Organ-on-a-Chip & Tissue-on-a-Chip Europe 2019 Agenda

Chairperson's Welcome and Introduction to the Conference
Nancy Allbritton, Kenan Professor of Chemistry and Biomedical Engineering and Chair of the Joint Department of Biomedical Engineering, University of North Carolina and North Carolina State University, United States of America

Optofluidic Labs-on-Chip For Single Molecule Analysis
Holger Schmidt, Narinder Kapany Professor of Electrical Engineering, University of California-Santa Cruz, United States of America

Lab-on-chip devices have long held the promise of providing a convenient and rapid way to analyze small amounts of biological samples. However, when pushed to the ultimate limit of single molecule sensitivity, the detection mechanism is often based on off-chip elements. I will discuss a chip-scale platform that offers both integrated optical and electrical single molecule analysis. Optical integration is achieved by using liquid-core waveguides interfaced with traditional photonic elements to implement advanced functionalities. Examples include multiplex detection of single viruses, simultaneous detection of proteins and nucleic acid biomarkers, and front-to-back sample handling and single DNA detection on a single chip. Electrical single molecule analysis is achieved by integration of solid-state nanopores. Novel nanopore detection capabilities such as feedback-controlled delivery of single molecules to a fluidic channel are demonstrated. The combination of both optical and electrical detection modalities results in a novel, high throughput platform for single molecule analysis.

Organs-on-Chips: Analytical Tools and Objects for Analysis
Elisabeth Verpoorte, Chair of Analytical Chemistry and Pharmaceutical Analysis, University of Groningen, Netherlands

Organs-on-chips (OoC) can be viewed as biological processing units, meant to replicate on a smaller scale the cell architecture and functioning of the organs they represent. OoC may be used to probe and thus better understand the mechanisms by which organs function. Mostly, though, they will be used as in vitro processing tools, to yield insight as to the fate of compounds like drugs or foodstuffs in vivo. Either way, merging analytical detection technologies with microfluidics and cell culture is key to the development of these systems for advanced in vitro analysis. Several examples of analytical approaches for monitoring liver slice behavior stemming from our labs will be presented in this presentation.

New Opportunities in Acoustofluidic Processing of Liquid Biopsies
Thomas Laurell, Professor, Lund University, Sweden

Blood sampling and molecular analysis of plasma is the most common clinical diagnostic modality in modern health care. With more advanced means to downstream processing, sub components of blood can be isolated, improving the diagnostic outcome. Detection and analysis of rare events is specifically an area where high performance isolation of sub components of blood is critical and where lab-on-a-chip technology has paved the way for several new approaches. Acoustic forces in combination with microfluidics has open new avenues for high performance blood cell separation, enabling isolation of cancer cells from blood, leukocyte sub populations and more recently also enrichment and purification of extracellular vesicles. Our initial efforts in isolating circulating tumor cells using acoustophoresis in clinical scale prostate cancer patients samples will be reported.

Although conventional acoustophoresis is limited in is ability to manipulate particles smaller than 1 um secondary acoustic effects can alleviate this short coming. Scattered ultrasound between larger particles, that can be retained in a local acoustic field in a microchannel, can be utilized to enrich submicron vesicles on the larger “trapping particles”. Based on this we have developed a microfluidic platform that enables isolation of extra cellular vesicles (EVs) from biofluids such a urine, blood plasma and cell culture medium without the need for ultracentrifugation. Further, benefits of isolating EVs by means of acoustic trapping is rapid processing that provides relatively high recoveries (up to 60-80%), yet handling small sample volumes (20-50 uL blood plasma), giving access to molecular profiling of extra cellular vesicles in biobanks. Our studies have demonstrated protein and miRNA profiling of blood plasma EVs from myocardial infarction patients as well as in urine samples from healthy donors. More recently we have been able to detect EV protein biomarker profiles in heart transplant patients with acute rejection which could not be detected in blood plasma.

Nanosensor Chips for the Single-Molecule Sequencing of DNA and RNA
Steve Soper, Foundation Distinguished Professor, Director, Center of BioModular Multi-Scale System for Precision Medicine, The University of Kansas, United States of America

We are generating a single-molecule DNA/RNA sequencing platform that can acquire sequencing information with high accuracy (>99%) at unprecedented throughputs (106 nt/s). The technology employs a fluidic chip populated with nanosensors that read the identity of individual mononucleotides from their characteristic flight-time through a 2-dimensional (2D) nanochannel (~50 nm in width and depth; >10 µm in length) and their current transient amplitudes. The nanosensors are fabricated in a thermoplastic via nanoimprint lithography (NIL). The mononucleotides are generated from an intact DNA fragment using a highly processive exonuclease, which is covalently anchored to a plastic support (500 nm in diameter) contained within a bioreactor that sequentially feeds mononucleotides into a 2D nanochannel. The identity of the mononucleotides is deduced from a molecular-dependent flight-time through the 2D nanochannel that is related to the electrophoretic mobility of that molecule. The flight time is read in a label-less fashion by measuring current transients (i.e., resistive pulse sensing) induced by a single mononucleotide when it travels through a constriction possessing molecular dimensions (<10 nm in diameter) and poised at the input/output ends of the flight tube. In this presentation, our efforts in building these nanosensors using NIL in thermoplastics will be discussed. We will also talk about the detection of single molecules using NIL-produced nanopores. Also, surface modifications of plastics for the immobilization of biologics, such as exonucleases, will be discussed and their enzymatic performance when surface immobilized. Finally, information on the manipulation of single DNA molecules using nanofluidic circuits that uses nano-scale features to shape electric fields will be presented.

Intestine on a Chip for Basic Biology and Patient-Specific Medicine
Nancy Allbritton, Kenan Professor of Chemistry and Biomedical Engineering and Chair of the Joint Department of Biomedical Engineering, University of North Carolina and North Carolina State University, United States of America

The ability to monitor and control the environment at the cellular and tissue level is one of the most promising applications for microengineered systems. Advances over the past decade in our ability to isolate and culture stem cells when combined with microengineering make it possible to conceive of physiologically functional systems that reconstitute whole organ physiology. These “organ-on-a-chip” platforms enable the establishment of the tissue interfaces necessary for organ function while providing exquisite control of experimental variables and sharing the richness of the intact organism. My group is at the forefront of marrying these advances in stem-cell culture with microengineering to create in vitro tissue mimics with a focus on the intestine. We constructed in vitro epithelium for human and mouse, small and large intestine that bear a stunning physical resemblance to the in vivo intestinal epithelium epithelia displaying arrays of polarized crypts. Most importantly this system recapitulates much of the physiology of the native intestinal epithelium. The system is constructed by developing a self-renewing monolayer of primary cells which is then shaped using microfabrication techniques. The platform enables application of the diverse chemical gradients (growth factors, morphogens, bacterial metabolites, food substances, and gases) thought to exist along the crypt-villus axis.  Application of gradients of microbiota-derived fermentation products across this tissue provided a direct demonstration that these products drive alterations in the size of the crypts’ proliferative and differentiated compartments as predicted to occur in vivo. The system also enables co-culture of anaerobic intestinal bacteria above a physiologic mucus layer. Using this platform, small intestinal biopsies from humans can be used to populate the constructs with cells producing patient-specific tissues for personalized medicine.

Emerging Technologies For Biohybrid Machines
Shoji Takeuchi, Professor, Center For International Research on Integrative Biomedical Systems (CIBiS), Institute of Industrial Science, The University of Tokyo, Japan

Engineers have created various kinds of machines such as smart phones, humanoid robots, self-driving cars, etc. However, there are still big hurdles to build a system that demonstrates attractive functions appearing in biological systems, such as single molecule recognition, highly efficient biomolecular production, biocompatibility and self-healing/self-reproducing ability etc…

To achieve this function, we have studied biohybrid machines that harnesses the living system within artificial systems. In this symposium, I would like to talk about a couple of our recent results regarding biohybrid sensors and actuators.

Towards Wearable Microfluidic Devices For Well-Being and Personalized Healthcare
Martyn Boutelle, Professor of Biomedical Sensors Engineering, Imperial College London, United Kingdom

Advances in microfluidic technologies, electronics and sensors, combined with the phenomenal growth in mobile computing power provided by current tablets and mobiles allow us to imaging finally taking the ‘lab-on-a-chip’ away from its laboratory full of control equipment. Through miniaturization and carefully engineered smart designs we can embed computer control and analytical best practice into portable even wearable devices that are able to compensate for shortcomings such as falling performance. These hybrid microfluidic systems appear to their target users as simple stable systems that tell them what they want to know. My group specializes in designing and building such microfluidic systems to meet the needs of acute critical care medicine. Key molecular markers are measured using both optical and electrochemical sensors and biosensors. We then work with clinical care teams to show proof of concept of the real-time continuous chemical information that microfluidic systems can produce. Our ultimate goal is that such systems can be used to monitor patients and guide therapy in a patient-specific, personalized way.

Human-on-a-Chip Systems For Use in Efficacy and Toxicological Investigations For Applications in Neurological Diseases
James Hickman, Professor, Nanoscience Technology, Chemistry, Biomolecular Science and Electrical Engineering, University of Central Florida; Chief Scientist, Hesperos, United States of America

One of the primary limitations in drug discovery and toxicology research is the lack of good model systems between the single cell level and animal or human systems. This is especially true for neurodegenerative diseases such as ALS and Alzheimer’s as well as spinal cord injury. In addition, with the banning of animals for toxicology testing in many industries body-on-a-chip systems to replace animals with human mimics is essential for product development and safety testing. Our research focus is on the establishment of functional in vitro systems to address this deficit where we seek to create organs and subsystems to model motor control, muscle function, myelination and cognitive function, as well as cardiac and liver subsystems. The idea is to integrate microsystems fabrication technology and surface modifications with protein and cellular components, for initiating and maintaining self-assembly and growth into biologically, mechanically and electronically interactive functional multi-component systems. Our advances in culturing adult rat, mouse and human mammalian spinal cord, hippocampal neurons, muscle and cardiac cells in a defined serum-free medium, suggest outstanding potential for answering questions related to maturation, aging, neurodegeneration and injury. A specific embodiment of this technology is the creation of a functional human NMJ system to understand ALS. We have investigated four mutations found in ALS patients; SOD1, FUS, TDP43 and C9ORF72. The models have demonstrated variations of the disease phenotype compared to WT for NMJ stability and functional dynamics. Results of these studies will be presented as well as preliminary results for reversal of the deficits. Examples will be given of some of the more advanced human-on-a-chip systems being developed for CNS and PNS disease applications as well as the results of six workshops held at NIH to explore what is needed for validation and qualification of these systems.

Advanced Organ-on-a-Chip Systems: Integrated Microphysiological Platforms Recapitulating Complex Human Tissue
Peter Loskill, Assistant Professor for Experimental Regenerative Medicine; Attract Group Leader Organ-on-a-Chip, Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Eberhard Karls University Tübingen, Germany

Drug discovery and development to date has relied on animal models, which are useful, but fail to resemble human physiology. The discovery of human induced pluripotent stem cells (hiPSC) has led to the emergence of a new paradigm of drug screening using human patient- and disease-specific organ/tissue-models. One promising approach to generate these models is by combining the hiPSC technology with microfluidic devices tailored to create microphysiological environments and recapitulate 3D tissue structure and function. Such organ-on-a-chip systems (OoCs) or microphysiological systems combine human genetic background, in vivo-like tissue structure, physiological functionality, and “vasculature-like” perfusion.

Using microfabrication techniques, we have developed a variety of OoCs that incorporate complex human 3D tissues and keep them viable and functional over multiple weeks, including “Retina-on-a-chip”, “Choroid-on-a-chip”, “Heart-on-a-chip”, “Pancreas-on-a-chip and a “White adipose tissue(WAT)-on-a-chip”. The OoCs generally consist of three functional components: organ-specific tissue chambers mimicking in vivo structure and microenvironment of the respective tissues; “vasculature-like” media channels enabling a precise and computationally predictable delivery of soluble compounds (nutrients, drugs, hormones); “endothelial-like” barriers protecting the tissues from shear forces while allowing diffusive transport. The small scale and accessibility for in situ analysis makes our OoCs amenable for both massive parallelization and integration into a high-content-screening approach.

The adoption of OoCs in industrial and non-specialized laboratories requires enabling technologies that are user-friendly and compatible with automated workflows. We have developed technologies for automated 3D tissue generation as well as for the flexible plug&play connection of individual OoCs into multi-organ-chips. These technologies paired with the versatility of our OoCs pave the way for applications in drug development, personalized medicine, toxicity screening, and mechanistic research.

“Cancer-on-a-Chip”: Basic and Translational Studies in Cancer Biology
Steven C. George, Edward Teller Distinguished Professor and Chair, Department of Biomedical Engineering, University of California-Davis, United States of America

Tissue engineering holds enormous potential to not only replace or restore function to a wide range of tissues, but also to capture and control 3D physiology in vitro (e.g., microphysiological systems or “organ-on-a-chip” technology).  The latter has important applications in the fields of drug development, toxicity screening, modeling tumor metastasis, and repairing damaged cardiac (heart) muscle. In order to replicate the complex 3D arrangement of cells and extracellular matrix (ECM), new human microphysiological systems must be developed.  The past decade has brought tremendous advances in our understanding of stem cell technology and microfabrication producing a rich environment to create an array of “organ-on-a-chip” designs.  Over the past six years we have developed novel microfluidic-based systems of 3D human microtissues (~ 1 mm³) that contain features such as perfused human microvessels, primary human cancer, mouse tumor organoids, stem cells, and spatiotemporal control of oxygen.  The technologies are part of two early start-up companies, Kino Biosciences and Immunovalent Therapeutics, based in Irvine, CA and St. Louis, MO.  This seminar will describe our approach and early results, including basic and translational studies in cancer biology.

Organ-on-a-Chip Technology as a Routine Tool in Drug Discovery and Development
Paul Vulto, Managing Director, MIMETAS, Netherlands

Organ-on-a-Chip technology is a new paradigm in drug testing. The technology has as its aim to raise the physiological relevance of traditional cell culture by combining this with microfluidic techniques. Organs-on-a-Chip are 3D tissues that capture the complexity of in vivo tissues including 3D morphology, extracellular matrix embedment, multiple cell types, vascular structure and perfusion flow.

In this presentation I will introduce culture and interrogation of complex 3D tissues, such as liver, kidney, gut and brain tissue. The tissues are grown on the OrganoPlate® platform, that allow culturing of over 40 tissues in parallel. The tissues are embedded in an extracellular matrix gel and comprise both stromal tissue, bloodvessel structures as well as epithelial barriers with clear apical and basal sides. The platform can be interrogated using automated imaging equipment as well as all standard equipment including fluorescence-based assays, immuno-histochemical staining, barrier integrity monitoring, transport studies, viability assays, qRT-PCR, ELISA’s and many others. Last but not least, operation of the platform is thus straightforward that an end-user who is not necessarily an expert in the field of microfluidics is able to perform the study. In this presentation I will show examples of Organ-on-a-Chip tissues for use in drug safety testing and disease modeling.

Human Vascular Microphysiological Systems to Model Genetic and Acquired Diseases
George Truskey, R. Eugene and Susie E. Goodson Professor of Biomedical Engineering, Duke University, United States of America

Cardiovascular disease represents the major cause of death in much of the world today.  Current in vitro models fail to recapitulate the complex tissue architecture and 3D structure of in vivo vasculature and animal models do not accurately model disease states. To address these limitations, we developed an endothelialized tissue engineered blood vessel (eTEBV) microphysiological system. Small-diameter eTEBVs (400-800 µm) were prepared by plastic compression and perfused at physiological shear stresses for 1- 5 weeks. The eTEBVs expressed von Willebrand factor and demonstrated endothelial cell (EC)-specific release of nitric oxide (NO). Acute stimulation by TNFa transiently inhibited ACh-induced relaxation and was eliminated by pre-exposure of eTEBVs to therapeutic doses of statins. Treatment of eTEBVs with enzyme-modified low-density lipoprotein (eLDL) caused activation of endothelial cells and promoted monocyte adhesion and transmigration. Further, treatment of eTEBVs with 50 µg/mL of eLDL for 96 hours caused monocytes to become foam cells and inhibited vasoactivity, indicators of early atherosclerosis. Monocyte accumulation and foam cell formation were inhibited by addition of lovastatin with eLDL. Using smooth muscle cells (SMCs) and ECs derived from induced pluripotent stem cells (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 SMCs from individuals with HGPS show reduced vasoactivity, increased medial wall thickness, calcification and apoptosis in comparison to eTEBVs fabricated with SMCs from normal individuals or primary MSCs.  HGPS viECs aligned with flow but exhibited reduced function compared to normal controls.  Relative to eTEBVs with healthy cells, HGPS eTEBVs showed reduced function and markers of cardiovascular disease associated with the endothelium. HGPS eTEBVs exhibited a reduction in both vasoconstriction and vasodilation and HGPS viECs produced VCAM-1 and E-selectin in eTEBVs with either healthy or HGPS viSMCs.  HGPS eTEBV function could be restored by addition of a farnesyl transferase inhibitor with or without a rapamycin analog. These results indicate that we can use human eTEBVs to model a variety of diseases in vitro.

Tomorrow Today: Organ-on-a-Chip Advances Towards Clinically Relevant Pharmaceutical and Medical in vitro Models
Peter Ertl, Professor of Lab-on-a-Chip Systems, Vienna University of Technology, Austria

Organ-on-a-chip technology offers the potential to recapitulate human physiology by keeping human cells in a precisely controlled and artificial tissue-like microenvironment. The current and potential advantages of organ-on-chips over conventional cell cultures systems and animal models have captured the attention of scientists, clinicians and policymakers as well as advocacy groups in the past few years. Recent advances in tissue engineering and stem cell research are also aiding the development of clinically relevant chip-based organ and diseases models with organ level physiology for drug screening, biomedical research and personalized medicine. Here, the latest advances in organ-on-a-chip technology are reviewed and future clinical applications.

Modeling the Embryonic Neural Tube in a Chip
Thomas Laurell, Professor, Lund University, Sweden

Studying the biology of the early embryonic development stages of the human brain poses severe challenges in terms of access to fetal brain tissue. Current strategies are either referred to studies on animal models or so called neurospheres organoids. Although more widely available, current animal models do not fully reflect the human biology, neither in terms of brain size nor in anatomy. Neural organoids (spheroids), on the other hand, can be generated in large quantities but with the draw back that the organization of the brain regions occurs randomly, in contrast to the spatially well-structured organization along the rostro caudal axis of the fetal brain, in the fore brain, midbrain and hind brain regions. To overcome these bottlenecks, we have developed an organ-on-a-chip (OOC) system that reproducibly generates a neural tissue from human embryonic stem cells with a rostro caudal organization modelling the human embryonic brain. A core of this organ-on-chip system is a gradient generator that produces a morphogen (gsk3i) gradient across the cell culture during the initial neural stem cell patterning phase. The obtained rostro caudal organization has been verified by RNA expression profiling of region specific genes and this OOC platform now provides a robust and reproducible platform to model the human fetal brain. The OOC-system design and performance will be presented and the outlook towards new opportunities to accomplish temporally resolved monitoring of cellular signaling in the course of brain development will be discussed.