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To enable accurate, high-throughput and longer-term studies of the immunopathogenesis of type 1 diabetes (T1D), we established three in-vitro islet-immune injury models by culturing spheroids derived from primary human islets with proinflammatory cytokines, activated peripheral blood mononuclear cells or HLA-A2-restricted preproinsulin-specific cytotoxic T lymphocytes. In all models, β-cell function declined as manifested by increased basal and decreased glucose-stimulated insulin release (GSIS), and decreased intracellular insulin content. Additional hallmarks of T1D progression such as loss of the first-phase insulin response (FFIR), increased proinsulin-to-insulin ratios, HLA-class I expression, and inflammatory cytokine release were also observed. Using these models, we show that liraglutide, a glucagon-like peptide 1 receptor agonist, prevented loss of GSIS under T1D-relevant stress, by preserving the FFIR and decreasing immune cell infiltration and cytokine secretion. Our results corroborate that liraglutide mediates an anti-inflammatory effect that aids in protecting β-cells from the immune-mediated attack that leads to T1D.

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 secre-tagogs. It provides a new tool for studying physiologically relevant dynamic insulin secretion at comparably low sample-to-sample variation and high temporal resolution.

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.

This work presents a new versatile method to integrate discrete sensor chips into open-microfluidic hanging-drop networks. As proof of concept, we integrated an electrical-impedance-spectroscopy (EIS) sensor unit into a hanging-drop platform for monitoring the size of microtissues. The fabrication and assembly method enables the integration of a large variety of silicon-based sensors, such as chemical sensors, biosensors or microelectrode arrays (MEAs), in open-microfluidic hanging-drop networks to perform studies on three-dimensional microtissues. © 2019 IEEE., 2019 IEEE 32nd International Conference on Micro Electro Mechanical Systems (MEMS), ISBN:978-1-7281-1610-5

The hanging-drop network (HDN) is a technology platform based on a completely open microfluidic network at the bottom of an inverted, surface-patterned substrate. The platform is predominantly used for the formation, culturing, and interaction of self-assembled spherical microtissues (spheroids) under precisely controlled flow conditions. Here, we describe design, fabrication, and operation of microfluidic hanging-drop networks.

Open microfluidic cell culturing devices offer new possibilities to simplify loading, culturing, and harvesting of individual cells or microtissues due to the fact that liquids and cells/microtissues are directly accessible. We present a complete workflow for microfluidic handling and culturing of individual cells and microtissue spheroids, which is based on the hanging-drop network concept: The open microfluidic devices are seamlessly combined with fluorescence-activated cell sorting (FACS), so that individual cells, including stem cells, can be directly sorted into specified culturing compartments in a fully automated way and at high accuracy. Moreover, already assembled microtissue spheroids can be loaded into the microfluidic structures by using a conventional pipet. Cell and microtissue culturing is then performed in hanging drops under controlled perfusion. On-chip drop size control measures were applied to stabilize the system. Cells and microtissue spheroids can be retrieved from the chip by using a parallelized transfer method. The presented methodology holds great promise for combinatorial screening of stem-cell and multicellular-spheroid cultures. [Figure: see text]

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.

Microfluidics is becoming a technology of growing interest to build miniature culturing systems, capable of mimicking tissue functions and multi-tissue interactions in so-called “body-on-a-chip ” applications while featuring integrated readout functionalities. This chapter presents a highly versatile, modular and scalable analytical platform technology, which combines microfluidic hanging-drop networks with multi-analyte biosensors for in situ monitoring of the metabolism of 3D microtissues. The microfluidic platform is based on the hanging -drop network technology, which has been designed for formation, cultivation, and analysis of fluidically interconnected organotypic spherical 3D microtissues that can be obtained from various different cell types. The sensor modules were designed as small glass plug-ins, which allow for convenient functionalization and calibration of the sensors and do not interfere with the microfluidic functions. They were placed in the ceiling substrate, from which the hanging drops that host the spheroid cultures were suspended. The detection of secreted lactate of single microtissue spheroids will be presented. Further, we will demonstrate that it is possible to monitor microtissue lactate secretion and glucose consumption in parallel.

We present a microfluidic chip for the culturing of 3D microtissues under varying perfusion conditions. The microtissues are individually immobilized at the bottom of hanging hydrogel microdrops and are located at defined positions inside the culturing chambers of the microfluidic network. The precision in positioning and the stability of the microtissues over time renders the platform ideal for multi-position, high-resolution time-lapse imaging and techniques involving cell tracking. At the same time, fabrication and operation of the chip is simple and robust and does not require dedicated equipment.

We present the integration of enzyme-based lactate biosensors into a microfluidic network of hanging drops, in which spherical microtissues can be formed and cultured over time. The addition of biosensors enables continuous in-situ monitoring of effects upon applying different culturing conditions or dosing compounds to the 3D tissue structures. The integration has been achieved by using a hybrid fabrication method, in which sensor modules on glass are embedded into a microfluidic PDMS structure. The lactate biosensors enabled real-time measurements of lactate produced by human colon carcinoma spheroids., Procedia Engineering, 87, EUROSENSORS 2014, the 28th European Conference on Solid-State Transducers

This work presents a highly versatile and scalable analytical platform for 3D microtissues. The device combines microfluidics for precise control of culture conditions with sensor technology for parallel and real-time monitoring of microtissue metabolism. The technology is based on an inverted, open microfluidic network. Interconnected hanging drops are formed underneath a patterned glass chip, which hosts the integrated microelectrode array. The electrodes can be individually functionalized and used as biosensors. The fabrication is simple, and the overall system can be expanded through the integration of larger numbers of hanging drops and more electrodes. The device can be used as a microfluidic platform for cultivation and continuous monitoring of 3D microtissues., 2017 IEEE 30th International Conference on Micro Electro Mechanical Systems (MEMS), ISBN:978-1-5090-5078-9, ISBN:978-1-5090-5079-6

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. [Figure: see text]

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., Microsystems & Nanoengineering, 2, ISSN:2096-1030, ISSN:2055-7434

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.

We present the integration of enzyme-based lactate and glucose biosensors into hanging-drop networks. Hangingdrop networks are an ideal microfluidic platform for formation, cultivation, and continuous and long-term observation of 3D microtissues. The implementation of biosensors enables in-situ online monitoring of the effects of different culturing conditions and compound dosages on the microtissues. A hybrid approach including glass sensor modules embedded into a microfluidic polydimethylsiloxane (PDMS)-based chip facilitates system integration. The biosensors enable real-time recording of lactate production and glucose consumption of human colon carcinoma spheroids., 2015 Transducers - 2015 18th International Conference on Solid-State Sensors, Actuators and Microsystems (TRANSDUCERS), ISBN:978-1-4799-8955-3

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.