In vitro grafting of hepatic spheroids and organoids on a microfluidic vascular bed.

With recent progress in modeling liver organogenesis and regeneration, the lack of vasculature is becoming the bottleneck in progressing our ability to model human hepatic tissues in vitro. Here, we introduce a platform for routine grafting of liver and other tissues on an in vitro grown microvascular bed. The platform consists of 64 microfluidic chips patterned underneath a 384-well microtiter plate. Each chip allows the formation of a microvascular bed between two main lateral vessels by inducing angiogenesis. Chips consist of an open-top microfluidic chamber, which enables addition of a target tissue by manual or robotic pipetting. Upon grafting a liver microtissue, the microvascular bed undergoes anastomosis, resulting in a stable, perfusable vascular network. Interactions with vasculature were found in spheroids and organoids upon 7 days of co-culture with space of Disse-like architecture in between hepatocytes and endothelium. Veno-occlusive disease was induced by azathioprine exposure, leading to impeded perfusion of the vascularized spheroid. The platform holds the potential to replace animals with an in vitro alternative for routine grafting of spheroids, organoids, or (patient-derived) explants.

Direct On-Chip Differentiation of Intestinal Tubules from Induced Pluripotent Stem Cells.

Intestinal organoids have emerged as the new paradigm for modelling the healthy and diseased intestine with patient-relevant properties. In this study, we show directed differentiation of induced pluripotent stem cells towards intestinal-like phenotype within a microfluidic device. iPSCs are cultured against a gel in microfluidic chips of the OrganoPlate, in which they undergo stepwise differentiation. Cells form a tubular structure, lose their stem cell markers and start expressing mature intestinal markers, including markers for Paneth cells, enterocytes and neuroendocrine cells. Tubes develop barrier properties as confirmed by transepithelial electrical resistance (TEER). Lastly, we show that tubules respond to pro-inflammatory cytokine triggers. The whole procedure for differentiation lasts 14 days, making it an efficient process to make patient-specific organoid tubules. We anticipate the usage of the platform for disease modelling and drug candidate screening.

Development of a Gut-On-A-Chip Model for High Throughput Disease Modeling and Drug Discovery.

A common bottleneck in any drug development process is finding sufficiently accurate models that capture key aspects of disease development and progression. Conventional drug screening models often rely on simple 2D culture systems that fail to recapitulate the complexity of the organ situation. In this study, we show the application of a robust high throughput 3D gut-on-a-chip model for investigating hallmarks of inflammatory bowel disease (IBD). Using the OrganoPlate platform, we subjected enterocyte-like cells to an immune-relevant inflammatory trigger in order to recapitulate key events of IBD and to further investigate the suitability of this model for compound discovery and target validation activities. The induction of inflammatory conditions caused a loss of barrier function of the intestinal epithelium and its activation by increased cytokine production, two events observed in IBD physiopathology. More importantly, anti-inflammatory compound exposure prevented the loss of barrier function and the increased cytokine release. Furthermore, knockdown of key inflammatory regulators RELA and MYD88 through on-chip adenoviral shRNA transduction alleviated IBD phenotype by decreasing cytokine production. In summary, we demonstrate the routine use of a gut-on-a-chip platform for disease-specific aspects modeling. The approach can be used for larger scale disease modeling, target validation and drug discovery purposes.

High-throughput Bronchus-on-a-Chip system for modeling the human bronchus.

Airway inflammation, a protective response in the human body, can disrupt normal organ function when chronic, as seen in chronic obstructive pulmonary disease (COPD) and asthma. Chronic bronchitis induces goblet cell hyperplasia and metaplasia, obstructing airflow. Traditional animal testing is often replaced by in vitro three-dimensional cultures of human epithelial cells to assess chronic cell responses. However, these cells are cultured horizontally, differing from the tubular structure of the human airway and failing to accurately reproduce airway stenosis. To address this, we developed the Bronchus-on-a-Chip (BoC) system. The BoC uses a novel microfluidic design in a standard laboratory plate, embedding 62 chips in one plate. Human bronchial epithelial cells were cultured against a collagen extracellular matrix for up to 35 days. Characterization included barrier integrity assays, microscopy, and histological examination. Cells successfully cultured in a tubular structure, with the apical side air-lifted. Epithelial cells differentiated into basal, ciliated, and secretory cells, mimicking human bronchial epithelium. Upon exposure to inducers of goblet cell hyperplasia and metaplasia, the BoC system showed mucus hyperproduction, replicating chronic epithelial responses. This BoC system enhances in vitro testing for bronchial inflammation, providing a more human-relevant and high-throughput method.

Vascular inflammation on a chip: A scalable platform for trans-endothelial electrical resistance and immune cell migration.

The vasculature system plays a critical role in inflammation processes in the body. Vascular inflammatory mechanisms are characterized by disruption of blood vessel wall permeability together with increased immune cell recruitment and migration. There is a critical need to develop models that fully recapitulate changes in vascular barrier permeability in response to inflammatory conditions. We developed a scalable platform for parallel measurements of trans epithelial electrical resistance (TEER) in 64 perfused microfluidic HUVEC tubules under inflammatory conditions. Over 250 tubules where exposed to Tumor necrosis factor alpha (TNFα) and interferon gamma (INF-γ) or human peripheral blood mononuclear cells. The inflammatory response was quantified based on changes TEER and expression of ICAM and VE-cadherin. We observed changes in barrier function in the presence of both inflammatory cytokines and human peripheral blood mononuclear cells, characterized by decreased TEER values, increase in ICAM expression as well changes in endothelial morphology. OrganoPlate 3-lane64 based HUVEC tubules provide a valuable tool for inflammatory studies in an automation compatible manner. Continuous TEER measurements enable long term, sensitive assays for barrier studies. We propose the use of our platform as a powerful tool for modelling endothelial inflammation in combination with immune cell interaction that can be used to screen targets and drugs to treat chronic vascular inflammation.

Culture and analysis of kidney tubuloids and perfused tubuloid cells-on-a-chip.

Advanced in vitro kidney models are of great importance to the study of renal physiology and disease. Kidney tubuloids can be established from primary cells derived from adult kidney tissue or urine. Tubuloids are three-dimensional multicellular structures that recapitulate tubular function and have been used to study infectious, malignant, metabolic, and genetic diseases. For tubuloids to more closely represent the in vivo kidney, they can be integrated into an organ-on-a-chip system that has a more physiological tubular architecture and allows flow and interaction with vasculature or epithelial and mesenchymal cells from other organs. Here, we describe a detailed protocol for establishing tubuloid cultures from tissue and urine (1-3 weeks), as well as for generating and characterizing tubuloid cell-derived three-dimensional tubular structures in a perfused microfluidic multi-chip platform (7 d). The combination of the two systems yields a powerful in vitro tool that better recapitulates the complexity of the kidney tubule with donor-specific properties.

An Intestine-on-a-Chip Model of Plug-and-Play Modularity to Study Inflammatory Processes.

Development of efficient drugs and therapies for the treatment of inflammatory conditions in the intestine is often hampered by the lack of reliable, robust, and high-throughput in vitro and in vivo models. Current models generally fail to recapitulate key aspects of the intestine, resulting in low translatability to the human situation. Here, an immunocompetent 3D perfused intestine-on-a-chip platform was developed and characterized for studying intestinal inflammation. Forty independent polarized 3D perfused epithelial tubular structures were grown from cells of mixed epithelial origin, including enterocytes (Caco-2) and goblet cells (HT29-MTX-E12). Immune cells THP-1 and MUTZ-3, which can be activated, were added to the system and assessed for cytokine release. Intestinal inflammation was mimicked through exposure to tumor necrosis factor-α (TNFα) and interleukin (IL)-1ß. The effects were quantified by measuring transepithelial electrical resistance (TEER) and proinflammatory cytokine secretion on the apical and basal sides. Cytokines induced an inflammatory state in the culture, as demonstrated by the impaired barrier function and increased IL-8 secretion. Exposure to the known anti-inflammatory drug TPCA-1 prevented the inflammatory state. The model provides biological modularity for key aspects of intestinal inflammation, making use of well-established cell lines. This allows robust assays that can be tailored in complexity to serve all preclinical stages in the drug discovery and development process.

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.

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.

One-year efficacy and safety of the Trufill DCS Orbit and Orbit Galaxy detachable coils in the endovascular treatment of intracranial aneurysms: Results from the TRULINE study.

Background and purpose No series reported the mid-term results of Trufill DCS Orbit and Orbit Galaxy detachable coils with independent evaluation. We present the one-year safety and efficacy of these coils in real-life routine clinical practice. Methods A total of 167 patients with 167 aneurysms (39.1% ruptured) were enrolled in the prospective TRULINE study. The primary endpoint was the safety, assessed by the combined morbidity-mortality rate observed since the time of the procedure and up to one-year follow-up. For safety, primary analyses were performed on intent-to-treat population (attempted coils procedure) and all adverse events have been reviewed by an independent Data Safety Monitoring Board. For efficacy, primary analyses were performed on the per-protocol population (patients treated with more than 70% of Trufill coils and not retreated during the follow-up period) and an independent core laboratory evaluated angiographic results. Results At one-year post-procedure, neurologic impairment was observed in 6.5% (95% confidence interval: 3.5-11.8) of the patients, and 2.6% (95% confidence interval: 1.0-6.8) had a permanent neurological deterioration. Three deaths were observed, unrelated to the procedure or coils. At one year, complete occlusion was seen in 52 aneurysms (54.2%), neck remnant in 28 aneurysms (29.2%), and aneurysm remnant in 16 aneurysms (16.7%). During the one-year follow-up, the overall incidence of recurrence was 30.2% with a mean interval of 13.8 ± 4.5 months and the retreatment for major recanalization was needed in nine patients (6.3%). Conclusions The TRULINE study confirms that endovascular coiling with Trufill DCS Orbit and Orbit Galaxy detachable coils is safe and effective.

High-throughput compound evaluation on 3D networks of neurons and glia in a microfluidic platform.

With great advances in the field of in vitro brain modelling, the challenge is now to implement these technologies for development and evaluation of new drug candidates. Here we demonstrate a method for culturing three-dimensional networks of spontaneously active neurons and supporting glial cells in a microfluidic platform. The high-throughput nature of the platform in combination with its compatibility with all standard laboratory equipment allows for parallel evaluation of compound effects.

Effects of training on the estimation of muscular moment in submaximal exercise.

The purpose of this study was to observe the effects of a submaximal isometric training program on estimation capacity at 25, 50, and 75% of maximal contraction in isometric action and at two angular velocities. The second purpose was to study the variability of isometric action. To achieve these purposes, participants carried out an isokinetic extension movement of the dominant lower limb during six test sessions and nine training sessions. Following the training program, estimation capacity in the different actions did not improve. However an improvement in performance was observed with a reduction in the variability of submaximal isometric actions. The proprioceptors activated in isometric action seemed to adapt to the training program itself which would promote better adaptation by a greater solicitation of internal feedback.