Eye-Mimicked Neural Network Composed of Photosensitive Neural Spheroids with Human Opsin Proteins.

An in vitro model, composed of the short-wavelength human opsins and rhodopsins, is created. Two types of photosensitive neural spheroids are transfected for selective reaction under bluish-purple and green lights. These are employed to two devices with intact neuron and neural-spheroid to study the interaction. By photostimulation, the photosensitive spheroid initiated photoactivation, and the signal generated from its body is transmitted to adjacent neural networks. Specifically, the signal traveled through the axon bundle in narrow gap from photosensitive spheroid to intact spheroid as an eye-to-brain model including optic nerve. The whole process with photosensitive spheroid is monitored by calcium ion detecting fluorescence images. The results of this study can be applied to examine vision restoration and novel photosensitive biological systems with spectral sensitivity.

Integration of reconfigurable microchannels into aligned three-dimensional neural networks for spatially controllable neuromodulation.

Anisotropically organized neural networks are indispensable routes for functional connectivity in the brain, which remains largely unknown. While prevailing animal models require additional preparation and stimulation-applying devices and have exhibited limited capabilities regarding localized stimulation, no in vitro platform exists that permits spatiotemporal control of chemo-stimulation in anisotropic three-dimensional (3D) neural networks. We present the integration of microchannels seamlessly into a fibril-aligned 3D scaffold by adapting a single fabrication principle. We investigated the underlying physics of elastic microchannels' ridges and interfacial sol-gel transition of collagen under compression to determine a critical window of geometry and strain. We demonstrated the spatiotemporally resolved neuromodulation in an aligned 3D neural network by local deliveries of KCl and Ca2+ signal inhibitors, such as tetrodotoxin, nifedipine, and mibefradil, and also visualized Ca2+ signal propagation with a speed of ~3.7 µm/s. We anticipate that our technology will pave the way to elucidate functional connectivity and neurological diseases associated with transsynaptic propagation.

Modulating and monitoring the functionality of corticostriatal circuits using an electrostimulable microfluidic device.

The central nervous system is organized into different neural circuits, each with particular functions and properties. Studying neural circuits is essential to understanding brain function and neuronal diseases. Microfluidic systems are widely used for reconstructing and studying neural circuits but still need improvement to allow modulation and monitoring of the physiological properties of circuits. In this study, we constructed an improved microfluidic device that supports the electrical modulation of neural circuits and proper reassembly. We demonstrated that our microfluidic device provides a platform for electrically modulating and monitoring the physiological function of neural circuits with genetic indicators for synaptic functionality in corticostriatal (CStr) circuits. In particular, our microfluidic device measures activity-driven Ca2+ dynamics using Ca2+ indicators (synaptophysin-GCaMP6f and Fluo5F-AM), as well as activity-driven synaptic transmission and retrieval using vGlut-pHluorin. Overall, our findings indicate that the improved microfluidic platform described here is an invaluable tool for studying the physiological properties of specific neural circuits.

Brain organoid-on-a-chip: A next-generation human brain avatar for recapitulating human brain physiology and pathology.

Neurodegenerative diseases and neurodevelopmental disorders have become increasingly prevalent; however, the development of new pharmaceuticals to treat these diseases has lagged. Animal models have been extensively utilized to identify underlying mechanisms and to validate drug efficacies, but they possess inherent limitations including genetic heterogeneity with humans. To overcome these limitations, human cell-based in vitro brain models including brain-on-a-chip and brain organoids have been developed. Each technique has distinct advantages and disadvantages in terms of the mimicry of structure and microenvironment, but each technique could not fully mimic the structure and functional aspects of the brain tissue. Recently, a brain organoid-on-a-chip (BOoC) platform has emerged, which merges brain-on-a-chip and brain organoids. BOoC can potentially reflect the detailed structure of the brain tissue, vascular structure, and circulation of fluid. Hence, we summarize recent advances in BOoC as a human brain avatar and discuss future perspectives. BOoC platform can pave the way for mechanistic studies and the development of pharmaceuticals to treat brain diseases in future.

Dynamics of axonal ß-actin mRNA in live hippocampal neurons.

Localization of mRNA facilitates spatiotemporally controlled protein expression in neurons. In axons, mRNA transport followed by local protein synthesis plays a critical role in axonal growth and guidance. However, it is not yet clearly understood how mRNA is transported to axonal subcellular sites and what regulates axonal mRNA localization. Using a transgenic mouse model in which endogenous ß-actin mRNA is fluorescently labeled, we investigated ß-actin mRNA movement in axons of hippocampal neurons. We cultured neurons in microfluidic devices to separate axons from dendrites and performed single-particle tracking of axonal ß-actin mRNA. Compared with dendritic ß-actin mRNA, axonal ß-actin mRNA showed less directed motion and exhibited mostly subdiffusive motion, especially near filopodia and boutons in mature dissociated hippocampal neurons. We found that axonal ß-actin mRNA was likely to colocalize with actin patches (APs), regions that have a high density of filamentous actin (F-actin) and are known to have a role in branch initiation. Moreover, simultaneous imaging of F-actin and axonal ß-actin mRNA in live neurons revealed that moving ß-actin mRNA tended to be docked in the APs. Our findings reveal that axonal ß-actin mRNA localization is facilitated by actin networks and suggest that localized ß-actin mRNA plays a potential role in axon branch formation.

Brain physiome: A concept bridging in vitro 3D brain models and in silico models for predicting drug toxicity in the brain.

In the last few decades, adverse reactions to pharmaceuticals have been evaluated using 2D in vitro models and animal models. However, with increasing computational power, and as the key drivers of cellular behavior have been identified, in silico models have emerged. These models are time-efficient and cost-effective, but the prediction of adverse reactions to unknown drugs using these models requires relevant experimental input. Accordingly, the physiome concept has emerged to bridge experimental datasets with in silico models. The brain physiome describes the systemic interactions of its components, which are organized into a multilevel hierarchy. Because of the limitations in obtaining experimental data corresponding to each physiome component from 2D in vitro models and animal models, 3D in vitro brain models, including brain organoids and brain-on-a-chip, have been developed. In this review, we present the concept of the brain physiome and its hierarchical organization, including cell- and tissue-level organizations. We also summarize recently developed 3D in vitro brain models and link them with the elements of the brain physiome as a guideline for dataset collection. The connection between in vitro 3D brain models and in silico modeling will lead to the establishment of cost-effective and time-efficient in silico models for the prediction of the safety of unknown drugs.

Three-Dimensional Axotomy and Regeneration on Open-Access Microfluidic Platform.

An increasing number of patients are suffering from central nervous system (CNS) injury, including spinal cord injury. However, no suitable treatment is available for such patients as yet. Various platforms have been utilized to recapitulate CNS injuries. However, animal models and in vitro two-dimensional (2D)-based cell culture platforms have limitations, such as genetic heterogeneity and loss of the neural-circuit ultrastructure. To overcome these limitations, we developed a method for performing axotomy on an open-access three-dimensional (3D) neuron-culture platform. In this platform, the 3D alignment of axons in the brain tissue was recapitulated. For direct access to the cultured axons, the bottom of the 3D neuron-culture device was disassembled, enabling exposure of the neuron-laden Matrigel to the outside. The mechanical damage to the axons was recapitulated by puncturing the neuron-laden Matrigel using a pin. Thus, precise axotomy of three-dimensionally aligned axons could be performed. Furthermore, it was possible to fill the punctuated area by re-injecting Matrigel. Consequently, neurites regenerated into re-injected Matrigel. Moreover, it was confirmed that astrocytes can be co-cultured on this open-access platform without interfering with the axon alignment. The proposed open-access platform is expected to be useful for developing treatment techniques for CNS injuries.

Engineered neural circuits for modeling brain physiology and neuropathology.

The neural circuits of the central nervous system are the regulatory pathways for feeling, motion control, learning, and memory, and their dysfunction is closely related to various neurodegenerative diseases. Despite the growing demand for the unraveling of the physiology and functional connectivity of the neural circuits, their fundamental investigation is hampered because of the inability to access the components of neural circuits and the complex microenvironment. As an alternative approach, in vitro human neural circuits show principles of in vivo human neuronal circuit function. They allow access to the cellular compartment and permit real-time monitoring of neural circuits. In this review, we summarize recent advances in reconstituted in vitro neural circuits using engineering techniques. To this end, we provide an overview of the fabrication techniques and methods for stimulation and measurement of in vitro neural circuits. Subsequently, representative examples of in vitro neural circuits are reviewed with a particular focus on the recapitulation of structures and functions observed in vivo, and we summarize their application in the study of various brain diseases. We believe that the in vitro neural circuits can help neuroscience and the neuropharmacology. STATEMENT OF SIGNIFICANCE: Despite the growing demand to unravel the physiology and functional connectivity of the neural circuits, the studies on the in vivo neural circuits are frequently limited due to the poor accessibility. Furthermore, single neuron-based analysis has an inherent limitation in that it does not reflect the full spectrum of the neural circuit physiology. As an alternative approach, in vitro engineered neural circuit models have arisen because they can recapitulate the structural and functional characteristics of in vivo neural circuits. These in vitro neural circuits allow the mimicking of dysregulation of the neural circuits, including neurodegenerative diseases and traumatic brain injury. Emerging in vitro engineered neural circuits will provide a better understanding of the (patho-)physiology of neural circuits.

Emerging Brain-Pathophysiology-Mimetic Platforms for Studying Neurodegenerative Diseases: Brain Organoids and Brains-on-a-Chip.

Neurodegenerative diseases are a group of disorders characterized by progressive degeneration of the structural and functional integrity of the central and peripheral nervous systems. Millions of people suffer from degenerative brain diseases worldwide, and the mortality continues to increase every year, causing a growing demand for knowledge of the underlying mechanisms and development of therapeutic targets. Conventional 2D-based cell culture platforms and animal models cannot fully recapitulate the pathophysiology, and this has limited the capability for estimating drug efficacy. Recently, engineered platforms, including brain organoids and brain-on-a-chip, have emerged. They mimic the physiology of brain tissue and reflect the fundamental pathophysiological signatures of neurodegenerative diseases, such as the accumulation of neurotoxic proteins, structural abnormalities, and functional loss. In this paper, recent advances in brain-mimetic platforms and their potential for modeling features of neurodegenerative diseases in vitro are reviewed. The development of a physiologically relevant model should help overcome unresolved neurodegenerative diseases.

Self-detachable UV-curable polymers for open-access microfluidic platforms.

This study presents an ultraviolet (UV)-curable polymer which is applicable to open-access microfluidic platforms. The UV-curable polymer was prepared by mixing trimethylolpropane triacrylate (TMPTA), 1,6-hexanediol diacrylate (HDDA), polyethylene glycol-diacrylate (PEG-DA), and Irgacure 184. The polymer resin is optically transparent before and after UV-assisted curing and showed good biocompatibility when culturing multiple types of cells on the nanopatterned polymer substrate. The polymer has good adhesion with poly(dimethylsiloxane) (PDMS) even under large deformation and showed a low swelling ratio when exposed to water, suggesting a possibility to be used as a substrate for an organ on a chip. Furthermore, because the polymers have controllable hydrolysis ability depending on the composition, long-term 3D cell culture and subsequent biological analysis with harvested cells are possible. The self-detachable synthesized UV-curable polymer may help the advancement of biomedical studies using in vitro cell culture.

Brain-on-a-chip: A history of development and future perspective.

Since the advent of organ-on-a-chip, many researchers have tried to mimic the physiology of human tissue on an engineered platform. In the case of brain tissue, structural connections and cell-cell interactions are important factors for brain function. The recent development of brain-on-a-chip is an effort to mimic those structural and functional aspects of brain tissue within a miniaturized engineered platform. From this perspective, we provide an overview of trace of brain-on-a-chip development, especially in terms of complexity and high-content/high-throughput screening capabilities, and future perspectives on more in vivo-like brain-on-a-chip development.

Optogenetic neuronal stimulation promotes axon outgrowth and myelination of motor neurons in a three-dimensional motor neuron-Schwann cell coculture model on a microfluidic biochip.

Axonal regeneration and remyelination of peripheral motor neurons (MNs) are critical for restoring neuromuscular motor function after injury or peripheral neuropathy. We examined whether optogenetically mediated light stimulation (OMLS) could enhance the axon outgrowth and myelination of MNs using three-dimensional motor neuron-Schwann cell (MN-SC) coculture on a microfluidic biochip. The biochip was designed to allow SCs to interact with the axons of MNs, while preventing direct contact between SCs and the cell bodies of MNs. Following coculture with SCs on the microfluidic biochip, MNs were transfected with a light-sensitive channelrhodopsin gene. Transfected MNs subjected to repeated light stimulation (20 Hz, 1 hr) produced significantly longer axons than nontransfected MNs. OMLS of MNs greatly increased the number of myelin basic protein (MBP)-expressing SCs, promoting the initiation of myelination of MNs. Ultrastructurally, OMLS of MNs markedly enhanced the thickness of the compact myelin sheath around the MN axons such that the average thickness was closer to that of the theoretical estimates in vivo. Thus, the MN-SC coculture model on a microfluidic biochip augmented by OMLS of MNs is a feasible platform for studying the relationship of neuronal activity with regrowth and remyelination.

Injured Axons Instruct Schwann Cells to Build Constricting Actin Spheres to Accelerate Axonal Disintegration.

After a peripheral nerve lesion, distal ends of injured axons disintegrate into small fragments that are subsequently cleared by Schwann cells and later by macrophages. Axonal debris clearing is an early step of the repair process that facilitates regeneration. We show here that Schwann cells promote distal cut axon disintegration for timely clearing. By combining cell-based and in vivo models of nerve lesion with mouse genetics, we show that this mechanism is induced by distal cut axons, which signal to Schwann cells through PlGF mediating the activation and upregulation of VEGFR1 in Schwann cells. In turn, VEGFR1 activates Pak1, leading to the formation of constricting actomyosin spheres along unfragmented distal cut axons to mediate their disintegration. Interestingly, oligodendrocytes can acquire a similar behavior as Schwann cells by enforced expression of VEGFR1. These results thus identify controllable molecular cues of a neuron-glia crosstalk essential for timely clearing of damaged axons.

Modeling neural circuit, blood-brain barrier, and myelination on a microfluidic 96 well plate.

Microfluidics have enabled a wide range of experimental possibilities in the field of neuroscience. Unfortunately, the wider scale adoption of polydimethylsiloxane (PDMS) based microfluidic devices faces challenges due to inherent material compatibility issues and lack of standardized manufacturable devices. In this work, we present an injection molded plastic array three-dimensional (3D) neuron culture platform (Neuro-IMPACT) made of polystyrene (PS) with a standard 96-well plate form factor that can recapitulate elements of both the central and peripheral nervous systems. A standardized in vitro platform for neuron culture will facilitate the development of new therapies for neurodegenerative diseases, as they would enable quantitative analysis based on imaging as well as biochemical analysis. To demonstrate the versatility of Neuro-IMPACT, we modeled physiologically relevant complex co-culture models such as a 3D neuronal network, blood-brain barrier, and myelination. The Neuro-IMPACT offers a high-throughput screening compatible platform with the ability to engineer the neuronal microenvironment to aid both basic and applied neuroscience research.

Reliable autapse formation using the single-cell patterning method.

Auto neuronal synapses, or autapses, are aberrant structures where the synaptic contact of a neuron forms onto its own branch. The functions of autapses, however, remain unknown. Here, we introduce a simple patterning method for capturing a single-cell, in which we maintained the isolated cell until it reached maturity, and developed arrays of autapses for electrophysiological analysis using multi-electrode arrays (MEA). The pattern arrays were formed by selective patterning of poly-L-lysine and various cell repellent materials. We tested the efficiency of single neuron pattern formed according to materials and pattern dimensions. Autapse formation was verified by immunostaining synaptic markers and physiological measurements via recordings from MEA. The results demonstrated that our multiscale patterning method increased the number of autapses consisting of a single neuron, which matured to connect onto themselves. The proposed patterning method (4.06 ± 0.33 isolated single-cells mm-2) is at least twelve times more efficient and productive than the spray method (0.31 ± 0.10 isolated single-cells mm-2). The spontaneous activity of a single neuron on the patterned MEA occured after 11 d in vitro. The single neuron activity consisted of bursts followed by spike trains (the burst rate was 2.56 min-1). This indicates that our method could be used for electrophysiological analysis, including MEA.

A Low Permeability Microfluidic Blood-Brain Barrier Platform with Direct Contact between Perfusable Vascular Network and Astrocytes.

A novel three dimensional blood brain barrier (BBB) platform was developed by independently supplying different types of media to separate cell types within a single device. One channel (vascular channel, VC) is connected to the inner lumen of the vascular network while the other supplies media to the neural cells (neural channel, NC). Compared to co-cultures supplied with only one type of medium (or 1:1 mixture), best barrier properties and viability were obtained with culturing HUVECs with endothelial growth medium (EGM) and neural cells with neurobasal medium supplemented with fetal bovine serum (NBMFBS) independently. The measured vascular network permeability were comparable to reported in vivo values (20 kDa FITC-dextran, 0.45 ± 0.11 × 10-6 cm/s; 70 kDa FITC-dextran, 0.36 ± 0.05 × 10-6 cm/s) and a higher degree of neurovascular interfacing (astrocytic contact with the vascular network, GFAP-CD31 stain overlap) and presence of synapses (stained with synaptophysin). The BBB platform can dependably imitate the perivascular network morphology and synaptic structures characteristic of the NVU. This microfluidic BBB model can find applications in screening pharmaceuticals that target the brain for in neurodegenerative diseases.

Rapid large area fabrication of multiscale through-hole membranes.

There are many proposed mechanisms by which single cells can be trapped; among them is the through-hole membrane for the characterization of individual microorganisms. Due to the small scale of the fabricated pores, the construction of through-hole membranes on a large scale and with relatively large areas faces many difficulties. This paper describes novel fabrication methods for a large-area, freestanding micro/nano through-hole membrane constructed from versatile membrane materials using through-hole membranes on a microfluidic chip (THMMC). This process can rapidly (<20 min) fabricate membranes with high fidelity multiscale hole size without residual layers. The through-hole site was easily customizable from the micro to the nanoscale, with a low or high aspect ratio giving rise to reliable membranes. Also, the rigidity and biocompatibility of the through-hole membrane are easily tunable by simple injection of versatile membrane materials to obtain a large area (up to 3600 mm2). Membranes produced in this manner were then applied as a proof of concept for the isolation, cultivation, and quantification of individual micro-algal cells for selection with respect to the growth rate, while controlling the quorum sensing mediated metabolic and proliferative changes.

3D tissue formation by stacking detachable cell sheets formed on nanofiber mesh.

We present a novel approach for assembling 3D tissue by layer-by-layer stacking of cell sheets formed on aligned nanofiber mesh. A rigid frame was used to repeatedly collect aligned electrospun PCL (polycaprolactone) nanofiber to form a mesh structure with average distance between fibers 6.4 µm. When human umbilical vein endothelial cells (HUVECs), human foreskin dermal fibroblasts, and skeletal muscle cells (C2C12) were cultured on the nanofiber mesh, they formed confluent monolayers and could be handled as continuous cell sheets with areas 3 × 3 cm2 or larger. Thicker 3D tissues have been formed by stacking multiple cell sheets collected on frames that can be nested (i.e. Matryoshka dolls) without any special tools. When cultured on the nanofiber mesh, skeletal muscle, C2C12 cells oriented along the direction of the nanofibers and differentiated into uniaxially aligned multinucleated myotube. Myotube cell sheets were stacked (upto 3 layers) in alternating or aligned directions to form thicker tissue with ∼50 µm thickness. Sandwiching HUVEC cell sheets with two dermal fibroblast cell sheets resulted in vascularized 3D tissue. HUVECs formed extensive networks and expressed CD31, a marker of endothelial cells. Cell sheets formed on nanofiber mesh have a number of advantages, including manipulation and stacking of multiple cell sheets for constructing 3D tissue and may find applications in a variety of tissue engineering applications.

Engineering-Aligned 3D Neural Circuit in Microfluidic Device.

The brain is one of the most important and complex organs in the human body. Although various neural network models have been proposed for in vitro 3D neuronal networks, it has been difficult to mimic functional and structural complexity of the in vitro neural circuit. Here, a microfluidic model of a simplified 3D neural circuit is reported. First, the microfluidic device is filled with Matrigel and continuous flow is delivered across the device during gelation. The fluidic flow aligns the extracellular matrix (ECM) components along the flow direction. Following the alignment of ECM fibers, neurites of primary rat cortical neurons are grown into the Matrigel at the average speed of 250 µm d(-1) and form axon bundles approximately 1500 µm in length at 6 days in vitro (DIV). Additionally, neural networks are developed from presynaptic to postsynaptic neurons at 14 DIV. The establishment of aligned 3D neural circuits is confirmed with the immunostaining of PSD-95 and synaptophysin and the observation of calcium signal transmission.