Motor unit chip, manufacturing method thereof and method of detecting pathological event using the same

Abstract

This application provides a motor unit chip, a method for manufacturing the motor unit chip, and a method for detecting pathological events using the motor unit chip. The motor unit chip includes a first hydrogel with parallel microgrooves on its surface; muscle cells disposed within the parallel microgrooves; a second hydrogel disposed on the first hydrogel and covering the muscle cells; and motor neurons laid flat on top of the second hydrogel. Because the motor unit is a 3D structure, the motor unit chip of this application makes the detection of pathological events easier.

Description

Motion unit chip, method for manufacturing the same, and method for detecting pathological events using the same The present invention relates to a biochip. Specifically, the present invention relates to a novel motor chip comprising a double-layer hydrogel and a motor. The motor chip can be used to study the function of the motor. The neuromuscular system consists of motor neurons (MNs) and the skeletal muscles they innervate. Superior MNs within the motor cortex project to inferior MNs (in the spinal cord), which then send signals to the innervated muscles, inducing muscle contraction. A motor unit is defined as a single α-motor neuron and the muscle fibers it innervates. MN axons are myelinated by Schwann cells. The neuromuscular junction (NMJ) is a highly specialized synapse formed between MN axon terminals and muscle fibers. Physiologically, axon terminals release acetylcholine (ACh) to stimulate postsynaptic ACh receptors (AChRs) on myotubes, inducing contraction. This neuromuscular system controls all voluntary movements, including basic activities such as breathing. Defects in MNs, skeletal muscles, or NMJs can lead to neuromuscular disorders (NMDs), a broad range of conditions such as amyotrophic lateral sclerosis, myopathy, muscular dystrophy, myasthenia gravis, and sarcopenia. The pathogenesis of most neuromuscular junction (NMD) disorders remains unclear. Furthermore, most NMDs remain incurable. Only two FDA-approved drugs are available for the treatment of amyotrophic lateral sclerosis (ALS): riluzole and edaravon; however, their clinical efficacy is suboptimal. Despite the availability of numerous in vitro neuromuscular junction (NMJ) models, new models specifically designed for studying motor units (MUs) have been needed over the past two decades. New bioengineering and cell-based technologies have driven the development of in vitro NMJ models. In particular, advances in culture techniques, biomaterials, and microfluidic devices have facilitated the transition from two-dimensional (2D) cultures to multicellular, three-dimensional (3D) model systems. The MNs and skeletal muscles used in these previous in vitro models were typically derived from dissociated explants, primary cells, commercially available cell lines, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs). For example, a "NMJ chip" assembled within a microfluidic component was created using human iPSCs for drug screening and precision medicine applications in neuromuscular diseases. This process takes approximately two to three months to complete and requires advanced equipment. NMJ models featuring cultured 3D skeletal muscle tissue are designed to study adult NMJ development and neuromuscular diseases. A 3D human trunk neuromuscular organoid system has been established and demonstrated to possess functional NMJ characteristics. However, most of these systems were developed for specific purposes and are relatively time-consuming and labor-intensive. There is a need for a more general, functional NMJ model that can be used in a wide range of applications. This application claims priority to U.S. Provisional Patent Application No. 63 / 615,352, filed on December 28, 2023. The entire contents of the above application are incorporated herein by reference. For the above-mentioned purpose, the present application provides a motor unit chip, which comprises: a first hydrogel having parallel microgrooves on its surface; muscle cells placed in the parallel microgrooves; a second hydrogel placed on the first hydrogel and covering the muscle cells; and motor neurons spread flat on top of the second hydrogel. Preferably, the first hydrogel comprises gelatin. Preferably, the thickness of the first hydrogel is 10 μm to 4 mm. Preferably, the second hydrogel comprises Matrigel and / or basement membrane-like matrix. Preferably, the second hydrogel has a thickness of 1 μm to 10 mm. Preferably, the width of the parallel micro-grooves is 2 to 100 μm, and / or the depth of the parallel micro-grooves is 2 to 40 μm. Preferably, the second hydrogel further comprises myelinated nerve fibers formed by Schwann cells. Preferably, the muscle cells, Schwann cells and motor neurons are derived from rodents. Preferably, the muscle cells, Schwann cells and motor neurons are derived from primates. For another purpose, the present application provides a method for manufacturing a chip of neuromuscular junctions, which includes: preparing a first hydrogel and forming parallel microgrooves on its surface; seeding myoblasts onto the first hydrogel; differentiating the myoblasts into muscle cells; loading a second hydrogel having Schwann cells onto the first hydrogel; and seeding and plating the motor neurons onto the second hydrogel having motor neuron differentiation medium. Preferably, the myoblasts include C2C12 cells and myoblasts derived from stem cells. Preferably, the motor neurons include MN1 cells and motor neurons derived from stem cells. Preferably, the Schwann cells include IMS32 cells and Schwann cells derived from stem cells. Preferably, the myoblasts are differentiated for 4 to 60 days. Preferably, the Schwann cells are pre-cultured for 6 to 60 days. Preferably, the motor neurons are pre-cultured for 6 to 60 days before being loaded into the second hydrogel. For another purpose, the present application provides a kit for manufacturing a neuromuscular junction chip, comprising: a myotube differentiation medium; a first hydrogel having parallel microgrooves on one surface; a second hydrogel; a motor neuron differentiation medium; and a neuromuscular maturation medium; wherein the first hydrogel comprises gelatin; and wherein the second hydrogel comprises Matrigel. Preferably, the kit further comprises myoblasts, motor neuron precursor cells and Schwann cells. On the other hand, the present application further provides a method for detecting pathological events using the above-mentioned motion unit chip, which includes: preparing the motion unit chip; and inducing pathological events in the motion unit chip; wherein the pathological events include amyotrophic lateral sclerosis, myasthenia gravis and peripheral neuropathy. As used in the description herein and the appended claims, the singular forms "a" and "an" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes more than one protein, and reference to "a compound" means more than one compound. The use of "comprise," "comprising," and "includes," "including" are interchangeable and are not intended to be limiting. It should also be further understood that while the term "comprising" is used in the description of various embodiments, those skilled in the art will understand that, in certain circumstances, the language "consisting essentially of" or "consisting of" may alternatively be used to describe an embodiment. When a numerical range is provided, it is understood that every intermediate integer of that value, and every tenth of every intermediate integer of that value, and every upper and lower limit of that range, and any other stated or intermediate values ​​in that stated range, are encompassed within the scope of the invention unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the scope of the invention, subject to any specifically excluded limit in the stated range. When the stated range includes one or both limits, ranges excluding (i) one or (ii) both of those limits are also encompassed within the scope of the invention. For example, "1 to 50" includes "2 to 25," "5 to 20," "25 to 50," "1 to 10," etc. All publications, patents, patent applications, and other references cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference were individually incorporated by reference for all purposes. It is to be understood that both the foregoing general description (including the accompanying drawings) and the following detailed description are exemplary and explanatory only and are not restrictive of the invention. In one embodiment, the present invention provides a motor unit chip comprising: a first hydrogel having parallel microgrooves on its surface; muscle cells disposed within the parallel microgrooves; a second hydrogel disposed on the first hydrogel and covering the muscle cells; and motor neurons layered on top of the second hydrogel. Because the muscle cells are placed within the grooves, muscles can grow as muscle fibers without clumping together, better simulating real muscle cells. In one embodiment, the first hydrogel preferably comprises gelatin. Preferably, the first hydrogel further comprises a prepolymer solution containing acrylamide (3%), bis-acrylamide (0.4%), and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (0.5%). The second hydrogel may also comprise Matrigel. Preferably, the second hydrogel further comprises alginate and / or a basement membrane-like matrix. Specifically, the second hydrogel may comprise DMEM, horse serum (2%), insulin (2 μg / ml), and penicillin / streptomycin (1%). In another embodiment, the hydrogel may comprise a collagen:Matrigel hydrogel ratio of 4:1. Rat tail type I collagen (Corning) was mixed on ice with 10× PBS, phenol red (as a pH indicator), 0.5 N NaOH, and water to produce a collagen concentration of 2 mg / ml at pH 7.4 for cell culture. In one embodiment, the thickness of the first hydrogel may be 10 μm to 4 mm, preferably 10 to 40 μm, and the thickness of the second hydrogel may be 1 μm to 10 mm, preferably 10 to 80 μm. In a specific embodiment, the width of the parallel microgrooves may be 2 to 100 μm, preferably 10 μm, and / or the depth of the parallel microgrooves may be 2 to 40 μm, preferably 10 μm. In one embodiment, the muscle cells, Schwann cells, and motor neurons can be derived from rodents, primates, or any other desired species. In one embodiment, the present invention provides a method for manufacturing a motor unit chip, comprising: preparing a first hydrogel and forming parallel microgrooves on the surface; seeding myoblasts onto the first hydrogel; differentiating the myoblasts into muscle cells; loading a second hydrogel containing Schwann cells onto the first hydrogel; and seeding and plating motor neurons and motor neuron differentiation medium onto the second hydrogel. In one embodiment, the myoblasts can be C2C12, myoblast cell lines, or myoblasts derived from stem cells. In one embodiment, the motor neurons can be MN1, motor neuron cell lines, or motor neurons derived from stem cells. In one embodiment, the Schwann cells can be IMS32, Schwann cell lines, or Schwann cells derived from stem cells. In one embodiment, the myoblasts can be differentiated for 4 to 60 days, preferably 4 to 8 days. In one embodiment, the Schwann cells can be pre-cultured for 6 to 60 days, preferably 6 to 10 days, before being loaded into the second hydrogel. In one embodiment, the motor neurons can be cultured for 6 to 60 days, preferably 6 to 10 days. In one embodiment, the present invention provides a kit for fabricating motor unit chips, comprising: myotube differentiation medium; a first hydrogel having parallel microgrooves on one surface; a second hydrogel; a motor neuron differentiation medium; and a neuromuscular maturation medium; wherein the first hydrogel comprises gelatin; and wherein the second hydrogel comprises Matrigel. In one embodiment, the kit may further comprise myoblasts, motor neuron precursor cells, and Schwann cells. Preferably, the first hydrogel may further comprise a prepolymer solution containing acrylamide (3%), bis-acrylamide (0.4%), and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methyl-propiophenone (0.5%), and the second hydrogel may comprise DMEM, horse serum (2%), insulin (2 μg / ml), and penicillin / streptomycin (1%). In another embodiment, the hydrogel may comprise a collagen:Matrigel hydrogel in a 4:1 ratio. Rat tail type I collagen (Corning) was mixed on ice with 10× PBS and phenol red (as a pH indicator), 0.5 N NaOH, and water for cell culture to yield a collagen concentration of 2 mg / ml, pH 7.4. In one embodiment, the present invention provides a method for detecting pathological events using a motion unit chip. The method comprises the following steps: preparing the motion unit chip; and inducing a pathological event in the motion unit chip; wherein the pathological event may include amyotrophic lateral sclerosis, myasthenia gravis, or peripheral neuropathy. The cell lines of muscle cells, motor neurons, or Wann cells of the motion unit chip may vary depending on the desired pathological event. In one embodiment, a specific drug may be added to the motion unit chip to detect its response. The present invention is not limited thereto, and chemical or physical control may also be performed as needed, such as peripheral neuropathy induced by chemotherapy, and drug screening for amyotrophic lateral sclerosis, peripheral neuropathy, myopathy, and sarcopenia. The present invention is described below with reference to various embodiments and accompanying drawings. Methods and Materials Cell lines and culture conditions The mouse C2C12 myoblast cell line was purchased from ATCC (Catalog No. CRL-1772). The mouse motor neuron cell line MN1 was kindly provided by Ahmet Hoke (The Johns Hopkins University, Baltimore, MD). The mouse Schwann cell line IMS32 was purchased from CosmoBio Co., LTD (Catalog No. PMC-SWN-IMS32C). All three cell lines were maintained in high-glucose Dulbecco's Modified Eagle Medium (DMEM) containing 2 mM L-glutamine (Thermo Fisher Scientific, Catalog No. 11965–092) and supplemented with 10% FBS (ALTANTA Biologics, Catalog No. S11150), 100 U / mL penicillin, and 100 mg / mL streptomycin (Thermo Fisher Scientific, Catalog No. 15140–122). Cells were maintained at 37°C in a humidified atmosphere with 5% CO. 2) Incubate the cells in a cell culture incubator. C2C12 cells were split using 0.25% trypsin (Thermo Fisher Scientific, Catalog No. 25200–056) after reaching approximately 70% confluence. MN1 cells were split using 0.05% trypsin (Thermo Fisher Scientific, Catalog No. 25200–054) after reaching approximately 90% confluence. IMS32 cells were split using 5 mM EDTA (Thermo Fisher Scientific, Catalog No. 15575-020) after reaching approximately 70–80% confluence. The manufacturing process of the motion unit wafer is shown in FIG1 and will be described in detail below. Impression ( Stamp )manufacture PDMS stamps were fabricated as described elsewhere. Briefly, a silicon wafer mold was treated with trichloromethylsilane (Millipore Sigma, catalog number 75-79-6) vapor as a release agent for 30 minutes. A PDMS mixture (PDMS:curing agent = 10:1, Sylgard 184, Dow Corning) was poured onto the master mold and cured at 60°C for 2 hours. The cured PDMS was cut from the master mold and trimmed with a clean blade to create a stamp. The PDMS stamp was then cleaned with oxygen plasma (radiation intensity: 200 W) for 10 minutes. Micromolding ( µ- Molding) Hydrogel Production Under sterile conditions, in sterile ddHO A 10% solution of Gelatin A (Sigma, catalog number G1890-100 g) was prepared in 2O. The solution was placed on a rotating stand at 65°C for 10 minutes, or until completely dissolved, after which it was removed from the stand to allow air bubbles to rise from the solution for 10 minutes. A 65°C hotplate (Apollo; digital heating and cooling plate) was used to create the µ-molded hydrogel and adhere it to a coverslip (Fisher Scientific, catalog number 1254580). Therefore, the µ-molded PDMS stamp, gelatin, and coverslip were heated to 65°C before molding. 18 µl of gelatin solution was carefully pipetted directly onto the stamp, followed by a coverslip quickly placed on top of the gelatin drop, ensuring complete coverage. The stamp was removed from the hotplate, and the completed sandwich was cooled at room temperature for 30 minutes. Under sterile conditions, the coverslip and hydrogel were carefully peeled from the stamp using forceps and placed into a 24-well plate. The hydrogel was exposed to UV light for 5 minutes. The gelatin hydrogel was cross-linked with 5% transglutaminase solution for 18 hours. Under sterile conditions, 1 g of powdered transglutaminase was mixed with 19 ml of autoclaved ddH 2O. The enzyme was placed on a rotating rack at 50 °C for 10 minutes to dissolve and allowed to cool to room temperature before filtration. The transglutaminase solution was filtered through a 0.2 µm filter. After the hydrogel was exposed to UV light for 5 minutes, the transglutaminase was added. The 24-well plate was covered and placed in a fume hood at room temperature for 18 hours. After 18 hours, the transglutaminase was aspirated. The enzyme was then removed by adding autoclaved ddH2O to the wells. Rinse the hydrogel three times with 20°C. For the final rinse, place the 24-well plate uncovered under UV light for 5 minutes. After UV exposure, the 24-well plate can be covered, placed in a sterile bag, and stored at 4°C until use. The depth and width can be varied as needed; in this example, the depth and width are 10 µm and 10 µm, respectively. MN1 Transfection The plasmid CLYBL-(Ef1a-SBP-LNGFR-T2A-mApple)(CAG-rtTA)-(TRE-hNIL) was purchased from Addgene (catalog no. 05842). MN1 cells were transfected using Lipofectamine 3000 (Thermo Fisher Scientific, cat. no. L3000-015) according to the manufacturer's instructions. One day after transfection, MN1 cells were added to the motor unit chip. Cell culture in motor unit chips On day 0, 2 × 10 4 C2C12 cells / cm 2 Cells were seeded onto the hydrogels in a 24-well dish and cultured in growth medium. Once confluent (approximately 2 days), myotube differentiation medium (DMEM (Thermo Fisher Scientific, catalog number 5H30074.2) with 2% horse serum, 100 U / mL penicillin, 100 mg / mL streptomycin, and 2 mM L-glutamine) was added. C2C12 cells were differentiated for 4 days, with myotube differentiation medium replenished every 2 days. On day 6, Schwann cell line IMS32 cells were digested with 0.05% trypsin and plated at 4 × 10 4 Resuspend the cells in ice-cold myotube differentiation medium at a density of 10 cells / ml. Mix Schwann cells with Matrigel at a 1:1 ratio and store on ice. Thaw Matrigel matrix (Corning, catalog number 356255) on ice overnight in a cold room before use. Remove the medium from the motor unit chip and add 18 µl of the mixture to the center of the hydrogel. Maintain the motor unit chip in a 37°C incubator for 3 minutes and add fresh myotube differentiation medium. On day 7, transfect 1 × 10 3 MN1 cells / cm 2 MN1 cells were added to each well of a 24-well plate above differentiated C2C12 cells. Before plating on the C2C12 cells, MN1 cells were transfected with either an empty vector control or GFP-Crbp1. MN1 cells were dispersed and plated on Matrigel matrix to dissociate and isolate the vertical structures of motor units. Co-cultures were maintained in MN Differentiation Medium 1 (DMEM supplemented with 100 U / mL penicillin, 100 mg / mL streptomycin, 2 mM L-glutamine, 1% MEM non-essential amino acids (NEAA; Thermo Fisher Scientific, catalog no. 1140–050), 2 µg / mL doxycycline (Sigma, catalog no. D9891), and 10 µL Y27632 dihydrochloride (R&D, catalog no. 1254 / 1) for 4 days, after which they were replaced with MN Differentiation Medium 2 (MN Differentiation Medium 1 without Y27632 dihydrochloride). MN Differentiation Medium was replenished every two days. Immunostaining For immunostaining, triplicate slides were fixed in 100% methanol at –20°C for 5 minutes. After three 5-minute washes in 1X PBS, the slides were blocked with 2% BSA for 1 hour at room temperature. The slides were incubated with primary antibodies against α-actinin (1:100; Cell Signaling, catalog no. 6487S), Tuj1 (1:1000; R and D, catalog no. MAB 1196), or myelin basic protein (MBP) (1:500; R and D, catalog no. MAB42282) in 1X PBS overnight at 4°C. After three 5-minute washes in 1X PBS, fluorescent secondary antibodies (1:1000) and fluorescent dye-conjugated µBTX (1:400; Biotium, catalog no. 00005) were incubated for 2 hours at room temperature in the dark. A solution of DAPI (1:1000; Thermal Fisher Scientific, catalog no. 62248) in 1X PBS was incubated at room temperature in the dark for 10 minutes. Slides were then washed three times with 1X PBS for 5 minutes each. Z-stack images were captured on an Olympus Fluoview FV1000 BX2 upright confocal microscope. Calcium imaging Triple cultures on 12.5 mm glass coverslips were placed in HEPES Hanks' buffer solution (HHBS) (5.6 mM glucose, 20 mM Hepes, 3 mM NaHCO 3. 0.3 mM Na 2HPO 4. 0.4 mM KH 2PO 4. 137mM NaCl, 5.0mM KCl, 4.9mM MgCl 2. 4.1 mM MgSO 4. 1.26 mM CaCl After brief washing in 5% PBS (pH = 7.4), the coverslips were incubated with a solution of fura-2AM in 0.04% pluronic acid in HHBS (final concentration 20 μM) for 30 min at 37 °C. After loading with the indicator, the coverslips were washed in fresh HBSS at 37 °C for 10 min and then transferred to an imaging chamber for microscopy experiments. Fura-2 imaging was performed on an Olympus IX71 microscope using a 20X objective. The excitation wavelength was selected using a galvanometer-driven single-photometer (8 nm slit width) coupled to a 75 W xenon arc lamp (Optoscan; Cairn Research). Intracellular Ca 2+ Concentration ([Ca 2+ ] i ) was monitored by sequentially exciting fura-2 at 340 and 380 nm (1 Hz). Single images were acquired at 610 nm to identify MNs expressing mApple within the field of view. Fluorescence images were captured using a cooled charge-coupled device camera (Cascade 512B; Roper Scientific) and Metaflu acquisition software (Molecular Devices). All fura-2 experiments were performed at room temperature in HBSS buffer. To depolarize MNs, extracellular K + The concentration was increased to 50 mM, where K + With Na in HHBS + For myotube stimulation, 10 μM ACh was applied by bath perfusion. A 60-second baseline of HHBS was obtained before stimulating each coverslip. + Stimulation was applied for 30 seconds, and ACh was applied for 1 minute. After stimulation, a 60-second washout was performed. A background image was acquired after each experiment; the coverslip was wiped clean, and 20 frames were captured using the same parameters as above. Calcium imaging data analysis Images were processed using MetaFluor Analyst to draw regions of interest (ROIs), subtract background, and generate a 340 / 380 ratio. MN ROIs were selected based on mApple performance and further validated by a blinded expert using morphology. Each differentiated MN had a cell body and a single enlarged axon terminal. Myotube ROIs were selected based on morphological criteria; myotubes exhibited a clear, elongated skeletal muscle fiber-like morphology. Given that myotubes spanned the entire length of the field of view, three ROIs were drawn for each individual myotube and averaged to represent a single cell. By subtracting the average baseline [Ca 2+ ] i Peak amplitude analysis was performed by comparing the peak amplitude to the maximum value reached after stimulation. Only responding cells were included in this peak amplitude analysis. Responses were defined by the following criteria: (1) a signal intensity of at least 450 (16-bit scale) after background subtraction, and (2) a peak [Ca 2+ ] i At least two standard deviations (SD) above baseline (average value for 60 seconds). The criterion of "1" is determined empirically to exclude cells with poor signal noise. [Ca 2+ ] i Calculations were performed in Microsoft Excel by averaging the 340 / 380 intensity ratios of MNs or myotubes that responded to stimulation in that field of view. Statistical analysis Data were analyzed using the Student's t-test. Statistical analysis was performed using Prism 8.0 (GraphPad, CA). All tests were performed at a significance level of p ≤ 0.05, and data are presented as mean ± standard error or mean ± SD. For statistical analysis of calcium imaging, normality of MN and myotube data was assessed using the D'Agostino-Pearson test. Myotube data showed normality, while MN data did not. Therefore, myotube data were subjected to a paired Student's t-test, while MN data were subjected to a nonparametric Wilcoxon signed-rank test. For each experiment, "n" was defined by a single field of view on a single coverslip. The percentage of responders was calculated as the number of responders in that field divided by the total number of MNs or myotubes. result The results of the above experiments are described in the following specific examples. 1. Motion unit chip The motor unit chip is specifically designed to mimic the MU (Figure 2). It is constructed using the three essential cell types of the MU: MNs, skeletal muscle, and Schwann cells. To ensure consistency across the motor unit chip, the inventors selected three well-studied mouse cell lines for each cell type: MN1 for MNs, IMS32 for Schwann cells, and C2C12 for myoblasts. Designing a microenvironment that mimics physiological conditions during MU development is crucial. This is achieved through the design of a two-layer matrix. A customized µ-molded gelatin hydrogel on the bottom surface provides optimized topological cues for the differentiation, orientation, and alignment of myoblasts (Figure 3). The upper Matrigel layer is designed to serve several purposes. First, the Matrigel layer physically separates myoblasts from MNs, thereby enhancing axonal elongation. Second, the Matrigel layer embeds Schwann cells, providing an extracellular matrix-like microenvironment for axon myelination (Schwann cell-MN axon interaction) (Figure 4). These factors contribute to the formation of the neural junction (MN-muscle cell interaction) (Figures 5A-C). After co-differentiation, MN axons can easily penetrate the Matrigel layer and reach the myotubes on the µ-molded hydrogel layer to form functional NMJs, while Schwann cells are used to form myelinated axons. The overall process is outlined in Figure 1. Before beginning the process, µ-molded hydrogels are prepared. On day 0, C2C12 myoblasts are seeded onto these µ-molded hydrogels and cultured in triplicate. After two days of culture in growth medium, myoblasts typically expand to >90% confluence; the medium is then replaced with fresh myotube differentiation medium, with medium changes occurring every two days thereafter. By day 6, most myoblasts have begun to differentiate. Schwann cells (IMS32) are mixed with Matrigel and added to the differentiated myoblasts. Also on day 6, in a separate dish, MN1 cells are transfected with a plasmid (Plasmid #105842; Addgene) expressing the mApple marker and the doxycycline-inducible factors Neurogenin 2 (NGN2), ISL LIM homeobox 1 (ISL1), and LIM homeobox 3 (LHX3). The growth factors and transcription factors encoded in this plasmid promote MN differentiation, resulting in a more uniform MN morphology. Without this plasmid, control MN1-derived neurons exhibit significant morphological changes, as shown in Figure 7. On day 7, >80% of MN1 cells were mApple-positive. These transfected MN1 cells were then plated on Matrigel in MN Differentiation Medium 1 to induce MN differentiation and axonal elongation. On day 11, the medium was changed to MN Differentiation Medium 2, which promotes NMJ formation. MN Differentiation Medium 2 was changed every two days. By day 14, functional MUs had formed and were ready for assay. 2. Matrigel Layers help MN Differentiation and myelination In this bilayer motor unit chip, the Matrigel layer is a key element that facilitates MN differentiation and myelination. To demonstrate the effects of Matrigel, experiments were performed as described in Figure 4(a). On day 6 (as in Figure 1), Schwann cells were mixed with (+) or without (-) Matrigel, and MN differentiation and myelination were examined on day 14. As shown in Figure 4(b), the typical multipolar morphology of spinal cord MNs was observed in the Matrigel (Tuj1; red) group. In this group, a single axon extended from each differentiated MN at one end of the cell body, and multiple dendrites branched from the other end. Importantly, the (+) Matrigel group produced significantly and consistently longer axons compared to the (-) Matrigel group (Figures 4(c), (d), and (e)). Schwann cell myelination of axons was examined by immunostaining with an antibody against myelin basic protein (MBP; green). As expected, in the (+) Matrigel group, MBP signaling was detected only in axons and axon terminals, but not in the cell bodies (Figure 4(d)). In contrast, in the (-) Matrigel group, the majority of MBP signaling was detected around the cell bodies, partially covering axons and axon terminals (Figure 4(e)). Taken together, these data demonstrate that a Matrigel layer containing Schwann cells is essential for efficient MN differentiation, axon elongation, and myelination. 3. µ- Molded hydrogels facilitate myotube differentiation and proper alignment The µ-molded hydrogels are crucial for myoblast differentiation and alignment. To demonstrate the effects of µ-molded hydrogels, C2C12 cells were seeded into untreated 24-well plates (uncoated control), plates coated with hydrogels, or plates coated with µ-molded hydrogels, as shown in Figure 4(a). Motor unit wafers were maintained as described in Figure 1 and examined for myotube differentiation on day 14. Myoblasts proliferated and differentiated into muscle cells, which then fused into multinucleated myotubes and expressed skeletal muscle markers such as sarcomeric α-actinin. α-actinin specifically marks Z-discs, a characteristic of myotubes (Figure 3), which define the individual functional units of skeletal muscle (called sarcomeres). As shown in Figure 3(b), myotube formation was observed in all experimental groups, but myotube morphology varied across these conditions. In both the control and hydrogel groups (left and center images; Figure 3(b)), myotubes appeared to lack proper alignment. Furthermore, on day 12, when contraction is typically observed, most myotubes in the control group detached from the surface. In contrast, the µ-molded hydrogel group displayed well-differentiated and aligned myotubes (right image, Figure 3(b)), which exhibited the expected contractility (Figure 9). To further assess the organizational characteristics of mature myotubes in the µ-molded hydrogel group, the inventors labeled the 3D cultures with an anti-α-actinin antibody (Figures 3(c), (d)). Mature myotubes in complete triplicate cultures resembled in vivo skeletal muscle fibers. As shown in Figure 3(c), α-actinin signal was concentrated at the Z-disc, where it cross-linked with thin filaments in adjacent sarcomeres (the basic contractile units of myotubes). Furthermore, in contrast to the typical central localization of nuclei in immature myotubes, the nuclei in these mature myotubes (DAPI; blue) were flattened and located adjacent to the myotube surface, as expected given the mature cylindrical structure of myotubes. Finally, at lower magnification (Figure 3(d)), these stripes and aligned myotubes had similar widths, approximately 15 to 25 µm, and were well elongated. In summary, µ-molded hydrogels facilitated the alignment of myotubes. The overall design of this motor unit wafer facilitates the formation of skeletal muscles with cylindrical, aligned, striated, unbranched, and extremely long myotubes (over 700 µm). 4. Functionality in motion unit chips NMJ Identification In addition to characterizing the morphological characteristics and biomarkers of the 3D cultures, the inventors also assessed the physiological function of the motor unit chip. The NMJ was investigated using the neural marker Tuj1 (red) to label presynaptic MNs and the AChR marker αBTX (green) to label postsynaptic compartments. As shown in Figure 5A, two sets of small branches from axon terminals form specialized presynapses (white and blue arrows, left panel), indicating that the MN innervates two myotubes (lower and upper panels). Importantly, while approximately eight to nine myotubes are visible in this field of view, only two of these tubes (located near the axon terminals) exhibit clear AChR clusters (stained with αBTX, middle panel). These AChR clusters were detected near the MN in this field of view, particularly at its axon terminals (right panel). This provides evidence that the MN recruits AChRs to its axon terminals to form the NMJ. In addition, NMJ formation, including enlarged axon terminals, small branches, and recruited AChR clusters, was gradually detected from differentiation day 12 ( Fig. 8 ) to day 14 ( Fig. 5A ). In summary, these results show that this motor unit chip can provide an in vitro model for studying NMJ development and formation. To further examine the function of NMJs in this motor unit chip, the inventors performed two functional assays: contractility and calcium imaging. The following section describes the contractility of functional MUs. Figure 9 shows myotube contraction under bright field illumination. The positions of the three MNs in this field of view (determined by mApple signals) are indicated by white arrows, as shown in Figure 9, and the five contracting myotubes are indicated by blue arrows. These myotubes contract in response to MN stimulation, indicating the formation of functional NMJs. Notably, the lower MN drives two rapidly contracting myotubes, while the two closely positioned MNs above drive synchronized, slowly contracting myotubes. This indicates the formation of individual MUs and functional NMJs that mediate MN-driven myotube contraction. To further elucidate MN-driven myotube contraction, the inventors cultured MNs at ultra-low density (2 × 10 2 cells / cm 2 ) were inoculated with mApple-labeled MNs. Figure 10 demonstrates contractility in this experiment. Representative images were captured and are shown in Figure 5B. The left image in Figure 5B shows the MN cell body (white arrow) and enlarged axon terminals (blue arrow). The right image in Figure 5B is an extract from Figure 10, capturing contracting myotubes and MNs under brightfield illumination using a Texas Red fluorescent filter to visualize both mApple-labeled MNs and myotubes in the same field of view. The contraction displacement in Figure 10 was quantified as in Figure 5C. Myotube contraction occurred, with maximum displacement located at the axon terminals (Figure 10 and Figure 5C). Furthermore, the frequency of MN-driven myotube contractions was significantly greater than that of spontaneous contractions (Figure 10 and Figure 5C). This further demonstrates that MNs drive myotube contraction in this motor unit wafer, indicating the formation of functional NMJs within the MU. 5. MN and myotubes Ca 2+ Imaging Next, the inventors used traditional calcium imaging methods to probe the functions of neurons (including MNs) and muscles (myotubes in MUs), thereby demonstrating the physiological functions of MNs and myotubes, two key components of MUs. Physiologically, MNs and myotubes respond to stimuli that cause neuronal firing and muscle contraction by increasing intracellular calcium concentration ([Ca 2+ ] i). Therefore, calcium imaging provides a suitable tool for studying MN and myotube function. The inventors used the fluorescent ratiometric calcium indicator fura-2AM to detect [Ca 2+ ] i Changes in [Ca 2+ ] i The changes in 340 nm / 380 nm ratio are shown. + Depolarization or treatment with 10 μM ACh induced [Ca 2+ ] i As expected, Schwann cells did not show any measurable changes in [Ca 2+ ] i The results indicate that the Schwann cells added to the triple culture did not exhibit an inappropriate response. This further supports the specificity of this improved triple culture model in terms of physiological relevance and specific functional responses of each component. Figure 6A shows representative images of a single field of view using brightfield illumination (upper left and upper right), fura-2 fluorescence (lower left), and mApple fluorescence (lower right). Brightfield images show that MNs (red arrows) are located on the top Matrigel layer of the motor unit wafer (upper left), while myotubes (blue arrows) are located on the bottom hydrogel layer of the motor unit wafer (upper right), mimicking the structure of MUs. Fura-2AM is taken up and labeled by MNs, Schwann cells, and myotubes. As described above, MNs were identified by the expression of mApple. Figure 6B shows the expression of MNs in response to 30 s, 50 mM K + Representative traces of responses to stimulation. High extracellular K+ concentrations can open voltage-gated Ca 2+ channels to depolarize neurons, leading to Ca 2+ The MN response showed a peak signal, followed by several oscillations at the peak amplitude before returning to baseline. In each independent experiment (n = 8 fields), 70% of MNs (43 / 62 cells) responded to 50 mM K +The depolarization-induced response increased from a baseline 340 / 380 ratio of 0.62 ± 0.06 to a peak value of 1.83 ± 1.41 (Figure 6C). Figure 6C(d) shows a representative trace of a typical myotube response to 10 µM ACh stimulation for 60 s. At this concentration, ACh has been shown to induce intracellular Ca 2+ Release and contraction. Myotube responses immediately after stimulation exhibit [Ca 2+ ] i ACh stimulation was observed in 8 independent experiments (n = 8 fields), with 89% of myotubes (24 / 27 cells) responding to ACh stimulation. The ACh-evoked response increased from a baseline 340 / 380 ratio of 0.68 ± 0.12 to a peak of 3.62 ± 2.73 (Figure 6C(e)). Furthermore, in at least two independent experiments, myotubes also exhibited physical contractions when stimulated with ACh (Figures 11A-11B and 12). Spontaneous contractions were also observed in some motor unit wafers (Figure 10). These data demonstrate that the key functional components of the MU—MNs and myotubes—exhibit measurable and significant [Ca] in response to physiological stimuli. 2+ ] i Increase. discuss This motor unit chip is specifically designed to mimic the MU and can be used to investigate a wide range of biological questions related to NMD, as well as for pharmacological studies (e.g., drug screening). These experiments demonstrate the advantages of a bilayered hydrogel design. This design utilizes an upper layer of Matrigel, which facilitates MN differentiation and timely myelination, and a lower layer of µ-molded hydrogel, which facilitates myotube differentiation, alignment, and innervation. This MU model is characterized based on multiple criteria, including morphological features, essential biomarkers of the NMJ, and MU functions such as contractility and calcium signaling. This new model has several positive features. First, it encompasses all three cell types of the MU: MNs, Schwann cells, and myotubes, with all three cell types exhibiting good differentiation and function. Second, the bilayer of µ-molded hydrogel / Matrigel provides a critical and desirable microenvironment that mimics the physiological conditions of the developing MU. Specifically, MNs and muscle cells are initially separate, and only when MN axons are myelinated by Schwann cells and penetrate the muscle layer where myotubes mature do MNs and muscle cells come into contact for co-differentiation and synapse formation. During this stage, MN axons innervate myotubes to form NMJs. To allow this to occur, Matrigel provides an extracellular matrix-like environment that separates MNs from muscle cells. This layer embeds Schwann cells that serve to myelinate the axons as MN axons extend through this layer. In addition, the µ-molded hydrogel creates a surface area that facilitates myotube differentiation and alignment, which is critical for organized myotube formation. A third advantage of this motor unit chip is that multiple parameters of individual MUs can be detected and quantified, such as axon length and frequency of myotube contractions. In addition, axon myelination, mature myotubes, and NMJ formation can all be assessed by immunostaining. These can be used to study pathological conditions associated with various NMDs, such as axonal degeneration, demyelination, cell death, muscle atrophy, and degeneration. Importantly, Ca 2+Imaging can be used to monitor the biological functions and properties of individual MNs and myotubes. Finally, by manipulating MNs or myoblasts, fundamental questions related to MUs can be investigated and NMD can be modeled in vitro. In previous studies, the inventors employed a simpler in vitro MU model (sans Schwann cells and Matrigel) consisting solely of MNs and myotubes on a µ-molded hydrogel to study NMJ formation. Using this motor unit chip, the inventors successfully demonstrated that the expression of the retinoic acid signaling protein, cellular retinoic acid binding protein 1 (CRABP1), in MNs influences axon length and AChR cluster formation. The requirement for CRABP1 for MU function in vivo was validated in Crabp1 knockout mice and confirmed using genetic rescue experiments. Therefore, this motor unit chip is a powerful tool for effectively identifying molecular players and dissecting signaling pathways critical for MU or NMJ formation and function. Given practical considerations (time and effort), this motor unit chip provides a reliable and effective in vitro MU model for a wide range of studies, including physiological and pharmacological studies. The formation of functional NMJs in motor unit wafers is also supported by data from the inventors' previous study, which reported a similar but simpler (only two cell types, MNs and C2C12) version of the co-culture system. This previous study examined the expression of the presynaptic vesicle protein SV2 and postsynaptic AChR at the NMJ. Expression of synaptic vesicle glycoprotein 2 indicates the ability of MNs to release neurotransmitters from axon terminals. To improve the physiological relevance of this previous model, the inventors in this study incorporated a Matrigel layer embedded with a third cell type crucial for the NMJ: Schwann cells. This significantly improved the model, as demonstrated by multiple measurements, including increased axon length, myelinated axons, and axon terminals. At the NMJ in this triple culture model (Figure 4(a)), the presence of two sets of small branches (white and blue arrows, left panel) from the axon terminals indicates that MNs innervate multiple myotubes. The morphology of these small branches indicates the potential of the NMJ to form a pretzel-like structure, which is a classic morphological feature of the NMJ, as shown in mouse models (Figure 4(a), left). In the postsynaptic compartment, AChR expression was clearly detected, mainly near the MN, especially in its axon terminals (Figure 4(a), Figure 8), indicating that the MN recruits AChRs to its axon terminals to form AChR clusters. The function of the NMJ was demonstrated using traditional real-time imaging methods that can detect Ca 2+ The dynamics of Ca reflect the functional responses of neurons (in this case, MNs) and muscles (in this case, myotubes). 2+Imaging data verified the relevant responses of key components in MU - MN (electrophysiological response) or myotube (contractility) after high potassium (KCl) or acetylcholine stimulation, intracellular Ca 2+ The increase in intracellular Ca 2+ The predicted increase was not observed. Therefore, each cellular component (MNs, myotubes, and Schwann cells) appropriately exhibited its biological response (or lack thereof) to stimulation, supporting the physiological relevance of this improved triple culture model. As shown in Figures 4(b) and 10, myotube contraction and maximum displacement occurred specifically at the axon terminals. Furthermore, the frequency of MN-driven myotube contractions was fivefold higher than the spontaneous contraction frequency of nearby myotubes without MNs (Figures 4(c) and 10). Taken together, these results support the formation of functional NMJs within motor units. One of the reasons for using a cell line to generate this MU model is consistency, simplicity, and time savings in the culture process. Whether appropriate human cell lines or stem cell systems (e.g., iPSC-derived MNs or muscle cells) can be similarly optimized in this system remains to be determined. 2+ In imaging experiments, due to the thickness of the Matrigel layer, it will be challenging to collect clear signals from MNs and myotubes simultaneously, as the cells reside in different layers (Figure 5A). This will limit the immediate assessment of MN excitation and MN-driven myotube contraction. However, advanced imaging tools may help to improve the temporal resolution of this assay. Although [Ca 2+ The percentage of MNs and myofibers that responded was quite high, but the magnitude of the response was variable. Possible explanations for this variability may be due to the challenges of accurately correcting for background fluorescence in 3D cell populations, variable drug application rates, and dye loading through these gel matrix layers. These issues may be addressed in future experiments by using genetically encoded Ca2+ inhibitors specific to specific cell types. 2+ Indicator, and local drug application by micropipette. 2+ The records demonstrate the functional feasibility of this new model system. Notable are the well-differentiated and well-organized mature myotubes within these motor unit wafers. These mature myotubes resemble skeletal muscle fibers in their morphology, including similar width and length, as well as their cylindrical, aligned, striated, and unbranched shapes. The µ-molded hydrogel facilitates myotube alignment, while Matrigel provides an extracellular matrix-like microenvironment to enhance myotube maturation. Furthermore, Matrigel protects the contracting myotubes, preventing them from detaching during long-term culture. in conclusion This application provides a process for engineering a structurally organized motor unit chip that can be reliably generated in a reasonably short time. This MU model has been validated in terms of morphology, basic biomarkers, and MU function (including contractility and calcium signaling). This new model has three key advantages. First, the entire MU (single MN) can be examined, and subcellular / functional details can be studied. Second, the further addition of Schwann cells allows for proper myelination, enabling detailed study of MN regulation of NMJ development / maturation and maintenance. Third, using this motor unit chip, a variety of in vitro disease models can be generated by manipulating each relevant cell type. Therefore, this novel motor unit chip is not only suitable for basic research, but also for disease modeling and drug screening. The above description is presented to enable one having ordinary skill in the art to make and use the present invention. Various modifications to the described embodiments will be apparent to one having ordinary skill in the art, and the general principles herein may be applied to other embodiments. Therefore, the present invention is not intended to be limited to the embodiments shown, but rather should be given the widest scope consistent with the principles and features described herein. It will be apparent to one having ordinary skill in the art that other modifications may be made to these embodiments without departing from the spirit and scope of the invention as defined by the appended claims. In particular, the entire disclosures of the following non-patent documents are incorporated herein by reference. References 1. Yu-Lung Lin, et al., "A novel 3D bilayer hydrogel tri-culture system for studying functional motor units", Cell & Bioscience (2023) 13:168. none FIG. 1 shows a manufacturing process of a motion unit wafer according to one embodiment of the present application. FIG. 2 shows a flow chart of forming a motion unit wafer according to one embodiment of the present application. Figure 3 shows that a µ-molded hydrogel, according to one embodiment of the present application, facilitates myotube differentiation and alignment. (a) Experimental flow chart for evaluating the effects of µ-molded hydrogels. As shown in Figure 1, on day 0, C2C12 cells were seeded onto 24-well plates (control group), hydrogels, and µ-molded hydrogels. Differentiated myotubes were identified on day 14. (b) Brightfield images show the morphology of differentiated myotubes in each group (scale bar, 200 μm). (c) Immunostaining images of the µ-molded hydrogel group identify mature myotubes as multinucleated (DAPI, blue) myocytes with sarcomere structures (α-actin, red). (d) At a lower magnification, immunostaining for α-actin (red) reveals aligned, elongated myotubes of similar width. Figure 4 shows that a Matrigel-Schwann cell layer, according to one embodiment of the present application, facilitates MN differentiation and myelination. (a) Flowchart of an experiment to determine the effects of the Matrigel layer. On day 6 in Figure 1, Schwann cells were mixed with the (+) Matrigel group but not with the (-) Matrigel group. Motor neuron differentiation and myelination were measured on day 14. (b) Immunostaining shows differentiated motor neurons in the (+) Matrigel group. Motor neuron morphology was visualized using the neural marker Tuj1 (red), and all nuclei were detected using DAPI (blue). (c) Quantification of axon length in the (+) Matrigel group (n=6) and the (-) Matrigel group (n=6). (d) and (e) Immunostaining shows myelination of axons and axon terminals, (d) with (+) Matrigel and (e) without (-) Matrigel. Myelin formation was detected using myelin basic protein (MBP, green). Results are expressed as mean ± SD, ***p ≤ 0.001, compared with the (+) Matrigel group. Figures 5A to 5C illustrate the identification of functional NMJs according to one embodiment of the present application. Figure 5A shows immunostaining of differentiated neuromuscular junctions (NMJs) surrounded by acetylcholine receptor clusters (αBTX, green signal) at motor neuron axon terminals (Tuj1, red; white and blue arrows). Figure 5B shows live cell images on the left, demonstrating the localization of motor neurons (mApple; white arrows) and their axon terminals (blue arrows) (scale bar, 200 µm). Live cell images from Figures 11A to 11B on the right show MN-driven contractions of axon terminals (blue arrows) as well as spontaneous contractions in the absence of motor neurons. Figure 5C shows quantitative data for the amplitude and frequency of myotube contractions in Figure 10. (d) Maximum myotube contraction displacement measured in the motor neuron (MN)-driven contraction group and the spontaneous contraction group (n = 5 / group). Results are expressed as mean ± SD, **p ≤ 0.01, compared to the MN-driven contraction group. 6A to 6C show a Ca according to one embodiment of the present application. 2+ Imaging results. Figure 6A shows representative fields of view of triplicate cultures using brightfield illumination (upper left and upper right), Fura-2 fluorescence (380 nm; lower left), and mApple fluorescence (605 nm; lower right). Red arrows represent motor neurons, and blue arrows represent myotubes. Figure 6B shows a 50 mM K + Representative traces of typical responses observed in MNs during stimulation. Figure 6C shows baseline and depolarization-induced (50 mM K + ) of [Ca 2+ ] i (n = 8) Bar graph of peak amplitude. P = 0.008. Wilcoxon matched-pairs signed-rank test. (d) Representative trace showing typical myofiber responses to ACh (10 μM) stimulation. (e) Baseline and ACh (10 μM)-induced [Ca] in myotubes. 2+ ] i (n = 8) Bar graph of peak amplitude, * p = 0.02, paired Student's t-test. Data presented are mean ± SD and * p < 0.05, ** p < 0.01. FIG7 shows the morphology of poorly differentiated MN1-derived MNs according to one embodiment of the present application. FIG8 shows an image of a motion unit chip on the 12th day of differentiation according to one embodiment of the present application. FIG9 shows MN-driven myotube contraction under bright field according to one embodiment of the present application. FIG10 shows individual MU- and MN-driven myotube contractions under bright field using a Texas Red fluorescence filter according to one embodiment of the present application. Figures 11A and 11B show a motor unit chip with spontaneous and ACh-induced contractions according to one embodiment of the present application. Figure 11A shows the 340 nm channel (left) and the pseudo-color of the fura-2 340 / 380 fluorescence ratio (right). Figure 11B shows [Ca 2+ ] i Initially, spontaneous contractions can be seen at baseline. ACh (10 µM) stimulates physical contractions (left) and [Ca 2+ ] i Increase. Figure 12 shows a triple culture of ACh-induced contraction according to one embodiment of the present application. (a) shows 340 nm fluorescence (left) and pseudo-color of fura-2 340 / 380 intensity ratio (right). (b) shows [Ca 2+ ] i ACh (10 µM) induced physical contraction (left) and [Ca 2+ ] i Increase.

Claims

1. A motor unit chip comprising: a first hydrogel having parallel microgrooves on its surface; a muscle cell disposed within the parallel microgrooves; a second hydrogel disposed on the first hydrogel and covering the muscle cell; and a motor neuron disposed on top of the second hydrogel.

2. The motion unit chip of claim 1, wherein the first hydrogel comprises gelatin.

3. The motion unit chip of claim 1, wherein the thickness of the first hydrogel is from 10 µm to 4 mm.

4. The motion unit chip of claim 1, wherein the second hydrogel comprises Matrigel and / or a basement membrane-like matrix.

5. The motion unit chip of claim 1, wherein the thickness of the second hydrogel is from 1 μm to 10 mm.

6. The motion unit wafer of claim 1, wherein the width of the parallel microtrench is 2 to 100 µm, and / or the depth of the parallel microtrench is 2 to 40 µm.

7. The motor unit chip of claim 4, wherein the second hydrogel further comprises myelinated nerve fibers formed by Schwann cells.

8. The motor unit chip of claim 7, wherein the muscle cells, Schwann cells and motor neurons are derived from rodents.

9. The motor unit chip of claim 7, wherein the muscle cells, Schwann cells and motor neurons are derived from primates.

10. A method of manufacturing a motor unit chip as claimed in claim 1, comprising: forming parallel microgrooves on the surface of a first hydrogel; seeding myoblasts onto the first hydrogel; differentiating the myoblasts into muscle cells; loading a second hydrogel containing Schwann cells onto the first hydrogel; and seeding the motor neuron and motor neuron differentiation culture medium onto the second hydrogel.

11. The method of claim 10, wherein the myoblast comprises C2C12 cells and myoblasts derived from stem cells.

12. The method of claim 10, wherein the motor neuron comprises MN1 cells and motor neurons derived from stem cells.

13. The method of claim 10, wherein the Schwann cells comprise IMS32 cells and Schwann cells derived from stem cells.

14. The method of claim 10, wherein the myoblasts differentiate over 4 to 60 days.

15. The method of claim 10, wherein the Schwann cells are pre-cultured for 6 to 60 days before being loaded onto the second hydrogel.

16. The method of claim 10, wherein the motor neuron is cultured for 6 to 60 days.

17. A kit for manufacturing motor unit wafers, comprising: a myotube differentiation culture medium; a first hydrogel comprising gelatin and having parallel microgrooves on one surface thereon; a second hydrogel comprising Matrigel; a motor neuron differentiation culture medium; and a neuromuscular maturation culture medium.

18. The kit of claim 17 further comprises myoblasts, motor neuron precursor cells and Schwann cells.

19. A method for detecting pathological events using a motor unit chip as claimed in claim 1, comprising: preparing a motor unit chip as claimed in claim 1; and inducing a pathological event in the motor unit chip; wherein the pathological event includes amyotrophic lateral sclerosis, myasthenia gravis, or peripheral neuropathy.

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