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Crosstalk of microbes with human gut epithelia and immune cells is crucial for gut health. However, there is no existing system for a long-term co-culture of human innate immune cells with epithelium and oxygen-intolerant commensal microbes, hindering the understanding of microbe-immune interactions in a controlled manner. Here, we established a gut epithelium-microbe-immune (GuMI) microphysiological system to maintain the long-term continuous co-culture of Faecalibacterium prausnitzii/Faecalibacterium duncaniae with colonic epithelium, antigen-presenting cells (APCs, herein dendritic cells and macrophages), and CD4+ naive T cells circulating underneath the colonic epithelium. In GuMI-APC condition, multiplex cytokine assays suggested that APCs contribute to the elevated level of cytokines and chemokines secreted into both apical and basolateral compartments compared to GuMI condition that lacks APC. In GuMI-APC with F. prausnitzii (GuMI-APC-FP), F. prausnitzii increased the transcription of pro-inflammatory genes such as toll-like receptor 1 (TLR1) and interferon alpha 1 (IFNA1) in the colonic epithelium, without a significant effect on cytokine secretion, compared to the GuMI-APC without bacteria (GuMI-APC-NB). In contrast, in the presence of CD4+ naive T cells (GuMI-APCT-FP), TLR1, IFNA1, and IDO1 transcription levels decreased with a simultaneous increase in F. prausnitzii-induced secretion of pro-inflammatory cytokines (e.g., IL8) compared to GuMI-APC-FP that lacks T cells. These results highlight the contribution of individual innate immune cells in regulating the immune response triggered by the gut commensal F. prausnitzii. The integration of defined p

The National Center for Advancing Translational Sciences (NCATS) Assay Guidance Manual (AGM) Workshop on 3D Tissue Models for Antiviral Drug Development, held virtually on 7-8 June 2022, provided comprehensive coverage of critical concepts intended to help scientists establish robust, reproducible, and scalable 3D tissue models to study viruses with pandemic potential. This workshop was organized by NCATS, the National Institute of Allergy and Infectious Diseases, and the Bill and Melinda Gates Foundation. During the workshop, scientific experts from academia, industry, and government provided an overview of 3D tissue models' utility and limitations, use of existing 3D tissue models for antiviral drug development, practical advice, best practices, and case studies about the application of available 3D tissue models to infectious disease modeling. This report includes a summary of each workshop session as well as a discussion of perspectives and challenges related to the use of 3D tissues in antiviral drug discovery.

Background. Pediatric gliomas comprise a diverse set of brain tumor entities that have substantial long-term ramifications for patient survival and quality of life. However, the study of these tumors is currently limited due to a lack of authentic models. Additionally, many aspects of pediatric brain tumor biology, such as tumor cell invasiveness, have been difficult to study with currently available tools. To address these issues, we developed a synthetic extracellular matrix (sECM)-based culture system to grow and study primary pediatric brain tumor cells. Methods. We developed a brain-like sECM material as a supportive scaffold for the culture of primary, patientderived pediatric glioma cells and established patient-derived cell lines. Primary juvenile brainstem-derived murine astrocytes were used as a feeder layer to support the growth of primary human tumor cells. Results. We found that our culture system facilitated the proliferation of various primary pediatric brain tumors, including low-grade gliomas, and enabled ex vivo testing of investigational therapeutics. Additionally, we found that tuning this sECM material allowed us to assess high-grade pediatric glioma cell invasion and evaluate therapeutic interventions targeting invasive behavior. Conclusion. Our sECM culture platform provides a multipurpose tool for pediatric brain tumor researchers that enables both a wide breadth of biological assays and the cultivation of diverse tumor types.

In vitro models of human liver functions are used across a diverse range of applications in preclinical drug development and disease modeling, with particular increasing interest in models that capture facets of liver inflammatory status. This study investigates how the interplay between biophysical and biochemical microenvironment cues influence phenotypic responses, including inflammation signatures, of primary human hepatocytes (PHH) cultured in a commercially available perfused bioreactor. A 3D printing-based alginate microwell system was designed to form thousands of hepatic spheroids in a scalable manner as a comparator 3D culture modality to the bioreactor. Soft, synthetic extracellular matrix (ECM) hydrogel scaffolds with biophysical properties mimicking features of liver were engineered to replace polystyrene scaffolds, and the biochemical microenvironment was modulated with a defined set of growth factors and signaling modulators. The supplemented media significantly increased tissue density, albumin secretion, and CYP3A4 activity but also upregulated inflammatory markers. Basal inflammatory markers were lower for cells maintained in ECM hydrogel scaffolds or spheroid formats than polystyrene scaffolds, while hydrogel scaffolds exhibited the most sensitive response to inflammation as assessed by multiplexed cytokine and RNA-seq analyses. Together, these engineered 3D liver microenvironments provide insights for probing human liver functions and inflammatory response in vitro.

AbstractInsulin receptor (IR) signaling is central to normal metabolic control and is dysregulated in metabolic diseases such as type 2 diabetes. We report here that IR is incorporated into dynamic clusters at the plasma membrane, in the cytoplasm and in the nucleus of human hepatocytes and adipocytes. Insulin stimulation promotes further incorporation of IR into these dynamic clusters in insulin-sensitive cells but not in insulin-resistant cells, where both IR accumulation and dynamic behavior are reduced. Treatment of insulin-resistant cells with metformin, a first-line drug used to treat type 2 diabetes, can rescue IR accumulation and the dynamic behavior of these clusters. This rescue is associated with metformin’s role in reducing reactive oxygen species that interfere with normal dynamics. These results indicate that changes in the physico-mechanical features of IR clusters contribute to insulin resistance and have implications for improved therapeutic approaches.

© 2020 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Adenomyosis remains an enigmatic disease in the clinical and research communities. The high prevalence, diversity of morphological and symptomatic presentations, array of potential etiological explanations, and variable response to existing interventions suggest that different subgroups of patients with distinguishable mechanistic drivers of disease may exist. These factors, combined with the weak links to genetic predisposition, make the entire spectrum of the human condition challenging to model in animals. Here, after an overview of current approaches, a vision for applying physiomimetic modeling to adenomyosis is presented. Physiomimetics combines a system's biology analysis of patient populations to generate hypotheses about mechanistic bases for stratification with in vitro patient avatars to test these hypotheses. A substantial foundation for three-dimensional (3D) tissue engineering of adenomyosis lesions exists in several disparate areas: epithelial organoid technology; synthetic biomaterials matrices for epithelial-stromal coculture; smooth muscle 3D tissue engineering; and microvascular tissue engineering. These approaches can potentially be combined with microfluidic platform technologies to model the lesion microenvironment and can potentially be coupled to other microorgan systems to examine systemic effects. In vitro patient-derived models are constructed to answer specific questions leading to target identification and validation in a manner that informs preclinical research and ultimately clinical trial design.

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.

Metastatic breast cancer remains a largely incurable and fatal disease with liver involvement bearing the worst prognosis. The danger is compounded by a subset of disseminated tumor cells that may lie dormant for years to decades before re-emerging as clinically detectable metastases. Pathophysiological signals can drive these tumor cells to emerge. Prior studies indicated CXCR3 ligands as being the predominant signals synergistically and significantly unregulated during inflammation in the gut-liver axis. Of the CXCR3 ligands, IP-10 (CXCL10) was the most abundant, correlated significantly with shortened survival of human breast cancer patients with metastatic disease and was highest in those with triple negative (TNBC) disease. Using a complex ex vivo all-human liver microphysiological (MPS) model of dormant-emergent metastatic progression, CXCR3 ligands were found to be elevated in actively growing populations of metastatic TNBC breast cancer cells whereas they remained similar to the tumor-free hepatic niche in those with dormant breast cancer cells. Subsequent stimulation of dormant breast cancer cells in the ex vivo metastatic liver MPS model with IP-10 triggered their emergence in a dose-dependent manner. Emergence was indicated to occur indirectly possibly via activation of the resident liver cells in the surrounding metastatic microenvironment, as stimulation of breast cancer cells with exogenous IP-10 did not significantly change their migratory, invasive or proliferative behavior. The findings reveal that IP-10 is capable of triggering the emergence of dormant breast cancer cells within the liver metastatic niche and identifies the IP-10/CXCR3 as a candidate targetable pathway for rational approaches aimed at maintaining dormancy., NIH (UH3TR000496), NIH (GM69668), NIH (GM63569), NIH (U01-CA215798), US Department of Defense (W81XWH-19-1-0494)

Microphysiological systems (MPS), comprising human cell cultured in formats that capture features of the three-dimensional (3D) microenvironments of native human organs under microperfusion, are promising tools for biomedical research. Here we report the development of a mesoscale physiological system (MePS) enabling the long-term 3D perfused culture of primary human hepatocytes at scales of over 106cells per MPS. A central feature of the MePS, which employs a commercially-available multiwell bioreactor for perfusion, is a novel scaffold comprising a dense network of nano- and micro-porous polymer channels, designed to provide appropriate convective and diffusive mass transfer of oxygen and other nutrients while maintaining physiological values of shear stress. The scaffold design is realized by a high resolution stereolithography fabrication process employing a novel resin. This new culture system sustains mesoscopic hepatic tissue-like cultures with greater hepatic functionality (assessed by albumin and urea synthesis, and CYP3A4 activity) and lower inflammation markers compared to comparable cultures on the commercial polystyrene scaffold. To illustrate applications to disease modeling, we established an insulin-resistant phenotype by exposing liver cells to hyperglycemic and hyperinsulinemic media. Future applications of the MePS include the co-culture of hepatocytes with resident immune cells and the integration with multiple organs to model complex liver-associated diseases.

Endometriosis remains a challenge to understand and to diagnose. This is an observational cross-sectional pilot study to characterize the gut and vaginal microbiome profiles among endometriosis patients and control subjects without the disease and to explore their potential use as a less-invasive diagnostic tool for endometriosis. Overall, 59 women were included, n = 35 with endometriosis and n = 24 controls. Rectal and vaginal samples were collected in two different periods of the menstrual cycle from all subjects. Gut and vaginal microbiomes from patients with different rASRM (revised American Society for Reproductive Medicine) endometriosis stages and controls were analyzed. Illumina sequencing libraries were constructed using a two-step 16S rRNA gene PCR amplicon approach. Correlations of 16S rRNA gene amplicon data with clinical metadata were conducted using a random forest-based machine-learning classification analysis. Distribution of vaginal CSTs (community state types) significantly differed between follicular and menstrual phases of the menstrual cycle (p = 0.021, Fisher’s exact test). Vaginal and rectal microbiome profiles and their association to severity of endometriosis (according to rASRM stages) were evaluated. Classification models built with machine-learning methods on the microbiota composition during follicular and menstrual phases of the cycle were built, and it was possible to accurately predict rASRM stages 1–2 verses rASRM stages 3–4 endometriosis. The feature contributing the most to this prediction was an OTU (operational taxonomic unit) from the genus Anaerococcus. Gut and vaginal microbiomes of women with endometriosis have been investigated. Our findings suggest for the first time that vaginal microbiome may predict stage of disease when endometriosis is present.

© 2020 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Adenomyosis remains an enigmatic disease in the clinical and research communities. The high prevalence, diversity of morphological and symptomatic presentations, array of potential etiological explanations, and variable response to existing interventions suggest that different subgroups of patients with distinguishable mechanistic drivers of disease may exist. These factors, combined with the weak links to genetic predisposition, make the entire spectrum of the human condition challenging to model in animals. Here, after an overview of current approaches, a vision for applying physiomimetic modeling to adenomyosis is presented. Physiomimetics combines a system's biology analysis of patient populations to generate hypotheses about mechanistic bases for stratification with in vitro patient avatars to test these hypotheses. A substantial foundation for three-dimensional (3D) tissue engineering of adenomyosis lesions exists in several disparate areas: epithelial organoid technology; synthetic biomaterials matrices for epithelial-stromal coculture; smooth muscle 3D tissue engineering; and microvascular tissue engineering. These approaches can potentially be combined with microfluidic platform technologies to model the lesion microenvironment and can potentially be coupled to other microorgan systems to examine systemic effects. In vitro patient-derived models are constructed to answer specific questions leading to target identification and validation in a manner that informs preclinical research and ultimately clinical trial design.

© 2020 The Authors Women's health concerns are generally underrepresented in basic and translational research, but reproductive health in particular has been hampered by a lack of understanding of basic uterine and menstrual physiology. Menstrual health is an integral part of overall health because between menarche and menopause, most women menstruate. Yet for tens of millions of women around the world, menstruation regularly and often catastrophically disrupts their physical, mental, and social well-being. Enhancing our understanding of the underlying phenomena involved in menstruation, abnormal uterine bleeding, and other menstruation-related disorders will move us closer to the goal of personalized care. Furthermore, a deeper mechanistic understanding of menstruation—a fast, scarless healing process in healthy individuals—will likely yield insights into a myriad of other diseases involving regulation of vascular function locally and systemically. We also recognize that many women now delay pregnancy and that there is an increasing desire for fertility and uterine preservation. In September 2018, the Gynecologic Health and Disease Branch of the Eunice Kennedy Shriver National Institute of Child Health and Human Development convened a 2-day meeting, “Menstruation: Science and Society” with an aim to “identify gaps and opportunities in menstruation science and to raise awareness of the need for more research in this field.” Experts in fields ranging from the evolutionary role of menstruation to basic endometrial biology (including omic analysis of the endometrium, stem cells and tissue engineering of the endometrium, endometrial microbiome, and abnormal uterine bleeding and fibroids) and translational medicine (imaging and sampling modalities, patient-focused analysis of menstrual disorders including abnormal uterine bleeding, smart technologies or applications and mobile health platforms) to societal challenges in health literacy and dissemination frameworks across

Slow progress in the fight against neurodegenerative diseases (NDs) motivates an urgent need for highly controlled in vitro systems to investigate organ-organ– and organ-immune–specific interactions relevant for disease pathophysiology. Of particular interest is the gut/microbiome-liver-brain axis for parsing out how genetic and environmental factors contribute to NDs. We have developed a mesofluidic platform technology to study gut-liver-cerebral interactions in the context of Parkinson’s disease (PD). It connects microphysiological systems (MPSs) of the primary human gut and liver with a human induced pluripotent stem cell–derived cerebral MPS in a systemically circulated common culture medium containing CD4 regulatory T and T helper 17 cells. We demonstrate this approach using a patient-derived cerebral MPS carrying the PD-causing A53T mutation, gaining two important findings: (i) that systemic interaction enhances features of in vivo–like behavior of cerebral MPSs, and (ii) that microbiome-associated short-chain fatty acids increase expression of pathology-associated pathways in PD. +

Metastatic breast cancer remains a largely incurable and fatal disease with liver involvement bearing the worst prognosis. The danger is compounded by a subset of disseminated tumor cells that may lie dormant for years to decades before re-emerging as clinically detectable metastases. Pathophysiological signals can drive these tumor cells to emerge. Prior studies indicated CXCR3 ligands as being the predominant signals synergistically and significantly unregulated during inflammation in the gut-liver axis. Of the CXCR3 ligands, IP-10 (CXCL10) was the most abundant, correlated significantly with shortened survival of human breast cancer patients with metastatic disease and was highest in those with triple negative (TNBC) disease. Using a complex ex vivo all-human liver microphysiological (MPS) model of dormant-emergent metastatic progression, CXCR3 ligands were found to be elevated in actively growing populations of metastatic TNBC breast cancer cells whereas they remained similar to the tumor-free hepatic niche in those with dormant breast cancer cells. Subsequent stimulation of dormant breast cancer cells in the ex vivo metastatic liver MPS model with IP-10 triggered their emergence in a dose-dependent manner. Emergence was indicated to occur indirectly possibly via activation of the resident liver cells in the surrounding metastatic microenvironment, as stimulation of breast cancer cells with exogenous IP-10 did not significantly change their migratory, invasive or proliferative behavior. The findings reveal that IP-10 is capable of triggering the emergence of dormant breast cancer cells within the liver metastatic niche and identifies the IP-10/CXCR3 as a candidate targetable pathway for rational approaches aimed at maintaining dormancy.

Background: The gut microbiome plays an important role in human health and disease. Gnotobiotic animal and in vitro cell-based models provide some informative insights into mechanistic crosstalk. However, there is no existing system for a long-term co-culture of a human colonic mucosal barrier with super oxygen-sensitive commensal microbes, hindering the study of human-microbe interactions in a controlled manner. Methods: Here, we investigated the effects of an abundant super oxygen-sensitive commensal anaerobe, Faecalibacterium prausnitzii, on a primary human mucosal barrier using a Gut-MIcrobiome (GuMI) physiome platform that we designed and fabricated. Findings: Long-term continuous co-culture of F. prausnitzii for two days with colon epithelia, enabled by continuous flow of completely anoxic apical media and aerobic basal media, resulted in a strictly anaerobic apical environment fostering growth of and butyrate production by F. prausnitzii, while maintaining a stable colon epithelial barrier. We identified elevated differentiation and hypoxia-responsive genes and pathways in the platform compared with conventional aerobic static culture of the colon epithelia, attributable to a combination of anaerobic environment and continuous medium replenishment. Furthermore, we demonstrated anti-inflammatory effects of F. prausnitzii through HDAC and the TLR-NFKB axis. Finally, we identified that butyrate largely contributes to the anti-inflammatory effects by downregulating TLR3 and TLR4. Conclusions: Our results are consistent with some clinical observations regarding F. prausnitzii, thus motivating further studies employing this platform with more complex engineered colon tissues for understanding the interaction between the human colonic mucosal barrier and microbiota, pathogens, or engineered bacteria.

Human-on-a-chip systems are rapidly advancing due to the availability of human stem cells from a variety of tissues, but publications have utilized mostly simple methods of biochemical analysis. Here, we apply mass spectrometry to a sophisticated multiorgan human-on-a-chip system for the comprehensive study of tolcapone metabolite profiling and metabolomics. The developed human-on-a-chip includes seven interacting microphysiological systems (MPSs), brain, pancreas, liver, lung, heart, gut, and endometrium, with a mixer chamber for systemic circulation and tolcapone dose. We investigated tolcapone metabolism by analyzing the circulating medium using mass spectrometry. Twelve tolcapone metabolites were identified, three of which are newly reported. These metabolites demonstrated that oxidation, reduction, and conjugation reactions were the most important routes of tolcapone metabolism. In parallel, metabolomics in brain MPS evaluated the tolcapone influences on endogenous pathways in human brain. Untargeted metabolomics identified 18 key biomarkers significantly changed in human brain MPS after tolcapone dosing, which were mainly associated with perturbation of tryptophan and phenylalanine metabolism (BH4 cycle), glycerophospholipid metabolism, energy metabolism, and aspartate metabolism. This is the first example of successfully combining drug metabolism, metabolomics, and cell engineering to capture complex human physiology and the multiorgan interactions; the results we present here could be a step toward using analytical chemistry to advance the utilization of human-on-a-chip for testing both drug efficacy and toxicity in a single system., U.S. Army Research Office & the Defense Advanced Research Project Agency (Cooperative Agreement no. W911NF-12-2-0039)

Although the association between the microbiome and IBD and liver diseases is known, the cause and effect remain elusive. By connecting human microphysiological systems of the gut, liver, and circulating Treg and Th17 cells, we created a multi-organ model of ulcerative colitis (UC) ex vivo. The approach shows microbiome-derived short-chain fatty acids (SCFAs) to either improve or worsen UC severity, depending on the involvement of effector CD4 T cells. Using multiomics, we found SCFAs increased production of ketone bodies, glycolysis, and lipogenesis, while markedly reducing innate immune activation of the UC gut. However, during acute T cell-mediated inflammation, SCFAs exacerbated CD4+ T cell-effector function, partially through metabolic reprograming, leading to gut barrier disruption and hepatic injury. These paradoxical findings underscore the emerging utility of human physiomimetic technology in combination with systems immunology to study causality and the fundamental entanglement of immunity, metabolism, and tissue homeostasis.

Vascularization is an important strategy to overcome diffusion limits and enable the formation of complex, physiologically relevant engineered tissues and organoids. Self-assembly is a technique to generate in vitro vascular networks, but engineering the necessary network morphology and function remains challenging. Here, autofluorescence multiphoton microscopy (aMPM), a label-free imaging technique, is used to quantitatively evaluate in vitro vascular network morphology. Vascular networks are generated using human embryonic stem cell–derived endothelial cells and primary human pericytes encapsulated in synthetic poly(ethylene glycol)-based hydrogels. Two custom-built bioreactors are used to generate distinct fluid flow patterns during vascular network formation: recirculating flow or continuous flow. aMPM is used to image these 3D vascular networks without the need for fixation, labels, or dyes. Image processing and analysis algorithms are developed to extract quantitative morphological parameters from these label-free images. It is observed with aMPM that both bioreactors promote formation of vascular networks with lower network anisotropy compared to static conditions, and the continuous flow bioreactor induces more branch points compared to static conditions. Importantly, these results agree with trends observed with immunocytochemistry. These studies demonstrate that aMPM allows label-free monitoring of vascular network morphology to streamline optimization of growth conditions and provide quality control of engineered tissues., National Institutes of Health (U.S.) (Grant R01 HL093282-01A1), National Institutes of Health (U.S.) (Grant 1U H2TR000506-01), National Institutes of Health (U.S.) (Grant 3UH2TR000506-02S1), National Institutes of Health (U.S.) (Grant 4U H3TR000506-03), National Institutes of Health (U.S.) (Grant R01CA205101), National Institutes of Health (U.S.) (Grant R01CA185747), National Institutes of Health (U.S.) (Grant R01CA211082), National Institutes of Health (U.S.) (Grant R01CA226526), National Science Foundation (U.S.) (Grant (CBET-1642287), Entertainment Industry Foundation. Stand Up to Cancer Colorectal Cancer Dream Team (Grant SU2C-AACR-IG-08-16), Stand Up To Cancer (Grant SU2C-AACR-IG-08-16)

Recent technological breakthroughs in our ability to derive and differentiate induced pluripotent stem cells, organoid biology, organ-on-chip assays, and 3-D bioprinting have all contributed to a heightened interest in the design, assembly, and manufacture of living systems with a broad range of potential uses. This white paper summarizes the state of the emerging field of "multi-cellular engineered living systems," which are composed of interacting cell populations. Recent accomplishments are described, focusing on current and potential applications, as well as barriers to future advances, and the outlook for longer term benefits and potential ethical issues that need to be considered., National Science Foundation (U.S.) Science Technology Center. Emergent Behaviors of Integrated Cellular Systems (Grant CBET0939511), National Cancer Institute (U.S.) (Grant U01 CA202177)

A clinically relevant risk factor forClostridioides difficile-associated dis-ease (CDAD) is recent antibiotic treatment. Although broad-spectrum antibioticshave been shown to disrupt the structure of the gut microbiota, some antibioticsappear to increase CDAD risk without being highly active against intestinal anaerobes, suggesting direct nonantimicrobial effects. We examined cell biological effectsof antibiotic exposure that may be involved in bacterial pathogenesis using aninvitrogermfree human colon epithelial culture model. We found a marked loss of mucosal barrier and immune function with exposure to the CDAD-associated antibiotics clindamycin and ciprofloxacin, distinct from the results of pretreatment with anantibiotic unassociated with CDAD, tigecycline, which did not reduce innate immuneor mucosal barrier functions. Importantly, pretreatment with CDAD-associated antibi-otics sensitized mucosal barriers to C. difficiletoxin activity in primary cell-derived enteroid monolayers. These data implicate commensal-independent gut mucosal barrier changes in the increased risk of CDAD with specific antibiotics and warrantfurther studies inin vivosystems. We anticipate this work to suggest potential ave-nues of research for host-directed treatment and preventive therapies for CDAD., National Institutes of Health (U.S.) (Grant 01-EB021908), National Cancer Institute (U.S.) (Grant P30-ES002109), David H. Koch Institute for Integrative Cancer Research at MIT (Support core Grant P30-CA14501)

© 2017 The liver plays critical roles in both homeostasis and pathology. It is the major site of drug metabolism in the body and, as such, a common target for drug-induced toxicity and is susceptible to a wide range of diseases. In contrast to other solid organs, the liver possesses the unique ability to regenerate. The physiological importance and plasticity of this organ make it a crucial system of study to better understand human physiology, disease, and response to exogenous compounds. These aspects have impelled many to develop liver tissue systems for study in isolation outside the body. Herein, we discuss these biologically engineered organoids and microphysiological systems. Keywords: Microphysiologic systems; Organoids; 3D culture systems, National Institutes of Health (U.S.) (Grant UH3TR000496), National Institutes of Health (U.S.) (Grant UH3TR000503)

The surfaces of many hollow or tubular tissues/organs in our respiratory, gastrointestinal, and urogenital tracts are covered by mucosa with folded patterns. The patterns are induced by mechanical instability of the mucosa under compression due to constrained growth. Recapitulating this folding process in vitro will facilitate the understanding and engineering of mucosa in various tissues/organs. However, scant attention has been paid to address the challenge of reproducing mucosal folding. Here we mimic the mucosal folding process using a cell-laden hydrogel film attached to a prestretched tough-hydrogel substrate. The cell-laden hydrogel constitutes a human epithelial cell lining on stromal component to recapitulate the physiological feature of a mucosa. Relaxation of the prestretched tough-hydrogel substrate applies compressive strains on the cell-laden hydrogel film, which undergoes mechanical instability and evolves into morphological patterns. We predict the conditions for mucosal folding as well as the morphology of and strain in the folded artificial mucosa using a combination of theory and simulation. The work not only provides a simple method to fold artificial mucosa but also demonstrates a paradigm in tissue engineering via harnessing mechanical instabilities guided by quantitative mechanics models. Keywords: mucosa; hydrogel; mechanical instabiity; tissue engineering; biomechanics, National Science Foundation (U.S.) (Award CMMI-1661627), United States. Office of Naval Research (Award N00014-17-1-2920)

With the rise of research utilizing microphysiological systems (MPSs), the need for tools that enable the physiological mimicking of the relevant cellular environment is vital. The limited ability to reproduce crucial features of the microenvironment, such as surrounding fluid flow and dynamic changes in biochemical stimuli, severely limits the types of experiments that can be carried out. Current equipment to achieve this, such as syringe and peristaltic pumps, is expensive, large, difficult to program and has limited potential for scalability. Here, we present a new pumping platform that is open-source, low-cost, modular, scalable, fully-programmable and easy to assemble that can be incorporated into cell culture systems to better recapitulate physiological environments. By controlling two commercially available piezoelectric pumps using a Raspberry Pi Zero microcontroller, the system is capable of producing arbitrary dynamic flow profiles with reliable flow rates ranging from 1 to 3000 µL/min as specified by an easily programmable Python-based script. We validated the accuracy of the flow rates, the use of time-varying profiles, and the practicality of the system by creating repeatable dynamic concentration profiles using a 3D-printed static micromixer., United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039)

Mucosal barrier tissues, comprising a layer of tightly-bonded epithelial cells in intimate molecular communication with an underlying matrix-rich stroma containing fibroblasts and immune cells, are prominent targets for drugs against infection, chronic inflammation, and other disease processes. Although human in vitro models of such barriers are needed for mechanistic studies and drug development, differences in extracellular matrix (ECM) needs of epithelial and stromal cells hinder efforts to create such models. Here, using the endometrium as an example mucosal barrier, we describe a synthetic, modular ECM hydrogel suitable for 3D functional co-culture, featuring components that can be remodeled by cells and that respond dynamically to sequester local cell-secreted ECM characteristic of each cell type. The synthetic hydrogel combines peptides with off-the-shelf reagents and is thus accessible to cell biology labs. Specifically, we first identified a single peptide as suitable for initial attachment of both endometrial epithelial and stromal cells using a 2D semi-empirical screen. Then, using a co-culture system of epithelial cells cultured on top of gel-encapsulated stromal cells, we show that inclusion of ECM-binding peptides in the hydrogel, along with the integrin-binding peptide, leads to enhanced accumulation of basement membrane beneath the epithelial layer and more fibrillar collagen matrix assembly by stromal cells over two weeks in culture. Importantly, endometrial co-cultures composed of either cell lines or primary cells displayed hormone-mediated differentiation as assessed by morphological changes and secretory protein production. A multiplex analysis of apical cytokine and growth factor secretion comparing cell lines and primary cells revealed strikingly different patterns, underscoring the importance of using primary cell models in analysis of cell-cell communication networks. In summary, we define a "one-size-fits-all" synthetic ECM that enables lon, John and Karinne Begg Fund, National Institutes of Health (T32 GM 008334 (Interdepartmental Biotechnology Training Program)), United States. Defense Advanced Research Projects Agency (Microphysiological Systems: Program W911NF-12-2-0039), Manton Foundation, National Science Foundation (U.S.) Science Technology Center. Emergent Behaviors of Integrated Cellular Systems, National Institutes of Health (grant DP3-DK097681), United States. Army Research Office. Institute for Collaborative Biotechnologies (grant W91NF-09-001), National Cancer Institute (U.S.) (Koch Institute Support (core) Grant P30-CA14051)

Microphysiological systems (MPS), consisting of tissue constructs, biomaterials, and culture media, aim to recapitulate relevant organ functions in vitro. MPS components are housed in fluidic hardware with operational protocols, such as periodic complete media replacement. Such batch-like operations provide relevant nutrients and remove waste products but also reset cell-secreted mediators (e.g. cytokines, hormones) and potentially limit exposure to drugs (and metabolites). While each component plays an essential role for tissue functionality, MPS-specific nutrient needs are not yet well-characterized nor utilized to operate MPSs at more physiologically-relevant conditions. MPS-specific nutrient needs for gut (immortalized cancer cells), liver (human primary hepatocytes) and cardiac (iPSC-derived cardiomyocytes) MPSs were experimentally quantified. In a long-term study of the gut MPS (10 days), this knowledge was used to design operational protocols to maintain glucose and lactate at desired levels. This quasi-steady state operation was experimentally validated by monitoring glucose and lactate as well as MPS functionality. In a theoretical study, nutrient needs of an integrated multi-MPS platform (gut, liver, cardiac MPSs) were computationally simulated to identify long-term quasi-steady state operations. This integrative experimental and computational approach demonstrates the utilization of quantitative multi-scale characterization of MPSs and incorporating MPS-specific information to establish more physiologically-relevant experimental operations., United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039), National Institutes of Health (U.S.) (U24TR001951), National Institutes of Health (U.S.). Microphysiological Systems Program (4-UH3-TR000496-03)

Increases in expression of ADAM10 and ADAM17 genes and proteins have been evaluated, but not validated as cancer biomarkers. Specific enzyme activities better reflect enzyme cellular functions, and might be better biomarkers than enzyme genes or proteins. However, no high throughput assay is available to test this possibility. Recent studies have developed the high throughput real-time proteolytic activity matrix analysis (PrAMA) that integrates the enzymatic processing of multiple enzyme substrates with mathematical-modeling computation. The original PrAMA measures with significant accuracy the activities of individual metalloproteinases expressed on live cells. To make the biomarker assay usable in clinical practice, we modified PrAMA by testing enzymatic activities in cell and tissue lysates supplemented with broad-spectrum non-MP enzyme inhibitors, and by maximizing the assay specificity using systematic mathematical-modeling analyses. The modified PrAMA accurately measured the absence and decreases of ADAM10 sheddase activity (ADAM10sa) and ADAM17sa in ADAM10-/-and ADAM17-/-mouse embryonic fibroblasts (MEFs), and ADAM10- and ADAM17-siRNA transfected human cancer cells, respectively. It also measured the restoration and inhibition of ADAM10sa in ADAM10-cDNA-transfected ADAM10-/-MEFs and GI254023X-treated human cancer cell and tissue lysates, respectively. Additionally, the modified PrAMA simultaneously quantified with significant accuracy ADAM10sa and ADAM17sa in multiple human tumor specimens, and showed the essential characteristics of a robust high throughput multiplex assay that could be broadly used in biomarker studies. Selectively measuring specific enzyme activities, this new clinically applicable assay is potentially superior to the standard protein- and gene-expression assays that do not distinguish active and inactive enzyme forms.

Background: Increases in expression of ADAM10 and ADAM17 genes and proteins are inconsistently found in cancer lesions, and are not validated as clinically useful biomarkers. The enzyme-specific proteolytic activities, which are solely mediated by the active mature enzymes, directly reflect enzyme cellular functions and might be superior biomarkers than the enzyme gene or protein expressions, which comprise the inactive proenzymes and active and inactivated mature enzymes. Methods: Using a recent modification of the proteolytic activity matrix analysis (PrAMA) measuring specific enzyme activities in cell and tissue lysates, we examined the specific sheddase activities of ADAM10 (ADAM10sa) and ADAM17 (ADAM17sa) in human non-small cell lung-carcinoma (NSCLC) cell lines, patient primary tumors and blood exosomes, and the noncancerous counterparts. Results: NSCLC cell lines and patient tumors and exosomes consistently showed significant increases of ADAM10sa relative to their normal, inflammatory and/or benign-tumor controls. Additionally, stage IA-IIB NSCLC primary tumors of patients who died of the disease exhibited greater increases of ADAM10sa than those of patients who survived 5 years following diagnosis and surgery. In contrast, NSCLC cell lines and patient tumors and exosomes did not display increases of ADAM17sa. Conclusions: This study is the first to investigate enzyme-specific proteolytic activities as potential cancer biomarkers. It provides a proof-of-concept that ADAM10sa could be a biomarker for NSCLC early detection and outcome prediction. To ascertain that ADAM10sa is a useful cancer biomarker, further robust clinical validation studies are needed., National Institutes of Health (U.S.) (R01 CA96504)

Microphysiological systems (MPS) provide relevant physiological environments in vitro for studies of pharmacokinetics, pharmacodynamics and biological mechanisms for translational research. Designing multi-MPS platforms is essential to study multi-organ systems. Typical design approaches, including direct and allometric scaling, scale each MPS individually and are based on relative sizes not function. This study's aim was to develop a new multi-functional scaling approach for integrated multi-MPS platform design for specific applications. We developed an optimization approach using mechanistic modeling and specification of an objective that considered multiple MPS functions, e.g., drug absorption and metabolism, simultaneously to identify system design parameters. This approach informed the design of two hypothetical multi-MPS platforms consisting of gut and liver (multi-MPS platform I) and gut, liver and kidney (multi-MPS platform II) to recapitulate in vivo drug exposures in vitro. This allows establishment of clinically relevant drug exposure-response relationships, a prerequisite for efficacy and toxicology assessment. Design parameters resulting from multi-functional scaling were compared to designs based on direct and allometric scaling. Human plasma time-concentration profiles of eight drugs were used to inform the designs, and profiles of an additional five drugs were calculated to test the designed platforms on an independent set. Multi-functional scaling yielded exposure times in good agreement with in vivo data, while direct and allometric scaling approaches resulted in short exposure durations. Multi-functional scaling allows appropriate scaling from in vivo to in vitro of multi-MPS platforms, and in the cases studied provides designs that better mimic in vivo exposures than standard MPS scaling methods., United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039), National Institutes of Health (U.S.). Microphysiological Systems Program (4-UH3-TR000496-03)

Methods to parse paracrine epithelial-stromal communication networks are a vital need in drug development, as disruption of these networks underlies diseases ranging from cancer to endometriosis. Here, we describe a modular, synthetic, and dissolvable extracellular matrix (MSD-ECM) hydrogel that fosters functional 3D epithelial-stromal co-culture, and that can be dissolved on-demand to recover cells and paracrine signaling proteins intact for subsequent analysis. Specifically, synthetic polymer hydrogels, modified with cell-interacting adhesion motifs and crosslinked with peptides that include a substrate for cell-mediated proteolytic remodeling, can be rapidly dissolved by an engineered version of the microbial transpeptidase Sortase A (SrtA) if the crosslinking peptide includes a SrtA substrate motif and a soluble second substrate. SrtA-mediated dissolution affected only 1 of 31 cytokines and growth factors assayed, whereas standard protease degradation methods destroyed about half of these same molecules. Using co-encapsulated endometrial epithelial and stromal cells as one model system, we show that the dynamic cytokine and growth factor response of co-cultures to an inflammatory cue is richer and more nuanced when measured from SrtA-dissolved gel microenvironments than from the culture supernate. This system employs accessible, reproducible reagents and facile protocols; hence, has potential as a tool in identifying and validating therapeutic targets in complex diseases., National Institutes of Health (U.S.) (R01EB010246), National Institutes of Health (U.S.) (UH2TR000496), Institute for Collaborative Biotechnologies (W911NF-09-0001), National Institutes of Health (U.S.) (T32GM008334), United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039), John and Karinne Begg Fund, Begg New Horizon Fund for Undergraduate Research at MIT, Massachusetts Institute of Technology. Biophysical Instrumentation Facility, Manton Foundation, Ludwig Postdoctoral Fellowship for Cancer Research, Swiss National Science Foundation (Postdoctoral Fellowship)

Traditional in vitro human liver cell culture models lose key hepatic functions such as metabolic activity during short-term culture. Advanced three-dimensional (3D) liver coculture platforms offer the potential for extended hepatocyte functionality and allow for the study of more complex biologic interactions, which can improve and refine human drug safety evaluations. Here, we use a perfusion flow 3D microreactor platform for the coculture of cryopreserved primary human hepatocytes and Kupffer cells to study the regulation of cytochrome P450 3A4 isoform (CYP3A4) activity by chronic interleukin 6 (IL-6)-mediated inflammation over 2 weeks. Hepatocyte cultures remained stable over 2 weeks, with consistent albumin production and basal IL-6 levels. Direct IL-6 stimulation that mimics an inflammatory state induced a dose-dependent suppression of CYP3A4 activity, an increase in C-reactive protein (CRP) secretion, and a decrease in shed soluble interleukin-6 receptor (IL-6R) levels, indicating expected hepatic IL-6 bioactivity. Tocilizumab, an anti-IL- 6R monoclonal antibody used to treat rheumatoid arthritis, has been demonstrated clinically to impact small molecule drug pharmacokinetics by modulating cytochrome P450 enzyme activities, an effect not observed in traditional hepatic cultures. We have now recapitulated the clinical observation in a 3D bioreactor system. Tocilizumab was shown to desuppress CYP3A4 activity while reducing the CRP concentration after 72 hours in the continued presence of IL-6. This change in CYP3A4 activity decreased the half-life and area under the curve up to the last measurable concentration (AUClast) of the small molecule CYP3A4 substrate simvastatin hydroxy acid, measured before and after tocilizumab treatment. We conclude that next-generation in vitro liver culture platforms are well suited for these types of long-term treatment studies and show promise for improved drug safety assessment., United States. Defense Advanced Research Projects Agency (W911NF-12-2-0039), National Institutes of Health (U.S.) (UH3TR000496)

In this work, we first describe the population variability in hepatic drug metabolism using cryopreserved hepatocytes from five different donors cultured in a perfused three-dimensional human liver microphysiological system, and then show how the resulting data can be integrated with a modeling and simulation framework to accomplish in vitro-in vivo translation. For each donor, metabolic depletion profiles of six compounds (phenacetin, diclofenac, lidocaine, ibuprofen, propranolol, and prednisolone) were measured, along with metabolite formation, mRNA levels of 90 metabolism-related genes, and markers of functional viability [lactate dehydrogenase (LDH) release, albumin, and urea production]. Drug depletion data were analyzed with mixed-effects modeling. Substantial interdonor variability was observed with respect to gene expression levels, drug metabolism, and other measured hepatocyte functions. Specifically, interdonor variability in intrinsic metabolic clearance ranged from 24.1% for phenacetin to 66.8% for propranolol (expressed as coefficient of variation). Albumin, urea, LDH, and cytochrome P450 mRNA levels were identified as significant predictors of in vitro metabolic clearance. Predicted clearance values from the liver microphysiological system were correlated with the observed in vivo values. A population physiologically based pharmacokinetic model was developed for lidocaine to illustrate the translation of the in vitro output to the observed pharmacokinetic variability in vivo. Stochastic simulations with this model successfully predicted the observed clinical concentration-time profiles and the associated population variability. This is the first study of population variability in drug metabolism in the context of a microphysiological system and has important implications for the use of these systems during the drug development process., United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (Grant W911NF-12-2-0039), National Institutes of Health (U.S.). Microphysiological Systems Program (Grant 4-UH3-TR000496-03)

Distant metastasis is the major cause of breast cancer-related mortality, commonly emerging clinically after 5 or more years of seeming 'cure'of the primary tumor, indicating a quiescent dormancy. The lack of relevant accessible model systems for metastasis that recreate this latent stage has hindered our understanding of the molecular basis and the development of therapies against these lethal outgrowths. We previously reported on the development of an all-human 3D ex vivo hepatic microphysiological system that reproduces several features of liver physiology and enables spontaneous dormancy in a subpopulation of breast cancer cells. However, we observed that the dormant cells were localized primarily within the 3D tissue, while the proliferative cells were in contact with the polystyrene scaffold. As matrix stiffness is known to drive inflammatory and malignant behaviors, we explored the occurrence of spontaneous tumor dormancy and inflammatory phenotype. The microphysiological system was retrofitted with PEGDa-SynKRGD hydrogel scaffolding, which is softer and differs in the interface with the tissue. The microphysiological system incorporated donor-matched primary human hepatocytes and non-parenchymal cells (NPCs), with MDA-MB-231 breast cancer cells. Hepatic tissue in hydrogel scaffolds secreted lower levels of pro-inflammatory analytes, and was more responsive to inflammatory stimuli. The proportion of tumor cells entering dormancy was markedly increased in the hydrogel-supported tissue compared to polystyrene. Interestingly, an unexpected differential response of dormant cells to varying chemotherapeutic doses was identified, which if reflective of patient pathophysiology, has important implications for patient dosing regimens. These findings highlight the metastatic microphysiological system fitted with hydrogel scaffolds as a critical tool in the assessment and development of therapeutic strategies to target dormant metastatic breast cancer.

Quantifying protein location and concentration is critical for understanding function in situ. Scaffold conjugated to environment-sensitive fluorophore (SuCESsFul) biosensors, in which a reporting fluorophore is conjugated to a binding scaffold, can, in principle, detect analytes of interest with high temporal and spatial resolution. However, their adoption has been limited due to the extensive empirical screening required for their development. We sought to establish design principles for this class of biosensor by characterizing over 400 biosensors based on various protein analytes, binding proteins, and fluorophores. We found that the brightest readouts are attained when a specific binding pocket for the fluorophore is present on the analyte. Also, interaction of the fluorophore with the binding protein it is conjugated to can raise background fluorescence, considerably limiting sensor dynamic range. Exploiting these two concepts, we designed biosensors that attain a 100-fold increase in fluorescence upon binding to analyte, an order of magnitude improvement over the previously best-reported SuCESsFul biosensor. These design principles should facilitate the development of improved SuCESsFul biosensors. Keywords: solvatochromism; Sso7d scaffold; sensors; protein engineering; directed evolution, National Science Foundation (U.S.) (Grant MCB-115803), National Institutes of Health (U.S.) (Grant U54CA112967), National Cancer Institute (U.S.) (Grant U54CA112967), National Institutes of Health (U.S.) (Grant R01 EB 010246)

"Mice are not little people" - a refrain becoming louder as the gaps between animal models and human disease become more apparent. At the same time, three emerging approaches are headed toward integration: powerful systems biology analysis of cell-cell and intracellular signaling networks in patient-derived samples; 3D tissue engineered models of human organ systems, often made from stem cells; and micro-fluidic and meso-fluidic devices that enable living systems to be sustained, perturbed and analyzed for weeks in culture. Integration of these rapidly moving fields has the potential to revolutionize development of therapeutics for complex, chronic diseases, including those that have weak genetic bases and substantial contributions from gene-environment interactions. Technical challenges in modeling complex diseases with "organs on chips" approaches include the need for relatively large tissue masses and organ-organ cross talk to capture systemic effects, such that current microfluidic formats often fail to capture the required scale and complexity for interconnected systems. These constraints drive development of new strategies for designing in vitro models, including perfusing organ models, as well as "mesofluidic" pumping and circulation in platforms connecting several organ systems, to achieve the appropriate physiological relevance. Keywords: organs-on-chips; 3D liver culture; perfusion; drug development; inflammation; organ crosstalk; tissue chip; intestine, United States. Defense Advanced Research Projects Agency (Award W911NF-12-2- 0039)), National Institutes of Health (U.S.) (Grant UH3TR000496)

Microphysiological systems (MPSs) are in vitro models that capture facets of in vivo organ function through use of specialized culture microenvironments, including 3D matrices and microperfusion. Here, we report an approach to co-culture multiple different MPSs linked together physiologically on re-useable, open-system microfluidic platforms that are compatible with the quantitative study of a range of compounds, including lipophilic drugs. We describe three different platform designs - "4-way", "7-way", and "10-way" - each accommodating a mixing chamber and up to 4, 7, or 10 MPSs. Platforms accommodate multiple different MPS flow configurations, each with internal re-circulation to enhance molecular exchange, and feature on-board pneumatically-driven pumps with independently programmable flow rates to provide precise control over both intra- and inter-MPS flow partitioning and drug distribution. We first developed a 4-MPS system, showing accurate prediction of secreted liver protein distribution and 2-week maintenance of phenotypic markers. We then developed 7-MPS and 10-MPS platforms, demonstrating reliable, robust operation and maintenance of MPS phenotypic function for 3 weeks (7-way) and 4 weeks (10-way) of continuous interaction, as well as PK analysis of diclofenac metabolism. This study illustrates several generalizable design and operational principles for implementing multi-MPS "physiome-on-a-chip" approaches in drug discovery., United States. Army Research Office (Grant W911NF-12-2-0039)

Described is the development and application of a versatile semisynthetic strategy, based on a combination of sortase-mediated coupling and tetrazine ligation chemistry, which can be exploited for the efficient incorporation of tunable functionality into chimeric recombinant proteins. To demonstrate the scope of the method, the assembly of a set of bivalent ligands, which integrate members of the epidermal growth factor (EGF) ligand family, is described. By using a series of bivalent EGFs with variable intraligand spacing, the differences in structure were correlated with the ability to bias signaling in the ErbB receptor family in a cell motility assay. Biasing away from EGFR-HER2 dimerization with a bivalent EGF was observed to reduce cell motility in an intraligand distance-dependent fashion, thus demonstrating the utility of the approach for acutely perturbing receptor-mediated cell signaling pathways., National Cancer Institute (U.S.). Integrative Cancer Biology Program (Grant U54-CA112967), National Institutes of Health (U.S.) (R01DE019523-13), Massachusetts Institute of Technology. Computational and Systems Biology Program. MIT-Merck Postdoctoral Fellowship, Swiss National Science Foundation (Postdoctoral Fellowship), National Institute of Environmental Health Sciences (Training Grant in Environmental Toxicology 5-T32-ES007020)

Synthetic extracellular matrices are widely used in regenerative medicine and as tools in building in vitro physiological culture models. Synthetic hydrogels display advantageous physical properties, but are challenging to modify with large peptides or proteins. Here, a facile, mild enzymatic postgrafting approach is presented. Sortase-mediated ligation was used to conjugate human epidermal growth factor fused to a GGG ligation motif (GGG-EGF) to poly(ethylene glycol) (PEG) hydrogels containing the sortase LPRTG substrate. The reversibility of the sortase reaction was then exploited to cleave tethered EGF from the hydrogels for analysis. Analyses of the reaction supernatant and the postligation hydrogels showed that the amount of tethered EGF increases with increasing LPRTG in the hydrogel or GGG-EGF in the supernatant. Sortase-tethered EGF was biologically active, as demonstrated by stimulation of DNA synthesis in primary human hepatocytes and endometrial epithelial cells. The simplicity, specificity, and reversibility of sortase-mediated ligation and cleavage reactions make it an attractive approach for modification of hydrogels., National Institutes of Health (U.S.) (5R01EB010246), National Institutes of Health (U.S.) (5UH2TR000496), Institute for Collaborative Biotechnologies (W911NF-09-0001), National Institutes of Health (U.S.) (1T32GM008334), United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039), Begg New Horizon Fund for Undergraduate Research at MIT, National Institutes of Health (U.S.) (Biotechnology Training Program NIH/NIGMS 5T32GM008334)), Biophysical Instrumentation Facility, Virginia and Daniel K. Ludwig Graduate Fellowship, Swiss National Science Foundation (Postdoctoral Fellowship)

Local recurrence is a common cause of treatment failure for patients with solid tumors. Intraoperative detection of microscopic residual cancer in the tumor bed could be used to decrease the risk of a positive surgical margin, reduce rates of reexcision, and tailor adjuvant therapy. We used a protease-activated fluorescent imaging probe, LUM015, to detect cancer in vivo in a mouse model of soft tissue sarcoma (STS) and ex vivo in a first-in-human phase 1 clinical trial. In mice, intravenous injection of LUM015 labeled tumor cells, and residual fluorescence within the tumor bed predicted local recurrence. In 15 patients with STS or breast cancer, intravenous injection of LUM015 before surgery was well tolerated. Imaging of resected human tissues showed that fluorescence from tumor was significantly higher than fluorescence from normal tissues. LUM015 biodistribution, pharmacokinetic profiles, and metabolism were similar in mouse and human subjects. Tissue concentrations of LUM015 and its metabolites, including fluorescently labeled lysine, demonstrated that LUM015 is selectively distributed to tumors where it is activated by proteases. Experiments in mice with a constitutively active PEGylated fluorescent imaging probe support a model where tumor-selective probe distribution is a determinant of increased fluorescence in cancer. These co-clinical studies suggest that the tumor specificity of protease-activated imaging probes, such as LUM015, is dependent on both biodistribution and enzyme activity. Our first-in-human data support future clinical trials of LUM015 and other protease-sensitive probes.

Investigation of the pharmacokinetics (PK) of a compound is of significant importance during the early stages of drug development, and therefore several in vitro systems are routinely employed for this purpose. However, the need for more physiologically realistic in vitro models has recently fueled the emerging field of tissue-engineered 3D cultures, also referred to as organs-on-chips, or microphysiological systems (MPSs). We have developed a novel fluidic platform that interconnects multiple MPSs, allowing PK studies in multi-organ in vitro systems along with the collection of high-content quantitative data. This platform was employed here to integrate a gut and a liver MPS together in continuous communication, and investigate simultaneously different PK processes taking place after oral drug administration in humans (e.g., intestinal permeability, hepatic metabolism). Measurement of tissue-specific phenotypic metrics indicated that gut and liver MPSs can be fluidically coupled with circulating common medium without compromising their functionality. The PK of diclofenac and hydrocortisone was investigated under different experimental perturbations, and results illustrate the robustness of this integrated system for quantitative PK studies. Mechanistic model-based analysis of the obtained data allowed the derivation of the intrinsic parameters (e.g., permeability, metabolic clearance) associated with the PK processes taking place in each MPS. Although these processes were not substantially affected by the gut-liver interaction, our results indicate that inter-MPS communication can have a modulating effect (hepatic metabolism upregulation). We envision that our integrative approach, which combines multi-cellular tissue models, multi-MPS platforms, and quantitative mechanistic modeling, will have broad applicability in pre-clinical drug development., United States. Defense Advanced Research Projects Agency (Grant W911NF-12-2-0039), National Institutes of Health (U.S.) (Grant 4-UH3-TR000496-03)

Investigation of the pharmacokinetics (PK) of a compound is of significant importance during the early stages of drug development, and therefore several in vitro systems are routinely employed for this purpose. However, the need for more physiologically realistic in vitro models has recently fueled the emerging field of tissue-engineered 3D cultures, also referred to as organs-on-chips, or microphysiological systems (MPSs). We have developed a novel fluidic platform that interconnects multiple MPSs, allowing PK studies in multi-organ in vitro systems along with the collection of high-content quantitative data. This platform was employed here to integrate a gut and a liver MPS together in continuous communication, and investigate simultaneously different PK processes taking place after oral drug administration in humans (e.g., intestinal permeability, hepatic metabolism). Measurement of tissue-specific phenotypic metrics indicated that gut and liver MPSs can be fluidically coupled with circulating common medium without compromising their functionality. The PK of diclofenac and hydrocortisone was investigated under different experimental perturbations, and results illustrate the robustness of this integrated system for quantitative PK studies. Mechanistic model-based analysis of the obtained data allowed the derivation of the intrinsic parameters (e.g., permeability, metabolic clearance) associated with the PK processes taking place in each MPS. Although these processes were not substantially affected by the gut-liver interaction, our results indicate that inter-MPS communication can have a modulating effect (hepatic metabolism upregulation). We envision that our integrative approach, which combines multi-cellular tissue models, multi-MPS platforms, and quantitative mechanistic modeling, will have broad applicability in pre-clinical drug development., United States. Defense Advanced Research Projects Agency (Grant W911NF-12-2- 0039), National Institutes of Health (U.S.) (Grant 4-UH3-TR000496-0)

Our goal in developing Microphysiological Systems (MPS) technology is to provide an improved approach for more predictive preclinical drug discovery via a highly integrated experimental/computational paradigm. Success will require quantitative characterization of MPSs and mechanistic analysis of experimental findings sufficient to translate resulting insights from in vitro to in vivo. We describe herein a systems pharmacology approach to MPS development and utilization that incorporates more mechanistic detail than traditional pharmacokinetic/pharmacodynamic (PK/PD) models. A series of studies illustrates diverse facets of our approach. First, we demonstrate two case studies: a PK data analysis and an inflammation response––focused on a single MPS, the liver/immune MPS. Building on the single MPS modeling, a theoretical investigation of a four-MPS interactome then provides a quantitative way to consider several pharmacological concepts such as absorption, distribution, metabolism, and excretion in the design of multi-MPS interactome operation and experiments., United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039), National Institutes of Health (U.S.) Microphysiological Systems Program (4-UH3-TR000496-03), Massachusetts Institute of Technology. Center for Environmental Health Sciences (NIEHS Grant P30-ES002109)

Background: PDZK1 is a four PDZ-domain containing cytoplasmic protein that binds to a variety of membrane proteins via their C-termini and can influence the abundance, localization and/or function of its target proteins. One of these targets in hepatocytes in vivo is the HDL receptor SR-BI. Normal hepatic expression of SR-BI protein requires PDZK1 - <5% of normal hepatic SR-BI is seen in the livers of PDZK1 knockout mice. Progress has been made in identifying features of PDZK1 required to control hepatic SR-BI in vivo using hepatic expression of wild-type and mutant forms of PDZK1 in wild-type and PDZK1 KO transgenic mice. Such in vivo studies are time consuming and expensive, and cannot readily be used to explore many features of the underlying molecular and cellular mechanisms. Methodology/Principal Findings: Here we have explored the potential to use either primary rodent hepatocytes in culture using 2D collagen gels with newly developed optimized conditions or PDZK1/SR-BI co-transfected cultured cell lines (COS, HEK293) for such studies. SR-BI and PDZK1 protein and mRNA expression levels fell rapidly in primary hepatocyte cultures, indicating this system does not adequately mimic hepatocytes in vivo for analysis of the PDZK1 dependence of SR-BI. Although PDZK1 did alter SR-BI protein expression in the cell lines, its influence was independent of SR-BI’s C-terminus, and thus is not likely to occur via the same mechanism as that which occurs in hepatocytes in vivo. Conclusions/Significance: Caution must be exercised in using primary hepatocytes or cultured cell lines when studying the mechanism underlying the regulation of hepatic SR-BI by PDZK1. It may be possible to use SR-BI and PDZK1 expression as sensitive markers for the in vivo-like state of hepatocytes to further improve primary hepatocyte cell culture conditions., National Institutes of Health (U.S.) (Grant HL052212), National Institutes of Health (U.S.) (Grant HL066105), National Institutes of Health (U.S.) (Grant ES015241), National Institutes of Health (U.S.) (Grant GM068762)

Author Manuscript 2012 May 21., Design of 3D scaffolds that can facilitate proper survival, proliferation, and differentiation of progenitor cells is a challenge for clinical applications involving large connective tissue defects. Cell migration within such scaffolds is a critical process governing tissue integration. Here, we examine effects of scaffold pore diameter, in concert with matrix stiffness and adhesivity, as independently tunable parameters that govern marrow-derived stem cell motility. We adopted an “inverse opal” processing technique to create synthetic scaffolds by crosslinking poly(ethylene glycol) at different densities (controlling matrix elastic moduli or stiffness) and small doses of a heterobifunctional monomer (controlling matrix adhesivity) around templating beads of different radii. As pore diameter was varied from 7 to 17 µm (i.e., from significantly smaller than the spherical cell diameter to approximately cell diameter), it displayed a profound effect on migration of these stem cells—including the degree to which motility was sensitive to changes in matrix stiffness and adhesivity. Surprisingly, the highest probability for substantive cell movement through pores was observed for an intermediate pore diameter, rather than the largest pore diameter, which exceeded cell diameter. The relationships between migration speed, displacement, and total path length were found to depend strongly on pore diameter. We attribute this dependence to convolution of pore diameter and void chamber diameter, yielding different geometric environments experienced by the cells within. Bioeng. 2011; 108:1181–1193, (National Institute of General Medical Sciences (U.S.) (NRSA Fellowship GM083472), National Institutes of Health (U.S.) (National Institute of General Medical Sciences (U.S.) Cell Migration Consortium Grant GM064346), National Science Foundation (U.S.) (CAREER CBET-0644846)

Human induced pluripotent stem cells (hiPSCs) have potential for personalized and regenerative medicine. While most of the methods using these cells have focused on deriving homogenous populations of specialized cells, there has been modest success in producing hiPSC-derived organotypic tissues or organoids. Here we present a novel approach for generating and then co-differentiating hiPSC-derived progenitors. With a genetically engineered pulse of GATA-binding protein 6 (GATA6) expression, we initiate rapid emergence of all three germ layers as a complex function of GATA6 expression levels and tissue context. Within 2 weeks we obtain a complex tissue that recapitulates early developmental processes and exhibits a liver bud-like phenotype, including haematopoietic and stromal cells as well as a neuronal niche. Collectively, our approach demonstrates derivation of complex tissues from hiPSCs using a single autologous hiPSCs as source and generates a range of stromal cells that co-develop with parenchymal cells to form tissues., National Science Foundation (U.S.). Emergent Behaviors of Integrated Cellular Systems (NSF CBET-0939511), Synthetic Biology Engineering Research Center, National Institutes of Health (U.S.) (P50GM098792), Ernst Schering Foundation, Swiss National Science Foundation

In vitro models that recapitulate the liver's structural and functional complexity could prolong hepatocellular viability and function to improve platforms for drug toxicity studies and understanding liver pathophysiology. Here, stereolithography (SLA) was employed to fabricate hydrogel scaffolds with open channels designed for post-seeding and perfused culture of primary hepatocytes that form 3D structures in a bioreactor. Photopolymerizable polyethylene glycol-based hydrogels were fabricated coupled to chemically activated, commercially available filters (polycarbonate and polyvinylidene fluoride) using a chemistry that permitted cell viability, and was robust enough to withstand perfused culture of up to 1 µL/s for at least 7 days. SLA energy dose, photoinitiator concentrations, and pretreatment conditions were screened to determine conditions that maximized cell viability and hydrogel bonding to the filter. Multiple open channel geometries were readily achieved, and included ellipses and rectangles. Rectangular open channels employed for subsequent studies had final dimensions on the order of 350 µm by 850 µm. Cell seeding densities and flow rates that promoted cell viability were determined. Perfused culture of primary hepatocytes in hydrogel scaffolds in the presence of soluble epidermal growth factor (EGF) prolonged the maintenance of albumin production throughout the 7-day culture relative to 2D controls. This technique of bonding hydrogel scaffolds can be employed to fabricate soft scaffolds for a number of bioreactor configurations and applications., National Institutes of Health (U.S.). National Center for Advancing Translational Sciences (5UH2TR000496-02), National Science Foundation (U.S.). Emergent Behaviors of Integrated Cellular Systems, National Science Foundation (U.S.). Integrative Graduate Education and Research Traineeship (Grant 0965918), United States. Defense Advanced Research Projects Agency (BAA-11-73 Microphysiological Systems W911NF-12-2-0039), National Science Foundation (U.S.). Graduate Research Fellowship (Grant DGE-1144245), Massachusetts Institute of Technology. Center for Environmental Health Sciences (National Institutes of Health (U.S.) P30-ES002109)

Strategies to combine aspirated marrow cells with scaffolds to treat connective tissue defects are gaining increasing clinical attention and use. In situations such as large defects where initial survival and proliferation of transplanted connective tissue progenitors (CTPs) are limiting, therapeutic outcomes might be improved by using the scaffold to deliver growth factors that promote the early stages of cell function in the graft. Signaling by the epidermal growth factor receptor (EGFR) plays a role in cell survival and has been implicated in bone development and homeostasis. Providing epidermal growth factor (EGF) in a scaffold-tethered format may sustain local delivery and shift EGFR signaling to pro-survival modes compared to soluble ligand. We therefore examined the effect of tethered EGF on osteogenic colony formation from human bone marrow aspirates in the context of three different adhesion environments using a total of 39 donors. We found that tethered EGF, but not soluble EGF, increased the numbers of colonies formed regardless of adhesion background, and that tethered EGF did not impair early stages of osteogenic differentiation., National Institute of General Medical Sciences (U.S.) (Grant NIH RO1 AR42997), National Institute of General Medical Sciences (U.S.) (Grant NIH RO1 AG024980), National Institute of General Medical Sciences (U.S.) (Grant NIH RO1 GM59870), National Institute of General Medical Sciences (U.S.) (Grant NIH DE019523)

Reliable in vitro human disease models that capture the complexity of in vivo tissue behaviors are crucial to gain mechanistic insights into human disease and enable the development of treatments that are effective across broad patient populations. The integration of stem cell technologies, tissue engineering, emerging biomaterials strategies and microfabrication processes, as well as computational and systems biology approaches, is enabling new tools to generate reliable in vitro systems to study the molecular basis of human disease and facilitate drug development. In this review, we discuss these recently developed tools and emphasize opportunities and challenges involved in combining these technologies toward regenerative science., National Institute for Biomedical Imaging and Bioengineering (U.S.) (Grant 5R01EB010246-02), National Center for Advancing Translational Sciences (U.S.) (Grant 1UH2TR000496), United States. Defense Advanced Research Projects Agency (Cooperative Agreement W911NF-12-2-0039)

Introduction Bone marrow mesenchymal stem cells/multipotent stromal cells (MSCs) are recruited to sites of injury and subsequently support regeneration through differentiation or paracrine activity. During periods of stress such as wound site implant or differentiation, MSCs are subjected to a variety of stressors that might activate pathways to improve cell survival and generate energy. In this study, we monitored MSC autophagy in response to the process of differentiation. Methods MSC autophagosome structures were observed by using transmission electron microscopy and a tandem green fluorescent protein-red fluorescent protein autophagic flux reporter to monitor the mammalian microtubule-associated protein-1 light chain 3 (LC3) turnover in real time. MSCs were differentiated by using standard osteogenic and adipogenic media, and autophagy was examined during short-term and long-term differentiation via immunoblots for LC3I and II. Autophagy was modulated during differentiation by using rapamycin and bafilomycin treatments to disrupt the autophagosome balance during the early stages of the differentiation process, and differentiation was monitored in the long term by using Von Kossa and Oil Red O staining as well as quantitative polymerase chain reaction analysis of typical differentiation markers. Results We found that undifferentiated MSCs showed an accumulation of a large number of undegraded autophagic vacuoles, with little autophagic turnover. Stimulation of autophagy with rapamycin led to rapid degradation of these autophagosomes and greatly increased rough endoplasmic reticulum size. Upon induction of osteogenic differentiation, MSC expression of LC3II, a common autophagosome marker, was lost within 12 hours, consistent with increased turnover. However, during adipogenic differentiation, drug treatment to alter the autophagosome balance during early differentiation led to changes in differentiation efficiency, with inhibited adipocyte formation following rapa, National Institute of General Medical Sciences (U.S.) (Grant GM069668), National Institute of Dental and Craniofacial Research (U.S.) (Grant DE019523), National Institute on Aging (Grant AG026389)