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.

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.

Insulin is released from pancreatic islets in a biphasic and pulsatile manner in response to elevated glucose levels. This highly dynamic insulin release can be studied in vitro with islet perifusion assays. Herein, a novel platform to perform glucose‐stimulated insulin secretion (GSIS) assays with single islets is presented for studying the dynamics of insulin release at high temporal resolution. A standardized human islet model is developed and a microfluidic hanging‐drop‐based perifusion system is engineered, which facilitates rapid glucose switching, minimal sample dilution, low analyte dispersion, and short sampling intervals. Human islet microtissues feature robust and long‐term glucose responsiveness and demonstrate reproducible dynamic GSIS with a prominent first phase and a sustained, pulsatile second phase. Perifusion of single islet microtissues produces a higher peak secretion rate, higher secretion during the first and second phases of insulin release, as well as more defined pulsations during the second phase in comparison to perifusion of pooled islets. The developed platform enables to study compound effects on both phases of insulin secretion as shown with two classes of insulin secretagogs. It provides a new tool for studying physiologically relevant dynamic insulin secretion at comparably low sample‐to‐sample variation and high temporal resolution.

Electrical impedance spectroscopy (EIS) as a label-free and noninvasive analysis method receives growing attention for monitoring three-dimensional tissue constructs. In this Article, we present the integration of an EIS readout function into the hanging-drop network platform, which has been designed for culturing microtissue spheroids in perfused multitissue configurations. Two pairs of microelectrodes have been implemented directly in the support of the hanging drops by using a small glass inlay inserted in the microfluidic structure. The pair of bigger electrodes is sensitive to the drop size and allows for drop size control over time. The pair of smaller electrodes is capable of monitoring, on the one hand, the size of microtissue spheroids to follow, for example, the growth of cancer microtissues, and, on the other hand, the beating of cardiac microtissues in situ. The presented results demonstrate the feasibility of an EIS readout within the framework of multifunctional hanging-drop networks.

The integration of metabolic competence in developmental toxicity assays in vitro is of fundamental importance to better predict adverse drug effects. Here, a microfluidic hanging‐drop platform is presented that seamlessly integrates liver metabolism into the embryonic stem cell test (EST). Primary human liver microtissues (hLiMTs) and embryoid bodies (EBs) are combined in the same fluidic network, so that hLiMT‐generated metabolites are directly transported to the EBs. Gravity‐driven flow through the network enables continuous intertissue communication, constant medium turnover, and, most importantly, immediate exchange of metabolites. As a proof of concept, the prodrug cyclophosphamide is investigated and a fourfold lower ID50 concentration (50% inhibition of EB differentiation) is found after biotransformation, which demonstrates the potentially adverse effects of metabolites on embryotoxicity. The metaEST platform provides a promising tool to increase the predictive power of the current EST assay by more comprehensively including and better reflecting physiological processes in in vitro tests.

Microfluidic hanging-drop networks enable culturing and analysis of 3D microtissue spheroids derived from different cell types under controlled perfusion and investigating inter-tissue communication in multi-tissue formats. In this paper we introduce a compact on-chip pumping approach for flow control in hanging-drop networks. The pump includes one pneumatic chamber located directly above one of the hanging drops and uses the surface tension at the liquid-air-interface for flow actuation. Control of the pneumatic protocol provides a wide range of unidirectional pulsatile and continuous flow profiles. With the proposed concept several independent hanging-drop networks can be operated in parallel with only one single pneumatic actuation line at high fidelity. Closed-loop medium circulation between different organ models for multi-tissue formats and multiple simultaneous assays in parallel are possible. Finally, we implemented a real-time feedback control-loop of the pump actuation based on the beating of a human iPS-derived cardiac microtissue cultured in the same system. This configuration allows for simulating physiological effects on the heart and their impact on flow circulation between the organ models on chip.

Technological advances in microfabrication techniques in combination with organotypic cell and tissue models have enabled the realization of microphysiological systems capable of recapitulating aspects of human physiology in vitro with great fidelity. Concurrently, a number of analysis techniques has been developed to probe and characterize these model systems. However, many assays are still performed off-line, which severely compromises the possibility of obtaining real-time information from the samples under examination, and which also limits the use of these platforms in high-throughput analysis. In this review, we focus on sensing and actuation schemes that have already been established or offer great potential to provide in situ detection or manipulation of relevant cell or tissue samples in microphysiological platforms. We will first describe methods that can be integrated in a straightforward way and that offer potential multiplexing and/or parallelization of sensing and actuation functions. These methods include electrical impedance spectroscopy, electrochemical biosensors, and the use of surface acoustic waves for manipulation and analysis of cells, tissue, and multicellular organisms. In the second part, we will describe two sensor approaches based on surface-plasmon resonance and mechanical resonators that have recently provided new characterization features for biological samples, although technological limitations for use in high-throughput applications still exist.

Safety assessment of the effects of developmental toxicants on pregnant women is challenging, and systemic effects in embryo–maternal interactions are largely unknown. However, most developmental toxicity studies rely on animal trials, while in vitro platforms that recapitulate the maternal–placental–embryonic axis are missing. Here, the development of a dedicated microfluidic device for co‐cultivation of a placental barrier and 3D embryoid bodies to enable systemic toxicity testing at the embryo–maternal interface is reported. The microfluidic platform features simple handling and recuperation of both tissue models, which facilitates post‐hoc in‐depth analysis at the tissue and single‐cell level. Gravity‐driven flow enables inter‐tissue communication through the liquid phase as well as simple and robust operation and renders the platform parallelizable. As a proof of concept and to demonstrate platform use for systemic embryotoxicity testing in vitro, maternal exposure to plastic microparticles is emulated, and microparticle effects on the embryo–placental co‐culture are investigated.