Towards a three-dimensional microfluidic liver platform for predicting drug efficacy and toxicity in humans

Although the process of drug development requires efficacy and toxicity testing in animals prior to human testing, animal models have limited ability to accurately predict human responses to xenobiotics and other insults. Societal pressures are also focusing on reduction of and, ultimately, replacement of animal testing. However, a variety of in vitro models, explored over the last decade, have not been powerful enough to replace animal models. New initiatives sponsored by several US federal agencies seek to address this problem by funding the development of physiologically relevant human organ models on microscopic chips. The eventual goal is to simulate a human-on-a-chip, by interconnecting the organ models, thereby replacing animal testing in drug discovery and development. As part of this initiative, we aim to build a three-dimensional human liver chip that mimics the acinus, the smallest functional unit of the liver, including its oxygen gradient. Our liver-on-a-chip platform will deliver a microfluidic three-dimensional co-culture environment with stable synthetic and enzymatic function for at least 4 weeks. Sentinel cells that contain fluorescent biosensors will be integrated into the chip to provide multiplexed, real-time readouts of key liver functions and pathology. We are also developing a database to manage experimental data and harness external information to interpret the multimodal data and create a predictive platform.


Introduction
Th e liver is the largest metabolically active organ and is important in the modifi cation and detoxifi cation of external agents, but is also susceptible to damage from these substances [1] and their metabolic intermediates. Liver toxicity and cardiotoxicity are the most cited reasons for both market withdrawal and failure during late-stage clinical testing of drugs [2,3]. Current animal models, as well as in vitro liver platforms, are poor predictors of human liver toxicities, with success rates as low as 40% [4,5]. Th e pharmaceutical industry is therefore in need of better tools for predicting these toxicities in early stages of development in order to reduce dangerous clinical outcomes and drug development costs. Th us in 2011 the National Institutes of Health, through the National Center for Advancing Translational Science, the US Food and Drug Administration, the Environmental Protection Agency and the Defense Advanced Research Projects Agency, announced a collaboration to develop microphysiological systems that model major organs of the human body. Key requirements for these models are: to facilitate the assessment of biomarkers, bioavailability, effi cacy, and toxicity of therapeutic agents prior to clinical trials; and to predict the safety, effi cacy, and pharmacokinetics of drug/vaccine candidates prior to their fi rst human use.
As participants in the National Center for Advancing Translational Science program, our laboratories at the University of Pittsburgh and at Massachusetts General Hospital are collaboratively developing a three-dimensional microfl uidic human liver acinus with integrated fl uorescent biosensors to improve prediction of human liver response to xenobiotic insults ( Figure 1). Fluorescence-based protein biosensors can detect altered levels of specifi c analytes and changes of subcellular protein localization with spatiotemporal resolution in response to drugs or external stimuli [6,7]. Th e platform comprises four vital cell types of the liver (hepatocytes, endothelial, stellate and Kupff er cells) arranged layer by layer with

Abstract
Although the process of drug development requires effi cacy and toxicity testing in animals prior to human testing, animal models have limited ability to accurately predict human responses to xenobiotics and other insults. Societal pressures are also focusing on reduction of and, ultimately, replacement of animal testing. However, a variety of in vitro models, explored over the last decade, have not been powerful enough to replace animal models. New initiatives sponsored by several US federal agencies seek to address this problem by funding the development of physiologically relevant human organ models on microscopic chips. The eventual goal is to simulate a human-on-a-chip, by interconnecting the organ models, thereby replacing animal testing in drug discovery and development. As part of this initiative, we aim to build a three-dimensional human liver chip that mimics the acinus, the smallest functional unit of the liver, including its oxygen gradient. Our liver-on-a-chip platform will deliver a microfl uidic three-dimensional co-culture environment with stable synthetic and enzymatic function for at least 4 weeks. Sentinel cells that contain fl uorescent biosensors will be integrated into the chip to provide multiplexed, real-time readouts of key liver functions and pathology. We are also developing a database to manage experimental data and harness external information to interpret the multimodal data and create a predictive platform.

© 2010 BioMed Central Ltd
Towards a three-dimensional microfl uidic liver platform for predicting drug effi cacy and toxicity in humans well-defi ned cell numbers and organization in a threedimensional microfl uidic environment mimicking a hepatic cord. A subset of hepatocytes and nonparen chymal cells (NPC) integrated into the device are biosensor sentinel cells, expressing fl uorescence-based biosensors of key cellular functions in order to provide quantitative, real-time reports of cell health and molecular modes of action [8]. Th is approach seeks to extend the predictive relationship between hepatotoxicity triggers (mitochondrial damage, activation of Kupff er cell, oxidative stress) and the fi nal manifestation of drug-induced liver injury (DILI) to long-term and real-time dynamics [9]. Since DILI can manifest as both acute and chronic eff ects, our liver platform is being developed to function for at least 4 weeks; a signifi cant technical challenge considering the rapid de-diff erentiation usually observed for primary cell cultures. Another key component of our approach is the develop ment of a database application that will access informa tion from major drugs, drug targets, bioassays and pathway databases. Related chemical, bioactivity, pre clinical and clinical data will be used for aiding the interpretation of microphysiology readouts and development of computational models to predict the safety and toxicity of new compounds. Th e database design aim is ultimately to integrate the other microphysiological organ systems to complete a human-on-a-chip database.

Capturing precise liver p hysiology in vitro
Th e liver comprises two major cell populations: parenchymal cells (hepatocytes) and NPCs, including endothelial cells, stellate cells and Kupff er cells among others. Th e inspiration for our platform is the liver sinusoid and the acinus, the smallest metabolic functional unit of the liver. Th ere are four crucial aspects of liver physiology captured in our design: a three-dimensional architecture; multiple cell types; physiological microcirculation; and zonation.
A model that aims to recapitulate the intricate autocrine and paracrine interaction between diff erent cells of the liver should include the major parenchymal cell and NPC types as well as a three-dimensional microcellular environment mimicking the acinus-sinusoid [10]. Th e three-dimensional architecture shaped by the collagen sandwich in tissue culture plates was an important development in the long-term stable metabolic and synthetic function of hepatocytes [11]. Co-cultures of liver cells can extend the stability and function even further, as well as modify the response to drugs as compared with monocultures of hepatocytes [12][13][14]. Kupff er cells, for example, are known to contribute to DILI during acetaminophen toxicity [15]. Th ese cocultures can also stabilize cells after unavoidable injury [13]. Recent results show that both human and rat hepatocytes are stable for over 3 months in a co-culture with their respective NPC fraction, with improved response to drug induction of clinically relevant cytochrome P450 enzymes [12], highlighting the importance of a coculture over standard monocultures. Further, arranging the co-cultures into sinusoid-like structures improves albumin secretion and cytochrome P450 activity for over 2 months [16]. Th e latter is important as evidence suggests reactive metabolites are often the cause of unanticipated DILI. Evidence further confi rms that threedimensional co-cultures, either on a scaff old or as a spheroid, improve both the longevity and toxicological response [12,17,18].
Microcirculatory fl ow can also modulate the response of hepatocytes [19] and of endothelial cells [20], and can form the oxygen zonation that is important for site-directed injury and the transport of nutrients and waste metabolites. However, when exposing cells to fl ow, it is important to emulate physiologically relevant conditions including appropriate shear stress, drug residence times, and chemical gradients. Th e microfl uidic approaches allow for such emulation as well as providing high-throughput test beds for pharmacokinetic and toxicity studies.

Modes of action to predict human hepatoxicity and approach
Th e exact molecular basis for many instances of DILI is unknown. A major goal of our project is to identify realtime functional responses associated with hepatotoxicity using sentinel cells [21]. We are developing the sentinel cells of various cell types to respond to several molecular signatures that are associated with hepatotoxicity [22]. Based on our previous experience with biosensors, we are developing hepatocyte sentinel cells to report metabolic alterations due to apoptosis, oxidative stress, phospholipidosis, and intrahepatic cholestasis caused by inhibition of bile effl ux transport, among others [7,23,24]. Kupff er cell activation in response to the injury trigger will be used to measure immune dysfunction that occurs as a result of reaction to a drug, a drug metabolite, or a protein altered by a reactive drug metabolite [25]. Stellate cells will have biosensors to monitor proliferation that results from fi brosis associated with cholestasis or mitochondrial failure [26]. Cells placed in the chip are also confi rmed for intermediary metabolism.
Our vision is to use a microfl uidic device where the hepatic and sentinel cells are arranged in parallel micro grooves in a sinusoid-like architecture (Figure 2A,B,C) [27]. Th e cells at the inlet of these channels consume oxygen, which generates an oxygen gradient along the fl ow direction of the device. Previous work on microfl uidic liver(s)-on-a-chip used feeder cells to stabilize cultures, but lacked long-term functional data and had no incorporation of NPCs in a three-dimensional architecture [28][29][30]. Although bioreactors include longer culture times and multiple cell types, they are not suited for microfl uidic format [31]. Our strategy is to seed the hepatocytes and add the NPCs sequentially. Th e cells will be proportioned in appropriate physiological ratios of 70 to 80% hepatocytes, 15 to 20% endothelial cells, 5 to 10% stellate cells and 5 to 10% Kupff er cells.
Our initial prototype for cell culture and parameter optimization is a cell culture chamber 10 mm long, 1.5 mm wide, and 100 μm tall, fabricated out of polydimethyl siloxane [32]. In preliminary work, approximately 12,000 seeded hepato cytes demonstrated stable hepatocyte function ality for 10 days of culture under a static (no-fl ow) condition, showing excellent viability ( Figure 2D,E). Although the device incorporates human cells, prelimi nary device optimization was carried out using primary rat liver cells. Similarly, polydimethylsiloxane used in prototypes will be replaced with more robust materials. Th e functional data are supported by periodic cell viability assays as well as visualization of the intracellular albumin ( Figure 2F).
A novel concept in our vision is to integrate multiple live, stable, and nondestructive cellular reporters of test drug eff ects to enable the early prediction of DILI using high content analysis. A subset of primary human liver cells is effi ciently transformed into sentinel cells by lentiviral gene delivery of fl uorescent protein biosensors. Long-term high content analysis can be carried out within the microfl uidic device without the need for offl ine analysis [33] (Figure 1). Th e sentinel cell data are quantifi able to assess the toxicity, metabolic functions, and key modes of action responses to drugs over time. Initial studies of sentinel cell primary human hepatocytes report increased reactive oxygen species ( Figure 2FG) and mitochondrial impairment ( Figure 2H,I) in response to known hepatotoxins versus a vehicle control. Importantly, these responses are time and concentration dependent, reproducible, quantitative and collected by nondestructive techniques. Sentinel cell data will be integrated with biochemical and metabolic measurements to monitor drug clearance and cytochrome P450 induction, glucose consumption, and urea and albumin secretions. Th e sentinel cell concept will advance the fi eld because it enables the study of primary cell heterotypic interactions due to drug response and enables the study of dynamic cellular response from within a functional liver model.
Central to the liver model, and indeed all of the organ models, is the organization and analysis of large and complex datasets to predict human organ effi cacy and safety [21]. We are constructing a database application to collect, manage, and analyze data from the real-time readouts, biochemical assays, and mass spectrometry. Beyond data management, the database will incorporate access to clinically relevant hepatotoxic and non hepatotoxic drug exposure results, and biochemical and metabolic activities in order to establish the concordance of the liver chip data with in vivo eff ects. To achieve this goal, a selection of reference compounds with available clinical data will be profi led in the liver chip, and the resulting dataset will be used to construct predictive models of human hepatotoxicity.

Conclusions and future directions
We are particularly interested in using the liver acinus module to investigate DILI, which leads to pathologies such as fi brotic scarring, fatty liver disease, reduced metabolic detoxifi cation, and even liver failure [1]. An important cause of DILI is human genetic variability that can lead to altered liver metabolism and response to drugs. Our long-term goal is to seed the liver microchip with induced pluripotent stem cell-derived adult-like hepatocytes to characterize and predict hepatotoxicity arising from distinct genetic backgrounds. Since our liver acinus module is modular and reconfi gurable, we plan to combine it with other organ platforms to begin assessing multiorgan toxicities. Key elimination organs that have direct interaction with the liver, such as the gut and kidney, are of particular importance because they can provide robust pharmacokinetic modeling. Th is plan fi ts our goals of: generating a microscale platform technology to support human physiological organ systems; maintaining the liver module at high, stable levels of function for at least 4 weeks; and utilizing real-time high content measure ments and a reference database to improve the prediction of human liver toxicity. Finally, sentinel biosensor cells can be developed for other organs and the general design of our database application is adaptable for other organ models.

Competing interests
The authors declare that they have no competing interests.