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.

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.

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.

Integrated Microphysiological Systems: Transferable Organ Models and Recirculating Flow.

Studying and understanding of tissue and disease mechanisms largely depend on the availability of suitable and representative biological model systems. These model systems should be carefully engineered and faithfully reproduce the biological system of interest to understand physiological effects, pharmacokinetics, and toxicity to better identify new drug compounds. By relying on microfluidics, microphysiological systems (MPSs) enable the precise control of culturing conditions and connections of advanced in vitro 3D organ models that better reproduce in vivo environments. This review focuses on transferable in vitro organ models and integrated MPSs that host these transferable biological units and enable interactions between different tissue types. Interchangeable and transferrable in vitro organ models allow for independent quality control of the biological model before system assembly and building MPS assays on demand. Due to the complexity and different maturation times of individual in vitro tissues, off-chip production and quality control entail improved stability and reproducibility of the systems and results, which is important for large-scale adoption of the technology. Lastly, the technical and biological challenges and open issues for realizing and implementing integrated MPSs with transferable in vitro organ models are discussed.