Novel 3D Culture Systems for Studies of Human Liver Function and Assessments of the Hepatotoxicity of Drugs and Drug Candidates

Abstract

The liver is an organ with critical importance for drug treatment as the disposition and response to a given drug is often determined by its hepatic metabolism. Patient-specific factors can entail increased susceptibility to drug-induced liver injury, which constitutes a major risk for drug development programs causing attrition of promising drug candidates or costly withdrawals in postmarketing stages. Hitherto, mainly animal studies and 2D hepatocyte systems have been used for the examination of human drug metabolism and toxicity. Yet, these models are far from satisfactory due to extensive species differences and because hepatocytes in 2D cultures rapidly dedifferentiate resulting in the loss of their hepatic phenotype and functionality. With the increasing comprehension that 3D cell culture systems more accurately reflect in vivo physiology, in the recent decade more and more research has focused on the development and optimization of various 3D culture strategies in an attempt to preserve liver properties in vitro. In this contribution, we critically review these developments, which have resulted in an arsenal of different static and perfused 3D models. These systems include sandwich-cultured hepatocytes, spheroid culture platforms, and various microfluidic liver or multiorgan biochips. Importantly, in many of these models hepatocytes maintain their phenotype for prolonged times, which allows probing the potential of newly developed chemical entities to cause chronic hepatotoxicity. Moreover, some platforms permit the investigation of drug action in specific genetic backgrounds or diseased hepatocytes, thereby significantly expanding the repertoire of tools to detect drug-induced liver injuries. It is concluded that the development of 3D liver models has hitherto been fruitful and that systems are now at hand whose sensitivity and specificity in detecting hepatotoxicity are superior to those of classical 2D culture systems. For the future, we highlight the need to develop more integrated coculture model systems to emulate immunotoxicities that arise due to complex interactions between hepatocytes and immune cells.

Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long‐term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics

“…Yet, the confidence in conventional human in vitro models, such as hepatoma cell lines or primary hepatocytes in 2D culture, is also limited, as these simple systems do not accurately mimic human liver biology and function. To overcome these hurdles, a plethora of advanced 3D hepatic in vitro models have been developed that permit the maintenance of hepatic phenotypes for extended periods (18). However, to obtain regulatory approval substantial evidence for increased translational confidence has to be presented, which requires comprehensive characterization of these novel culture paradigms, as well as an extensive data and experience base (45).…”

“…To overcome these obstacles, various hepatic 3-dimensional (3D) systems have been developed in which cultured hepatocytes remain viable and functional for prolonged times (17, 18). PHHs cultured in 3D cellular aggregates termed spheroids present a functionally and phenotypically stable, versatile system in which bile canaliculi are formed and hepatocytes retain their periportal and perivenous phenotypes (19, 20).…”

Transcriptional, Functional, and Mechanistic Comparisons of Stem Cell–Derived Hepatocytes, HepaRG Cells, and Three-Dimensional Human Hepatocyte Spheroids as Predictive In Vitro Systems for Drug-Induced Liver Injury

“…However, their rapid dedifferentiation in conventional two-dimensional (2D) monolayer cultures, paralleled by a loss of hepatic functionality, renders them unsuitable for long-term studies and significantly impairs their predictive power for DILI risk (Gerets et al, 2012; Lauschke et al, 2016c; Sison-Young et al, 2016; Heslop et al, 2017). To prevent dedifferentiation, an array of three-dimensional (3D) culture techniques has been developed in which hepatic phenotypes are maintained for extended periods of time (Lauschke et al, 2016a). One promising strategy is the culture of PHHs as 3D spheroidal aggregates in which hepatocyte-specific functions can be retained for several weeks (Bell et al, 2016), thus enabling repeated-exposure experiments.…”

Utility of human hepatocyte spheroids without feeder cells for evaluation of hepatotoxicity

“…Organ-on-a-chip platforms can imitate the hemodynamics of the in vivo liver by perfusion, and generate efficient nutrient exchange and shear stress at the in vitro setting (Domansky et al, 2010). On the other hand, such 3D-culture methods are difficult to operate, leading to their being unsuitable for large-scale screening of new chemical compounds (Lauschke et al, 2016). Manipulating hepatocytes in the Cell-able ® is rather simple and fulfills the terms of high throughput screening for exclusion of hepatotoxins.…”