physiological conditions, cells within tissues and organs create an optimal tissue-specific ECM including collagen, fibronectin, laminin, glycosaminoglycans (GAGs), proteoglycans (PGs) and non-soluble growth factors. Thus, we hypothesized that using tissue-specific ECM components derived from the tissue where somatic cells reside might improve primary cell tissue culture. When tissue-specific ECM components matched to skin, skeletal muscle, and liver tissue were employed, cell adhesion efficiencies, growth rates, morphology, and phenotypes of cells derived were greatly improved [20]. In addition, when cell and ECM types were mismatched, growth rates and cellular function were not optimal [18]. Thus, subtle differences in ECM composition among tissue types can affect cellular interactions in a lineage-specific manner. Certain decellularization and oxidation procedures for whole organs or tissues can optimize a 3D collagen matrix (such as bladder submucosa or tendon), with high porosity and minimal retention of cellular components while maintaining the ECM architecture and content [19], [21]. To further develop such a culture platform for liver cell expansion, we tested different methods to fabricate liver ECM discs and to determine how culture on a liver-specific ECM impacted the long-term expansion and maintenance of function of human hepatocytes. Also, we sought to understand how the decellularization method influenced the composition of the liver ECM and affected cell expansion and function. The water wash method removed almost all cellular and nuclear material from hepatic tissues, maintained key elements of the ECM, and best supported cell growth and function of liver cells.
Section snippets
Hepatocytes and liver tissue
We used HepG2 cells (ATCC, Manassas, VA) and human primary hepatocytes (Invitrogen, Carlsbad, CA) for this study. HepG2 cells, a human liver carcinoma cell line, were mainly used to optimize the decellularized liver ECM discs and confirm cell viability and cell-matrix interaction when the cells were cultured on the discs treated with six methods (see 2.2). Human primary hepatocytes were employed to test albumin secretion function. To obtain ECM from liver tissue, 15 fresh porcine livers were
Decellularized liver ECM
To optimize the decellularized liver ECM discs for liver cell culture, six wash methods (Table 1) were used. Grossly, the ECM discs maintained their original round shape throughout all wash protocols after decellularization. Although creating a rougher surface and greater porosity on the discs, all PAA treatments caused the discs to shrink significantly compared to their original size (p < 0.05) (Table 2). The water-wash decellularization method generated less shrinkage and created more
Discussion
Current culture systems for primary hepatocyte growth need improvement. Our previous study with 2D ECM systems [18] and 3D ECM in other tissues [5], [6], [8] served as the basis for the present study, in which we tested different methods to produce liver ECM discs for liver cell growth. The use of water wash followed by detergents during the decellularization procedure produced optimal liver ECM discs for cell growth, with uniform thicknesses and even surfaces. The water-decellularized ECM
Conclusions
In this study, we developed a micro-scale system for culture of human hepatocytes. This methodology provides a simple and inexpensive means of producing a liver-specific ECM-based culture substrate that enhances hepatocyte growth and function compared to traditional 2D and collagen gel sandwich cultures. Our data indicate that interactions of hepatocytes with 3D tissue-specific ECM improve cell attachment, survival, growth, and long-term viability of the highly functional phenotypes of liver
Acknowledgment
The authors thank Dr Jennifer Olson and Ms. Karen Klein for their editorial assistance. This study was supported, in part, by the Telemedicine and Advanced Technology Research Center (TATRC) at the U.S. Army Medical Research and Materiel Command (USAMRMC) through award W81XWH-07-1-0718.
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Ren Lang, Matthew Stern made equal contributions.