Phenotypic screening in Organ-on-a-Chip systems: a 1537 kinase inhibitor library screen on a 3D angiogenesis assay.

Modern drug development increasingly requires comprehensive models that can be utilized in the earliest stages of compound and target discovery. Here we report a phenotypic screening exercise in a high-throughput Organ-on-a-Chip setup. We assessed the inhibitory effect of 1537 protein kinase inhibitors in an angiogenesis assay. Over 4000 micro-vessels were grown under perfusion flow in microfluidic chips, exposed to a cocktail of pro-angiogenic factors and subsequently exposed to the respective kinase inhibitors. Efficacy of compounds was evaluated by reduced angiogenic sprouting, whereas reduced integrity of the main micro-vessel was taken as a measure for toxicity. The screen yielded 53 hits with high anti-angiogenicity and low toxicity, of which 44 were previously unassociated with angiogenic pathways. This study demonstrates that Organ-on-a-Chip models can be screened in high numbers to identify novel compounds and targets. This will ultimately reduce bias in early-stage drug development and increases probability to identify first in class compounds and targets for today's intractable diseases.

Healthy and diseased placental barrier on-a-chip models suitable for standardized studies.

Pathologies associated with uteroplacental hypoxia, such as preeclampsia are among the leading causes of maternal and perinatal morbidity in the world. Its fundamental mechanisms are yet poorly understood due to a lack of good experimental models. Here we report an in vitro model of the placental barrier, based on co-culture of trophoblasts and endothelial cells against a collagen extracellular matrix in a microfluidic platform. The model yields a functional syncytium with barrier properties, polarization, secretion of relevant extracellular membrane components, thinning of the materno-fetal space, hormone secretion, and transporter function. The model is exposed to low oxygen conditions and perfusion flow is modulated to induce a pathological environment. This results in reduced barrier function, hormone secretion, and microvilli as well as an increased nuclei count, characteristics of preeclamptic placentas. The model is implemented in a titer plate-based microfluidic platform fully amenable to high-throughput screening. We thus believe this model could aid mechanistic understanding of preeclampsia and other placental pathologies associated with hypoxia/ischemia, as well as support future development of effective therapies through target and compound screening campaigns. STATEMENT OF SIGNIFICANCE: The human placenta is a unique organ sustaining fetal growth but is also the source of severe pathologies, such as preeclampsia. Though leading cause of perinatal mortality in the world, preeclampsia remains untreatable due to a lack of relevant in vitro placenta models. To better understand the pathology, we have developed 3D placental barrier models in a microfluidic device. The platform allows parallel culture of 40 perfused physiological miniaturized placental barriers, comprising a differentiated syncytium and endothelium that have been validated for transporter functions. Exposure to a hypoxic and ischemic environment enabled the mimicking of preeclamptic characteristics in high-throughput, which we believe could lead to a better understanding of the pathology as well as support future effective therapies development.

Gut-on-a-Chip Models: Current and Future Perspectives for Host-Microbial Interactions Research.

The intestine contains the largest microbial community in the human body, the gut microbiome. Increasing evidence suggests that it plays a crucial role in maintaining overall health. However, while many studies have found a correlation between certain diseases and changes in the microbiome, the impact of different microbial compositions on the gut and the mechanisms by which they contribute to disease are not well understood. Traditional pre-clinical models, such as cell culture or animal models, are limited in their ability to mimic the complexity of human physiology. New mechanistic models, such as organ-on-a-chip, are being developed to address this issue. These models provide a more accurate representation of human physiology and could help bridge the gap between clinical and pre-clinical studies. Gut-on-chip models allow researchers to better understand the underlying mechanisms of disease and the effect of different microbial compositions on the gut. They can help to move the field from correlation to causation and accelerate the development of new treatments for diseases associated with changes in the gut microbiome. This review will discuss current and future perspectives of gut-on-chip models to study host-microbial interactions.

A versatile multiplexed assay to quantify intracellular ROS and cell viability in 3D on-a-chip models.

Reactive oxygen species (ROS) have different properties and biological functions. They contribute to cell signaling and, in excessive amounts, to oxidative stress (OS). Although ROS is pivotal in a wide number of physiological systems and pathophysiological processes, direct quantification in vivo is quite challenging and mainly limited to in vitro studies. Even though advanced in vitro cell culture techniques, like on-a-chip culture, have overcome the lack of crucial in vivo-like physiological aspects in 2D culture, the majority of in vitro ROS quantification studies are generally performed in 2D. Here we report the development, application, and validation of a multiplexed assay to quantify ROS and cell viability in organ-on-a-chip models. The assay utilizes three dyes to stain live cells for ROS, dead cells, and DNA. Confocal images were analyzed to quantify ROS probes and determine the number of nuclei and dead cells. We found that, in contrast to what has been reported with 2D cell culture, on-a-chip models are more prone to scavenge ROS rather than accumulate them. The assay is sensitive enough to distinguish between different phenotypes of endothelial cells (ECs) based on the level of OS to detect higher level in tumor than normal cells. Our results indicate that the use of physiologically relevant models and this assay could help unravelling the mechanisms behind OS and ROS accumulation. A further step could be taken in data analysis by implementing AI in the pipeline to also analyze images for morphological changes to have an even broader view of OS mechanism.

Nephrotoxicity and Kidney Transport Assessment on 3D Perfused Proximal Tubules.

Proximal tubules in the kidney play a crucial role in reabsorbing and eliminating substrates from the body into the urine, leading to high local concentrations of xenobiotics. This makes the proximal tubule a major target for drug toxicity that needs to be evaluated during the drug development process. Here, we describe an advanced in vitro model consisting of fully polarized renal proximal tubular epithelial cells cultured in a microfluidic system. Up to 40 leak-tight tubules were cultured on this platform that provides access to the basolateral as well as the apical side of the epithelial cells. Exposure to the nephrotoxicant cisplatin caused a dose-dependent disruption of the epithelial barrier, a decrease in viability, an increase in effluent LDH activity, and changes in expression of tight-junction marker zona-occludence 1, actin, and DNA-damage marker H2A.X, as detected by immunostaining. Activity and inhibition of the efflux pumps P-glycoprotein (P-gp) and multidrug resistance protein (MRP) were demonstrated using fluorescence-based transporter assays. In addition, the transepithelial transport function from the basolateral to the apical side of the proximal tubule was studied. The apparent permeability of the fluorescent P-gp substrate rhodamine 123 was decreased by 35% by co-incubation with cyclosporin A. Furthermore, the activity of the glucose transporter SGLT2 was demonstrated using the fluorescent glucose analog 6-NBDG which was sensitive to inhibition by phlorizin. Our results demonstrate that we developed a functional 3D perfused proximal tubule model with advanced renal epithelial characteristics that can be used for drug screening studies.

Therapy response testing of breast cancer in a 3D high-throughput perfused microfluidic platform.

BACKGROUND: Breast cancer is the most common invasive cancer among women. Currently, there are only a few models used for therapy selection, and they are often poor predictors of therapeutic response or take months to set up and assay. In this report, we introduce a microfluidic OrganoPlate® platform for extracellular matrix (ECM) embedded tumor culture under perfusion as an initial study designed to investigate the feasibility of adapting this technology for therapy selection. METHODS: The triple negative breast cancer cell lines MDA-MB-453, MDA-MB-231 and HCC1937 were selected based on their different BRCA1 and P53 status, and were seeded in the platform. We evaluate seeding densities, ECM composition (Matrigel®, BME2rgf, collagen I) and biomechanical (perfusion vs static) conditions. We then exposed the cells to a series of anti-cancer drugs (paclitaxel, olaparib, cisplatin) and compared their responses to those in 2D cultures. Finally, we generated cisplatin dose responses in 3D cultures of breast cancer cells derived from 2 PDX models. RESULTS: The microfluidic platform allows the simultaneous culture of 96 perfused micro tissues, using limited amounts of material, enabling drug screening of patient-derived material. 3D cell culture viability is improved by constant perfusion of the medium. Furthermore, the drug response of these triple negative breast cancer cells was attenuated by culture in 3D and differed from that observed in 2D substrates. CONCLUSIONS: We have investigated the use of a high-throughput organ-on-a-chip platform to select therapies. Our results have raised the possibility to use this technology in personalized medicine to support selection of appropriate drugs and to predict response to therapy in a real time fashion.

Membrane-free culture and real-time barrier integrity assessment of perfused intestinal epithelium tubes.

In vitro models that better reflect in vivo epithelial barrier (patho-)physiology are urgently required to predict adverse drug effects. Here we introduce extracellular matrix-supported intestinal tubules in perfused microfluidic devices, exhibiting tissue polarization and transporter expression. Forty leak-tight tubules are cultured in parallel on a single plate and their response to pharmacological stimuli is recorded over 125 h using automated imaging techniques. A study comprising 357 gut tubes is performed, of which 93% are leak tight before exposure. EC50-time curves could be extracted that provide insight into both concentration and exposure time response. Full compatibility with standard equipment and user-friendly operation make this Organ-on-a-Chip platform readily applicable in routine laboratories.Efforts to determine the effects of drugs on epithelial barriers could benefit from better in vitro models. Here the authors develop a microfluidic device supporting the growth and function of extracellular matrix-supported intestinal tubules, and evaluate the effect of staurosporine and acetylsalicylic acid on barrier integrity.

Kidney-on-a-Chip Technology for Drug-Induced Nephrotoxicity Screening.

Improved model systems to predict drug efficacy, interactions, and drug-induced kidney injury (DIKI) are crucially needed in drug development. Organ-on-a-chip technology is a suitable in vitro system because it reproduces the 3D microenvironment. A kidney-on-a-chip can mimic the structural, mechanical, transport, absorptive, and physiological properties of the human kidney. In this review we address the application of state-of-the-art microfluidic culturing techniques, with a focus on culturing kidney proximal tubules, that are promising for the detection of biomarkers that predict drug interactions and DIKI. We also discuss high-throughput screening and the challenges for in vitro to in vivo extrapolation (IVIVE) that will need to be overcome for successful implementation.

Regenerating the cell resistance of micromolded PEG hydrogels.

Polydimethylsiloxane stamp materials used during soft lithography undermine the non-fouling behaviour of bio-inert PEG-based hydrogels, resulting in increased protein adsorption and cell adhesion and migration on the gel. This previously unreported phenomenon undermines the function of lab-on-a-chip devices that require the device to be bio-inert, and slows the implementation of promising micromolding and imprinting methods for 3D culture and commercial cell culture systems. We illustrate that the degree of cell adhesion and protein adsorption to the gels correlates with the amount of residual stamp material remaining at the hydrogel interface after fabrication. After identifying this previously unreported phenomenon, we screened multiple polymer cleaning/fabrication techniques in order to maintain/restore the non-fouling properties of the gels including PDMS curing and extraction, use of other common soft lithography stamp materials, post-fabrication cleaning of the hydrogels, and changing the composition of the hydrogel. The optimal solution was determined to be incorporation of reactive sites into the hydrogel during micromolding followed by grafting of PEG macromers to these sites post-fabrication. This treatment resulted in micromolded hydrogels with robust cell resistant properties. Broadly, this work identifies and solves a previously unreported problem in hydrogel micromolding, and specifically reports the development of a cell culture platform that when combined with video microscopy enables high-resolution in situ study of single cell behaviour during in vitro culture.

Enhanced ex vivo expansion of adult mesenchymal stem cells by fetal mesenchymal stem cell ECM.

Large-scale expansion of highly functional adult human mesenchymal stem cells (aMSCs) remains technologically challenging as aMSCs lose self renewal capacity and multipotency during traditional long-term culture and their quality/quantity declines with donor age and disease. Identification of culture conditions enabling prolonged expansion and rejuvenation would have dramatic impact in regenerative medicine. aMSC-derived decellularized extracellular matrix (ECM) has been shown to provide such microenvironment which promotes MSC self renewal and "stemness". Since previous studies have demonstrated superior proliferation and osteogenic potential of human fetal MSCs (fMSCs), we hypothesize that their ECM may promote expansion of clinically relevant aMSCs. We demonstrated that aMSCs were more proliferative (∼ 1.6 ×) on fMSC-derived ECM than aMSC-derived ECMs and traditional tissue culture wares (TCPS). These aMSCs were smaller and more uniform in size (median ± interquartile range: 15.5 ± 4.1 µm versus 17.2 ± 5.0 µm and 15.5 ± 4.1 µm for aMSC ECM and TCPS respectively), exhibited the necessary biomarker signatures, and stained positive for osteogenic, adipogenic and chondrogenic expressions; indications that they maintained multipotency during culture. Furthermore, fMSC ECM improved the proliferation (∼ 2.2 ×), size (19.6 ± 11.9 µm vs 30.2 ± 14.5 µm) and differentiation potential in late-passaged aMSCs compared to TCPS. In conclusion, we have established fMSC ECM as a promising cell culture platform for ex vivo expansion of aMSCs.

Surface characteristics of acrylic modified polysulfone membranes improves renal proximal tubule cell adhesion and spreading.

Current polyvinylpyrrolidone-modified polysulfone (PVP-PSU) membranes in haemodialysers do not facilitate the attachment and proliferation of renal proximal tubule cells (RPTCs). For bioartificial kidney (BAK) development expensive extracellular matrices are employed to ensure the PVP-PSU membranes can serve as a substrate for RPTCs. In this study we modified PSU using an acrylic monomer (am-PSU) and polymerization using ultraviolet irradiation. We demonstrated that on adjusting the PSU or acrylic content of the membranes the wettability and surface chemistry were altered, and this affected the amount of fibronectin (Fn) that was adsorbed onto the membranes. Using an integrin blocking assay we ascertained that Fn is an important extracellular matrix component that mediates RPTC attachment. The amount of Fn adsorbed also led to different bioresponses of RPTCs, which were evaluated using attachment and proliferation assays and qualitative quantification of vinculin, focal adhesion kinase, zonula occludens and Na(+)/K(+) ATPase. Our optimized membrane, am-PSU1 (21.4% C-O groups, 19.1% PVP-PSU; contact angle 71.5-80.80, PVP-PSU: 52.4-67.50), supports a confluent monolayer of RPTCs and prevents creatinine and inulin diffusion from the apical to the basal side, meeting the requirements for application in BAKs. However, further in vivo evaluation to assess the full functionality of RPTCs on am-PSU1 is required.

Bio-mimetic surface engineering of plasmid-loaded nanoparticles for active intracellular trafficking by actin comet-tail motility.

Intracellular transport after endosomal escape presents one of the major barriers for efficient non-viral gene delivery because plasmid DNA and synthetic nanoparticulate carriers suffer from significantly restricted diffusion in the cytoplasm. We postulate that forces generated by actin polymerization, a mechanism used by several bacterial pathogens such as Listeria monocytogenes, can be harnessed to propel nanoparticles within the cytoplasm and thereby overcome diffusional limitations associated with gene transport in the cell cytoplasm. In this work, we synthesized and characterized plasmid DNA-containing nanoparticles modified with ActA protein, the single protein in L. monocytogenes responsible for activating actin polymerization and initiating actin comet-tail propulsion. The motility of the ActA-modified nanoparticles was assessed in Xenopus laevis cytoplasmic extract supplemented with fluorescently labeled actin. Nanoparticle motility was monitored using multi-color, time-lapse fluorescence microscopy for the formation of actin comet tails attached to the fluorescently labeled vehicle. We observed particle motility with velocities approximately 0.06 microm/s with anionic-charged plasmid carriers formed from either poly(lactic-co-glycolic acid) (PLGA) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes, but interestingly not with cationic particles assembled by encapsulation of plasmid with either polyethylenimine (PEI) or 1,2-dioleoyl-3-trimethylammonium-propane/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOTAP/DOPE) lipids. Control particles coated with albumin instead of ActA also showed no motility. Taken together, we have demonstrated the feasibility of translating the comet-tail propulsion mechanism to synthetic drug carriers as a potential approach to overcome intracellular transport barriers, and also have identified appropriate gene delivery systems that can be employed for this mechanism.

3-D tissue culture systems for the evaluation and optimization of nanoparticle-based drug carriers.

Nanoparticle carriers are attractive vehicles for a variety of drug delivery applications. In order to evaluate nanoparticle formulations for biological efficacy, monolayer cell cultures are typically used as in vitro testing platforms. However, these studies sometimes poorly predict the efficacy of the drug in vivo. The poor in vitro and in vivo correlation may be attributed in part to the inability of two-dimensional cultures to reproduce extracellular barriers, and may also be due to differences in cell phenotype between cells cultured as monolayers and cells in native tissue. In order to more accurately predict in vivo results, it is desirable to test nanoparticle therapeutics in cells cultured in three-dimensional (3-D) models that mimic in vivo conditions. In this review, we discuss some 3-D culture systems that have been used to assess nanoparticle delivery and highlight several implications for nanoparticle design garnered from studies using these systems. While our focus will be on nanoparticle drug formulations, many of the systems discussed here could, or have been, used for the assessment of small molecule or peptide/protein drugs. We also offer some examples of advancements in 3-D culture that could provide even more highly predictive data for designing nanoparticle therapeutics for in vivo applications.

A perfusable 3D cell-matrix tissue culture chamber for in situ evaluation of nanoparticle vehicle penetration and transport.

A key factor in gene or drug therapy is the development of carriers that can efficiently reach targeted cells from a distal administration. In many gene/drug delivery studies, results obtained in 2D cultures fail to translate to similar results in vivo. In this work, we developed a perfusable 3D chamber for studying nanoparticle penetration and transport in cell-gel soft tissue cultures. The compartmented chamber is made of a polydimethylsiloxane (PDMS) top layer with the chamber features, created using micromachined lithography, bonded to a bottom glass coverslip. A solution of cells embedded in a hydrogel is loaded in the chamber between PDMS posts that serve as anchors to the cell-matrix at the gel-media interface. The chamber offers the following unique features: (i) rapid fabrication and simplicity in assembly, (ii) direct in situ cell imaging in a plane normal to the direction of flow or action, (iii) an easily configurable and controllable environment conducive cell culture under static or interstitial flow conditions, and (iv) facile recovery of live cells from chambers for post-experimental analysis. To assess the chamber, we delivered fluorescently labeled nanoparticles of three distinct sizes to cells-embedded Matrigels in the 3D chamber under flow and static conditions. Penetration of nanoparticles were enhanced under interstitial flow while live cell imaging and flow cytometry of recovered cells revealed particle size restrictions to efficient delivery. Although designed for delivery studies, the chamber is versatile and can be easily modified. Thus it may have broad applications for biological, tissue engineering, and therapeutic studies.

Determination of nanoparticle vehicle unpackaging by MR imaging of a T(2) magnetic relaxation switch.

Non-invasive imaging of gene and drug delivery is an important tool in understanding the biodistribution and pharmacokinetics of vectors after in vivo administration. In this work, we demonstrate the utility of a multifunctional delivery vector comprised of polyethylenimine conjugated to ultrasmall, superparamagnetic iron oxide (USPIO). The conjugate (USPIO-PEI) is capable of complexing plasmid DNA into nanoparticles (SPIO-polyplex) with diameters approximately 100 nm and protecting the DNA from nuclease degradation. SPIO-polyplexes transfect cells with high efficiency and low toxicity. In addition, the T2 relaxation time of water enhanced by USPIO is shown to be a function of the packaging state of the vector. Thus, this material integrates capabilities of gene delivery with magnetic resonance (MR) contrast and also provides an MR-based read-out for vector unpackaging.

Mechanisms of interstitial flow-induced remodeling of fibroblast-collagen cultures.

Interstitial fluid flow, critical for macromolecular transport, was recently shown to drive fibroblast differentiation and perpendicular cell and matrix alignment in 3D collagen cultures. Here we explore the mechanisms underlying this flow-induced cell and collagen alignment. Cell and matrix alignment was assessed from 3D confocal reflectance stacks using a Fast Fourier Transform method. We found that human dermal and lung fibroblasts align perpendicular to flow in the range of 5-13 mum/s (0.1-0.3 dyn/cm(2)) in collagen; however, neither cells nor matrix fibers align in fibrin cultures, which unlike collagen, is covalently cross-linked and generally degraded by cell fibrinolysis. We also found that even acellular collagen matrices align weakly upon exposure to flow. Matrix alignment begins within 12 h of flow onset and continues, along with cell alignment, over 48 h. Together, these data suggest that interstitial flow first induces collagen fiber alignment, providing contact guidance for the cells to orient along the aligned matrix; later, the aligned cells further remodel and align their surrounding matrix fibers. These findings help elucidate the effects of interstitial flow on cells in matrices and have relevance physiologically in tissue remodeling and in tissue engineering applications.

Interstitial fluid flow induces myofibroblast differentiation and collagen alignment in vitro.

The differentiation of fibroblasts to contractile myofibroblasts, which is characterized by de novo expression of alpha-smooth muscle actin (alpha-SMA), is crucial for wound healing and a hallmark of tissue scarring and fibrosis. These processes often follow inflammatory events, particularly in soft tissues such as skin, lung and liver. Although inflammatory cells and damaged epithelium can release transforming growth factor beta1 (TGF-beta1), which largely mediates myofibroblast differentiation, the biophysical environment of inflammation and tissue regeneration, namely increased interstitial flow owing to vessel hyperpermeability and/or angiogenesis, may also play a role. We demonstrate that low levels of interstitial (3D) flow induce fibroblast-to-myofibroblast differentiation as well as collagen alignment and fibroblast proliferation, all in the absence of exogenous mediators. These effects were associated with TGF-beta1 induction, and could be eliminated with TGF-beta1 blocking antibodies. Furthermore, alpha1beta1 integrin was seen to play an important role in the specific response to flow, as its inhibition prevented fibroblast differentiation and subsequent collagen alignment but did not block their ability to contract the gel in a separate floating gel assay. This study suggests that the biophysical environment that often precedes fibrosis, such as swelling, increased microvascular permeability and increased lymphatic drainage--all which involve interstitial fluid flow--may itself play an important role in fibrogenesis.

Interstitial flow differentially stimulates blood and lymphatic endothelial cell morphogenesis in vitro.

Whereas high shearing flows are known to induce endothelial cell remodeling, we show here that very low interstitial flow rates trigger endothelial cell morphogenesis in 3D cultures. Interstitial flow is a functionally critical component of the circulation, and we have recently observed that it plays a regulatory role in lymphangiogenesis; here we investigate interstitial flow as a powerful morphoregulatory stimulant. We exposed both lymphatic and blood endothelial cells (LECs and BECs) to interstitial flow in 3D collagen gels as well as simple shear flow in 2D monolayers. We found that under interstitial flow (average 10 microm/s for 6 days), both cell types underwent drastic morphologic changes from static conditions: LECs formed large vacuoles and long extensions, while BECs formed multicellular branched lumen-containing networks. Under planar shear (20 dyn/cm2 for 24 h), LECs downregulated their cell-cell adhesions compared to BECs but did not differ morphologically; both aligned with flow as expected. The significance of these findings is twofold: first, they identify an important role of interstitial flow for in vitro microvascular organization and stabilization, and second, they demonstrate for the first time notable differences between LEC and BEC response to the biophysical environment, reflecting some of their functional differences in vivo.

Fibroblast alignment under interstitial fluid flow using a novel 3-D tissue culture model.

Interstitial flow is an important component of the microcirculation and interstitial environment, yet its effects on cell organization and tissue architecture are poorly understood, in part due to the lack of in vitro models. To examine the effects of interstitial flow on cell morphology and matrix remodeling, we developed a tissue culture model that physically supports soft tissue cultures and allows microscopic visualization of cells within the three-dimensional matrix. In addition, pressure-flow relationships can be continuously monitored to evaluate the bulk hydraulic resistance as an indicator of changes in the overall matrix integrity. We observed that cells such as human dermal fibroblasts aligned perpendicular to the direction of interstitial flow. In contrast, fibroblasts in static three-dimensional controls remained randomly oriented, whereas cells subjected to fluid shear as a two-dimensional monolayer regressed. Also, the dynamic measurements of hydraulic conductivity suggest reorganization toward a steady state. These primary findings help establish the importance of interstitial flow on the biology of tissue organization and interstitial fluid balance.