Integration of multiple three-dimensional microtissues into microfluidic networks enables new insights in how different organs or tissues of an organism interact. Here, we present a platform that extends the hanging-drop technology, used for multi-cellular spheroid formation, to multifunctional complex microfluidic networks. Engineered as completely open, 'hanging' microfluidic system at the bottom of a substrate, the platform features high flexibility in microtissue arrangements and interconnections, while fabrication is simple and operation robust. Multiple spheroids of different cell types are formed in parallel on the same platform; the different tissues are then connected in physiological order for multi-tissue experiments through reconfiguration of the fluidic network. Liquid flow is precisely controlled through the hanging drops, which enable nutrient supply, substance dosage and inter-organ metabolic communication. The possibility to perform parallelized microtissue formation on the same chip that is subsequently used for complex multi-tissue experiments renders the developed platform a promising technology for 'body-on-a-chip'-related research.

The organ-on-a-chip (OoC) is an intriguing scientific and technological development in which biology is coupled with microtechnology 1,2 to mimic key aspects of human physiology. The chip takes the form of a microfluidic device containing networks of hair-fine microchannels for guiding and manipulating minute volumes (picolitres up to millilitres) of solution [3][4][5] . The organ is a more relatable term that refers to the miniature tissues grown and residing in the microfluidic chips, which can recapitulate one or more tissue-specific functions. Although they are much simpler than native tissues and organs, scientists have discovered that these systems can often serve as effective mimics of human physiology and disease. OoCs comprise advanced in vitro technology that enables experimentation with biological cells and tissues outside the body. This is achieved by containing them inside vessels conditioned to sustain a reasonable semblance of the in vivo environment, from a biochemical and physical point of view. Working on the microscale lends a unique opportunity to attain a higher level of control over the microenvironment that ensures tissue life support, as well as a means to directly observe cell and tissue behaviour.The OoC is a relatively recent addition to the toolbox of model biological systems available to life science researchers to probe aspects of human pathophysiology and disease. These systems cover a spectrum of physiological relevance, with 2D cell cultures the least relevant, followed in increasing order by 3D cell cultures, organoids and OoCs. Unsurprisingly, the use of model organisms such as mice and Drosophila physiologically exceeds engineered tissue approaches 6,7 . While biological complexity increases with physiological relevance in model organisms, this unfortunately leads to increased experimental difficulty. In vivo physiological processes are, in many ways, the least accessible to direct investigation in mice, humans and other mammals, despite significant advances in in vivo imaging. However, 2D and 3D cell cultures, such as spheroids and stem cell-derived organoids, sacrifice some aspects of in vivo relevance to facilitate experimentation. The OoC may be regarded as a bridging technology, offering the ability to work with complex cell cultures, while providing better engineered microenvironments to maximize the model.Following on from early concepts, including animal-on-a-chip 8 , body-on-a-chip 9 and breathing lung-on-a-chip 10 , research in the OoC and microphysiological systems fields has grown exponentially; evidenced by numerous excellent reviews published recently 1,2,11 . Recognition of OoC technology now extends far beyond university laboratories, driven by a need to better understand the human physiology underlying health and disease, and to find new approaches to improve the human condition. The World Economic Forum, for instance, selected the OoC as one of the top ten emerging technologies in 2016 (ref. 12

Summary The recent advent of microphysiological systems – microfluidic biomimetic devices that aspire to emulate the biology of human tissues, organs and circulation in vitro – is envisaged to enable a global paradigm shift in drug development. An extraordinary US governmental initiative and various dedicated research programs in Europe and Asia have led recently to the first cutting-edge achievements of human single-organ and multi-organ engineering based on microphysiological systems. The expectation is that test systems established on this basis would model various disease stages, and predict toxicity, immunogenicity, ADME profiles and treatment efficacy prior to clinical testing. Consequently, this technology could significantly affect the way drug substances are developed in the future. Furthermore, microphysiological system-based assays may revolutionize our current global programs of prioritization of hazard characterization for any new substances to be used, for example, in agriculture, food, ecosystems or cosmetics, thus, replacing laboratory animal models used currently. Thirty-five experts from academia, industry and regulatory bodies present here the results of an intensive workshop (held in June 2015, Berlin, Germany). They review the status quo of microphysiological systems available today against industry needs, and assess the broad variety of approaches with fit-for-purpose potential in the drug development cycle. Feasible technical solutions to reach the next levels of human biology in vitro are proposed. Furthermore, key organ-on-a-chip case studies, as well as various national and international programs are highlighted. Finally, a roadmap into the future is outlined, to allow for more predictive and regulatory-accepted substance testing on a global scale.

purpose. The application of fluid flow (dynamic) for the physiological nutrition of the tissues and the creation of microenvironmental biomolecular gradients and relevant mechanical cues (e.g., shear stress) is a major aspect of these systems, differentiating them from conventional (static) cell and tissue cultures. This review uses the term MPS exclusively for microfluidic sys- Introduction Definitions and terminologyMicrophysiological systems (MPS) are microfluidic devices capable of emulating human (or any other animal species') biology in vitro at the smallest biologically acceptable scale, defined by t 4 Workshop Report*

Surface redox activities, oxygen evolution reaction (OER), oxidation of formic acid (FA), and anodic stability were investigated and compared for IrO 2 electrodes prepared by two techniques: the thermal decomposition of H 2 IrCl 6 precursor (TDIROF) and the anodic oxidation of metallic iridium (AIROF). Surface redox activities involved on the AIROF were found to be much faster than those involved on the TDIROF. Concerning the oxygen evolution reaction, both films show a similar mechanism and specific electrocatalytic activities. The situation seems to be different for FA oxidation. In fact, on TDIROF, the oxidation of FA and the OER compete involving the same surface redox couple Ir(VI)/Ir(IV) contrary to FA oxidation on AIROF, where the Ir(V)/Ir(IV) surface redox couple is involved. Finally, electrode stability measurements have shown that contrary to TDIROF, which are very stable under anodic polarization, the AIROF are rapidly corroded under anodic treatment. This corrosion is enhanced even further in the presence of formic acid.

Microfluidics is becoming a technology of growing interest for building microphysiological systems with integrated read-out functionalities. Here we present the integration of enzyme-based multi-analyte biosensors into a multi-tissue culture platform for 'body-on-a-chip' applications. The microfluidic platform is based on the technology of hanging-drop networks, which is designed for the formation, cultivation, and analysis of fluidically interconnected organotypic spherical three-dimensional (3D) microtissues of multiple cell types. The sensor modules were designed as small glass plug-ins featuring four platinum working electrodes, a platinum counter electrode, and an Ag/AgCl reference electrode. They were placed directly into the ceiling substrate from which the hanging drops that host the spheroid cultures are suspended. The electrodes were functionalized with oxidase enzymes to enable continuous monitoring of lactate and glucose through amperometry. The biosensors featured high sensitivities of 322 ± 41 nA mM − 1 mm − 2 for glucose and 443 ± 37 nA mM − 1 mm − 2 for lactate; the corresponding limits of detection were below 10 μM. The proposed technology enabled tissue-size-dependent, real-time detection of lactate secretion from single human colon cancer microtissues cultured in the hanging drops. Furthermore, glucose consumption and lactate secretion were monitored in parallel, and the impact of different culture conditions on the metabolism of cancer microtissues was recorded in real-time.

Rational development of more physiologic in vitro models includes the design of robust and flexible 3D-microtissue-based multi-tissue devices, which allow for tissue-tissue interactions. The developed device consists of multiple microchambers interconnected by microchannels. Pre-formed spherical microtissues are loaded into the microchambers and cultured under continuous perfusion. Gravity-driven flow is generated from on-chip reservoirs through automated chip-tilting without any need for additional tubing and external pumps. This tilting concept allows for operating up to 48 devices in parallel in order to test various drug concentrations with a sufficient number of replicates. For a proof of concept, rat liver and colorectal tumor microtissues were interconnected on the chip and cultured during 8 days in the presence of the pro-drug cyclophosphamide. Cyclophosphamide has a significant impact on tumor growth but only after bio-activation by the liver. This effect was only observed in the perfused and interconnected co-cultures of different microtissue types on-chip, whereas the discontinuous transfer of supernatant via pipetting from static liver microtissues that have been treated with cyclophosphamide did not significantly affect tumor growth. The results indicate the utility and multi-tissue functionality of this platform. The importance of continuous medium circulation and tissue interaction is highlighted.

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