Patent Application
20250215371
The present disclosure relates to a biochip. Particularly, the present disclosure relates to a novel motor unit chip comprising bilayer hydrogel and motor unit. The motor unit chip may be used for studying functions of the motor unit.
A Neuromuscular system includes motor neurons (MNs) and their innervated skeletal muscles. The upper MNs in the motor cortex project to the lower (spinal) MNs which then send signals to the innervated muscles to induce muscle contraction. The motor unit are defined as the single alpha motor neuron and the muscle fibers it innervate. The axons of MNs are myelinated by Schwann cells. Neuromuscular junctions (NMJs) are highly specialized synapses that form between the MN axon terminal and myofibers. Physiologically, axon terminals release acetylcholine (ACh) to stimulate postsynaptic ACh receptors (AChR) on myotubes to induce contraction. This neuromuscular system controls all voluntary movements including essential activities like breathing. Deficits in MNs, skeletal muscles, or NMJs lead to neuromuscular disorders (NMDs) that span a spectrum of diseases such as amyotrophic lateral sclerosis, myopathy, muscular dystrophy, myasthenia gravis, and age-related sarcopenia.
The pathological mechanisms underlying most NMDs remain unclear. Furthermore, there is still no cure for most NMDs. Only two FDA (Food and Drug Administration) approved drugs, Riluzole and Edaravon, are available for ALS treatment; but their clinical efficacy is not satisfactory. Although there are already many in vitro neuromuscular junction models, a novel model specifically designed for studying motor units (MUs) is still needed in the last two decades, novel bioengineering and cellular technologies have advanced the development of in vitro NMJ models. In particular, progress in culture technologies, biomaterials, and microfluidic devices has facilitated the advancement from two-dimensional cultures to multicellular three-dimensional (3D) model systems. The MNs and skeletal muscles used in these previous in vitro models were typically obtained from dissociated explants, primary cells, commercial cell lines, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs), etc.
For example, a “NMJ chip” assembled within a microfluidic device was established using human iPSCs for drug screening and precision medicine application in neuromuscular diseases. This procedure takes about 2-3 months to complete and requires advanced equipment. A NMJ model with cultured 3D skeletal muscle tissues was designed to study adult human NMJ development and neuromuscular diseases. A 3D human trunk neuromuscular organoids system was established and shown to exhibit functional NMJ features.
However, these systems were established mostly for certain specific objectives and are relatively time-and effort-intensive. A more generalized, functional NMJ model that can be used for a wide spectrum of applications is still needed.
For the above mentioned purpose, the present disclosure provides a motor unit chip, comprising: a first hydrogel having parallel micro grooves on a surface; a muscle cell disposed in the parallel micro grooves; a second hydrogel disposed on the first hydrogel and covering the muscle cell; and a motor neuron plated on top of the second hydrogel.
Preferably, the first hydrogel comprises Gelatin.
Preferably, the thickness of the first hydrogel is from 10 μm to 4 mm.
Preferably, the second hydrogel comprises Matrigel and/or basement membrane-like matrix.
Preferably, the thickness of the second hydrogel is from 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 fiber formed from Schwann cell.
Preferably, the muscle cell, Schwann cell and the motor neuron are derived from rodent.
Preferably, the muscle cell, Schwann cell and the motor neuron are derived from primate.
For another purpose, the present disclosure provides a method for manufacturing the neuromuscular junction chip, comprising: preparing the first hydrogel with forming the parallel micro grooves on the surface; seeding a myoblast onto the first hydrogel; differentiating the myoblast to the muscle cell; loading the second hydrogel with Schwann cell onto the first hydrogel; and seeding and tiling the motor neuron on the second hydrogel with a motor neuron differentiation medium.
Preferably, the myoblast comprises C2C12 cell and stem cell derived myoblasts.
Preferably, the motor neuron comprises MN1 cell and stem cell derived motor neurons.
Preferably, the Schwann cell comprises IMS32 cell and stem cell derived Schwann cell.
Preferably, the myoblast is differentiated for 4 to 60 days.
Preferably, the Schwann cell is pre-cultured for 6 to 60 days.
Preferably, the motor neuron is cultured for 6 to 60 days before loading into the second hydrogel.
For still another purpose, the present disclosure provides a kit for manufacturing a neuromuscular junction chip, comprising: a myotube differentiation medium; first hydrogel having parallel micro grooves on a surface; a second hydrogel; a motor neuron differentiation medium; and a neuromuscular maturation medium; wherein the first hydrogel comprises gelatin; wherein the second hydrogel comprises Matrigel.
Preferably, the kit further comprises myoblast, motor neuron precursor cell and Schwann cell.
On the other hand, the present disclosure further provides a method of detecting pathological event using the above-mentioned motor unit chip, comprising: preparing the motor unit chip; and inducing the pathological event in the motor unit chip; wherein the pathological event comprises amyotrophic lateral sclerosis, myasthenia gravis, and peripheral neuropathies.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
For the descriptions herein and the appended claims, the singular forms “a”, and “an” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a protein” includes more than one protein, and reference to “a compound” refers to more than one compound. The use of “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.”
Where a range of values is provided, unless the context clearly dictates otherwise, it is understood that each intervening integer of the value, and each tenth of each intervening integer of the value, unless the context clearly dictates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding (i) either or (ii) both of those included limits are also included in 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 documents referenced in this disclosure are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference herein for all purposes.
It is to be understood that both the foregoing general description, including the drawings, and the following detailed description are exemplary and explanatory only and are not restrictive of this disclosure.
In one embodiment, the present disclosure provides a motor unit chip, comprising: a first hydrogel having parallel micro grooves on a surface; a muscle cell disposed in the parallel micro grooves; a second hydrogel disposed on the first hydrogel and covering the muscle cell; and a motor neuron plated on top of the second hydrogel. Since the muscle cells are disposed in the grooves, the muscles can grow in the form of muscle fibers without clumping together, thus better simulating real muscle cells.
In a particular embodiment, the first hydrogel may preferably comprise Gelatin, preferably, the first hydrogel may further comprises a prepolymer solution containing acrylamide (3%), bis-acrylamide (0.4%), and 2-hydroxy-4′-(2-hydroxyethoxy)-2-meth-ylpropiophenone (0.5%). The second hydrogel may comprise Matrigel, preferably, the second hydrogel may further comprise alginate and/or basement membrane-like matrix. Particularly, 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 with phenol red (serving as pH indicator), 0.5 N NaOH and water for cell culture in order to yield a collagen concentration of 2 mg/ml at a pH of 7.4.
In one embodiment, the thickness of first hydrogel may be from 10 μm to 4 mm, preferably 10 to 40 μm, and the thickness of second hydrogel may be from 1 μm to 10 mm, preferably 10 to 80 μm. In a particular embodiment, the width of the parallel micro grooves may be 2 to 100 μm, preferably 10 μm, and/or the depth of the parallel micro grooves may be 2 to 40 μm, preferably 10 μm.
In one embodiment, the muscle cell, Schwann cell and the motor neuron may be derived from rodent, primate or any other species needed.
In one embodiment, the present disclosure provides a method for manufacturing the motor unit chip, comprising: preparing the first hydrogel with forming the parallel micro grooves on the surface; seeding a myoblast onto the first hydrogel; differentiating the myoblast to the muscle cell; loading the second hydrogel with Schwann cell onto the first hydrogel; and seeding and tiling the motor neuron on the second hydrogel with a motor neuron differentiation medium. In a particular embodiment, the myoblast may be C2C12, myoblast cell lines or stem cell derived myoblasts. In a particular embodiment, the motor neuron may be MN1, motor neuron cell lines or stem cell derived motor neurons. In a particular embodiment, the Schwann cell may be IMS32, Schwann cell lines or stem cell derived Schwann cells.
In one embodiment, the myoblast may be differentiated for 4 to 60 days, preferably, 4 to 8 days. In one embodiment, the Schwann cell may be pre-cultured for 6 to 60 days before loading into the second hydrogel, preferably, may be pre-cultured for 6 to 10 days. In one embodiment, the motor neuron may be cultured for 6 to 60 days, preferably, may be cultured for 6 to 10 days.
In one embodiment, the present disclosure provides a kit for manufacturing a motor unit chip, comprising: a myotube differentiation medium; a first hydrogel having parallel micro grooves on a surface; a second hydrogel; a motor neuron differentiation medium; and a neuromuscular maturation medium; wherein the first hydrogel comprises gelatin; wherein the second hydrogel comprises Matrigel. In a particular embodiment, the kit may further comprises myoblast, motor neuron precursor cell and Schwann cell. Preferably, the first hydrogel may further comprise a prepolymer solution containing acrylamide (3%), bis-acrylamide (0.4%), and 2-hydroxy-4′-(2-hydroxyethoxy)-2-meth-ylpropiophenone (0.5%), 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 with phenol red (serving as pH indicator), 0.5 N NaOH and water for cell culture in order to yield a collagen concentration of 2 mg/ml at a pH of 7.4.
In one embodiment, the present disclosure provides a method of detecting pathological event using the motor unit chip. The method comprises the following steps: preparing the motor unit chip; and inducing the pathological event in the motor unit chip; wherein the pathological event may comprise amyotrophic lateral sclerosis, myasthenia gravis, and peripheral neuropathies. The cell line of the muscle cell, motor neuron or the Schwann cell of the motor unit chip may be different according to the desired pathological event. In one embodiment, particular medicine may be added into the motor unit ship to detect the reaction thereof. The present disclosure is not limited to this, chemical or physical control may also be performed based on the needs, such as chemotherapy-induced peripheral neuropathy and drug screening for amyotrophic lateral sclerosis, peripheral neuropathies, myopathies, and sarcopenia.
Hereinafter, the present disclosure will be explained with embodiments with drawings.
The mouse C2C12 myoblast cell line was purchase from ATCC (Cat. no. CRL-1772). The mouse motor neuron cell line MN1 were kindly provided by Ahmet Hoke (The Johns Hopkins University, Baltimore, MD). The mouse Schwann cell line IMS32 were purchased from CosmoBio Co., LTD (Cat no. PMC-SWN-IMS32C). The three Cell lines were maintained in high glucose Dulbecco's Modified Eagle Medium (DMEM) with 2 mM L-glutamine (Thermo Fisher Scientific, cat. no. 11965-092) supplemented with 10% FBS (ALTANTA Biologics, cat. no. S11150), 100 U/mL penicillin and 100 mg/mL streptomycin (Thermo Fisher Scientific, cat. no. 15140-122). Cells were maintained at 37° C. in a humidified 5% CO2 cell culture incubator. The C2C12 Cells were sub-cultured using 0.25% trypsin (Thermo Fisher Scientific, cat. no. 25200-056) upon reaching approximately 70% confluence. The MN1 cells were sub-cultured using 0.05% trypsin (Thermo Fisher Scientific, cat. no. 25200-054) upon reaching approximately 90% confluence. The IMS32 cells were sub-cultured using 5 mM EDTA (Thermo Fisher Scientific, cat. no. 15575-020) upon reaching approximately 70-80% confluence.
The procedure for manufacturing the motor unit chip is illustrated in
PDMS stamps were fabricated as described elsewhere. Briefly, the silicon wafer mold was treated with trichloromethyl silane (Millipore Sigma, cat. no. 75-79-6) vapor for 30 mins as a releasing agent. PDMS mixture (PDMS: curing agent=10:1, Sylgard 184, Dow Corning) was poured onto the master mold and cured for 2 h. at 60° C. The cured PDMS was excised from the master mold and trimmed with a clean razor blade to generate the stamp. The PDMS stamp was then cleaned with oxygen plasma (irradiation intensity: 200 W) for 10 mins.
Under sterile conditions, prepare a 10% solution of gelatin A (Sigma, cat. no. G1890-100 g) was prepared in sterile ddH2O. The solution was placed on a rotating rack at 65° C. for 10 mins or until thoroughly dissolved, then removed from rotation to allow bubbles to rise out of the solution for 10 mins. A 65° C. hotplate (Apollo; Digital Heating and Chilling plate) was used to create the μ-molded hydrogel and adhere it to the cover glass (Fisher Scientific, cat. no. 1254580), so μ-mold PDMS stamps, gelatin, and coverslip glass were warmed to 65° C. before molding. 18 μl of gelatin solution was carefully pipetted directly onto the stamp, then quickly place the cover glass on top of the gelatin droplet ensuring full coverage of the glass. The stamps were removed from the hotplate and the entire sandwich was allowed to cool at room temperature for 30 mins. Under sterile conditions, the cover glass and hydrogel were carefully peeled off from the stamp using forceps and placed in a 24-well plate. The hydrogels were exposed to UV for 5 mins. The gelatin hydrogel was cross-linked with a 5% transglutaminase solution for 18 h. 1 g of powdered transglutaminase was mixed with 19 ml of autoclaved ddH2O under sterile conditions. The enzyme was placed on a rotating rack at 50° C. for 10 mins to dissolve and allowed to cool at room temperature before filtration. The transglutaminase solution was filtered through a 0.2 μm filter. After hydrogels have had 5 mins under UV, they were ready for the addition of transglutaminase. Leave the 24-well plate covered in the hood at room temperature for 18 h. After 18 h, transglutaminase was suctioned out. The hydrogel was then rinsed three times by adding autoclaved ddH2O to the well. On the last rinse, the 24-well was placed under UV uncovered for 5 mins. Following UV exposure, the 24-well plate can be covered, placed in a sterile bag, and stored at 4° C. until use. The depth and the width may be different according to the needs, in this embodiment, the depth is 10 μm, and the width is 10 μm.
MN1 Transfection
Plasmid CLYBL-(Ef1a-SBP-LNGFR-T2A-mApple) (CAG-rtTA)-(TRE-hNIL)) was purchased from Addgene (cat. no. 05842). MN1 cells transfection was performed by Lipofectamine 3000 (Thermo Fisher Scientific, cat. no. L3000-015) following the manufacturer's instruction. MN1 cells were added to the motor unit chip 1 day after transfection.
Cell Culture in the Motor Unit Chip
On day 0, 2×104 C2C12 cells/cm2 were seeded on hydrogels in 24-well plates and cultured in growth medium. Once confluent (about 2 day), myotube differentiation medium (DMEM with 2% horse serum (Thermo Fisher Scientific, cat. no. 5H30074.2), 100 U/mL penicillin, 100 mg/mL streptomycin and 2 mM L-glutamine) was added. C2C12 were differentiated for 4 days and the myotube differentiation medium was replenished every 2 days. On day 6, Schwann cell line IMS32 was trypsinized by 0.05% trypsin and re-suspended in cold myotube differentiation medium in the density of 4×104 cells/ml. Schwann cells were mixed with Matrigel in a 1-to-1 ratio and then kept on ice. Matrigel Matrix (Corning, cat. no. 356255) was thaw in ice in cold room over night before use. The medium in the motor unit chip was removed and 18 μl of the mixture was added on the center of the hydrogel. The motor unit chip was kept in 37° C. incubator for 3 mins and added with the fresh myotube differentiation medium. On day 7, the transfected 1×103 MN1 cells/cm2 were added to each well of 24-well, on top of the differentiated C2C12 cells. MN1 cells were previously transfected with either empty vector control or GFP-Crabp1 prior to seeding onto C2C12 cells. The MN1 cells were spread and plated on the Matrigel Matrix for separating them to obtain vertical structure of motor unit. The co-culture was maintained in MN differentiation medium 1 (DMEM media with 100 U/mL penicillin, 100 mg/mL streptomycin, 2 mM L-glutamine, 1% MEM non-essential amino acids (NEAA; Thermo Fisher Scientific, cat. no. 11140-050), 2 μg/ml doxycycline (Sigma, cat. no. D9891) and 10 um Y27632 Dihydrochloride (R&D, cat no. 1254/1) for 4 days, then changed to MN differentiation medium 2 (MN differentiation medium 1 without Y27632 Dihydrochloride). The MN differentiation mediums was replenished every two days.
Immunostaining
For Immunostaining, the tri-culture slides were fixed in 100% methanol for 5 mins at −20° C. After three times of 5 mins 1XPBS wash, 2% BSA was added for 1 h at room temperature for blocking. Primary antibodies, α-actinin (1:100; Cell Signaling, cat. no. 6487S), Tuj1 (1:1000; R and D, cat. no. MAB 1196) or myelin basic protein MBP (1:500; R and D, Cat. No. MAB42282) in 1XPBS were incubated overnight at 4° C. After three times of 5 mins 1XPBS wash, fluorescent secondary antibodies (1:1000), and μBTX conjugated to a fluorescent dye (1:400; Biotium, Cat No. 00005) were incubated for 2 h in dark at room temperature. The DAPI (1:1000; Thermal Fisher Scientific, Cat. No. 62248) in 1×PBS were incubated for 10 mins in dark at room temperature. The slide then was washed three times of 5 mins 1xPBS. Z-stack images were captured on Olympus Fluoview FV1000 BX2 Upright Confocal.
Tri-cultures on 12.5 mm glass coverslips were briefly washed in HEPES Hanks' Buffer Solution (HHBS) (5.6 mM glucose, 20 mM Hepes, 3 mM NaHCO3, 0.3 mM Na2HPO4, 0.4 mM KH2PO4, 137 mM NaCl, 5.0 mM KCl, 4.9 mM MgCl2, 4.1 mM MgSO4, 1.26 mM CaCl2, pH=7.4) and then the coverslip was incubated with fura-2 AM in 0.04% pluronic acid dissolved in HHBS to a final concentration of 20 μM for 30 mins at 37° C. After loading indicator, coverslips were washed in fresh HBSS for 10 mins at 37° C. and then transferred to an imaging chamber for microscopy experiments. Fura-2 imaging was performed on an Olympus IX71 microscope using a 20× objective lens. Excitation wavelength was selected with a galvanometer-driven monochromator (8 nm slit width) coupled to a 75 W xenon arc lamp (Optoscan; Cairn Research). Intracellular Ca2+ concentration ([Ca2+]i) was monitored by sequential excitation (1 Hz) of fura-2 at 340 and 380 nm. A single image was acquired at 610 nm to identify mApple expressing MNs in the field. Fluorescent images were captured using a cooled charge-coupled device camera (Cascade 512B; Roper Scientific) and Metafluor acquisition software (Molecular Devices). All fura-2 experiments were performed in HBSS buffer at room temperature. To depolarize MNs the extracellular K+ concentration was increased to 50 mM with K+ exchanged reciprocally for Na+in HHBS. For myotube stimulation 10 μM ACh was applied by bath perfusion. A 60 s baseline in HHBS was acquired before stimulation of each coverslip. High K+ was applied for 30 s and ACh for 1 min. After stimulus application, a washout of 60 s was performed. Background images were acquired after each experiment; the coverslip was wiped clean, then 20 frames were acquired using the same parameters as above.
Images were processed using MetaFluor Analyst to draw regions of interest (ROIs), subtract background, and generate 340/380 ratios. MN ROIs were selected based on mApple expression and further verified by morphology by a blinded expert. Differentiated MNs each has a cell body and a single enlarged axon terminal. Myotube ROIs were selected based on morphological criteria; myotubes exhibit a distinct elongated, skeletal muscle fiber like morphology. Given that myotubes can span the total length of the field, three ROIs for each individual myotube were drawn and averaged to represent a single cell. Peak amplitude analysis was performed by comparing the average baseline [Ca2+]i to the maximum reached after stimulation. Only responding cells were included in peak amplitude analysis. A response was defined by the following criteria: (1) a signal intensity of at least 450 (16-bit scale) after background subtraction, and (2) a peak [Ca2+]i at least two standard deviations (SD) above baseline (60 s average). Criterion “1” was empirically determined to exclude cells with poor signal to noise ratio. [Ca2+]i calculations were performed in Microsoft Excel by averaging 340/380 intensity ratios for MNs or myotubes in the field that responded to stimulation.
Data were analyzed using the Student's t test. Statistical analyses were 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 as indicated. For statistical analyses of calcium image, MN and myotube data were assessed for normality using the D′Agostino-Pearson normality test. Myotube data exhibited normality, whereas MN did not. Therefore, myotube data was subjected to pair Student's t-test and MN data were subjected to the non-parametric Wilcoxon signed-rank test. For each experiment “n” was defined by a single microscopic field on a single coverslip. Percent responders was calculated using number of responders/total MNs or myotubes in the field.
The results of the above-mentioned experiments are described in the following particular embodiments.
The motor unit chip is specifically designed for modeling the MUs (
The entire procedure is depicted in
On day 6, most of the myoblasts began to differentiate. Schwann cells (IMS32) were mixed with Matrigel and added on top of differentiating myoblasts. Also on day 6, in a separate culture dish, MN1 cells were transfected with an expression vector (Plasmid #105842; Addgene) expressing mApple marker and doxycycline-inducible factors Neurogenin 2 (NGN2), ISL LIM Homeobox 1 (ISL1), and LIM Homeobox 3 (LHX3). Growth factors and transcription factors encoded in this plasmid facilitated MN differentiation, resulting in a more uniform MN morphology. Without this plasmid, control MN1-derived neurons varied greatly in morphology as shown in
In this bilayer motor unit chip, the Matrigel layer is a critical element contributing to MN differentiation and myelination. To demonstrate the effect of Matrigel, an experiment was carried out as described in
The μ-molded hydrogel is critical for myoblast differentiation and alignment. To demonstrate the effects of μ-molded hydrogel, C2C12 cells were seeded into an untreated 24-well plate (uncoated Control), a hydrogel-coated plate, or a μ-molded hydrogel coated plate as described in
As shown in
To further evaluate the organizational features of mature myotubes in the μ-molded hydrogel group, we labeled the 3D culture with an antibody against α-actinin (
In addition to characterizing the morphological features and biomarkers of the 3D culture, we also evaluated the physiological function of this motor unit chip. NMJs were studied using a neural marker Tuj1 (Red) to label the pre-synaptic MNs, and an AChR marker αBTX (green) to label the post-synaptic compartments. As shown in
Together, these results show that this motor unit chip provides an in vitro model for studying NMJ development and formation. To further examine the functionality of NMJs in this motor unit chip, we performed two functional assays: contractility, and calcium imaging. The following section describes contractility of functional MUs.
To further illustrate MN-driven myotube contraction, we seeded mApple-marked MNs in an ultra-low density (2×102 cells/cm2) on day 7.
We next demonstrated the physiological function of two key components of the MUs, MNs and myotubes, using a conventional calcium imaging method to probe the functionality of neurons (including MNs) and muscles (myotubes in MUs). Physiologically, MNs and myotubes respond to stimuli that evoke neuronal firing and muscle contraction both with increases in the intracellular calcium concentration ([Ca2+]i). Therefore, calcium imaging provides an appropriate tool to study the functions of MNs and myotubes. We employed a fluorescent, ratiometric calcium indicator, fura-2AM, to detect changes in [Ca2+]i following exposure to a depolarizing stimulus (for MNs) or neurotransmitter acetylcholine (ACh) (for myotubes). Changes in [Ca2+]i are represented by changes in the 340 nm/380 nm ratio. It appeared that depolarization with 50 mM extracellular K+ or treatment with 10 μM ACh evoked measurable changes in [Ca2+]i in MNs and myotubes, respectively. As expected, the Schwann cells did not show any changes in [Ca2+]i under these treatments, indicating that the added Schwann cells in the tri-culture did not exhibit inappropriate response. This further supports the specificity of this improved tri-culture model with respect to physiologically relevance and the specific functional response of each component.
Furthermore, in at least two independent experiments myotubes also exhibited physical contraction when stimulated with ACh (
This motor unit chip is specifically designed for modeling MUs that can be used to study a wide spectrum of biological problems associated with NMD and in pharmacological studies such as drug screening. These experiments demonstrate the benefits of the bilayer hydrogel design. This design exploits an upper layer, Matrigel layer, that contributes to MN differentiation and timely myelination, and a lower layer, μ-molded hydrogel, that contributes to myotube differentiation, alignment and innervation. The feature of this MU model is demonstrated according to multiple criteria including morphological characteristics, essential biomarkers of NMJ, and MU function such as contractility and calcium signaling. There are several positive features of this new model. First, this model contains the three cell types of a MU, including MNs, Schwann cells, and myotubes, and all three cell types are well differentiated and functional. Secondly, the bilayer μ-molded hydrogel/Matrigel provides a critically needed micro-environment mimicking the physiological condition of a developing MU. Specifically, MNs and muscle cells are separated initially, and then make contact for co-differentiation and synapse formation only when MN axons are myelinated by Schwann cells and penetrate to the muscle layer where myotubes are maturing. At this stage, MN axons innervate myotubes to form NMJs. For this to happen, Matrigel provides an environment resembling the extracellular matrix that separates MNs from muscle cells. This layer embeds Schwann cells for axon myelination when MN axons extend through this layer. Further, μ-molded hydrogel forms a surface area facilitating differentiation and the alignment of myotubes, which is critical to the formation of organized myotubes. A third advantage of this motor unit chip is that multiple parameters of individual MUs can be examined and quantified, such as axon length and the frequency of myotube contraction. Moreover, axon myelination, mature myotube, NMJ formation can all be assessed by immunostaining. These could be useful in studying pathological conditions associated with various NMDs, such as axonal regression, demyelination, cell death, muscle atrophy, and degradation. Importantly, Ca2+ imaging can be used to monitor the biological function/property of individual MNs and myotubes. Finally, by manipulating MNs or myoblasts, it is possible to study fundamental questions related to MUs and to model NMDs in vitro. In our previous study, we employed a simpler (without Schwann cells and Matrigel) in vitro MU model containing only MNs and myotubes on μ-molded hydrogel to study NMJ formation. Using this motor unit chip, we have successfully demonstrated that the expression level of a retinoic acid signaling protein, Cellular Retinoic Acid Binding Protein 1 (CRABP1) in MNs affected the length of axons and AChR cluster formation. The requirement for CRABP1 in MU functionality in vivo was validated in Crabp1 knockout mice and confirmed using gene rescue experiments. Therefore, this motor unit chip can be a powerful tool to efficiently identify molecular players and dissect signaling pathways critical to formation and function of MUs or NMJs. For a practical consideration (time and effort), this motor unit chip provides a reliable and efficient in vitro MU model for numerous studies including physiological and pharmacological investigations.
The formation of functional NMJs in the motor unit chip is also supported by data shown in our previous study where a similar, but simpler (only two cell types, MN and C2C12), version of co-culture system was reported. This earlier study has detected the expression of pre-synaptic vesicle protein SV2 and postsynaptic AChR in NMJs. The expression of synaptic vesicle glycoprotein-2 would indicate the ability of MNs to release neurotransmitter from the axon terminals. To improve the physiological relevance of this earlier model, in this current study, we added the Matrigel layer embedded with a third cell type important for NMJ, i.e. Schwann cells. This has apparently improved this model as demonstrated in multiple measurements including extended axon length, myelinated axons, and axon terminals. In the NMJ of this tri-culture model (
One purpose of using cell lines to generate this MU model is for the sake of consistency, ease in culture procedure and timesaving. It remains to be determined whether appropriate human cell lines, or stem cell systems, such as iPSC differentiated MNs or muscle cells, can be similarly optimized for this system. Secondly, in Ca2+ imaging experiments, due to the thickness of Matrigel layer, it will be challenging to collect clear signals from both MNs and myotubes at the same time because the cells reside in different layers (
It is worthy to note the well differentiated and organized mature myotubes in this motor unit chip. The mature myotubes are similar to skeletal muscle fibers, including their morphology, similar width, length and a cylindrical, aligned, striated, and unbranched shape. The μ-molded hydrogel contributes to the alignment of myotubes and the Matrigel may provide the extracellular matrix-like microenvironment to improve myotube maturation. Additionally, the Matrigel also protects the contracting myotubes, preventing them from detachment in a long-term culture.
We report a procedure to engineer a structurally organized motor unit chip that can be reliably generated within a reasonably short period of time. This MU model is validated with regards to morphology, essential biomarkers and the function of a MU including contractility and calcium signaling. Three key advantages are noted in this new model. First, the entire MU (single MN) can be examined, and subcellular/functional details can be studied. Secondly, the further included Schwann cells allows proper myelination to occur, enabling detailed studies of MN regulation with regards to NMJ development/maturation and maintenance. Thirdly, using this motor unit chip, it is possible to generate various in vitro disease models by manipulating each relevant cell type. Thus, this new motor unit chip is appropriate not only for basic research, but also for disease modeling and drug screening.
The description is presented to enable one of ordinary skill in the art to make and use the invention. Various modifications to the described embodiments are readily apparent to those persons skilled in the art and the generic principles herein can be applied to other embodiments. Thus, the present invention is not intended to be limited to the embodiments shown but is to be accorded the widest scope consistent with the principles and features described herein. It is readily apparent to one skilled in the art that other modifications can be made to the embodiments without departing from the spirit and scope of the invention as defined by the appended claims.
Particularly, the entire disclosure of following non-patent literatures is hereby incorporated by reference.
This application claims the priority of U.S. Provisional Patent Applications 63/615,352 filed on Dec. 28, 2023. The entirety of the aforementioned application is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63615352 | Dec 2023 | US |
