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Numerous diseases, including age‐related macular degeneration (AMD), arise from the blood‐retinal barrier and blood vessel abnormalities in the eye; unfortunately, there is a lack of reliable in vitro models for their systematic study. This study describes a high‐throughput microphysiological system (MPS) designed to model the outer Blood‐Retinal Barrier (oBRB). The MPS platform is engineered to integrate seamlessly with high‐content screening technologies, utilizing a design with a single oBRB model incorporating RPE (retina pigment epithelial cells) and endothelial cell co‐culture to fit within a single 96‐well. Arranged in the standard 96‐well plate format, the platform allows high‐throughput assessment of barrier integrity through 3D confocal imaging (ZO‐1 staining), Trans Epithelial Electrical Resistance (TEER), and permeability measurements. The oBRB model enables the investigation of crosstalk among different cell types in co‐culture. This includes assessing changes in the barrier integrity of the Retinal Pigment Epithelium (RPE) monolayer and investigating neovascularization events resulting from endothelial cell remodeling. The platform is positioned for utility in drug discovery and development efforts targeting diseases involving oBRB damage and choroidal neovascularization, such as age‐related macular degeneration. [ABSTRACT FROM AUTHOR]

The average cost to bring a new drug from its initial discovery to a patient's bedside is estimated to surpass $2 billion and requires over a decade of research and development. There is a need for new drug screening technologies that can parse drug candidates with increased likelihood of clinical utility early in development in order to increase the cost‐effectiveness of this pipeline. For example, during the COVID‐19 pandemic, resources were rapidly mobilized to identify effective therapeutic treatments but many lead antiviral compounds failed to demonstrate efficacy when progressed to human trials. To address the lack of predictive preclinical drug screening tools, PREDICT96‐ALI, a high‐throughput (n = 96) microphysiological system (MPS) that recapitulates primary human tracheobronchial tissue,is adapted for the evaluation of differential antiviral efficacy of native SARS‐CoV‐2 variants of concern. Here, PREDICT96‐ALI resolves both the differential viral kinetics between variants and the efficacy of antiviral compounds over a range of drug doses. PREDICT96‐ALI is able to distinguish clinically efficacious antiviral therapies like remdesivir and nirmatrelvir from promising lead compounds that do not show clinical efficacy. Importantly, results from this proof‐of‐concept study track with known clinical outcomes, demonstrate the feasibility of this technology as a prognostic drug discovery tool. [ABSTRACT FROM AUTHOR]

A microphysiological system (MPS) is an in vitro culture technology that reproduces the physiological microenvironment and functionality of humans and is expected to be applied for drug screening. In this study, we developed an MPS for the structured culture of human iPSC-derived sensory neurons and then predicted drug-induced neurotoxicity by morphological deep learning. Using human iPSC-derived sensory neurons, after the administration of representative anti-cancer drugs, the toxic effects on soma and axons were evaluated by an AI model with neurite images. Significant toxicity was detected in positive drugs and could be classified by different effects on soma or axons, suggesting that the current method provides an effective evaluation of chemotherapy-induced peripheral neuropathy. The results of neurofilament light chain expression changes in the MPS device also agreed with clinical reports. Therefore, the present MPS combined with morphological deep learning is a useful platform for in vitro peripheral neurotoxicity assessment. [ABSTRACT FROM AUTHOR]

Abstract Hepatic spheroids are of high interest in basic research, drug discovery and cell therapy. Existing methods for spheroid culture present advantages and drawbacks. An alternative technology is explored: the hepatic spheroid formation and culture in an acoustofluidic chip, using HepaRG cell line. Spheroid formation and morphology, cell viability, genetic stability, and hepatic functions are analyzed after 6 days of culture in acoustic levitation. They are compared to 2D culture and non-levitated 3D cultures. Sizes of the 25 spheroids created in a single acoustofluidic microphysiological system are homogeneous. The acoustic parameters in our system do not induce cell mortality nor DNA damage. Spheroids are cohesive and dense. From a functional point of view, hepatic spheroids obtained by acoustic levitation exhibit polarity markers, secrete albumin and express hepatic genes at higher levels compared to 2D and low attachment 3D cultures. In conclusion, this microphysiological system proves not only to be suitable for long-term culture of hepatic spheroids, but also to favor differentiation and functionality within 6 days of culture.

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.

Abstract Introduction Microphysiological systems (MPS) offer simulation of (patho)physiological parameters. Investigation includes items which lead to fibrosis and calcification in development and progress of calcific aortic valve disease, based e.g. on culturing of isolated valvular interstitial cells (VICs). Hypoxia regulated by hypoxia inducible factors impacts pathological differentiation in aortic valve (AV) disease. This is mimicked via an MPS implemented oxygenator in combination with calcification inducing medium supplementation. Methods Human valvular interstitial cells were isolated and dynamically cultured in MPS at hypoxic, normoxic, arterial blood oxygen concentration and cell incubator condition. Expression profile of fibrosis and calcification markers was monitored and calcification was quantified in induction and control media with and without hypoxia and in comparison to statically cultured counterparts. Results Hypoxic 24-hour culture of human VICs leads to HIF1α nuclear localization and induction of EGLN1, EGLN3 and LDHA mRNA expression but does not directly impact expression of fibrosis and calcification markers. Dependent on medium formulation, induction medium induces monolayer calcification and elevates RUNX2, ACTA2 and FN1 but reduces SOX9 mRNA expression in dynamic and static MPS culture. But combining hypoxic oxygen concentration leads to higher calcification potential of human VICs in calcification and standard medium formulation dynamically cultured for 96 h. Conclusion In hypoxic oxygen concentration an increased human VIC calcification in 2D VIC culture in an oxygenator assisted MPS was detected. Oxygen regulation therefore can be combined with calcification induction media to monitor additional effects of pathological marker expression. Validation of oxygenator dependent VIC behavior envisions future advancement and transfer to long term aortic valve tissue culture MPS.

Abstract This study focuses on the use of pulsed electric fields (PEF) in microfluidics for controlled cell studies. The commonly used material for soft lithography, polydimethylsiloxane (PDMS), does not fully ensure the necessary chemical and mechanical resistance in these systems. Integration of specific analytical measurement setups into microphysiological systems (MPS) are also challenging. We present an off-stoichiometry thiol-ene (OSTE)-based microchip, containing integrated electrodes for PEF and transepithelial electrical resistance (TEER) measurement and the equipment to monitor pH and oxygen concentration in situ. The effectiveness of the MPS was empirically demonstrated through PEF treatment of the C6 cells. The effects of PEF treatment on cell viability and permeability to the fluorescent dye DapI were tested in two modes: stop flow and continuous flow. The maximum permeability was achieved at 1.8 kV/cm with 16 pulses in stop flow mode and 64 pulses per cell in continuous flow mode, without compromising cell viability. Two integrated sensors detected changes in oxygen concentration before and after the PEF treatment, and the pH shifted towards alkalinity following PEF treatment. Therefore, our proof-of-concept technology serves as an MPS for PEF treatment of mammalian cells, enabling in situ physiological monitoring.

Abstract Organs-on-chips are microphysiological systems that allow to replicate the key functions of human organs and accelerate the innovation in life sciences including disease modeling, drug development, and precision medicine. However, due to the lack of standards in their definition, structural design, cell source, model construction, and functional validation, a wide range of translational application of organs-on-chips remains a challenging. “Organs-on-chips: Intestine” is the first group standard on human intestine-on-a-chip in China, jointly agreed and released by the experts from the Chinese Society of Biotechnology on 29th April 2024. This standard specifies the scope, terminology, definitions, technical requirements, detection methods, and quality control in building the human intestinal model on a chip. The publication of this group standard will guide the institutional establishment, acceptance and execution of proper practical protocols and accelerate the international standardization of intestine-on-a-chip for translational applications.

Hepatic spheroids are of high interest in basic research, drug discovery and cell therapy. Existing methods for spheroid culture present advantages and drawbacks. An alternative technology is explored: the hepatic spheroid formation and culture in an acoustofluidic chip, using HepaRG cell line. Spheroid formation and morphology, cell viability, genetic stability, and hepatic functions are analyzed after 6 days of culture in acoustic levitation. They are compared to 2D culture and non-levitated 3D cultures. Sizes of the 25 spheroids created in a single acoustofluidic microphysiological system are homogeneous. The acoustic parameters in our system do not induce cell mortality nor DNA damage. Spheroids are cohesive and dense. From a functional point of view, hepatic spheroids obtained by acoustic levitation exhibit polarity markers, secrete albumin and express hepatic genes at higher levels compared to 2D and low attachment 3D cultures. In conclusion, this microphysiological system proves not only to be suitable for long-term culture of hepatic spheroids, but also to favor differentiation and functionality within 6 days of culture. [ABSTRACT FROM AUTHOR]

Calcific aortic valve disease (CAVD) is one of the most common forms of valvulopathy, with a 50 % elevated risk of a fatal cardiovascular event, and greater than 15,000 annual deaths in North America alone. The treatment standard is valve replacement as early diagnostic, mitigation, and drug strategies remain underdeveloped. The development of early diagnostic and therapeutic strategies requires the fabrication of effective in vitro valve mimetic models to elucidate early CAVD mechanisms. In this study, we developed a multilayered physiologically relevant 3D valve-on-chip (VOC) system that incorporated aortic valve mimetic extracellular matrix (ECM), porcine aortic valve interstitial cell (VIC) and endothelial cell (VEC) co-culture and dynamic mechanical stimuli. Collagen and glycosaminoglycan (GAG) based hydrogels were assembled in a bilayer to mimic healthy or diseased compositions of the native fibrosa and spongiosa. Multiphoton imaging and proteomic analysis of healthy and diseased VOCs were performed. Collagen-based bilayered hydrogel maintained the phenotype of the VICs. Proteins related to cellular processes like cell cycle progression, cholesterol biosynthesis, and protein homeostasis were found to be significantly altered and correlated with changes in cell metabolism in diseased VOCs. This study suggested that diseased VOCs may represent an early, adaptive disease initiation stage, which was corroborated by human aortic valve proteomic assessment. In this study, we developed a collagen-based bilayered hydrogel to mimic healthy or diseased compositions of the native fibrosa and spongiosa layers. When the gels were assembled in a VOC with VECs and VICs, the diseased VOCs revealed key insights about the CAVD initiation process. Calcific aortic valve disease (CAVD) elevates the risk of death due to cardiovascular pathophysiology by 50 %, however, prevention and mitigation strategies are lacking, clinically. Developing tools to assess early disease would significantly aid in the prevention of disease and in the development of therapeutics. Previously, studies have utilized collagen and glycosaminoglycan-based hydrogels for valve cell co-cultures, valve cell co-cultures in dynamic environments, and inorganic polymer-based multilayered hydrogels; however, these approaches have not been combined to make a physiologically relevant model for CAVD studies. We fabricated a bi-layered hydrogel that closely mimics the aortic valve and used it for valve cell co-culture in a dynamic platform to gain mechanistic insights into the CAVD initiation process using proteomic and multiphoton imaging assessment. [Display omitted] [ABSTRACT FROM AUTHOR]

Enteric neurons are critical in maintaining organ homeostasis within the small intestine, and their dysregulation are implicated in gastrointestinal disorders and neurodegenerative diseases. Most in vitro models lack enteric innervation, limiting basic discovery and disease modeling research. Here, a high‐throughput 3D microphysiological system (MPS), or organ chip is presented that supports a primary epithelial monolayer interfacing directly with encapsulated primary enteric neurons. The device features twelve 3D MPSs per device and gravity‐driven flow via a laboratory rocker to induce biomimetic shear stress on the epithelium culture and provide continuous nutrient presentation. Intestinal and neural tissue exhibited expected morphologies. Neural gene upregulation in the epithelium suggests RNA contamination from proximal enteric neurons extending neurites toward the epithelial monolayer. With the enteric nervous system (ENS), barrier integrity significantly increased for both TEER and permeability assays, a 1.25‐fold greater resistance and 10% lower permeability as compared to epithelium cultured alone. The presence of the ENS resulted in a significant (1.4‐fold) reduction in epidermal growth factor (EGF). Additionally, several key epithelial genes are compared between duodenal tissue and epithelial monolayers with and without neurons present. Results demonstrated changes in cytokine gene expression and WNT pathways, highlighting innervation is essential to create more biomimetic and physiologically relevant in vitro models. [ABSTRACT FROM AUTHOR]

Coculture of intestinal bacteria with primary human intestinal epithelium provides a valuable tool for investigating host‐colon bacterial interactions and for testing and screening therapeutics. However, most current intestinal model systems lack key physiological features of the in vivo colon, such as both a proper oxygen microenvironment and a mucus layer. In this work, a new in vitro colonic microphysiological system is demonstrated with a cell‐derived, functional mucus that closely resembles the in vivo colonic mucosa and apical microenvironment by employing an anaerobic air‐liquid interface culture. The human primary colon epithelial cells in this new in vitro system exhibit high cell viability (>98%) with ≈100 µm thick functional mucus layer on average. Successful coculture of model anaerobic gut bacterial strains Lactobacillus rhamnosus GG and Anaerobutyricum hallii without loss in human cell viability demonstrates that this new model can be an invaluable tool for future studies of the impact of commensal and pathogenic bacteria on the colon. [ABSTRACT FROM AUTHOR]

The vascular system, essential for human physiology, is vital for transporting nutrients, oxygen, and waste. Since vascular structures are involved in various disease pathogeneses and exhibit different morphologies depending on the organ, researchers have endeavored to develop organ-specific vascular models. While animal models possess sophisticated vascular morphologies, they exhibit significant discrepancies from human tissues due to species differences, which limits their applicability. To overcome the limitations arising from these discrepancies and the oversimplification of 2D dish cultures, microphysiological systems (MPS) have emerged as a promising alternative. These systems more accurately mimic the human microenvironment by incorporating cell interactions, physical stimuli, and extracellular matrix components, thus facilitating enhanced tissue differentiation and functionality. Importantly, MPS often utilize human-derived cells, greatly reducing disparities between model and patient responses. This review focuses on recent advancements in MPS, particularly in modeling the human organ-specific vascular system, and discusses their potential in biological adaptation. [ABSTRACT FROM AUTHOR]

Fetal membrane (amniochorion), the innermost lining of the intrauterine cavity, surround the fetus and enclose amniotic fluid. Unlike unidirectional blood flow, amniotic fluid subtly rocks back and forth, and thus, the innermost amnion epithelial cells are continuously exposed to low levels of shear stress from fluid undulation. Here, we tested the impact of fluid motion on amnion epithelial cells (AECs) as a bearer of force impact and their potential vulnerability to cytopathologic changes that can destabilize fetal membrane functions. A previously developed amnion membrane (AM) organ-on-chip (OOC) was utilized but with dynamic flow to culture human fetal amnion membrane cells. The applied flow was modulated to perfuse culture media back and forth for 48 h to mimic fluid motion. A static culture condition was used as a negative control, and oxidative stress (OS) condition was used as a positive control representing pathophysiological changes. The impacts of fluidic motion were evaluated by measuring cell viability, cellular transition, and inflammation. Additionally, scanning electron microscopy (SEM) imaging was performed to observe microvilli formation. The results show that regardless of the applied flow rate, AECs and AMCs maintained their viability, morphology, innate meta-state, and low production of pro-inflammatory cytokines. E-cadherin expression and microvilli formation in the AECs were upregulated in a flow rate-dependent fashion; however, this did not impact cellular morphology or cellular transition or inflammation. OS treatment induced a mesenchymal morphology, significantly higher vimentin to cytokeratin 18 (CK-18) ratio, and pro-inflammatory cytokine production in AECs, whereas AMCs did not respond in any significant manner. Fluid motion and shear stress, if any, did not impact AEC cell function and did not cause inflammation. Thus, when using an amnion membrane OOC model, the inclusion of a dynamic flow environment is not necessary to mimic in utero physiologic cellular conditions of an amnion membrane. [ABSTRACT FROM AUTHOR]

Three-dimensional (3D) cultured cells, such as spheroids and organoids, have been recognized as highly relevant in vitro tissue models. In addition, recent advances in microfabrication techniques have facilitated the development of microphysiological systems (MPS), in which cells are cultured in a microfluidic device that mimics the physiological environment and promotes the development of tissue-like structures. To make effective use of these in vitro models, techniques are needed that can assess the activities of the cells. Electrochemical sensors are promising due to their ease of miniaturization, label-free and real-time measurement of cell-derived molecules. Here we review the electrochemical assessment of 3D cultured cells using different types of electrochemical devices such as probe electrodes, electrode arrays, and electrochemical imaging methods. In addition, recent advances in the integration of electrochemical sensors into MPS are presented. Finally, the challenges and prospects for advancements in 3D culture systems integrated with electrochemical devices are discussed. [ABSTRACT FROM AUTHOR]

Introduction: Microphysiological systems (MPS) offer simulation of (patho)physiological parameters. Investigation includes items which lead to fibrosis and calcification in development and progress of calcific aortic valve disease, based e.g. on culturing of isolated valvular interstitial cells (VICs). Hypoxia regulated by hypoxia inducible factors impacts pathological differentiation in aortic valve (AV) disease. This is mimicked via an MPS implemented oxygenator in combination with calcification inducing medium supplementation. Methods: Human valvular interstitial cells were isolated and dynamically cultured in MPS at hypoxic, normoxic, arterial blood oxygen concentration and cell incubator condition. Expression profile of fibrosis and calcification markers was monitored and calcification was quantified in induction and control media with and without hypoxia and in comparison to statically cultured counterparts. Results: Hypoxic 24-hour culture of human VICs leads to HIF1α nuclear localization and induction of EGLN1, EGLN3 and LDHA mRNA expression but does not directly impact expression of fibrosis and calcification markers. Dependent on medium formulation, induction medium induces monolayer calcification and elevates RUNX2, ACTA2 and FN1 but reduces SOX9 mRNA expression in dynamic and static MPS culture. But combining hypoxic oxygen concentration leads to higher calcification potential of human VICs in calcification and standard medium formulation dynamically cultured for 96 h. Conclusion: In hypoxic oxygen concentration an increased human VIC calcification in 2D VIC culture in an oxygenator assisted MPS was detected. Oxygen regulation therefore can be combined with calcification induction media to monitor additional effects of pathological marker expression. Validation of oxygenator dependent VIC behavior envisions future advancement and transfer to long term aortic valve tissue culture MPS. [ABSTRACT FROM AUTHOR]

This study focuses on the use of pulsed electric fields (PEF) in microfluidics for controlled cell studies. The commonly used material for soft lithography, polydimethylsiloxane (PDMS), does not fully ensure the necessary chemical and mechanical resistance in these systems. Integration of specific analytical measurement setups into microphysiological systems (MPS) are also challenging. We present an off-stoichiometry thiol-ene (OSTE)-based microchip, containing integrated electrodes for PEF and transepithelial electrical resistance (TEER) measurement and the equipment to monitor pH and oxygen concentration in situ. The effectiveness of the MPS was empirically demonstrated through PEF treatment of the C6 cells. The effects of PEF treatment on cell viability and permeability to the fluorescent dye DapI were tested in two modes: stop flow and continuous flow. The maximum permeability was achieved at 1.8 kV/cm with 16 pulses in stop flow mode and 64 pulses per cell in continuous flow mode, without compromising cell viability. Two integrated sensors detected changes in oxygen concentration before and after the PEF treatment, and the pH shifted towards alkalinity following PEF treatment. Therefore, our proof-of-concept technology serves as an MPS for PEF treatment of mammalian cells, enabling in situ physiological monitoring. [ABSTRACT FROM AUTHOR]

Organs-on-chips are microphysiological systems that allow to replicate the key functions of human organs and accelerate the innovation in life sciences including disease modeling, drug development, and precision medicine. However, due to the lack of standards in their definition, structural design, cell source, model construction, and functional validation, a wide range of translational application of organs-on-chips remains a challenging. "Organs-on-chips: Intestine" is the first group standard on human intestine-on-a-chip in China, jointly agreed and released by the experts from the Chinese Society of Biotechnology on 29th April 2024. This standard specifies the scope, terminology, definitions, technical requirements, detection methods, and quality control in building the human intestinal model on a chip. The publication of this group standard will guide the institutional establishment, acceptance and execution of proper practical protocols and accelerate the international standardization of intestine-on-a-chip for translational applications. [ABSTRACT FROM AUTHOR]

Cancer immunotherapy focuses on the use of patients' adaptive immune systems to combat cancer. In the past decade, FDA has approved many immunotherapy products for cancer patients who suffer from primary tumors, tumor relapse, and metastases. However, these immunotherapies still show resistance in many patients and often lead to inconsistent responses in patients due to variations in tumor genetic mutations and tumor immune microenvironment. Microfluidics‐based organ‐on‐a‐chip technologies or microphysiological systems have opened new ways that can provide relatively fast screening for personalized immunotherapy and help researchers and clinicians understand tumor‐immune interactions in a patient‐specific manner. They also have the potential to overcome the limitations of traditional drug screening and testing, given the models provide a more realistic 3D microenvironment with better controllability, reproducibility, and physiological relevance. This review focuses on the cutting‐edge microphysiological organ‐on‐a‐chip devices developed in recent years for studying cancer immunity and testing cancer immunotherapeutic agents, as well as some of the largest challenges of translating this technology to clinical applications in immunotherapy and personalized medicine. [ABSTRACT FROM AUTHOR]

Introduction: As the body's first line of defense against disease and infection, neutrophils must efficiently navigate to sites of inflammation; however, neutrophil dysregulation contributes to the pathogenesis of numerous diseases that leave people susceptible to infections. Many of these diseases are also associated with changes to the protein composition of the extracellular matrix. While it is known that neutrophils and endothelial cells, which play a key role in neutrophil activation, are sensitive to the mechanical and structural properties of the extracellular matrix, our understanding of how protein composition in the matrix affects the neutrophil response to infection is incomplete. Methods: To investigate the effects of extracellular matrix composition on the neutrophil response to infection, we used an infection-on-a-chip microfluidic device that replicates a portion of a blood vessel endothelium surrounded by a model extracellular matrix. Model blood vessels were fabricated by seeding human umbilical vein endothelial cells on 2, 4, or 6 mg/mL type I collagen hydrogels. Primary human neutrophils were loaded into the endothelial lumens and stimulated by adding the bacterial pathogen Pseudomonas aeruginosa to the surrounding matrix. Results: Collagen concentration did not affect the cell density or barrier function of the endothelial lumens. Upon infectious challenge, we found greater neutrophil extravasation into the 4 mg/mL collagen gels compared to the 6 mg/mL collagen gels. We further found that extravasated neutrophils had the highest migration speed and distance in 2mg/mL gels and that these values decreased with increasing collagen concentration. However, these phenomena were not observed in the absence of an endothelial lumen. Lastly, no differences in the percent of extravasated neutrophils producing reactive oxygen species were observed across the various collagen concentrations. Discussion: Our study suggests that neutrophil extravasation and migration in response to an infectious challenge are regulated by collagen concentration in an endothelial cell-dependent manner. The results demonstrate how the mechanical and structural aspects of the tissue microenvironment affect the neutrophil response to infection. Additionally, these findings underscore the importance of developing and using microphysiological systems for studying the regulatory factors that govern the neutrophil response. [ABSTRACT FROM AUTHOR]

Endothelial dysfunction is a critical feature of acute respiratory distress syndrome (ARDS) associated with higher disease severity and worse outcomes. Preclinical in vivo models of sepsis and ARDS have failed to yield useful therapies in humans, perhaps due to interspecies differences in inflammatory responses and heterogeneity of human host responses. Use of microphysiological systems (MPS) to investigate lung endothelial function may shed light on underlying mechanisms and targeted treatments for ARDS. We assessed the response to plasma from critically ill sepsis patients in our lung endothelial MPS through measurement of endothelial permeability, expression of adhesion molecules, and inflammatory cytokine secretion. Sepsis plasma induced areas of endothelial cell (EC) contraction, loss of cellular coverage, and luminal defects. EC barrier function was significantly worse following incubation with sepsis plasma compared to healthy plasma. EC ICAM‐1 expression, IL‐6 and soluble ICAM‐1 secretion increased significantly more after incubation with sepsis plasma compared with healthy plasma. Plasma from sepsis patients who developed ARDS further increased IL‐6 and sICAM‐1 compared to plasma from sepsis patients without ARDS and healthy plasma. Our results demonstrate the proof of concept that lung endothelial MPS can enable interrogation of specific mechanisms of endothelial dysfunction that promote ARDS in sepsis patients. [ABSTRACT FROM AUTHOR]

Microphysiological systems (MPS) and Organs-on-Chips (OoCs) hold significant potential for replicating complex human biological processes in vitro. However, their widespread adoption by industry and regulatory bodies depends on effective qualification to demonstrate that these models are fit for purpose. Many models developed in academia are not initially designed with qualification in mind, which limits their future implementation in end-user settings. Here, we explore to which extent aspects of qualification can already be performed during early development stages of MPS and OoCs. Through a case study of our blood-perfused Vessel-on-Chip model, we emphasize key elements such as defining a clear context-of-use, establishing relevant readouts, ensuring model robustness, and addressing inherent limitations. By considering qualification early in development, researchers can streamline the progression of MPS and OoCs, facilitating their adoption in biomedical, pharmaceutical, and toxicological research. In addition, all in vitro methods must be independent of animal-derived materials to be considered fully fit for purpose. Ultimately, early qualification efforts can enhance the availability, reliability, and regulatory as well as ethical acceptance of these emerging New Approach Methodologies.

3D human tissue models/microphysiological systems (3D/MPS) can represent human physiology. Linking organ models can improve biological fidelity and predictive capability. 3D/MPS may be infected with HIV, enabling studies of pathogenesis, host–virus interactions, inflammation, neuropathogenesis, and HIV reservoirs. Integrating immune cells or lymphoid tissues into 3D/MPS facilitates study of HIV immune responses, leukocyte depletion, immune ontogeny, immunotherapies, and vaccines. Application of 3D/MPS to therapeutic development facilitates sophisticated preclinical pharmacology, in silico modeling, predictions of human response and safety, and evaluation of potency of biologics. Tissue sources and/or modifications permit study of unique conditions, including TB co-infection, substance use, and special populations (e.g., medically fragile, pregnant or lactating people, and children). Three-dimensional (3D) human tissue models/microphysiological systems (e.g., organs-on-chips, organoids, and tissue explants) model HIV and related comorbidities and have potential to address critical questions, including characterization of viral reservoirs, insufficient innate and adaptive immune responses, biomarker discovery and evaluation, medical complexity with comorbidities (e.g., tuberculosis and SARS-CoV-2), and protection and transmission during pregnancy and birth. Composed of multiple primary or stem cell-derived cell types organized in a dedicated 3D space, these systems hold unique promise for better reproducing human physiology, advancing therapeutic development, and bridging the human–animal model translational gap. Here, we discuss the promises and achievements with 3D human tissue models in HIV and comorbidity research, along with remaining barriers with respect to cell biology, virology, immunology, and regulatory issues. [ABSTRACT FROM AUTHOR]

Hepatic physiology depends on the liver's complex structural composition which among others, provides high oxygen supply rates, locally differential oxygen tension, endothelial paracrine signaling, as well as residual hemodynamic shear stress to resident hepatocytes. While functional improvements were shown by implementing these factors into hepatic culture systems, direct cause‐effect relationships are often not well characterized—obfuscating their individual contribution in more complex microphysiological systems. By comparing increasingly complex hepatic in vitro culture systems that gradually implement these parameters, we investigate the influence of the cellular microenvironment to overall hepatic functionality in pharmacological applications. Here, hepatocytes were modulated in terms of oxygen tension and supplementation, endothelial coculture, and exposure to fluid shear stress delineated from oxygen influx. Results from transcriptomic and metabolomic evaluation indicate that particularly oxygen supply rates are critical to enhance cellular functionality—with cellular drug metabolism remaining comparable to physiological conditions after prolonged static culture. Endothelial signaling was found to be a major contributor to differential phenotype formation known as metabolic zonation, indicated by WNT pathway activity. Lastly, oxygen‐delineated shear stress was identified to direct cellular fate towards increased hepatic plasticity and regenerative phenotypes at the cost of drug metabolic functionality – in line with regenerative effects observed in vivo. With these results, we provide a systematic evaluation of critical parameters and their impact in hepatic systems. Given their adherence to physiological effects in vivo, this highlights the importance of their implementation in biomimetic devices, such as organ‐on‐a‐chip systems. Considering recent advances in basic liver biology, direct translation of physiological structures into in vitro models is a promising strategy to expand the capabilities of pharmacological models. [ABSTRACT FROM AUTHOR]

Acoustic levitation, which allows contactless manipulation of micro‐objects with ultrasounds, is a promising technique for spheroids formation and culture. This acoustofluidic technique favors cell–cell interactions, away from the walls of the chip, which leads to the spontaneous self‐organization of cells. Using this approach, we generated spheroids of mesenchymal stromal cells, hepatic and endothelial cells, and showed that long‐term culture of cells in acoustic levitation is feasible. We also demonstrated that this self‐organization and its dynamics depended weakly on the acoustic parameters but were strongly dependent on the levitated cell type. Moreover, spheroid organization was modified by actin cytoskeleton inhibitors or calcium‐mediated interaction inhibitors. Our results confirmed that acoustic levitation is a rising technique for fundamental research and biotechnological industrial application in the rapidly growing field of microphysiological systems. It allowed easily obtaining spheroids of specific and predictable shape and size, which could be cultivated over several days, without requiring hydrogels or extracellular matrix. [ABSTRACT FROM AUTHOR]

Abstract Coculture of intestinal bacteria with primary human intestinal epithelium provides a valuable tool for investigating host‐colon bacterial interactions and for testing and screening therapeutics. However, most current intestinal model systems lack key physiological features of the in vivo colon, such as both a proper oxygen microenvironment and a mucus layer. In this work, a new in vitro colonic microphysiological system is demonstrated with a cell‐derived, functional mucus that closely resembles the in vivo colonic mucosa and apical microenvironment by employing an anaerobic air‐liquid interface culture. The human primary colon epithelial cells in this new in vitro system exhibit high cell viability (>98%) with ≈100 µm thick functional mucus layer on average. Successful coculture of model anaerobic gut bacterial strains Lactobacillus rhamnosus GG and Anaerobutyricum hallii without loss in human cell viability demonstrates that this new model can be an invaluable tool for future studies of the impact of commensal and pathogenic bacteria on the colon.

Microphysiological systems (MPSs) have attracted increasing attention as a method for simulating in vitro drug efficiency. In particular, the interaction between liver and intestine tissues is one of the primary targets since they are closely involved in drug absorption and metabolism. However, most of the intestine-liver MPSs reported previously require pumps, electrodes, and porous membranes for co-culture of cells and evaluation of intestinal permeability (i.e., Trans-Epithelial Electrical Resistance, TEER), requiring complex manufacturing processes and operations. In this study, we report an all-polydimethylsiloxane (PDMS) co-culture microfluidic device, connecting microchamber-based paracellular transport assay on gut microtissues to liver tissues matured on the same device. On one side of the device, HepaRG cells are confined within thin parallel grooves that induce their differentiation into hepatocytes. The other side of the device is connected with microchannels to the liver side and includes the gut tissues, organized above microchambers. Such microchambers allow the evaluation of paracellular permeability by fluorescence imaging. Thanks to the microfluidic device we investigated changes in intestinal permeability induced by differentiated hepatocyte excretion and found that Caco-2 permeability was decreased when co-culture with HepaRG. Due to its simplicity and straightforward implementation, this method is anticipated as an innovative and efficient approach to assess tissue barrier function in multi-organ on-chip experiments.

Abstract Endothelial dysfunction is a critical feature of acute respiratory distress syndrome (ARDS) associated with higher disease severity and worse outcomes. Preclinical in vivo models of sepsis and ARDS have failed to yield useful therapies in humans, perhaps due to interspecies differences in inflammatory responses and heterogeneity of human host responses. Use of microphysiological systems (MPS) to investigate lung endothelial function may shed light on underlying mechanisms and targeted treatments for ARDS. We assessed the response to plasma from critically ill sepsis patients in our lung endothelial MPS through measurement of endothelial permeability, expression of adhesion molecules, and inflammatory cytokine secretion. Sepsis plasma induced areas of endothelial cell (EC) contraction, loss of cellular coverage, and luminal defects. EC barrier function was significantly worse following incubation with sepsis plasma compared to healthy plasma. EC ICAM‐1 expression, IL‐6 and soluble ICAM‐1 secretion increased significantly more after incubation with sepsis plasma compared with healthy plasma. Plasma from sepsis patients who developed ARDS further increased IL‐6 and sICAM‐1 compared to plasma from sepsis patients without ARDS and healthy plasma. Our results demonstrate the proof of concept that lung endothelial MPS can enable interrogation of specific mechanisms of endothelial dysfunction that promote ARDS in sepsis patients.

IntroductionAs the body’s first line of defense against disease and infection, neutrophils must efficiently navigate to sites of inflammation; however, neutrophil dysregulation contributes to the pathogenesis of numerous diseases that leave people susceptible to infections. Many of these diseases are also associated with changes to the protein composition of the extracellular matrix. While it is known that neutrophils and endothelial cells, which play a key role in neutrophil activation, are sensitive to the mechanical and structural properties of the extracellular matrix, our understanding of how protein composition in the matrix affects the neutrophil response to infection is incomplete.MethodsTo investigate the effects of extracellular matrix composition on the neutrophil response to infection, we used an infection-on-a-chip microfluidic device that replicates a portion of a blood vessel endothelium surrounded by a model extracellular matrix. Model blood vessels were fabricated by seeding human umbilical vein endothelial cells on 2, 4, or 6 mg/mL type I collagen hydrogels. Primary human neutrophils were loaded into the endothelial lumens and stimulated by adding the bacterial pathogen Pseudomonas aeruginosa to the surrounding matrix.ResultsCollagen concentration did not affect the cell density or barrier function of the endothelial lumens. Upon infectious challenge, we found greater neutrophil extravasation into the 4 mg/mL collagen gels compared to the 6 mg/mL collagen gels. We further found that extravasated neutrophils had the highest migration speed and distance in 2mg/mL gels and that these values decreased with increasing collagen concentration. However, these phenomena were not observed in the absence of an endothelial lumen. Lastly, no differences in the percent of extravasated neutrophils producing reactive oxygen species were observed across the various collagen concentrations.DiscussionOur study suggests that neutrophil extravasation and migration in response to an infectious challenge are regulated by collagen concentration in an endothelial cell-dependent manner. The results demonstrate how the mechanical and structural aspects of the tissue microenvironment affect the neutrophil response to infection. Additionally, these findings underscore the importance of developing and using microphysiological systems for studying the regulatory factors that govern the neutrophil response.

A microphysiological system (MPS) is an in vitro culture technology that reproduces the physiological microenvironment and functionality of humans and is expected to be applied for drug screening. In this study, we developed an MPS for the structured culture of human iPSC-derived sensory neurons and then predicted drug-induced neurotoxicity by morphological deep learning. Using human iPSC-derived sensory neurons, after the administration of representative anti-cancer drugs, the toxic effects on soma and axons were evaluated by an AI model with neurite images. Significant toxicity was detected in positive drugs and could be classified by different effects on soma or axons, suggesting that the current method provides an effective evaluation of chemotherapy-induced peripheral neuropathy. The results of neurofilament light chain expression changes in the MPS device also agreed with clinical reports. Therefore, the present MPS combined with morphological deep learning is a useful platform for in vitro peripheral neurotoxicity assessment.

Drug-induced proximal tubule (PT) injury remains a serious safety concern throughout drug development. Traditional in vitro 2-dimensional (2D) and preclinical in vivo models often fail to predict drug-related injuries presented in clinical trials. Various 3-dimensional (3D) microphysiological systems (MPSs) have been developed to mimic physiologically relevant properties, enabling them to be more predictive toward nephrotoxicity. To explore the capabilities of an MPS across species, we compared cytotoxicity in hRPTEC/TERT1s and rat primary proximal tubular epithelial cells (rPPTECs) following exposure to zoledronic acid and ibandronate (62.5–500 µM), and antibiotic polymyxin B (PMB) (50 and 250 µM, respectively). For comparison, we investigated cytotoxicity using 2D cultured hRPTEC/TERT1s and rPPTECs following exposure to the same drugs, including overlapping concentrations, as their 3D counterparts. Regardless of the in vitro model, bisphosphonate-exposed rPPTECs exhibited cytotoxicity quicker than hRPTEC/TERT1s. PMB was less sensitive toward nephrotoxicity in rPPTECs than hRPTEC/TERT1s, demonstrating differences in species sensitivity within both 3D and 2D models. Generally, 2D cultured cells experienced faster drug-induced cytotoxicity compared to the MPSs, suggesting that MPSs can be advantageous for longer-term drug-exposure studies, if warranted. Furthermore, ibandronate-exposed hRPTEC/TERT1s and rPPTECs produced higher levels of inflammatory and kidney injury biomarkers compared to zoledronic acid, indicating that ibandronate induces acute kidney injury, but also a potential protective response since ibandronate is less toxic than zoledronic acid. Our study suggests that the MPS model can be used for preclinical screening of compounds prior to animal studies and human clinical trials. [ABSTRACT FROM AUTHOR]

Female breast cancer accounts for 15.2% of all new cancer cases in the United States, with a continuing increase in incidence despite efforts to discover new targeted therapies. With an approximate failure rate of 85% for therapies in the early phases of clinical trials, there is a need for more translatable, new preclinical in vitro models that include cellular heterogeneity, extracellular matrix, and human-derived biomaterials. Specifically, adipose tissue and its resident cell populations have been identified as necessary attributes for current preclinical models. Adipose-derived stromal/stem cells (ASCs) and mature adipocytes are a normal part of the breast tissue composition and not only contribute to normal breast physiology but also play a significant role in breast cancer pathophysiology. Given the recognized pro-tumorigenic role of adipocytes in tumor progression, there remains a need to enhance the complexity of current models and account for the contribution of the components that exist within the adipose stromal environment to breast tumorigenesis. This review article captures the current landscape of preclinical breast cancer models with a focus on breast cancer microphysiological system (MPS) models and their counterpart patient-derived xenograft (PDX) models to capture patient diversity as they relate to adipose tissue. [ABSTRACT FROM AUTHOR]

Pancreatic islet transplantation presents a promising therapy for individuals suffering from type 1 diabetes. To maintain the function of transplanted islets in vivo, it is imperative to induce angiogenesis. However, the mechanisms underlying angiogenesis triggered by islets remain unclear. In this study, we introduced a microphysiological system to study the angiogenic capacity and dynamics of individual islets. The system, which features an open‐top structure, uniquely facilitates the inoculation of islets and the longitudinal observation of vascular formation in in vivo like microenvironment with islet‐endothelial cell communication. By leveraging our system, we discovered notable islet−islet heterogeneity in the angiogenic capacity. Transcriptomic analysis of the vascularized islets revealed that islets with high angiogenic capacity exhibited upregulation of genes related to insulin secretion and downregulation of genes related to angiogenesis and fibroblasts. In conclusion, our microfluidic approach is effective in characterizing the vascular formation of individual islets and holds great promise for elucidating the angiogenic mechanisms that enhance islet transplantation therapy. [ABSTRACT FROM AUTHOR]

Intestinal absorption is a complex process involving the permeability of the epithelial barrier, efflux transporter activity, and intestinal metabolism. Identifying the key factors that govern intestinal absorption for each investigational drug is crucial. To assess and predict intestinal absorption in humans, it is necessary to leverage appropriate in vitro systems. Traditionally, Caco-2 monolayer systems and intestinal Ussing chamber studies have been considered the 'gold standard' for studying intestinal absorption. However, these methods have limitations that hinder their universal use in drug discovery and development. Recently, there has been an increasing number of reports on complex in vitro models (CIVMs) using human intestinal organoids derived from intestinal tissue specimens or iPSC-derived enterocytes plated on 2D or 3D in microphysiological systems. These CIVMs provide a more physiologically relevant representation of key ADME-related proteins compared to conventional in vitro methods. They hold great promise for use in drug discovery and development due to their ability to replicate the expressions and functions of these proteins. This review highlights recent advances in gut CIVMs employing intestinal organoid model systems compared to conventional methods. It is important to note that each CIVM should be tailored to the investigational drug properties and research questions at hand. [ABSTRACT FROM AUTHOR]

Impaired white adipose tissue (WAT) function has been recognized as a critical early event in obesity-driven disorders, but high buoyancy, fragility, and heterogeneity of primary adipocytes have largely prevented their use in drug discovery efforts highlighting the need for human stem cell-based approaches. Here, human stem cells are utilized to derive metabolically functional 3D adipose tissue (iADIPO) in a microphysiological system (MPS). Surprisingly, previously reported WAT differentiation approaches create insulin resistant WAT ill-suited for type-2 diabetes mellitus drug discovery. Using three independent insulin sensitivity assays, i.e., glucose and fatty acid uptake and suppression of lipolysis, as the functional readouts new differentiation conditions yielding hormonally responsive iADIPO are derived. Through concomitant optimization of an iADIPO-MPS, it is abled to obtain WAT with more unilocular and significantly larger (≈40%) lipid droplets compared to iADIPO in 2D culture, increased insulin responsiveness of glucose uptake (≈2-3 fold), fatty acid uptake (≈3-6 fold), and ≈40% suppressing of stimulated lipolysis giving a dynamic range that is competent to current in vivo and ex vivo models, allowing to identify both insulin sensitizers and desensitizers.

Abstract Background Calcific aortic valve disease (CAVD) causes an increasing health burden in the 21st century due to aging population. The complex pathophysiology remains to be understood to develop novel prevention and treatment strategies. Microphysiological systems (MPSs), also known as organ-on-chip or lab-on-a-chip systems, proved promising in bridging in vitro and in vivo approaches by applying integer AV tissue and modelling biomechanical microenvironment. This study introduces a novel MPS comprising different micropumps in conjunction with a tissue-incubation-chamber (TIC) for long-term porcine and human AV incubation (pAV, hAV). Results Tissue cultures in two different MPS setups were compared and validated by a bimodal viability analysis and extracellular matrix transformation assessment. The MPS-TIC conjunction proved applicable for incubation periods of 14–26 days. An increased metabolic rate was detected for pulsatile dynamic MPS culture compared to static condition indicated by increased LDH intensity. ECM changes such as an increase of collagen fibre content in line with tissue contraction and mass reduction, also observed in early CAVD, were detected in MPS-TIC culture, as well as an increase of collagen fibre content. Glycosaminoglycans remained stable, no significant alterations of α-SMA or CD31 epitopes and no accumulation of calciumhydroxyapatite were observed after 14 days of incubation. Conclusions The presented ex vivo MPS allows long-term AV tissue incubation and will be adopted for future investigation of CAVD pathophysiology, also implementing human tissues. The bimodal viability assessment and ECM analyses approve reliability of ex vivo CAVD investigation and comparability of parallel tissue segments with different treatment strategies regarding the AV (patho)physiology.

The skin is the largest organ in the human body, and histologically consists of the epidermis, dermis and subcutaneous tissue. Humans maintain a cooperative symbiotic relationship with their skin microbiota, a complex community of bacteria, fungi and viruses that live on the surface of the skin, and which act as a barrier to protect the body from the inside and outside. The skin is a 'habitat' and vast 'ecosystem' inhabited by countless microbes; as such, relationships have been forged through millions of years of coevolution. It is not surprising then that microbes are key participants in shaping and maintaining essential physiological processes. In addition to maintaining barrier function, the unique symbiotic microbiota that colonizes the skin increases the immune response and provides protection against pathogenic microbes. This review examines our current understanding of skin microbes in shaping and enhancing the skin barrier, as well as skin microbiome–host interactions and their roles in skin diseases, such as atopic dermatitis (AD). We also report on the current status of AD therapeutic drugs that target the skin microbiome, related research on current therapeutic strategies, and the limitations and future considerations of skin microbiome research. In particular, as a future strategy, we discuss the need for a skin‐on‐a‐chip‐based microphysiological system research model amenable to biomimetic in vitro studies and human skin equivalent models, including skin appendages. [ABSTRACT FROM AUTHOR]

Human heart tissues grown as three-dimensional spheroids and consisting of different cardiac cell types derived from pluripotent stem cells (hiPSCs) recapitulate aspects of human physiology better than standard two-dimensional models in vitro. They typically consist of less than 5000 cells and are used to measure contraction kinetics although not contraction force. By contrast, engineered heart tissues (EHTs) formed around two flexible pillars, can measure contraction force but conventional EHTs often require between 0.5 and 2 million cells. This makes large-scale screening of many EHTs costly. Our goals here were (i) to create a physiologically relevant model that required fewer cells than standard EHTs making them less expensive, and (ii) to ensure that this miniaturized model retained correct functionality. We demonstrated that fully functional EHTs could be generated from physiologically relevant combinations of hiPSC-derived cardiomyocytes (70%), cardiac fibroblasts (15%) and cardiac endothelial cells (15%), using as few as 1.6 × 104 cells. Our results showed that these EHTs were viable and functional up to 14 days after formation. The EHTs could be electrically paced in the frequency range between 0.6 and 3 Hz, with the optimum between 0.6 and 2 Hz. This was consistent across three downscaled EHT sizes tested. These findings suggest that miniaturized EHTs could represent a cost-effective microphysiological system for disease modelling and examining drug responses particularly in secondary screens for drug discovery. • Co-culture based on three cardiac cell types derived from hiPSCs form functional Engineered Heart Tissues (EHTs). • Miniaturized EHTs with only 16,000 cells maintain functionality. • Three-fold miniaturized EHTs maintain the same contraction kinetics as their larger counterparts. • Downscaling of EHTs does not decrease force delivered per cardiomyocyte. [ABSTRACT FROM AUTHOR]

Since every biological system requires capillaries to support its oxygenation, design of engineered preclinical models of such systems, for example, vascularized microphysiological systems (vMPS) have gained attention enhancing the physiological relevance of human biology and therapies. But the physiology and function of formed vessels in the vMPS is currently assessed by non‐standardized, user‐dependent, and simple morphological metrics that poorly relate to the fundamental function of oxygenation of organs. Here, a chained neural network is engineered and trained using morphological metrics derived from a diverse set of vMPS representing random combinations of factors that influence the vascular network architecture of a tissue. This machine‐learned algorithm outputs a singular measure, termed as vascular network quality index (VNQI). Cross‐correlation of morphological metrics and VNQI against measured oxygen levels within vMPS revealed that VNQI correlated the most with oxygen measurements. VNQI is sensitive to the determinants of vascular networks and it consistently correlates better to the measured oxygen than morphological metrics alone. Finally, the VNQI is positively associated with the functional outcomes of cell transplantation therapies, shown in the vascularized islet‐chip challenged with hypoxia. Therefore, adoption of this tool will amplify the predictions and enable standardization of organ‐chips, transplant models, and other cell biosystems. [ABSTRACT FROM AUTHOR]

Several anticancer drugs used in cancer therapy induce chemotherapy-induced peripheral neuropathy (CIPN), leading to dose reduction or therapy cessation. Consequently, there is a demand for an in vitro assessment method to predict CIPN and mechanisms of action (MoA) in drug candidate compounds. In this study, a method assessing the toxic effects of anticancer drugs on soma and axons using deep learning image analysis is developed, culturing primary rat dorsal root ganglion neurons with a microphysiological system (MPS) that separates soma from neural processes and training two artificial intelligence (AI) models on soma and axonal area images. Exposing the control compound DMSO, negative compound sucrose, and known CIPN-causing drugs (paclitaxel, vincristine, oxaliplatin, suramin, bortezomib) for 24 h, results show the somatic area-learning AI detected significant cytotoxicity for paclitaxel (* p < 0.05) and oxaliplatin (* p < 0.05). In addition, axonal area-learning AI detected significant axonopathy with paclitaxel (* p < 0.05) and vincristine (* p < 0.05). Combining these models, we detected significant toxicity in all CIPN-causing drugs (** p < 0.01) and could classify anticancer drugs based on their different MoA on neurons, suggesting that the combination of MPS-based culture segregating soma and axonal areas and AI image analysis of each area provides an effective evaluation method to predict CIPN from low concentrations and infer the MoA. [ABSTRACT FROM AUTHOR]

Metastatic renal cell carcinoma (RCC) remains an incurable disease for most patients highlighting an urgent need for new treatments. However, the preclinical investigation of new therapies is limited by traditional two-dimensional (2D) cultures which do not recapitulate the properties of tumor cells within a collagen extracellular matrix (ECM), while human tumor xenografts are time-consuming, expensive and lack adaptive immune cells. We report a rapid and economical human microphysiological system (“RCC-on-a-chip”) to investigate therapies targeting RCC spheroids in a 3D collagen ECM. We first demonstrate that culture of RCC cell lines A498 and RCC4 in a 3D collagen ECM more faithfully reproduces the gene expression program of primary RCC tumors compared to 2D culture. We next used bortezomib as a cytotoxin to develop automated quantification of dose-dependent tumor spheroid killing. We observed that viable RCC spheroids exhibited collective migration within the ECM and demonstrated that our 3D system can be used to identify compounds that inhibit spheroid collective migration without inducing cell death. Finally, we demonstrate the RCC-on-a-chip as a platform to model the trafficking of tumor-reactive T cells into the ECM and observed antigen-specific A498 spheroid killing by engineered human CD8+ T cells expressing an ROR1-specific chimeric antigen receptor. In summary, the phenotypic differences between the 3D versus 2D environments, rapid imaging-based readout, and the ability to carefully study the impact of individual variables with quantitative rigor will encourage adoption of the RCC-on-a-chip system for testing a wide range of emerging therapies for RCC.

Abstract Since every biological system requires capillaries to support its oxygenation, design of engineered preclinical models of such systems, for example, vascularized microphysiological systems (vMPS) have gained attention enhancing the physiological relevance of human biology and therapies. But the physiology and function of formed vessels in the vMPS is currently assessed by non‐standardized, user‐dependent, and simple morphological metrics that poorly relate to the fundamental function of oxygenation of organs. Here, a chained neural network is engineered and trained using morphological metrics derived from a diverse set of vMPS representing random combinations of factors that influence the vascular network architecture of a tissue. This machine‐learned algorithm outputs a singular measure, termed as vascular network quality index (VNQI). Cross‐correlation of morphological metrics and VNQI against measured oxygen levels within vMPS revealed that VNQI correlated the most with oxygen measurements. VNQI is sensitive to the determinants of vascular networks and it consistently correlates better to the measured oxygen than morphological metrics alone. Finally, the VNQI is positively associated with the functional outcomes of cell transplantation therapies, shown in the vascularized islet‐chip challenged with hypoxia. Therefore, adoption of this tool will amplify the predictions and enable standardization of organ‐chips, transplant models, and other cell biosystems.

Background: Calcific aortic valve disease (CAVD) causes an increasing health burden in the 21st century due to aging population. The complex pathophysiology remains to be understood to develop novel prevention and treatment strategies. Microphysiological systems (MPSs), also known as organ-on-chip or lab-on-a-chip systems, proved promising in bridging in vitro and in vivo approaches by applying integer AV tissue and modelling biomechanical microenvironment. This study introduces a novel MPS comprising different micropumps in conjunction with a tissue-incubation-chamber (TIC) for long-term porcine and human AV incubation (pAV, hAV). Results: Tissue cultures in two different MPS setups were compared and validated by a bimodal viability analysis and extracellular matrix transformation assessment. The MPS-TIC conjunction proved applicable for incubation periods of 14–26 days. An increased metabolic rate was detected for pulsatile dynamic MPS culture compared to static condition indicated by increased LDH intensity. ECM changes such as an increase of collagen fibre content in line with tissue contraction and mass reduction, also observed in early CAVD, were detected in MPS-TIC culture, as well as an increase of collagen fibre content. Glycosaminoglycans remained stable, no significant alterations of α-SMA or CD31 epitopes and no accumulation of calciumhydroxyapatite were observed after 14 days of incubation. Conclusions: The presented ex vivo MPS allows long-term AV tissue incubation and will be adopted for future investigation of CAVD pathophysiology, also implementing human tissues. The bimodal viability assessment and ECM analyses approve reliability of ex vivo CAVD investigation and comparability of parallel tissue segments with different treatment strategies regarding the AV (patho)physiology. [ABSTRACT FROM AUTHOR]

For ethical, economical, and scientific reasons, animal experimentation, used to evaluate the potential neurotoxicity of chemicals before their release in the market, needs to be replaced by new approach methodologies. To illustrate the use of new approach methodologies, the human induced pluripotent stem cell-derived 3D model BrainSpheres was acutely (48 h) or repeatedly (7 days) exposed to amiodarone (0.625-15 µM), a lipophilic antiarrhythmic drug reported to have deleterious effects on the nervous system. Neurotoxicity was assessed using transcriptomics, the immunohistochemistry of cell type-specific markers, and real-time reverse transcription-polymerase chain reaction for various genes involved in the lipid metabolism. By integrating distribution kinetics modeling with neurotoxicity readouts, we show that the observed time- and concentrationdependent increase in the neurotoxic effects of amiodarone is driven by the cellular accumulation of amiodarone after repeated dosing. The development of a compartmental in vitro distribution kinetics model allowed us to predict the change in cell-associated concentrations in BrainSpheres with time and for different exposure scenarios. The results suggest that human cells are intrinsically more sensitive to amiodarone than rodent cells. Amiodaroneinduced regulation of lipid metabolism genes was observed in brain cells for the first time. Astrocytes appeared to be the most sensitive human brain cell type in vitro. In conclusion, assessing readouts at different molecular levels after the repeat dosing of human induced pluripotent stem cell-derived BrainSpheres in combination with the compartmental modeling of in vitro kinetics provides a mechanistic means to assess neurotoxicity pathways and refine chemical safety assessment for humans. [ABSTRACT FROM AUTHOR]

Angiogenesis is a complex process that is required for development and tissue regeneration and it may be affected by many pathological conditions. Chemicals and drugs can impact formation and maintenance of the vascular networks; these effects may be both desirable (e.g., anti-cancer drugs) or unwanted (e.g., side effects of drugs). A number of in vivo and in vitro models exist for studies of angiogenesis and endothelial cell function, including organ-on-a-chip microphysiological systems. An arrayed organ-on-a-chip platform on a 96-well plate footprint that incorporates perfused microvessels, with and without tumors, was recently developed and it was shown that survival of the surrounding tissue was dependent on delivery of nutrients through the vessels. Here we describe a technology transfer of this complex microphysiological model between laboratories and demonstrate that reproducibility and robustness of these tissue chip-enabled experiments depend primarily on the source of the endothelial cells. The model was highly reproducible between laboratories and was used to demonstrate the advantages of the perfusable vascular networks for drug safety evaluation. As a proof-of-concept, we tested Fluorouracil (1-1,000 μM), Vincristine (1-1,000 nM), and Sorafenib (0.1-100 μM), in the perfusable and non-perfusable micro-organs, and in a colon cancer-containing micro-tumor model. Tissue chip experiments were compared to the traditional monolayer cultures of endothelial or tumor cells. These studies showed that human in vitro vascularized micro-organ and micro-tumor models are reproducible organ-on-a-chip platforms for studies of anticancer drugs. The data from the 3D models confirmed advantages of the physiological environment as compared to 2D cell cultures. We demonstrated how these models can be translated into practice by verifying that the endothelial cell source and passage are critical elements for establishing a perfusable model.

Background and Aims: Hypoxia in the intestinal epithelium can be caused by acute ischemic events or chronic inflammation in which immune cell infiltration produces inflammatory hypoxia starving the mucosa of oxygen. The epithelium has the capacity to regenerate after some ischemic and inflammatory conditions suggesting that intestinal stem cells (ISCs) are highly tolerant to acute and chronic hypoxia; however, the impact of hypoxia on human ISC (hISC) function has not been reported. Here we present a new microphysiological system (MPS) to investigate how hypoxia affects hISCs from healthy donors and test the hypothesis that prolonged hypoxia modulates how hISCs respond to inflammation-associated interleukins (ILs). Methods: hISCs were exposed to

Abstract Background Sprouting angiogenesis is an important mechanism for morphogenetic phenomena, including organ development, wound healing, and tissue regeneration. In regenerative medicine, therapeutic angiogenesis is a clinical solution for recovery from ischemic diseases. Mesenchymal stem cells (MSCs) have been clinically used given their pro-angiogenic effects. MSCs are reported to promote angiogenesis by differentiating into pericytes or other vascular cells or through cell–cell communication using multiple protein–protein interactions. However, how MSCs physically contact and move around ECs to keep the sprouting angiogenesis active remains unknown. Methods We proposed a novel framework of EC–MSC crosstalk analysis using human umbilical vein endothelial cells (HUVECs) and MSCs obtained from mice subcutaneous adipose tissue on a 3D in vitro model, microvessel-on-a-chip, which allows cell-to-tissue level study. The microvessels were fabricated and cultured for 10 days in a collagen matrix where MSCs were embedded. Results Immunofluorescence imaging using a confocal laser microscope showed that MSCs smoothed the surface of the microvessel and elongated the angiogenic sprouts by binding to the microvessel’s specific microstructures. Additionally, three-dimensional modeling of HUVEC–MSC intersections revealed that MSCs were selectively located around protrusions or roots of angiogenic sprouts, whose surface curvature was excessively low or high, respectively. Conclusions The combination of our microvessel-on-a-chip system for 3D co-culture and image-based crosstalk analysis demonstrated that MSCs are selectively localized to concave–convex surfaces on scaffold structures and that they are responsible for the activation and stabilization of capillary vessels.