Integration of Functional Human Auditory Neural Circuits Based on a 3D Carbon Nanotube System.

The physiological interactions between the peripheral and central auditory systems are crucial for auditory information transmission and perception, while reliable models for auditory neural circuits are currently lacking. To address this issue, mouse and human neural pathways are generated by utilizing a carbon nanotube nanofiber system. The super-aligned pattern of the scaffold renders the axons of the bipolar and multipolar neurons extending in a parallel direction. In addition, the electrical conductivity of the scaffold maintains the electrophysiological activity of the primary mouse auditory neurons. The mouse and human primary neurons from peripheral and central auditory units in the system are then co-cultured and showed that the two kinds of neurons form synaptic connections. Moreover, neural progenitor cells of the cochlea and auditory cortex are derived from human embryos to generate region-specific organoids and these organoids are assembled in the nanofiber-combined 3D system. Using optogenetic stimulation, calcium imaging, and electrophysiological recording, it is revealed that functional synaptic connections are formed between peripheral neurons and central neurons, as evidenced by calcium spiking and postsynaptic currents. The auditory circuit model will enable the study of the auditory neural pathway and advance the search for treatment strategies for disorders of neuronal connectivity in sensorineural hearing loss.

Electroacoustic Responsive Cochlea-on-a-Chip.

Organ-on-chips can highly simulate the complex physiological functions of organs, exhibiting broad application prospects in developmental research, disease simulation, as well as new drug research and development. However, there is still less concern about effectively constructing cochlea-on-chips. Here, a novel cochlear organoids-integrated conductive hydrogel biohybrid system with cochlear implant electroacoustic stimulation (EAS) for cochlea-on-a-chip construction and high-throughput drug screening, is presented. Benefiting from the superior biocompatibility and electrical property of conductive hydrogel, together with cochlear implant EAS, the inner ear progenitor cells can proliferate and spontaneously shape into spheres, finally forming cochlear organoids with good cell viability and structurally mature hair cells. By incorporating these progenitor cells-encapsulated hydrogels into a microfluidic-based cochlea-on-a-chip with culture chambers and a concentration gradient generator, a dynamic and high-throughput evaluation of inner ear disease-related drugs is demonstrated. These results indicate that the proposed cochlea-on-a-chip platform has great application potential in organoid cultivation and deafness drug evaluation.

Increased mitophagy protects cochlear hair cells from aminoglycoside-induced damage.

Aminoglycosides exhibit ototoxicity by damaging mitochondria, which in turn generate reactive oxygen species that induce hair cell death and subsequent hearing loss. It is well known that damaged mitochondria are degraded by mitophagy, an important mitochondrial quality control system that maintains mitochondrial homeostasis and ensures cell survival. However, it is unclear whether dysregulation of mitophagy contributes to aminoglycoside-induced hair cell injury. In the current study, we found that PINK1-PRKN-mediated mitophagy was impaired in neomycin-treated hair cells. Our data suggested that mitochondrial recruitment of PRKN and phagophore recognition of damaged mitochondria during mitophagy were blocked following neomycin treatment. In addition, the degradation of damaged mitochondria by lysosomes was significantly decreased as indicated by the mitophagic flux reporter mt-mKeima. Moreover, we demonstrated that neomycin disrupted mitophagy through transcriptional inhibition of Pink1 expression, the key initiator of mitophagy. Moreover, we found that neomycin impaired mitophagy by inducing ATF3 expression. Importantly, treatment with a mitophagy activator could rescue neomycin-treated hair cells by increasing mitophagy, indicating that genetic modulation or drug intervention in mitophagy may have therapeutic potential for aminoglycoside-induced hearing loss.Abbreviations: AAV: adeno-associated virus; ABR: auditory brainstem response; ATF3: activating transcription factor 3; ATOH1/MATH1: atonal bHLH transcription factor 1; BafA1: bafilomycin A1; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; COX4I1/COXIV: cytochrome c oxidase subunit 4I1; CTBP2/RIBEYE: C-terminal binding protein 2; DFP: deferiprone; EGFP: enhanced green fluorescent protein; FOXO3: forkhead box O3; GRIA2/GLUR2: glutamate receptor, ionotropic, AMPA2 (alpha 2); HC: hair cell; HSPD1/HSP60: heat shock protein 1 (chaperonin); IHC: inner hair cell; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MYO7A: myosin VIIA; OPTN: optineurin; OMM: outer mitochondrial membrane; PRKN: parkin RBR E3 ubiquitin protein ligase; PINK1: PTEN induced putative kinase 1; RT-qPCR: real-time quantitative polymerase chain reaction; TOMM20/TOM20: translocase of outer mitochondrial membrane 20; TUNEL: Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling; USP30: ubiquitin specific peptidase 30; XBP1: X-box binding protein 1.

3D Ti3C2Tx MXene-Matrigel with Electroacoustic Stimulation to Promote the Growth of Spiral Ganglion Neurons.

Cochlear implantation has become the most effective treatment method for patients with profound and total hearing loss. However, its therapeutic efficacy is dependent on the number and normal physiological function of cochlear implant-targeted spiral ganglion neurons (SGNs). Electrical stimulation can be used as an effective cue to regulate the morphology and function of excitatory cells. Therefore, it is important to develop an efficient cochlear implant electroacoustic stimulation (EAS) system to study the behavior of SGNs. In this work, we present an electrical stimulation system constructed by combining a cochlear implant and a conductive Ti3C2Tx MXene-matrigel hydrogel. SGNs were cultured in the Ti3C2Tx MXene-matrigel hydrogel and exposed to electrical stimulation transduced by the cochlear implant. It was demonstrated that low-frequency stimulation promoted the growth cone development and neurite outgrowth of SGNs as well as signal transmission between cells. This work may have potential value for the clinical application of the Ti3C2Tx MXene hydrogel to optimize the postoperative listening effect of cochlear implantation and benefit people with sensorineural hearing loss.

GelMA-MXene hydrogel nerve conduits with microgrooves for spinal cord injury repair.

Repair of spinal cord injury (SCI) depends on microenvironment improvement and the reconnection between injured axons and regenerated neurons. Here, we fabricate a GelMA-MXene hydrogel nerve conduit with electrical conductivity and internal-facing longitudinal grooves and explore its function in SCI repair. It is found that the resultant grooved GelMA-MXene hydrogel could effectively promote the neural stem cells (NSCs) adhesion, directed proliferation and differentiation in vitro. Additionally, when the GelMA-MXene conduit loaded with NSCs (GMN) is implanted into the injured spinal cord site, effective repair capability for the complete transection of SCI was demonstrated. The GMN group shows remarkable nerve recovery and significantly higher BBB scores in comparison to the other groups. Therefore, GMN with the microgroove structure and loaded with NSCs is a promising strategy in treating SCI.

Ti3 C2 Tx MXene Composite 3D Hydrogel Potentiates mTOR Signaling to Promote the Generation of Functional Hair Cells in Cochlea Organoids.

Organoids have certain cellular composition and physiological features in common with real organs, making them promising models of organ formation, function, and diseases. However, Matrigel, the commonly used animal-derived matrices in which they are developed, has limitations in mechanical adjustability and providing complex physicochemical signals. Here, the incorporation of Ti3 C2 Tx MXene nanomaterial into Matrigel regulates the properties of Matrigel and exhibits satisfactory biocompatibility. The Ti3 C2 Tx MXene Matrigel composites (MXene-Matrigel) regulate the development of Cochlear Organoids (Cochlea-Orgs), particularly in promoting the formation and maturation of organoid hair cells. Additionally, regenerated hair cells in MXene-Matrigel are functional and exhibit better electrophysiological properties compared to hair cells in Matrigel. MXene-Matrigel potentiates the amycin (mTOR) signaling pathway to promote hair cell differentiation, and mTOR signaling inhibition restrains hair cell differentiation. Moreover, MXene-Matrigel facilitates innervation establishment between regenerated hair cells and spiral ganglion neurons (SGNs) growing from the Cochlea modiolus in a co-culture system, as well as promotes synapse formation efficiency. The approach overcomes some limitations of the Matrigel-dependent culture system and greatly accelerates the application of nanomaterials in organoid development and research on therapies for hearing loss.

Two-dimensional Ti3C2Tx MXene promotes electrophysiological maturation of neural circuits.

BACKGROUND: The ideal neural interface or scaffold for stem cell therapy shall have good biocompatibility promoting survival, maturation and integration of neural stem cells (NSCs) in targeted brain regions. The unique electrical, hydrophilic and surface-modifiable properties of Ti3C2Tx MXene make it an attractive substrate, but little is known about how it interacts with NSCs during development and maturation. RESULTS: In this study, we cultured NSCs on Ti3C2Tx MXene and examined its effects on morphological and electrophysiological properties of NSC-derived neurons. With a combination of immunostaining and patch-clamp recording, we found that Ti3C2Tx MXene promotes NSCs differentiation and neurite growth, increases voltage-gated current of Ca2+ but not Na+ or K+ in matured neurons, boosts their spiking without changing their passive membrane properties, and enhances synaptic transmission between them. CONCLUSIONS: These results expand our understanding of interaction between Ti3C2Tx MXene and NSCs and provide a critical line of evidence for using Ti3C2Tx MXene in neural interface or scaffold in stem cell therapy.

Graphene Substrates Promote the Differentiation of Inner Ear Lgr5+ Progenitor Cells Into Hair Cells.

The ideal treatment for sensory hearing loss is to regenerate inner ear hair cells (HCs) through stem cell therapy, thereby restoring the function and structure of the cochlea. Previous studies have found that Lgr5+ supporting cells (SCs) in the inner ear can regenerate HCs, thus being considered inner ear progenitor cells. In addition to traditional biochemical factors, physical factors such as electrical conductivity also play a crucial role in the regulation of stem cell proliferation and differentiation. In this study, the graphene substrates were used to culture Lgr5+ progenitor cells and investigated their regulatory effects on cells. It was demonstrated that the graphene substrates displayed great cytocompatibility for Lgr5+ progenitors and promoted their sphere-forming ability. Moreover, more Myosin7a+ cells were found on the graphene substrates compared with tissue culture polystyrene (TCPS). These results suggest that graphene is an efficient interface that can promote the differentiation of Lgr5+ progenitors into HCs, which is great significance for its future application in combination with Lgr5+ cells to regenerate HCs in the inner ear.

Conductive Nerve Guidance Conduits Based on Morpho Butterfly Wings for Peripheral Nerve Repair.

Peripheral nerve injury (PNI), causing loss of sensory and motor function, is a complex and challenging disease in the clinic due to the restricted regeneration capacity. Nerve guidance conduits (NGCs) have become a promising substitute for peripheral nerve regeneration, but their efficacy is often limited. Here, inspired by the physiological structures of peripheral nerves, we present a conductive topological scaffold for nerve repair by modifying Morpho butterfly wing with reduced graphene oxide (rGO) nanosheets and methacrylated gelatin (GelMA) hydrogel encapsulated brain-derived neurotrophic factor (BDNF). Benefiting from the biocompatibility of GelMA hydrogel, the conductivity of rGO and parallel nanoridge structures of wing scales, PC12 cells, and neural stem cells grown on the modified wing have an increased neurite length with guided cellular orientation. In addition, the NGCs are successfully obtained by manually rolling up the scaffolds and exhibited great performance in repairing 10 mm sciatic nerve defects in rats, and we believe that the NGCs can be applied in reparing longer nerve defects in the future by further optimization. We also demonstrate the feasibility of electrically conductive NGCs based on the rGO/BDNF/GelMA-integrated Morpho butterfly wing as functional nerve regeneration conduits, which may have potential value for application in repairing peripheral nerve injuries.

2D Ti3C2TxMXene couples electrical stimulation to promote proliferation and neural differentiation of neural stem cells.

Preclinical studies involving stem cells require efficient physiochemical regulations on the fate of such cells. Because of their unique planar structure, metallic conductivity, and flexible surface functionalization, MXenes show potential for modulating stem cell fate. Here, the Ti3C2TxMXenenanosheets are dispersed on tissue culture polystyrene (TCPS). When primary mouse neural stem cells (NSCs) are cultured on laminin-coated Ti3C2TxMXene film, they form stable adhesion, retain their proliferative ability, and show extensive spreading of terminal extensions. With respect to their functional activity, NSCs cultured on Ti3C2TxMXene films form more active and synchronous network activity than those cultured on TCPS substrates. Moreover, Ti3C2TxMXene film significantly promotes the neural differentiation and the neurons have longer neurites and greater numbers of branch points and branch tips. NSC-derived neurons grown on the Ti3C2Tx MXene film preserved normal synapse development. Finally, electrical stimulation coupled with Ti3C2TxMXene film significantly enhances the proliferation of NSCs. These results indicate that Ti3C2TxMXene is an efficient interface for the proliferation and neural differentiation of NSC and the maturation of NSC-derived neurons, which expands the potential uses of the MXene family of materials and provides new strategies for stem cell studies. STATEMENT OF SIGNIFICANCE: The 2DTi3C2TxMXenenanosheets were applied to be an interface for regulating neural stem cells (NSCs). NSCs cultured on Ti3C2TxMXene film possessed higher proliferative ability with higher and more synchronous electrical activities. Moreover, Ti3C2TxMXene film significantly promoted the neural differentiation ratio of NSCs, and the neurons derived from NSCs cultured on Ti3C2TxMXene film had longer neurites and greater numbers of branch points and branch tips.When electrical stimulation was applied to NSCs via the Ti3C2TxMXene film, it significantly enhanced the proliferation of NSCs. This work expands the potential uses of the MXene family of materials and provides new strategies for stem cell studies.

Cochlear implant-based electric-acoustic stimulation modulates neural stem cell-derived neural regeneration.

Cochlear implantation is considered to be the best therapeutic method for profound sensorineural hearing loss, but insufficient numbers of functional spiral ganglion neurons hinder the clinical effects of cochlear implantation. Stem cell transplantation has the potential to provide novel strategies for spiral ganglion neuron regeneration after injury. However, some obstacles still need to be overcome, such as low survival and uncontrolled differentiation. Several novel technologies show promise for modulating neural stem cell behaviors to address these issues. Here, a device capable of electrical stimulation was designed by combining a cochlear implant with a graphene substrate. Neural stem cells (NSCs) were cultured on the graphene substrate and subjected to electrical stimulation transduced from sound waves detected by the cochlear implant. Cell behaviors were studied, and this device showed good biocompatibility for NSCs. More importantly, electric-acoustic stimulation with higher frequencies and amplitudes induced NSC death and apoptosis, and electric-acoustic stimulation could promote NSCs to proliferate and differentiate into neurons only when low-frequency stimulation was supplied. The present study provides experimental evidence for understanding the regulatory role of electric-acoustic stimulation on NSCs and highlights the potentials of the above-mentioned device in stem cell therapy for hearing loss treatment.

Superparamagnetic Iron Oxide Nanoparticles: Cytotoxicity, Metabolism, and Cellular Behavior in Biomedicine Applications.

Superparamagnetic iron oxide nanoparticles (SPIONs) have been widely investigated and applied in the field of biomedicine due to their excellent superparamagnetic properties and reliable traceability. However, with the optimization of core composition, shell types and transfection agents, the cytotoxicity and metabolism of different SPIONs have great differences, and the labeled cells also show different cellular behaviors. Therefore, a holistic review of the construction and application of SPIONs is desired. This review focuses the advances of SPIONs in the field of biomedicine in recent years. After summarizing the toxicity of different SPIONs, the uptake, distribution and metabolism of SPIONs in vitro were discussed. Then, the regulation of labeled-cells behavior is outlined. Furthermore, the major challenges in the optimization process of SPIONs and insights on its future developments are proposed.

Topographically Conductive Butterfly Wing Substrates for Directed Spiral Ganglion Neuron Growth.

Spiral ganglion neuron (SGN) degeneration can lead to severe hearing loss, and the directional regeneration of SGNs has shown great potential for improving the efficacy of auditory therapy. Here, a novel 3D conductive microstructure with surface topologies is presented by integrating superaligned carbon-nanotube sheets (SA-CNTs) onto Morpho Menelaus butterfly wings for SGN culture. The parallel groove-like topological structures of M. Menelaus wings induce the cultured cells to grow along the direction of its ridges. The excellent conductivity of SA-CNTs significantly improves the efficiency of cellular information conduction. When integrating the SA-CNTs with M. Menelaus wings, the SA-CNTs are aligned in parallel with the M. Menelaus ridges, which further strengthens the consistency of the surface topography in the composite substrate. The SA-CNTs integrated onto butterfly wings provide powerful physical signals and regulate the behavior of SGNs, including cell survival, adhesion, neurite outgrowth, and synapse formation. These features indicate the possibility of directed regeneration after auditory nerve injury.

Neurite Extension and Orientation of Spiral Ganglion Neurons Can Be Directed by Superparamagnetic Iron Oxide Nanoparticles in a Magnetic Field.

INTRODUCTION: Neuroregeneration is a major challenge in neuroscience for treating degenerative diseases and for repairing injured nerves. Numerous studies have shown the importance of physical stimulation for neuronal growth and development, and here we report an approach for the physical guidance of neuron orientation and neurite growth using superparamagnetic iron oxide (SPIO) nanoparticles and magnetic fields (MFs). METHODS: SPIO nanoparticles were synthesized by classic chemical co-precipitation methods and then characterized by transmission electron microscope, dynamic light scattering, and vibrating sample magnetometer. The cytotoxicity of the prepared SPIO nanoparticles and MF was determined using CCK-8 assay and LIVE/DEAD assay. The immunofluorescence images were captured by a laser scanning confocal microscopy. Cell migration was evaluated using the wound healing assay. RESULTS: The prepared SPIO nanoparticles showed a narrow size distribution, low cytotoxicity, and superparamagnetism. SPIO nanoparticles coated with poly-L-lysine could be internalized by spiral ganglion neurons (SGNs) and showed no cytotoxicity at concentrations less than 300 µg/mL. The neurite extension of SGNs was promoted after internalizing SPIO nanoparticles with or without an external MF, and this might be due to the promotion of growth cone development. It was also confirmed that SPIO can regulate cell migration and can direct neurite outgrowth in SGNs preferentially along the direction imposed by an external MF. CONCLUSION: Our results provide a fundamental understanding of the regulation of cell behaviors under physical cues and suggest alternative treatments for sensorineural hearing loss caused by the degeneration of SGNs.

Ordered inverse-opal scaffold based on bionic transpiration to create a biomimetic spine.

The availability of functional spinal cord scaffolds for nerve tissue engineering (NTE) strategies is an urgent clinical demand for spinal transplantation. However, effective transplanted spinal cord scaffolds are restricted by poor mechanical integrity, topological cues, complex processing, or other properties. Hence, this work aims to fabricate a new three-dimensional (3D) scaffold with electrically micropatterned materials for structural spinal mimicry. Inspired by plant transpiration, the scaffold templates are formed by self-assembled colloidal crystals in a glass capillary after the solvent evaporates gradually. Replicated from bionic transpiration photonic crystal templates, the specific 3D conductive inter-surface ordered microstructures are fabricated through carbonization and corrosion. Nerve cell reconstruction on columnar scaffolds indicated that these conductive porous materials were of excellent biocompatibility. Meanwhile, due to the homogeneously interconnected architecture characteristics, the inverse opal structures facilitated the connection and information transmission between nerve cells. Statistics on the number and length of neural neurites indicated that the microstructures with uniform pores guided nerve cell neurite growth and development. These biomimetic spine properties make them potential alternative scaffolds for nerve tissue engineering.

Biomimetic 3D bacterial cellulose-graphene foam hybrid scaffold regulates neural stem cell proliferation and differentiation.

Neural stem cell (NSC)-based therapy is a promising candidate for treating neurodegenerative diseases and the preclinical researches call an urgent need for regulating the growth and differentiation of such cells. The recognition that three-dimensional culture has the potential to be a biologically significant system has stimulated an extraordinary impetus for scientific researches in tissue engineering and regenerative medicine. Here, A novel scaffold for culturing NSCs, three-dimensional bacterial cellulose-graphene foam (3D-BC/G), which was prepared via in situ bacterial cellulose interfacial polymerization on the skeleton surface of porous graphene foam has been reported. 3D-BC/G not only supports NSC growth and adhesion, but also maintains NSC stemness and enhances their proliferative capacity. Further phenotypic analysis indicated that 3D-BC/G induces NSCs to selectively differentiate into neurons, forming a neural network in a short amount of time. The scaffold has good biocompatibility with primary cortical neurons enhancing the neuronal network activities. To explore the underlying mechanisms, RNA-Seq analysis to identify genes and signaling pathways was performed and it suggests that 3D-BC/G offers a more promising three-dimensional conductive substrate for NSC research and neural tissue engineering, and the repertoire of gene expression serves as a basis for further studies to better understand NSC biology.

Superparamagnetic Iron Oxide Nanoparticles and Static Magnetic Field Regulate Neural Stem Cell Proliferation.

Neural stem cells (NSCs) transplantation is a promising approach for the treatment of various neurodegenerative diseases. Superparamagnetic iron oxide nanoparticles (SPIOs) are reported to modulate stem cell behaviors and are used for medical imaging. However, the detailed effects of SPIOs under the presence of static magnetic field (SMF) on NSCs are not well elucidated. In this study, it was found that SPIOs could enter the cells within 24 h, while they were mainly distributed in the lysosomes. SPIO exhibited good adhesion and excellent biocompatibility at concentrations below 500 µg/ml. In addition, SPIOs were able to promote NSC proliferation in the absence of SMF. In contrast, the high intensity of SMF (145 ± 10 mT) inhibited the expansion ability of NSCs. Our results demonstrate that SPIOs with SMF could promote NSC proliferation, which could have profound significance for tissue engineering and regenerative medicine for SPIO applications.

Transcriptomic profiling of neural stem cell differentiation on graphene substrates.

Graphene exhibits excellent mechanical strength, electrical conductivity and good biocompatibility, which make it a suitable candidate as a neural interfacing material in regenerative medicine and tissue engineering. Graphene is reported to promote both of neural stem cells (NSCs) proliferation and differentiation. However, the transcriptomes of 2D graphene-regulated NSC differentiation have not yet been investigated. To identify candidate genes, on which graphene may affect, we used next-generation RNA sequencing to analyze the transcriptome of NSCs differentiated for 21 days on a graphene substrate. These NSCs displayed highly enriched and differentially expressed genes compared with traditional cell culture in vitro. Of these, we identified motor protein genes that might regulate NSC differentiation, including cytoplasmic dynein and axonemal dynein genes, Ccdc108, Dnah5, and Dnah11. Furthermore, we analyzed the cell signaling pathway genes that might regulate NSC differentiation, and we constructed a protein-protein interaction network for the genes that are differentially expressed in NSCs on graphene compared to commercial tissue culture polystyrene substrates. We have identified genes potentially regulating the differentiation and migration of NSCs on graphene substrates, and our findings provide mechanistic evidence for the biological activities of graphene, especially in view of graphene-stem cell interactions.

Two-Photon Image Tracking of Neural Stem Cells via Iridium Complexes Encapsulated in Polymeric Nanospheres.

Iridium(III) complexes have been shown to be promising probes in two-photon imaging to real-time track the transplanted cells in stem-cell-based therapy. Here, we report on polymeric nanocapsules loaded with red phosphorescence dye of bis(2-methyldibenzo[f,h]quinoxaline) (acetylacetonate) iridium(III) (Ir(MDQ)2acac) with excellent stability created by the double emulsion method. The Ir(MDQ)2acac nanocapsules present high biocompatibility and an efficient fluorescent labeling rate when incubated with cultured mouse neural stem cells (NSCs). More importantly, the Ir(MDQ)2acac nanocapsules had both one- and two-photon imaging properties with stable phosphorescence lasting for 72 h. Furthermore, data from in vivo tracking in nude mice demonstrated that the photoluminescence from Ir(MDQ)2acac nanocapsules in NSCs could be stably monitored for up to 21 days. Our data shed light on the potential clinical application of iridium complexes encapsulated in polymeric nanospheres for two-photon imaging in real-time tracking of the transplanted stem cells.

Development and Application of Cochlear Implant-Based Electric-Acoustic Stimulation of Spiral Ganglion Neurons.

Cochlear implants are currently the most effective treatment for profound sensorineural hearing loss. However, their therapeutic effect is limited by the survival and proper physiological function of spiral ganglion neurons (SGNs), which are targeted by the cochlear implant. It is therefore critical to explore the mechanism behind the effect of electric-acoustic stimulation (EAS) on the targeted SGNs. In this work, a biocompatible cochlear implant/graphene EAS system was created by combining a cochlear implant to provide the electrically transformed sound stimulation with graphene as the conductive neural interface. SGNs were cultured on the graphene and exposed to EAS from the cochlear implant. Neurite extension of SGNs was accelerated with long-term stimulation, which might contribute to the development of growth cones. Our system allows us to study the effects of cochlear implants on SGNs in a low-cost and time-saving way, and this might provide profound insights into the use of cochlear implants and thus be of benefit to the populations suffering from sensorineural hearing loss.