类器官；芯片；肠道；脑

类器官作为三维微型器官模型，能够高度模拟体内器官的结构与功能，在疾病建模、药物筛选等领域彰显出巨大潜力。不过，其培养过程中存在的稳定性难题，极大地限制了实际应用。类器官的稳定性体现在两个维度：一方面是培养环境的稳态保持，涉及基质构成、生长因子、机械刺激等外源性要素；另一方面是细胞的内在稳态，包含遗传稳定性、代谢均衡以及细胞异质性等内源性要素。本文全面综述了维持类器官培养环境及内源稳定性的前沿手段，包括工程化基质、血管化技术、标准化流程、菌群平衡等方面，通过批判性评估各技术的优劣提出整合性策略，并对其临床转化前景进行了展望。

类器官装置(Organoid)是一种体外培养的细胞聚集体，可以模拟特定器官的结构和功能。与传统的细胞培养模型相比，类器官装置更为复杂和接近真实生物体，它由来自特定器官的多种类型的细胞组成，并通过合适的支架材料和生长因子提供支持和刺激。作为一种新兴的研究工具，类器官装置在疾病模型研究、药物研发和个性化医学领域具有重要的意义和潜力。随着技术的不断进步和应用的扩大，相信类器官装置将在医学研究和临床实践中发挥越来越重要的作用，为疾病治疗和药物开发带来新的突破。目前，国内外的类器官装置研究取得了显著进展，并涉及多个器官系统。主要的关键技术有自组装、微流控技术和三维打印技术；材料有生物降解支架和仿生材料；使用方法包括细胞分化、多细胞类型组装和功能刺激等。文章主要通过说明类器官装置设计原理，多腔室类器官装置建模以及类器官装置在各个领域的应用，进而总结类器官装置的应用前景和未来可能遇到的挑战，并对未来的发展方向做一个展望。

Before a lead compound goes through a clinical trial, preclinical studies utilize two-dimensional (2D) in vitro models and animal models to study the pharmacodynamics and pharmacokinetics of that lead compound. However, these current preclinical studies may not accurately represent the efficacy and safety of a lead compound in humans, as there has been a high failure rate of drugs that enter clinical trials. All of these failures and the associated costs demonstrate a need for more representative models of human organ systems for screening in the preclinical phase of drug development. In this study, we review the recent advances in in vitro modeling including three-dimensional (3D) organoids, 3D microfabrication, and 3D bioprinting for various organs including the heart, kidney, lung, gastrointestinal tract (intestine-gut-stomach), liver, placenta, adipose, retina, bone, and brain as well as multiorgan models. The availability of organ-on-chip models provides a wealth of opportunities to understand the pathogenesis of human diseases and provide a potentially better model to screen a drug, as these models utilize a dynamic 3D environment similar to the human body. Although there are limitations of organ-on-chip models, the emergence of new technologies have refined their capability for translational research as well as precision medicine.

We are entering an era of medicine where increasingly sophisticated data will be obtained from patients to determine proper diagnosis, predict outcomes and direct therapies. We predict that the most valuable data will be produced by systems that are highly dynamic in both time and space. Three-dimensional (3D) organoids are poised to be such a highly valuable system for a variety of gastrointestinal (GI) diseases. In the lab, organoids have emerged as powerful systems to model molecular and cellular processes orchestrating natural and pathophysiological human tissue formation in remarkable detail. Preclinical studies have impressively demonstrated that these organs-in-a-dish can be used to model immunological, neoplastic, metabolic or infectious GI disorders by taking advantage of patient-derived material. Technological breakthroughs now allow to study cellular communication and molecular mechanisms of interorgan cross-talk in health and disease including communication along for example, the gut-brain axis or gut-liver axis. Despite considerable success in culturing classical 3D organoids from various parts of the GI tract, some challenges remain to develop these systems to best help patients. Novel platforms such as organ-on-a-chip, engineered biomimetic systems including engineered organoids, micromanufacturing, bioprinting and enhanced rigour and reproducibility will open improved avenues for tissue engineering, as well as regenerative and personalised medicine. This review will highlight some of the established methods and also some exciting novel perspectives on organoids in the fields of gastroenterology. At present, this field is poised to move forward and impact many currently intractable GI diseases in the form of novel diagnostics and therapeutics.

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The human gut microbiome has emerged as a key player in the bidirectional communication of the gut-brain axis, affecting various aspects of homeostasis and pathophysiology. Until recently, the majority of studies that seek to explore the mechanisms underlying the microbiome-gut-brain axis cross-talk, relied almost exclusively on animal models, and particularly gnotobiotic mice. Despite the great progress made with these models, various limitations, including ethical considerations and interspecies differences that limit the translatability of data to human systems, pushed researchers to seek for alternatives. Over the past decades, the field of in vitro modelling of tissues has experienced tremendous growth, thanks to advances in 3D cell biology, materials, science and bioengineering, pushing further the borders of our ability to more faithfully emulate the in vivo situation. The discovery of stem cells has offered a new source of cells, while their use in generating gastrointestinal and brain organoids, among other tissues, has enabled the development of novel 3D tissues that better mimic the native tissue structure and function, compared with traditional assays. In parallel, organs-on-chips technology and bioengineered tissues have emerged as highly promising alternatives to animal models for a wide range of applications. Here, we discuss how recent advances and trends in this area can be applied in host-microbe and host-pathogen interaction studies. In addition, we highlight paradigm shifts in engineering more robust human microbiome-gut-brain axis models and their potential to expand our understanding of this complex system and hence explore novel, microbiome-based therapeutic approaches.

Cell culture is an important and necessary process in drug discovery, cancer research, as well as stem cell study. Most cells are currently cultured using two-dimensional (2D) methods but new and improved methods that implement three-dimensional (3D) cell culturing techniques suggest compelling evidence that much more advanced experiments can be performed yielding valuable insights. When performing 3D cell culture experiments, the cell environment can be manipulated to mimic that of a cell <i>in vivo</i> and provide more accurate data about cell-to-cell interactions, tumor characteristics, drug discovery, metabolic profiling, stem cell research, and other types of diseases. Scaffold based techniques such as hydrogel-based support, polymeric hard material-based support, hydrophilic glass fiber, and organoids are employed, and each provide their own advantages and applications. Likewise, there are also scaffold free techniques used such as hanging drop microplates, magnetic levitation, and spheroid microplates with ultra-low attachment coating. 3D cell culture has the potential to provide alternative ways to study organ behavior via the use of organoids and is expected to eventually bridge the gap between 2D cell culture and animal models. The present review compares 2D cell culture to 3D cell culture, provides the details surrounding the different 3D culture techniques, as well as focuses on the present and future applications of 3D cell culture.

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Tremendous progress has been made in the past decade regarding our understanding of the gut microbiome's role in human health. Currently, however, a comprehensive and focused review marrying the two distinct fields of gut microbiome and material research is lacking. To bridge the gap, the current paper discusses critical aspects of the rapidly emerging research topic of "material engineering in the gut microbiome and human health." By engaging scientists with diverse backgrounds in biomaterials, gut-microbiome axis, neuroscience, synthetic biology, tissue engineering, and biosensing in a dialogue, our goal is to accelerate the development of research tools for gut microbiome research and the development of therapeutics that target the gut microbiome. For this purpose, state-of-the-art knowledge is presented here on biomaterial technologies that facilitate the study, analysis, and manipulation of the gut microbiome, including intestinal organoids, gut-on-chip models, hydrogels for spatial mapping of gut microbiome compositions, microbiome biosensors, and oral bacteria delivery systems. In addition, a discussion is provided regarding the microbiome-gut-brain axis and the critical roles that biomaterials can play to investigate and regulate the axis. Lastly, perspectives are provided regarding future directions on how to develop and use novel biomaterials in gut microbiome research, as well as essential regulatory rules in clinical translation. In this way, we hope to inspire research into future biomaterial technologies to advance gut microbiome research and gut microbiome-based theragnostics.

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Organoids are simple tissue-engineered cell-based in vitro models that recapitulate many aspects of the complex structure and function of the corresponding in vivo tissue. They can be dissected and interrogated for fundamental mechanistic studies on development, regeneration and repair in human tissues, and can also be used in diagnostics, disease modelling, drug discovery and personalized medicine. Organoids are derived from either pluripotent or tissue-resident stem (embryonic or adult) or progenitor or differentiated cells from healthy or diseased tissues, such as tumours. To date, numerous organoid engineering strategies that support organoid culture and growth, proliferation, differentiation and maturation have been reported. This Primer highlights the rationale underlying the selection and development of these materials and methods to control the cellular/tissue niche; and therefore, the structure and function of the engineered organoid. We also discuss key considerations for generating robust organoids, such as those related to cell isolation and seeding, matrix and soluble factor selection, physical cues and integration. The general standards for data quality, reproducibility and deposition within the organoid community are also outlined. Lastly, we conclude by elaborating on the limitations of organoids in different applications, and the key priorities in organoid engineering for the coming years.

Here we describe a method for fabricating a primary human Small Intestine-on-a-Chip (Intestine Chip) containing epithelial cells isolated from healthy regions of intestinal biopsies. The primary epithelial cells are expanded as 3D organoids, dissociated, and cultured on a porous membrane within a microfluidic device with human intestinal microvascular endothelium cultured in a parallel microchannel under flow and cyclic deformation. In the Intestine Chip, the epithelium forms villi-like projections lined by polarized epithelial cells that undergo multi-lineage differentiation similar to that of intestinal organoids, however, these cells expose their apical surfaces to an open lumen and interface with endothelium. Transcriptomic analysis also indicates that the Intestine Chip more closely mimics whole human duodenum in vivo when compared to the duodenal organoids used to create the chips. Because fluids flowing through the lumen of the Intestine Chip can be collected continuously, sequential analysis of fluid samples can be used to quantify nutrient digestion, mucus secretion and establishment of intestinal barrier function over a period of multiple days in vitro. The Intestine Chip therefore may be useful as a research tool for applications where normal intestinal function is crucial, including studies of metabolism, nutrition, infection, and drug pharmacokinetics, as well as personalized medicine.

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Organ-on-a-chip systems are miniaturized microfluidic 3D human tissue and organ models designed to recapitulate the important biological and physiological parameters of their in vivo counterparts. They have recently emerged as a viable platform for personalized medicine and drug screening. These in vitro models, featuring biomimetic compositions, architectures, and functions, are expected to replace the conventional planar, static cell cultures and bridge the gap between the currently used preclinical animal models and the human body. Multiple organoid models may be further connected together through the microfluidics in a similar manner in which they are arranged in vivo, providing the capability to analyze multiorgan interactions. Although a wide variety of human organ-on-a-chip models have been created, there are limited efforts on the integration of multisensor systems. However, in situ continual measuring is critical in precise assessment of the microenvironment parameters and the dynamic responses of the organs to pharmaceutical compounds over extended periods of time. In addition, automated and noninvasive capability is strongly desired for long-term monitoring. Here, we report a fully integrated modular physical, biochemical, and optical sensing platform through a fluidics-routing breadboard, which operates organ-on-a-chip units in a continual, dynamic, and automated manner. We believe that this platform technology has paved a potential avenue to promote the performance of current organ-on-a-chip models in drug screening by integrating a multitude of real-time sensors to achieve automated in situ monitoring of biophysical and biochemical parameters.

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The past few decades have shown significant advancement as complex in vitro humanized systems have substituted animal trials and 2D in vitro studies. 3D humanized platforms mimic the organs of interest with their stimulations (physical, electrical, chemical, and mechanical). Organ-on-chip devices, including in vitro modelling of 3D organoids, 3D microfabrication, and 3D bioprinted platforms, play an essential role in drug discovery, testing, and assessment. In this article, a thorough review is provided of the latest advancements in the area of organ-on-chip devices targeting liver, kidney, lung, gut, heart, skin, and brain mimicry devices for drug discovery, development, and/or assessment. The current strategies, fabrication methods, and the specific application of each device, as well as the advantages and disadvantages, are presented for each reported platform. This comprehensive review also provides some insights on the challenges and future perspectives for the further advancement of each organ-on-chip device.

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Changes in the human gastrointestinal microbiome are associated with several diseases. To infer causality, experiments in representative models are essential, but widely used animal models exhibit limitations. Here we present a modular, microfluidics-based model (HuMiX, human-microbial crosstalk), which allows co-culture of human and microbial cells under conditions representative of the gastrointestinal human-microbe interface. We demonstrate the ability of HuMiX to recapitulate in vivo transcriptional, metabolic and immunological responses in human intestinal epithelial cells following their co-culture with the commensal Lactobacillus rhamnosus GG (LGG) grown under anaerobic conditions. In addition, we show that the co-culture of human epithelial cells with the obligate anaerobe Bacteroides caccae and LGG results in a transcriptional response, which is distinct from that of a co-culture solely comprising LGG. HuMiX facilitates investigations of host-microbe molecular interactions and provides insights into a range of fundamental research questions linking the gastrointestinal microbiome to human health and disease.

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Nanoparticles (NPs) have become one of the most popular objects of scientific study during the past decades. However, despite wealth of study reports, still there is a gap, particularly in health toxicology studies, underlying mechanisms, and related evaluation models to deeply understanding the NPs risk effects. In this review, we first present a comprehensive landscape of the applications of NPs on health, especially addressing the role of NPs in medical diagnosis, therapy. Then, the toxicity of NPs on health systems is introduced. We describe in detail the effects of NPs on various systems, including respiratory, nervous, endocrine, immune, and reproductive systems, and the carcinogenicity of NPs. Furthermore, we unravels the underlying mechanisms of NPs including ROS accumulation, mitochondrial damage, inflammatory reaction, apoptosis, DNA damage, cell cycle, and epigenetic regulation. In addition, the classical study models such as cell lines and mice and the emerging models such as 3D organoids used for evaluating the toxicity or scientific study are both introduced. Overall, this review presents a critical summary and evaluation of the state of understanding of NPs, giving readers more better understanding of the NPs toxicology to remedy key gaps in knowledge and techniques.

For the past century, experimental data obtained from animal studies have been required by reviewers of scientific articles and grant applications to validate the physiological relevance of in vitro results. At the same time, pharmaceutical researchers and regulatory agencies recognize that results from preclinical animal models frequently fail to predict drug responses in humans. This <i>Progress Report</i> reviews recent advances in human organ-on-a-chip (Organ Chip) microfluidic culture technology, both with single Organ Chips and fluidically coupled human "Body-on-Chips" platforms, which demonstrate their ability to recapitulate human physiology and disease states, as well as human patient responses to clinically relevant drug pharmacokinetic exposures, with higher fidelity than other in vitro models or animal studies. These findings raise the question of whether continuing to require results of animal testing for publication or grant funding still makes scientific or ethical sense, and if more physiologically relevant human Organ Chip models might better serve this purpose. This issue is addressed in this article in context of the history of the field, and advantages and disadvantages of Organ Chip approaches versus animal models are discussed that should be considered by the wider research community.

Irritable bowel syndrome (IBS) is a functional disorder which affects a large proportion of the population globally. The precise etiology of IBS is still unknown, although consensus understanding proposes IBS to be of multifactorial origin with yet undefined subtypes. Genetic and epigenetic factors, stress-related nervous and endocrine systems, immune dysregulation and the brain-gut axis seem to be contributing factors that predispose individuals to IBS. In addition to food hypersensitivity, toxins and adverse life events, chronic infections and dysbiotic gut microbiota have been suggested to trigger IBS symptoms in tandem with the predisposing factors. This review will summarize the pathophysiology of IBS and the role of gut microbiota in relation to IBS. Current methodologies for microbiome studies in IBS such as genome sequencing, metagenomics, culturomics and animal models will be discussed. The myriad of therapy options such as immunoglobulins (immune-based therapy), probiotics and prebiotics, dietary modifications including FODMAP restriction diet and gluten-free diet, as well as fecal transplantation will be reviewed. Finally this review will highlight future directions in IBS therapy research, including identification of new molecular targets, application of 3-D gut model, gut-on-a-chip and personalized therapy.

Abstract Organoids are three‐dimensional (3D) miniaturized versions of organs or tissues that are derived from cells with stem potential and can self‐organize and differentiate into 3D cell masses, recapitulating the morphology and functions of their in vivo counterparts. Organoid culture is an emerging 3D culture technology, and organoids derived from various organs and tissues, such as the brain, lung, heart, liver, and kidney, have been generated. Compared with traditional bidimensional culture, organoid culture systems have the unique advantage of conserving parental gene expression and mutation characteristics, as well as long‐term maintenance of the function and biological characteristics of the parental cells in vitro. All these features of organoids open up new opportunities for drug discovery, large‐scale drug screening, and precision medicine. Another major application of organoids is disease modeling, and especially various hereditary diseases that are difficult to model in vitro have been modeled with organoids by combining genome editing technologies. Herein, we introduce the development and current advances in the organoid technology field. We focus on the applications of organoids in basic biology and clinical research, and also highlight their limitations and future perspectives. We hope that this review can provide a valuable reference for the developments and applications of organoids.

The gut communicates with the brain in a variety of ways known as the gut-brain axis (GBA), which is known to affect neurophysiological functions as well as neuronal disorders. Exosomes capable of passing through the blood-brain-barrier (BBB) have received attention as a mediator of gut-brain signaling and drug delivery vehicles. In conventional well plate-based experiments, it is difficult to observe the exosome movement in real time. Here, we developed a microfluidic-based GBA chip for co-culturing gut epithelial cells and neuronal cells and simultaneously observing exosome transport. The GBA-chip is aimed to mimic the <i>in vivo</i> situation of convective flow in blood vessels and convective and diffusive transport in the tissue interstitium. Here, fluorescence-labeled exosome was produced by transfection of HEK-293T cells with CD63-GFP plasmid. We observed in real time the secretion of CD63-GFP-exosomes by the transfected HEK-293T cells in the chip, and transport of the exosomes to neuronal cells and analyzed the dynamics of GFP-exosome movement. Our model is expected to enhance understanding of the roles of exosome in GBA.

In this work, we developed a microfluidic device that can recapitulate the anatomical layout of vagal afferent neurons in vitro. We demonstrated two physiologically-relevant applications of the platforms: retrograde transport and electrophysiological response. We expect this tool to enable controlled studies on the role of vagal afferent neurons in the gut-brain axis.

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Inflammatory bowel diseases (IBD) are complex chronic inflammatory disorders of the gastrointestinal (GI) tract. Recent evidence suggests that the gut-brain axis may be pivotal in gastrointestinal and neurological diseases, especially IBD. Here, we present the first proof of concept for a microfluidic technology to model bilateral neuro-immunological communication. We designed a device composed of three compartments with an asymmetric channel that allows the isolation of soma and neurites thanks to microchannels and creates an in vitro synaptic compartment. Human-induced pluripotent stem cell-derived cortical glutamatergic neurons were maintained in soma compartments for up to 21 days. We performed a localized addition of dendritic cells (MoDCs) to either the soma or synaptic compartment. The microfluidic device was coupled with microelectrode arrays (MEAs) to assess the impact on the electrophysiological activity of neurons while adding dendritic cells. Our data highlight that an electrophysiologic signal is transmitted between two compartments of glutamatergic neurons linked by synapses in a bottom-up way when soma is exposed to primed dendritic cells. In conclusion, our study authenticates communication between dendritic cells and neurons in inflammatory conditions such as IBD. This platform opens the way to complexification with gut components to reach a device for pharmacological compound screening by blocking the gut-brain axis at a mucosal level and may help patients.

The oxygen gradient across the intestine influences intestinal physiology and the microbial environment of the microbiome. The microbiome releases metabolites that communicate with enterochromaffin cells, neuronal cells, and resident immune cells to facilitate the bidirectional communication across the gut-brain axis. Measuring communication between various cell types within the intestine could provide essential information about key regulators of gut and brain health; however, the microbial environment of the intestine is heavily dependent on the physiological oxygen gradient that exists across the intestinal wall. Likewise, there exist a need for methods which enable real-time monitoring of intestinal signaling <i>ex vivo</i> yet this remains challenging due to the inability to adequately culture intestinal tissue <i>ex vivo</i> while also exposing the appropriate locations of the intestine for probe insertion and monitoring. Here, we designed and fabricated a 3D printed microfluidic device to maintain the oxygen gradient across precision cut murine intestinal slices with the capability to couple to external neurochemical recording techniques. The gradient is maintained from outlets below while allowing access to the slice from above for detection with fast scan cyclic voltammetry (FSCV) and carbon-fiber microelectrodes. A series of 11 outlet ports were designed to lay underneath the slice which were connected to channels to deliver oxygenated <i>vs.</i> deoxygenated media. Outlet ports were designed in an oval shape where deoxygenated media was delivered to the center of the slice and oxygenated media is delivered to the outer portion of the slice to mimic the location of oxygen across the intestine. An oxygen sensitive fluorescent dye, tris(2,2'-bipyridyl)dichlororuthenium(II), was used to characterize the tunability of the gradient. Viability of the tissue was confirmed by both fluorescence microscopy and FSCV. Additionally, we measured simultaneous serotonin and melatonin signaling with FSCV in the intestine for the first time. Overall, this chip provides a significant advance in our ability to culture intestinal slices <i>ex vivo</i> with the added benefit of direct access for measurements and imaging.

Systemic absorption and metabolism of drugs in the small intestine, metabolism by the liver as well as excretion by the kidney are key determinants of efficacy and safety for therapeutic candidates. However, these systemic responses of applied substances lack in most in vitro assays. In this study, a microphysiological system maintaining the functionality of four organs over 28 days in co-culture has been established at a minute but standardized microsystem scale. Preformed human intestine and skin models have been integrated into the four-organ-chip on standard cell culture inserts at a size 100,000-fold smaller than their human counterpart organs. A 3D-based spheroid, equivalent to ten liver lobules, mimics liver function. Finally, a barrier segregating the media flow through the organs from fluids excreted by the kidney has been generated by a polymeric membrane covered by a monolayer of human proximal tubule epithelial cells. A peristaltic on-chip micropump ensures pulsatile media flow interconnecting the four tissue culture compartments through microfluidic channels. A second microfluidic circuit ensures drainage of the fluid excreted through the kidney epithelial cell layer. This four-organ-chip system assures near to physiological fluid-to-tissue ratios. In-depth metabolic and gene analysis revealed the establishment of reproducible homeostasis among the co-cultures within two to four days, sustainable over at least 28 days independent of the individual human cell line or tissue donor background used for each organ equivalent. Lastly, 3D imaging two-photon microscopy visualised details of spatiotemporal segregation of the two microfluidic flows by proximal tubule epithelia. To our knowledge, this study is the first approach to establish a system for in vitro microfluidic ADME profiling and repeated dose systemic toxicity testing of drug candidates over 28 days.

This study introduces an innovative nanophotonic biosensor system designed to explore exosome dynamics within the gut-brain axis, highlighting the bidirectional and biochemical communication between the gastrointestinal tract and the central nervous system. The proposed system incorporates coculture environments for various cell types, microfluidic control of exosomes, and super-resolution imaging capabilities for both exosomes and live cells. While enabling real-time observation of long-range exosome dynamics within the gut-brain-axis-on-a-chip, this approach offers superior spatial resolution for visualizing individual exosomes in both donor and recipient cells. Through precise microfluidic manipulation, exosomes are observed as they are secreted from donor cells, transported within the chip, and interact with recipient cells in a coculture environment, mimicking the communication process occurring in the gut-brain axis. The dynamics of exosome transport within the gut-brain axis model are expected to improve the understanding of their biological functions and potential applications.

The gastrointestinal tract, in particular the gut, is composed of several roughly tubular-shapedorgans (e.g., small intestine, colon), each with a unique function, luminal (i.e., innermost cavity) microbiome, and multiple layers with complex cellular interactions (e.g., enteric nervous system (ENS)). Advancements in understanding the role of the gut microbiome (found in the lumen) and the gut-brain axis in local (e.g., intestinal bowel disease) and systemic disease (e.g., metabolic disease) constitutes an exciting avenue for new therapies. In vitro disease modeling - or laboratory based recapitulation of illness with cultured cells - offers an approach to test at scale for new treatment options. Microfluidic platforms (devices with micrometer-scale channels to house cells) offer an exciting improvement over traditional in vitro modeling through the option of increased complexity and scalability due to their tailored engineering. This work details the development and characterization of two disease models: a traditional in vitro approach of Parkinson's disease (PD) in the ENS and a microfluidic chip modeling acute bacterial toleration by ileal epithelial populations. PD represents the second most prevalent neurodegenerative disease. It is believed to occur via prion-like spread and aggregation of toxic alpha-synuclein (a-Syn) fibrils. As symptom onset correlates with loss of neurons, research in identifying and treating the source of initial a-Syn fibrils has led research to the gut. Within PD gut-pathogenesis, bacterial infection has been identified as one of several factors potentially inducing initial disease pathology. Development of in vitro and microfluidic disease models may offer mechanistic insight and enable future work targeting therapeutic or prophylactic interventions. Here, we assess primary rat enteric neuron responses to the preformed fibril (PFF) PD disease model with modulatory assessments of the bacterial products lipopolysaccharide (LPS) and butyrate to model infection and probiotic effects respectively. Additionally, we model acute bacterial tolerance of the epithelial barrier and evaluate differences in physiological responses. To date, limited work has assessed enteric neuron PD responses. We utilized live-imaging,immunostaining, and image analysis to quantify differences in PFF retention (via fluorescent tag) and a-Syn aggregation antibody average intensity between cultures. PFF dosage was uptaken and retained during the culture period in enteric neurons by PFF fluorescent-tag intensity and reduced by butyrate administration. Cortical and enteric neurons responded to PFF dosage with increased aggregate conformation-specific a-Syn average intensity within the neural morphology region. LPS dosage resulted in increased enteric neuron average a-Syn intensity. Enteric neuron growth cones dosed with PFFs exhibited significantly increased filopodia and filopodia activity. Filopodia count normalized activity was unaffected, indicating changes to growth cone morphology, but not dynamics. Spontaneous action potential (spike) counts normalized to 5 minutes of recording (measured via microelectrode array) were not significantly altered by PFF dosage. However, dopamine stimulated (PD-relevant) spike counts were significantly increased in untreated controls, but not PFF dosed cultures. Next, we designed, tested, and characterized host-bacterial interaction using the cut-and-assemblemicrofluidic platform. Long-term modeling of intestinal host-bacterial interactions in vitro to date have been limited to microfluidic chips under perfusion, although limited work has evaluated differences in bacterial tolerance between flow and traditional static cultures or cell line and primary cells. Improved modeling of bacterial toleration and invasion offers potential tie-in value to model theoretical PD pathogenesis. We observe that acute (48 hour) bacterial tolerance is established from perfusion, and that primary cells overall tolerate bacterial co-culture. Contrarily, traditional cell line static cultures are mostly lost before 48 hours of co-culture. These cultures experience rapid apical bacterial expansion within 14 hours, associated with significantly increased malondialdehyde (MDA) lipid oxidative damage at 24 hours. Comparatively, flow and primary cultures have significantly less MDA. Finally, primary culture secreted mucin production in is substantially elevated compared to cell lines (median ~50X), pointing to one potential mechanism for acute tolerance. This work has relevance for future microfluidic study of Parkinson's disease on chip, where ahumanized microfluidic co-culture may enable modeling of bacterial infection and PD pathogenesis on chip.--Author's abstract

The recent advent of microphysiological systems - microfluidic biomimetic devices that aspire to emulate the biology of human tissues, organs and circulation in vitro - is envisaged to enable a global paradigm shift in drug development. An extraordinary US governmental initiative and various dedicated research programs in Europe and Asia have led recently to the first cutting-edge achievements of human single-organ and multi-organ engineering based on microphysiological systems. The expectation is that test systems established on this basis would model various disease stages, and predict toxicity, immunogenicity, ADME profiles and treatment efficacy prior to clinical testing. Consequently, this technology could significantly affect the way drug substances are developed in the future. Furthermore, microphysiological system-based assays may revolutionize our current global programs of prioritization of hazard characterization for any new substances to be used, for example, in agriculture, food, ecosystems or cosmetics, thus, replacing laboratory animal models used currently. Thirty-six experts from academia, industry and regulatory bodies present here the results of an intensive workshop (held in June 2015, Berlin, Germany). They review the status quo of microphysiological systems available today against industry needs, and assess the broad variety of approaches with fit-for-purpose potential in the drug development cycle. Feasible technical solutions to reach the next levels of human biology in vitro are proposed. Furthermore, key organ-on-a-chip case studies, as well as various national and international programs are highlighted. Finally, a roadmap into the future is outlined, to allow for more predictive and regulatory-accepted substance testing on a global scale.

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Possible strategy to integrate pre-vascularized organoid and <italic>in vitro</italic> capillary bed on a microfluidic based platform, aiming for establishing perfused vasculature throughout organoids <italic>in vitro</italic>.

Research in cell biology greatly relies on cell-based <i>in vitro</i> assays and models that facilitate the investigation and understanding of specific biological events and processes under different conditions. The quality of such experimental models and particularly the level at which they represent cell behavior in the native tissue, is of critical importance for our understanding of cell interactions within tissues and organs. Conventionally, <i>in vitro</i> models are based on experimental manipulation of mammalian cells, grown as monolayers on flat, two-dimensional (2D) substrates. Despite the amazing progress and discoveries achieved with flat biology models, our ability to translate biological insights has been limited, since the 2D environment does not reflect the physiological behavior of cells in real tissues. Advances in 3D cell biology and engineering have led to the development of a new generation of cell culture formats that can better recapitulate the <i>in vivo</i> microenvironment, allowing us to examine cells and their interactions in a more biomimetic context. Modern biomedical research has at its disposal novel technological approaches that promote development of more sophisticated and robust tissue engineering <i>in vitro</i> models, including scaffold- or hydrogel-based formats, organotypic cultures, and organs-on-chips. Even though such systems are necessarily simplified to capture a particular range of physiology, their ability to model specific processes of human biology is greatly valued for their potential to close the gap between conventional animal studies and human (patho-) physiology. Here, we review recent advances in 3D biomimetic cultures, focusing on the technological bricks available to develop more physiologically relevant <i>in vitro</i> models of human tissues. By highlighting applications and examples of several physiological and disease models, we identify the limitations and challenges which the field needs to address in order to more effectively incorporate synthetic biomimetic culture platforms into biomedical research.

Neurodegenerative diseases are progressive degenerative conditions characterized by the functional deterioration and ultimate loss of neurons. These incurable and debilitating diseases affect millions of people worldwide, and therefore represent a major global health challenge with severe implications for individuals and society. Recently, several neuroprotective drugs have failed in human clinical trials despite promising pre-clinical data, suggesting that conventional cell cultures and animal models cannot precisely replicate human pathophysiology. To bridge the gap between animal and human studies, three-dimensional cell culture models have been developed from human or animal cells, allowing the effects of new therapies to be predicted more accurately by closely replicating some aspects of the brain environment, mimicking neuronal and glial cell interactions, and incorporating the effects of blood flow. In this review, we discuss the relative merits of different cerebral models, from traditional cell cultures to the latest high-throughput three-dimensional systems. We discuss their advantages and disadvantages as well as their potential to investigate the complex mechanisms of human neurodegenerative diseases. We focus on <i>in vitro</i> models of the most frequent age-related neurodegenerative disorders, such as Parkinson's disease, Alzheimer's disease and prion disease, and on multiple sclerosis, a chronic inflammatory neurodegenerative disease affecting young adults.

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Organ-on-chip (OOC) systems recapitulate key biological processes and responses <i>in vitro</i> exhibited by cells, tissues, and organs <i>in vivo</i>. Accordingly, these models of both health and disease hold great promise for improving fundamental research, drug development, personalized medicine, and testing of pharmaceuticals, food substances, pollutants etc. Cells within the body are exposed to biomechanical stimuli, the nature of which is tissue specific and may change with disease or injury. These biomechanical stimuli regulate cell behavior and can amplify, annul, or even reverse the response to a given biochemical cue or drug candidate. As such, the application of an appropriate physiological or pathological biomechanical environment is essential for the successful recapitulation of <i>in vivo</i> behavior in OOC models. Here we review the current range of commercially available OOC platforms which incorporate active biomechanical stimulation. We highlight recent findings demonstrating the importance of including mechanical stimuli in models used for drug development and outline emerging factors which regulate the cellular response to the biomechanical environment. We explore the incorporation of mechanical stimuli in different organ models and identify areas where further research and development is required. Challenges associated with the integration of mechanics alongside other OOC requirements including scaling to increase throughput and diagnostic imaging are discussed. In summary, compelling evidence demonstrates that the incorporation of biomechanical stimuli in these OOC or microphysiological systems is key to fully replicating <i>in vivo</i> physiology in health and disease.

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The pharmaceutical industry is continuing to face high research and development (R&D) costs and low overall success rates of clinical compounds during drug development. There is an increasing demand for development and validation of healthy or disease-relevant and physiological human cellular models that can be implemented in early-stage discovery, thereby shifting attrition of future therapeutics to a point in discovery at which the costs are significantly lower. There needs to be a paradigm shift in the early drug discovery phase (which is lengthy and costly), away from simplistic cellular models that show an inability to effectively and efficiently reproduce healthy or human disease-relevant states to steer target and compound selection for safety, pharmacology, and efficacy questions. This perspective article covers the various stages of early drug discovery from target identification (ID) and validation to the hit/lead discovery phase, lead optimization, and preclinical safety. We outline key aspects that should be considered when developing, qualifying, and implementing complex in vitro models (CIVMs) during these phases, because criteria such as cell types (e.g., cell lines, primary cells, stem cells, and tissue), platform (e.g., spheroids, scaffolds or hydrogels, organoids, microphysiological systems, and bioprinting), throughput, automation, and single and multiplexing endpoints will vary. The article emphasizes the need to adequately qualify these CIVMs such that they are suitable for various applications (e.g., context of use) of drug discovery and translational research. The article ends looking to the future, in which there is an increase in combining computational modeling, artificial intelligence and machine learning (AI/ML), and CIVMs.

The last few decades have witnessed diversified in vitro models to recapitulate the architecture and function of living organs or tissues and contribute immensely to advances in life science. Two novel 3D cell culture models: 1) Organoid, promoted mainly by the developments of stem cell biology and 2) Organ-on-a-chip, enhanced primarily due to microfluidic technology, have emerged as two promising approaches to advance the understanding of basic biological principles and clinical treatments. This review describes the comparable distinct differences between these two models and provides more insights into their complementarity and integration to recognize their merits and limitations for applicable fields. The convergence of the two approaches to produce multi-organoid-on-a-chip or human organoid-on-a-chip is emerging as a new approach for building 3D models with higher physiological relevance. Furthermore, rapid advancements in 3D printing and numerical simulations, which facilitate the design, manufacture, and results-translation of 3D cell culture models, can also serve as novel tools to promote the development and propagation of organoid and organ-on-a-chip systems. Current technological challenges and limitations, as well as expert recommendations and future solutions to address the promising combinations by incorporating organoids, organ-on-a-chip, 3D printing, and numerical simulation, are also summarized.

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The number of successful drug development projects has been stagnant for decades despite major breakthroughs in chemistry, molecular biology, and genetics. Unreliable target identification and poor translatability of preclinical models have been identified as major causes of failure. To improve predictions of clinical efficacy and safety, interest has shifted to three-dimensional culture methods in which human cells can retain many physiologically and functionally relevant phenotypes for extended periods of time. Here, we review the state of the art of available organotypic culture techniques and critically review emerging models of human tissues with key importance for pharmacokinetics, pharmacodynamics, and toxicity. In addition, developments in bioprinting and microfluidic multiorgan cultures to emulate systemic drug disposition are summarized. We close by highlighting important trends regarding the fabrication of organotypic culture platforms and the choice of platform material to limit drug absorption and polymer leaching while supporting the phenotypic maintenance of cultured cells and allowing for scalable device fabrication. We conclude that organotypic and microphysiological human tissue models constitute promising systems to promote drug discovery and development by facilitating drug target identification and improving the preclinical evaluation of drug toxicity and pharmacokinetics. There is, however, a critical need for further validation, benchmarking, and consolidation efforts ideally conducted in intersectoral multicenter settings to accelerate acceptance of these novel models as reliable tools for translational pharmacology and toxicology. SIGNIFICANCE STATEMENT: Organotypic and microphysiological culture of human cells has emerged as a promising tool for preclinical drug discovery and development that might be able to narrow the translation gap. This review discusses recent technological and methodological advancements and the use of these systems for hit discovery and the evaluation of toxicity, clearance, and absorption of lead compounds.

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We describe a human "Body-on-a-chip" device (or microphysiological system) that could be used to emulate drug distribution, metabolism, and action in the body. It is based upon a physiologically based pharmacokinetic-pharmacodynamic (PBPK-PD) model, where multiple chambers representing different organs are connected with fluidic channels to mimic multi-organ interactions within the body. Here we describe a pumpless 14 chamber (13 organs) microfluidic cell culture device that provides a separation between barrier and nonbarrier types of cell cultures. Our barrier chamber layer (skin, GI tract, and lung) allows for direct access and/or exposures to chemical or biological reagents forcing these reagents to pass through a barrier of cells established on a microfabricated membrane before exposing the nonbarrier tissue chambers (fat, kidney, heart, adrenal glands, liver, spleen, pancreas, bone marrow, brain, muscle) or entering the microfluidic circulation within the device. Our nonbarrier tissue chambers were created as three-dimensional configurations by resuspending cells in hydrogel (PGMatrix). We used cell lines to represent five of these organs (barrier lines-A549 [lung] and Caco2 [GI]) (nonbarrier lines-HepG2 C3A [liver], Meg01 [bone marrow], and HK2 [kidney]). The dimensions of our straight duct-like channels to each organ chamber were designed to provide the appropriate flow of a culture medium. The organ volumes and organ flow rates that have been reported for an average human male were used to estimate the desired fluid retention times in each organ chamber. The flow through the channels was induced by gravity on a custom programmed rocker platform which enabled pumpless operation and minimized bubble entrapment. The purpose of this paper is to describe the design and operation of a 14 chamber multi-organ system representing 13 tissues/organs with both barrier and nonbarrier tissue chambers and to study the interactive responses among the various cell lines. We demonstrate that five different cell lines survived with high viability (above 85%) for 7 days. We compared the individual observed flow rates to the compartments to the desired or estimated flow rates. This work demonstrates the feasibility of constructing, operating and maintaining a simple, gravity-driven, multi-organ microphysiological system with the capability of measuring cellular functions such as CYP1A1 and CYP3A4 activities, albumin release, urea, maintenance of tight junctions, and presence of surfactant for a sustained period. Biotechnol. Bioeng. 2016;113: 2213-2227. © 2016 Wiley Periodicals, Inc.

Drug development is a lengthy and costly process that proceeds through several stages from target identification to lead discovery and optimization, preclinical validation and clinical trials culminating in approval for clinical use. An important step in this process is high-throughput screening (HTS) of small compound libraries for lead identification. Currently, the majority of cell-based HTS is being carried out on cultured cells propagated in two-dimensions (2D) on plastic surfaces optimized for tissue culture. At the same time, compelling evidence suggests that cells cultured in these non-physiological conditions are not representative of cells residing in the complex microenvironment of a tissue. This discrepancy is thought to be a significant contributor to the high failure rate in drug discovery, where only a low percentage of drugs investigated ever make it through the gamut of testing and approval to the market. Thus, three-dimensional (3D) cell culture technologies that more closely resemble <i>in vivo</i> cell environments are now being pursued with intensity as they are expected to accommodate better precision in drug discovery. Here we will review common approaches to 3D culture, discuss the significance of 3D cultures in drug resistance and drug repositioning and address some of the challenges of applying 3D cell cultures to high-throughput drug discovery.

Microvascular networks support metabolic activity and define microenvironmental conditions within tissues in health and pathology. Recapitulation of functional microvascular structures in vitro could provide a platform for the study of complex vascular phenomena, including angiogenesis and thrombosis. We have engineered living microvascular networks in three-dimensional tissue scaffolds and demonstrated their biofunctionality in vitro. We describe the lithographic technique used to form endothelialized microfluidic vessels within a native collagen matrix; we characterize the morphology, mass transfer processes, and long-term stability of the endothelium; we elucidate the angiogenic activities of the endothelia and differential interactions with perivascular cells seeded in the collagen bulk; and we demonstrate the nonthrombotic nature of the vascular endothelium and its transition to a prothrombotic state during an inflammatory response. The success of these microvascular networks in recapitulating these phenomena points to the broad potential of this platform for the study of cardiovascular biology and pathophysiology.

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Organoids are prototypes of human organs derived from cultured human stem cells. They provide a reliable and accurate experimental model to study the physical mechanisms underlying the early developmental stages of human organs morphogenesis and, in particular, the early morphogenesis of the cortex. Here, we propose a mathematical model to elucidate the role played by two mechanisms which have been experimentally proven to be crucial in shaping human brain organoids: the contraction of the inner core of the organoid and the microstructural remodeling of the outer cortex. Our results show that both mechanisms are crucial for the final shape of the organoid and can explain the origin of brain pathologies such as lissencephaly (smooth brain).

Recent advances in brain organoid technology are exciting new ways, which have the potential to change the way how doctors and researchers understand and treat cerebral diseases. Despite the remarkable use of brain organoids derived from human stem cells in new drug testing, disease modeling, and scientific research, it is still heavily time-consuming work to observe and analyze the internal structure, cells, and neural inside the organoid by humans, specifically no standard quantitative analysis method combined growing AI technology for brain organoid. In this paper, an automated computer-assisted analysis method is proposed for brain organoid slice channels tagged with different fluorescent. We applied the method on two channels of two group microscopy images and the experiment result shows an obvious difference between Wild Type and Mutant Type cerebral organoids.

Recent advances have enabled the study of human brain development using brain organoids derived from stem cells. Quantifying cellular processes like mitosis in these organoids offers insights into neurodevelopmental disorders, but the manual analysis is time-consuming, and existing datasets lack specific details for brain organoid studies. We introduce BOrg, a dataset designed to study mitotic events in the embryonic development of the brain using confocal microscopy images of brain organoids. BOrg utilizes an efficient annotation pipeline with sparse point annotations and techniques that minimize expert effort, overcoming limitations of standard deep learning approaches on sparse data. We adapt and benchmark state-of-the-art object detection and cell counting models on BOrg for detecting and analyzing mitotic cells across prophase, metaphase, anaphase, and telophase stages. Our results demonstrate these adapted models significantly improve mitosis analysis efficiency and accuracy for brain organoid research compared to existing methods. BOrg facilitates the development of automated tools to quantify statistics like mitosis rates, aiding mechanistic studies of neurodevelopmental processes and disorders. Data and code are available at https://github.com/awaisrauf/borg.

Understanding the mechanics of brain embryogenesis can provide insights on pathologies related to brain development, such as lissencephaly, a genetic disease which cause a reduction of the number of cerebral sulci. Recent experiments on brain organoids have confirmed that gyrification, i.e. the formation of the folded structures of the brain, is triggered by the inhomo-geneous growth of the peripheral region. However, the rheology of these cellular aggregates and the mechanics of lissencephaly are still matter of debate. In this work, we develop a mathematical model of brain organoids based on the theory of morpho-elasticity. We describe them as non-linear elastic bodies, composed of a disk surrounded by a growing layer called cortex. The external boundary is subjected to a tissue surface tension due the intercellular adhesion forces. We show that the resulting surface energy is relevant at the small length scales of brain organoids and significantly affects the mechanics of cellular aggregates. We perform a linear stability analysis of the radially symmetric configuration and we study the post-buckling behaviour through finite element simulations. We find that the process of gyrification is triggered by the cortex growth and modulated by the competition between two length scales: the radius of the organoid and the capillary length due to surface tension. We show that a solid model can reproduce the results of the in-vitro experiments. Furthermore, we prove that the lack of brain sulci in lissencephaly is caused by a reduction of the cell stiffness: the softening of the organoid strengthens the role of surface tension, delaying or even inhibiting the onset of a mechanical instability at the free boundary.

We present an analytical and numerical investigation of the activity-induced hydrodynamic instabilities in model brain organoids. While several mechanisms have been introduced to explain the experimental observation of surface instabilities in brain organoids, the role of activity has been largely overlooked. Our results show that the active stress generated by the cells can be a, previously overlooked, contributor to the emergence of surface deformations in brain organoids.

Brain organoids recapitulate a number of brain properties, including neuronal diversity. However, do they recapitulate brain structure? Using a hydrodynamic description for cell nuclei as particles interacting initially via an effective, attractive force as mediated by the respective, surrounding cytoskeletons, we quantify structure development in brain organoids to determine what physical mechanism regulates the number of cortex-core structures. Regions of cell nuclei overdensity in the linear regime drive the initial seeding for cortex-core structures, which ultimately develop in the non-linear regime, as inferred by the emergent form of an effective interaction between cell nuclei and with the extracellular environment, as mediated by a dynamic cytoskeleton. Individual cortex-core structures then provide a basis upon which we build an extended version of the buckling without bending morphogenesis (BWBM) model, with its proliferating cortex and constraining core, to predict foliations/folds of the cortex in the presence of a nonlinearity due to cortical cells actively regulating strain. In doing so, we obtain asymmetric foliations/folds with respect to the trough (sulci) and the crest (gyri). In addition to laying new groundwork for the design of more familiar and less familiar brain structures, the hydrodynamic description for cell nuclei during the initial stages of brain organoid development provides an intriguing quantitative connection with large-scale structure formation in the universe.

Stem cell-derived organoids are a promising tool to model native human tissues as they resemble human organs functionally and structurally compared to traditional monolayer cell-based assays. For instance, colon organoids can spontaneously develop crypt-like structures similar to those found in the native colon. While analyzing the structural development of organoids can be a valuable readout, using traditional image analysis tools makes it challenging because of the heterogeneities and the abstract nature of organoid morphologies. To address this limitation, we developed and validated a deep learning-based image analysis tool, named D-CryptO, for the classification of organoid morphology. D-CryptO can automatically assess the crypt formation and opacity of colorectal organoids from brightfield images to determine the extent of organoid structural maturity. To validate this tool, changes in organoid morphology were analyzed during organoid passaging and short-term forskolin stimulation. To further demonstrate the potential of D-CryptO for drug testing, organoid structures were analyzed following treatments with a panel of chemotherapeutic drugs. With D-CryptO, subtle variations in how colon organoids responded to the different chemotherapeutic drugs were detected, which suggest potentially distinct mechanisms of action. This tool could be expanded to other organoid types, like intestinal organoids, to facilitate 3D tissue morphological analysis.

Fourier ptychographic microscopy (FPM) is a promising quantitative phase imaging technique that enables high-resolution, label-free imaging over a large field-of-view. Here, we present the first application of FPM for the quantitative analysis of human brain organoid slices, providing a powerful, cost-effective, and label-free enhancement to the current gold-standard fluorescence microscopy. Brain organoids, prepared as thin (5 micrometer) slices, were imaged with a custom-built FPM system consisting of a standard light microscope (4x, 0.2 NA objective) and a 7x7 LED array. This configuration achieved a synthetic numerical aperture of 0.54 and a spatial resolution of approximately 488 nm across an area of 2.077 x 3.65 mm. Fluorescence microscopy was used in parallel for neurons, astrocytes, and nuclei labeling, providing rich fluorescence imaging. Moreover, we designed an automated method to merge classical resolution fluorescence images to visualize the whole brain organoid and align it with the numerically increased space-bandwidth product FPM image. The provided alignment method enables rich phase-fluorescence correlative imaging. Based on the segmentation performed on the stitched fluorescence images, we devised a quantitative phase analysis revealing a higher mean optical thickness of the nuclei versus astrocytes and neurons. Notably, nuclei located in neurogenic regions consistently exhibited significantly higher phase values (optical path difference) compared to nuclei elsewhere, suggesting cell-type-specific biophysical signatures. The label-free, quantitative, and high-throughput capabilities of the FPM approach demonstrated here make it a powerful and accessible tool for future structural and functional studies of whole-section brain organoid development and disease modeling studies.

Tumor organoid-on-a-chip platforms represent a cutting-edge fusion of patient-derived organoids with microfluidic technologies, offering unprecedented capabilities for personalized cancer research. These systems overcome limitations of conventional models by enabling precise control over the tumor microenvironment, including nutrient gradients, fluid flow, and immune interactions. Tumor organoids recapitulate patient-specific tumor heterogeneity and genetic landscapes, while microfluidic chips provide dynamic perfusion and mechanical stimuli, enhancing physiological relevance. Together, they facilitate advanced applications such as high-throughput drug screening, immunotherapy testing, and metastasis modeling, showing superior predictive power for clinical outcomes. Despite challenges in standardization, scalability, and integration of complex tumor components, ongoing advances in hydrogel engineering, automation, and artificial intelligence are poised to accelerate their clinical translation. This review highlights current technologies, applications, and future directions of tumor organoid-on-a-chip systems, emphasizing their transformative potential in precision oncology.

In biological and medical research, scientists now routinely acquire microscopy images of hundreds of morphologically heterogeneous organoids and are then faced with the task of finding patterns in the image collection, i.e., subsets of organoids that appear similar and potentially represent the same morphological class. We adopt models and algorithms for correlating organoid images, i.e., for quantifying the similarity in appearance and geometry of the organoids they depict, and for clustering organoid images by consolidating conflicting correlations. For correlating organoid images, we adopt and compare two alternatives, a partial quadratic assignment problem and a twin network. For clustering organoid images, we employ the correlation clustering problem. Empirically, we learn the parameters of these models, infer a clustering of organoid images, and quantify the accuracy of the inferred clusters, with respect to a training set and a test set we contribute of state-of-the-art light microscopy images of organoids clustered manually by biologists.

Recent numerical analyses to optimize the design of microfluidic devices for more effective entrapment or segregation of surrogate circulating tumor cells (CTCs) from healthy cells have been reported in the literature without concurrently accommodating the non-Newtonian nature of the body fluid and the non-uniform geometric shapes of the CTCs. Through a series of two-dimensional proof-of-concept simulations with increased levels of complexity (e.g., number of particles, inline obstacles), we investigated the validity of the assumptions of the Newtonian fluid behavior for pseudoplastic fluids and the circular particle shape for different-shaped particles (DSPs) in the context of microfluidics-facilitated shape-based segregation of particles. Simulations with a single DSP revealed that even in the absence of internal geometric complexities of a microfluidics channel, the aforementioned assumptions led to 0.11-0.21W (W is the channel length) errors in lateral displacements of DSPs, up to 3-20% errors in their velocities, and 3-5% errors in their travel times. When these assumptions were applied in simulations involving multiple DSPs in inertial microfluidics with inline obstacles, errors in the lateral displacements of DSPs were as high as 0.78W and in their travel times up to 23%, which led to different (un)symmetric flow and segregation patterns of DSPs. Thus, the fluid type and particle shape should be included in numerical models and experiments to assess the performance of microfluidics for targeted cell (e.g., CTCs) harvesting.

We give a general, physical description of ``induced-charge electro-osmosis'' (ICEO), the nonlinear electrokinetic slip at a polarizable surface, in the context of some new techniques for microfluidic pumping and mixing. ICEO generalizes ``AC electro-osmosis'' at micro-electrode arrays to various dielectric and conducting structures in weak DC or AC electric fields. The basic effect produces micro-vortices to enhance mixing in microfluidic devices, while various broken symmetries -- controlled potential, irregular shape, non-uniform surface properties, and field gradients -- can be exploited to produce streaming flows. Although we emphasize the qualitative picture of ICEO, we also briefly describe the mathematical theory (for thin double layers and weak fields) and apply it to a metal cylinder with a dielectric coating in a suddenly applied DC field.

In this contribution we review recent efforts on investigations of the effect of (apparent) boundary slip by utilizing lattice Boltzmann simulations. We demonstrate the applicability of the method to treat fundamental questions in microfluidics by investigating fluid flow in hydrophobic and rough microchannels as well as over surfaces covered by nano- or microscale gas bubbles.

Arrays of H-shape microfluidic channels connecting two different fluidic reservoirs have been built with silicon/SU8 microfabrication technologies utilized in production of thermal inkjet printheads. The fluids are delivered to the channels via slots etched through the silicon wafer. Every H-shape channel comprises four thermal inkjet resistors, one in each of the four legs. The resistors vaporize water and generate drive bubbles that pump the fluids from the bulk reservoirs into and out of the channels. By varying relative frequencies of the four pumps, input fluids can be routed to any part of the network in any proportion. Several fluidic operations including dilution, mixing, dynamic valving, and routing have been demonstrated. Thus, a fully integrated microfluidic switchboard that does not require external sources of mechanical power has been achieved. A matrix formalism to describe flow in complex switchboards has been developed and tested.

Synthetic molecular communication (SMC) is a key enabler for future healthcare systems in which Internet of Bio-Nano-Things (IoBNT) devices facilitate the continuous monitoring of a patient's biochemical signals. To close the loop between sensing and actuation, both the detection and the generation of in-body molecular communication (MC) signals is key. However, generating signals inside the human body, e.g., via synthetic nanodevices, poses a challenge in SMC, due to technological obstacles as well as legal, safety, and ethical issues. Hence, this paper considers an SMC system in which signals are generated indirectly via the modulation of a natural in-body MC system, namely the gut-brain axis (GBA). Therapeutic GBA modulation is already established as treatment for neurological diseases, e.g., drug refractory epilepsy (DRE), and performed via the administration of nutritional supplements or specific diets. However, the molecular signaling pathways that mediate the effect of such treatments are mostly unknown. Consequently, existing treatments are standardized or designed heuristically and able to help only some patients while failing to help others. In this paper, we propose to leverage personal health data, e.g., gathered by in-body IoBNT devices, to design more versatile and robust GBA modulation-based treatments as compared to the existing ones. To show the feasibility of our approach, we define a catalog of theoretical requirements for therapeutic GBA modulation. Then, we propose a machine learning model to verify these requirements for practical scenarios when only limited data on the GBA modulation exists. By evaluating the proposed model on several datasets, we confirm its excellent accuracy in identifying different modulators of the GBA. Finally, we utilize the proposed model to identify specific modulatory pathways that play an important role for therapeutic GBA modulation.

Optical techniques are finding widespread use in analytical chemistry for chemical and bio-chemical analysis. During the past decade, there has been an increasing emphasis on miniaturization of chemical analysis systems and naturally this has stimulated a large effort in integrating microfluidics and optics in lab-on-a-chip microsystems. This development is partly defining the emerging field of optofluidics. Scaling analysis and experiments have demonstrated the advantage of micro-scale devices over their macroscopic counterparts for a number of chemical applications. However, from an optical point of view, miniaturized devices suffer dramatically from the reduced optical path compared to macroscale experiments, e.g. in a cuvette. Obviously, the reduced optical path complicates the application of optical techniques in lab-on-a-chip systems. In this paper we theoretically discuss how a strongly dispersive photonic crystal environment may be used to enhance the light-matter interactions, thus potentially compensating for the reduced optical path in lab-on-a-chip systems. Combining electromagnetic perturbation theory with full-wave electromagnetic simulations we address the prospects for achieving slow-light enhancement of Beer-Lambert-Bouguer absorption, photonic band-gap based refractometry, and high-Q cavity sensing.

Incorporating nanomaterials into food products provides key benefits, including extended shelf life, improved safety, and enhanced quality and texture. These innovations could help tackle major challenges in modern food systems, such as reducing waste and enhancing food quality and safety. However, potential toxicity remains a concern, compounded by the lack of physiologically relevant models for assessing ingested nanomaterials. Traditional in vitro and in vivo approaches often fail to mimic gastrointestinal complexity, resulting in inconsistent and non predictive nanotoxicity data that hinder accurate risk assessment of nano enabled foods. To address this gap, this review evaluates the potential of microphysiological systems (MPS), particularly gut-targeted MPS, for modeling gastrointestinal nanoparticle exposure. It examines how MPS technologies replicate key physiological processes relevant to food specific risk assessment, including intestinal barrier function, microbiota immune interactions, and gut organ communication. A comparative analysis of technological advances and their applications in nanotoxicology explores how MPS can be better adapted for nanofood safety evaluation.

Intestinal enteroendocrine cells secrete hormones that are vital for the regulation of glucose metabolism but their differentiation from intestinal stem cells is not fully understood. Asymmetric stem cell divisions have been linked to intestinal stem cell homeostasis and secretory fate commitment. We monitored cell divisions using 4D live cell imaging of cultured intestinal crypts to characterize division modes by means of measurable features such as orientation or shape. A statistical analysis of these measurements requires annotation of mitosis events, which is currently a tedious and time-consuming task that has to be performed manually. To assist data processing, we developed a learning based method to automatically detect mitosis events. The method contains a dual-phase framework for joint detection of dividing cells (mothers) and their progeny (daughters). In the first phase we detect mother and daughters independently using Hough Forest whilst in the second phase we associate mother and daughters by modelling their joint probability as Conditional Random Field (CRF). The method has been evaluated on 32 movies and has achieved an AUC of 72%, which can be used in conjunction with manual correction and dramatically speed up the processing pipeline.

Neural tissues of the central nervous system are among the softest and most fragile in the human body, protected from mechanical perturbation by the skull and the spine. In contrast, the enteric nervous system is embedded in a compliant, contractile tissue and subject to chronic, high-magnitude mechanical stress. Do neurons and glia of the enteric nervous system display specific mechanical properties to withstand these forces? Using nano-indentation combined with immunohistochemistry and second harmonic generation imaging of collagen, we discovered that enteric ganglia in adult mice are an order of magnitude more resistant to deformation than brain tissue. We found that glia-rich regions in ganglia have a similar stiffness to neuron-rich regions and to the surrounding smooth muscle, of ~3 kPa at 3 $μ$m indentation depth and of ~7 kPa at 8 $μ$m depth. Differences in the adhesion strength of the different tissue layers to the glass indenter were scarce. The collagen shell surrounding ganglia and inter-ganglionic fibers may play a key role in strengthening the enteric nervous system to resist the manifold mechanical challenges it faces.

The brain and gut are sensory organs responsible for sensing, transmitting, integrating, and responding to signals from the internal and external environment. In-depth analysis of brain-gut axis interactions is important for human health and disease prevention. Current research on the brain-gut axis primarily relies on animal models. However, animal models make it difficult to study disease mechanisms due to inherent species differences, and the reproducibility of experiments is poor because of individual animal variations, which leads to a significant limitation of real-time sensory responses. Organ-on-a-chip platforms provide an innovative approach for disease treatment and personalized research by replicating brain and gut ecosystems in vitro. This enables a precise understanding of their biological functions and physiological responses. In this article, we examine the history and most current developments in brain, gut, and gut-brain chips. The importance of these systems for understanding pathophysiology and developing new drugs is emphasized throughout the review. This article also addresses future directions and present issues with the advancement and application of gut-brain-on-a-chip technologies.

Patient responses to immune checkpoint inhibitors can be influenced by the gastrointestinal microbiome. Mouse models can be used to study microbiome-host crosstalk, yet their utility is constrained by substantial anatomical, functional, immunological and microbial differences between mice and humans. Here we show that a gut-on-a-chip system mimicking the architecture and functionality of the human intestine by including faecal microbiome and peristaltic-like movements recapitulates microbiome-host interactions and predicts responses to immune checkpoint inhibitors in patients with melanoma. The system is composed of a vascular channel seeded with human microvascular endothelial cells and an intestinal channel with intestinal organoids derived from human induced pluripotent stem cells, with the two channels separated by a collagen matrix. By incorporating faecal samples from patients with melanoma into the intestinal channel and by performing multiomic analyses, we uncovered epithelium-specific biomarkers and microbial factors that correlate with clinical outcomes in patients with melanoma and that the microbiome of non-responders has a reduced ability to buffer cellular stress and self-renew. The gut-on-a-chip model may help identify prognostic biomarkers and therapeutic targets.

The establishment of a three-dimensional (3D) epithelial structure and cytodifferentiation in vitro is necessary to recapitulate in vivo-relevant structure and function of the human intestine. Here, we describe an experimental protocol to build an organomimetic gut-on-a-chip microdevice that allows inducing 3D morphogenesis of human intestinal epithelium using Caco-2 cells or intestinal organoid cells. Under physiological flow and physical motions, intestinal epithelium spontaneously recreates 3D epithelial morphology in a gut-on-a-chip that offers enhanced mucus production, epithelial barrier, and longitudinal host-microbe co-culture. This protocol may provide implementable strategies to advance traditional in vitro static cultures, human microbiome studies, and pharmacological testing.

In drug discovery, human organ-on-a-chip (organ chip) technology has emerged as an essential tool for preclinical testing, offering a realistic representation of human physiology, real-time monitoring, and disease modeling. Polydimethylsiloxane (PDMS) is commonly used in organ chip fabrication owing to its biocompatibility, flexibility, transparency, and ability to replicate features down to the nanoscale. However, the porous nature of PDMS leads to unintended absorption of small molecules, critically affecting the drug response analysis. Addressing this challenge, the precision drug testing organ chip (PreD chip) is introduced, an innovative platform engineered to minimize small molecule absorption while facilitating cell culture. This chip features a PDMS microchannel wall coated with a perfluoropolyether-based lubricant, providing slipperiness and antifouling properties. It also incorporates an ECM-coated semi-porous membrane that supports robust multicellular cultures. The PreD chip demonstrates its outstanding antifouling properties and resistance to various biological fluids, small molecule drugs, and plasma proteins. In simulating the human gut barrier, the PreD chip demonstrates highly enhanced sensitivity in tests for dexamethasone toxicity and is highly effective in assessing drug transport across the human blood-brain barrier. These findings emphasize the potential of the PreD chip in advancing organ chip-based drug testing methodologies.

Perfluoroalkyl substances (PFASs), persistent environmental contaminants linked to neurodevelopmental toxicity, cannot be adequately modeled by traditional

Microphysiological systems (MPSs) have emerged as alternatives to animal testing in drug development, following the FDA Modernization Act 2.0. Double-layer channel-type MPS chips with porous membranes are widely used for modeling various organs, including the intestines, blood-brain barrier, renal tubules, and lungs. However, these chips faced challenges owing to optical interference caused by light scattering from the porous membrane, which hinders cell observation. Trans-epithelial electrical resistance (TEER) measurement offers a non-invasive method for assessing barrier integrity in these chips. However, existing electrode-integrated MPS chips for TEER measurement have non-uniform current densities, leading to compromised measurement accuracy. Additionally, chips made from polydimethylsiloxane have been associated with drug absorption issues. This study developed an electrode-integrated MPS chip for TEER measurement with a uniform current distribution and minimal drug absorption. Through a finite element method simulation, electrode patterns were optimized and incorporated into a polyethylene terephthalate (PET)-based chip. The device was fabricated by laminating PET films, porous membranes, and patterned gold electrodes. The chip's performance was evaluated using a perfused Caco-2 intestinal model. TEER levels increased and peaked on day 5 when cells formed a monolayer, and then they decreased with the development of villi-like structures. Concurrently, capacitance increased, indicating microvilli formation. Exposure to staurosporine resulted in a dose-dependent reduction in TEER, which was validated by immunostaining, indicating a disruption of the tight junction. This study presents a TEER measurement MPS platform with a uniform current density and reduced drug absorption, thereby enhancing TEER measurement reliability. This system effectively monitors barrier integrity and drug responses, demonstrating its potential for non-animal drug-testing applications.

The gut-brain axis (GBA) interaction is important for human health and disease prevention. Organ chips are considered a solution for GBA research. Three-dimensional (3D) cultures and microfluidics engineered in an organ chip could improve the scientific knowledge in the GBA interactions field. In this study, a novel organ chip is developed, which achieves multicellular three-dimensional cultivation by utilizing a decellularized matrix. In addition, this paper reports the rapid prototyping process of the GBA microfluidic chip in polydimethylsiloxane (PDMS) using 3D printing interconnecting poly(ethylene/vinyl acetate) (PEVA) microchannel templates. In comparison to the static culture system of the transwell model, the intestinal epithelial barrier (IEB) and blood-brain barrier (BBB) models on our chip demonstrated superior barrier function and the efflux functionality of transporters under appropriate fluidic conditions. Additionally, it is observed that butyrate protected against BBB dysfunction induced by gut-derived lipopolysaccharide (LPS) via enhancing intestinal barrier function. These results demonstrate that this multicellular, three-dimensional cultivation integrated with a fluidic shear stress simulation chip offers a promising tool for gut-brain interaction study to predict therapy of intestinal and neurological disorders.

The interaction between the gut and the liver, often known as the gut-liver axis, play crucial roles in modulating the body's responses to the xenobiotics as well as progression of diseases. Dysfunction of the axis can cause metabolic disorders as well as obesity, diabetes, and fatty liver disease. During the progression of such diseases, inflammatory responses involving the immune system also play an important part. In this study, we developed a three-tissue microphysiological system (MPS) that can accommodate three different cell types in separated compartments connected via fluidic channels in a microfluidic device. Using computational fluid dynamics, geometry of fluidic channels and flow conditions were optimized for seeding and culturing different cell types in the three-tissue MPS. Caco-2 (gut), RAW264.7 (immune), and HepG2 (liver) cells were seeded and cultured in the chip. Stimulation of the gut cells in the MPS with lipopolysaccharide (LPS) resulted in induction of inflammatory response and production of nitric oxide (NO) in all connected chambers. The anti-inflammatory effect of luteolin was demonstrated. Our study demonstrates that the three-tissue MPS can recapitulate the inflammatory responses involving the gut, liver and immune cells.

The blood-brain barrier (BBB) is essential for central nervous system homeostasis, but most current

To improve our understanding of how the central nervous system functions in health and disease, we report the development of an integrated chip for studying the effects of the neurotransmitters dopamine and serotonin on adult rat hippocampal progenitor cell (AHPC) neurospheroids. This chip allows dopamine or serotonin located in one chamber to diffuse to AHPC neurospheroids cultured in an adjacent chamber through a built-in diffusion barrier created by an array of intentionally misaligned micropillars. The gaps among the micropillars are filled with porous poly(ethylene glycol) (PEG) gel to tune the permeability of the diffusion barrier. An electrochemical sensor is also integrated within the chamber where the neurospheroids can be cultured, thereby allowing monitoring of the concentrations of dopamine or serotonin. Experiments show that concentrations of the neurotransmitters inside the neurospheroid chamber can be increased over a period of several hours to over 10 days by controlling the compositions of the PEG gel inside the diffusion barrier. The AHPC neurospheroids cultured in the chip remain highly viable following dopamine or serotonin treatment. Cell proliferation and neuronal differentiation have also been observed following treatment, revealing that the AHPC neurospheroids are a valuable

The possible pathogenic impact of pro-inflammatory molecules produced by the gut microbiota is one of the hypotheses considered at the basis of the biomolecular dialogue governing the microbiota-gut-brain axis. Among these molecules, lipopolysaccharides (LPS) produced by Gram-negative gut microbiota strains may have a potential key role due to their toxic effects in both the gut and the brain. In this work, we engineered a new dynamic fluidic system, the MINERVA device (MI-device), with the potential to advance the current knowledge of the biological mechanisms regulating the microbiota-gut molecular crosstalk. The MI-device supported the growth of bacteria that are part of the intestinal microbiota under dynamic conditions within a 3D moving mucus model, with features comparable to the physiological conditions (storage modulus of 80 ± 19 Pa, network mesh size of 41 ± 3 nm), without affecting their viability (

Spheroids and organoids have attracted significant attention as innovative models for disease modeling and drug screening. By employing diverse types of spheroids or organoids, it is feasible to establish microphysiological systems that enhance the precision of disease modeling and offer more dependable and comprehensive drug screening. High-throughput microphysiological systems that support optional, parallel testing of multiple drugs have promising applications in personalized medical treatment and drug research. However, establishing such a system is highly challenging and requires a multidisciplinary approach. This study introduces a dynamic Microphysiological System Chip Platform (MSCP) with multiple functional microstructures that encompass the mentioned advantages. We developed a high-throughput lung cancer spheroids model and an intestine-liver-heart-lung cancer microphysiological system for conducting parallel testing on four anti-lung cancer drugs, demonstrating the feasibility of the MSCP. This microphysiological system combines microscale and macroscale biomimetics to enable a comprehensive assessment of drug efficacy and side effects. Moreover, the microphysiological system enables evaluation of the real pharmacological effect of drug molecules reaching the target lesion after absorption by normal organs through fluid-based physiological communication. The MSCP could serves as a valuable platform for microphysiological system research, making significant contributions to disease modeling, drug development, and personalized medical treatment.

Microphysiological systems play a pivotal role in progressing toward a global paradigm shift in drug development. Here, we designed a four-organ-chip interconnecting miniaturized human intestine, liver, brain and kidney equivalents. All four organ models were predifferentiated from induced pluripotent stem cells from the same healthy donor and integrated into the microphysiological system. The coculture of the four autologous tissue models in one common medium deprived of tissue specific growth factors was successful over 14-days. Although there were no added growth factors present in the coculture medium, the intestine, liver and neuronal model maintained defined marker expression. Only the renal model was overgrown by coexisting cells and did not further differentiate. This model platform will pave the way for autologous coculture cross-talk assays, disease induction and subsequent drug testing.

To replicate key physiological barriers in vitro, we utilized the CELLBLOKS® modular microphysiological system. Specifically, human cerebral microvascular endothelial hCMEC/D3 cells, human retinal pigment epithelial (HRPE) cells, and rat small intestinal IEC-6 cells were grown in CELLBLOKS® to mimic the blood-brain (BBB), blood-cerebrospinal fluid (BCSFB), and intestinal (IB) barriers, respectively. Eugenol is an essential oil component known to permeate the central nervous system (CNS) in vivo after intravenous and oral administrations; it was therefore used for simulated intravenous and oral administrations into the CELLBLOKS® system, using also celiprolol as negative control compound, since it is known for its poor ability to permeate in the CNS from the bloodstream. In particular, the intravenous administration (systemic) of the compounds was simulated by their direct addition to the bloodstream-like lower channel of CELLBLOKS® (basolateral side of both CSFB and IB; apical side for BBB), whereas their oral administration was simulated by apical addition to IEC-6. Permeation measurements, via HPLC, across physiological barriers cultured in CELLBLOKS® demonstrated that, following both simulated oral and systemic administration, eugenol crosses the mimicked BBB and the BCSFB indiscriminately; conversely, the permeation of celiprolol across these barriers results strongly limited in comparison to eugenol. To assess downstream neuroactivity, dopaminergic neuron-like PC12 cells were cultured on NANOSTACKS™ inserts and incorporated into the BBB and BCSFB blocks. After simulated intravenous and oral administrations, significant eugenol-induced dopamine release by PC12 cells was evidenced both in BBB- and BCSFB-delimited neuronal-like compartments. These results validate the CELLBLOKS® and NANOSTACKS™ platforms as robust tools characterized by low costs, high reproducibility and ease of manipulation for in vitro studies of brain targeting of new drugs. This system requires two weeks culture period to be ready for the simulation in vitro of IB, BBB, BCFSB and neuronal tissues, appearing useful in limiting pre-clinical animal testing.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic. Clinical evidence suggests that the intestine is another high-risk organ for SARS-CoV-2 infection besides the lungs. However, a model that can accurately reflect the response of the human intestine to the virus is still lacking. Here, we created an intestinal infection model on a chip that allows the recapitulation of human relevant intestinal pathophysiology induced by SARS-CoV-2 at organ level. This microengineered gut-on-chip reconstitutes the key features of the intestinal epithelium-vascular endothelium barrier through the three-dimensional (3D) co-culture of human intestinal epithelial, mucin-secreting, and vascular endothelial cells under physiological fluid flow. The intestinal epithelium showed permissiveness for viral infection and obvious morphological changes with injury of intestinal villi, dispersed distribution of mucus-secreting cells, and reduced expression of tight junction (E-cadherin), indicating the destruction of the intestinal barrier integrity caused by virus. Moreover, the vascular endothelium exhibited abnormal cell morphology, with disrupted adherent junctions. Transcriptional analysis revealed abnormal RNA and protein metabolism, as well as activated immune responses in both epithelial and endothelial cells after viral infection (e.g., upregulated cytokine genes), which may contribute to the injury of the intestinal barrier associated with gastrointestinal symptoms. This human organ system can partially mirror intestinal barrier injury and the human response to viral infection, which is not possible in existing

Infectious diseases present tremendous challenges to human progress and public health. The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the associated coronavirus disease 2019 (COVID-19) pandemic continue to pose an imminent threat to humanity. These infectious diseases highlight the importance of developing innovative strategies to study disease pathogenesis and protect human health. Although conventional

Environmental pollutants; bioaerosols, chemicals and micro/nano-plastics (MNPs) can disrupt the mechano-biological processes of the human body, impairing structural, cellular, and molecular functions. Such disruptions may ultimately contribute to the onset of toxicity-associated pharmacokinetic disorders. Herein, we highlighted our iPSCs or patient-derived brain, lung, liver and intestinal organoid models, iPSCs-differentiated alveolar epithelial cell (AEC) and brain microvascular endothelial cell (BMVEC) barriers, and their OoC models to evaluate the effect of kinds of exposomes. We successfully characterized SPC+ AECs and CD31+ BMVECs with highly transepithelial electrical resistance values as a gold standart, IBA1+ microglia and CD31+ endothelial cells enriched, cortical plate structured advanced matured functional brain organoids, MUC1+ lung-like organoids, EPCAM+/ALB+ liver organoids and human crypt-derived intestinal organoids (Figure 1). Subsequently, we developed integratable organ-specific OoC models utilizing a layer-by-layer fabrication approach to enable co-culture systems of cells and organoids. Our advanced bioengineered models demonstrated that environmental exposures significantly compromised barrier integrity, leading to increased translocation across the tissue construct, reduced cell-organoid viability, and dysregulated expression of inflammatory cytokines and immune cell activity. The resilience of human physiological barriers can be effectively modelled using humanized bioengineering platforms that emulate the dynamic mechanical and biochemical forces present

The complexity of integrating microbiota into clinical pharmacology, environmental toxicology, and opioid studies arises from bidirectional and multiscale interactions between humans and their many microbiota, notably those of the gut. Hosts and each microbiota are governed by distinct central dogmas, with genetics influencing transcriptomics, proteomics, and metabolomics. Each microbiota's metabolome differentially modulates its own and the host's multi-omics. Exogenous compounds (e.g., drugs and toxins), often affect host multi-omics differently than microbiota multi-omics, shifting the balance between drug efficacy and toxicity. The complexity of the host-microbiota connection has been informed by current methods of in vitro bacterial cultures and in vivo mouse models, but they fail to elucidate mechanistic details. Together, in vitro organ-on-chip microphysiological models, multi-omics, and in silico computational models have the potential to supplement the established methods to help clinical pharmacologists and environmental toxicologists unravel the myriad of connections between the gut microbiota and host health and disease.

A 'gut-brain axis' is an intricate bidirectional connection between the gut and the central nervous system, serving as a key pathway for signal exchange. However, current in vitro models do not fully capture these dynamic interactions, limiting mechanistic insight and therapeutic testing. Here, we show a 3D human gut-brain-vascular microphysiological platform that integrates lumenized villus-like intestinal barrier, blood vascular-astrocyte interactions, and brain tissue to model circulation-mediated crosstalk between the gut and brain. Using this system, we demonstrate gut-to-brain signaling by delivering bacterial-derived toxins to the gut compartment, which traverse the gut and neurovascular barriers and trigger neuroinflammatory responses and tau-associated pathology in the brain tissue. Conversely, we show that Alzheimer's- and Parkinson's-relevant stimuli applied to the brain compartment elicit neuroinflammation and disrupt both vascular and intestinal barrier integrity, indicating brain-to-gut feedback. Together, our platform provides a human-relevant tool to dissect mechanisms of bidirectional gut-brain communication and to evaluate therapeutic strategies for neurogastrointestinal disease.

Organ interactions resulting from drug, metabolite or xenobiotic transport between organs are key components of human metabolism that impact therapeutic action and toxic side effects. Preclinical animal testing often fails to predict adverse outcomes arising from sequential, multi-organ metabolism of drugs and xenobiotics. Human microphysiological systems (MPS) can model these interactions and are predicted to dramatically improve the efficiency of the drug development process. In this study, five human MPS models were evaluated for functional coupling, defined as the determination of organ interactions via an in vivo-like sequential, organ-to-organ transfer of media. MPS models representing the major absorption, metabolism and clearance organs (the jejunum, liver and kidney) were evaluated, along with skeletal muscle and neurovascular models. Three compounds were evaluated for organ-specific processing: terfenadine for pharmacokinetics (PK) and toxicity; trimethylamine (TMA) as a potentially toxic microbiome metabolite; and vitamin D3. We show that the organ-specific processing of these compounds was consistent with clinical data, and discovered that trimethylamine-N-oxide (TMAO) crosses the blood-brain barrier. These studies demonstrate the potential of human MPS for multi-organ toxicity and absorption, distribution, metabolism and excretion (ADME), provide guidance for physically coupling MPS, and offer an approach to coupling MPS with distinct media and perfusion requirements.

Halofuginone hydrobromide has shown potent antiviral efficacy against a variety of viruses such as SARS-CoV-2, dengue, or chikungunya virus, and has, therefore, been hypothesized to have broad-spectrum antiviral activity. In this paper, we tested this broad-spectrum antiviral activity of Halofuginone hydrobomide against viruses from different families (Picornaviridae, Herpesviridae, Orthomyxoviridae, Coronaviridae, and Flaviviridae). To this end, we used relevant human models of the airway and intestinal epithelium and regionalized neural organoids. Halofuginone hydrobomide showed antiviral activity against SARS-CoV-2 in the airway epithelium with no toxicity at equivalent concentrations used in human clinical trials but not against any of the other tested viruses.