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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.

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.

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

Summary: During the last decade, organs-on-chips or organoids microphysiological analysis platforms (MAP) have garnered attention in the practical applications of disease models, drug discovery, and developmental biology. Research on pregnant women has firm limitations due to ethical issues; thus, remodelling human pregnancy in vitro is highly beneficial for treatment modality development via disease remodelling or drug monitoring. This review highlights current efforts in bioengineering devices to reproduce human pregnancy and emphasises the significant convergence of biology, engineering, and maternal–foetal medicine. First, we review recent achievements in culturing cells from tissues involved in pregnancy; specifically, trophoblasts from the placenta. Second, we highlight developments in the reconstitution of pregnancy-related female reproductive organs across several structural and functional interpretations. Last, we examine research on the fundamental comprehension of pregnancy-associated diseases to find bioengineering solutions. Recreating human pregnancy through an engineered model is naturally complex; nevertheless, challenges are inevitable to progress precision medicine.

Nanofiber (NF) membranes have been highlighted as functional materials for biomedical applications owing to their high surface-to-volume ratios, high permeabilities, and extracellular matrix-like biomimetic structures. Because many in vitro platforms for biomedical applications are made of thermoplastic polymers (TP), a simple and leak-free method for bonding NF membranes onto TP platforms is essential. Here, we propose a facile but leak-free localized thermal bonding method for integrating 2D or 3D-structured NF membrane onto a TP supporting substrate while preserving the pristine nanofibrous structure of the membrane, based on localized preheating of the substrate. A methodology for determining the optimal preheating temperature was devised based on a numerical simulation model considering the melting temperature of the NF material and was experimentally validated by evaluating bonding stability and durability under cell culture conditions. The thermally-bonded interface between the NF membrane and TP substrate was maintained stably for 3 weeks allowing the successful construction of an intestinal barrier model. The applicability of the localized thermal bonding method was also demonstrated on various combinations of TP materials (e.g., polystyrene and polymethylmethacrylate) and geometries of the supporting substrate, including a culture insert and microfluidic chip. We expect the proposed localized thermal bonding method to contribute toward broadening and realizing the practical applications of functional NF membranes in various biomedical fields.

Atmospheric pollutants, including particulate matters, nanoparticles, bioaerosols, and some chemicals, have posed serious threats to the environment and the human’s health. The lungs are the responsible organs for providing the interface between the circulatory system and the external environment, where pollutant particles can deposit or penetrate into bloodstream circulation. Conventional studies to decipher the mechanism underlying air pollution and human health are quite limited, due to the lack of reliable models that can reproduce in vivo features of lung tissues after pollutants exposure. In the past decade, advanced near-to-native lung chips, combining cell biology with bioengineered technology, present a new strategy for atmospheric pollutants assessment and narrow the gap between 2D cell culture and in vivo animal models. In this review, the key features of artificial lung chips and the cutting-edge technologies of the lung chip manufacture are introduced. The recent progresses of lung chip technologies for atmospheric pollutants exposure assessment are summarized and highlighted. We further discuss the current challenges and the future opportunities of the development of advanced lung chips and their potential utilities in atmospheric pollutants associated toxicity testing and drug screening.

Pain is the predominant symptom of osteoarthritis (OA) that drives patients to seek medical care. Currently, there are no pharmacological treatments that can reverse or halt the progression of OA. Safe and efficacious medications for long-term management of OA pain are also unavailable. Understanding the mechanisms behind OA pain generation at onset and over time is critical for developing effective treatments. In this narrative review, we first summarize our current knowledge on the innervation of the knee joint, and then discuss the molecular mechanism(s) currently thought to underlie OA pain. In particular, we focus on the contribution of each joint component to the generation of pain. Next, the current experimental models for studying OA pain are summarized, and the methods to assess pain in rodents are presented. The potential application of emerging microphysiological systems in OA pain research is especially highlighted. Lastly, we discuss the current challenge in standardizing models and the selection of appropriate systems to address specific questions.

Oxidative stress is caused by an imbalance between the generation and detoxification of reactive oxygen and nitrogen species (ROS/RNS). This imbalance plays an important role in brain aging and age-related neurodegenerative diseases. In the context of Parkinson's disease (PD), the sensitivity of dopaminergic neurons in the substantia nigra pars compacta to oxidative stress is considered a key factor of PD pathogenesis. Here we study the effect of different oxidative stress-inducing compounds (6-OHDA, MPTP or MPP+) on the population of dopaminergic neurons in an iPSC-derived human brain 3D model (aka BrainSpheres). Treatment with 6-OHDA, MPTP or MPP+ at 4 weeks of differentiation disrupted the dopaminergic neuronal phenotype in BrainSpheres at (50, 5000, 1000 μM respectively). 6-OHDA increased ROS production and decreased mitochondrial function most efficiently. It further induced the greatest changes in gene expression and metabolites related to oxidative stress and mitochondrial dysfunction. Co-culturing BrainSpheres with an endothelial barrier using a transwell system allowed the assessment of differential penetration capacities of the tested compounds and the damage they caused in the dopaminergic neurons within the BrainSpheres In conclusion, treatment with compounds known to induce PD-like phenotypes in vivo caused molecular deficits and loss of dopaminergic neurons in the BrainSphere model. This approach therefore recapitulates common animal models of neurodegenerative processes in PD at similarly high doses. The relevance as tool for drug discovery is discussed.

Background: 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., Methods: 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., Results: 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., Conclusions: 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., Statement of Significance: 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

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 Ca 2+ 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., Competing Interests: Declaration of competing interest Authors Bettina Lickiss, Matthias Gossmann, Jan Hunker and Peter Linder are employed by innoVitro GmbH, the manufacturer of the FLEXcyte 96 consumables and co-developer of the FLEXcyte 96 used to compile this manuscript. Authors Ulrich Thomas, Elena Dragicevic and Sonja Stoelzle-Feix are employed by Nanion Technologies GmbH, the manufacturer of the FLEXcyte 96 used to compile this manuscript. Authors Jamie Bhagwan, Hardeep Lotay, Steven Broadbent and Jan Turner are employed by Axol Bioscience Ltd., the manufacturer of axoCells™ Ventricular and Atrial Cardiomyocytes used in this study., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Microfluidic devices are widely used for biomedical applications but there is still a lack of affordable, reliable and user-friendly systems for transferring microfluidic chips from an incubator to a microscope while maintaining physiological conditions when performing microscopy. The presented carrier represents a cost-effective option for sustaining environmental conditions of microfluidic chips in combination with minimizing the device manipulation required for reagent injection, media exchange or sample collection. The carrier, which has the outer dimension of a standard well plate size, contains an integrated perfusion system that can recirculate the media using piezo pumps, operated in either continuous or intermittent modes (50–1000 µl/min). Furthermore, a film resistive heater made from 37 µm-thick copper wires, including temperature feedback control, was used to maintain the microfluidic chip temperature at 37 °C when outside the incubator. The heater characterisation showed a uniform temperature distribution along the chip channel for perfusion flow rates up to 10 µl/min. To demonstrate the feasibility of our platform for long term cell culture monitoring, mouse brain endothelial cells (bEnd.3) were repeatedly monitored for a period of 10 days, demonstrating a system with both the versatility and the potential for long imaging in microphysiological system cell cultures.

Chimeric antigen receptor (CAR) cells are genetically engineered immune cells that specifically target tumor-associated antigens and have revolutionized cancer treatment, particularly in hematological malignancies, with ongoing investigations into their potential applications in solid tumors. This review provides a comprehensive overview of the current status and challenges in drug metabolism and pharmacokinetics (DMPK) for CAR cell therapy, specifically emphasizing on quantitative modeling and simulation (M&S). Furthermore, the recent advances in quantitative model analysis have been reviewed, ranging from clinical data characterization to mechanism-based modeling that connects in vitro and in vivo nonclinical and clinical study data. Additionally, the future perspectives and areas for improvement in CAR cell therapy translation have been reviewed. This includes using formulation quality considerations, characterization of appropriate animal models, refinement of in vitro models for bottom-up approaches, and enhancement of quantitative bioanalytical methodology. Addressing these challenges within a DMPK framework is pivotal in facilitating the translation of CAR cell therapy, ultimately enhancing the patients' lives through efficient CAR cell therapies., Competing Interests: Declaration of competing interest AG, YM, MN, SI, and SY are employees of Takeda Pharmaceutical Company Limited., (Copyright © 2024 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: T.B.Z, A.B, P.V, C.C.V and J.J.S, declare that they are employees and equity holders at TwinStrand Biosciences, Inc. and are authors on one or more Duplex Sequencing-related patents., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

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 × 10 4  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., Competing Interests: Declaration of competing Interest CM is co-founder of Pluriomics (now Ncardia) and BM is co-founder and CTO of Demcon Biovitronix. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (© 2018 Published by Elsevier B.V.)