Gas-modulating microcapsules for spatiotemporal control of hypoxia.

Oxygen is a vital molecule involved in regulating development, homeostasis, and disease. The oxygen levels in tissue vary from 1 to 14% with deviations from homeostasis impacting regulation of various physiological processes. In this work, we developed an approach to encapsulate enzymes at high loading capacity, which precisely controls the oxygen content in cell culture. Here, a single microcapsule is able to locally perturb the oxygen balance, and varying the concentration and distribution of matrix-embedded microcapsules provides spatiotemporal control. We demonstrate attenuation of hypoxia signaling in populations of stem cells, cancer cells, endothelial cells, cancer spheroids, and intestinal organoids. Varying capsule placement, media formulation, and timing of replenishment yields tunable oxygen gradients, with concurrent spatial growth and morphogenesis in a single well. Capsule containing hydrogel films applied to chick chorioallantoic membranes encourages neovascularization, providing scope for topical treatments or hydrogel wound dressings. This platform can be used in a variety of formats, including deposition in hydrogels, as granular solids for 3D bioprinting, and as injectable biomaterials. Overall, this platform's simplicity and flexibility will prove useful for fundamental studies of oxygen-mediated processes in virtually any in vitro or in vivo format, with scope for inclusion in biomedical materials for treating injury or disease.

Biomimetic Vasculatures by 3D-Printed Porous Molds.

Despite recent advances in biofabrication, recapitulating complex architectures of cell-laden vascular constructs remains challenging. To date, biofabricated vascular models have not yet realized four fundamental attributes of native vasculatures simultaneously: freestanding, branching, multilayered, and perfusable. In this work, a microfluidics-enabled molding technique combined with coaxial bioprinting to fabricate anatomically relevant, cell-laden vascular models consisting of hydrogels is developed. By using 3D porous molds of poly(ethylene glycol) diacrylate as casting templates that gradually release calcium ions as a crosslinking agent, freestanding, and perfusable vascular constructs of complex geometries are fabricated. The bioinks can be tailored to improve the compatibility with specific vascular cells and to tune the mechanical modulus mimicking native blood vessels. Crucially, the integration of relevant vascular cells (such as smooth muscle cells and endothelial cells) in a multilayer and biomimetic configuration is highlighted. It is also demonstrated that the fabricated freestanding vessels are amenable for testing percutaneous coronary interventions (i.e., drug-eluting balloons and stents) under physiological mechanical states such as stretching and bending. Overall, a versatile fabrication technique with multifaceted possibilities of generating biomimetic vascular models that can benefit future research in mechanistic understanding of cardiovascular diseases and the development of therapeutic interventions is introduced.

Determination of critical shear stress for maturation of human pluripotent stem cell-derived endothelial cells towards an arterial subtype.

Human pluripotent stem cell-derived endothelial cells (hPSC-ECs) present an attractive alternative to primary EC sources for vascular grafting. However, there is a need to mature them towards either an arterial or venous subtype. A vital environmental factor involved in the arteriovenous specification of ECs during early embryonic development is fluid shear stress; therefore, there have been attempts to employ adult arterial shear stress conditions to mature hPSC-ECs. However, hPSC-ECs are naïve to fluid shear stress, and their shear responses are still not well understood. Here, we used a multiplex microfluidic platform to systematically investigate the dose-time shear responses on hPSC-EC morphology and arterial-venous phenotypes over a range of magnitudes coincidental with physiological levels of embryonic and adult vasculatures. The device comprised of six parallel cell culture chambers that were individually linked to flow-setting resistance channels, allowing us to simultaneously apply shear stress ranging from 0.4 to 15 dyne/cm 2 . We found that hPSC-ECs required up to 40 hr of shear exposure to elicit a stable phenotypic change. Cell alignment was visible at shear stress <1 dyne/cm 2 , which was independent of shear stress magnitude and duration of exposure. We discovered that the arterial markers NOTCH1 and EphrinB2 exhibited a dose-dependent increase in a similar manner beyond a threshold level of 3.8 dyne/cm 2 , whereas the venous markers COUP-TFII and EphB4 expression remained relatively constant across different magnitudes. These findings indicated that hPSC-ECs were sensitive to relatively low magnitudes of shear stress, and a critical level of ~4 dyne/cm 2 was sufficient to preferentially enhance their maturation into an arterial phenotype for future vascular tissue engineering applications.

A pump-free microfluidic 3D perfusion platform for the efficient differentiation of human hepatocyte-like cells.

The practical application of microfluidic liver models for in vitro drug testing is partly hampered by their reliance on human primary hepatocytes, which are limited in number and have batch-to-batch variation. Human stem cell-derived hepatocytes offer an attractive alternative cell source, although their 3D differentiation and maturation in a microfluidic platform have not yet been demonstrated. We develop a pump-free microfluidic 3D perfusion platform to achieve long-term and efficient differentiation of human liver progenitor cells into hepatocyte-like cells (HLCs). The device contains a micropillar array to immobilize cells three-dimensionally in a central cell culture compartment flanked by two side perfusion channels. Constant pump-free medium perfusion is accomplished by controlling the differential heights of horizontally orientated inlet and outlet media reservoirs. Computational fluid dynamic simulation is used to estimate the hydrostatic pressure heads required to achieve different perfusion flow rates, which are experimentally validated by micro-particle image velocimetry, as well as viability and functional assessments in a primary rat hepatocyte model. We perform on-chip differentiation of HepaRG, a human bipotent progenitor cell, and discover that 3D microperfusion greatly enhances the hepatocyte differentiation efficiency over static 2D and 3D cultures. However, HepaRG progenitor cells are highly sensitive to the time-point at which microperfusion is applied. Isolated HepaRG cells that are primed as static 3D spheroids before being subjected to microperfusion yield a significantly higher proportion of HLCs (92%) than direct microperfusion of isolated HepaRG cells (62%). This platform potentially offers a simple and efficient means to develop highly functional microfluidic liver models incorporating human stem cell-derived HLCs. Biotechnol. Bioeng. 2017;114: 2360-2370. © 2017 Wiley Periodicals, Inc.

Functionally Enhanced Human Stem Cell Derived Hepatocytes in Galactosylated Cellulosic Sponges for Hepatotoxicity Testing.

Pluripotent stem cell derived hepatocyte-like cells (hPSC-HLCs) are an attractive alternative to primary human hepatocytes (PHHs) used in applications ranging from therapeutics to drug safety testing studies. It would be critical to improve and maintain mature hepatocyte functions of the hPSC-HLCs, especially for long-term studies. If 3D culture systems were to be used for such purposes, it would be important that the system can support formation and maintenance of optimal-sized spheroids for long periods of time, and can also be directly deployed in liver drug testing assays. We report the use of 3-dimensional (3D) cellulosic scaffold system for the culture of hPSC-HLCs. The scaffold has a macroporous network which helps to control the formation and maintenance of the spheroids for weeks. Our results show that culturing hPSC-HLCs in 3D cellulosic scaffolds increases functionality, as demonstrated by improved urea production and hepatic marker expression. In addition, hPSC-HLCs in the scaffolds exhibit a more mature phenotype, as shown by enhanced cytochrome P450 activity and induction. This enables the system to show a higher sensitivity to hepatotoxicants and a higher degree of similarity to PHHs when compared to conventional 2D systems. These results suggest that 3D cellulosic scaffolds are ideal for the long-term cultures needed to mature hPSC-HLCs. The mature hPSC-HLCs with improved cellular function can be continually maintained in the scaffolds and directly used for hepatotoxicity assays, making this system highly attractive for drug testing applications.

Development of a Probability-Based In Vitro Eye Irritation Screening Platform.

Traditional eye irritation assessments, which rely on animal models or ex vivo tissues, face limitations due to ethical concerns, costs, and low throughput. Although numerous in vitro tests have been developed, none have successfully reconciled the need for high experimental throughput with the accurate prediction of irritation potential, attributable to the complexity of irritation mechanisms. Simple cell models, while suitable for high-throughput screening, offer limited mechanistic insights, contrasting with more physiologically relevant but less scalable complex organotypic corneal tissue constructs. This study presents a novel strategy to enhance the predictive accuracy of screening-compatible simple cell models in eye irritation testing. Our method combines the results of two in vitro assays-cell apoptosis and nociceptor (TRPV1) activation-using micropatterned chips to partition human corneal epithelial cells into numerous discrete small populations. Following exposure to test compounds, we measure apoptosis and nociceptor activation responses. The large datasets collected from the cell micropatterns facilitate binarization and statistical fitting to calculate a mathematical probability, which assesses the compound's potential to cause eye irritation. This method potentially enables the amalgamation of multiple mechanistic readouts into a singular index, providing a more accurate and reliable prediction of eye irritation potential in a format amenable to high-throughput screening.

An AI-assisted integrated, scalable, single-cell phenomic-transcriptomic platform to elucidate intratumor heterogeneity against immune response.

We present a novel framework combining single-cell phenotypic data with single-cell transcriptomic analysis to identify factors underpinning heterogeneity in antitumor immune response. We developed a pairwise, tumor-immune discretized interaction assay between natural killer (NK-92MI) cells and patient-derived head and neck squamous cell carcinoma (HNSCC) cell lines on a microfluidic cell-trapping platform. Furthermore we generated a deep-learning computer vision algorithm that is capable of automating the acquisition and analysis of a large, live-cell imaging data set (>1 million) of paired tumor-immune interactions spanning a time course of 24 h across multiple HNSCC lines (n = 10). Finally, we combined the response data measured by Kaplan-Meier survival analysis against NK-mediated killing with downstream single-cell transcriptomic analysis to interrogate molecular signatures associated with NK-effector response. As proof-of-concept for the proposed framework, we efficiently identified MHC class I-driven cytotoxic resistance as a key mechanism for immune evasion in nonresponders, while enhanced expression of cell adhesion molecules was found to be correlated with sensitivity against NK-mediated cytotoxicity. We conclude that this integrated, data-driven phenotypic approach holds tremendous promise in advancing the rapid identification of new mechanisms and therapeutic targets related to immune evasion and response.

Liver click dECM hydrogels for engineering hepatic microenvironments.

Decellularized extracellular matrix (dECM) hydrogels provide tissue-specific microenvironments which accommodate physiological cellular phenotypes in 3D in vitro cell cultures. However, their formation hinges on collagen fibrillogenesis, a complex process which limits regulation of physicochemical properties. Hence, achieving reproducible results with dECM hydrogels poses as a challenge. Here, we demonstrate that thiolation of solubilized liver dECM enables rapid formation of covalently crosslinked hydrogels via Michael type addition, allowing for precise control over mechanical properties and superior organotypic biological activity. Investigation of various decellularization methodologies revealed that treatment of liver tissue with Triton X-100 and ammonium hydroxide resulted in near complete DNA removal with significant retention of the native liver proteome. Chemical functionalization of pepsin-solubilized liver dECMs via 1-ethyl-3(3-dimethylamino)propyl carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling of L-Cysteine created thiolated liver dECM (dECM-SH), which rapidly reacted with 4-arm polyethylene glycol (PEG)-maleimide to form optically clear hydrogels under controlled conditions. Importantly, Young's moduli could be precisely tuned between 1 - 7 kPa by varying polymer concentrations, enabling close replication of healthy and fibrotic liver conditions in in vitro cell cultures. Click dECM-SH hydrogels were cytocompatible, supported growth of HepG2 and HepaRG liver cells, and promoted liver-specific functional phenotypes as evidenced by increased metabolic activity, as well CYP1A2 and CYP3A4 activity and excretory function when compared to monolayer culture and collagen-based hydrogels. Our findings demonstrate that click-functionalized dECM hydrogels offer a highly controlled, reproducible alternative to conventional tissue-derived hydrogels for in vitro cell culture applications. STATEMENT OF SIGNIFICANCE: Traditional dECM hydrogels face challenges in reproducibility and mechanical property control due to variable crosslinking processes. We introduce a click hydrogel based on porcine liver decellularized extracellular matrix (dECM) that circumnavigates these challenges. After optimizing liver decellularization for ECM retention, we integrated thiol-functionalized liver dECM with polyethylene-glycol derivatives through Michael-type addition click chemistry, enabling rapid, room-temperature gelation. This offers enhanced control over the hydrogel's mechanical and biochemical properties. The resultant click dECM hydrogels mimic the liver's natural ECM and exhibit greater mechanical tunability and handling ease, facilitating their application in high-throughput and industrial settings. Moreover, these hydrogels significantly improve the function of HepaRG-derived hepatocytes in 3D culture, presenting an advancement for liver tissue cell culture models for drug testing applications.

The FDA modernisation act 2.0: Bringing non-animal technologies to the regulatory table.

The FDA modernisation Act 2.0 marks a game-changing legislation enabling drug registration without the absolute requirement for the use of animals in safety toxicology assessment. We discuss landmark developments in the legislation under which the FDA operates and consider the implications of this most recent chapter in the evolution of the drug regulation pathway, focussing on new opportunities to embed microphysiological systems.

Controlling Microenvironments with Organs-on-Chips for Osteoarthritis Modelling.

Osteoarthritis (OA) remains a prevalent disease affecting more than 20% of the global population, resulting in morbidity and lower quality of life for patients. The study of OA pathophysiology remains predominantly in animal models due to the complexities of mimicking the physiological environment surrounding the joint tissue. Recent development in microfluidic organ-on-chip (OoC) systems have demonstrated various techniques to mimic and modulate tissue physiological environments. Adaptations of these techniques have demonstrated success in capturing a joint tissue's tissue physiology for studying the mechanism of OA. Adapting these techniques and strategies can help create human-specific in vitro models that recapitulate the cellular processes involved in OA. This review aims to comprehensively summarise various demonstrations of microfluidic platforms in mimicking joint microenvironments for future platform design iterations.

Vat photopolymerization 3D printed microfluidic devices for organ-on-a-chip applications.

Organs-on-a-chip, or OoCs, are microfluidic tissue culture devices with micro-scaled architectures that repeatedly achieve biomimicry of biological phenomena. They are well positioned to become the primary pre-clinical testing modality as they possess high translational value. Current methods of fabrication have facilitated the development of many custom OoCs that have generated promising results. However, the reliance on microfabrication and soft lithographic fabrication techniques has limited their prototyping turnover rate and scalability. Additive manufacturing, known commonly as 3D printing, shows promise to expedite this prototyping process, while also making fabrication easier and more reproducible. We briefly introduce common 3D printing modalities before identifying two sub-types of vat photopolymerization - stereolithography (SLA) and digital light processing (DLP) - as the most advantageous fabrication methods for the future of OoC development. We then outline the motivations for shifting to 3D printing, the requirements for 3D printed OoCs to be competitive with the current state of the art, and several considerations for achieving successful 3D printed OoC devices touching on design and fabrication techniques, including a survey of commercial and custom 3D printers and resins. In all, we aim to form a guide for the end-user to facilitate the in-house generation of 3D printed OoCs, along with the future translation of these important devices.

Preferential adhesion of bacterial cells onto top- and bottom-mounted nanostructured surfaces under flow conditions.

The bactericidal effect of biomimetic nanostructured surfaces has been known for a long time, with recent data suggesting an enhanced efficiency of the nanostructured surfaces under fluid shear. While some of the influential factors on the bactericidal effect of nanostructured surfaces under fluid shear are understood, there are numerous important factors yet to be studied, which is essential for the successful implementation of this technology in industrial applications. Among those influential factors, the orientation of the nanostructured surface can play an important role in bacterial cell adhesion onto surfaces. Gravitational effects can become dominant under low flow velocities, making the diffusive transport of bacterial cells more prominent than the advective transport. However, the role of nanostructure orientation in determining its bactericidal efficiency under flow conditions is still not clear. In this study, we analysed the effect of surface orientation of nanostructured surfaces, along with bacterial cell concentration, fluid flow rate, and the duration of time which the surface is exposed to flow, on bacterial adhesion and viability on these surfaces. Two surface orientations, with one on the top and the other on the bottom of a flow channel, were studied. Under flow conditions, the bactericidal efficacy of the nanostructured surface is both orientation and bacterial species dependent. The effects of cell concentration, fluid flow rate, and exposure time on cell adhesion are independent of the nanostructured surface orientation. Fluid shear showed a species-dependent effect on bacterial adhesion, while the effects of concentration and exposure time on bacterial cell adhesion are independent of the bacterial species. Moreover, bacterial cells demonstrate preferential adhesion onto surfaces based on the surface orientation, and these effects are species dependent. These results outline the capabilities and limitations of nanostructures under flow conditions. This provides valuable insights into the applications of nanostructures in medical or industrial sectors, which are associated with overlaying fluid flow.

3D-printed biomimetic scaffolds with precisely controlled and tunable structures guide cell migration and promote regeneration of osteochondral defect.

Untreated osteochondral defects will develop into osteoarthritis, affecting patients' quality of life. Since articular cartilage and subchondral bone exhibit distinct biological characteristics, repairing osteochondral defects remains a major challenge. Previous studies have tried to fabricate multilayer scaffolds with traditional methods or 3D printing technology. However, the efficacy is unsatisfactory because of poor control over internal structures or a lack of integrity between adjacent layers, severely compromising repair outcomes. Therefore, there is a need for a biomimetic scaffold that can simultaneously boost osteochondral defect regeneration in both structure and function. Herein, an integrated bilayer scaffold with precisely controlled structures is successfully 3D-printed in one step via digital light processing (DLP) technology. The upper layer has both 'lotus- and radial-' distribution pores, and the bottom layer has 'lotus-' pores to guide and facilitate the migration of chondrocytes and bone marrow mesenchymal stem cells, respectively, to the defect area. Tuning pore sizes could modulate the mechanical properties of scaffolds easily. Results show that 3D-printed porous structures allow significantly more cells to infiltrate into the area of 'lotus- and radial-' distribution pores during cell migration assay, subcutaneous implantation, andin situtransplantation, which are essential for osteochondral repair. Transplantation of this 3D-printed bilayer scaffold exhibits a promising osteochondral repair effect in rabbits. Incorporation of Kartogenin into the upper layer of scaffolds further induces better cartilage formation. Combining small molecules/drugs and precisely size-controlled and layer-specific porous structure via DLP technology, this 3D-printed bilayer scaffold is expected to be a potential strategy for osteochondral regeneration.

A User-Centric 3D-Printed Modular Peristaltic Pump for Microfluidic Perfusion Applications.

Microfluidic organ-on-a-chip (OoC) technology has enabled studies on dynamic physiological conditions as well as being deployed in drug testing applications. A microfluidic pump is an essential component to perform perfusion cell culture in OoC devices. However, it is challenging to have a single pump that can fulfil both the customization function needed to mimic a myriad of physiological flow rates and profiles found in vivo and multiplexing requirements (i.e., low cost, small footprint) for drug testing operations. The advent of 3D printing technology and open-source programmable electronic controllers presents an opportunity to democratize the fabrication of mini-peristaltic pumps suitable for microfluidic applications at a fraction of the cost of commercial microfluidic pumps. However, existing 3D-printed peristaltic pumps have mainly focused on demonstrating the feasibility of using 3D printing to fabricate the structural components of the pump and neglected user experience and customization capability. Here, we present a user-centric programmable 3D-printed mini-peristaltic pump with a compact design and low manufacturing cost (~USD 175) suitable for perfusion OoC culture applications. The pump consists of a user-friendly, wired electronic module that controls the operation of a peristaltic pump module. The peristaltic pump module comprises an air-sealed stepper motor connected to a 3D-printed peristaltic assembly, which can withstand the high-humidity environment of a cell culture incubator. We demonstrated that this pump allows users to either program the electronic module or use different-sized tubing to deliver a wide range of flow rates and flow profiles. The pump also has multiplexing capability as it can accommodate multiple tubing. The performance and user-friendliness of this low-cost, compact pump can be easily deployed for various OoC applications.

FEZ1 participates in human embryonic brain development by modulating neuronal progenitor subpopulation specification and migrations.

Mutations in the human fasciculation and elongation protein zeta 1 (FEZ1) gene are found in schizophrenia and Jacobsen syndrome patients. Here, using human cerebral organoids (hCOs), we show that FEZ1 expression is turned on early during brain development and is detectable in both neuroprogenitor subtypes and immature neurons. FEZ1 deletion disrupts expression of neuronal and synaptic development genes. Using single-cell RNA sequencing, we detected abnormal expansion of homeodomain-only protein homeobox (HOPX)- outer radial glia (oRG), concurrent with a reduction of HOPX+ oRG, in FEZ1-null hCOs. HOPX- oRGs show higher cell mobility as compared to HOPX+ oRGs. Ectopic localization of neuroprogenitors to the outer layer is seen in FEZ1-null hCOs. Anomalous encroachment of TBR2+ intermediate progenitors into CTIP2+ deep layer neurons further indicated abnormalities in cortical layer formation these hCOs. Collectively, our findings highlight the involvement of FEZ1 in early cortical brain development and how it contributes to neurodevelopmental disorders.

Fluid shear stress primes mouse embryonic stem cells for differentiation in a self-renewing environment via heparan sulfate proteoglycans transduction.

Shear stress is a ubiquitous environmental cue experienced by stem cells when they are being differentiated or expanded in perfusion cultures. However, its role in modulating self-renewing stem cell phenotypes is unclear, since shear is usually only studied in the context of cardiovascular differentiation. We used a multiplex microfluidic array, which overcomes the limitations of macroperfusion systems in shear application throughput and precision, to initiate a comprehensive, quantitative study of shear effects on self-renewing mouse embryonic stem cells (mESCs), where shear stresses varying by >1000 times (0.016-16 dyn/cm(2)) are applied simultaneously. When compared with static controls in the presence or absence of a saturated soluble environment (i.e., mESC-conditioned medium), we ascertained that flow-induced shear stress specifically up-regulates the epiblast marker Fgf5. Epiblast-state transition in mESCs involves heparan sulfate proteoglycans (HSPGs), which have also been shown to transduce shear stress in endothelial cells. By disrupting (with sulfation inhibitors and heparinase) and partially reconstituting (with heparin) HSPG function, we show that mESCs also mechanically sense shear stress via HSPGs to modulate Fgf5 expression. This study demonstrates that self-renewing mESCs possess the molecular machinery to sense shear stress and provides quantitative shear application benchmarks for future scalable stem cell culture systems.

Design and fabrication of micro/nanofluidics devices and systems.

This chapter provides an overview of the science, engineering, and design methods required in the development of micro/nanofluidic devices. Section 2 provides the scientific background of fluid mechanics and physical phenomena in micro/nanoscale. Section 3 gives a brief overview of the existing fabrication techniques employed in micro/nanofluidics. The techniques are grouped into three categories: (1) subtractive manufacturing, (2) formative manufacturing, and (3) additive manufacturing. The advantages and disadvantages of each manufacturing technique are also discussed. Implementation of the fluidic devices beyond laboratory demonstrations is not trivial, which requires a good understanding of the problems of interest and the end-users. To that end, Section 4 introduces the design thinking approach and its application to develop micro/nanofluidic devices. Finally, Section 5 concludes the chapter with future outlooks.

Fluid Flow Induces Differential Detachment of Live and Dead Bacterial Cells from Nanostructured Surfaces.

Nanotopographic surfaces are proven to be successful in killing bacterial cells upon contact. This non-chemical bactericidal property has paved an alternative way of fighting bacterial colonization and associated problems, especially the issue of bacteria evolving resistance against antibiotic and antiseptic agents. Recent advancements in nanotopographic bactericidal surfaces have made them suitable for many applications in medical and industrial sectors. The bactericidal effect of nanotopographic surfaces is classically studied under static conditions, but the actual potential applications do have fluid flow in them. In this study, we have studied how fluid flow can affect the adherence of bacterial cells on nanotopographic surfaces. Gram-positive and Gram-negative bacterial species were tested under varying fluid flow rates for their retention and viability after flow exposure. The total number of adherent cells for both species was reduced in the presence of flow, but there was no flowrate dependency. There was a significant reduction in the number of live cells remaining on nanotopographic surfaces with an increasing flowrate for both species. Conversely, we observed a flowrate-independent increase in the number of adherent dead cells. Our results indicated that the presence of flow differentially affected the adherent live and dead bacterial cells on nanotopographic surfaces. This could be because dead bacterial cells were physically pierced by the nano-features, whereas live cells adhered via physiochemical interactions with the surface. Therefore, fluid shear was insufficient to overcome adhesion forces between the surface and dead cells. Furthermore, hydrodynamic forces due to the flow can cause more planktonic and detached live cells to collide with nano-features on the surface, causing more cells to lyse. These results show that nanotopographic surfaces do not have self-cleaning ability as opposed to natural bactericidal nanotopographic surfaces, and nanotopographic surfaces tend to perform better under flow conditions. These findings are highly useful for developing and optimizing nanotopographic surfaces for medical and industrial applications.

Integration of a microfluidic multicellular coculture array with machine learning analysis to predict adverse cutaneous drug reactions.

Adverse cutaneous reactions are potentially life-threatening skin side effects caused by drugs administered into the human body. The availability of a human-specific in vitro platform that can prospectively screen drugs and predict this risk is therefore of great importance to drug safety. However, since adverse cutaneous drug reactions are mediated by at least 2 distinct mechanisms, both involving systemic interactions between liver, immune and dermal tissues, existing in vitro skin models have not been able to comprehensively recapitulate these complex, multi-cellular interactions to predict the skin-sensitization potential of drugs. Here, we report a novel in vitro drug screening platform, which comprises a microfluidic multicellular coculture array (MCA) to model different mechanisms-of-action using a collection of simplistic cellular assays. The resultant readouts are then integrated with a machine-learning algorithm to predict the skin sensitizing potential of systemic drugs. The MCA consists of 4 cell culture compartments connected by diffusion microchannels to enable crosstalk between hepatocytes that generate drug metabolites, antigen-presenting cells (APCs) that detect the immunogenicity of the drug metabolites, and keratinocytes and dermal fibroblasts, which collectively determine drug metabolite-induced FasL-mediated apoptosis. A single drug screen using the MCA can simultaneously generate 5 readouts, which are integrated using support vector machine (SVM) and principal component analysis (PCA) to classify and visualize the drugs as skin sensitizers or non-skin sensitizers. The predictive performance of the MCA and SVM classification algorithm is then validated through a pilot screen of 11 drugs labelled by the US Food and Drug Administration (FDA), including 7 skin-sensitizing and 4 non-skin sensitizing drugs, using stratified 4-fold cross-validation (CV) on SVM. The predictive performance of our in vitro model achieves an average of 87.5% accuracy (correct prediction rate), 75% specificity (prediction rate of true negative drugs), and 100% sensitivity (prediction rate of true positive drugs). We then employ the MCA and the SVM training algorithm to prospectively identify the skin-sensitizing likelihood and mechanism-of-action for obeticholic acid (OCA), a farnesoid X receptor (FXR) agonist which has undergone clinical trials for non-alcoholic steatohepatitis (NASH) with well-documented cutaneous side effects.

Bactericidal Efficacy of Nanostructured Surfaces Increases under Flow Conditions.

Bacterial colonization on solid surfaces creates enormous problems across various industries causing billions of dollars' worth of economic damages and costing human lives. Biomimicking nanostructured surfaces have demonstrated a promising future in mitigating bacterial colonization and related issues. The importance of this non-chemical method has been elevated due to bacterial evolvement into antibiotic and antiseptic-resistant strains. However, bacterial attachment and viability on nanostructured surfaces under fluid flow conditions has not been investigated thoroughly. In this study, attachment and viability of Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) on a model nanostructured surface were studied under fluid flow conditions. A wide range of flow rates resulting in a broad spectrum of fluid wall shear stress on a nanostructured surface representing various application conditions were experimentally investigated. The bacterial suspension was pumped through a custom-designed microfluidic device (MFD) that contains a sterile Ti-6Al-4V substrate. The surface of the titanium substrate was modified using a hydrothermal synthesis process to fabricate the nanowire structure on the surface. The results of the current study show that the fluid flow significantly reduces bacterial adhesion onto nanostructured surfaces and significantly reduces the viability of adherent cells. Interestingly, the bactericidal efficacy of the nanostructured surface was increased under the flow by ∼1.5-fold against P. aeruginosa and ∼3-fold against S. aureus under static conditions. The bactericidal efficacy had no dependency on the fluid wall shear stress level. However, trends in the dead-cell count with the fluid wall shear were slightly different between the two species. These findings will be highly useful in developing and optimizing nanostructures in the laboratory as well as translating them into successful industrial applications. These findings may be used to develop antibacterial surfaces on biomedical equipment such as catheters and vascular stents or industrial applications such as ship hulls and pipelines where bacterial colonization is a great challenge.