Search

The superior clinical therapeutic effects and broad applications of bispecific T cell engagers (BiTEs) has transformed treatment strategies in oncology. While benefits of the novel immunotherapies are indisputable, the safety aspects require to be intensively investigated. Considering challenges of animal models for accessing efficacy and safety of biologics leading to failed clinical trials, there is unmet need for human disease relevant systems, which facilitate development of new clinical candidates. We developed a high-throughput, 384-well-format co-culturing platform for combined assessment of anti-tumor efficacy and liver safety of immunotherapeutics. As a model of liver we employed 3D spheroids composed of primary human hepatocytes and Kupffer cells with preserved metabolic and inflammatory functions. In parallel, the solid tumor model was aggerated of human cancer cell lines (HCT116-GFP) and primary cancer associated fibroblasts, mimicking the tumor microenvironment. Both models were cultured in automation-friendly wells pairwise interconnected with microfluidic channels. Gravity-driven tubeless flow ensured tissue-tissue interaction. To evaluate our system, we treated 3D spheroid models with runimotamab (HER2xCD3 BiTE) in the presence of peripheral blood mononuclear cells (PBMCs). Tumor viability and growth was assessed by fluorescence measurements, while liver toxicity and function were monitored by release of liver aminotransferases ALT/AST, albumin secretion and live confocal microscopy. Immune cell activation was assessed by a cytokine bead array. The treatment with runimotamab resulted in a significant decrease of fluorescence and size of tumor spheroids. At the same time, we observed an induced secretion of cytokines with a peak of expression of IL-2, TNFα after 24h and INFγ, IL-6 and IL-17A after 48h. Cytokine release was coupled with elevated levels of clinically relevant liver damage biomarkers ALT and AST with peak at 72h timepoint. This data suggests potential risk of cytokine release syndrome (CRS) followed by liver damage. Interestingly, redosing of the antibody was not followed by another release of liver enzymes, similarly to clinical observations. In summary, we have developed a human disease relevant, high-throughput platform for the evaluation of novel immunotherapies closely emulating clinical results. Automation-compatible with up to 192 co-culture conditions per plate it represents a powerful tool for clinical candidate development. Citation Format: Michal Rudnik, Ozlem Yavaş Grining, Simon Hutter, Frauke Greve, Daniela Ortiz Franyuti, Ramona Matheis, Ekaterina Breous-Nystrom, Olivier Frey. Combined investigation of anti-tumor efficacy and liver safety of bispecific T cell engagers in immune-competent high-throughput co-culturing platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2747.

Emerging studies have demonstrated that innate immune checkpoints, regulating Tumor-associated Macrophage (TAM) phagocytic activity, play a crucial role in immune escape and survival of cancer cells. CD47-SIRPα axis is the best studied example of the “don’t eat me” signal, frequently overexpressed on cancer cell. Targeting of CD47 protein has shown impressive results in clinical trials in hematologic malignancies but remained challenging for solid tumors therapy. Therefore, tools facilitating the development of novel innate immune checkpoint inhibitors are of great interest. To address that unmet need, we established a high-throughput, 384-well-format assay for efficacy assessment of compounds inducing macrophage phagocytosis. As a model of solid tumor, we employed 3D spheroids aggregated of human cancer cell lines (A375, HCT116, MCF7, SKOV3) expressing GFP or PDX material (LXFA1647 lung adenocarcinoma, MEXF2106 melanoma) and cancer associated fibroblasts, as well as Monocyte-Derived Macrophages (MDMs) which altogether were co-aggregated to form spheroids mimicking the tumor microenvironment and immune-competence. In parallel MDMs were co-cultured with matured spheroid to emulate tumor infiltration. Both models were cultured in automation and High Content imaging compatible Akura™ 384-well plate. To evaluate our system, we targeted CD47-SIRPα axis by treatment of 3D spheroid models with αCD47 antibody (H6D12) in combination with atezolizumab (αPD-L1 antibody). Tumor viability and growth were assessed by fluorescence measurements and ATP level. Phagocytosis of fluorescently labeled tumor cells was imaged by confocal microscopy and quantified by inhouse developed analysis pipeline. Immune cell activation was evaluated by a cytokine bead array. The treatment of cell line-based 3D models with αCD47 antibody resulted in an increased number of phagocytic macrophages followed by a decrease of fluorescence and size of tumor spheroids in a dose-dependent manner. Moreover, we observed an induced secretion of TNFα, IL-6, IL-10 and IL-RA after 48h. Similarly, in PDX-based models, we observed decreased spheroid size and ATP level at day 7. Notably, αCD47 antibody-induced level of cytokines was further increased in combination with atezolizumab. To conclude, we have developed a human disease relevant, high-throughput assay for the evaluation of novel therapeutic modalities targeting phagocytosis. Due to compatibility with microscopy and most of the biochemical assays, it represents a powerful tool for clinical candidate development. Citation Format: Michal Rudnik, Özlem Yavaş Grining, Nadezhda Rotankova, Silvan Strebel, Francesca Chiovaro, Olivier Frey, Irina Agarkova, Wolfgang Moritz. High-throughput, high content Imaging assay for assessing solid tumor phagocytosis in 3D spheroid model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4558.

Organs-on-chips (OoCs) are systems containing engineered or natural miniature tissues grown inside microfluidic chips. To better mimic human physiology, the chips are designed to control cell microenvironments and maintain tissue-specific functions. Combining advances in tissue engineering and microfabrication, OoCs have gained interest as a next-generation experimental platform to investigate human pathophysiology and the effect of therapeutics in the body. There are as many examples of OoCs as there are applications, making it difficult for new researchers to understand what makes one OoC more suited to an application than another. This Primer is intended to give an introduction to the aspects of OoC that need to be considered when developing an application-specific OoC. The Primer covers guiding principles and considerations to design, fabricate and operate an OoC, as well as subsequent assaying techniques to extract biological information from OoC devices. Alongside this is a discussion of current and future applications of OoC technology, to inform design and operational decisions during the implementation of OoC systems.

Microphysiological systems hold the promise to increase the predictive and translational power of in vitro substance testing owing to their faithful recapitulation of human physiology. However, the implementation of academic developments in industrial settings remains challenging. We present an injection-molded microfluidic microtissue (MT) culture chip that features two channels with 10 MT compartments each and that was designed in compliance with microtiter plate standard formats. Polystyrene as a chip material enables reliable, large-scale production and precise control over experimental conditions due to low adsorption or absorption of small, hydrophobic molecules at or into the plastic material in comparison with predecessor chips made of polydimethylsiloxane. The chip is operated by tilting, which actuates gravity-driven flow between reservoirs at both ends of every channel, so that the system does not require external tubing or pumps. The flow rate can be modulated by adjusting the tilting angle on demand. The top-open design of the MT compartment enables efficient MT loading using standard or advanced pipetting equipment, ensures oxygen availability in the chip, and allows for high-resolution imaging. Every channel can be loaded with up to 10 identical or different MTs, as demonstrated by culturing liver and tumor MTs in the same medium channel on the chip.

Three-dimensional (3D) spheroid models are rapidly gaining favor for drug discovery applications due to their improved morphological characteristics, cellular complexity, long lifespan in culture, and higher physiological relevance relative to two-dimensional (2D) cell culture models. High-content imaging (HCI) of 3D spheroid models has the potential to provide valuable information to help researchers untangle disease pathophysiology and assess novel therapies more effectively. The transition from 2D monolayer models to dense 3D spheroids in HCI applications is not trivial, however, and requires 3D-optimized protocols, instrumentation, and resources. Here, we discuss considerations for moving from 2D to 3D models and present a framework for HCI and analysis of 3D spheroid models in a drug discovery setting. We combined scaffold-free, multicellular spheroid models with scalable, automation-compatible plate technology enabling image-based applications ranging from high-throughput screening to more complex, lower-throughput microphysiological systems of organ networks. We used this framework in three case studies: investigation of lipid droplet accumulation in a human liver nonalcoholic steatohepatitis (NASH) model, real-time immune cell interactions in a multicellular 3D lung cancer model, and a high-throughput screening application using a 3D co-culture model of gastric carcinoma to assess dose-dependent drug efficacy and specificity. The results of these proof-of-concept studies demonstrate the potential for high-resolution image-based analysis of 3D spheroid models for drug discovery applications, and confirm that cell-level and temporal-spatial analyses that fully exploit multicellular features of spheroid models are not only possible but soon will be routine practice in drug discovery workflows.

Drug-drug interactions (DDIs) occur when the pharmacological activity of one drug is altered by a second drug. As multimorbidity and polypharmacotherapy are becoming more common due to the increasing age of the population, the risk of DDIs is massively increasing. Therefore, in vitro testing methods are needed to capture such multiorgan events. Here, a scalable, gravity-driven microfluidic system featuring 3D microtissues (MTs) that represent different organs for the prediction of drug-drug interactions is used. Human liver microtissues (hLiMTs) are combined with tumor microtissues (TuMTs) and treated with drug combinations that are known to cause DDIs in vivo. The testing system is able to capture and quantify DDIs upon co-administration of the anticancer prodrugs cyclophosphamide or ifosfamide with the antiretroviral drug ritonavir. Dosage of ritonavir inhibits hepatic metabolization of the two prodrugs to different extents and decreases their efficacy in acting on TuMTs. The flexible MT compartment design of the system, the use of polystyrene as chip material, and the assembly of several chips in stackable plates offer the potential to significantly advance preclinical substance testing. The possibility of testing a broad variety of drug combinations to identify possible DDIs will improve the drug development process and increase patient safety.

A major obstacle in the development of efficient therapies for progressive liver fibrosis is the lack of representative in vitro models of liver fibrosis to aid in understanding the mechanisms of the disease and to promote the development of pharmaceuticals. Our aim was to develop a relevant in vitro mouse liver fibrosis model, based on the central hypothesis that liver fibrosis in vitro cannot be studied using only hepatic stellate cells (HSCs)-the main producer of scar tissue during fibrosis-, but requires cultures in which at least hepatocytes are integrated. We established robust methods to generate co-culture spheroids from freshly isolated mouse hepatocytes and HSCs. Characteristics and functionality of these spheroids were analyzed by qPCR of cell-type specific markers, CYP induction and immunohistochemistry. Compound toxicity was determined by ATP-assays. Hepatocytes and HSCs maintained their cell-type specific marker expression over a 15-day culture period without major hepatocyte dedifferentiation or HSC activation. Exposure of spheroids to TGFβ can directly activate HSCs, while acetaminophen exposure mounts a hepatocyte damage dependent activation of HSCs. Pharmaceuticals with known anti-fibrotic properties, such as Valproic acid and Verteporfin, reduce HSC activation in response to hepatocyte damage in these cultures. A comparison between the fibrotic response of the spheroid co-cultures and in vivo activated HSCs showed that these 3D co-cultures are more representative than the commonly used 2D HSC monocultures. Finally, we showed that the 3D cultures can be integrated in microfluidic chips. We conclude that our hepatocyte-stellate cell-spheroid cultures are a robust in vitro model of liver fibrosis. This model could be used to further unravel the mechanism of HSC activation and facilitate the discovery of, or testing for novel anti-fibrotic compounds, as these spheroids better reproduce HSC in vivo activation compared to the more traditional 2D mono-culture models.

The advancement of microfluidic technology for cell cultures has given rise to a wide spectrum of new in vitro models mimicking the physiological functions of organs. These microphysiological systems are often considered the next step toward more comprehensive and representative in vitro testing systems in research and drug discovery. Fluidically interconnecting different organ models into networks enables the study of organ cross-talk and extraction of systemic information that is typically only available through animal testing. Constructing such systems involves multiple challenges, and research groups have developed numerous approaches with different levels of complexity in this field.

The first microfluidic microphysiological systems (MPS) entered the academic scene more than 15 years ago and were considered an enabling technology to human (patho)biology in vitro and, therefore, provide alternative approaches to laboratory animals in pharmaceutical drug development and academic research. Nowadays, the field generates more than a thousand scientific publications per year. Despite the MPS hype in academia and by platform providers, which says this technology is about to reshape the entire in vitro culture landscape in basic and applied research, MPS approaches have neither been widely adopted by the pharmaceutical industry yet nor reached regulated drug authorization processes at all. Here, 46 leading experts from all stakeholders - academia, MPS supplier industry, pharmaceutical and consumer products industries, and leading regulatory agencies - worldwide have analyzed existing challenges and hurdles along the MPS-based assay life cycle in a second workshop of this kind in June 2019. They identified that the level of qualification of MPS-based assays for a given context of use and a communication gap between stakeholders are the major challenges for industrial adoption by end-users. Finally, a regulatory acceptance dilemma exists against that background. This t4 report elaborates on these findings in detail and summarizes solutions how to overcome the roadblocks. It provides recommendations and a roadmap towards regulatory accepted MPS-based models and assays for patients' benefit and further laboratory animal reduction in drug development. Finally, experts highlighted the potential of MPS-based human disease models to feedback into laboratory animal replacement in basic life science research.

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.

Microtissue spheroids in microfluidic devices are increasingly used to establish novel in vitro organ models of the human body. As the spheroids are comparably sizable, it is difficult to monitor larger numbers of them by optical means. Therefore, electrical impedance spectroscopy (EIS) emerges as a viable alternative to probing spheroid properties. Current spheroid EIS systems are, however, not suitable for investigating multiple spheroids in parallel over extended time in an automated fashion. Here we address this issue by presenting an automated, multiplexed EIS (AMEIS) platform for impedance analysis in a microfluidic setting. The system was used to continuously monitor the effect of the anticancer drug fluorouracil (5-FU) on HCT116 cancer spheroids. Simultaneous EIS monitoring of up to 15 spheroids was performed in parallel over 4 days at a temporal resolution of 2 min without any need for pumps. The measurements were continuous in nature, and the setup was kept in a standard incubator under controlled conditions during the measurements. A baseline normalization method to improve robustness and to reduce the influence of slow changes in the medium conductivity on the spheroid EIS readings has been developed and validated by experiments and means of a finite-element model. The same method and platform was then used for online monitoring of cardiac spheroids. The beating frequency of each cardiac spheroid could be read out in a completely automated fashion. The developed system constitutes a promising method for simultaneously evaluating drug impact and/or toxic effects on multiple microtissue spheroids. [Figure: see text]

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]

Surface redox activities, oxygen evolution reaction (OER), oxidation of formic acid (FA), and anodic stability were investigated and compared for IrO2 electrodes prepared by two techniques: the thermal decomposition of H2IrCl6 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.

Growth rate is a widely studied parameter for various cell-based biological studies. Growth rates of cell populations can be monitored in chemostats and micro-chemostats, where nutrients are continuously replenished. Here, we present an integrated microfluidic platform that enables long-term culturing of non-adherent cells as well as parallel and mutually independent continuous monitoring of (i) growth rates of cells by means of impedance measurements and of (ii) specific other cellular events by means of high-resolution optical or fluorescence microscopy. Yeast colonies were grown in a monolayer under culturing pads, which enabled high-resolution microscopy, as all cells were in the same focal plane. Upon cell growth and division, cells leaving the culturing area passed over a pair of electrodes and were counted through impedance measurements. The impedance data could then be used to directly determine the growth rates of the cells in the culturing area. The integration of multiple culturing chambers with sensing electrodes enabled multiplexed long-term monitoring of growth rates of different yeast strains in parallel. As a demonstration, we modulated the growth rates of engineered yeast strains using calcium. The results indicated that impedance measurements provide a label-free readout method to continuously monitor the changes in the growth rates of the cells without compromising high-resolution optical imaging of single cells., Microsystems & Nanoengineering, 4, ISSN:2096-1030, ISSN:2055-7434

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.

This chapter describes a microfluidic device that enables immobilization and culturing of single rod-shaped S. pombe cells in a stand-up mode. The wide-band electrical impedance spectroscopy (EIS) has been integrated in the microfluidic device to continuously measure cell growth of single S. pombe cells. Cell growth curves showing cellular and intracellular features at high spatiotemporal resolution can be obtained from EIS signals. The features include longitudinal cell elongation in the G2 phase, mitosis, and cell division during an entire cell cycle of S. pombe cells. Microfluidics-based EIS systems provide, hence, a tool for dynamic single-cell studies.

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.

In this article, we present a microfluidic platform, compatible with conventional 96-well formats, that enables facile and parallelized culturing and testing of spherical microtissues in a standard incubator. The platform can accommodate multiple microtissues (up to 66) of different cell types, formed externally by using the hanging-drop method, and enables microtissue interconnection through microfluidic channels for continuous media perfusion or dosage of substances. The platform contains 11 separate channels, and each channel has six tissue compartments. Primary rat liver tissues were cultured over 8 days, and multiple tumor tissues (HCT116) were exposed to various concentrations of 5-fluorouracil for platform characterization.

We present an integrated microfluidic system, which combines long-term cell culturing with electrical impedance spectroscopy (EIS) on a single chip. Non-adherent cells, such as yeast (S. cerevisiae), can be grown under constant media perfusion in a 2D layer under clamping pads that enable long-term time-lapse microscopy within the same focal plane. The addition of EIS provides a complementary method for continuous in-situ analysis of single cells and has been used here to monitor the growth rate of cell colonies in real time., Procedia Engineering, 120, Eurosensors 2015

Time-lapse fluorescence imaging of yeast cells allows the study of multiple fluorescent targets in single cells, but is often hampered by the tedious cultivation using agar pads or glass bottom wells. Here, we describe the fabrication and operation of a microfluidic device for long-term imaging of yeast cells under constant or changing media conditions. The device allows acquisition of high quality images as cells are fixed in a two-dimensional imaging plane. Four yeast strains can be analyzed simultaneously over several days while up to four different media can be flushed through the chip. The microfluidic device does not rely on specialized equipment for its operation. To illustrate the use of the chip in DNA damage research, we show how common readouts for DNA damage or genomic instability behave upon induction with genotoxic chemicals (MMS, HU) or induction of a single double-strand break using induced CRISPR-Cas9 expression.

We present a microfluidic tilting platform with gravity-driven flow that enables culturing of three-dimensional microtissues under in vivo-like drug dosing regimens. In contrast to traditional, constant dosing regimens, the chip allows for gradual changes in substance concentrations. Dosing gradients are generated through an asymmetric Y-junction of microfluidic channels of different widths. Changes in the chip operation parameters, e.g., different tilting angles, enable to alter the drug dosage on demand. The concentration changes over time matched the predicted values, and preformed microtissues could be cultured in the chip system for at least 24 h. The chip represents an easy-to-handle tool for toxicity and efficacy testing of transient drug concentrations changes., Proceedings, 1 (4), ISSN:2504-3900

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.

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

Microelectrode arrays offer the potential to electrochemically monitor concentrations of molecules at high spatial resolution. However, current systems are limited in the number of sensor sites, signal resolution, and throughput. Here, we present a fully integrated complementary metal oxide semiconductor (CMOS) system with an array of 32 × 32 working electrodes to perform electrochemical measurements like amperometry and voltammetry. The array consists of platinum electrodes with a center-to-center distance of 100 μm and electrode diameters of 5 to 50 μm. Currents in the range from 10 μA down to pA can be measured. The current is digitized by sigma-delta converters at a maximum resolution of 13.3 bits. The integrated noise is 220 fA for a bandwidth of 100 Hz, allowing for detection of pA currents. Currents can be continuously acquired at up to 1 kHz bandwidth, or the whole array can be read out rapidly at a frame rate of up to 90 Hz. The results of the electrical characterization meet the requirements of a wide range of electrochemical methods including cyclic voltammograms and amperometric images of high spatial and temporal resolution.

Surface modification of microelectrodes is a central step in the development of microsensors and microsensor arrays. Here, we present an electrodeposition scheme based on voltage pulses. Key features of this method are uniformity in the deposited electrode coatings, flexibility in the overall deposition area, i.e., the sizes and number of the electrodes to be coated, and precise control of the surface texture. Deposition and characterization of four different materials are demonstrated, including layers of high-surface-area platinum, gold, conducting polymer poly(ethylenedioxythiophene), also known as PEDOT, and the non-conducting polymer poly(phenylenediamine), also known as PPD. The depositions were conducted using a fully integrated complementary metal-oxide-semiconductor (CMOS) chip with an array of 1024 microelectrodes. The pulsed potentiostatic deposition scheme is particularly suitable for functionalization of individual electrodes or electrode subsets of large integrated microelectrode arrays: the required deposition waveforms are readily available in an integrated system, the same deposition parameters can be used to functionalize the surface of either single electrodes or large arrays of thousands of electrodes, and the deposition method proved to be robust and reproducible for all materials tested.

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

We present a microfluidic cell-culture chip that enables trapping, cultivation and release of selected individual cells. The chip is fabricated by a simple hybrid glass-SU-8-PDMS approach, which produces a completely transparent microfluidic system amenable to optical inspection. Single cells are trapped in a microfluidic channel using mild suction at defined cell immobilization orifices, where they are cultivated under controlled environmental conditions. Cells of interest can be individually and independently released for further downstream analysis by applying a negative dielectrophoretic force via the respective electrodes located at each immobilization site. The combination of hydrodynamic cell-trapping and dielectrophoretic methods for cell releasing enables highly versatile single-cell manipulation in an array-based format. Computational fluid dynamics simulations were performed to estimate the properties of the system during cell trapping and releasing. Polystyrene beads and yeast cells have been used to investigate and characterize the different functions and to demonstrate biological compatibility and viability of the platform for single-cell applications in research areas such as systems biology.

In this paper, the IrO2 electrode preparation technique consisting in thermal treatment of pure Ir in air was used in order to produce an IrO2-based microelectrode-array (TOIROF-MEA) with low capacitive background current. The TOIROF-MEA was characterized using Fe(CN)64-/3- as model redox couple. It was found that very low concentrations can be detected; this feature makes TOIROF-MEA suitable for analytical applications. Keywords: Microelectrode-array, IrO2, Capacitive current, Thermal treatment of pure Ir

Silicon biosensor microprobes for the in vivo detection of neurotransmitters are equipped with removable clips for an on-chip functionalisation technique that allows a simple and comfortable but very local and reproducible deposition of sensitive layers on dedicated microelectrodes. It can been seen as an alternative to high-priced low-volume dispensing equipment or electro-chemical setups since not only coatings of a) several electrodes in parallel or b) closely spaced electrodes with different substances are possible, but – additionally – it can be performed directly before use with a conventional micropipette.

As a complement and alternative to optical methods, wide-band electrical impedance spectroscopy (EIS) enables multi-parameter, label-free and real-time detection of cellular and subcellular features. We report on a microfluidics-based system designed to reliably capture single rod-shaped Schizosaccharomyces pombe cells by applying suction through orifices in a channel wall. The system enables subsequent culturing of immobilized cells in an upright position, while dynamic changes in cell-cycle state and morphology were continuously monitored through EIS over a broad frequency range. Besides measuring cell growth, clear impedance signals for nuclear division have been obtained. The EIS system has been characterized with respect to sensitivity and detection limits. The spatial resolution in measuring cell length was 0.25 μm, which corresponds to approximately a 5-min interval of cell growth under standard conditions. The comprehensive impedance data sets were also used to determine the occurrence of nuclear division and cytokinesis. The obtained results have been validated through concurrent confocal imaging and plausibilized through comparison with finite-element modeling data. The possibility to monitor cellular and intracellular features of single S. pombe cells during the cell cycle at high spatiotemporal resolution renders the presented microfluidics-based EIS system a suitable tool for dynamic single-cell investigations., Scientific Reports, 5, ISSN:2045-2322

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.

Optical long-term observation of individual cells, combined with modern data analysis tools, allows for a detailed study of cell-to-cell variability, heredity, and differentiation. We developed a microfluidic device featuring facile cell loading, simple and robust operation, and which is amenable to high-resolution life-cell imaging. Different cell strains can be grown in parallel in the device under constant or changing media perfusion without cross-talk between the cell ensembles. The culturing chamber has been optimized for use with nonadherent cells, such as Saccharomyces cerevisiae, and enables controlled colony growth over multiple generations under aerobic or anaerobic conditions. Small changes in the layout will make the device also useable with bacteria or mammalian cells. The platform can be readily set up in every laboratory with minimal additional requirements and can be operated without technology training. [Figure: see text]

We present a new on-chip pumping approach for microfluidic hanging-drop networks that are used for experiments with 3D microtissue spheroids. The pump includes a pneumatic chamber located directly above one of the hanging drops and uses the liquid-air-interface for flow-control. With this approach several independent hanging drop networks (HDN) can be operated in parallel with only one single pneumatic actuation line. The pump concept enables closed-loop medium circulation between different organ models for body-on-a-chip applications and allows for multiple simultaneous assays in parallel., 2015 28th IEEE International Conference on Micro Electro Mechanical Systems (MEMS), ISBN:978-1-4799-7955-4, ISBN:978-1-4799-7956-1

Here, we present electrical impedance spectroscopy (EIS) data of human iPS-derived cardiac 3D spheroids with electric stimulation integrated in a hanging drop network. Microscopy videos of the beating spheroids were correlated with synchronously obtained EIS recordings. For stimulation, the spheroid was exposed to a continuous sinusoidal electric field - in contrast to traditional pulse trains. This stimulating field was supplied via the same electrodes that were used for the EIS recordings. Our measurements revealed a beating frequency modulation upon tuning the stimulation signal amplitude., 2015 28th IEEE International Conference on Micro Electro Mechanical Systems (MEMS), ISBN:978-1-4799-7955-4

Rational development of more physiologic in vitro models includes the design of robust and flexible 3Dmicrotissue-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. (C) 2015 Elsevier B.V. All rights reserved.

All metabolic activities operate within a narrow pH range that is controlled by the CO2-bicarbonate buffering system. We hypothesized that pH could serve as surrogate signal to monitor and respond to the physiological state. By functionally rewiring the human proton-activated cell-surface receptor TDAG8 to chimeric promoters, we created a synthetic signaling cascade that precisely monitors extracellular pH within the physiological range. The synthetic pH sensor could be adjusted by organic acids as well as gaseous CO2 that shifts the CO2-bicarbonate balance toward hydrogen ions. This enabled the design of gas-programmable logic gates, provided remote control of cellular behavior inside microfluidic devices, and allowed for CO2-triggered production of biopharmaceuticals in standard bioreactors. When implanting cells containing the synthetic pH sensor linked to production of insulin into type 1 diabetic mice developing diabetic ketoacidosis, the prosthetic network automatically scored acidic pH and coordinated an insulin expression response that corrected ketoacidosis.