Towards a microfluidic H295R steroidogenesis assay-biocompatibility study and steroid detection on a thiol-ene-based chip.

The development of cell-based microfluidic assays offers exciting new opportunities in toxicity testing, allowing for integration of new functionalities, automation, and high throughput in comparison to traditional well-plate assays. As endocrine disruption caused by environmental chemicals and pharmaceuticals represents a growing global health burden, the purpose of the current study was to contribute towards the miniaturization of the H295R steroidogenesis assay, from the well-plate to the microfluidic format. Microfluidic chip fabrication with the established well-plate material polystyrene (PS) is expensive and complicated; PDMS and thiol-ene were therefore tested as potential chip materials for microfluidic H295R cell culture, and evaluated in terms of cell attachment, cell viability, and steroid synthesis in the absence and presence of collagen surface modification. Additionally, spike-recovery experiments were performed, to investigate potential steroid adsorption to chip materials. Cell aggregation with poor steroid recoveries was observed for PDMS, while cells formed monolayer cultures on the thiol-ene chip material, with cell viability and steroid synthesis comparable to cells grown on a PS surface. As thiol-ene overall displayed more favorable properties for H295R cell culture, a microfluidic chip design and corresponding cell seeding procedure were successfully developed, achieving repeatable and uniform cell distribution in microfluidic channels. Finally, H295R perfusion culture on thiol-ene chips was investigated at different flow rates (20, 10, and 2.5 µL/min), and 13 steroids were detected in eluting cell medium over 48 h at the lowest flow rate. The presented work and results pave the way for a time-resolved microfluidic H295R steroidogenesis assay.

The Cultivation Modality and Barrier Maturity Modulate the Toxicity of Industrial Zinc Oxide and Titanium Dioxide Nanoparticles on Nasal, Buccal, Bronchial, and Alveolar Mucosa Cell-Derived Barrier Models.

As common industrial by-products, airborne engineered nanomaterials are considered important environmental toxins to monitor due to their potential health risks to humans and animals. The main uptake routes of airborne nanoparticles are nasal and/or oral inhalation, which are known to enable the transfer of nanomaterials into the bloodstream resulting in the rapid distribution throughout the human body. Consequently, mucosal barriers present in the nose, buccal, and lung have been identified and intensively studied as the key tissue barrier to nanoparticle translocation. Despite decades of research, surprisingly little is known about the differences among various mucosa tissue types to tolerate nanoparticle exposures. One limitation in comparing nanotoxicological data sets can be linked to a lack of harmonization and standardization of cell-based assays, where (a) different cultivation conditions such as an air-liquid interface or submerged cultures, (b) varying barrier maturity, and (c) diverse media substitutes have been used. The current comparative nanotoxicological study, therefore, aims at analyzing the toxic effects of nanomaterials on four human mucosa barrier models including nasal (RPMI2650), buccal (TR146), alveolar (A549), and bronchial (Calu-3) mucosal cell lines to better understand the modulating effects of tissue maturity, cultivation conditions, and tissue type using standard transwell cultivations at liquid-liquid and air-liquid interfaces. Overall, cell size, confluency, tight junction localization, and cell viability as well as barrier formation using 50% and 100% confluency was monitored using trans-epithelial-electrical resistance (TEER) measurements and resazurin-based Presto Blue assays of immature (e.g., 5 days) and mature (e.g., 22 days) cultures in the presence and absence of corticosteroids such as hydrocortisone. Results of our study show that cellular viability in response to increasing nanoparticle exposure scenarios is highly compound and cell-type specific (TR146 6 ± 0.7% at 2 mM ZnO (ZnO) vs. ~90% at 2 mM TiO2 (TiO2) for 24 h; Calu3 93.9 ± 4.21% at 2 mM ZnO vs. ~100% at 2 mM TiO2). Nanoparticle-induced cytotoxic effects under air-liquid cultivation conditions declined in RPMI2650, A549, TR146, and Calu-3 cells (~0.7 to ~0.2-fold), with increasing 50 to 100% barrier maturity under the influence of ZnO (2 mM). Cell viability in early and late mucosa barriers where hardly influenced by TiO2 as well as most cell types did not fall below 77% viability when added to Individual ALI cultures. Fully maturated bronchial mucosal cell barrier models cultivated under ALI conditions showed less tolerance to acute ZnO nanoparticle exposures (~50% remaining viability at 2 mM ZnO for 24 h) than the similarly treated but more robust nasal (~74%), buccal (~73%), and alveolar (~82%) cell-based models.

Galectin network in osteoarthritis: galectin-4 programs a pathogenic signature of gene and effector expression in human chondrocytes in vitro.

Galectin-4 (Gal-4) is a member of the galectin family, which have been identified as galactose-binding proteins. Gal-4 possesses two tandem repeat carbohydrate recognition domains and acts as a cross-linking bridge in sulfatide-dependent glycoprotein routing. We herein document its upregulation in osteoarthritis (OA) in correlation with the extent of cartilage degradation in vivo. Primary human OA chondrocytes in vitro respond to carbohydrate-inhibitable Gal-4 binding with the upregulation of pro-degradative/-inflammatory proteins such as interleukin-1ß (IL-1ß) and matrix metalloproteinase-13 (MMP-13), as documented by RT-qPCR-based mRNA profiling and transcriptome data processing. Activation of p65 by phosphorylation of Ser536 within the NF-κB pathway and the effect of three p65 inhibitors on Gal-4 activity support downstream involvement of such signaling. In 3D (pellet) cultures, Gal-4 presence causes morphological and biochemical signs of degradation. Taken together, our findings strongly support the concept of galectins acting as a network in OA pathogenesis and suggest that blocking their activity in disease progression may become clinically relevant in the future.

A microfluidic microparticle-labeled impedance sensor array for enhancing immunoassay sensitivity.

An impedimetric biosensor is used to measure electrical impedance changes in the presence of biomolecules from sinusoidal input voltages. In this paper, we present a new portable impedance-based biosensor platform to improve the sensitivity of immunoassays with microparticles as a label. Using a 2 × 4 interdigitated electrode array with a 10/10 µm electrode/gap and a miniaturized impedance analyzer, we performed immunoassays with microparticles by integrating a microfluidic channel to evaluate signal enhancement. First, to understand the material dependency of microparticles on the sensor array, magnetic, silica, and polystyrene microparticles were tested. Among these microparticles, magnetic microparticles presented a high signal enhancement with relevant stability from the sensor array. With the magnetic microparticles, we demonstrate a series of immunoassays to detect human tumor necrosis factor (TNF-α) and compare the level of signal enhancement by measuring the limit of detection (LOD). With the microparticles, we achieved over ten times improvement of LOD from sandwich immunoassays. By incorporating with sample preparation and flow manipulation systems, this impedance sensor array can be utilized for digital diagnostics for a real sample-in answer-out system.

Monitoring the neurotransmitter release of human midbrain organoids using a redox cycling microsensor as a novel tool for personalized Parkinson's disease modelling and drug screening.

In this study, we have aimed at developing a novel electrochemical sensing approach capable of detecting dopamine, the main biomarker in Parkinson's disease, within the highly complex cell culture matrix of human midbrain organoids in a non-invasive and label-free manner. With its ability to generate organotypic structures in vitro, induced pluripotent stem cell technology has provided the basis for the development of advanced patient-derived disease models. These include models of the human midbrain, the affected region in the neurodegenerative disorder Parkinson's disease. Up to now, however, the analysis of so-called human midbrain organoids has relied on time-consuming and invasive strategies, incapable of monitoring organoid development. Using a redox-cycling approach in combination with a 3-mercaptopropionic acid self-assembled monolayer modification enabled the increase of sensor selectivity and sensitivity towards dopamine, while simultaneously reducing matrix-mediated interferences. In this work, we demonstrate the ability to detect and monitor even small differences in dopamine release between healthy and Parkinson`s disease-specific midbrain organoids over prolonged cultivation periods, which was additionally verified using liquid chromatography-multiple reaction monitoring mass spectrometry. Furthermore, the detection of a phenotypic rescue in midbrain organoids carrying a pathogenic mutation in leucine-rich repeat kinase 2, upon treatment with the leucine-rich repeat kinase 2 inhibitor II underlines the practical implementability of our sensing approach for drug screening applications as well as personalized disease modelling.

Downscaling screening cultures in a multifunctional bioreactor array-on-a-chip for speeding up optimization of yeast-based lactic acid bioproduction.

A key challenge for bioprocess engineering is the identification of the optimum process conditions for the production of biochemical and biopharmaceutical compounds using prokaryotic as well as eukaryotic cell factories. Shake flasks and bench-scale bioreactor systems are still the golden standard in the early stage of bioprocess development, though they are known to be expensive, time-consuming, and labor-intensive as well as lacking the throughput for efficient production optimizations. To bridge the technological gap between bioprocess optimization and upscaling, we have developed a microfluidic bioreactor array to reduce time and costs, and to increase throughput compared with traditional lab-scale culture strategies. We present a multifunctional microfluidic device containing 12 individual bioreactors (Vt = 15 µl) in a 26 mm × 76 mm area with in-line biosensing of dissolved oxygen and biomass concentration. Following initial device characterization, the bioreactor lab-on-a-chip was used in a proof-of-principle study to identify the most productive cell line for lactic acid production out of two engineered yeast strains, evaluating whether it could reduce the time needed for collecting meaningful data compared with shake flasks cultures. Results of the study showed significant difference in the strains' productivity within 3 hr of operation exhibiting a 4- to 6-fold higher lactic acid production, thus pointing at the potential of microfluidic technology as effective screening tool for fast and parallelizable industrial bioprocess development.

Combinatorial in Vitro and in Silico Approach To Describe Shear-Force Dependent Uptake of Nanoparticles in Microfluidic Vascular Models.

In the present work, we combine experimental and computational methods to define the critical shear stress as an alternative parameter for nanotoxicological and nanomedical evaluations using an in vitro microfluidic vascular model. We demonstrate that our complementary in vitro and in silico approach is well suited to assess the fluid flow velocity above which clathrin-mediated (active) nanoparticle uptake per cell decreases drastically although higher numbers of nanoparticles per cell are introduced. Results of our study revealed a critical shear stress of 1.8 dyn/cm2, where maximum active cellular nanoparticle uptake took place, followed by a 70% decrease in uptake of 249 nm nanoparticles at 10 dyn/cm2, respectively. The observed nonlinear relationship between flow velocity and nanoparticle uptake strongly suggests that fluid mechanical forces also need to be considered in order to predict potential in vivo distribution, bioaccumulation, and clearance of nanomaterials and novel nanodrugs.

Simultaneous Determination of Oxygen and pH Inside Microfluidic Devices Using Core-Shell Nanosensors.

A powerful online analysis setup for the simultaneous detection of oxygen and pH is presented. It features core-shell nanosensors, which enable contactless and inexpensive read-out using adapted oxygen meters via modified dual lifetime referencing in the frequency domain (phase shift measurements). Lipophilic indicator dyes were incorporated into core-shell structured poly(styrene-block-vinylpyrrolidone) nanoparticles (average diameter = 180 nm) yielding oxygen nanosensors and pH nanosensors by applying different preparation protocols. The oxygen indicator platinum(II) meso-tetra(4-fluorophenyl) tetrabenzoporphyrin (PtTPTBPF) was entrapped into the polystyrene core (oxygen nanosensors) and a pH sensitive BF2-chelated tetraarylazadipyrromethene dye (aza-BODIPY) was incorporated into the polyvinylpyrrolidone shell (pH nanosensors). The brightness of the pH nanoparticles was increased by more than 3 times using a light harvesting system. The nanosensors have several advantages such as being excitable with red light, emitting in the near-infrared spectral region, showing a high stability in aqueous media even at high particle concentrations, high ionic strength, or high protein concentrations and are spectrally compatible with the used read-out device. The resolution for oxygen of the setup is 0.5-2.0 hPa (approximately 0.02-0.08 mg/L of dissolved oxygen) at low oxygen concentrations (<50 hPa) and 4-8 hPa (approximately 0.16-0.32 mg/L of dissolved oxygen) at ambient air oxygen concentrations (approximately 200 hPa at 980 mbar air pressure) at room temperature. The pH resolution is 0.03-0.1 pH units within the dynamic range (apparent pKa 7.23 ± 1.0) of the nanosensors. The sensors were used for online monitoring of pH changes during the enzymatic transformation of Penicillin G to 6-aminopenicillanic acid catalyzed by Penicillin G acylase in miniaturized stirred batch reactors or continuous flow microreactors.

Monitoring cellular stress responses using integrated high-frequency impedance spectroscopy and time-resolved ELISA.

We have developed a lab-on-a-chip system for continuous and non-invasive monitoring of microfluidic cell cultures using integrated high-frequency contactless impedance spectroscopy. Electrically insulated microfabricated interdigitated electrode structures were embedded into four individually addressable microchambers to reliably and reproducibly detect cell-substrate interactions, cell viability and metabolic activity. While silicon nitride passivated sensor substrates provided a homogeneous cell culture surface that minimized cell orientation along interdigitated electrode structures, the application of high-frequency AC fields reduced the impact of the 300 nm thick passivation layer on sensor sensitivity. The additional implementation of multivariate data analysis methods such as partial least square (PLS) for high-frequency impedance spectra provided unambiguous information on intracellular pathway activation, up and down-regulation of protein synthesis as well as global cellular stress responses. A comparative cell analysis using connective tissue fibroblasts showed that high-frequency contactless impedance spectroscopy and time-resolved quantification of IL-6 secretion using ELISA provided similar results following stimulation with circulating pro-inflammatory cytokines IL-1ß and TNFα. The combination of microfluidics with contactless impedance sensing and time-resolved quantification of stress factor release will provide biologist with a new tool to (a) establish a variety of uniform cell culture surfaces that feature complex biochemistries, micro- and nanopatterns; and (b) to simultaneously characterize cell responses under physiologically relevant conditions using a complementary non-invasive cell analysis method.

An update on microfluidic multi-organ-on-a-chip systems for reproducing drug pharmacokinetics: the current state-of-the-art.

INTRODUCTION: Advances in the accessibility of manufacturing technologies and iPSC-based modeling have accelerated the overall progress of organs-on-a-chip. Notably, the progress in multi-organ systems is not progressing with equal speed, indicating that there are still major technological barriers to overcome that may include biological relevance, technological usability as well as overall accessibility. AREAS COVERED: We here review the progress in the field of multi-tissue- and body-on-a-chip pre and post- SARS-CoV-2 pandemic and review five selected studies with increasingly complex multi-organ chips aiming at pharmacological studies. EXPERT OPINION: We discuss future and necessary advances in the field of multi-organ chips including how to overcome challenges regarding cell diversity, improved culture conditions, model translatability as well as sensor integrations to enable microsystems to cover organ-organ interactions in not only toxicokinetic but more importantly pharmacodynamic and -kinetic studies.

Development of a Flexible Sensor-Integrated Tissue Patch to Monitor Early Organ Rejection Processes Using Impedance Spectroscopy.

Heart failure represents a primary cause of hospitalization and mortality in both developed and developing countries, often necessitating heart transplantation as the only viable recovery path. Despite advances in transplantation medicine, organ rejection remains a significant post-operative challenge, traditionally monitored through invasive endomyocardial biopsies (EMB). This study introduces a rapid prototyping approach to organ rejection monitoring via a sensor-integrated flexible patch, employing electrical impedance spectroscopy (EIS) for the non-invasive, continuous assessment of resistive and capacitive changes indicative of tissue rejection processes. Utilizing titanium-dioxide-coated electrodes for contactless impedance sensing, this method aims to mitigate the limitations associated with EMB, including procedural risks and the psychological burden on patients. The biosensor's design features, including electrode passivation and three-dimensional microelectrode protrusions, facilitate effective monitoring of cardiac rejection by aligning with the heart's curvature and responding to muscle contractions. Evaluation of sensor performance utilized SPICE simulations, scanning electron microscopy, and cyclic voltammetry, alongside experimental validation using chicken heart tissue to simulate healthy and rejected states. The study highlights the potential of EIS in reducing the need for invasive biopsy procedures and offering a promising avenue for early detection and monitoring of organ rejection, with implications for patient care and healthcare resource utilization.

Using Rapid Prototyping to Develop a Cell-Based Platform with Electrical Impedance Sensor Membranes for In Vitro RPMI2650 Nasal Nanotoxicology Monitoring.

Due to advances in additive manufacturing and prototyping, affordable and rapid microfluidic sensor-integrated assays can be fabricated using additive manufacturing, xurography and electrode shadow masking to create versatile platform technologies aimed toward qualitative assessment of acute cytotoxic or cytolytic events using stand-alone biochip platforms in the context of environmental risk assessment. In the current study, we established a nasal mucosa biosensing platform using RPMI2650 mucosa cells inside a membrane-integrated impedance-sensing biochip using exclusively rapid prototyping technologies. In a final proof-of-concept, we applied this biosensing platform to create human cell models of nasal mucosa for monitoring the acute cytotoxic effect of zinc oxide reference nanoparticles. Our data generated with the biochip platform successfully monitored the acute toxicity and cytolytic activity of 6 mM zinc oxide nanoparticles, which was non-invasively monitored as a negative impedance slope on nasal epithelial models, demonstrating the feasibility of rapid prototyping technologies such as additive manufacturing and xurography for cell-based platform development.

Monitoring dynamic interactions of tumor cells with tissue and immune cells in a lab-on-a-chip.

A complementary cell analysis method has been developed to assess the dynamic interactions of tumor cells with resident tissue and immune cells using optical light scattering and impedance sensing to shed light on tumor cell behavior. The combination of electroanalytical and optical biosensing technologies integrated in a lab-on-a-chip allows for continuous, label-free, and noninvasive probing of dynamic cell-to-cell interactions between adherent and nonadherent cocultures, thus providing real-time insights into tumor cell responses under physiologically relevant conditions. While the study of adherent cocultures is important for the understanding and suppression of metastatic invasion, the analysis of tumor cell interactions with nonadherent immune cells plays a vital role in cancer immunotherapy research. For the first time, the direct cell-to-cell interactions of tumor cells with bead-activated primary T cells were continuously assessed using an effector cell to target a cell ratio of 10:1.

Bioinspired human stomach-on-a-chip with in vivo like function and architecture.

The lack of biomimetic in vitro models capable of reproducing the complex architecture and the dynamic environment of the gastric mucosa, delay the development of diagnostic and therapeutic tools. Recent advances in microengineering made possible the fabrication of bioinspired microdevices capable of replicating the physiological properties of an organ, inside a microfluidics chip. Herein, a bioinspired stomach-on-a-chip (SoC) device is described, supporting peristalsis-like motion and reconstituting organ-level epithelial architecture and function. The device simulates the upper epithelial interface, representing the three innermost layers of the gastric mucosa, namely the epithelial barrier, the basement membrane and the lamina propria. The dynamic environment imparted by mechanical actuation of the flexible on-chip cell culture substrate, was the main driver in the development of epithelial polarization and differentiation traits characteristic of the native gastric mucosa, and allowed partial recapitulation of gastric barrier function. These traits were not affected by the addition of a mesenchymal population to the system, which was able to remodel the surrounding extracellular matrix, nor by the potential epithelial-mesenchymal cross-talk. The engineered platform highlights the importance of addressing the mechanical microenvironment of the native organ, to potentiate an organ-level response of the artificial tissue. The proposed SoC represents an appealing tool in personalized medicine, with bio-relevance for the study of gastric diseases and an alternative to current animal models.

Microfluidic device for enhancement and analysis of osteoblast differentiation in three-dimensional cell cultures.

Three-dimensional (3D) cell cultures are to date the gold standard in biomedical research fields due to their enhanced biological functions compared to conventional two-dimensional (2D) cultures. 3D cell spheroids, as well as organoids, are better suited to replicate tissue functions, which enables their use both as in vitro models for basic research and toxicology, as well as building blocks used in tissue/organ biofabrication approaches. Culturing 3D spheroids from bone-derived cells is an emerging technology for both disease modelling and drug screening applications. Bone tissue models are mainly limited by the implementation of sophisticated devices and procedures that can foster a tissue-specific 3D cell microenvironment along with a dynamic cultivation regime. In this study, we consequently developed, optimized and characterized an advanced perfused microfluidic platform to improve the reliability of 3D bone cell cultivation and to enhance aspects of bone tissue maturation in vitro. Moreover, biomechanical stimulation generated by fluid flow inside the arrayed chamber, was used to mimic a more dynamic cell environment emulating a highly vascularized bone we expected to improve the osteogenic 3D microenvironment in the developed multifunctional spheroid-array platform. The optimized 3D cell culture protocols in our murine bone-on-a-chip spheroid model exhibited increased mineralization and viability compared to static conditions. As a proof-of-concept, we successfully confirmed on the beneficial effects of a dynamic culture environment on osteogenesis and used our platform for analysis of bone-derived spheroids produced from primary human pre-osteoblasts. To conclude, the newly developed system represents a powerful tool for studying human bone patho/physiology in vitro under more relevant and dynamic culture conditions converging the advantages of microfluidic platforms with multi-spheroid array technologies.

Automated analysis of mitochondrial dimensions in mesenchymal stem cells: Current methods and future perspectives.

As centre of energy production and key regulators of metabolic and cellular signaling pathways, the integrity of mitochondria is essential for mesenchymal stem cell function in tissue regeneration. Alterations in the size, shape and structural organization of mitochondria are correlated with the physiological state of the cell and its environment and could be used as diagnostic biomarkers. Therefore, high-throughput experimental and computational techniques are crucial to ensure adequate correlations between mitochondrial function and disease phenotypes. The emerge of microfluidic technologies can address the shortcomings of traditional methods to determine mitochondrial dimensions for diagnostic and therapeutic use. This review discusses optical detection methods compatible with microfluidics to measure mitochondrial dynamics and their potential for clinical stem cell research targeting mitochondrial dysfunction.

Rapid identification of in vitro cell toxicity using an electrochemical membrane screening platform.

This study compares the performance and output of an electrochemical phospholipid membrane platform against respective in vitro cell-based toxicity testing methods using three toxicants of different biological action (chlorpromazine (CPZ), colchicine (COL) and methyl methanesulphonate (MMS)). Human cell lines from seven different tissues (lung, liver, kidney, placenta, intestine, immune system) were used to validate this physicochemical testing system. For the cell-based systems, the effective concentration at 50 % cell death (EC50) values are calculated. For the membrane sensor, a limit of detection (LoD) value was extracted as a quantitative parameter describing the minimum concentration of toxicant which significantly affects the structure of the phospholipid sensor membrane layer. LoD values were found to align well with the EC50 values when acute cell viability was used as an end-point and showed a similar toxicity ranking of the tested toxicants. Using the colony forming efficiency (CFE) or DNA damage as end-point, a different order of toxicity ranking was observed. The results of this study showed that the electrochemical membrane sensor generates a parameter relating to biomembrane damage, which is the predominant factor in decreasing cell viability when in vitro models are acutely exposed to toxicants. These results lead the way to using electrochemical membrane-based sensors for rapid relevant preliminary toxicity screens.

Emerging Biosensor Trends in Organ-on-a-Chip.

Organ-on-a-chip technology is ideally suited to cultivate and analyze 2D/3D cell cultures, organoids, and other tissue analogues in vitro, because these microphysiological systems have been shown to generate architectures, structural organization, and functions that closely resemble their respective human tissues and organs. Although great efforts have been undertaken to demonstrate organotypic cell behavior, proper cell-to-cell communication, and tissue interactions in recent years, the integration of biosensing strategies into organ-on-a-chip platforms is still in its infancy. While a multitude of micro-, nano-, and biosensors are well established and could be easily adapted for organ-on-a-chip models, to date only a handful of analytical approaches (aside from microscopical techniques) have been combined with organ-on-a-chip technology. This chapter aims to summarize current efforts and survey the progress that has been made in integrating analytical techniques that are being implemented for organ-, multi-organ-, and body-on-a-chip systems based on electrochemical and optical sensors.

Fingerprinting Metabolic Activity and Tissue Integrity of 3D Lung Cancer Spheroids under Gold Nanowire Treatment.

Inadequacy of most animal models for drug efficacy assessments has led to the development of improved in vitro models capable of mimicking in vivo exposure scenarios. Among others, 3D multicellular spheroid technology is considered to be one of the promising alternatives in the pharmaceutical drug discovery process. In addition to its physiological relevance, this method fulfills high-throughput and low-cost requirements for preclinical cell-based assays. Despite the increasing applications of spheroid technology in pharmaceutical screening, its application, in nanotoxicity testing is still in its infancy due to the limited penetration and uptake rates into 3D-cell assemblies. To gain a better understanding of gold nanowires (AuNWs) interactions with 3D spheroids, a comparative study of 2D monolayer cultures and 3D multicellular spheroids was conducted using two lung cancer cell lines (A549 and PC9). Cell apoptosis (live/dead assay), metabolic activity, and spheroid integrity were evaluated following exposure to AuNWs at different dose-time manners. Results revealed a distinct different cellular response between 2D and 3D cell cultures during AuNWs treatment including metabolic rates, cell viability, dose-response curves and, uptake rates. Our data also highlighted further need for more physiologically relevant tissue models to investigate in depth nanomaterial-biology interactions. It is important to note that higher concentrations of AuNWs with lower exposure times and lower concentrations of AuNWs with higher exposure times of 3 days resulted in the loss of spheroid integrity by disrupting cell-cell contacts. These findings could help to increase the understanding of AuNWs-induced toxicity on tissue levels and also contribute to the establishment of new analytical approaches for toxicological and drug screening studies.

A Progress Report and Roadmap for Microphysiological Systems and Organ-On-A-Chip Technologies to Be More Predictive Models in Human (Knee) Osteoarthritis.

Osteoarthritis (OA), a chronic debilitating joint disease affecting hundreds of million people globally, is associated with significant pain and socioeconomic costs. Current treatment modalities are palliative and unable to stop the progressive degeneration of articular cartilage in OA. Scientific attention has shifted from the historical view of OA as a wear-and-tear cartilage disorder to its recognition as a whole-joint disease, highlighting the contribution of other knee joint tissues in OA pathogenesis. Despite much progress in the field of microfluidic systems/organs-on-a-chip in other research fields, current in vitro models in use do not yet accurately reflect the complexity of the OA pathophenotype. In this review, we provide: 1) a detailed overview of the most significant recent developments in the field of microsystems approaches for OA modeling, and 2) an OA-pathophysiology-based bioengineering roadmap for the requirements of the next generation of more predictive and authentic microscale systems fit for the purpose of not only disease modeling but also of drug screening to potentially allow OA animal model reduction and replacement in the near future.