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Microphysiological Systems Emerging as Powerful Tools for Drug Discovery


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Given that only one in ten preclinical candidates in Phase I trials are likely to gain market approval, better predictors of clinical success are urgently needed (1). Translating findings from in vitro studies to in vivo settings remains a challenge because of species differences in pharmacokinetics and pharmacodynamics (PKPD), oversimplified in vitro models, and an insufficient understanding of the underlying pathophysiology. Termination is often attributed to safety issues identified in animal studies, which could be minimised with more accurate predictions of absorption, distribution, metabolism, and excretion (ADME) profiles (2). While 2D monolayer cell culture experiments and animal models are deeply embedded in the pharmaceutical infrastructure, clear gaps, inefficiencies, and inaccuracies exist, creating a need for new alternative and complementary research models (3). At the cross-section of bioengineering and cell biology lies a new approach to drug discovery and development, which is being pursued to help overcome the notoriously low clinical success rate. Microphysiological systems (MPS) are an emerging class of in vitro models expected to expedite drug discovery by providing robust physiologically relevant data at critical stages of R&D (4).
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Improving Drug Discovery Models
A new class of
in vitro
model is expected to assist drug discovery by providing robust
physiologically relevant data at critical stages of R&D
Systems Emerging as
Powerful Tools for
Drug Discovery
Given that only one in ten preclinical candidates in Phase I
trials are likely to gain market approval, better predictors of
clinical success are urgently needed (1). Translating findings
from in vitro studies to in vivo settings remains a challenge
because of species differences in pharmacokinetics and
pharmacodynamics (PKPD), oversimplified in vitro models,
and an insufficient understanding of the underlying
pathophysiology. Termination is often attributed to safety
issues identified in animal studies, which could be
minimised with more accurate predictions of absorption,
distribution, metabolism, and excretion (ADME) profiles (2).
While 2D monolayer cell culture experiments and animal
models are deeply embedded in the pharmaceutical
infrastructure, clear gaps, inefficiencies, and inaccuracies
exist, creating a need for new alternative and complementary
research models (3). At the cross-section of bioengineering
and cell biology lies a new approach to drug discovery and
development, which is being pursued to help overcome
the notoriously low clinical success rate. Microphysiological
systems (MPS) are an emerging class of in vitro models
expected to expedite drug discovery by providing robust
physiologically relevant data at critical stages of R&D (4).
Micro-Engineering Meets Physiology
MPS encompass a range of platforms that mimic aspects of
organ function by using microengineering technology, often
combined with 3D microenvironments (5). Such systems
have been reported as 3D spheroids, organoids, organ-
on-a-chip, multi-organs on chips, static micropatterned
technologies, and physiome-on-a-chip models (5-6). In
these platforms, living cells and microfluidic technologies are
combined with some form of drug delivery, stimulation, and/
or sensing tools. Organ-on-a-chip (OOC) models can exist as
single systems or as connected units that simulate elements
of organ crosstalk. MPS build on concepts harnessed by
traditional two-dimensional assays and include design
features that improve physiological relevance, such as (2):
A 3D microenvironment within a biopolymer or tissue-
derived matrix
Mechanical cues that mimic those found in vivo, such as
stretch cues and perfusion, to provide shear stress
Multiple cell types
The ability to introduce a concentration gradient
A diverse array of design and manufacturing approaches lie
behind these compact and adaptable systems. Computer-
aided design tools are used to generate digital 3D designs
for microfluidic and microelectronic systems, which can be
imported into 3D printing software (also known as ‘additive
manufacturing’). A range of 3D printing methods exist in
the production of tissue-engineering scaffolds; extrusion-
based 3D printing is an established method that directly
deposits thermoplastic or thermosetting materials using a
layer-by-layer process (7-8). In contrast, stereolithography
is employed to print entire microfluidicsystems and
Dr Audrey Dubourg at CN Bio
48 European Biopharmaceutical Review | July 2020
harnesses light and
photoreactive material to
cause spatially-controlled
A diverse array of intricate
components may be
incorporated among
microfluidic channels in an
MPS, such as micropumping
systems, mixing chambers,
synthetic matrices, sensors
(that may be integrated to
online data recorders), valves,
and independently controllable
pneumatic lines (3, 6).
Pathways for cell crosstalk
must be established for
multi-organ MPS, which
may involve soluble factors
or cell migration across
matrices. Tunable flow rates, intra- and inter-MPS mixing and
distribution, and adjustable oxygenation levels provide a high
level of flexibility for researchers to optimise cell viability or ask
experimental questions.
Single and Multi-Organ MPS in ADME Profiling
The applications of OOC models and other MPS are diverse
– just like their manufacturing and design approaches.
Organoids, on-chip models, and other MPS have been
developed for most tissues, and provide unprecedented
opportunities for toxicity testing, personalised medicine,
and studies of PKPD and disease mechanisms. Given their
importance in drug development, significant efforts have been
directed at developing models for absorption and metabolism.
Assays for intestinal drug absorption typically employ colon
adenocarcinoma (Caco-2) cells in a static, 2D monoculture.
Despite their popularity, Caco-2 assays suffer from inherent
limitations that result in a severe under-prediction of
paracellular drug transport (9). Innovative organ-on-a-chip
technologies provide opportunities to overcome this, as in
vivo conditions can be replicated more closely. Improving the
gut MPS epithelial barrier integrity is a high priority, and this
can be assessed by measuring the transepithelial electrical
resistance (10). Towards this goal, Caco-2 cells have been
co-cultured with other intestinal cells, such as goblet-like
mucosal cells, to provide further complexity and complement
dynamically perfused models (5, 10).
The value of perfused hepatocytes in a single-organ MPS was
demonstrated in a toxicology study that captured the effects of
a well-characterised hepatotoxin and revealed novel insights
into the (previously underestimated) toxicity of its analogue (11).
Metabolites formed in a dose-dependent manner, similar to
those seen in a patient overdose, and albumin secretion and
glutathione depletion were measured to assess hepatocyte
function and toxicity, respectively. While the capacity to assess
drug metabolism and toxicological response is a significant
step forward, researchers are aware that an MPS comprised
of a single cell type will not offer a complete solution for
all metabolism studies. To provide access to organ-like
models that more closely reflect the complexity of the liver
microarchitecture in vivo, co-culture models have been
created using multiple cell types
Single- and multi-organ MPS technologies are designed to mimic
specific aspects of organ function and/or crosstalk, rather than
reproduce an entire organ or human body (10). For example,
fully capturing the complexity of kidney function may be out of
reach for renal excretion-related studies, yet progress has been
made towards developing on-chip models and primary kidney
tubule epithelial organoids for studies of specific aspects of
kidney physiology (12-13). Multi-organ MPS can provide insights
on interactions between organs and enable different processes
to be studied simultaneously; incorporating liver tissue or other
organs susceptible to toxicity provides a unique opportunity to
study efficacy and toxicity concurrently (3-4).
Particular emphasis has been placed on mimicking gut-
liver interactions, which are critical to the prediction of
drug disposition, efficacy, toxicity, and the elucidation of
pathophysiological mechanisms. The simulation of gut-
liver crosstalk has been achieved to some degree in MPS,
as evidenced by gut-mediated suppression of hepatic
CYP7A1, an enzyme central to bile acid synthesis (5).
Interconnected MPS that incorporate multiple cell types can
help fill in the gaps in ADME profiling. For example, data on
drug distribution can be obtained indirectly by combining
observations from studies of intestinal permeability, hepatic
metabolism, drug vehicles, carrier proteins, and efflux/influx
membrane pumps (14).
Figure 1: The future of MPS technologies; from single-organ to patient-on-a-chip for personalised medicine
Source: Modied from
et al,
dose Gut
Liver scaffold
European Biopharmaceutical Review | July 2020
To support PKPD modelling in multi-MPS, an experimental
design approach known as quantitative systems
pharmacology (QSP) is applied. QSP uses computational
models to describe the complex interactions within multi-
MPS, while considering both physical dynamics (e.g., media
volumes and flow rates) and biological dynamics (e.g.,
hormone production and release) (6). The ability to measure
aspects of ADME in MPS has created many opportunities
across drug development, from disease modelling through to
drug repurposing (see Figure 2).
MPS in Drug Discovery: Finding a Niche
Although safety assessment and ADME profiling is the
primary context for MPS technologies, there are a number
of other ways in which these research models could improve
the efficiency of drug development. To ensure that MPS
development aligns with the greatest needs in the industry,
these opportunities have been considered in depth.
Ultimately, MPS technology innovators aim to improve the
predictivity of drug efficacy and safety for new and existing
drugs (drug repurposing). In turn, this improves the clinical
success rate and accelerates drug development, alleviates
costs associated with drug failures, and reduces the risk to
clinical trial participants. MPS have the potential to greatly
benefit the health sector and identifying specific gaps in
current preclinical research is critical to realising this goal.
Opportunities for MPS applications lie in disease modelling
and phenotypic screening, to help identify and rank new and
known (including orphan drugs and failed compounds that
are available for repurposing) compound candidates (2).
Improved models are being sought for conditions that are
poorly served by animal models (e.g., hepatitis B infection)
and to enable host genetic studies, modelling of drug
treatment responses, and the identification of biomarkers that
could be used to monitor drug treatments (15). Advanced
in vitro models are under development to support studies of
highly prevalent diseases with well-established impacts on
public health, such as non-alcoholic steatohepatitis (NASH).
Microtissue models of human NASH can demonstrate key
hallmarks of disease, providing opportunities to elucidate
pathophysiological mechanisms on a cellular level (16).
By improving predictivity to identify risk over standard tools,
or by providing a more comprehensive model that is not
otherwise available, MPS are expected to fill many gaps. The
ability to reveal toxicities that would otherwise go undetected,
or reveal changes in cell function that precede adverse drug
events would be of significant value (2). However, to maximise
the potential of MPS, insights gathered from these advanced
in vitro models should be considered alongside in vivo data
for comparison. MPS could complement in vivo models by (2):
Clarifying mechanisms underlying apparent adverse events
revealed by animal models
Providing confidence to data derived from human-based
MPS, using animal-based chips
Settling differences found in animal models, e.g.,
when preclinical lesions are found in one of two
preclinical species
Figure 2: Current opportunities for MPS across the drug discovery process
Source: Modied from Hughes et al, 2017
Discovery Development
Disease modelling Drug metabolism and
pharmacokinetics safety
Clinical trial design and stratification, precision medicine
Pre-clinical Ph I Ph II Ph III Approved
Short-medium duration (2 days to 2 weeks)
cultures of human primary cells or stem cells
under well controlled conditions to simulate
tissue homeostasis or tissue remodelling
Continuous feeds/sampling for temporal control of
culture conditions e.g., to test complex dosing regimes
or circadian cycles
Extended human MPs cultures
(1+ months) and multi-organ studies
e.g., for investigational toxicology
Fluidic Human
Human specific modalities
Immunotherapy Viral vector
drug delivery
Gene therapies
Inter-organ cross talk
Adaptive immune
Target organ for
drug efficacy…
…linked to target
organ for toxicity
reproductive cycle
Stem cell
Post-approval studies
toxicology Drug rescue
Fluidic and mechanical
stimulation of disease
Basic biological
research and target ID Inflammatory
diseases Metabolic syndrome
and NASH
European Biopharmaceutical Review | July 2020
Organoids are being actively pursued for use in cancer
research, where the consideration of inter- and intra-patient
heterogeneity is critical to therapy development. Equally,
there could be benefits to reducing variability in certain
settings, by using cells from the same individual to create
MPS for studies of multiple doses, drugs, and timepoints (2).
Looking Ahead: What is Needed for the
Successful Implementation of MPS?
Establishing translational relevance will be critical to the
successful integration of MPS in preclinical research.
Developers and researchers must clearly demonstrate
benefits over existing models, while communicating
effectively with other stakeholders to identify and navigate
challenges, needs, and approaches to validation (2).
Technology needs to be developed with the end-user
in mind, with a design that maximises usability and
reproducibility. Offering high-throughput capabilities that
are compatible with automation provides an incentive to
researchers who would benefit from efficiency gains and
reduced labour costs. In some cases, MPS could also
reduce the cost of animal trials, cells, and reagents, as
smaller volumes are needed for many microfluidic devices.
Significant efforts have been directed at extending the
lifespan of MPS models to provide a greater window for
long-term experiments, enabling compound dosing and
observations of disease progression; in vitro models of
intestinal barrier function and liver disease models have
been maintained for a number of weeks (5, 16).
After finding their way into research settings across
the globe, single- and multi-organ MPS are emerging
as powerful tools for drug discovery and development,
from disease modelling to drug repurposing. To improve
the chances of clinical success, the pharma industry is
currently evaluating and adopting these technologies, while
technology developers continue in their pursuit of advancing
MPS for use in drug discovery.
1. Visit:
2. Fabre K et al, Introduction to a manuscript series on the
characterization and use of microphysiological systems
(MPS) in pharmaceutical safety and ADME applications,
Lab Chip 20(6): pp1,049-57, 2020
3. Hughes D et al, Opportunities and challenges in the wider
adoption of liver and interconnected microphysiological
systems, Exp Biol Med 242(16): pp1,593-604, 2017
4. Ribeiro A et al, Liver microphysiological systems for
predicting and evaluating drug effects, Clin Pharmacol
Ther 106(1): pp139-47, 2019
5. Chen W et al, Integrated gut/liver microphysiological
systems elucidates inflammatory inter-tissue crosstalk,
Biotechnol Bioeng 114(11): pp2,648-59, 2017
6. Edington CD et al, Interconnected microphysiological
systems for quantitative biology and pharmacology
studies, Sci Rep 8(1): p4,530, 2018
7. Sochol RD et al, 3D printed microfluidics and
microelectronics, Microelectronic Engineering 189: pp52-
68, 2018
8. Vaezi M et al, Extrusion-based 3D printing technologies
for 3D scaffold engineering, Functional 3D Tissue
Engineering Scaffolds: pp235-54, 2018
9. DiMarco RL et al, Improvement of paracellular transport in
the caco-2 drug screening model using protein-engineered
substrates, Biomaterials 129: pp152-62, 2017
10. Tsamandouras N et al, Integrated gut and liver
microphysiological systems for quantitative in vitro
pharmacokinetic studies, AAPS J 19(5): pp1,499-512,
11. Rowe C et al, Perfused human hepatocyte microtissues
identify reactive metabolite-forming and mitochondria-
perturbing hepatotoxins, Toxicol In Vitro 46: pp29-38,
12. Ashammakhi N et al, Kidney-on-a-chip: untapped
opportunities, Kidney Int 94(6): pp1,073-86, 2018
13. Schutgens F et al, Tubuloids derived from human adult
kidney and urine for personalized disease modelling, Nat
Biotechnol 37(3), pp303-13, 2019
14. Wikswo J, The relevance and potential roles of
microphysiological systems in biology and medicine, Exp
Biol Med 239(9): pp1,061-72, 2014
15. Ortega-Prieto AM et al, 3D microfluidic liver cultures
as a physiological preclinical tool for hepatitis B virus
infection, Nat Commun 9(1): p682, 2018
16. Kostrzewski T et al, A microphysiological system for
studying nonalcoholic steatohepatitis, Hepatology
Communications 4(1): pp77-91, 2020
Dr Audrey Dubourg is CN Bio’s Product Manager for their
PhysioMimix™ Organ-On-Chip lab benchtop platform, which enables
researchers to model human biology in the lab through rapid and
predictive 3D tissue-based studies harnessing microfluidic technology.
Audrey has an extensive research background in microbiology and
parasitology, with significant experience in molecular biology techniques
and 3D mammalian cell culture employing MPS technologies. Audrey
gained her PhD at the University of East Anglia, UK and carried out a
postdoctoral scholarship at the University of California, US.
European Biopharmaceutical Review | July 2020
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Full-text available
Liver plays a major role in drug metabolism and is one of the main sites of drug adverse effects. Microphysiological systems (MPS), also known as organs‐on‐a‐chip, are a class of microfluidic platforms that recreate properties of tissue microenvironments. Among different properties, the liver microenvironment is three‐dimensional, fluid flows around its cells, and different cell types regulate its function. Liver MPS aim to recreate these properties and enable drug testing and measurement of functional endpoints. Tests with these systems have demonstrated their potential for predicting clinical drug effects. Properties of liver MPS that improve the physiology of cell culture are reviewed, specifically focusing on the importance of recreating a physiological microenvironment to evaluate and model drug effects. Advances in modeling hepatic function by leveraging MPS are addressed, noting the need for standardization in the use, quality control, and interpretation of data from these systems. This article is protected by copyright. All rights reserved.
Full-text available
Adult stem cell-derived organoids are three-dimensional epithelial structures that recapitulate fundamental aspects of their organ of origin. We describe conditions for the long-term growth of primary kidney tubular epithelial organoids, or ‘tubuloids’. The cultures are established from human and mouse kidney tissue and can be expanded for at least 20 passages (>6 months) while retaining a normal number of chromosomes. In addition, cultures can be established from human urine. Human tubuloids represent proximal as well as distal nephron segments, as evidenced by gene expression, immunofluorescence and tubular functional analyses. We apply tubuloids to model infectious, malignant and hereditary kidney diseases in a personalized fashion. BK virus infection of tubuloids recapitulates in vivo phenomena. Tubuloids are established from Wilms tumors. Kidney tubuloids derived from the urine of a subject with cystic fibrosis allow ex vivo assessment of treatment efficacy. Finally, tubuloids cultured on microfluidic organ-on-a-chip plates adopt a tubular conformation and display active (trans-)epithelial transport function. © 2019, The Author(s), under exclusive licence to Springer Nature America, Inc.
Full-text available
Microphysiological systems (MPSs) are in vitro models that capture facets of in vivo organ function through use of specialized culture microenvironments, including 3D matrices and microperfusion. Here, we report an approach to co-culture multiple different MPSs linked together physiologically on re-useable, open-system microfluidic platforms that are compatible with the quantitative study of a range of compounds, including lipophilic drugs. We describe three different platform designs - "4-way", "7-way", and "10-way" - each accommodating a mixing chamber and up to 4, 7, or 10 MPSs. Platforms accommodate multiple different MPS flow configurations, each with internal re-circulation to enhance molecular exchange, and feature on-board pneumatically-driven pumps with independently programmable flow rates to provide precise control over both intra- and inter-MPS flow partitioning and drug distribution. We first developed a 4-MPS system, showing accurate prediction of secreted liver protein distribution and 2-week maintenance of phenotypic markers. We then developed 7-MPS and 10-MPS platforms, demonstrating reliable, robust operation and maintenance of MPS phenotypic function for 3 weeks (7-way) and 4 weeks (10-way) of continuous interaction, as well as PK analysis of diclofenac metabolism. This study illustrates several generalizable design and operational principles for implementing multi-MPS "physiome-on-a-chip" approaches in drug discovery.
Full-text available
With more than 240 million people infected, hepatitis B virus (HBV) is a major health concern. The inability to mimic the complexity of the liver using cell lines and regular primary human hepatocyte (PHH) cultures pose significant limitations for studying host/pathogen interactions. Here, we describe a 3D microfluidic PHH system permissive to HBV infection, which can be maintained for at least 40 days. This system enables the recapitulation of all steps of the HBV life cycle, including the replication of patient-derived HBV and the maintenance of HBV cccDNA. We show that innate immune and cytokine responses following infection with HBV mimic those observed in HBV-infected patients, thus allowing the dis-section of pathways important for immune evasion and validation of biomarkers. Additionally, we demonstrate that the co-culture of PHH with other non-parenchymal cells enables the identification of the cellular origin of immune effectors, thus providing a valuable preclinical platform for HBV research.
Full-text available
Investigation of the pharmacokinetics (PK) of a compound is of significant importance during the early stages of drug development, and therefore several in vitro systems are routinely employed for this purpose. However, the need for more physiologically realistic in vitro models has recently fueled the emerging field of tissue-engineered 3D cultures, also referred to as organs-on-chips, or microphysiological systems (MPSs). We have developed a novel fluidic platform that interconnects multiple MPSs, allowing PK studies in multi-organ in vitro systems along with the collection of high-content quantitative data. This platform was employed here to integrate a gut and a liver MPS together in continuous communication, and investigate simultaneously different PK processes taking place after oral drug administration in humans (e.g., intestinal permeability, hepatic metabolism). Measurement of tissue-specific phenotypic metrics indicated that gut and liver MPSs can be fluidically coupled with circulating common medium without compromising their functionality. The PK of diclofenac and hydrocortisone was investigated under different experimental perturbations, and results illustrate the robustness of this integrated system for quantitative PK studies. Mechanistic model-based analysis of the obtained data allowed the derivation of the intrinsic parameters (e.g., permeability, metabolic clearance) associated with the PK processes taking place in each MPS. Although these processes were not substantially affected by the gut-liver interaction, our results indicate that inter-MPS communication can have a modulating effect (hepatic metabolism upregulation). We envision that our integrative approach, which combines multi-cellular tissue models, multi-MPS platforms, and quantitative mechanistic modeling, will have broad applicability in pre-clinical drug development.
Full-text available
A capability for analyzing complex cellular communication among tissues is important in drug discovery and development, and in vitro technologies for doing so are required for human applications. A prominent instance is communication between the gut and the liver, whereby perturbations of one tissue can influence behavior of the other. Here, we present a study on human gut-liver tissue interactions under normal and inflammatory contexts, via an integrative multi-organ platform comprising human liver (hepatocytes and Kupffer cells) and intestinal (enterocyte, goblet cells, and dendritic cells) models. Our results demonstrated long-term (>2 weeks) maintenance of intestinal (e.g., barrier integrity) and hepatic (e.g., albumin) functions in baseline interaction. Gene expression data comparing liver in interaction with gut, versus isolation, revealed modulation of bile acid metabolism. Intestinal FGF19 secretion and associated inhibition of hepatic CYP7A1 expression provided evidence of physiologically relevant gut-liver crosstalk. Moreover, significant non-linear modulation of cytokine responses was observed under inflammatory gut-liver interaction; for example, production of CXCR3 ligands (CXCL9,10,11) was synergistically enhanced. RNA-seq analysis revealed significant upregulation of IFNα/β/γ signaling during inflammatory gut-liver crosstalk, with these pathways implicated in the synergistic CXCR3 chemokine production. Exacerbated inflammatory response in gut-liver interaction also negatively affected tissue-specific functions (e.g., liver metabolism). These findings illustrate how an integrated multi-tissue platform can generate insights useful for understanding complex pathophysiological processes such as inflammatory organ crosstalk. This article is protected by copyright. All rights reserved.
Safety related drug failures continue to be a challenge for pharmaceutical companies despite the numerous complex and lengthyin vitro assays and in vivo studies that make up the typical safety...
The organs-on-a-chip technology has shown strong promise in mimicking the complexity of native tissues in vitro and ex vivo, and recently significant advances have been made in applying this technology to studies of the kidney and its diseases. Individual components of the nephron, including the glomerulus, proximal tubule, and distal tubule/medullary collecting duct, have been successfully mimicked using organs-on-a-chip technology and yielding strong promises in advancing the field of ex vivo drug toxicity testing and augmenting renal replacement therapies. Although these models show promise over 2-dimensional cell systems in recapitulating important nephron features in vitro, nephron functions, such as tubular secretion, intracellular metabolism, and renin and vitamin D production, as well as prostaglandin synthesis are still poorly recapitulated in on-chip models. Moreover, construction of multiple-renal-components-on-a-chip models, in which various structures and cells of the renal system interact with each other, has remained a challenge. Overall, on-chip models show promise in advancing models of normal and pathological renal physiology, in predicting nephrotoxicity, and in advancing treatment of chronic kidney diseases.
Different fabrication methods from traditional chemical engineering methods to advanced additive manufacturing (AM) are used for fabrication of tissue engineering (TE) scaffolds. The traditional techniques are subjected to limitations such as manual intervention, and inconsistent and inflexible processing procedures. In addition, the traditional techniques usually cannot control pore size, pore geometry, and spatial distribution of pores properly. To complement these limitations, there has been a trend in recent years to fabricate TE scaffolds using AM processes directly (3D printing of final scaffold) or indirectly (making negative scaffold to be used as a mold). In particular, extrusion-based AM systems have been widely used for fabrication of TE scaffolds due to their ability of processing different biomaterials, their possibility of manufacturing scaffolds in a cell-friendly environment, their high reproducibility and flexibility, and their simple process control in comparison with other AM techniques. In this chapter, the applications of extrusion-based 3D printing techniques are reviewed. In addition, an in-depth discussion on solvent-based extrusion freeforming (SEF) method, recent advances in printing bioceramic scaffolds, and its new application to make polyether-ether-ketone (PEEK)-based composites are presented.
Hepatotoxins cause liver damage via many mechanisms but the formation of reactive metabolites and/or damage to liver mitochondria are commonly implicated. We assess 3D human primary hepatocyte microtissues as a platform for hepatotoxicity studies with reactive metabolite-forming and mitochondria-perturbing compounds. We show that microtissues formed from cryopreserved human hepatocytes had bile canaliculi, transcribed mRNA from genes associated with xenobiotic metabolism and expressed functional cytochrome P450 enzymes. Hierarchical clustering was used to distinguish dose-dependent hepatotoxicity elicited by clozapine, fialuridine and acetaminophen (APAP) from control cultures and less liver-damaging compounds, olanzapine and entecavir. The regio-isomer of acetaminophen, N-acetyl-meta-aminophenol (AMAP) clustered with the hepatotoxic compounds. The principal metabolites of APAP were formed and dose-dependent changes in metabolite profile similar to those seen in patient overdose was observed. The toxicological profile of APAP was indistinguishable from that of AMAP, confirming AMAP as a human hepatotoxin. Tissue oxygen consumption rate was significantly decreased within 2h of exposure to APAP or AMAP, concomitant with glutathione depletion. These data highlight the potential utility of perfused metabolically functional human liver microtissues in drug development and mechanistic toxicology.