Three-dimensional culture of hepatocytes on porcine liver tissue-derived extracellular matrix

Abstract

There is currently no optimal system to expand and maintain the function of human adult hepatocytes in culture. Recent studies have demonstrated that specific tissue-derived extracellular matrix (ECM) can serve as a culture substrate and that cells tend to proliferate and differentiate best on ECM derived from their tissue of origin. The goal of this study was to investigate whether three-dimensional (3D) ECM derived from porcine liver can facilitate the growth and maintenance of physiological functions of liver cells. Optimized decellularization/oxidation procedures removed up to 93% of the cellular components from porcine liver tissue and preserved key molecular components in the ECM, including collagen-I, -III, and -IV, proteoglycans, glycosaminoglycans, fibronectin, elastin, and laminin. When HepG2 cells or human hepatocytes were seeded onto ECM discs, uniform multi-layer constructs of both cell types were formed. Dynamic culture conditions yielded better cellular infiltration into the ECM discs. Human hepatocytes cultured on ECM discs expressed significantly higher levels of albumin over a 21-day culture period compared to cells cultured in traditional polystyrene cultureware or in a collagen gel “sandwich”. The culture of hepatocytes on 3D liver-specific ECM resulted in considerably improved cell growth and maintained cell function; therefore, this system could potentially be used in liver tissue regeneration, drug discovery or toxicology studies.

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.