Organ-on-a-Chip, Tissue-on-a-Chip Europe 2018 Agenda

Conference Chairman Welcome and Opening Remarks
Richard Chasen Spero, CEO, Redbud Labs, United States of America

Nanoplasmonic Biosensors: From Innovative Materials to Multimode Sensing with Integrated Microdevices
Amy Shen, Professor and Provost, Okinawa Institute of Science and Technology Graduate University, Japan

Gold nanostructures are a highly attractive class of materials with unique electrochemical and optical sensing properties. Recent developments have greatly improved the sensitivity of optical sensors based on metal nanostructured arrays. We introduce the localized surface plasmon resonance (LSPR) sensors and describe how its exquisite sensitivity to size, shape and environment can be harnessed to detect molecular binding events. We then describe recent progress in three areas representing the most significant challenges: integration of LSPR with complementary electrochemical techniques, long term live-cell biosensing and practical development of sensors and instrumentation for routine use and high-throughput detection. As an example we will demonstrate a novel refractive index and charge sensitive device integrated with nanoplasmonic islands to develop nano-metal-insulator-semiconductor (nMIS) junctions. The developed sensor facilitates simultaneous detection of charge and mass changes on the nanoislands due to biomolecule binding. A brief insight on microcontact printing to functionalize proteins on nanoplasmonic sensors will also be discussed. The developed nanosensors can readily be adopted for multiplexed and high throughput label-free immunoassay systems, further driving innovations in biomedical and healthcare research.

Towards Wearable Real-Time Clinical Monitoring Using Microfluidic Devices
Martyn Boutelle, Professor of Biomedical Sensors Engineering, Imperial College London, United Kingdom

Modern acute critical care medicine is increasingly seeking to protect vulnerable tissue from damage by monitoring the patterns of physical, electrical and chemical changes taking place in tissue – so called multimodal monitoring. Such patterns of molecular changes offer the exciting possibility of allowing clinicians to detect changes in patient condition and to guide therapy on an individualized basis in real time. Microfluidic lab-on-chip devices coupled to tissue sampling using microdialysis provide an important new way for measuring real-time chemical changes as the low volume flow rates of microdialysis probes are ideally matched to the length scales of microfluidic devices. In this presentation, I will describe the combination of miniature electrochemical sensors and biosensors with 3D printed microfluidic devices for transplant organ and patient monitoring. Concentrations of key biomarker molecules can then be determined continuously using either optically or electrochemically, using amperometric, potentiometic and array sensors.  Wireless devices allow analysis to take place close to the patient. Droplet-based microfluidics, by digitizing the dialysis stream into discrete low volume samples, both minimizes dispersion allowing very rapid concentration changes to be measured, and allows rapid transport of samples between patient and analysis chip. This talk will overview successful design, optimization, automatic-calibration and use of both continuous flow and droplet-based microfluidic analysis systems for real-time clinical monitoring, using clinical examples from our recent work.

Silicon Nanotechnology Meets Biology (Smaller and Wetter is Better)
Gregory Timp, Keough-Hesburgh Professor of Electrical Engineering & Systems Biology, The University of Notre Dame, United States of America

According to Moore’s law, the scaling of silicon integrated circuits is supposed to reach the 5 nm-node sometime after 2020, although the schedule is still problematic due to the astronomical cost and atomically precise line-rules. On the other hand, biology has been performing cost-effectively using proteins the size of 5 nm (and smaller) that fold with atomic precision for 4.28 billion years now—it is a robust and proven technology, albeit wet. In this talk, it is argued that there is still “plenty of room at the bottom” for improving performance if silicon nanotechnology is adapted to biology. With silicon nanotechnology it is now within our grasp to create an interface to biology on a nanometer-scale. Three examples of such interfaces are proffered. The first is a liquid flow cell that works like an envelope made from 30 nm-thick silicon nitride membranes, which can hold and sustain living cells in medium and yet fits inside a Scanning Transmission Electron Microscope (STEM). In a STEM, the liquid cell can be used to visualize and track live cell physiology like a phage infecting a bacterium with nucleic acids at 5 nm resolution. The second is a nanometer-diameter pore sputtered through a silicon nitride membrane 10-nm-thick that can be used to transfect cells precisely with nucleic acids to affect gene expression in them and, under different bias conditions, detect protein secretions from single cells with single molecule sensitivity. The secretions inform on the cell phenotype and offer a molecular diagnosis of disease. Finally, the third interface is a sub-nanometer-diameter pore, which is about the size of an amino acid residue, in either silicon dioxide or silicon nitride membranes ranging from 6 to 10 nm-thick.  Sub-nanopores like this have been used to read the primary structure of a protein, i.e. the amino acid sequence, with low fidelity, but with single molecule sensitivity, vastly outstripping the sensitivity of conventional methods for sequencing such as mass spectrometry. Taken altogether, the prospects are dazzling for a new type of integrated circuit that incorporates biology with state-of-the-art silicon electronics.

Extracellular Vesicles (EVs) and Cell Free DNA (cfDNA) as Blood-based Biomarkers: Plastic-based Microfluidics for their Enrichment and Analysis
Steve Soper, Foundation Distinguished Professor, Director, Center of BioModular Multi-Scale System for Precision Medicine, The University of Kansas, United States of America

While there are a plethora of different blood-based markers, EVs are generating significant interests due to their relatively high abundance (~10¹³ particles per mL of blood) and the information they carry. EVs contain a diverse array of nucleic acids, such as mRNA, lncRNA, and miRNA that can be used for disease management. In addition to EVs, cfDNA also are biomarkers that can be used to help manage different disease states using the mutations they possess that can have high diagnostic value. In spite of the relatively high abundance of cfDNA in diseased patients (~160 ng/mL), the extraction and enrichment of cfDNA has been inefficient, even by commercial kits, due to the low abundance of the tumor bearing DNA fragments (<0.01%) and the short nature of these fragments, especially cancer-related cfDNA (as small as 50 bp). In this presentation, we will discuss the design, fabrication and analytical figures-of-merit of a microfluidic device that can serve the dual purpose for the affinity-based selection of EVs and the solid phase extraction of cfDNA directly from plasma using the same device. The microfluidic is made from a plastic that can be injection molded to produce high quality devices at low cost. For EVs, the device is made cyclic olefin copolymer (COC) is UV/O3 activated to allow for the efficient immobilization of affinity agents to the surface of the device. In the case of cfDNA, the device is made from COC as well, but is only UV/O3 activated (i.e., no affinity agents used). Information will be provided as to the ability to molecularly profile the cargo contained within the affinity-selected EVs, in particular mRNA expression profiling. We will also discuss the use of this microfluidic to isolate with high recovery cfDNA from plasma samples with size selection capabilities. The isolated cfDNA could be queried for mutations using an allele-specific ligation detection reaction at a mutant to wild-type ratio <0.1%.

Microphysiological Models Relying on Emergence of Multi-Cellular Engineered Living Systems
Roger Kamm, Cecil and Ida Green Distinguished Professor of Biological and Mechanical Engineering, Massachusetts Institute of Technology (MIT), United States of America

Recent work from many labs has demonstrated the unique capability of cells placed in 3D culture to self-organize into functional units and organ-like systems.  In some cases, pluripotent cells can be induced to differentiate down independent pathways, leading to an organoid.  In others, interacting units can be generated, often from iPS cells, and ‘engineered’ to interact in a way that recapitulates certain aspects of in vivo function or disease.  Such models have tremendous potential both to gain new insight into disease processes and for moderate throughput drug screening.  In this talk I will describe several models developed in our lab including a neuromuscular junction, blood-brain barrier, and vascularized skeletal muscle, addressing some of the design principles they have in common, the future potential, and barriers to progress.

Microscale Flow Patterning
Moran Bercovici, Associate Professor, Faculty of Mechanical Engineering; Head, Technion Microfluidic Technologies Laboratory, Technion, Israel Institute of Technology, Israel

The ability to manipulate fluids at the microscale is a key element of any lab-on-a-chip platform, enabling core functionalities such as liquid mixing, splitting and transport of molecules and particles. Lab-on-a-chip devices are commonly divided in two main families: continuous phase devices, and discrete phase (droplets) devices. While a large number of mechanisms are available for precise control of droplets on a large scale, microscale control of continuous phases remains a substantial challenge. In a traditional continuous-flow microfluidic device, fluids are pumped actively (e.g. by pressure gradients, electro-osmotic flow) or passively (e.g. capillary driven) through a fixed microfluidic network, making the device geometry and functionality intimately dependent on one another (e.g. DLD, inertial mixer, H-separator, etc.). The advent of on-chip microfluidic valves brought more flexibility in routing fluids through microfluidic networks, adding a dynamic dimension to the static geometrical network. However, the number of degrees of freedom of valve-based systems is restricted by their dependence on bulky pneumatic lines (regulators, pressure systems, controllers), which are difficult to scale down in size and cost. In this talk I will present our ongoing work leveraging non-uniform EOF and thermocapillary flows to control flow patterns in microfluidic chambers.  By setting the spatial distribution of surface potential or a spatial temperature distribution, we demonstrate the ability to dictate desired flow patterns without the use of physical walls. We believe that such flow control concepts will help break the existing link between geometry and functionality, bringing new capabilities to on-chip analytical methods.

Functional Coupling of Human Pancreatic Islet Microtissues and Liver Spheroids in a Novel Micro-physiological Multi-organ System For Studies of Human Metabolic Diseases in Drug Discovery
Tommy Andersson, Drug Metabolism and Pharmacokinetics, Cardiovascular and Metabolic Diseases, AstraZeneca, Gothenburg, Sweden

Multi organ-on-a-chip technologies, emulating human physiology and mimicking human disease states, could aid preclinical efforts by providing relevant translational models to validate targets and test tool compounds early in drug discovery. Such models have the potential to improve translation to patients, decrease time spent in early clinical programs and reduce the need for animal models. Rodent studies have shown that insulin resistance causes hepatocytes to produce secreted factors that influence the islets. Whether similar cross talk exist in man remains to be determined. We therefore decided to develop a human liver - pancreatic islets chip model. Pancreatic islets and liver spheroids were applied in a two-organ microfluidic chip supplied by TissUse™ that allows cross talk between both organs. We have demonstrated that the model responds in a physiological way to a glucose load by increasing insulin secretion, which stimulates glucose consumption by the liver spheroids.  Both islet and liver spheroids show stable functionality as indicated by insulin secretion, albumin production and glucose consumption over the experimental period of two weeks. Initial results indicate that the liver spheroids can be made insulin resistant, and thus represent a relevant metabolic disease model.

Human Organs-on-Chips
Andries D. van der Meer, Associate Professor, Scientific lead, Organ-on-Chip Center Twente, University of Twente, Netherlands

Organs-on-chips are plastic microdevices the size of a USB-stick with microchannels and small chambers that are filled with liquid. The devices contain multiple human cell types which are cultured in a technologically controlled microenvironment that artificially mimics aspects of the human body like morphology, movement, flow, electrical stimuli and liquid gradients. The resulting device emulates human organ functions and can be used to study biomedical phenomena in the lab. By using patient-specific cells, blood samples, biometrics and imaging data to develop ‘personalized organs-on-chips’, we can study health and disease in models that are relevant for specific patients. The power of such a ‘personalized’ approach is demonstrated in our application of organs-on-chips in studies of thrombosis and vascular biology.

Human Vascular Microphysiological System Models for Drug Testing and Disease Modeling
George Truskey, R. Eugene and Susie E. Goodson Professor of Biomedical Engineering, Duke University, United States of America

We developed an endothelialized tissue engineered blood vessel (eTEBV) microphysiological system by rapid generation of small-diameter vessels (400-800 µm) by plastic compression. TEBVs were mechanically strong enough to allow endothelialization and perfusion at physiological shear stresses immediately after fabrication.  eTEBVs perfused at physiological shear stresses for 1- 5 weeks expressed von Willebrand factor (vWF) and demonstrated EC-specific release of NO, indicating a confluent layer of ECs. After 1-5 weeks of perfusion, eTEBVs exhibited dose-dependent contraction and relaxation following exposure to phenylephrine and acetylcholine (ACh), respectively. In contrast, TEBVs without ECs or eTEBVs pre-treated with the NO synthase inhibitor L-NG-Nitroarginine methyl ester underwent vasoconstriction in response to ACh consistent with vasodilation by EC release of NO. TEBVs elicited reversible activation following acute stimulation by TNFa which transiently inhibited ACh-induced relaxation, and was eliminated by pre-exposure of eTEBVs to therapeutic doses of statins. TNFa treatment also promoted monocyte adhesion and transmigration.  Using smooth muscle cells and endothelial cells derived from iPSCs, we produced a functional eTEBV model of Hutchison-Gilford Progeria Syndrome (HGPS), a rare, accelerated aging disorder caused by an altered form of the lamin A (LMNA) gene termed progerin.  eTEBVs fabricated with smooth muscle cells from individuals with HGPS show reduced vasoactivity, increased medial wall thickness, increased calcification and apoptosis in comparison to eTEBVs fabricated with smooth muscle cells from normal individuals or primary MSCs.  In addition, treatment with the rapamycin analog, RAD001, increased HGPS TEBV vasoactivity and reduces some disease symptoms.  These results indicate that we can use human eTEBVs to model diseases in vitro.

Lung-on-Chip Models of the Healthy and Diseased Lung Parenchyma
Olivier Guenat, Head, Organs-on-Chip Technologies, ARTORG Center for Biomedical Engineering Research, University of Bern-Switzerland, Switzerland

The complexity of the lung can be illustrated by its delicate tree-like architecture that ends with the alveolar sacs, where the gas exchanges take place. This whole environment is subjected to a cyclic, mechanical constraint induced by the respiratory movements. We recently reported about an advanced in-vitro model of the lung parenchyma that mimics the key aspects of the lung alveolar environment in an unprecedented way (Stucki et al., Lab Chip, 2015). The system reproduces the three-dimensional mechanical strain induced by the respiratory movements, the air-liquid interface and the ultra-thin barrier, using an elastic membrane made of a 3um thin PDMS porous (3um pores) layer on which cells can be cultured on both sides. We could demonstrate that the physiological mechanical stress significantly affects a number of barrier functions, such as the transport of molecules though the barrier. Furthermore, in view to make the system widely accessible, great care was taken to make it robust and simple to use. In contrast to the lung-on-chip reported by the Wyss Institute in Boston (Huh et al. Science 2010), our device mimics the three-dimensional movements induced by the respiration (instead of a unidirectional stress in the Wyss lung-on-chip). We also demonstrated that the three-dimensional breathing motions of the alveolar barrier can be monitored in real time with a micro-impedance tomography system (Mermoud et al., Sensors and Actuators B: 2018). The development of the lung-on-chip was awarded with a number of prizes, including the Venturekick awards, a nomination at the Swiss Medtech Award in 2017 and is currently under development at AlveoliX, a start-up aimed at bringing organs-on-chip on the market. Recently, we reported about another part of the lung alveolar barrier, the lung microvasculature.  Lung endothelial cells and lung pericytes seeded in a micro-engineered environment filled with fibrin gel, self-assemble to build a network of perfusable and contractile microvessels of only a few tens of micrometers in diameter (Bichsel et al., Tissue Eng. A, 2015). We could demonstrate for the first time in-vitro that microvessels contract upon exposure to a vasoconstrictor. This model, made of primary human cells of the lung is of great relevance for the investigation of pathomechanism of lung diseases.

Validating Microphysiological Systems
Danilo Tagle, Director, Office of Special Initiatives, National Center for Advancing Translational Sciences at the NIH (NCATS), United States of America

Concurrent Toxicity and Efficacy Evaluations in Multi-Organ Functional Human-on-a-Chip Systems
James Hickman, Professor, Nanoscience Technology, Chemistry, Biomolecular Science and Electrical Engineering, University of Central Florida; Chief Scientist, Hesperos, United States of America

The current drug development process is inefficient, taking years from target compound identification to marketable drug, and costs up to 2 billion dollars during the process. The considerable attrition-rate of drug candidates at all stages of development to a significant extent arises from the poor predictive nature of preclinical models for efficacy and toxicity, especially the inability to translate efficacy between preclinical and clinical situations. Systems capable of directly measuring organ function, biomarker release, and most importantly the synergistic interactions between organs, especially the generation of liver metabolites would be ideal. Body-on-a-chip systems utilize function based cell models that accurately capture and predict multi organ complexity in response to administered compound within correctly scaled and physiologically relevant platforms. This data can then theoretically be used to produce relevant modeling information related to drug responses in clinical settings, and thereby provide accurate predictions of a compound’s patient specific toxicology and efficacy. We have demonstrated functional human models to evaluate multi-organ toxicity in 2-, 3- and 4-organ systems under continuous flow conditions in a serum-free defined medium utilizing a pumpless platform for 28 days with cardiac, muscle, cancer, neuronal and liver modules. The pharmacological relevance of these integrated modules was evaluated with drugs and compared to human and animal data and these results will be presented. There currently is also a focus at the NIH, FDA and EMA to understand how one could validate these systems such that qualification could be granted for their use to augment and possibly replace animal studies. This talk will also give results of six workshops held at NIH as a collaboration between the American Institute for Medical and Biological Engineering (AIMBE) and NIH to explore what is needed for validation and qualification of these new systems.

Engineering Multicompartment Organ-Chip Systems
Elisabeth Verpoorte, Chair of Analytical Chemistry and Pharmaceutical Analysis, University of Groningen, Netherlands

The miniaturized total chemical analysis system has been successfully repurposed over the past twenty years or so for engineering cellular microenvironments which more faithfully mimic in vivo conditions for cell- and tissue-based studies. Organs-on-a-chip are a recent outgrowth of this effort, comprising systems that extend beyond microfluidic perfusion culture to allow the establishment of microphysiological systems comprising interconnected multiorgan systems. This presentation will consider a couple of examples of such systems out of our labs in Groningen for the elucidation of organ (inter)actions in the gastrointestinal tract and the liver.

Organs-on-Chips: Microengineered Systems for Safety Assessment & Disease Modeling
Geraldine A Hamilton, President/Chief Scientific Officer, Emulate Inc, United States of America

Microengineered Organs-Chips show physiological functions consistent with normal living human or animal cells in vivo. Each Organ-Chip is composed of a clear flexible polymer about the size of a AA battery that contains hollow channels lined by living human cells. The cells are cultured under continuous flow and mechanical forces thereby recreating key factors known to influence cell function in vivo.  Cells cultured  under continuously perfused, engineered 3D microenvironments go beyond conventional 3D in vitro models by recapitulating in vivo intercellular interactions, spatiotemporal gradients, vascular perfusion, and mechanical microenvironments. Integrating cells within Organs-on-Chips, enables the study of normal physiology and pathophysiology in an organ-specific context. We have developed different Organ-Chips, such as, Liver-Chip, Lung-Chip, and Intestine-Chip.  We have further demonstrated new advancements in functionality of these systems and their potential across multiple applications relevant to drug discovery and development including: understanding disease mechanisms, efficacy testing, safety assessment, and in de-risking mechanistic concerns using various organ systems. In this presentation, we highlight data to demonstrate the utility of the system for understanding disease mechanisms and predicting safety of drugs.

Microfluidics within a Well: Injection-Molded Plastic Array for 3D Culture
Noo Li Jeon, Professor, Seoul National University, Korea South

Polydimethylsiloxane (PDMS) has been widely used in fabricating microfluidic devices for prototyping and proof-of-concept experiments. Due to several material limitations, PDMS has not been widely adopted for commercial applications that require large-scale production. This presentation will describe a mass-producible Injection-Molded Plastic Array for 3D Culture Tissue (IMPACT) platform that incorporates a novel microfluidic design to integrate patterned 3D cell culture within a single 96-well. Multiple hydrogel patterns can be sequentially assembled by utilizing capillary-guided flow along wedges and narrow gaps designed within the well. The patterning is quick and robust. It is as simple as placing a liquid droplet on any part of the wedge structure to obtain spontaneous patterning within 1 second. Optimal dimensionless parameters required for successful capillary loading have been determined. The IMPACT platform was used to co-culture HUVEC and LF, and to obtain angiogenic sprouts with lumens and tip cells that are comparable to those observed in PDMS based devices. This platform is expected to have a wide range of applications related to biological discovery, tissue engineering and drug screening, and can be used in a variety of scales from lab bench to automated equipment.

Cell Fiber Technology For 3D Organ-on-a-Chip Application
Shoji Takeuchi, Professor, Center For International Research on Integrative Biomedical Systems (CIBiS), Institute of Industrial Science, The University of Tokyo, Japan