Apparatus and method for creating an in vitro model of the neuromuscular junction

Abstract

A cell culture device and associated method for preparing an in vitro model of a neuromuscular junction (NMJ) is provided. The cell culture device has a cell culture layer (22) including a neuronal channel for receiving neuronal cells therein and a neuromuscular junction chamber in fluid communication with the neuronal channel for receiving muscle cells therein to allow co-culture of neuronal and muscle cells and form an NMJ. The neuronal channel includes a first portion having a first partial cross-section and a second portion downstream of the first portion for holding neuronal cells and having a second partial cross-section smaller than the first partial cross-section. The neuromuscular junction chamber can optionally include a support for culturing muscle cells therebetween. The in vitro model can be used, for example, to analyze biological material from a patient or to diagnose a condition.

Description

[Technical field]

[0001] The technical field relates generally to cell culture techniques, and more particularly to systems, devices and methods for preparing in vitro models of the neuromuscular junction. [Background technology]

[0002] The neuromuscular junction (NMJ) is the synaptic connection between the end of a motor nerve and a muscle, e.g., skeletal, cardiac, or smooth muscle. The NMJ is the site of transmission of action potentials from nerve to muscle. The NMJ is also a susceptible site for many diseases and the site of action of various pharmacological agents. The NMJ governs muscle contraction from the central nervous system to the muscle fiber, acting as an interface that converts action potentials from the presynaptic motor neuron into contraction of the postsynaptic muscle fiber to enable muscle contraction. More specifically, when an action potential reaches the presynaptic motor neuron and voltage-gated calcium channels are activated, calcium flows into the neuron, triggering the diffusion of acetylcholine (ACh) across the synaptic cleft to acetylcholine receptors (AChRs) on the postsynaptic muscle fiber, opening cation channels and resulting in depolarization of the muscle fiber.

[0003] The NMJ is affected in the early stages of many neurodegenerative and neuroimmune diseases. The NMJ is also the target of several toxicants, toxins and neuropharmaceuticals. Therefore, designing appropriate experimental NMJ models is crucial not only to generate efficient and reliable approaches to study NMJ development and function, but also to create conditions that recapitulate distinct features of the disease.

[0004] However, efforts to date have proven difficult to systematically study and manipulate the NMJ in living subjects. Animal models have been used to study the NMJ to date, but there are differences in human synapses compared to synapses in other mammals that may affect the transferability of results from animal models to humans. Additionally, in vitro models of the NMJ, both mammalian and non-mammalian, have been developed in an attempt to mimic the in vivo environment for culturing muscle cells and motor neurons, but these current models also have various limitations. Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, there is a need to improve in vitro models of the NMJ, which have been developed using both healthy and diseased cells, to enable mechanistic studies and drug development. [Means for solving the problem]

[0006] According to one embodiment, a cell culture device for preparing an in vitro model of a neuromuscular junction is provided, the cell culture device comprising a cell culture layer having a nerve cell inlet configured to receive nerve cells therein, first and second muscle cell reservoirs, at least one of the first and second muscle cell reservoirs configured to have muscle cells seeded therein, a neuromuscular junction chamber extending between and in fluid communication with the first and second muscle cell reservoirs to allow co-culture of nerve cells and muscle cells to form a neuromuscular junction, and a nerve cell channel extending between and in fluid communication with the nerve cell inlet and the neuromuscular junction chamber, the nerve cell channel including a first portion having a first partial cross-section sized to hold a nerve cell body of a nerve cell and a second portion downstream of the first portion, the second portion having a second partial cross-section smaller than the first partial cross-section and sized to block entry into the nerve cell body.

[0007] In some embodiments, the first portion consists of a first partial side wall, a first partial front wall, and a first partial rear wall, and the second portion consists of a second partial side wall, a second partial front wall, and a second partial rear wall.

[0008] In some embodiments, the transition of the sidewall from the first portion sidewall to the second portion sidewall is substantially straight such that the neuronal channel has substantially the same channel width between the first portion and the second portion.

[0009] In some embodiments, the first portion sidewall and the second portion sidewall are provided such that the neuronal channel has a smaller channel width in the second portion compared to the first portion.

[0010] In some embodiments, the cell culture device further comprises a cell culture dish, cell culture plate or microscope slide having a cell culture layer receiving surface on which the cell culture layer is deposited.

[0011] In some embodiments, the first partial back wall is provided by a cell culture layer receiving surface of a cell culture dish, cell culture plate, or microscope slide.

[0012] In some embodiments, the first partial back wall and the second partial back wall are provided by a cell culture layer receiving surface of a cell culture dish, a cell culture plate, or a microscope slide.

[0013] In some embodiments, the second anterior wall is provided inwardly from the first anterior wall such that the neuronal channel has a smaller channel height in the second portion compared to the first portion, thereby at least partially providing a second partial cross-section that is smaller compared to the first partial cross-section.

[0014] In some embodiments, the second posterior wall is provided inwardly from the first posterior wall such that the neuronal channel has a smaller channel height in the second portion compared to the first portion, thereby providing a second partial cross-section that is smaller compared to the first partial cross-section.

[0015] In some embodiments, at least one of the second anterior wall and the second posterior wall is disposed inwardly from the first anterior wall and the first posterior wall, respectively, and the neuronal channel has a smaller channel height in the second portion compared to the first portion, thereby providing a smaller cross-section of the second portion compared to the cross-section of the first portion.

[0016] In some embodiments, the height of the channel varies from the first portion to the second portion according to a ratio ranging from about 2 to about 12.

[0017] In some embodiments, the height of the channel varies from the first portion to the second portion according to a ratio ranging from about 3 to about 9.

[0018] In some embodiments, the first portion of the neuronal channel consists of an inwardly converging frustoconical converging portion.

[0019] In some embodiments, the first portion of the neuronal channel consists of an inwardly converging truncated pyramidal converging portion.

[0020] In some embodiments, the transition from the first partial anterior wall to the second partial anterior wall comprises a gradual change that defines an abutting wall that extends laterally across at least a portion of the neuronal channel.

[0021] In some embodiments, the abutting walls converge inwardly toward the centerline of the neuronal channel.

[0022] In some embodiments, the abutment wall includes a curvature.

[0023] In some embodiments, the adjacent walls are substantially flat.

[0024] In some embodiments, the abutment wall comprises a plurality of inwardly converging planar surfaces.

[0025] In some embodiments, the cell culture device further comprises a first support post disposed in the neuromuscular junction chamber adjacent to the first muscle cell reservoir and a second support post disposed in the neuromuscular junction chamber adjacent to the second muscle cell reservoir, the first and second supports extending upwardly and serving as respective anchoring positions for the muscle cells.

[0026] In some embodiments, the cell culture device further comprises a gel seeding inlet in fluid communication with the neuromuscular junction chamber and opposite the neuronal cell inlet, the gel seeding inlet configured to seed a gel into the neuromuscular junction chamber.

[0027] In some embodiments, the neuronal cells are provided as a cluster of neuronal cell bodies and include axons extending away from the cell bodies, and a first portion of the neuronal channel is configured to receive the cluster of cell bodies and a second portion of the neuronal channel is configured to direct the axons to the neuromuscular junction chamber.

[0028] In some embodiments, the second portion of the neuronal channel comprises microchannels for directing axonal growth.

[0029] In some embodiments, the neural cells are provided as neurospheres.

[0030] In some embodiments, the neural cells are provided as neuroorganoids.

[0031] In some embodiments, the neural cells comprise motor neurons.

[0032] In one embodiment, at least one of the neuron inlet, the first muscle cell reservoir, and the second muscle cell reservoir is configured to receive a test substance therein.

[0033] In some embodiments, the cell culture device further comprises an electrode disposed adjacent the cell culture layer.

[0034] In some embodiments, the electrode forms part of an electrode layer.

[0035] In some embodiments, the electrode layer underlies or overlaps the cell culture layer.

[0036] In some embodiments, the electrode layer is integrated with the cell culture layer.

[0037] In some embodiments, the electrode comprises a plurality of electrodes.

[0038] In some embodiments, the electrodes are comprised of at least one of a metal electrode, a metal oxide electrode, a carbon electrode, a multi-electrode array, and a field effect transistor detector.

[0039] In some embodiments, the electrodes are configured to stimulate neurons.

[0040] In some embodiments, the electrodes are configured to stimulate muscle cells.

[0041] In some embodiments, the electrodes are configured to at least one of collect, record, measure, and detect a neuronal response to the stimulus.

[0042] In some embodiments, the electrodes are configured to at least one of collect, record, measure, and detect a response of muscle cells to a stimulus.

[0043] In some embodiments, the cell culture device further comprises an electronic device in ohmic contact with the electrodes.

[0044] In some embodiments, the cell culture device further comprises a sensor configured to stimulate the neuronal cells, measure a response from the neuronal cells to the stimulation, provide an output or receive an input.

[0045] In some embodiments, the cell culture device further comprises a sensor configured to stimulate muscle cells and measure a response from the neural cells to the stimulation, provide an output or receive an input.

[0046] In some embodiments, the sensor comprises an optical or electrical transducer.

[0047] In some embodiments, the second portion of the neuronal channel includes a longitudinal support to prevent downstream migration towards the neuromuscular junction chamber.

[0048] In some embodiments, the first portion of the neuron channel includes an hourglass shaped portion to prevent upstream movement toward the neuron inlet.

[0049] In some embodiments, a first portion of the neuronal channel includes a post to block upstream movement toward the neuronal entrance.

[0050] In some embodiments, the cell culture device comprises one or more of the features defined and / or described and / or illustrated herein.

[0051] According to another aspect, a cell culture device for preparing an in vitro model of a neuromuscular junction is provided, the cell culture device comprising: a cell culture layer, the cell culture layer comprising a neuronal channel configured to receive neuronal cells therein, the neuronal channel having a first portion with a first cross-section and a second portion having a second cross-section smaller than the first cross-section and disposed downstream of the first portion; and a neuromuscular junction chamber in fluid communication with the neuronal channel and configured to receive muscle cells therein to enable co-culture of the neuronal cells and form a neuromuscular junction, the neuromuscular junction chamber having first and second struts spaced apart on either side of the neuronal channel for culturing the muscle cells therebetween.

[0052] In some embodiments, the first portion consists of a first partial side wall, a first partial front wall, and a first partial rear wall, and the second portion consists of a second partial side wall, a second partial front wall, and a second partial rear wall.

[0053] In some embodiments, the transition of the sidewall from the first portion sidewall to the second portion sidewall is substantially straight, such that the neuronal channel has substantially the same channel width between the first portion and the second portion.

[0054] In some embodiments, the sidewalls of the first portion and the sidewalls of the second portion are provided such that the neuronal channel has a smaller channel width in the second portion compared to the first portion.

[0055] In some embodiments, the cell culture device further comprises a cell culture dish, cell culture plate or microscope slide having a cell culture layer receiving surface on which the cell culture layer is deposited.

[0056] In some embodiments, the first partial back wall is provided by a cell culture layer receiving surface of a cell culture dish, cell culture plate, or microscope slide.

[0057] In some embodiments, the first partial back wall and the second partial back wall are provided by a cell culture layer receiving surface of a cell culture dish, a cell culture plate, or a microscope slide.

[0058] In some embodiments, the second front wall is disposed inwardly from the first front wall such that the neuronal channel has a smaller channel height in the second portion compared to the first portion, thereby at least partially providing a second partial cross-section that is smaller compared to the first partial cross-section.

[0059] In some embodiments, the second rear wall is disposed inwardly from the first rear wall such that the neuronal channel has a smaller channel height in the second portion compared to the first portion, thereby providing a smaller second partial cross-section compared to the first partial cross-section.

[0060] In some embodiments, at least one of the second anterior wall and the second posterior wall is disposed inwardly from the first anterior wall and the first posterior wall, respectively, and the neuronal channel has a smaller channel height in the second portion compared to the first portion, thereby providing a smaller second partial cross-section compared to the first partial cross-section.

[0061] In some embodiments, the height of the channel varies from the first portion to the second portion according to a ratio ranging from about 2 to about 12.

[0062] In some embodiments, the height of the channel varies from the first portion to the second portion according to a ratio ranging from about 3 to about 9.

[0063] In some embodiments, the first portion of the neuronal channel comprises an inwardly converging, frustoconical converging portion.

[0064] In some embodiments, the first portion of the neuronal channel comprises an inwardly converging, truncated pyramidal converging portion.

[0065] In some embodiments, the transition from the first partial anterior wall to the second partial anterior wall comprises a gradual change that defines an abutting wall that extends laterally across a portion of the neuronal channel.

[0066] In some embodiments, the abutting walls converge inwardly toward the centerline of the neuronal channel.

[0067] In some embodiments, the abutment wall includes a curvature.

[0068] In some embodiments, the abutment wall is substantially flat.

[0069] In some embodiments, the abutment wall includes a plurality of inwardly converging planar surfaces.

[0070] In some embodiments, the cell culture layer further comprises a gel seeding inlet in fluid communication with the neuromuscular junction chamber and opposite the neuronal cell inlet for seeding a gel into the neuromuscular junction chamber.

[0071] In some embodiments, the neurons are provided as a cluster of neuronal cell bodies and axons extending away from the cell bodies, and a first portion of the neuronal channel is configured to receive the cluster of cell bodies and a second portion of the neuronal channel is configured to direct the axons to the neuromuscular junction chamber.

[0072] In some embodiments, the second portion of the neuronal channel includes microchannels for directing axonal growth.

[0073] In some embodiments, the neural cells are provided as neurospheres.

[0074] In some embodiments, the neural cells are provided as neuroorganoids.

[0075] In some embodiments, the neural cells comprise motor neurons.

[0076] In some embodiments, the neuronal inlet is configured to receive a test substance therein such that the test substance reaches the neuromuscular junction chamber.

[0077] In some embodiments, the second portion of the neuronal channel includes a longitudinal support to prevent downstream migration toward the neuromuscular junction chamber.

[0078] In some embodiments, the first portion of the neuron channel comprises an hourglass shape to prevent upstream migration towards the neuron inlet.

[0079] In some embodiments, a first portion of the neuronal channel includes struts to prevent upstream migration toward the neuronal inlet.

[0080] In some embodiments, the cell culture device comprises one or more of the features defined herein and / or described and / or illustrated herein.

[0081] According to another aspect, a method for preparing an in vitro model of a neuromuscular junction is provided, comprising the steps of: providing neurons to a neuronal channel in a cell culture layer, the neuronal channel having a first portion with a first cross-section and a second portion having a second cross-section smaller than the first cross-section and located downstream of the first portion; and providing muscle cells to a neuromuscular junction chamber in the cell culture layer and in fluid communication with the neuronal channel to allow co-culture of the neurons and muscle cells and form a neuromuscular junction.

[0082] In some embodiments, providing the muscle cells to the neuromuscular junction chamber comprises providing the muscle cells suspended in a gel solution.

[0083] In some embodiments, the gel solution comprises an extracellular membrane matrix (ECM) material.

[0084] In some embodiments, the gel solution comprises one or more of Matrigel™, Geltrex™, fibrin, or collagen.

[0085] In some embodiments, the neural cells are provided as a three-dimensional neural assembly of neural cells.

[0086] In some embodiments, the three-dimensional neural cell aggregate comprises at least one of a neurosphere, a spheroid, a neural aggregate, or a neural organoid.

[0087] According to another embodiment, a method is provided for analyzing biological material from a patient using an in vitro model of a neuromuscular junction, the method comprising the steps of: providing neuronal cells to a neuronal channel in a cell culture layer, the neuronal channel having a first portion with a first cross-section and a second portion having a second cross-section smaller than the first cross-section and located downstream of the first portion; providing muscle cells to a neuromuscular junction chamber in the cell culture layer and in fluid communication with the neuronal channel to enable co-culture of the neuronal cells and the muscle cells and form a neuromuscular junction; contacting at least one of the neuronal cells and the muscle cells with biological material from the patient; and measuring a functional parameter associated with the neuromuscular junction.

[0088] In some embodiments, the functional parameters include at least one of contractile force from a muscle cell, contractile duration of a muscle cell, neuronal cell viability, muscle cell viability, neuronal cell viability, muscle cell viability, and an electrical signal from a neuronal or muscle cell.

[0089] In some embodiments, the method further comprises adding a test substance to at least one of the neuronal channel and the neuromuscular junction chamber, and determining a functional parameter related to the neuromuscular junction is performed following addition of the test substance.

[0090] In some embodiments, the test substance comprises at least one of a biological material and a chemical material.

[0091] In some embodiments, the biological material comprises at least one of an antibody, an antibody fragment, an antigen, an antigen fragment, a toxin, an enzyme, a DNA fragment, an RNA fragment, a virus, and a bacterium.

[0092] In some embodiments, the chemical material includes at least one of a drug and a chemical compound.

[0093] According to another embodiment, a method for analyzing a neuromuscular junction is provided, comprising the steps of: providing a neuronal cell channel in a cell culture layer, the neuronal cell channel having a first portion with a first partial cross-section and a second portion with a second partial cross-section smaller than the first partial cross-section and downstream of the first portion; providing a muscle cell in a neuromuscular junction chamber in the cell culture layer and in fluid communication with the neuronal cell channel to enable co-culture of the neuronal cell and the muscle cell and form a neuromuscular junction; contacting at least one of the neuronal cell and the muscle cell with a test substance; and measuring a functional parameter associated with the neuromuscular junction.

[0094] In some embodiments, the functional parameters include at least one of contractile force from a muscle cell, contractile duration of a muscle cell, neuronal cell viability, muscle cell survival, neuronal cell viability, muscle cell survival rate, and an electrical signal from a neuronal or muscle cell.

[0095] In some embodiments, the test substance comprises at least one of a biological material and a chemical material.

[0096] In some embodiments, the biological material comprises at least one of a biological fluid or biological tissue from a patient, an antibody, an antibody fragment, an antigen, an antigen fragment, a toxin, an enzyme, a DNA fragment, an RNA fragment, a virus, and a bacteria.

[0097] In some embodiments, the chemical material includes at least one of a drug and a chemical compound.

[0098] According to another aspect, a method of diagnosing a patient condition using an in vitro model of a neuromuscular junction is provided, comprising the steps of: providing biological material from a patient to a cell culture device including a cell culture layer having a neuronal channel configured to receive neuronal cells therein, the neuronal channel having a first portion having a first partial cross-section and a second portion having a second partial cross-section smaller than the first partial cross-section and provided downstream of the first portion, and a neuromuscular junction chamber in fluid communication with the neuronal channel, the neuromuscular junction chamber configured to receive muscle cells therein to enable co-culture of the neuronal cells and the muscle cells and to form a neuromuscular junction, and determining a functional parameter associated with the neuromuscular junction, the functional parameter providing information related to the patient condition.

[0099] In some embodiments, the functional parameters include at least one of contractile force from a muscle cell, contractile duration of a muscle cell, neuronal cell viability, muscle cell survival, neuronal cell viability, muscle cell survival rate, and electrical signals from a neuronal or muscle cell.

[0100] In some embodiments, the method further comprises adding a test substance to at least one of the neuronal channel and the neuromuscular junction chamber.

[0101] In some embodiments, the step of determining a functional parameter associated with the neuromuscular junction is carried out after addition of the test substance.

[0102] In some embodiments, the step of determining a functional parameter associated with the neuromuscular junction is carried out before and after addition of the test substance.

[0103] In some embodiments, the method includes one or more features defined herein and / or described and / or illustrated herein.

[0104] The accompanying figures illustrate various features, aspects and embodiments of the technology described herein. [Brief description of the drawings]

[0105] [Figure 1] FIG. 1 is a bottom view of a cell culture device comprising a cell culture layer having a neuron inlet, a first muscle cell reservoir, a second muscle cell reservoir, a neuron channel with abutting walls, and a neuromuscular junction chamber with two posts. [Diagram 2] FIG. 2 is a front view of the cell culture device of FIG. [Figure 3A] 3A is a bottom perspective view of the cell culture device of FIG. 1. FIG. [Figure 3B] FIG. 3B is an enlarged view of a portion of the cell culture device of FIG. 3B, showing a portion of the neuron inlet, a portion of the first muscle cell reservoir, and a portion of the second muscle cell reservoir, the neuromuscular junction chamber, the neuron channel, and two struts. [Figure 4] FIG. 4 is a front perspective view of the cell culture device of FIG. [Diagram 5] 5 is another front perspective view of the cell culture device of FIG. 1. FIG. [Figure 6A] 6A is a front perspective view of the cell culture device and cell culture dish of FIG. 1. FIG. [Figure 6B] FIG. 6B is a front perspective view of the cell culture device and cell culture dish shown in FIG. 6A, with the cell culture device shown disposed on the cell culture layer receiving surface of the cell culture dish. [Figure 7]FIG. 7 is an enlarged bottom view of a portion of the cell culture device, showing a portion of the neuronal cell inlet, a portion of the first muscle cell reservoir, a portion of the second muscle cell reservoir, the neuromuscular junction chamber, the neuronal cell channel, neurospheres and muscle cells. [Figure 8] FIG. 8 is a bottom view of a cell culture device including two neuromuscular junction preparation units, each including a cup-shaped neuronal channel and a neuromuscular junction chamber including two posts. [Figure 9] FIG. 9 is a bottom view of a cell culture device including two neuromuscular junction preparation units, each including a cup-shaped neuronal channel and a neuromuscular junction chamber. [Figure 10] FIG. 10 is an enlarged front perspective view of a mold for manufacturing the cell culture device shown in FIG. [Figure 11] FIG. 11 is a bottom view of a cell culture device including two neuromuscular junction preparation units, each of which includes a neuronal channel with an abutment, and a neuromuscular junction chamber including two posts. [Figure 12] FIG. 12 is a bottom view of a cell culture device including two neuromuscular junction preparation units, each including a neuronal channel with an abutment, and a neuromuscular junction chamber. [Figure 13] FIG. 13 is an enlarged front perspective view of a mold for manufacturing the cell culture device shown in FIG. [Figure 14] FIG. 14 is a bottom view of a cell culture device comprising a cell culture layer having a neuron inlet, a first muscle cell reservoir, a second muscle cell reservoir, a neuron channel with an abutment, and a neuromuscular junction chamber with two posts. [Figure 15] FIG. 15 is a bottom view of a cell culture device comprising a neuron inlet, a first muscle cell reservoir, a second muscle cell reservoir, a neuron channel with an abutment, a neuromuscular junction chamber with two posts, and a cell culture layer with a gel seeding inlet. [Figure 16]FIG. 16 is an enlarged front perspective view of a mold for manufacturing the cell culture device shown in FIG. [Figure 17] FIG. 17 is a bottom view of a cell culture device comprising a neuron inlet, a first muscle cell reservoir, a second muscle cell reservoir, a neuron channel having an abutment and including a microchannel, a neuromuscular junction chamber including two posts, and a cell culture layer having a gel seeding inlet. [Figure 18] FIG. 18 is an enlarged front perspective view of the cell culture device of FIG. [Figure 19] FIG. 19 is a bottom view of a cell culture device including a cell culture layer having a neuronal cell inlet, a first muscle cell reservoir, a second muscle cell reservoir, a neuronal channel including first and second channels, and a neuromuscular junction chamber including two cylinders. [Figure 20] FIG. 20 is a bottom view of a cell culture device comprising a cell culture layer having a neuronal cell inlet, a first muscle cell reservoir, a second muscle cell reservoir, a neuronal channel comprising first and second channels, and a neuromuscular junction chamber comprising two elliptical cylinders. [Figure 21] FIG. 21 is an enlarged front perspective view of a mold for manufacturing the cell culture device shown in FIG. [Figure 22] FIG. 22 is a bottom view of a cell culture device comprising a cell culture layer having a neuron inlet, a first muscle cell reservoir, a second muscle cell reservoir, a neuron channel with abutting walls, and a neuromuscular junction chamber with two posts. [Figure 23] FIG. 23 is an enlarged front perspective view of a mold for manufacturing the cell culture device shown in FIG. [Figure 24] FIG. 24 is a top view of a cell culture layer with 20 NMJ preparation units. [Diagram 25] FIG. 25 is a top view of a cell culture layer containing 64 NMJ preparation units. [Figure 26] FIG. 26 is a top view of a cell culture layer containing 88 NMJ regulatory units. [Figure 27]FIG. 27 is an exploded perspective view of a cell culture layer in combination with a cell culture plate including a multigrid layer and a base layer. [Figure 28] FIG. 28 is a top view of a cell culture layer in combination with a cell culture plate including a multigrid layer and a base layer. [Figure 28A] FIG. 28A is an enlarged top view of a portion of the cell culture layer and cell culture plate of FIG. 28, the portion having an NMJ preparation unit containing two neuronal inlets. [Figure 28B] FIG. 28B is an enlarged top view of another portion of the cell culture layer and cell culture plate of FIG. 28, the portion having an NMJ preparation unit containing a single neuronal inlet. [Figure 29A] FIG. 29A is a bottom view of a cell culture device including a cell culture layer having a neuronal cell inlet, a first muscle cell reservoir, a second muscle cell reservoir, a neuronal cell channel including an abutting wall, and a neuromuscular junction chamber having two struts and a longitudinally extending support provided in a second portion of the neuronal cell channel. [Figure 29B] FIG. 29B is a bottom perspective view of the cell culture device of FIG. 29A. [Figure 29C] FIG. 29C is an enlarged view of the neuronal channel of the cell culture layer of FIG. 29A. [Figure 30A] FIG. 30A is a bottom view of a cell culture device including a cell culture layer having a neuronal cell inlet, a first muscle cell reservoir, a second muscle cell reservoir, a neuronal cell channel with abutting walls, and a neuromuscular junction chamber having two struts and a longitudinally extending support provided in a second portion of the neuronal cell channel. [Figure 30B] FIG. 30B is a bottom perspective view of the cell culture device of FIG. 30A. [Figure 30C] FIG. 30C is an enlarged view of the neuronal channel of the cell culture layer of FIG. 30A. [Figure 31A]FIG. 31A is a bottom view of a cell culture device comprising a cell culture layer having a neuronal cell inlet, a first muscle cell reservoir, a second muscle cell reservoir, a neuronal channel with abutting walls and an hourglass-shaped portion, and a neuromuscular junction chamber including two posts. [Figure 31B] FIG. 31B is a bottom perspective view of the cell culture device of FIG. 31A. [Figure 31C] FIG. 31C is an enlarged view of the neuronal channel of the cell culture layer of FIG. 31A. [Figure 32A] FIG. 32A is a bottom view of a cell culture device including a cell culture layer having a neuronal cell inlet, a first muscle cell reservoir, a second muscle cell reservoir, a neuronal cell channel with abutting walls, and a neuromuscular junction chamber including two struts and a post in a first portion of the neuronal cell channel. [Figure 32B] FIG. 32B is a bottom perspective view of the cell culture device of FIG. 32A. [Figure 32C] FIG. 32C is an enlarged view of the neuronal channel of the cell culture layer of FIG. 32A. [Figure 33A] FIG. 33A is a bottom view of a cell culture device including a cell culture layer having a neuronal cell inlet, a first muscle cell reservoir, a second muscle cell reservoir, a neuronal cell channel with abutting walls, and a neuromuscular junction chamber including two struts and a post in a first portion of the neuronal channel. [Figure 33B] FIG. 33B is a bottom perspective view of the cell culture device of FIG. 33A. [Figure 33C] FIG. 33C is an enlarged view of the neuronal channel of the cell culture layer of FIG. 33A. [Diagram 34] FIG. 34 is an exploded perspective view of a cell culture layer that combines a cell culture plate including a multigrid layer and a base layer with an electrode layer disposed below the cell culture layer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0106] The technology described herein relates to systems, devices and methods for preparing in vitro models of the neuromuscular junction (NMJ). The in vitro models described herein can be used for a wide range of cellular assays including compound screening, compound discovery, screening of patient-derived samples, safety, and efficacy testing, etc. The in vitro models described herein can also be used to perform mechanistic studies related to the development and maturation of NMJs, and mechanisms involved in the regulation and function of NMJs, as well as to model neurological diseases and disorders, immune and neuroimmune diseases, etc.

[0107] The in vitro models of the NMJ described herein can be obtained as a result of the predetermined configuration and interaction of various reservoirs, or chambers, provided in the cell culture layer, which can be used in cooperation with any other surface, such as a cell culture plate, cell culture dish, microscope slide, or a surface containing a sensor. Furthermore, the neural cells used in the preparation of the in vitro models of the NMJ are generally provided as three-dimensional neural cell aggregates according to various organizations, such as neurospheres, neurospheroids, neural aggregates, neuroorganoids, clusters, which contain only neural cells or are combined with one or more cell types, such as neurons and astrocytes, neurons and glia, etc. The size and configuration of certain features of the cultured cell layer can be determined at least in part according to the features of the neural cell aggregate, particularly its size.

[0108] For example, in some embodiments, a cell culture device for preparing an in vitro model of the NMJ can have a cell culture layer including a nerve cell inlet configured to receive nerve cells therein and first and second muscle cell reservoirs. At least one of the first and second muscle cell reservoirs is configured to have muscle cells seeded therein. The cell culture layer further includes a neuromuscular junction chamber extending between and in fluid communication with the first and second muscle cell reservoirs to allow for co-culture of nerve cells and muscle cells and subsequent formation of a neuromuscular junction. In some embodiments, supports can be provided within the neuromuscular junction chamber to promote growth of muscle cell bundles therebetween.

[0109] To provide fluid communication between the neuronal inlet and the neuromuscular junction chamber, the cell culture layer includes a neuronal channel extending between the neuronal inlet and the neuromuscular junction chamber. The neuronal channel includes a first portion and a second portion, the first portion being provided adjacent to the neuronal inlet and the second portion being provided adjacent to the neuromuscular junction chamber. In other words, the second portion is provided downstream of the first portion. The first portion has a first partial cross-section and the second portion has a second partial cross-section, the second partial cross-section being smaller than the first partial cross-section. More specifically, the first portion can have a first partial cross-section sized to retain the neuronal cell body therein, while the second portion can have a second partial cross-section sized to prevent the neuronal cell body from entering therein, i.e., to prevent the cell body from moving downstream of the first portion.

[0110] As mentioned above, neural cells can be assembled to form neural cell aggregates, such as neurospheres, neurospheroids, neuroorganoids, or clusters, containing only neural cells or in combination with one or more cell types, e.g., neural cells and astrocytes, neural cells and glia, etc. In such a scenario, neural cells, with or without additional cells, can be introduced into the cell culture layer via the neural cell inlet and migrate toward the first portion of the neural channel. As mentioned above, the first portion of the neural channel can be sized to hold the soma of the neural cell in a predetermined position within the neural channel without reaching the neuromuscular junction chamber, while the axon can extend therefrom, i.e., away from the first portion, into the second portion to reach the neuromuscular junction chamber and interact with the muscle cells to form the NMJ.

[0111] It will be understood that in the context of this specification, location terms such as "top", "bottom", "left", "right", "inwardly", "outwardly", "vertical", etc., should be taken in the context of the figure and should not be considered limiting unless otherwise indicated. When referring to length, for example in the context of axon length, it should be understood to refer to a measure along the horizontal axis. When referring to height, for example in the context of the height of the neuronal channels of a cell culture layer as described herein, it should be understood to refer to a measure along the vertical axis when the cell culture layer is placed substantially horizontally, for example on a cell culture plate, cell culture dish or microscope slide. The term "front" is intended to mean the orientation when the cell culture device is placed on a substantially horizontal surface facing the user, and the term "bottom" is intended to mean the opposite orientation when a user views the cell culture device from below. The term "outwardly" is intended to refer to features that extend outwardly of a reference axis. The term "inwardly" is intended to refer to features that extend inwardly of a reference axis.

[0112] In order to provide a more concise description, some of the quantitative expressions given herein may be modified with the term "about". Regardless of whether the term "about" is explicitly used, it is understood that all amounts given herein are meant to refer to the actual given value, and also to refer to an approximation to such a given value that is reasonably estimated based on the ordinary skill of a person skilled in the art, including an approximation based on experimental and / or measurement conditions for such a given value.

[0113] Various embodiments of the cell culture device will now be described in more detail.

[0114] Cell culture device With reference to Figures 1 to 23, various embodiments of a cell culture device 20 are shown. In each embodiment shown, the cell culture device 20 includes a cell culture layer 22. The cell culture layer 22 can take various forms and generally includes a predefined compartment that can be configured to receive a given type of cell. Figures 1 to 7 show various views of an embodiment of the cell culture layer 22, and Figures 8 to 23 show alternative embodiments of the cell culture layer 22. It should be noted that throughout the figures, the same reference numbers refer to similar components. Furthermore, for the sake of simplicity and clarity, i.e., to not overwhelm the figures with reference numbers, not all figures include references to all components and features, and references to some components and features are found only in one figure, while components and features of the present disclosure illustrated in other figures can be readily inferred therefrom. The illustrated embodiments, geometric configurations, materials mentioned, and / or dimensions shown in the figures are arbitrary and are given for illustrative purposes only.

[0115] 1-23, in the illustrated embodiment, each cell culture layer 22 includes a neuron inlet 24, a first muscle cell reservoir 26, and a second muscle cell reservoir 28. The first muscle cell reservoir 26 is spaced longitudinally from the second muscle cell reservoir 28, i.e., spaced along the x-axis of the cell culture layer 22. The neuron inlet 24 is offset from the first and second muscle cell reservoirs 26, 28, i.e., between and spaced therefrom along the y-axis, such that the combination of the neuron inlet 24 and the first and second muscle cell reservoirs 26, 28 forms a triangular shape.

[0116] The cell culture layer 22 further includes a neuromuscular junction chamber 30 extending between the first and second muscle cell reservoirs 26, 28, and a neuronal channel 32 extending between the neuronal inlet 24 and the neuromuscular junction chamber 30. The neuromuscular junction chamber 30 is configured to be in fluid communication with the first and second muscle cell reservoirs 26, 28, and with the neuronal inlet 24 via the neuronal channel 32. In the illustrated embodiment, the neuronal channel 32 extends substantially perpendicular to the neuromuscular junction chamber 30. In other embodiments, the neuronal channel 32 can extend at an angle other than 90° relative to the neuromuscular junction chamber 30, i.e., with the neuronal inlet 24 disposed closer to the first muscle cell reservoir 26 than the second muscle cell reservoir 28, or with the neuronal inlet 24 disposed closer to the second muscle cell reservoir 28 than the first muscle cell reservoir 26. Thus, it should be understood that the location of the nerve cell inlet 24 in the cell culture layer 22 relative to the first and second muscle cell reservoirs 26, 28 may differ from the embodiment shown in Figures 1-23.

[0117] The cell culture layer 22 can be made of any suitable polymeric material into which the neuron inlet 24, the first muscle cell reservoir 26, the second muscle cell reservoir 28, the neuromuscular junction chamber 30 and the neuron channel 32 can be engraved, stamped or molded. Examples of materials suitable for manufacturing the cell culture layer 22 include, but are not limited to, polystyrene (PS), cycloolefin copolymer (COC), cycloolefin polymer (COP), polymethylmethacrylate (PMMA), polycarbonate (PC), polyethylene (PE), polyethylene terephthalate (PET), polyamide (Nylon™), polypropylene (PP), polyetheretherketone (PEEK), Teflon™, polydimethylsiloxane (PDMS), and / or thermoplastic elastomers (TPE), as well as synthetic and biological materials such as hydrogels, gelatin, collagen, chitosan, etc. In some embodiments, the cell culture layer 22 can be made of a polymeric material that is transparent to light to facilitate optical analysis and visualization of the neurons in the neuronal channel 32 and the muscle cells and axons in the neuromuscular junction chamber 30.

[0118] The cell culture layer 22 can be inserted into a cell culture dish, a tray of a cell culture plate, a microscope slide, or into a well of a multi-well cell culture plate, such as those commonly available commercially. In other words, the cell culture layer 22 can be configured to be placed on a cell culture layer receiving surface of a cell culture dish, a well of a cell culture plate, or a cell culture layer receiving surface of a microscope slide, or any other type of vessel that can be suitable for receiving the cell culture layer 22.

[0119] The cell culture layer 22 shown in Figures 1 to 23 includes what may be referred to as a single NMJ preparation unit, which includes a neuron inlet 24, first and second muscle cell reservoirs 26, 28, a neuromuscular junction chamber 30, and a neuron channel 32. In alternative embodiments, the cell culture layer 22 may include multiple NMJ preparation units. The multiple NMJ preparation units may be provided, for example, on a rectangular cell culture layer that may be inserted onto a cell culture layer receiving surface of a cell culture plate. In some embodiments, the cell culture plate may be a cell culture plate that conforms to the American Society for Laboratory Automation and Screening Standards (ANSI / SLAS) microplate standard. The cell culture plate may also be used in conjunction with a multiwell grid layer that may be positioned on top of the cell culture layer and has a number of wells that correspond to the number of NMJ preparation units in the cell culture layer. The multiwell grid layer may conform to the ANSI / SLAS microplate standard. Depending on the needs of the user, the multi-well grid layer can be configured with any number of wells, for example, 6, 12, 24, 48, 96, 384, or 1536 wells. Figures 24 to 26 show examples of cell culture layers 22 containing 20, 64, and 88 NMJ preparation units 21, respectively. In the embodiment shown in Figure 26, the NMJ preparation units 21 are arranged in an alternating manner along the x-axis, with a first one of the NMJ preparation units 21 in the top left corner and a second one of the NMJ preparation units 21 positioned longitudinally adjacent and below the first one of the NMJ preparation units 21 along the x-axis (full-body arrangement). In an embodiment, alternating upward and downward orientation of the NMJ preparation units 21 can provide more NMJ preparation units 21 in a cell culture layer with a given surface area. FIG. 27 is an exploded view showing an example of a combination of cell culture layer 22 and cell culture plate 120, where cell culture plate 120 is composed of a multi-well grid layer 122 and a base layer .

[0120] In the embodiment shown in Figure 6B, cell culture layer 22 is shown adhered onto cell culture layer receiving surface 34 of bottom wall 38 of cell culture dish 36 shown in Figure 6A. In some embodiments, and as illustrated in Figure 6A, cell culture dish 36 can have a central notch for receiving a glass bottom that acts as bottom wall 38, and cell culture layer 22 can be adhered to the glass bottom. When cell culture layer 22 is used with a multi-well cell culture plate, cell culture layer 22 is inserted on its cell culture layer receiving surface into the corresponding well of the cell culture plate.

[0121] The cell culture layer 22 can be reversibly or irreversibly attached to the cell culture layer receiving surface using any suitable method or technique, including, but not limited to, compression, surface bonding, ultrasonic welding, thermocompression bonding, plasma bonding, solvent-assisted bonding, laser-assisted bonding, or bonding using an adhesive or double-sided adhesive tape.

[0122] Alternatively, in some embodiments, the cell culture layer 22 can be fabricated integrally with the bottom wall 38 of the cell culture dish or cell culture plate. When the cell culture layer 22 forms an integral part of the cell culture dish or cell culture plate, the resulting device can be manufactured as a single unit. Examples of materials suitable for fabricating the cell culture layer 22 integral with the bottom wall of the cell culture plate include, but are not limited to, polystyrene (PS), cycloolefin copolymer (COC), cycloolefin polymer (COP), polymethylmethacrylate (PMMA), polycarbonate (PC), polyethylene (PE), polyethylene terephthalate (PET), polyamide (Nylon™), polypropylene or polyetheretherketone (PEEK), Teflon™, polydimethylsiloxane (PDMS), and / or thermoplastic elastomers (TPE), as well as synthetic and biological materials such as hydrogels, gelatin, collagen, chitosan, etc.

[0123] 1-23, cell culture layer 22 is configured to extend substantially horizontally when placed on a cell culture dish, cell culture plate, or microscope slide. Although we refer to cell culture layer 22 as extending substantially horizontally, it should be understood that cell culture layer 22 may be provided at a slight angle relative to the cell culture layer receiving surface of the cell culture dish, cell culture plate, or microscope slide on which it is deposited. In some embodiments, providing cell culture layer 22 at a slight angle relative to the cell culture layer receiving surface of the cell culture dish, cell culture plate, or microscope slide on which it is deposited allows selected fluids and / or cells present in a given one of the compartments to migrate in a preferential direction.

[0124] The cell culture layer 22 can have various sizes and configurations. The size and configuration of the cell culture layer 22 can be adapted to the size and configuration of the vessel of a cell culture dish or the well of a cell culture plate into which the cell culture layer 22 is intended to be inserted. For example, in the case of an embodiment in which the cell culture layer 22 is intended to be inserted into a cell culture dish, the size of the cell culture layer 22 can be determined to fit within the vessel formed by the cell culture dish, as shown in Figures 6A to 6B. In the case in which the cell culture layer 22 is intended to be inserted into the well of a multi-well cell culture plate, the size of the cell culture layer 22 can be adapted, for example, by shrinking, so that the cell culture layer 22 can fit within the corresponding well of the multi-well cell culture plate.

[0125] The neuronal inlet 24 is configured to receive the infusion fluid and the neuronal cells therein. Examples of neuronal cell types that can be used to create an in vitro model of the NMJ include mammalian neuronal cells, such as rodent embryonic neuronal cells, and neuronal cells derived from induced pluripotent stem cells, such as human induced pluripotent stem cells. When creating an in vitro model of the NMJ, the option to culture different types of neuronal cells can increase the versatility of the resulting model, which in turn can provide a wider range of opportunities for different industrial needs. The use of cells of human origin is also beneficial in providing reproducible and accurate results and facilitating the testing of drugs and compounds to human applications. In some embodiments, diseased neuronal cells from a patient can be used to mimic a given pathology and / or to evaluate the effect of a given drug on a particular patient. For example, in some embodiments, neural cells from a patient suffering from a neurological disorder such as amyotrophic lateral sclerosis (ALS), and / or a myopathy such as Duchenne muscular dystrophy, or an autoimmune disease neuromuscular disease such as myasthenia gravis, or any other nerve, muscle, neurodegenerative disease or disorder, can be harvested and cultured to form one or more neurospheres that can then be introduced into the neural cell inlet 24 of the cell culture layer 22 as described herein. It should be understood that any type of neural cell, whether healthy or diseased, that can be cultured to form aggregates such as neurospheres is within the scope of the present specification.

[0126] The inlet fluid may be a cell culture medium that allows for survival and / or proliferation of neural cells. In some embodiments, the neural cell inlet 24 is further configured to receive a test substance therein such that neural cells present in the neural cell inlet 24 and / or in the neural cell channel 32 may be exposed to such test substance. As used herein, a test substance may be any type of substance desired to be tested to assess the response of the NMJ to the test substance, including, for example, bodily fluids (e.g., plasma, serum, blood, urine, etc.) or cells from a patient. The test substance may take a variety of forms, such as a liquid, a suspension, etc.

[0127] Either or both of the first and second muscle cell reservoirs 26, 28 can be configured to receive muscle cell reservoir fluid and muscle cells. The muscle cells can be dissociated from organs that make up the muscular system, such as the heart (cardiomyocytes), limb or body wall muscles (skeletal muscle cells), visceral muscles (smooth muscle cells), and / or cells differentiated from pluripotent cells, such as embryonic stem cells or induced pluripotent stem cell derived muscle cells. As described above with respect to the types of neural cells that can be used, diseased muscle cells from patients can also be used to mimic a given pathology and / or to evaluate the effect of a given drug on that particular patient. For example, in some embodiments, muscle cells from patients suffering from a neurological disorder, such as amyotrophic lateral sclerosis (ALS), and / or a myopathy, such as Duchenne muscular dystrophy, or other neural, muscular, neurodegenerative disease or disorder can be used. It is understood that any type of muscle cell, whether healthy or diseased, is within the scope of the present specification.

[0128] The muscle cell reservoir fluid can be the same as or different from the inlet fluid. The muscle cell reservoir fluid can be a cell culture medium that allows for the survival and / or proliferation of muscle cells received within the first and second muscle cell reservoirs 26, 28 and within the neuromuscular junction chamber 30 of the cell culture layer 22.

[0129] In some embodiments, the muscle cell reservoir fluid can be a gel solution, such as a gel solution including Matrigel™, Geltrex™, fibrin, collagen, or other types of basement membrane matrix or extracellular matrix (ECM), and the muscle cells can be provided suspended in the gel solution. The gel solution and suspended muscle cells can be seeded into one of the first and second muscle cell reservoirs 26, 28. The gel solution and suspended muscle cells can then be transferred from one of the first and second muscle cell reservoirs 26, 28 to the other of the first and second muscle cell reservoirs 26, 28 via the neuromuscular junction chamber 30. After a period of time, i.e., an incubation period, if the myocyte reservoir fluid is provided as a gel solution, the myocyte ladder solution polymerizes into a hydrogel and the myocytes can remodel the hydrogel and self-assemble into muscle tissue / fibers (also called muscle bundles) that form within the neuromuscular junction chamber 30 and, optionally, between the struts, if present. In some embodiments, this configuration of the first and second myocyte reservoirs 26, 28, neuromuscular junction chamber 30, neuronal channel 32 and neuronal inlet 24 allows neuronal extensions to have a substantially well-defined path to grow within the neuronal channel 32 and reach the neuromuscular junction chamber 30.

[0130] In other words, this configuration of the first and second muscle cell reservoirs 26, 28, the neuromuscular junction chamber 30, the neuronal channel 32 and the neuronal inlet 24 allows two types of flows to be established: a first flow from the first muscle cell reservoir 26 through the neuromuscular junction chamber 30 to the second muscle cell reservoir 28 (or vice versa) and a second flow from the neuronal channel 32 to the neuromuscular junction chamber 30. The first flow allows for the formation and maintenance of muscle tissue / fibers formed within the neuromuscular junction chamber 30 and for the muscle tissue / fibers to be treated with any desired test substance, such as a chemical or drug. The second flow allows for the neuronal cell extensions to be directed towards the muscle tissue / fibers so that innervation can occur to form a neuromuscular junction and also allows for the neuronal cells to be treated with any desired test substance, such as a chemical or drug, among other functions. In some embodiments, the flow rate of the second flow can be less than the flow rate of the first flow or vice versa.

[0131] Either the first and second muscle cell reservoirs 26, 28, or both, or the neuromuscular junction chamber 30 itself, are further configured to receive a test substance therein, such that the muscle cells present in the neuromuscular junction chamber 30 and that will ultimately form the NMJ are exposed to such test substance. The test substance can be any type of substance that one desires to test for to assess the response of the NMJ to the test substance, such as toxins, antibodies, chemicals, biological materials, patient-derived fluids (plasma, serum, blood, urine, etc.), parasites, bacteria, viruses, and / or cells. The test substance can take a variety of forms, such as liquids, suspensions, and the like. Thus, the cell culture layer 22 allows for testing of substances, such as toxins, antibodies, chemicals, biological materials, drugs, patient-derived fluids (plasma, serum, blood, urine, etc.), parasites, bacteria, viruses, and / or cells, in selected compartments of the cell culture layer 22, such as the neuromuscular junction chamber 30 where the NMJ is formed. Thus, the test substance can include biological and / or chemical materials. If the test substance comprises a biological material, it can include any type of biological material for which it is desired to determine a response from an in vitro model of the NMJ that can be obtained using the cell culture apparatus 20 described herein, such as, for example, a biological fluid or tissue from a patient, an antibody or antibody fragment, an antigen or antigen fragment, a toxin, an enzyme, a DNA or RNA fragment, a virus, or a bacterium. If the test substance comprises a chemical, it can include any type of chemical for which it is desired to determine a response from an in vitro model of the NMJ that can be obtained using the cell culture apparatus 20 described herein, such as, for example, a drug, or any chemical compound. The in vitro model of the NMJ can be used, for example, to test the potency of a toxin and / or the toxicity of a compound, such as a chemical.

[0132] In some embodiments, the test substance may be a toxin, and the toxin may include a botulinum toxin. A botulinum toxin has a variety of applications, including in the pharmaceutical, cosmetic, and food industries. In some embodiments, the cell culture device described herein may be used to test the neurotoxicity, potency, and / or safety of a botulinum toxin, for example, as part of a quality control operation. Also, various products may be tested to determine whether a botulinum toxin is present. It is to be understood that the above examples are given for illustrative purposes only, and that various other applications for testing a botulinum toxin are within the scope of the present specification.

[0133] The in vitro model of the NMJ, which can be obtained using the cell culture device 20 described herein, can be used to analyze the effect of a test substance on the functionality of the NMJ by measuring functional parameters associated with the NMJ. Examples of functional parameters can include, but are not limited to, at least one of contractile force from a muscle cell, contractile duration of a muscle cell, neuronal viability, muscle cell viability, neuronal viability, muscle cell viability, calcium signal indicators, and electrical signals from a neuronal or muscle cell. As discussed above, the test substance can include biological material from a patient, and / or a chemical.

[0134] For example, in an embodiment of the cell culture device 20 used to develop an in vitro model of the NMJ, biological material obtained from a patient can be tested using the in vitro model of the NMJ. The in vitro model of the NMJ can be used, for example, to test serum from a patient with an autoimmune disease, such as myasthenia gravis, to assess the presence of one or more specific antibodies or compounds that may affect the function of the NMJ. The biological material can be taken from the patient after the patient has been administered a predetermined drug. In such a scenario, the biological material removed from the patient can include patient-specific substances, such as, for example, antibodies, metabolites, or active components of a drug, resulting in a predetermined combination of this patient-specific substance with this particular patient. This predetermined combination can then be tested at the NMJ, for example, to assess the response of the NMJ to this predetermined combination. Alternatively, biological material can be taken even if the patient has not previously been administered a drug, and the biological material can be tested at the NMJ to assess the response of the NMJ. In some embodiments, it is desired to compare the response of the NMJ to a given combination of patient-specific substances with this particular patient with the response of the NMJ to biological material taken from the same patient, but without the patient having been administered the drug before. According to this embodiment, it is possible to determine whether a drug administered to a patient affects the NMJ, such as affecting the function of the NMJ, which can then contribute to determining whether the drug provides a therapeutic benefit to the patient. This method may be considered similar to a crossover study design, where two types of biological material are tested consecutively in an in vitro model of the NMJ in the same patient.

[0135] In another embodiment of the cell culture device 20 used to develop an in vitro model of the NMJ, a first patient group (which may include one or more patients) is administered a predetermined agent and a second patient group (which may include one or more patients) is not administered the predetermined agent. In such a scenario, the corresponding biological material may be withdrawn or removed from one or more patients of the first patient group. And the corresponding biological material may be withdrawn or removed from one or more patients of the second patient group. The effect of the corresponding biological material of the first patient group on the NMJ can then be evaluated, and the effect of the corresponding biological material of the second patient group on the NMJ can be evaluated in parallel. The respective effects of the corresponding biological material of the first patient group and the second patient group can then be compared, similar to a crossover study design.

[0136] In yet another embodiment, the cell culture device 20 can be used as a diagnostic tool to determine whether a patient is suffering from a given condition, such as an autoimmune or alloimmune disease. In such a scenario, a sample of biological material can be taken from a patient and the biological material can be tested in an in vitro model of the NMJ obtained using the cell culture device 20 to evaluate the effect of the biological material on the NMJ. After the biological material from the patient is added to the in vitro model of the NMJ, NMJ function can be evaluated to determine whether the biological material affects NMJ function. Examples of endpoints that can be evaluated to evaluate NMJ function include, but are not limited to, the degree and / or duration of muscle contraction after subjecting the NMJ to neuronal stimulation, as well as determining whether the addition of biological material from a patient to a test tube model of the NMJ caused neuronal degeneration. For example, certain autoimmune and autoimmune diseases can result in the body producing antibodies that can impair the function of the NMJ. By allowing testing of the effect on NMJ function of biological material from patients with or suspected of having an autoimmune disease, the in vitro models of the NMJ obtained using the cell culture device 20 described herein can be used to assess whether these antibodies affect the NMJ and, if so, whether the patient is likely to suffer from a given condition and / or whether a given drug may be useful in treating the patient's condition.

[0137] The above scenarios are described as examples of applications in which the cell culture device 20 and the resulting in vitro model of the NMJ can be used as diagnostic tools, and it is understood that the cell culture device 20 and the resulting in vitro model of the NMJ can be used to diagnose other types of diseases or conditions involving the NMJ.

[0138] Returning to the figures, the neuron inlet 24 and the first and second muscle cell reservoirs 26, 28 shown in Figure 3 are open to the atmosphere to allow for the introduction of various fluids, cells, and optionally test substances to selected compartments of the cell culture layer 22. The neuron inlet 24 and the first and second muscle cell reservoirs 26, 28 are thus through-holes extending throughout the entire thickness of the cell culture layer 22. Once the cell culture layer 22 is deposited on the cell culture layer receiving surface of a cell culture plate, cell culture dish, or microscope slide, the cell culture layer receiving surface serves as a back wall of the neuron inlet 24 and the first and second muscle cell reservoirs 26, 28, where fluids introduced into the neuron inlet 24 and the first and second muscle cell reservoirs 26, 28 can remain. In an alternative embodiment, at least one of the neuron inlet 24 and the first and second muscle cell reservoirs 26, 28 may include a back wall provided by the cell culture layer 22 itself and thus do not extend through the entire thickness of the cell culture layer 22.

[0139] In some embodiments, the first and second muscle cell reservoirs 26, 28 can each have a diameter ranging from about 2 mm to about 6 mm. In some embodiments, the nerve cell inlet 24 can have a diameter ranging from about 2 mm to about 10 mm. In some embodiments, the cell culture layer 22 can have a diameter ranging from about 8 mm to about 30 mm. It should be understood that these dimensions are given for illustrative purposes only in a scenario in which the cell culture layer 22 is configured to be inserted into a cell culture dish, and other dimensions of the above-mentioned compartments and the cell culture layer 24 are of course possible. For example, if the cell culture layer is inserted into a well of a multi-well cell culture plate, the dimensions of the cell culture layer can be adapted so that the cell culture layer fits within a given well of the multi-well cell culture plate.

[0140] In the embodiment shown in Figures 1-8, 10, 11, and 13-23, the neuromuscular junction chamber 30 includes a first strut 40 and a second strut 42 disposed in a spatially separated relationship from one another. The first strut 40 is disposed closer to the first myocyte reservoir 26 than to the second myocyte reservoir 28, and the second strut 42 is disposed closer to the second myocyte reservoir 28 than to the first myocyte reservoir 26. The first and second struts 40, 42 may act as anchoring points for assembling muscle cells therebetween and allowing the formation of attached muscle fibers and / or muscle tissue. The struts 40, 42 may vary in shape and size. For example, the struts may have a substantially cylindrical shape with a circular cross-section, thereby forming a regular cylinder. In other embodiments, as shown in Figure 20, either or both of the struts may have an elliptical cross-section, thereby forming an elliptical cylinder. In other embodiments, one or both of the struts can form an oblique cylinder. Other strut configurations and shapes are possible, and any physical structure capable of anchoring muscle fibers is suitable. For example, in some embodiments, either or both of the first strut 40 and the second strut 42 can include a wire. The wire can be a flexible wire that can be deflected in response to muscle cell movement at the NMJ, such as muscle cell contraction. The wire can be made of metal, polymer, or other suitable material.

[0141] In other embodiments, the struts may be omitted, for example as shown in Figures 9 and 12.

[0142] In some embodiments, such as those illustrated in Figures 1 to 7, the struts 40, 42 can have a diameter ranging from about 0.5 mm to about 10 mm. Other dimensions of the struts are possible, depending, for example, on the intended use of the cell culture layer 22 and whether the cell culture layer 22 is intended to be used with a cell culture plate, a cell culture dish, or a microscope slide. The distance between the first and second struts 40, 42 can vary depending on the desired length of the muscle fibers extending therebetween. Alternatively, the distance between the first and second struts 40, 42 can be kept within a certain range to ensure the formation of viable and sufficiently thick, i.e., not too thin, muscle fibers, thus promoting muscle fiber growth. The struts 40, 42 can be configured to remain in position even when subjected to a force. That is, the struts can be configured not to flex when subjected to a predetermined force. Alternatively, the struts 40, 42 can be configured to flex when subjected to a predetermined force.

[0143] In some embodiments, where at least one of the first and second posts 40, 42 is a deflectable post, e.g., a deflectable wire or other type of deflectable post, the degree of deflection and / or duration of deflection of at least one of the first and second posts 40, 42 may be evaluated in response to contraction of muscle cells at the NMJ initiated following, e.g., electrical, chemical or optical stimulation. Such configuration of at least one of the first and second posts 40, 42 allows the cell culture device 20 to be used to test various test substances, e.g., biological or chemical materials, for their effect on the functionality of the NMJ, i.e., on functional parameters of the NMJ. In turn, knowing the effect of the test substance on the functionality of the NMJ can contribute to determining whether the test substance has a desirable effect on the NMJ or, conversely, an undesirable effect on the NMJ.

[0144] Neuronal Channels Details regarding the neuronal channel 32 shown in Figures 1 to 23 will now be provided.

[0145] The neuron channel 32 extends between and is in fluid communication with the neuron inlet 24 and the neuromuscular junction chamber 30. In some embodiments, and as shown in FIGS. 1-23, the neuron channel 32 can be joined to the neuromuscular junction chamber 30 at a central region of the neuromuscular junction chamber 30. In some embodiments, the neuron channel 32 can be joined to the neuromuscular junction chamber 30 at a location equidistant from the first muscle cell reservoir 26 and the second muscle cell reservoir 28. If struts, such as struts 40, 42, are present in the neuromuscular junction chamber 30, the neuron channel 32 can be joined to the neuromuscular junction chamber 30 at a central region between the two struts of the neuromuscular junction chamber 30. In some embodiments, the neuron channel 32 can be joined to the neuromuscular junction chamber 30 at a location equidistant from both the first and second struts. 1, the centerline of the neuron cell channel 32 joins the neuromuscular junction chamber 30 at a distance from the first strut 40 that is substantially equal to its distance from the second strut 42. Alternatively, the neuron cell channel 32 can join the neuromuscular junction chamber 30 at a location other than the central region of the neuromuscular junction chamber 30, i.e., closer to one of the first or second muscle cell reservoirs 26, 28.

[0146] The neuron channel 32 includes a first portion 44 having a first partial cross-section and a second portion 46 downstream of the first portion 44 and having a second partial cross-section. As shown in Figures 2 and 5, the neuron channel 32 is enclosed within the cell culture layer 22, with a first end 48 of the neuron channel 32 opening into the neuron inlet 24 and a second end 50 of the neuron channel 32 opening into the neuromuscular junction chamber 30. It should be appreciated that in another embodiment, the neuron channel 32 can be configured as an open-top neuron channel.

[0147] The first portion 44 of the neuronal channel 32 includes a first partial side wall 52, a first partial front wall 54, and a first partial rear wall (not shown in FIG. 3B). The second portion 46 of the neuronal channel 32 includes a second partial side wall 56, a second partial front wall 58, and a second partial rear wall (not shown in FIG. 3B). In some embodiments, the first partial rear wall and the second partial rear wall may be provided by a cell culture layer receiving surface of a cell culture dish, cell culture plate, or microscope slide in which the cell culture layer 22 is to be used. This type of embodiment is shown in FIGS. 1 to 7, which show that the rear surface of the cell culture layer 22 is open when the cell culture layer 22 is not in contact with the cell culture layer receiving surface of the cell culture dish, cell culture plate, or microscope slide in which the cell culture layer 22 is to be used. In such an embodiment, the cell culture layer receiving surface provides confinement of the neuronal channel 32 along this plane. In other embodiments, the first partial rear wall and the second partial rear wall may be integral with the cell culture layer 22, i.e., form part of the cell culture layer 22.

[0148] When the cell culture layer 22 is configured for use with three-dimensional neuronal aggregates or clusters of neuronal cells, such as neurospheres, spheroids, neural aggregates, or neuroorganoids, the first portion 44 of the neuronal channel 32 can be sized and configured to receive the neurospheres, neural aggregates, or neuroorganoids therein. For simplicity, the three-dimensional neuronal aggregates are collectively referred to as "neurospheres" throughout this specification. Thus, when neurospheres are referred to herein, it should be understood to mean to include all types of three-dimensional neuronal aggregates. In other words, the first portion cross-section of the first portion 44 of the neuronal channel 32 is selected to be at least large enough to receive one or more neurospheres therein. In that regard, the cross-section of the first portion can be considered to correspond to the lateral area defined by the first portion sidewall 52, the first portion anterior wall 54, and the first portion posterior wall. In this manner, one or more neurospheres can be introduced into the neuron inlet 24 and moved toward the neuron channel 32, with the portion of the neurosphere containing the neuron cell body remaining in the first portion 44 of the neuron channel 32. FIG. 7 shows an example of the first portion 44 of the neuron channel 32.

[0149] Although not shown in the figures, in some embodiments, the NMJ preparation unit can include a plurality of neuronal channels 32 arranged in a side-by-side, spaced apart relationship, each of the neuronal channels 32 having a respective second end 50 that opens into the neuromuscular junction chamber 30.

[0150] In order to retain the portion of the neurosphere containing the neuronal cell body within the first portion 44 of the neuronal channel 32, the cross section of the second portion is smaller than the cross section of the first portion, and its size is determined to prevent the ingress of the portion of the neurosphere containing the neuronal cell body within the second portion 46, i.e., to prevent the ingress of the neuronal cell body within the second portion 46. The second portion 46 is further sized to accommodate the axon, allow the axon to extend away from the neuronal cell body, and direct the axon to the neuromuscular junction chamber 30 to reach the muscle cell cultured therein. FIG. 7 shows an example of an axon 62 extending from a neurosphere 60 located in the first portion 44 of the neuronal channel 32, extending into the second portion 46 of the neuronal channel 32, entering the neuromuscular junction chamber 30, and reaching a muscle cell 64 extending between the first strut 40 and the second strut 42. The second partial cross-section can be considered to correspond to a cross-sectional area defined by the second partial side wall 56, the second partial front wall 58 and the second partial rear wall. The reduced second partial cross-section compared to the first partial cross-section can be achieved in various ways, which will be described in more detail below.

[0151] 1 through 7, 22, and 23, the neuronal channel 32 can have a substantially constant channel width w (shown in FIG. 3B) along the x-axis throughout its length as determined along the y-axis. In other words, the first portion sidewall 52 can transition to the second portion sidewall 56 in a substantially linear manner such that the neuronal channel 32 has substantially the same channel width w between the first portion 44 and the second portion 46. The constant channel width w of the neuronal channel 32 can be the result of the neuronal channel 32 being formed as a rectangular prism having a substantially constant width along the x-axis throughout its length as determined along the y-axis of the cell culture layer 22, or the result of the neuronal channel 32 being formed as a cylinder having a substantially constant diameter along the x-axis throughout its length as determined along the y-axis of the cell culture layer 22. The width can be selected to be large enough to accommodate one or more neurospheres therein while being small enough to stabilize one or more neurospheres therein. For example, the channel width or diameter of the first portion 44 may be determined to obtain a predetermined ratio between the channel width of the first portion 44 and the width of the neurosphere. For example, in some embodiments, the ratio of the width or diameter of the neurosphere to the channel width may range from about 1.25 to about 2. This ratio range is given by way of example only, since the three-dimensional neuronal assemblies can take on various forms and sizes, and the size of the neuronal channel 32 is adapted accordingly. One purpose of the size of the neuronal channel 32 is to allow for stabilization of one or more three-dimensional neuronal assemblies contained therein, while allowing for efficient cell culture medium flow between the neuronal channel 32 and the neuromuscular junction chamber 30. Furthermore, the first portion 44 has a size and configuration to accommodate at least one neurosphere therein and prevent the cell bodies of the neurosphere from migrating downstream to the second portion 46 of the neuronal channel 32.

[0152] As mentioned above, the second portion 46 of the neuronal channel 32 has a second partial cross-section that is smaller than the first partial cross-section of the first portion 44 of the neuronal channel 32. In Figs. 1 to 7, the reduction in cross-section is achieved by a reduction in the channel height h of the second portion 46 compared to the channel height h of the first portion 44. To that end, the second partial front wall 58 is disposed medially, i.e., along the z-axis toward the second partial front wall, compared to the first partial front wall 54. This offset of the second partial front wall 58 relative to the first partial front wall 54 defines an abutment wall 66 within the neuronal channel 32. The location of the abutment wall 66 can be considered as the location of the transition from the first portion 44 to the second portion 46. The abutment wall 66 can function as a surface on which one or more neurospheres can rest or lean. The degree of reduction in channel height h in the second portion 46 of the neuronal channel 32 can be determined to prevent one or more neurospheres from migrating downstream within the second portion 46 while allowing axons of one or more neurospheres to extend within the second portion 46 through the second end 50 of the neuronal channel 32 to reach the neuromuscular junction chamber 30.

[0153] In the embodiment shown in Figures 1 to 7, the abutment wall 66 is formed to converge inwardly toward the centerline of the neuronal channel 32 along the x-axis. The convergence of the abutment wall 66 toward the centerline of the neuronal channel 32 may contribute to stabilizing one or more neurospheres within the first portion 44 of the neuronal channel 32. As shown in Figures 3A and 3B, the convergence of the abutment wall 66 toward the centerline of the neuronal channel 32 may be achieved by a series of multiple substantially linear planes oriented toward the centerline. Alternatively, in some embodiments, the convergence of the abutment wall 66 toward the centerline of the neuronal channel 32 may be achieved by a curved abutment wall, i.e., a concave abutment wall (see, e.g., Figures 8 to 10). In still other embodiments, the abutment wall 66 may be substantially flat (see, e.g., Figures 11 to 16). Whether the abutment wall 66 is substantially flat, includes multiple substantially linear planes oriented toward the centerline of the neuronal channel 32, or is concave, the abutment wall 66 does not include an opening, but rather is continuous. That is, it should be understood that the abutment wall 66 does not have any openings.

[0154] In other words, the decrease in the magnitude of the channel height h from the first portion 44 to the second portion 46 may be such that at some location along the length of the neuronal channel 32, i.e., along the y-axis, the cross-section of the neuronal channel 32 becomes too small for the cell bodies of one or more neurospheres to move further down the y-axis of the neuronal channel 32 such that the cell bodies of the one or more neurospheres are retained upstream of the second portion 46. In some embodiments, the ratio of the channel height h of the first portion 44 to the channel height h of the second portion 46 may range from about 2 to about 12, about 3 to about 9, or about 3 to about 7. In some embodiments, the channel height h of the first portion 44 may range from about 0.02 mm to about 1.2 mm, and the channel height h of the second portion 46 may range from about 0.01 mm to about 1 mm. In an exemplary embodiment, the channel height h of the first portion 44 may range from about 0.5 mm to about 1 mm, and the channel height h of the second portion 46 may range from about 0.01 mm to about 0.5 mm. It should be understood that these dimensions are given for illustrative purposes only, and that in other embodiments, the dimensions of the first portion 44 and second portion 46 of the neuronal channel 32 may be higher or lower depending on the intended use of the cell culture device.

[0155] In alternative embodiments, in addition to a reduction in channel height h, a reduced cross-section of the second portion compared to the cross-section of the first portion may further be achieved by a reduction in channel width w as shown in Figures 8 to 10. In such embodiments, the channel width w may thus be greater in the first portion 44 compared to the channel width w in the second portion 46. The transition from the first portion 44 to the second portion 46 may be achieved, for example, by a step change, a sloping wall or a curved wall. When the transition from the first portion 44 to the second portion 46 is achieved, for example, by a step change, the step change may include one or more rounded edges.

[0156] In the embodiments shown in Figures 1 to 7, 11 to 16, and 19 to 23, the reduction in channel height h from the first portion 44 to the second portion 46 is achieved by providing the second partial front wall 58 inwardly along the z-axis from the second partial rear wall 54, i.e., closer to the second partial rear wall compared to the location of the first partial front wall 54 relative to the first partial rear wall. In some embodiments, the reduction in channel height h can be further achieved by providing the second partial rear wall inwardly along the z-axis toward the centerline of the neuronal channel 32. This type of embodiment can be achieved when the second partial rear wall is provided by the cell culture layer 22 itself, rather than by the cell culture layer receiving surface of a cell culture plate, cell culture dish, or microscope slide. In still other embodiments, the reduction in channel height h can be achieved only by providing the second partial rear wall inwardly along the z-axis toward the centerline of the neuronal channel 32.

[0157] When the abutment wall 66 is concave, the combination of the first portion side wall 52 and the abutment wall 66 can form a cup as shown in Figures 8 to 10. The size and configuration of the cup can be adapted to match the size and shape of the neurosphere according to the size and shape of the neurosphere intended to be received within the cup. For example, in the case of a substantially circular neurosphere, the first portion, i.e., the cup, can be sized such that the cell bodies of the neurosphere lean gently against the abutment wall 66 and the axons extend into the second portion 46 downstream of the first portion 44.

[0158] electrode layer In some embodiments, the cell culture device 20 may further include an electrode or electrodes. The electrode or electrodes may be provided to directly or indirectly contact or electrically communicate with the neuronal cells, muscle cells, and / or neuromuscular junctions formed in the neuromuscular junction chamber 30, and / or axons extending into the neuronal channel 32, and / or neuronal extensions. The electrode or electrodes may take the form of an electrode layer that may be placed under or overlaid on the cell culture layer 22 to allow electrical communication with the neuronal cells and / or muscle cells. Alternatively, the electrode or electrodes may be provided in the vicinity of the cell culture layer 22, without necessarily being placed under or overlaid on the cell culture layer 22. When the electrode layer is provided in the vicinity of the cells, the distance between the electrode(s) of the electrode layer and the cells may be, for example, in the micrometer or millimeter range. In yet other embodiments, electrodes may be provided for insertion into culture material, such as neurons growing in the neuron channel 32 and / or neurons and muscle cells growing in the neuromuscular junction chamber 30. In such embodiments, electrodes may be provided within one or more compartments of the neuromuscular junction preparation unit, for example, on the top or bottom surface of one or more compartments of the neuromuscular junction preparation unit and / or on the side walls of one or more compartments of the neuromuscular junction preparation unit.

[0159] In some embodiments, the electrode layer can be disposed on a cell culture layer receiving surface, for example, a cell culture plate, cell culture dish, or microscope slide, and the cell culture layer 22 is disposed on the electrode layer. FIG. 34 shows an example of a cell culture device 120 including a multiwell grid layer 122 and a base layer 124, with the cell culture layer 22 and electrode layer 126 disposed between the multiwell grid layer 122 and the base layer 124. Alternatively, the cell culture layer 20 can be received on a cell culture layer receiving surface, for example, a cell culture plate, cell culture dish, or microscope slide, and the electrode layer can be disposed on the cell culture layer 20. In yet other embodiments, a first electrode layer can be disposed on a cell culture layer receiving surface, for example, a cell culture plate, cell culture dish, or microscope slide, the cell culture layer 22 can be disposed on the electrode layer, and a second electrode layer can be disposed on the cell culture layer 20. In still other embodiments, an electrode or electrodes can be incorporated into the cell culture layer 20. In yet other embodiments, the electrodes may be provided as spikes or needles to facilitate contact with the neurons growing in the neuronal channel 32 and / or the neuronal and muscle cells growing in the neuromuscular junction chamber 30.

[0160] In some embodiments, multiple electrode layers may be provided according to any combination of the above-mentioned locations.

[0161] It is understood that the electrode layer can be provided in close proximity to, or in direct or indirect contact with, the in vitro model NMJ. The proximity of the electrode layer to the neuronal channel 32 and / or neurons growing within the neuromuscular junction chamber 30 facilitates the application of electrical stimulation to the NMJ and the recording of electrical signals from the NMJ, thereby allowing for the measurement of responses from muscle cells to stimulation of the neuronal cells, e.g., by contraction, or responses from neurons to stimulation of the muscle cells. By combining the electrodes with the cell culture layer 22, neuronal and muscle cell function can be determined at the in vitro model NMJ, e.g., in response to electrical impulses or calcium signals, and the overall function of the NMJ can be determined.

[0162] In some embodiments, the same electrode can be configured to perform different actions sequentially. For example, the actions can be collecting, recording, measuring, and / or detecting a cellular response to a stimulus. For example, an electrode can be configured to collect a signal at a given time point, and at a subsequent time point, the electrode can be configured to provide an electrical signal. In some embodiments, the electrode can be configured to detect an optical signal or an electrical signal.

[0163] In some embodiments, the distribution of electrodes across the surface of the electrode layer can be such that the electrodes are not provided randomly across the cell culture layer 22, but rather follow the orientation of the neuronal outgrowths growing within the neuronal channel 32 or the orientation of the muscle cells extending between the first and second struts 40, 42. The distribution of electrodes can also include a first set of electrodes that follow the orientation of the neuronal outgrowths growing within the neuronal channel 32 and a second set of electrodes that follow the orientation of the muscle cells extending between the first and second struts 40, 42. Providing the electrodes in such a configuration can provide an organized format of electrodes, e.g., for stimulation of neuronal or muscle cells or for detection of signals from neuronal or muscle cells, and thus better target the function of the electrodes.

[0164] In some embodiments, the electrodes can include at least one metal electrode, at least one metal oxide electrode, at least one carbon electrode, a multi-electrode array, and / or at least one field effect transistor detector.

[0165] In some embodiments, the cell culture device 20 can include any other type of sensor capable of stimulating cells or measuring the response of cells to a stimulus, e.g., the sensor can stimulate neurons and / or muscle cells, measure the response from neurons and / or muscle cells to a stimulus, provide an output, and / or receive an input. Examples of sensors can include, e.g., optical sensors or transducers, chemical sensors, and electrical sensors or transducers. The sensor can be provided according to any of the embodiments described above with respect to the electrodes.

[0166] Thus, the cell culture device can include any type of electrodes and / or sensors in contact with the neuronal cells growing in the neuronal channel 32 and / or the neuronal and muscle cells growing in the neuromuscular junction chamber 30 and configured to provide an output to and / or receive an output from the neuronal and / or muscle cells. The output provided or received can be any one of a chemical output, an electrical output, and a physical output.

[0167] In some embodiments, the cell culture device 20 can include an electrode set disposed in proximity to the in vitro temporomandibular joint model. The electrode set can include at least one electrode configured to collect electrical signals associated with at least a portion or at least one compartment of the in vitro model of the NMJ. The electrode set can take the form of an electrode layer as described above, but can also take different forms. The electrode set can include more electrodes. The electrodes allow for providing electrical readouts consisting of one or more of electrograms, impedance spectroscopy, voltammetry, and amperometry.

[0168] In some embodiments, the cell culture device 20 can include electronic devices in ohmic contact with the electrodes described above. The electronic devices can include, for example, sensing devices or stimulators, and can be configured to provide electrical readouts comprising one or more of electrograms, impedance spectroscopy, voltammetry, and amperometry. The electronic devices can be located, for example, in or near reservoirs of a cell culture dish or wells of a cell culture plate.

[0169] Alternative embodiments of neuronal channels Alternative embodiments of neuronal channel configurations will now be described in more detail.

[0170] As discussed above, when the abutment wall 66 is concave, the combination of the first portion side wall 52 and the abutment wall 66 can form a cup. Figure 10 illustrates an exemplary embodiment of the first portion 44 having a cup shape. Although not shown in Figure 10, it should be understood that the second portion 46 can include a second portion anterior wall or a second portion posterior wall, or both, disposed inwardly toward the centerline of the neuronal channel 32 along the z-axis such that the second portion 46 has a reduced channel height h compared to the channel height of the first portion 44.

[0171] In some embodiments, the first portion 44 can include a converging portion that is frustoconical. In other embodiments, the first portion 44 can include a converging portion that is frustopyramidal. In such embodiments, the transition from the first portion 44 to the second portion 46 can be considered to be located where the cross-section of the neuronal channel 32 becomes small enough to prevent one or more neurospheres from continuing to migrate further toward the neuromuscular junction chamber 30.

[0172] 15 and 17, in some embodiments, the cell culture layer 22 can further include a gel seeding inlet 68 in fluid communication with the neuromuscular junction chamber 30 via a gel seeding channel 70. The gel seeding inlet 68 and associated gel seeding channel 70 can contribute to facilitating the production of neuromuscular junctions embedded in a predetermined matrix following addition of a gel solution to the gel seeding inlet 68. Thus, when the gel seeding inlet 68 is present, a gel solution, which can include, for example, Matrigel™, Geltrex™, fibrin, collagen, or other types of basement membrane matrix or extracellular membrane matrix (ECM), and suspended muscle cells can be seeded into the gel seeding inlet 68, and optionally, the gel solution can also be seeded into one of the first and second muscle cell reservoirs 26, 28 or into both the first and second muscle cell reservoirs 26, 28. In some embodiments, the presence of a gel seeding inlet 68 facilitates providing a greater degree of control over the introduction and seeding of muscle cells present within the neuromuscular junction chamber 30.

[0173] 17 and 18, in some embodiments, the second portion 46 of the neuron channel 32 can include a microchannel 72. The microchannel 72 in the second portion 46 has a reduced channel height h compared to the channel height h of the first portion 44. As used herein, a "microchannel" refers to a channel having, for example, a width of about 3 μm to about 500 μm, a height of about 3 μm to about 500 μm, and a cross-sectional area of ​​about 9 μm. 2 to about 0.25μm 2It is meant to be in the range of 0.1 mm to 1.0 mm. However, it is important to note that these ranges are given by way of example only, and other dimensions of the microchannels may also be implemented. In some embodiments, the presence of the microchannels 72 can provide a preferential direction for axonal growth towards the neuromuscular junction chamber 30 and facilitate retention of the cell bodies of one or more neurospheres within the first portion 44 of the neuronal channel 32. When the second portion 46 includes microchannels, techniques such as, for example, microfabrication or additive manufacturing can be used to fabricate the cell culture layer 22. These techniques can also be used when fabricating the cell culture layer 22 without microchannels.

[0174] 19-21, the second portion 46 may have a reduced channel height h compared to the channel height h of the first portion 44, while including one or more channels located in a peripheral region of the second portion 46 of the neuronal channel 32. More particularly, referring to FIG. 21, the second portion 46 includes a first channel 74 located on a first side thereof and a second channel 76 located on a second side thereof opposite the first side, the first channel 74 and the second channel 76 being separated by a solid region extending throughout the entire channel height h of the second portion 46. In this embodiment, the cell bodies of one or more neurospheres may rest against the abutment wall 66, and axons extending from one or more neurospheres may extend within the first and second channels 74, 76 to reach the neuromuscular junction chamber 30 via the second channel openings 78a, 78b, respectively.

[0175] The diagrams shown in Figures 10, 13, 16, 18, 21, and 23 are schematic representations of corresponding molds that may be used to fabricate a given cell culture layer. It will therefore be understood that to obtain a representation of a given cell culture layer, voids must be considered as filled and solid features must be considered as voids and interpreted accordingly.

[0176] FIG. 28 shows an example of a cell culture layer 22 for use with a cell culture plate 120 that includes a multi-well grid layer 122 and a base layer (not shown). In the illustrated embodiment, the cell culture layer 22 includes two types of NMJ preparation units. The NMJ preparation unit identified with "B" is similar to the NMJ preparation unit shown in FIG. 1. The NMJ preparation unit identified with "A" differs from the one identified with "B" in that it includes two neuronal inlets 24. Due to the presence of the two neuronal inlets 24, the neuronal channel 32 includes two first portions 44 that join into a single second portion 46 downstream of the two first portions 44. Thus, each of the first portions 44 of the neuronal channel 32 is connected to a corresponding one of the neuronal inlets 34.

[0177] FIG. 29 shows an example of a cell culture device 20 including a cell culture layer 22 including a neuron inlet 24, a first muscle cell reservoir 26, a second muscle cell reservoir 28, and a neuron channel 32 extending between the neuron inlet 24 and the neuromuscular junction chamber 30. The neuron channel 32 includes a first portion 44 having a first partial cross-section and a second portion 46 downstream of the first portion 44 and having a second partial cross-section. In this embodiment, the second portion 46 of the neuron channel 32 includes longitudinally extending supports 80 configured as rectangular supports 82. The longitudinally extending supports 80 are spaced apart from one another to define a channel therebetween. The longitudinally extending supports 80 extend the neuron channel 32 in the longitudinal direction, further contributing to reducing the cross-section of the second portion 46 of the neuron channel 32 while allowing the neuron extensions of the neurosphere to reach the neuromuscular junction chamber 30. In some embodiments, the presence of the longitudinally extending supports 80 can help to further stabilize the portion of the neurosphere containing the neuronal cell body relative to the first portion 44 of the neuronal channel 32, i.e., prevent its downstream migration towards the neuromuscular junction chamber 30. In this embodiment, the rectangular supports 82 have respective upstream cross-sections 86 that are substantially aligned with the abutting walls 66 of the neuronal channel 32. It should be appreciated that in other embodiments, the respective upstream cross-sections 86 can be aligned differently relative to the abutting walls 66 of the neuronal channel 32.

[0178] Figure 30 illustrates an embodiment similar to Figure 29 in which the longitudinal supports 80 are provided as triangular supports 84. In this embodiment, apexes 88 (or tops) of the triangular supports 84 are provided to coincide with respective transitions between two adjacent faces of the abutting walls 66 of the neuronal channel 32. It should be appreciated that in other embodiments, each apex 88 may be aligned differently relative to the abutting walls 66 of the neuronal channel 32.

[0179] FIG. 31 shows an example of a cell culture device 20 including a cell culture layer 22 including a neuron inlet 24, a first muscle cell reservoir 26, a second muscle cell reservoir 28, and a neuron channel 32 extending between the neuron inlet 24 and a neuromuscular junction chamber 30. The neuron channel 32 includes a first portion 44 having a first partial cross-section and a second portion 46 downstream of the first portion 44 and having a second partial cross-section. In this embodiment, the first portion 44 of the neuron channel 32 includes an hourglass-shaped portion 90. The hourglass-shaped portion 90 contributes to reducing the width w of the first portion 44 of the neuron channel 32. In some embodiments, the presence of the hourglass-shaped portion 90 can contribute to further stabilizing the portion of the neurosphere including the neuron cell body in the first portion 44 of the neuron channel 32 by preventing migration upstream toward the neuron inlet 24.

[0180] 32 and 33 show an example of a cell culture device 20 including a cell culture layer 22 including a neuron inlet 24, a first muscle cell reservoir 26, a second muscle cell reservoir 28, and a neuron channel 32 extending between the neuron inlet 24 and a neuromuscular junction chamber 30. The neuron channel 32 includes a first portion 44 having a first partial cross section and a second portion 46 downstream of the first portion 44 and having a second partial cross section. In this embodiment, the first portion 44 of the neuron channel 32 includes posts 92 configured as triangular pillars, the posts 92 being spaced apart from one another. The posts 92 extend downward from an upper surface of the first portion 44 of the neuron channel 32 (with reference to when the cell culture layer is deposited on a substantially horizontal surface) and are provided at predetermined locations along the length of the neuron channel 32 such that a neurosphere is positioned between the posts 92 and the abutting wall 66. Thus, the post 92 can further contribute to stabilizing the neurosphere in the neuronal channel 32. More specifically, the portion of the neurosphere including the neuronal cell body in the first portion 44 of the neuronal channel 32 can be maintained by preventing it from migrating upstream toward the neuronal inlet 24. The shape of the post 92 can vary. In the embodiment shown in FIG. 32, the post 92 is in the shape of a regular triangular prism (including an equilateral triangle at the base), while in the embodiment shown in FIG. 33, the post 92 is in the shape of an irregular triangular prism (including an irregular triangle at the base). It should be understood that in other embodiments, the post 92 can be shaped differently from those shown in FIGS. 32 and 33. For example, the post 92 can be in the shape of a rectangular prism or a cylinder.

[0181] Several alternative embodiments have been described and illustrated herein. The implementations of the technology described above are intended to be exemplary only. Those skilled in the art will appreciate that the features of the individual embodiments and possible combinations and variations of the components are possible. Those skilled in the art will further appreciate that any embodiment may be provided in any combination with other embodiments disclosed herein. It will be appreciated that the technology may be embodied in other specific forms without departing from its central characteristics. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, and the technology is not to be limited to the detailed description given herein. Thus, while specific embodiments have been illustrated and described, it will be understood that numerous modifications are possible.

Claims

1. A cell culture apparatus for preparing an in vitro model of the neuromuscular junction, The device comprises a cell culture layer (22), and the cell culture layer is (a) within which there is a nerve cell entrance configured to receive nerve cells, (b) First and second myocyte reservoirs, wherein at least one of the first and second myocyte reservoirs is configured to seed myocytes therein, (c) A neuromuscular junction chamber extending between the first and second muscle cell reservoirs and in fluid communication with them, in order to form the neuromuscular junction and enable co-culture of the nerve cells and the muscle cells, (d) A nerve cell channel extending between the nerve cell inlet and the neuromuscular junction chamber and communicating with them fluidly, wherein the nerve cell channel is (i) A first portion (44) having a first partial cross-section of a size that holds the nerve cell body of the nerve cell, (ii) A second portion (46) provided downstream of the first portion, the second portion having a second cross-section smaller than the cross-section of the first portion, the second cross-section being sized to prevent the nerve cell body from entering the second portion, and a nerve cell channel including the second portion A cell culture apparatus characterized by having the following:

2. The cell culture apparatus according to claim 1, characterized in that the first portion has a first partial side wall, a first partial front wall, and a first partial rear wall, and the second portion has a second partial side wall, a second partial front wall, and a second partial rear wall.

3. The cell culture apparatus according to claim 2, characterized in that the transition of the side wall from the first partial side wall to the second partial side wall is substantially linear, and the nerve cell channel has substantially the same channel width between the first and second portions.

4. The cell culture apparatus according to claim 2, characterized in that the first and second partial sidewalls are provided such that the nerve cell channels have a smaller channel width in the second portion compared to the first portion.

5. The cell culture apparatus according to claim 2, further comprising a cell culture dish, cell culture plate, or microscope slide having a cell culture layer receiving surface on which the cell culture layer is deposited.

6. The cell culture apparatus according to claim 5, characterized in that the first partial rear wall is provided by the cell culture layer receiving surface of the cell culture dish, the cell culture plate, or the microscope slide.

7. The cell culture apparatus according to claim 5, characterized in that the first partial posterior wall and the second partial posterior wall are provided by the cell culture layer receiving surface of the cell culture dish, the cell culture plate, or the microscope slide.

8. The cell culture apparatus according to claim 2, characterized in that the second anterior wall is provided inward from the first anterior wall, and the nerve cell channel has a smaller channel height in the second portion compared to the first portion, thereby providing at least partially a smaller second portion cross-section compared to the first portion cross-section.

9. The cell culture apparatus according to claim 2, characterized in that the second posterior wall is provided inward of the first posterior wall, and the nerve cell channel has a smaller channel height in the second portion compared to the first portion, thereby providing a smaller second portion cross-section compared to the first portion cross-section.

10. The cell culture apparatus according to claim 2, characterized in that the first portion of the nerve cell channel includes a frustoconical convergence portion that converges inward, or a frustopyroconical convergence portion that converges inward.

11. The cell culture apparatus according to any one of claims 2 to 10, characterized in that the transition from the first partial anterior wall to the second partial anterior wall includes a stepwise change that defines a contact wall extending laterally across at least a portion of the neuronal channel.

12. The cell culture apparatus according to claim 11, characterized in that the contact wall converges inward toward the central line of the nerve cell channel.

13. A cell culture apparatus according to any one of claims 1 to 10, further comprising a first support provided in the neuromuscular junction chamber adjacent to the first myocyte reservoir, and a second support provided in the neuromuscular junction chamber adjacent to the second myocyte reservoir, wherein the first and second supports extend upward and function as fixing positions for the respective myocytes.

14. The cell culture apparatus according to any one of claims 1 to 10, further comprising a gel seeding inlet that is in fluid communication with the neuromuscular junction chamber and faces the nerve cell inlet, wherein the gel seeding inlet is configured to seed a gel into the neuromuscular junction chamber.

15. The cell culture apparatus according to any one of claims 1 to 10, characterized in that the nerve cells are provided as a cluster of nerve cell bodies, including axons extending from the cell bodies, the first portion of the nerve cell channel is configured to receive the cluster of cell bodies, and the second portion of the nerve cell channel is configured to direct the axons toward the neuromuscular junction chamber.

16. The cell culture apparatus according to any one of claims 1 to 10, characterized in that the second portion of the nerve cell channel includes a microchannel for inducing axon growth.

17. The cell culture apparatus according to claim 1, characterized in that at least one of the nerve cell inlet, the first muscle cell reservoir, and the second muscle cell reservoir is configured to accept a test substance into it.

18. The cell culture apparatus according to claim 1, further comprising an electrode provided in close proximity to the cell culture layer.

19. The cell culture apparatus according to claim 18, characterized in that the electrode forms a part of the electrode layer.

20. The cell culture apparatus according to claim 19, characterized in that the electrode layer is disposed below the cell culture layer or superimposed on the cell culture layer.

21. The cell culture apparatus according to claim 19, characterized in that the electrode layer is integrated with the cell culture layer.

22. The cell culture apparatus according to any one of claims 18 to 21, further comprising an electronic device ohmic-connected to the electrode.

23. The cell culture apparatus according to claim 1, further comprising a sensor configured to stimulate the nerve cells, measure the response from the nerve cells to the stimulation, provide an output, or receive an input.

24. The cell culture apparatus according to claim 1, further comprising a sensor configured to stimulate the muscle cells, measure the response from nerve cells to the stimulation, provide an output, or receive an input.

25. The cell culture apparatus according to claim 23 or 24, characterized in that the sensor includes an optical or electrical transducer.

26. A cell culture apparatus for preparing an in vitro model of the neuromuscular junction, The cell culture layer comprises a cell culture layer, (a) a nerve cell channel configured to receive nerve cells, (i) A first portion having a first partial cross-section, (ii) A second portion having a smaller cross-section than the first portion and provided downstream of the first portion A neuronal channel having, (b) A neuromuscular junction chamber configured to receive muscle cells in order to fluidly communicate with the nerve cell channels, enable co-culture of the nerve cells and the muscle cells, and form the neuromuscular junction, comprising first and second supports spaced apart on both sides of the nerve cell channels for culturing the muscle cells between them, and A cell culture apparatus characterized by having the following:

27. The cell culture apparatus according to claim 26, characterized in that the first portion includes a first partial side wall, a first partial anterior wall, and a first partial posterior wall, and the second portion includes a second partial side wall, a second partial anterior wall, and a second partial posterior wall.

28. The cell culture apparatus according to claim 27, characterized in that the transition from the first partial sidewall to the second partial sidewall is substantially linear, and the neuronal channel has substantially the same channel width between the first and second portions.

29. The cell culture apparatus according to claim 27, characterized in that the first and second partial sidewalls are provided such that the nerve cell channels have a smaller channel width in the second portion compared to the first portion.

30. The cell culture apparatus according to claim 27, further comprising a cell culture dish, cell culture plate, or microscope slide having a cell culture layer receiving surface on which the cell culture layer is deposited.

31. The cell culture apparatus according to claim 30, characterized in that the first partial rear wall is provided by the cell culture layer receiving surface of the cell culture dish, the cell culture plate, or the microscope slide.

32. The cell culture apparatus according to claim 30, characterized in that the first partial posterior wall and the second partial posterior wall are provided by the cell culture layer receiving surface of the cell culture dish, the cell culture plate, or the microscope slide.

33. The cell culture apparatus according to claim 27, characterized in that the second anterior wall is provided inward from the first anterior wall such that the nerve cell channels have a smaller channel height in the second portion compared to the first portion, thereby providing at least partially a second portion cross-section that is smaller than the first portion cross-section.

34. The cell culture apparatus according to claim 27, characterized in that the second posterior wall is provided inward from the first posterior wall such that the nerve cell channels have a smaller channel height in the second portion compared to the first portion, thereby providing a smaller second portion cross-section compared to the first portion cross-section.

35. The cell culture apparatus according to claim 27, characterized in that the first portion of the nerve cell channel has a frustoconical convergence portion that converges inward, or a frustopyroconical convergence portion that converges inward.

36. The cell culture apparatus according to any one of claims 27 to 35, characterized in that the transition from the first partial anterior wall to the second partial anterior wall includes a stepwise change that defines a contact wall extending laterally across a portion of the neuronal channel.

37. The cell culture apparatus according to claim 36, characterized in that the contact wall converges inward toward the central line of the nerve cell channel.

38. The cell culture apparatus according to any one of claims 27 to 35, further comprising a gel seeding inlet that is in fluid communication with the neuromuscular junction chamber, faces the nerve cell inlet, and seedes a gel into the neuromuscular junction chamber.

39. The cell culture apparatus according to any one of claims 27 to 35, wherein the nerve cells are provided as clusters of nerve cell bodies and axons extending away from the cell bodies, the first portion of the nerve cell channel is configured to receive the clusters of cell bodies, and the second portion of the nerve cell channel is configured to direct the axons toward the neuromuscular junction chamber.

40. The cell culture apparatus according to any one of claims 27 to 35, characterized in that the second portion of the nerve cell channel includes a microchannel for inducing axon growth.

41. The cell culture apparatus according to any one of claims 27 to 35, characterized in that the nerve cell entrance is configured to receive the test substance into it so that the test substance reaches the neuromuscular junction chamber.

42. A method for analyzing a patient's biological material using an in vitro model of the neuromuscular junction, (a) A step of supplying nerve cells to nerve cell channels provided in a cell culture layer, wherein the nerve cell channels are (i) A first portion having a first partial cross-section, (ii) A process comprising: a second portion having a smaller cross-section than the first portion and provided downstream of the first portion; (b) A step of supplying muscle cells to a neuromuscular junction chamber provided in the cell culture layer and in fluid communication with the nerve cell channel in order to enable co-culture of the nerve cells and the muscle cells and to form the neuromuscular junction, (c) A step of bringing at least one of the nerve cells and muscle cells into contact with the biological material from the patient, (d) A step of measuring functional parameters related to the neuromuscular junction. A method for providing this.

43. The method according to 42, further comprising the step of adding a test substance to at least one of the nerve cell channel and the neuromuscular junction chamber, wherein the step of determining the functional parameters relating to the neuromuscular junction is performed following the addition of the test substance.

44. The method according to 43, characterized in that the test substance comprises at least one of a biological material and a chemical material.

45. A method for diagnosing a patient's condition using an in vitro model of the neuromuscular junction, (a) A step of supplying biological material from a patient to a cell culture device, wherein the cell culture device has a cell culture layer, and the cell culture layer is (i) A nerve cell channel configured to receive nerve cells inside, wherein the nerve cell channel has a first portion having a first partial cross-section, and a second portion having a second partial cross-section smaller than the first portion and provided downstream of the first portion, (ii) A neuromuscular junction chamber configured to be in fluid communication with the nerve cell channel and to accept muscle cells therein, the neuromuscular junction chamber enabling co-culture of the nerve cells and the muscle cells and forming the neuromuscular junction, comprising the steps of: (b) A step of determining functional parameters related to the neuromuscular junction, wherein the functional parameters provide information related to the patient's condition. A method for providing this.

46. The method according to claim 45, further comprising the step of adding a test substance to at least one of the nerve cell channel and the neuromuscular junction chamber.

47. The method according to 46, characterized in that the step of determining the functional parameters related to the neuromuscular junction is performed following the addition of the test substance.

48. The method according to 45, characterized in that the step of determining the functional parameters related to the neuromuscular junction is performed before and after the addition of the test substance.

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