The blood-brain barrier (BBB) regulates molecular trafficking, protects against pathogens, and prevents efficient drug delivery to the brain. Models to date failed to reproduce the human anatomical complexity of brain barriers, contributing to misleading results in clinical trials. To overcome these limitations, a novel 3-dimensional BBB microvascular network model was developed via vasculogenesis to accurately replicate the in vivo neurovascular organization. This microfluidic system includes human induced pluripotent stem cell-derived endothelial cells, brain pericytes, and astrocytes as self-assembled vascular networks in fibrin gel. Gene expression of membrane transporters, tight junction and extracellular matrix proteins, was consistent with computational analysis of geometrical structures and quantitative immunocytochemistry, indicating BBB maturation and microenvironment remodelling. Confocal microscopy validated microvessel-pericyte/astrocyte dynamic contact-interactions. The BBB model exhibited perfusable and selective microvasculature, with permeability lower than conventional in vitro models, and similar to in vivo measurements in rat brain. This robust and physiologically relevant BBB microvascular model offers an innovative and valuable platform for drug discovery to predict neuro-therapeutic transport efficacy in pre-clinical applications as well as recapitulate patient-specific and pathological neurovascular functions in neurodegenerative disease.

3D physiological models of neuromuscular junctions could facilitate drug screening and research on pathogenesis of ALS.

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Neurovascular coupling plays a key role in the pathogenesis of neurodegenerative disorders including motor neuron disease (MND). In vitro models provide an opportunity to understand the pathogenesis of MND, and offer the potential for drug screening. Here, we describe a new 3D microvascular and neuronal network model in a microfluidic platform to investigate interactions between these two systems. Both 3D networks were established by co-culturing human embryonic stem (ES)-derived MN spheroids and endothelial cells (ECs) in microfluidic devices. Co-culture with ECs improves neurite elongation and neuronal connectivity as measured by Ca2+ oscillation. This improvement was regulated not only by paracrine signals such as brain-derived neurotrophic factor secreted by ECs but also through direct cell-cell interactions via the delta-notch pathway, promoting neuron differentiation and neuroprotection. Bi-directional signaling was observed in that the neural networks also affected vascular network formation under perfusion culture. This in vitro model could enable investigations of neuro-vascular coupling, essential to understanding the pathogenesis of neurodegenerative diseases including MNDs such as amyotrophic lateral sclerosis.

Microfluidic devices enable novel means of emulating neurodegenerative disease pathophysiology in vitro. These organ-on-a-chip systems can potentially reduce animal testing and substitute (or augment) simple 2D culture systems. Reconstituting critical features of neurodegenerative diseases in a biomimetic system using microfluidics can thereby accelerate drug discovery and improve our understanding of the mechanisms of several currently incurable diseases. This review describes latest advances in modeling neurodegenerative diseases in the central nervous system and the peripheral nervous system. First, this study summarizes fundamental advantages of microfluidic devices in the creation of compartmentalized cell culture microenvironments for the co-culture of neurons, glial cells, endothelial cells, and skeletal muscle cells and in their recapitulation of spatiotemporal chemical gradients and mechanical microenvironments. Then, this reviews neurodegenerative-disease-on-a-chip models focusing on Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Finally, this study discusses about current drawbacks of these models and strategies that may overcome them. These organ-on-chip technologies can be useful to be the first line of testing line in drug development and toxicology studies, which can contribute significantly to minimize the phase of animal testing steps.

Proteins are typically denatured and aggregated by heating at near-boiling temperature. Exceptions to this principle include highly disordered and heat-resistant proteins found in extremophiles, which help these organisms tolerate extreme conditions such as drying, freezing, and high salinity. In contrast, the functions of heat-soluble proteins in non-extremophilic organisms including humans remain largely unexplored. Here, we report that heatresistant obscure (Hero) proteins, which remain soluble after boiling at 95˚C, are widespread in Drosophila and humans. Hero proteins are hydrophilic and highly charged, and function to stabilize various "client" proteins, protecting them from denaturation even under stress conditions such as heat shock, desiccation, and exposure to organic solvents. Hero proteins can also block several different types of pathological protein aggregations in cells and in Drosophila strains that model neurodegenerative diseases. Moreover, Hero proteins can extend life span of Drosophila. Our study reveals that organisms naturally use Hero proteins as molecular shields to stabilize protein functions, highlighting their biotechnological and therapeutic potential.

A major challenge in tissue engineering is to reproduce the native 3D microvascular architecture fundamental for in vivo functions. Current approaches still lack a network of perfusable vessels with native 3D structural organization. Here we present a new method combining self-assembled monolayer (SAM)-based cell transfer and gelatin methacrylate hydrogel photopatterning techniques for microengineering vascular structures. Human umbilical vein cell (HUVEC) transfer from oligopeptide SAM-coated surfaces to the hydrogel revealed two SAM desorption mechanisms: photoinduced and electrochemically triggered. The former, occurs concomitantly to hydrogel photocrosslinking, and resulted in efficient (>97%) monolayer transfer. The latter, prompted by additional potential application, preserved cell morphology and maintained high transfer efficiency of VE-cadherin positive monolayers over longer culture periods. This approach was also applied to transfer HUVECs to 3D geometrically defined vascular-like structures in hydrogels, which were then maintained in perfusion culture for 15 days. As a step toward more complex constructs, a cell-laden hydrogel layer was photopatterned around the endothelialized channel to mimic the vascular smooth muscle structure of distal arterioles. This study shows that the coupling of the SAM-based cell transfer and hydrogel photocrosslinking could potentially open up new avenues in engineering more complex, vascularized tissue constructs for regenerative medicine and tissue engineering applications.

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