A Versatile Intestine-on-Chip System for Deciphering the Immunopathogenesis of Inflammatory Bowel Disease.

The multifactorial nature of inflammatory bowel disease (IBD) necessitates reliable and practical experimental models to elucidate its etiology and pathogenesis. To model the intestinal microenvironment at the onset of IBD in vitro, it is important to incorporate relevant cellular and noncellular components before inducing stepwise pathogenic developments. A novel intestine-on-chip system for investigating multiple aspects of IBD's immunopathogenesis is presented. The system includes an array of tight and polarized barrier models formed from intestinal epithelial cells on an in-vivo-like subepithelial matrix within one week. The dynamic remodeling of the subepithelial matrix by cells or their secretome demonstrates the physiological relevance of the on-chip barrier models. The system design enables introduction of various immune cell types and inflammatory stimuli at specific locations in the same barrier model, which facilitates investigations of the distinct roles of each cell type in intestinal inflammation development. It is showed that inflammatory behavior manifests in an upregulated expression of inflammatory markers and cytokines (TNF-α). The neutralizing effect of the anti-inflammatory antibody Infliximab on levels of TNF-α and its inducible cytokines could be explicitly shown. Overall, an innovative approach to systematically developing a microphysiological system to comprehend immune-system-mediated disorders of IBD and to identify new therapeutic strategies is presented.

Controlling bead and cell mobility in a recirculating hanging-drop network.

Integrating flowing cells, such as immune cells or circulating tumour cells, within a microphysiological system is crucial for body-on-a-chip applications. However, ensuring unimpeded recirculation of cells is a significant challenge. Closed microfluidic devices have a no-slip boundary condition along channel walls and a defined chip geometry (laminar flow) that hinders the ability to freely control cell flow. Open microfluidic devices, where the bottom device boundary is an air-liquid interface (ALI), e.g., hanging drop networks (HDNs), offer the advantage of an easily-actuatable fluid-phase geometry, where cells can either flow or stagnate. In this paper, we optimized a hanging-drop-integrated pneumatic-pump system for closed-loop recirculation of particles (i.e., beads or cells). Experiments with both beads and cells in cell culture medium initially resulted in particle stagnation, which was suggestive of a pseudo-no-slip boundary condition at the ALI. Transmission electron microscopy and dynamic light scattering measurements of the ALI suggested that aggregation of submicron-scale cell-culture-medium components is the cause of the pseudo-no-slip boundary condition. We used the finite element method to study the forces on particles at the ALI and to optimize HDN design (drop aperture) and operation (drop height) parameters. Based on this analysis, we report a phase diagram delineating the conditions for free flow or stagnation of particles at the ALI of hanging drops. Using our experimental setup with 3.5 mm drop apertures, we conducted particle flow experiments while actuating drop heights. We confirmed the ability to control the flow or stagnation of particles by actuating the height of hanging drops: a drop height over 300 µm led to particle stagnation and a drop height under 300 µm allowed for particle flow. This particle-flow control, combined with the ease of integrating scaffold-free organ models (microtissues or organoids) in HDNs, constitutes the basis for an experimental setup enabling the control of the residence time of single cells around 3D organ models.

Microphysiological Drug-Testing Platform for Identifying Responses to Prodrug Treatment in Primary Leukemia.

Despite increasing survival rates of pediatric leukemia patients over the past decades, the outcome of some leukemia subtypes has remained dismal. Drug sensitivity and resistance testing on patient-derived leukemia samples provide important information to tailor treatments for high-risk patients. However, currently used well-based drug screening platforms have limitations in predicting the effects of prodrugs, a class of therapeutics that require metabolic activation to become effective. To address this issue, a microphysiological drug-testing platform is developed that enables co-culturing of patient-derived leukemia cells, human bone marrow mesenchymal stromal cells, and human liver microtissues within the same microfluidic platform. This platform also enables to control the physical interaction between the diverse cell types. Herein, it is made possible to recapitulate hepatic prodrug activation of ifosfamide in their platform, which is very difficult in traditional well-based assays. By testing the susceptibility of primary patient-derived leukemia samples to the prodrug ifosfamide, sample-specific sensitivities to ifosfamide in primary leukemia samples are identified. The microfluidic platform is found to enable the recapitulation of physiologically relevant conditions and the testing of prodrugs including short-lived and unstable metabolites. The platform holds great potential for clinical translation and precision chemotherapy selection.

speedingCARs: accelerating the engineering of CAR T cells by signaling domain shuffling and single-cell sequencing.

Chimeric antigen receptors (CARs) consist of an antigen-binding region fused to intracellular signaling domains, enabling customized T cell responses against targets. Despite their major role in T cell activation, effector function and persistence, only a small set of immune signaling domains have been explored. Here we present speedingCARs, an integrated method for engineering CAR T cells via signaling domain shuffling and pooled functional screening. Leveraging the inherent modularity of natural signaling domains, we generate a library of 180 unique CAR variants genomically integrated into primary human T cells by CRISPR-Cas9. In vitro tumor cell co-culture, followed by single-cell RNA sequencing (scRNA-seq) and single-cell CAR sequencing (scCAR-seq), enables high-throughput screening for identifying several variants with tumor killing properties and T cell phenotypes markedly different from standard CARs. Mapping of the CAR scRNA-seq data onto that of tumor infiltrating lymphocytes further helps guide the selection of variants. These results thus help expand the CAR signaling domain combination space, and supports speedingCARs as a tool for the engineering of CARs for potential therapeutic development.

Modeling and measuring glucose diffusion and consumption by colorectal cancer spheroids in hanging drops using integrated biosensors.

As 3D in vitro tissue models become more pervasive, their built-in nutrient, metabolite, compound, and waste gradients increase biological relevance at the cost of analysis simplicity. Investigating these gradients and the resulting metabolic heterogeneity requires invasive and time-consuming methods. An alternative is using electrochemical biosensors and measuring concentrations around the tissue model to obtain size-dependent metabolism data. With our hanging-drop-integrated enzymatic glucose biosensors, we conducted current measurements within hanging-drop compartments hosting spheroids formed from the human colorectal carcinoma cell line HCT116. We developed a physics-based mathematical model of analyte consumption and transport, considering (1) diffusion and enzymatic conversion of glucose to form hydrogen peroxide (H2O2) by the glucose-oxidase-based hydrogel functionalization of our biosensors at the microscale; (2) H2O2 oxidation at the electrode surface, leading to amperometric H2O2 readout; (3) glucose diffusion and glucose consumption by cancer cells in a spherical tissue model at the microscale; (4) glucose and H2O2 transport in our hanging-drop compartments at the macroscale; and (5) solvent evaporation, leading to glucose and H2O2 upconcentration. Our model relates the measured currents to the glucose concentrations generating the currents. The low limit of detection of our biosensors (0.4 ± 0.1 µM), combined with our current-fitting method, enabled us to reveal glucose dynamics within our system. By measuring glucose dynamics in hanging-drop compartments populated by cancer spheroids of various sizes, we could infer glucose distributions within the spheroid, which will help translate in vitro 3D tissue model results to in vivo.

An Immunocompetent Microphysiological System to Simultaneously Investigate Effects of Anti-Tumor Natural Killer Cells on Tumor and Cardiac Microtissues.

Existing first-line cancer therapies often fail to cope with the heterogeneity and complexity of cancers, so that new therapeutic approaches are urgently needed. Among novel alternative therapies, adoptive cell therapy (ACT) has emerged as a promising cancer treatment in recent years. The limited clinical applications of ACT, despite its advantages over standard-of-care therapies, can be attributed to (i) time-consuming and cost-intensive procedures to screen for potent anti-tumor immune cells and the corresponding targets, (ii) difficulties to translate in-vitro and animal-derived in-vivo efficacies to clinical efficacy in humans, and (iii) the lack of systemic methods for the safety assessment of ACT. Suitable experimental models and testing platforms have the potential to accelerate the development of ACT. Immunocompetent microphysiological systems (iMPS) are microfluidic platforms that enable complex interactions of advanced tissue models with different immune cell types, bridging the gap between in-vitro and in-vivo studies. Here, we present a proof-of-concept iMPS that supports a triple culture of three-dimensional (3D) colorectal tumor microtissues, 3D cardiac microtissues, and human-derived natural killer (NK) cells in the same microfluidic network. Different aspects of tumor-NK cell interactions were characterized using this iMPS including: (i) direct interaction and NK cell-mediated tumor killing, (ii) the development of an inflammatory milieu through enrichment of soluble pro-inflammatory chemokines and cytokines, and (iii) secondary effects on healthy cardiac microtissues. We found a specific NK cell-mediated tumor-killing activity and elevated levels of tumor- and NK cell-derived chemokines and cytokines, indicating crosstalk and development of an inflammatory milieu. While viability and morphological integrity of cardiac microtissues remained mostly unaffected, we were able to detect alterations in their beating behavior, which shows the potential of iMPS for both, efficacy and early safety testing of new candidate ACTs.

A Microphysiological Cell-Culturing System for Pharmacokinetic Drug Exposure and High-Resolution Imaging of Arrays of 3D Microtissues.

Understanding the pharmacokinetic/pharmacodynamic (PK/PD)-relationship of a drug candidate is key to determine effective, yet safe treatment regimens for patients. However, current testing strategies are inefficient in characterizing in vivo responses to fluctuating drug concentrations during multi-day treatment cycles. Methods based on animal models are resource-intensive and require time, while traditional in vitro cell-culturing methods usually do not provide temporally-resolved information on the effects of in vivo-like drug exposure scenarios. To address this issue, we developed a microfluidic system to 1) culture arrays of three-dimensional spheroids in vitro, to 2) apply specific dynamic drug exposure profiles, and to 3) in-situ analyze spheroid growth and the invoked drug effects in 3D by means of 2-photon microscopy at tissue and single-cell level. Spheroids of fluorescently-labeled T-47D breast cancer cells were monitored under perfusion-culture conditions at short time intervals over three days and exposed to either three 24 h-PK-cycles or a dose-matched constant concentration of the phosphatidylinositol 3-kinase inhibitor BYL719. While the overall efficacy of the two treatment regimens was similar, spheroids exposed to the PK profile displayed cycle-dependent oscillations between regression and regrowth. Spheroids treated with a constant BYL719 concentration regressed at a steady, albeit slower rate. At a single-cell level, the cell density in BYL719-treated spheroids oscillated in a concentration-dependent manner. Our system represents a versatile tool for in-depth preclinical characterization of PK/PD parameters, as it enables an evaluation of drug efficacy and/or toxicity under realistic exposure conditions.

Predicting Metabolism-Related Drug-Drug Interactions Using a Microphysiological Multitissue System.

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.

Parallelized Wireless Sensing System for Continuous Monitoring of Microtissue Spheroids.

Currently, the use of electrical readout methods for the investigation of microtissue spheroids in combination with lab automation tools is hindered by the cable connections that are required to interrogate the on-chip-integrated electrodes. To overcome this limitation, we developed a wireless sensor scheme, which can detect the size variation of microtissues during long-term culturing and drug exposure assays. The sensor system includes an interrogation board, which is composed of an inductor-capacitor (LC) readout circuit, and the tissue culture platform with integrated split-ring sensors. The magnetic coupling between the LC circuit and the sensors enables the interrogation of the on-chip sensors without any wire connection to the culture platform. By optimizing the sensor dimensions and the LC resonance frequencies, we were able to avoid cross talk between neighboring sensors. We integrated 12 tissue compartments on a standard microscopy slide with a sensor-to-sensor pitch of 9 mm, which is in accordance with standard 96-well plate dimensions. As a proof-of-concept experiment for the developed system, we monitored continuously and during more than four days the growth inhibition of colon cancer microtissue spheroids that had been exposed to different concentrations of doxorubicin, a chemotherapeutic compound. The stability of the measurements during long-term culturing and the compatibility of the sensor scheme with standard lab equipment offer great potential for automated electrical microtissue spheroid characterization.

Combining In Vivo and Organotypic In Vitro Approaches to Assess the Human Relevance of Basimglurant (RG7090), a Potential CAR Activator.

Basimglurant (RG7090), a small molecule under development to treat certain forms of depression, demonstrated foci of altered hepatocytes in a long-term rodent-toxicity study. Additional evidence pointed toward the activation of the constitutive androstane receptor (CAR), an established promoter of nongenotoxic and rodent-specific hepatic tumors. This mode of action and the potential human relevance was explored in vivo using rodent and cynomolgus monkey models and in vitro using murine and human liver spheroids. Wild type (WT) and CAR/pregnane X receptor (PXR) knockout mice (CAR/PXR KO) were exposed to RG7090 for 8 consecutive days. Analysis of liver lysates revealed induction of Cyp2b mRNA and enzyme activity, a known activation marker of CAR, in WT but not in CAR/PXR KO animals. A series of proliferative genes were upregulated in WT mice only, and immunohistochemistry data showed increased cell proliferation exclusively in WT mice. In addition, primary mouse liver spheroids were challenged with RG7090 in the presence or absence of modified antisense oligonucleotides inhibiting CAR and/or PXR mRNA, showing a concentration-dependent Cyp2b mRNA induction only if CAR was not repressed. On the contrary, neither human liver spheroids nor cynomolgus monkeys exposed to RG7090 triggered CYP2B mRNA upregulation. Our data suggested RG7090 to be a rodent-specific CAR activator, and that CAR activation and its downstream processes were involved in the foci of altered hepatocytes formation detected in vivo. Furthermore, we demonstrated the potential of a new in vitro approach using liver spheroids and antisense oligonucleotides for CAR knockdown experiments, which could eventually replace in vivo investigations using CAR/PXR KO mice.

Tubing-Free Microfluidic Microtissue Culture System Featuring Gradual, in vivo-Like Substance Exposure Profiles.

In vitro screening methods for compound efficacy and toxicity to date mostly include cell or tissue exposure to preset constant compound concentrations over a defined testing period. Such concentration profiles, however, do not represent realistic in vivo situations after substance uptake. Absorption, distribution, metabolism and excretion of administered substances in an organism or human body entail gradually changing pharmacokinetic concentration profiles. As concentration profile dynamics can influence drug effects on the target tissues, it is important to be able to reproduce realistic concentration profiles in in vitro systems. We present a novel design that can be integrated in tubing-free, microfluidic culture chips. These chips are actuated by tilting so that gravity-driven flow and perfusion of culture chambers can be established between reservoirs at both ends of a microfluidic channel. The design enables the realization of in vivo-like substance exposure scenarios. Compound gradients are generated through an asymmetric Y-junction of channels with different hydrodynamic resistances. Six microtissues (MTs) can be cultured and exposed in compartments along the channel. Changes of the chip design or operation parameters enable to alter the dosing profile over a large range. Modulation of, e.g., the tilting angle, changes the slope of the dosing curves, so that concentration curves can be attained that resemble the pharmacokinetic characteristics of common substances in a human body. Human colorectal cancer (HCT 116) MTs were exposed to both, gradually decreasing and constant concentrations of Staurosporine. Measurements of apoptosis induction and viability after 5 h and 24 h showed different short- and long-term responses of the MTs to dynamic and linear dosing regimes.

Scalable Microfluidic Platform for Flexible Configuration of and Experiments with Microtissue Multiorgan Models.

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.

Smart Cell Culture Systems: Integration of Sensors and Actuators into Microphysiological Systems.

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.

Automated, Multiplexed Electrical Impedance Spectroscopy Platform for Continuous Monitoring of Microtissue Spheroids.

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.

Multi-analyte biosensor interface for real-time monitoring of 3D microtissue spheroids in hanging-drop networks.

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.

Electrical Impedance Spectroscopy for Microtissue Spheroid Analysis in Hanging-Drop Networks.

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.

Mitotic cells contract actomyosin cortex and generate pressure to round against or escape epithelial confinement.

Little is known about how mitotic cells round against epithelial confinement. Here, we engineer micropillar arrays that subject cells to lateral mechanical confinement similar to that experienced in epithelia. If generating sufficient force to deform the pillars, rounding epithelial (MDCK) cells can create space to divide. However, if mitotic cells cannot create sufficient space, their rounding force, which is generated by actomyosin contraction and hydrostatic pressure, pushes the cell out of confinement. After conducting mitosis in an unperturbed manner, both daughter cells return to the confinement of the pillars. Cells that cannot round against nor escape confinement cannot orient their mitotic spindles and more likely undergo apoptosis. The results highlight how spatially constrained epithelial cells prepare for mitosis: either they are strong enough to round up or they must escape. The ability to escape from confinement and reintegrate after mitosis appears to be a basic property of epithelial cells.

Microfluidics-based single-step preparation of injection-ready polymeric nanosystems for medical imaging and drug delivery.

Translation of therapeutic polymeric nanosystems to patients and industry requires simplified, reproducible and scalable methods for assembly and loading. A single-step in-line process based on nanocoprecipitation of oxazoline-siloxane block copolymers in flow-focusing poly(dimethylsiloxane) microfluidics was designed to manufacture injection-ready nanosystems. Nanosystem characteristics could be controlled by copolymer concentration, block length and chemistry, microchannel geometry, flow rate, aqueous/organic flow rate ratio and payload concentration. The well-tolerated nanosystems exhibited differential cell binding and payload delivery and could confer sensitivity to photodynamic therapy to HeLa cancer cells. Such injection-ready nanosystems carrying drugs, diagnostic or functional materials may facilitate translation to clinical application.

Versatile, simple-to-use microfluidic cell-culturing chip for long-term, high-resolution, time-lapse imaging.

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.

3D spherical microtissues and microfluidic technology for multi-tissue experiments and analysis.

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.