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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]

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]

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]

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]

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]

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]

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]

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]

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]

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]

Disorders of the synovial joint, such as osteoarthritis (OA) and rheumatoid arthritis (RA), afflict a substantial proportion of the global population. However, current clinical management has not been focused on fully restoring the native function of joints. Organ-on-chip (OoC), also called a microphysiological system, which typically accommodates multiple human cell-derived tissues/organs under physiological culture conditions, is an emerging platform that potentially overcomes the limitations of current models in developing therapeutics. Herein, we review major steps in the generation of OoCs for studying arthritis, discuss the challenges faced when these novel platforms enter the next phase of development and application, and present the potential for OoC technology to investigate the pathogenesis of joint diseases and the development of efficacious therapies. A recently developed human stem cell-derived, joint-mimicking organ-on-chip (OoC) comprising four 3D tissues demonstrated its ability to model joint inflammation and test potential disease-modifying drugs. Joint OoCs developed thus far fall into four broad categories: those modeling inflammation, simulating mechanical stimuli, recapitulating immune response, and studying genetic disposition. Accumulating evidence reveals the active, bidirectional crosstalk between different joint elements, highlighting the importance of incorporating multiple tissue components in joint OoCs. Recent advances in induced pluripotent stem cell-based tissue engineering pave the way for building personalized joint OoC to address patient heterogeneity. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is a painful and disabling joint disease affecting millions worldwide. The lack of clinically relevant models limits our ability to predict therapeutic outcomes prior to clinical trials, where most drugs fail. Therefore, there is a need for a model that accurately recapitulates the whole-joint disease nature of OA in humans. Emerging microphysiological systems provide a new opportunity. We recently established a miniature knee joint system, known as the miniJoint, in which human bone-marrow-derived mesenchymal stem cells (hBMSCs) were used to create an osteochondral complex, synovial-like fibrous tissue, and adipose tissue analogs. In this study, we explored the potential of the miniJoint in developing novel treatments for OA by testing the hypothesis that co-treatment with anti-inflammation and chondroinducing agents can suppress joint inflammation and associated cartilage degradation. Specifically, we created a "synovitis"-relevant OA model in the miniJoint by treating synovial-like tissues with interleukin-1β (IL-1β), and then a combined treatment of oligodeoxynucleotides (ODNs) suppressing the nuclear factor kappa beta (NF-κB) genetic pathway and bone morphogenic protein-7 (BMP-7) was introduced. The combined treatment with BMP-7 and ODNs reduced inflammation in the synovial-like fibrous tissue and showed an increase in glycosaminoglycan formation in the cartilage portion of the osteochondral complex. For the first time, this study demonstrated the potential of the miniJoint in developing disease-modifying OA drugs. The therapeutic efficacy of co-treatment with NF-κB ODNs and BMP-7 can be further validated in future clinical studies. [ABSTRACT FROM AUTHOR]

Introduction: Obesity is associated with increased breast cancer incidence, recurrence, and mortality. Adipocytes and adipose-derived stem cells (ASCs), two resident cell types in adipose tissue, accelerate the early stages of breast cancer progression. It remains unclear whether obesity plays a role in the subsequent escape of malignant breast cancer cells into the local circulation. Methods: We engineered models of human breast tumors with adipose stroma that exhibited different obesity-specific alterations. We used these models to assess the invasion and escape of breast cancer cells into an empty, blind-ended cavity (as a mimic of a lymphatic vessel) for up to sixteen days. Results: Lean and obese donor-derived adipose stroma hastened escape to similar extents. Moreover, a hypertrophic adipose stroma did not affect the rate of adipose-induced escape. When admixed directly into the model tumors, lean and obese donor-derived ASCs hastened escape similarly. Conclusions: This study demonstrates that the presence of adipose cells, independently of the obesity status of the adipose tissue donor, hastens the escape of human breast cancer cells in multiple models of obesity-associated breast cancer. [ABSTRACT FROM AUTHOR]

Simple Summary: Organ-on-a-chip models, or organ chips, are tiny devices designed to accurately recreate the natural physiology and mechanical forces that cells experience in the human body. Similar to computer microchips, though carrying fluid through channels instead of an electric current, organ-chips are lined with living human cells and their tiny channels can reproduce blood and/or airflow just as in the human body. Their flexibility allows the chips to recreate breathing motions or undergo muscle contractions. This new and rapidly expanding field of research provides a unique opportunity to build models of human organs and to study how cancer cells develop and spread within them. As these chips are simpler and cheaper than animal models of cancer, they could significantly increase the speed of drug discovery and testing in cancer research. This exciting potential has led to the rapid development of this technology in the United Kingdom, with active research on a range of cancer types. This review covers the broad sweep of organ-chip research in the UK, and the network of researchers and companies being developed. Finally, it concludes with a perspective on the future directions in the field as researchers aim to bring about a leap forward in cancer therapies. Organ-on-chip systems are capable of replicating complex tissue structures and physiological phenomena. The fine control of biochemical and biomechanical cues within these microphysiological systems provides opportunities for cancer researchers to build complex models of the tumour microenvironment. Interest in applying organ chips to investigate mechanisms such as metastatsis and to test therapeutics has grown rapidly, and this review draws together the published research using these microfluidic platforms to study cancer. We focus on both in-house systems and commercial platforms being used in the UK for fundamental discovery science and therapeutics testing. We cover the wide variety of cancers being investigated, ranging from common carcinomas to rare sarcomas, as well as secondary cancers. We also cover the broad sweep of different matrix microenvironments, physiological mechanical stimuli and immunological effects being replicated in these models. We examine microfluidic models specifically, rather than organoids or complex tissue or cell co-cultures, which have been reviewed elsewhere. However, there is increasing interest in incorporating organoids, spheroids and other tissue cultures into microfluidic organ chips and this overlap is included. Our review includes a commentary on cancer organ-chip models being developed and used in the UK, including work conducted by members of the UK Organ-on-a-Chip Technologies Network. We conclude with a reflection on the likely future of this rapidly expanding field of oncological research. [ABSTRACT FROM AUTHOR]

The article in Advanced Materials Technologies introduces a high-throughput microphysiological platform for modeling the outer blood-retinal barrier in a 96-well format. This system allows for the evaluation of retinal barrier function, cellular interactions, gene expression, and protein secretion, making it valuable for drug screening and studying retinal diseases like age-related macular degeneration. The research was conducted by a team of authors led by Sanghee Yoo and Noo Li Jeon. [Extracted from the article]

To compensate the limitation of animal models, new models were proposed for drug safety evaluation to refine and reduce existing models. To mimic drug absorption and metabolism and predict toxicokinetic and toxic effects in an in vitro intestinal-liver microphysiological system (MPS), we constructed an intestinal-liver-on-chip and detected the acute liver injury process after an overdose of acetaminophen (APAP). Caco-2 and HT29-MTX-E12 cell lines were utilized to establish intestinal equivalents, along with HepG2, HUVEC-T1, and THP-1 induced by PMA and human hepatic stellate cell to establish liver equivalents. The APAP concentration was determined using high-performance liquid chromatography, and the toxicokinetic parameters were fitted using the non-compartmental analysis method by Phoenix. Changes in liver injury biomarkers aspartate aminotransferase and alanine aminotransferase, and liver function marker albumin indicated that the short-term culture of the two organs-on-chip model was stable for 4 days. Reactive oxygen species signaling was enhanced after APAP administration, along with decreased mitochondrial membrane potential, activated caspase-3, and enhanced p53 signaling, indicating a toxic response induced by APAP overdose. In the gut-liver MPS model, we fitted the toxicokinetic parameters and simulated the hepatotoxicity procedure following an APAP overdose, which will facilitate the organ-on-chips application in drug toxicity assays. An illustration of the gut-liver microphysiological system and a proof-of-concept for drug absorption and subsequent hepatotoxic examination with acetaminophen as a prototypical model compound. [Display omitted] [ABSTRACT FROM AUTHOR]

Well plates are widely used in biological experiments, particularly in pharmaceutical sciences and cell biology. Its popularity stems from its versatility to support a variety of fluorescent markers for high throughput monitoring of cellular activities. However, using fluorescent markers in traditional well plates has its own challenges, namely, they can be potentially toxic to cells, and thus, may perturb their biological functions; and it is difficult to monitor multiple analytes concurrently and in real-time inside each well. This paper presents a fully instrumented microphysiological system with integrated sensors (IMSIS) with a similar well format. Each well in the microphysiological system has a set of sensors for monitoring multiple metabolic analytes in real-time. The IMSIS platform is supported by integrated bioelectronic circuits and a graphical user interface for easy user configuration and monitoring. The system has integrated microfluidics to maintain its microphysiological environment within each well. The IMSIS platform currently incorporates O 2 , H 2 O 2 , and pH sensors inside each well, allowing up to six wells to perform concurrent measurements in real-time. Furthermore, the architecture is scalable to achieve an even higher level of throughput. The miniaturized design ensures portability, suitable for small offices and field applications. The IMSIS platform was successfully used to monitor in real-time the mitochondrial functions of live bovine embryos in O 2 consumption, H 2 O 2 release as an indication of ROS production, and extracellular acidity changes before and after the introduction of external substrates. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Introduction: Lymphatic vasculature provides a route for metastasis to secondary sites in the body. The role of the lymphatic endothelium in mediating the entry of breast cancer cells into the vasculature remains unclear. Methods: In this study, we formed aggregates of MDA-MB-231 human breast carcinoma cells next to human microvascular lymphatic endothelial cell (LEC)-lined cavities in type I collagen gels to model breast microtumors and lymphatic vessels, respectively. We tracked invasion and escape of breast microtumors into engineered lymphatics or empty cavities under matched flow rates for up to sixteen days. Results: After coming into contact with a lymphatic vessel, tumor cells escape by moving between the endothelium and the collagen wall, between endothelial cells, and/or into the endothelial lumen. Over time, tumor cells replace the LECs within the vessel wall and create regions devoid of endothelium. The presence of lymphatic endothelium slows breast tumor invasion and escape, and addition of LEC-conditioned medium to tumors is sufficient to reproduce nearly all of these inhibitory effects. Conclusions: This work sheds light on the interactions between breast cancer cells and lymphatic endothelium during vascular escape and reveals an inhibitory role for the lymphatic endothelium in breast tumor invasion and escape. [ABSTRACT FROM AUTHOR]

Tumor metastasis via the circulation requires crossing the vascular barrier twice: first, during intravasation when tumor cells disseminate from the primary site through proximal vasculature, and second, during extravasation, when tumor cells exit the circulation to form distant metastatic seeds. During these key metastatic events, chemomechanical signaling between tumor cells and endothelial cells elicits reciprocal changes in cell morphology and behavior that are necessary to breach the vessel wall. Existing experimental systems have provided a limited understanding of the diverse mechanisms underlying tumor-endothelial interactions during intravasation and extravasation. Recent advances in microphysiological systems have revolutionized the ability to generate miniaturized human tissues with tailored three-dimensional architectures, physiological cell interfaces, and precise chemical and physical microenvironments. By doing so, microphysiological systems enable experimental access to complex morphogenic processes associated with human tumor progression with unprecedented resolution and biological control. Here, we discuss recent examples in which microphysiological systems have been leveraged to reveal new mechanistic insight into cellular and molecular control systems operating at the tumor-endothelial interface during intravasation and extravasation. [ABSTRACT FROM AUTHOR]

Self‐organized microvascular networks (MVNs) have become key to the development of many microphysiological models. However, the self‐organizing nature of this process combined with variations between types or batches of endothelial cells (ECs) often lead to inconsistency or failure to form functional MVNs. Because interstitial flow (IF) has been reported to play a beneficial role in angiogenesis, vasculogenesis, and 3D capillary morphogenesis, the role IF plays during neovessel formation in a customized single‐channel microfluidic chip for which IF has been fully characterized is systematically investigated. Compared to static conditions, MVNs formed under IF have higher vessel density and diameters and greater network perfusability. Through a series of inhibitory experiments, this study demonstrates that IF treatment improves vasculogenesis by ECs through upregulation of matrix metalloproteinase‐2 (MMP‐2). This study then successfully implements a novel strategy involving the interplay between IF and MMP‐2 inhibitor to regulate morphological parameters of the self‐organized MVNs, with vascular permeability and perfusability well maintained. The revealed mechanism and proposed methodology are further validated with a brain MVN model. The findings and methods have the potential to be widely utilized to boost the development of various organotypic MVNs and can be incorporated into related bioengineering applications where perfusable vasculature is desired. [ABSTRACT FROM AUTHOR]

Physiologically relevant and broadly applicable liver cell culture platforms are of great importance in both drug development and disease modeling. Organ-on-a-chip systems offer a promising alternative to conventional, static two-dimensional (2-D) cultures, providing much-needed cues such as perfusion, shear stress, and three-dimensional (3-D) cell-cell communication. However, such devices cover a broad range of complexity both in manufacture and in implementation. In this review, we summarize the key features of the human liver that should be reflected in a physiologically relevant liver-on-a-chip model. We also discuss different material properties of importance in producing liver-on-a-chip devices and summarize recent and current progress in the field, highlighting different types of devices at different levels of complexity. [ABSTRACT FROM AUTHOR]

Diseases of the knee joint such as osteoarthritis (OA) affect all joint elements. An in vitro human cell‐derived microphysiological system capable of simulating intraarticular tissue crosstalk is desirable for studying etiologies/pathogenesis of joint diseases and testing potential therapeutics. Herein, a human mesenchymal stem cell‐derived miniature joint system (miniJoint) is generated, in which engineered osteochondral complex, synovial‐like fibrous tissue, and adipose tissue are integrated into a microfluidics‐enabled bioreactor. This novel design facilitates different tissues communicating while still maintaining their respective phenotypes. The miniJoint exhibits physiologically relevant changes when exposed to interleukin‐1β mediated inflammation, which are similar to observations in joint diseases in humans. The potential of the miniJoint in predicting in vivo efficacy of drug treatment is confirmed by testing the "therapeutic effect" of the nonsteroidal anti‐inflammatory drug, naproxen, as well as four other potential disease‐modifying OA drugs. The data demonstrate that the miniJoint recapitulates complex tissue interactions, thus providing a robust organ chip model for the study of joint pathology and the development of novel therapeutic interventions. [ABSTRACT FROM AUTHOR]

Per - and poly-fluoroalkyl substances (PFAS) have a wide range of elimination half-lives (days to years) in humans, thought to be in part due to variation in proximal tubule reabsorption. While human biomonitoring studies provide important data for some PFAS, renal clearance (CL renal) predictions for hundreds of PFAS in commerce requires experimental studies with in vitro models and physiologically-based in vitro -to- in vivo extrapolation (IVIVE). Options for studying renal proximal tubule pharmacokinetics include cultures of renal proximal tubule epithelial cells (RPTECs) and/or microphysiological systems. This study aimed to compare CL renal predictions for PFAS using in vitro models of varying complexity (96-well plates, static 24-well Transwells and a fluidic microphysiological model, all using human telomerase reverse transcriptase-immortalized and OAT1-overexpressing RPTECs combined with in silico physiologically-based IVIVE. Three PFAS were tested: one with a long half-life (PFOS) and two with shorter half-lives (PFHxA and PFBS). PFAS were added either individually (5 μM) or as a mixture (2 μM of each substance) for 48 h. Bayesian methods were used to fit concentrations measured in media and cells to a three-compartmental model to obtain the in vitro permeability rates, which were then used as inputs for a physiologically-based IVIVE model to estimate in vivo CL renal. Our predictions for human CL renal of PFAS were highly concordant with available values from in vivo human studies. The relative values of CL renal between slow- and faster-clearance PFAS were most highly concordant between predictions from 2D culture and corresponding in vivo values. However, the predictions from the more complex model (with or without flow) exhibited greater concordance with absolute CL renal. Overall, we conclude that a combined in vitro-in silico workflow can predict absolute CL renal values, and effectively distinguish between PFAS with slow and faster clearance, thereby allowing prioritization of PFAS with a greater potential for bioaccumulation in humans. • Some PFAS can bioaccumulate in humans, in part due to slow renal clearance CL renal. • We test use of 2D culture and MPS models to measure CL renal for three PFAS. • Combined with physiologically-based IVIVE, we can accurately predict CL renal. • Our in vitro-in silico workflow can prioritize PFAS that bioaccumulate in humans. [ABSTRACT FROM AUTHOR]

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) have found utility for conducting in vitro drug screening and disease modelling to gain crucial insights into pharmacology or disease phenotype. However, diseases such as atrial fibrillation, affecting >33 M people worldwide, demonstrate the need for cardiac subtype-specific cells. Here, we sought to investigate the base characteristics and pharmacological differences between commercially available chamber-specific atrial or ventricular hiPSC-CMs seeded onto ultra-thin, flexible PDMS membranes to simultaneously measure contractility in a 96 multi-well format. We investigated the effects of GPCR agonists (acetylcholine and carbachol), a Ca2+ channel agonist (S-Bay K8644), an HCN channel antagonist (ivabradine) and K+ channel antagonists (4-AP and vernakalant). We observed differential effects between atrial and ventricular hiPSC-CMs on contractile properties including beat rate, beat duration, contractile force and evidence of arrhythmias at a range of concentrations. As an excerpt of the compound analysis, S-Bay K8644 treatment showed an induced concentration-dependent transient increase in beat duration of atrial hiPSC-CMs, whereas ventricular cells showed a physiological increase in beat rate over time. Carbachol treatment produced marked effects on atrial cells, such as increased beat duration alongside a decrease in beat rate over time, but only minimal effects on ventricular cardiomyocytes. In the context of this chamber-specific pharmacology, we not only add to contractile characterization of hiPSC-CMs but propose a multi-well platform for medium-throughput early compound screening. Overall, these insights illustrate the key pharmacological differences between chamber-specific cardiomyocytes and their application on a multi-well contractility platform to gain insights for in vitro cardiac liability studies and disease modelling. [ABSTRACT FROM AUTHOR]

Human mesenchymal stem cells (MSCs) have attracted attention as a major cellular source for regenerative medicine and preclinical studies. Three-dimensional (3D) cell culture systems can be used to construct physiologically relevant MSC spheroids with enhanced tissue regeneration capacity. However, what aspect of the MSC spheroids retains the regeneration capacity is not yet fully understood. Here, we induced osteoblast differentiation of MSC spheroids and investigated their respiratory activity using electrochemiluminescence (ECL) imaging developed in our previous study. In parallel, the ability of MSC spheroids to induce angiogenesis was investigated in a microfluidic device with a co-culture of endothelial cells. We found that the osteogenic differentiation of MSCs induced changes in their metabolic activity (respiratory activity of the spheroids decreased during differentiation) and lead to the loss of their angiogenesis-inducing ability. In other words, it is suggested that maintaining the stem cell nature with in the MSC spheroids is an important factor for tissue regeneration. In this study, we demonstrated the effective use of ECL imaging and microfluidic systems for the evaluation of MSC spheroids, providing new insights into stem cell tissue engineering. [Display omitted] [ABSTRACT FROM AUTHOR]

Progressive multifocal leukoencephalopathy (PML) is a frequent neurological complication in immunosuppressed patients. PML is caused by the JC virus (JCV), a neurotropic DNA polyomavirus that infects oligodendrocytes and astrocytes, causing inflammation and demyelination which lead to neurological dysfunction. The pathogenesis of PML is poorly understood due to the lack of in vitro or animal models to study mechanisms of disease as the virus most efficiently infects only human cells. We developed a human-derived brain organotypic system (also called brain organoid) to model JCV infection. The model was developed by using human-induced pluripotent stem cells (iPSC) and culturing them in 3D to generate an organotypic model containing neurons, astrocytes, and oligodendrocytes which recapitulates aspects of the environment of the human brain. We infected the brain organoids with the JCV MAD4 strain or cerebrospinal fluid of a patient with PML. The organoids were assessed for evidence of infection by qPCR, immunofluorescence, and electron microscopy at 1, 2, and 3 weeks post-exposure. JCV infection in both JCV MAD4 strain and PML CSF-exposed brain organoids was confirmed by immunocytochemical studies demonstrating viral antigens and electron microscopy showing virion particles in the nuclear compartment of oligodendrocytes and astrocytes. No evidence of neuronal infection was visualized. Infection was also demonstrated by JCV qPCR in the virus-exposed organoids and their media. In conclusion, the brain organoid model of JCV infection establishes a human model suitable for studying the mechanisms of JCV infection and pathogenesis of PML and may facilitate the exploration of therapeutic approaches. [ABSTRACT FROM AUTHOR]

Introduction: Approximately 20–25% of human breast tumors are found within an adipose, rather than fibrous, stroma. Adipose stroma is associated with an increased risk of lymph node metastasis, but the causal association between adipose stroma and metastatic progression in human breast cancer remains unclear. Methods: We used micropatterned type I collagen gels to engineer ~3-mm-long microscale human breast tumors within a stroma that contains adipocytes and adipose-derived stem cells (ASCs) (collectively, "adipose cells"). Invasion and escape of human breast cancer cells into an empty 120-μm-diameter lymphatic-like cavity was used to model interstitial invasion and vascular escape in the presence of adipose cell-derived factors for up to 16 days. Results: We found that adipose cells hasten invasion and escape by 1–2 days and 2–3 days, respectively. These effects were mediated by soluble factors secreted by the adipose cells, and these factors acted directly on tumor cells. Surprisingly, tumor invasion and escape were more strongly induced by ASCs than by adipocytes. Conclusions: This work reveals that both adipocytes and ASCs accelerate the interstitial invasion and escape of human breast cancer cells, and sheds light on the link between adipose stroma and lymphatic metastasis in human breast cancer. [ABSTRACT FROM AUTHOR]

The liver microphysiological system (MPS) model is an in-vitro culture method that mimics physiological blood flow, which enhances basal cellular functions. However, the liver MPS model has not been tested in the preclinical stage because of its obscure utility. It can overcome the major problem of conventional systems--rapid loss of mitochondrial activity in cultured hepatocytes due to limited oxygen supply--by supplying oxygen to cultured hepatocytes using a perfusion device. In this study, we developed a new perfusion culture system that can detect mitochondrial toxicity. Primary mouse hepatocytes were cultured under perfusion condition for 48 hr. The hepatocytes showed increased oxygen consumption and reduced lactate release. These results indicated that the ATP-production pathway was switched from glycolysis to mitochondrial oxidative phosphorylation in the perfusion culture system. Furthermore, ATP levels were considerably reduced in the perfusion culture system after exposure to phenformin, a mitochondrial complex I inhibitor. To summarize, the perfusion culture system could improve the mitochondrial activity in primary mouse hepatocytes, and thus, has potential implications in the detection of mitochondrial toxicity. [ABSTRACT FROM AUTHOR]

Microphysiological systems (MPSs), based on microfabrication technologies and cell culture, can faithfully recapitulate the complex physiology of various tissues. However, 3D tissues formed using MPS have limitations in size and accessibility; their use in regenerative medicine is, therefore, still challenging. Here, an MPS‐inspired scale‐up vascularized engineered tissue construct that can be used in regenerative medicine is designed. Endothelial cell‐laden hydrogels are sandwiched between two through‐hole membranes. The microhole array in the through‐hole membranes enables the molecular transport across the hydrogel layer, allowing long‐term cell culture. Furthermore, the time‐controlled delamination of through‐hole membranes enables the harvesting of cell‐cultured hydrogel constructs without damaging the capillary network. Importantly, when the tissue constructs are implanted in a mouse ischemic model, they protect against necrosis and promoted functional recovery to a greater extent than implanted cells, hydrogels, and simple gel–cell mixtures. [ABSTRACT FROM AUTHOR]

BACKGROUND/AIM: Tissue pathogenesis of aortic valve (AV) stenosis is research focus in cardiac surgery. Model limitations of conventional 2D culture of human or porcine valvular interstitial/endothelial cells (VIC/VECs) isolated from aortic valve tissues but also limited ability of (small) animal models to reflect human (patho)physiological situation in AV position raise the need to establish an in vitro setup using AV tissues. Resulting aim is to approximate (patho)physiological conditions in a dynamic pulsatile Microphysiological System (MPS) to culture human and porcine AV tissue with preservation of tissue viability but also defined ECM composition. MATERIALS/METHODS: A tissue incubation chamber (TIC) was designed to implement human or porcine tissues (3×5 mm2) in a dynamic pulsatile culture in conventional cell culture ambience in a MPS. Cell viability assays based on lactate dehydrogenase (LDH)-release or resazurin-conversion were tested for applicability in the system and applied for a culture period of 14 days with interval evaluation of tissue viability on every other day. Resazurin-assay setup was compared in static vs. dynamic culture using varying substance saturation settings (50–300μM), incubation times and tissue masses and was consequently adapted. RESULTS: Sterile dynamic culture of human and porcine AV tissue segments was established at a pulsatile flow rate range of 0.9–13.4μl/s. Implementation of tissues was realized by stitching the material in a thermoplastic polyurethane (TPU)-ring and insertion in the TIC-MPS-system. Culture volume of 2 ml caused LDH dilution not detectable in standard membrane integrity assay setup. Therefore, detection of resazurin-conversion of viable tissue was investigated. Optimal incubation time for viability conversion was determined at two hours at a saturated concentration of 300μM resazurin. Measurement in static conditions was shown to offer comparable results as dynamic condition but allowing optimal handling and TIC sterilization protocols for long term culture. Preliminary results revealed favourable porcine AV tissue viability over a 14 day period confirmed via resazurin-assay comparing statically cultured tissue counterparts. CONCLUSIONS: Human and porcine AV tissue can be dynamically cultured in a TIC-MPS with monitoring of tissue viability using an adapted resazurin-assay setup. Preliminary results reveal advantageous viability of porcine AV tissues after dynamic TIC-MPS culture compared to static control. [ABSTRACT FROM AUTHOR]

Currently, there is no test system, whether in vitro or in vivo , capable of examining all endpoints required for genotoxicity evaluation used in pre-clinical drug safety assessment. The objective of this study was to develop a model which could assess all the required endpoints and possesses robust human metabolic activity, that could be used in a streamlined, animal-free manner. Liver-on-chip (LOC) models have intrinsic human metabolic activity that mimics the in vivo environment, making it a preferred test system. For our assay, the LOC was assembled using primary human hepatocytes or HepaRG cells, in a MPS-T12 plate, maintained under microfluidic flow conditions using the PhysioMimix® Microphysiological System (MPS), and co-cultured with human lymphoblastoid (TK6) cells in transwells. This system allows for interaction between two compartments and for the analysis of three different genotoxic endpoints, i.e. DNA strand breaks (comet assay) in hepatocytes, chromosome loss or damage (micronucleus assay) and mutation (Duplex Sequencing) in TK6 cells. Both compartments were treated at 0, 24 and 45 h with two direct genotoxicants: methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS), and two genotoxicants requiring metabolic activation: benzo[ a ]pyrene (B[ a ]P) and cyclophosphamide (CP). Assessment of cytochrome activity, RNA expression, albumin, urea and lactate dehydrogenase production, demonstrated functional metabolic capacities. Genotoxicity responses were observed for all endpoints with MMS and EMS. Increases in the micronucleus and mutations (MF) frequencies were also observed with CP, and %Tail DNA with B[ a ]P, indicating the metabolic competency of the test system. CP did not exhibit an increase in the %Tail DNA, which is in line with in vivo data. However, B[ a ]P did not exhibit an increase in the % micronucleus and MF, which might require an optimization of the test system. In conclusion, this proof-of-principle experiment suggests that LOC-MPS technology is a promising tool for in vitro hazard identification genotoxicants. • LOC built with primary hepatocytes or HepaRG and maintained under microfluidic flow. • LOC co-cultured with TK6 cells for comet and micronucleus/mutagenicity analysis. • Statistically significant increases observed for all endpoints with MMS and EMS. • Increases in the micronuclei/mutation frequency with CP and %Tail DNA with B[ a ]P. • This study suggests that LOC-MPS is a promising tool for genotoxicity assessment. [ABSTRACT FROM AUTHOR]

The liver is the most commonly involved organ in metastases from a wide variety of solid tumors. The use of biologically and cellularly complex liver tissue systems have shown that tumor cell behavior and therapeutic responses are modulated within the liver microenvironment and in ways distinct from the behaviors in the primary locations. These microphysiological systems have provided unexpected and powerful insights into the tumor cell biology of metastasis. However, neither the tumor nor the liver exist in an isolated tissue situation, having to function within a complete body and respond to systemic events as well as those in other organs. To examine the influence of one organ on the function of other tissues, microphysiological systems are being linked. Herein, we discuss extending this concept to tumor metastases by integrating complex models of the primary tumor with the liver metastatic environment. In addition, inflammatory organs and the immune system can be incorporated into these multi-organ systems to probe the effects on tumor behavior and cancer treatments. [ABSTRACT FROM AUTHOR]

[Display omitted] 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 in vitro culture models. It provides a unique and rapid platform to accelerate COVID-19 research and develop novel therapies. [ABSTRACT FROM AUTHOR]

Introduction: Interstitial hypertension, a rise in interstitial fluid pressure, is a common feature of many solid tumors as they progress to an invasive state. It is currently unclear whether this elevated pressure alters the probability that tumor cells eventually escape into a neighboring blood or lymphatic vessel. Methods: In this study, we analyze the escape of MDA-MB-231 human breast tumor cells from a ~3-mm-long preformed aggregate into a 120-μm-diameter empty cavity in a micromolded type I collagen gel. The "micro-tumors" were located within ~300 μm of one or two cavities. Pressures of ~0.65 cm H2O were applied only to the tumor ("interstitial hypertension") or to its adjacent cavity. Results: This work shows that interstitial hypertension suppresses escape into the adjacent cavity, but not because tumor cells respond directly to the pressure profile. Instead, hypertension alters the chemical microenvironment at the tumor margin to one that hampers escape. Administration of tumor interstitial fluid phenocopies the effects of hypertension. Conclusions: This work uncovers a link between tumor pressure, interstitial flow, and tumor cell escape in MDA-MB-231 cells, and suggests that interstitial hypertension serves to hinder further progression to metastatic escape. [ABSTRACT FROM AUTHOR]

The pharmaceutical industry has been desperately searching for efficient drug discovery methods. Organ-on-a-Chip, a cutting-edge technology that can emulate the physiological environment and functionality of human organs on a chip for disease modeling and drug testing, shows great potential for revolutionizing the drug development pipeline. However, successful translation of this novel engineering platform into routine pharmacological and medical scenarios remains to be realized. In this review, we discuss how the Organ-on-a-Chip technology can have critical roles in different preclinical stages of drug development and highlight the current challenges in translation and commercialization of this technology for the pharmacological and medical end-users. Moreover, this review sheds light on the future developmental trends and need for a next-generation Organ-on-a-Chip platform to bridge the gap between animal studies and clinical trials for the pharmaceutical industry. Organ-on-a-Chip is a promising interdisciplinary technique emulating in vivo physiology and pathology for in vitro disease modeling, drug screening, and precision medicine. The Organ-on-a-Chip technology can be organically incorporated into the drug development pipeline from early drug discovery to preclinical screening, testing, and translation of new drugs, which bridges the gap between animal studies and clinical trials involving human subjects. The future development of personalized Organ-on-a-Chip and continuous integration of novel engineering tools (e.g., automation handling, 3D printing. and in situ multisensors) and biological concepts (e.g., patient-specific induced pluripotent stem cells and organoids) into Organ-on-a-Chip platform will unprecedentedly promote its biomedical applications. [ABSTRACT FROM AUTHOR]

While conventional in vitro culture systems and animal models have been used to study the pathogenesis of viral infections and to facilitate development of vaccines and therapeutics for viral diseases, models that can accurately recapitulate human responses to infection are still lacking. Human organ-on-a-chip (Organ Chip) microfluidic culture devices that recapitulate tissue–tissue interfaces, fluid flows, mechanical cues, and organ-level physiology have been developed to narrow the gap between in vitro experimental models and human pathophysiology. Here, we describe how recent developments in Organ Chips have enabled re-creation of complex pathophysiological features of human viral infections in vitro. Microfluidic Organ Chip culture devices are emerging alternatives to conventional in vitro and animal models due to their ability to replicate many structural and functional features of human physiology and disease states. Recent innovations demonstrate that Organ Chip technology is a promising strategy for virology studies where there have been successes in reproducing various viral disease phenotypes. Organ Chips have enabled investigation of many aspects of viral infection, including virus–host interactions, viral therapy-resistance evolution, and development of new antiviral therapeutics, as well as underlying pathogenesis. As Organ Chip-based assays provide accessibility to study virus-induced diseases in real time and at high resolution, they can open new avenues to uncover viral pathogenesis in a human-relevant environment and may eventually enable development of novel therapeutics and vaccines. [ABSTRACT FROM AUTHOR]

The blood-retinal barrier (BRB) protects the retina by maintaining an adequate microenvironment for neuronal function. Alterations of the junctional complex of the BRB and consequent BRB breakdown in disease contribute to a loss of neuronal signaling and vision loss. As new therapeutics are being developed to prevent or restore barrier function, it is critical to implement physiologically relevant in vitro models that recapitulate the important features of barrier biology to improve disease modeling, target validation, and toxicity assessment. New directions in organ-on-a-chip technology are enabling more sophisticated 3-dimensional models with flow, multicellularity, and control over microenvironmental properties. By capturing additional biological complexity, organs-on-chip can help approach actual tissue organization and function and offer additional tools to model and study disease compared with traditional 2-dimensional cell culture. This review describes the current state of barrier biology and barrier function in ocular diseases, describes recent advances in organ-on-a-chip design for modeling the BRB, and discusses the potential of such models for ophthalmic drug discovery and development. [ABSTRACT FROM AUTHOR]

Abstract This study describes the characterization of pharmacokinetic (PK) properties of acetaminophen (APAP) in the Two-Organ-Chip platform (2-OC), a two-chamber device able to cultivate 3D tissues under flow. The APAP intestinal absorption and hepatic metabolism were emulated by human intestine and liver equivalents respectively. The intestinal barrier was produced using Caco-2 and HT-29 cells. The liver spheroids were produced with HepaRG and HHSTeC cells. Cell viability and toxicity were assessed by MTT assay, histology, confocal immunohistochemistry, and multiparametric high content analysis. Gene expression of intestine and liver equivalents were assessed by real-time PCR. Three assemblies of Microphysiological System (MPS) were applied: Intestine 2-OC, Liver 2-OC, and Intestine/Liver 2-OC. The oral administration was emulated by APAP placement over the apical side of the intestinal barrier and the intravenous routes were mimic by the application in the medium. Samples were analyzed by HPLC/UV. APAP 12 μM or 2 μM treatment did not induce cytotoxicity for the intestinal barrier (24 h time-point) or for the liver spheroids 12 h time-point), respectively. All preparations showed slower APAP absorption than reported for humans: Peak time (Tmax) = 12 h for Intestine 2-OC and 6 h for Intestine/Liver 2-OC in both static and dynamic conditions, against reported Tmax of 0,33 to 1,4 h after oral administration to humans. APAP metabolism was also slower than reported for humans. The APAP half-life (T 1/2) was 12 h in the dynamic Liver 2-OC, against T 1/2 = 2 ± 0,4 h reported for humans. Samples taken from the Liver 2-OC static preparation did not show APAP concentration decrease. These findings show the MPS capability and potential to emulate human PK properties and highlight the critical role of mechanical stimulus over cell functionality, especially by demonstrating the clear positive influence of the microfluidic flow over the liver equivalents metabolic performance. Highlights • Flow-induced a robust improvement of the liver equivalents metabolic activity. • Intestinal absorption and hepatic metabolism could be emulated in the 2-OC MPS. • Intestine/Liver 2-OC has great potential in providing data transposable to humans. [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 FROM AUTHOR]

Abstract Microphysiological systems are an emerging field of biomimetic technologies which utilize microfluidics to reconstitute tissue functions within an in vitro environment. Microfluidic organ-on-a-chip platforms exist at the intersection of biology and engineering, straddling the forefront of cutting edge biological research, fluidics, and microfabrication technologies to emulate complex in vitro tissue systems within a highly controllable, accessible, and observable, in vitro environment. Although the advance of cell culture technologies from comparatively rudimentary conventional 2D petri dish based cultures to sophisticated 3D tissue engineering may seem futuristic, the microfluidic engineering concepts behind organ-on-a-chip technologies have deep historical roots which span over half a century. In this paper, we will discuss the shortcomings of conventional cell culture and organ modeling platforms which contributed to the demand for in vitro tissue engineering, as well as provide a background in the technological foundations of microfluidics. We will further recount a brief history of microfluidic cell culture, with emphasis on important developments in fabrication practices and methods of engineering biologically relevant microenvironmental conditions, before discussing the present state of organ specific microfluidic platforms and the future directions that the field may take to overcome present challenges. Graphical abstract Unlabelled Image [ABSTRACT FROM AUTHOR]

Physiologically based pharmacokinetic (PBPK) modeling and simulation approaches are beginning to be integrated into drug development and approval processes because they enable key pharmacokinetic (PK) parameters to be predicted from in vitro data. However, these approaches are hampered by many limitations, including an inability to incorporate organ-specific differentials in drug clearance, distribution, and absorption that result from differences in cell uptake, transport, and metabolism. Moreover, such approaches are generally unable to provide insight into pharmacodynamic (PD) parameters. Recent development of microfluidic Organ-on-a-Chip (Organ Chip) cell culture devices that recapitulate tissue-tissue interfaces, vascular perfusion, and organ-level functionality offer the ability to overcome these limitations when multiple Organ Chips are linked via their endothelium-lined vascular channels. Here, we discuss successes and challenges in the use of existing culture models and vascularized Organ Chips for PBPK and PD modeling of human drug responses, as well as in vitro to in vivo extrapolation (IVIVE) of these results, and how these approaches might advance drug development and regulatory review processes in the future. [ABSTRACT FROM AUTHOR]

Hypoxia plays an essential role in the pathogenesis of various liver diseases, and albumin is one of the important biomarkers secreted by the liver. In this study, we developed an albumin monitoring system composed of hepatic hypoxia-on-a-chip and an albumin sensor to study liver function change due to hypoxia. In hepatic hypoxia-on-a-chip, we vertically stack an oxygen-scavenging channel on a liver on a chip with a thin gas-permeable membrane in the middle. This unique design of the hepatic hypoxia-on-a-chip can help to induce hypoxia quickly, attaining <5% within 10 min. An electrochemical albumin sensor was fabricated based on the covalent immobilization of antibodies on the Au electrode to monitor albumin secreting function on the hepatic hypoxia-on-a-chip. Standard albumin samples spiked in PBS, and culture media were measured by the electrochemical impedance spectroscopy using the fabricated immunosensor. The LOD was calculated to be 10 ag/mL in both cases. Using the electrochemical albumin sensor, we measured albumin secretion in normoxia and hypoxia in the chips. The albumin concentration decreased to 27% after 24 h in hypoxia compared to normoxia. This response was consistent with physiological studies. With technical refinements, the present albumin monitoring system can be a powerful tool in studying hepatic hypoxia with real-time liver function monitoring. [Display omitted] • Hepatic hypoxia-on-a-chip was developed based on the vertically stacked oxygen-scavenging channel. • Hepatic hypoxia-on-a-chip induces stable hypoxia within 10 min. • Electrochemical biosensor was developed to monitor albumin secreting function on the hepatic hypoxia-on-a-chip. • Albumin concentration was monitored to be decreased to 27% after 24 h in hypoxia compared to normoxia. [ABSTRACT FROM AUTHOR]

Microphysiological systems (MPS) are 2D or 3D multicellular constructs able to mimic tissue microenvironments. The latest models encompass a range of techniques, including co-culturing of various cell types, utilization of scaffolds and extracellular matrix materials, perfusion systems, 3D culture methods, 3D bioprinting, organ-on-a-chip technology, and examination of tissue structures. Several human brain 3D cultures or brain MPS (BMPS) have emerged in the last decade. These organoids or spheroids are 3D culture systems derived from induced pluripotent cells or embryonic stem cells that contain neuronal and glial populations and recapitulate structural and physiological aspects of the human brain. BMPS have been introduced recently in the study and modeling of neuroinfectious diseases and have proven to be useful in establishing neurotropism of viral infections, cell-pathogen interactions needed for infection, assessing cytopathological effects, genomic and proteomic profiles, and screening therapeutic compounds. Here we review the different methodologies of organoids used in neuroinfectious diseases including spheroids, guided and unguided protocols as well as microglia and blood-brain barrier containing models, their specific applications, and limitations. The review provides an overview of the models existing for specific infections including Zika, Dengue, JC virus, Japanese encephalitis, measles, herpes, SARS-CoV2, and influenza viruses among others, and provide useful concepts in the modeling of disease and antiviral agent screening. [ABSTRACT FROM AUTHOR]

Background: Our understanding of the multiple roles exosomes play during tumor progression is still very poor and the contribution of the normal tissue derived exosomes in distant seeding and tumor outgrowth has also not been widely appreciated. Methods: Using our all-human liver microphysiological system (MPS) platform as a model to closely recapitulate the early metastatic events, we isolated exosomes from both tumor cells and liver microenvironment. Results: We observed that while priming of the hepatic niche (HepN) with MDA-231 breast cancer derived exosomes facilitated seeding of the cancer cells in the liver, subsequent tumor outgrowth was diminished; this was consistent with increased entry into dormancy. We found that hepatic niche (HepN) derived exosomes contribute significantly to the exosome pool and are distinguished from cancer derived exosomes based on their size, protein and miRNA content. By Ingenuity Pathway Analysis (IPA) of the miRNA content of the HepN, MDA-231/HepN and MDA-231 cells we showed that the HepN derived exosomes affect the breast cancer cells by suppressing pathways involved in cancer cell proliferation and invasion. More importantly exposure of MDA-231 and MDA-468 cells to purified normal HepN derived exosomes, induced changes in the cells consistent with a Mesenchymal to Epithelial reverting Transition (MErT). miRNA arrays performed on MDA-231 treated with Hum Hep/NPC derived exosomes showed significant changes in the levels of a select number of miRNAs involved in epithelial cell differentiation and miRNAs, such as miR186, miR23a and miR205, from our top and bottom bins have previously been reported to regulate E-cadherin transcription and MErT induction in various cancer types. Consistently HepN derived exosome treatment of breast and prostate cancer lines lead to a transient induction of E-cadherin and ZO-1 at the protein level and a more epithelial-like morphology of the cells. Conclusions: Collectively our data revealed a novel mechanism of regulation of the metastatic cascade, showing a wellorchestrated, timely controlled crosstalk between the cancer cells and the HepN and implicating for the first time the normal tissue/HepN derived exosomes in enabling seeding and entry into dormancy of the cancer cells at the metastatic site. [ABSTRACT FROM AUTHOR]

Organoids are emerging in vitro models of human physiology. Neural models require the evaluation of functional activity of single cells and networks, which is commonly measured by microelectrode arrays. The characteristics of organoids clash with existing in vitro or in vivo microelectrode arrays. With inspiration from implantable mesh electronics and growth of organoids on polymer scaffolds, we fabricated suspended hammock-like mesh microelectrode arrays for neural organoids. We have demonstrated the growth of organoids enveloping these meshes and the culture of organoids on meshes for up to one year. Furthermore, we present proof-of-principle recordings of spontaneous electrical activity across the volume of an organoid. Our concept enables a new class of microelectrode arrays for in vitro models of three-dimensional electrically active tissue. [ABSTRACT FROM AUTHOR]