A human liver microphysiology platform for investigating physiology, drug safety, and disease models

Abstract

This paper describes the development and characterization of a microphysiology platform for drug safety and efficacy in liver models of disease that includes a human, 3D, microfluidic, four-cell, sequentially layered, self-assembly liver model (SQL-SAL); fluorescent protein biosensors for mechanistic readouts; as well as a microphysiology system database (MPS-Db) to manage, analyze, and model data. The goal of our approach is to create the simplest design in terms of cells, matrix materials, and microfluidic device parameters that will support a physiologically relevant liver model that is robust and reproducible for at least 28 days for stand-alone liver studies and microfluidic integration with other organs-on-chips. The current SQL-SAL uses primary human hepatocytes along with human endothelial (EA.hy926), immune (U937) and stellate (LX-2) cells in physiological ratios and is viable for at least 28 days under continuous flow. Approximately, 20% of primary hepatocytes and/or stellate cells contain fluorescent protein biosensors (called sentinel cells) to measure apoptosis, reactive oxygen species (ROS) and/or cell location by high content analysis (HCA). In addition, drugs, drug metabolites, albumin, urea and lactate dehydrogenase (LDH) are monitored in the efflux media. Exposure to 180μM troglitazone or 210μM nimesulide produced acute toxicity within 2–4 days, whereas 28μM troglitazone produced a gradual and much delayed toxic response over 21 days, concordant with known mechanisms of toxicity, while 600μM caffeine had no effect. Immune-mediated toxicity was demonstrated with trovafloxacin with lipopolysaccharide (LPS), but not levofloxacin with LPS. The SQL-SAL exhibited early fibrotic activation in response to 30nM methotrexate, indicated by increased stellate cell migration, expression of alpha-smooth muscle actin and collagen, type 1, alpha 2. Data collected from the in vitro model can be integrated into a database with access to related chemical, bioactivity, preclinical and clinical information uploaded from external databases for constructing predictive models.

Modeling Therapeutic Antibody–Small Molecule Drug-Drug Interactions Using a Three-Dimensional Perfusable Human Liver Coculture Platform

“…Advanced in vitro systems in combination with in silico modeling approaches continue to drive improvements in the early drug development process. As these in vitro models increase in complexity and approach greater physiologic relevance, their application should continue to expand from drug PK and toxicity evaluations to pharmacodynamic and human disease models (Ananthanarayanan et al, 2014;Wheeler et al, 2014;Vernetti et al, 2016).…”

Liver metastases: Microenvironments andex-vivomodels

“…Desirable features include: (i) inclusion of all resident hepatic cells necessary for liver functioning (hepatocytes and the various NPCs), (ii) controllable flow to provide the physiologically relevant perfusion shear stimulation, oxygenation, nutrients replenishment, waste product removal and extended culture periods, (iii) scaffolding or matrices that support a 3D multicellular microenvironment by encouraging cell self-organization and generation of macroscopic tissue morphology, and (iv) the ability to be assayed for a variety of real-time mechanistic readouts (e.g. genomic, phenotypic, mass spectroscopy, cell biochemical, and media secretion-based assays) (106–108, 110, 113, 114). The technologies and fabrication techniques are developing fast (107, 115), and although each system described below captures many of the features of liver (Table 1), it is unlikely that a single ex vivo model of liver will meet all the requirements for all applications of liver biology in research and industry.…”

“…The first generation SQL-SAL utilizes a commercially available TEMS single channel, microfluidic device manufactured by Nortis, Inc (108). The model is constructed using cryopreserved human hepatocytes and three human NPC cell lines (LSECs (EA.hy926), macrophages (U937) and HSCs (LX-2)) at physiological ratios.…”

Facilitating the commercialization and use of organ platforms generated by the microphysiological systems (Tissue Chip) program through public–private partnerships

“…The TCs may be used as a tool for mechanistic studies with a reduced number of variables [12]; they may be developed for preliminary or efficient dose–response testing for efficacy and off-target effects, thus reducing the cost of drug development [19]; or TCs developed from human cells can serve as a preclinical approach to identify compounds whose efficacy or toxicity is different in animal vs. human tissue [4], [8], [9], [20]. As an example of the latter, TCs can illustrate significant differences in hepatotoxicity in animals vs. humans [21], [22].…”