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.

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.

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.

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.

Reconfigurable microfluidic hanging drop network for multi-tissue interaction and analysis.

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.