Modelling and Prevention of Acute Kidney Injury through Ischemia and Reperfusion in a Combined Human Renal Proximal Tubule/Blood Vessel-on-a-Chip.

Background: Renal ischemia/reperfusion injury (rIRI) is one of the major causes of AKI. Although animal models are suitable for investigating systemic symptoms of AKI, they are limited in translatability. Human in vitro models are crucial in giving mechanistic insights into rIRI; however, they miss out on crucial aspects such as reperfusion injury and the multitissue aspect of AKI. Methods: We advanced the current renal proximal tubule-on-a-chip model to a coculture model with a perfused endothelial vessel separated by an extracellular matrix. The coculture was characterized for its three-dimensional structure, protein expression, and response to nephrotoxins. Then, rIRI was captured through control of oxygen levels, nutrient availability, and perfusion flow settings. Injury was quantified through morphologic assessment, caspase-3/7 activation, and cell viability. Results: The combination of low oxygen, reduced glucose, and interrupted flow was potent to disturb the proximal tubules. This effect was strongly amplified upon reperfusion. Endothelial vessels were less sensitive to the ischemia-reperfusion parameters. Adenosine treatment showed a protective effect on the disruption of the epithelium and on the caspase-3/7 activation. Conclusions: A human in vitro rIRI model was developed using a coculture of a proximal tubule and blood vessel on-a-chip, which was used to characterize the renoprotective effect of adenosine. The robustness of the model and assays in combination with the throughput of the platform make it ideal to advance pathophysiological research and enable the development of novel therapeutic modalities.

A Novel In Vitro Membrane Permeability Methodology Using Three-dimensional Caco-2 Tubules in a Microphysiological System Which Better Mimics In Vivo Physiological Conditions.

The aim of this study was to develop an in vitro drug permeability methodology which mimics the gastrointestinal environment more accurately than conventional 2D methodologies through a three-dimensional (3D) Caco-2 tubules using a microphysiological system. Such a system offers significant advantages, including accelerated cellular polarization and more accurate mimicry of the in vivo environment. This methodology was confirmed by measuring the permeability of propranolol as a model compound, and subsequently applied to those of solifenacin and bile acids for a comprehensive understanding of permeability for the drug product in the human gastrointestinal tract. To protect the Caco-2 tubules from bile acid toxicity, a mucus layer was applied on the surface of Caco-2 tubules and it enables to use simulated intestinal fluid. The assessment using propranolol reproduced results equivalent to those obtained from conventional methodology, while that using solifenacin indicated fluctuations in the permeability of solifenacin due to various factors, including interaction with bile acids. We therefore suggest that this model will serve as an alternative testing system for measuring drug absorption in an environment closely resembling that of the human gastrointestinal tract.

Modeling ischemic stroke in a triculture neurovascular unit on-a-chip.

BACKGROUND: In ischemic stroke, the function of the cerebral vasculature is impaired. This vascular structure is formed by the so-called neurovascular unit (NVU). A better understanding of the mechanisms involved in NVU dysfunction and recovery may lead to new insights for the development of highly sought therapeutic approaches. To date, there remains an unmet need for complex human in vitro models of the NVU to study ischemic events seen in the human brain. METHODS: We here describe the development of a human NVU on-a-chip model using a platform that allows culture of 40 chips in parallel. The model comprises a perfused vessel of primary human brain endothelial cells in co-culture with induced pluripotent stem cell derived astrocytes and neurons. Ischemic stroke was mimicked using a threefold approach that combines chemical hypoxia, hypoglycemia, and halted perfusion. RESULTS: Immunofluorescent staining confirmed expression of endothelial adherens and tight junction proteins, as well as astrocytic and neuronal markers. In addition, the model expresses relevant brain endothelial transporters and shows spontaneous neuronal firing. The NVU on-a-chip model demonstrates tight barrier function, evidenced by retention of small molecule sodium fluorescein in its lumen. Exposure to the toxic compound staurosporine disrupted the endothelial barrier, causing reduced transepithelial electrical resistance and increased permeability to sodium fluorescein. Under stroke mimicking conditions, brain endothelial cells showed strongly reduced barrier function (35-fold higher apparent permeability) and 7.3-fold decreased mitochondrial potential. Furthermore, levels of adenosine triphosphate were significantly reduced on both the blood- and the brain side of the model (4.8-fold and 11.7-fold reduction, respectively). CONCLUSIONS: The NVU on-a-chip model presented here can be used for fundamental studies of NVU function in stroke and other neurological diseases and for investigation of potential restorative therapies to fight neurological disorders. Due to the platform's relatively high throughput and compatibility with automation, the model holds potential for drug compound screening.

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.

A perfused human blood-brain barrier on-a-chip for high-throughput assessment of barrier function and antibody transport.

BACKGROUND: Receptor-mediated transcytosis is one of the major routes for drug delivery of large molecules into the brain. The aim of this study was to develop a novel model of the human blood-brain barrier (BBB) in a high-throughput microfluidic device. This model can be used to assess passage of large biopharmaceuticals, such as therapeutic antibodies, across the BBB. METHODS: The model comprises human cell lines of brain endothelial cells, astrocytes, and pericytes in a two-lane or three-lane microfluidic platform that harbors 96 or 40 chips, respectively, in a 384-well plate format. In each chip, a perfused vessel of brain endothelial cells was grown against an extracellular matrix gel, which was patterned by means of surface tension techniques. Astrocytes and pericytes were added on the other side of the gel to complete the BBB on-a-chip model. Barrier function of the model was studied using fluorescent barrier integrity assays. To test antibody transcytosis, the lumen of the model's endothelial vessel was perfused with an anti-transferrin receptor antibody or with a control antibody. The levels of antibody that penetrated to the basal compartment were quantified using a mesoscale discovery assay. RESULTS: The perfused BBB on-a-chip model shows presence of adherens and tight junctions and severely limits the passage of a 20 kDa FITC-dextran dye. Penetration of the antibody targeting the human transferrin receptor (MEM-189) was markedly higher than penetration of the control antibody (apparent permeability of 2.9 × 10-5 versus 1.6 × 10-5 cm/min, respectively). CONCLUSIONS: We demonstrate successful integration of a human BBB microfluidic model in a high-throughput plate-based format that can be used for drug screening purposes. This in vitro model shows sufficient barrier function to study the passage of large molecules and is sensitive to differences in antibody penetration, which could support discovery and engineering of BBB-shuttle technologies.

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.

Site-specific functionalization of proteins and their applications to therapeutic antibodies.

Protein modifications are often required to study structure and function relationships. Instead of the random labeling of lysine residues, methods have been developed to (sequence) specific label proteins. Next to chemical modifications, tools to integrate new chemical groups for bioorthogonal reactions have been applied. Alternatively, proteins can also be selectively modified by enzymes. Herein we review the methods available for site-specific modification of proteins and their applications for therapeutic antibodies.