Tissue analysis system, co-culture casting system, and method for functional interrogation of co-cultured tissue

Abstract

The tissue analysis system and the co-culture casting system comprise at least a porous co-culture casting plate and a muscular tissue casting plate. The use method comprises the following steps: inoculating a plurality of neuron culture zones of a porous co-culture casting plate with a living neuron material, inoculating a plurality of muscle culture zones of a muscle tissue casting plate with a living muscle material, and culturing a plurality of co-culture tissues in the porous co-culture casting plate, these co-cultured tissues comprise a muscle cell culture cultured from a living muscle material and in situ neurospheres matured from the living neuron material in cell aggregation characteristics, and constricting the innervated portion of the living muscle material in each co-cultured tissue by stimulating the living neuron material.

Description

Cross-references to related applications

[0001] This patent application claims priority to U.S. Provisional Application No. 63 / 508,551, filed June 16, 2023, and U.S. Provisional Application No. 63 / 621,771, filed January 17, 2024, the entire disclosure of which is incorporated herein by reference.

[0002] Government License Rights Statement

[0003] This invention was made with government support under license TR004795 granted by the National Institutes of Health in the United States. The government holds certain rights in this invention. Technical Field

[0004] This disclosure relates to the field of tissue engineering, such as modeling in neuromuscular biology. Background Technology

[0005] Within the human body, the musculoskeletal system is responsible for a range of vital processes, from breathing to movement. Control of muscle tissue within the musculoskeletal system is exercised by the central nervous system through neuromuscular junctions and related structures (known as the neuromuscular system). Therefore, diseases of the neuromuscular system lead to serious, progressive diseases similar to those of the musculoskeletal system. Current models of the neuromuscular system rely on animal models, which have significant limitations and cannot generate effective treatments for clinical use. Therefore, neuromuscular system models built in a controlled, engineered environment using human cells will enable the study of disease mechanisms, the discovery of treatments, and the screening of compounds for toxicity.

[0006] To this end, previous work has attempted to develop neuromuscular junction (“NMJ”) models by culturing motor neurons and skeletal muscle cells in two-dimensional and three-dimensional devices. However, each known model has limitations that hinder its widespread adoption in profitable drug discovery pathways. These limitations include non-scalable manufacturing techniques, poor reproducibility between different laboratories, the high level of technical expertise required for assembly, low-throughput measurement techniques, low-throughput seeding methods, low biomimicry, and a narrow measurement window due to low innervation percentage.

[0007] Specifically, the initial models utilized simple two-dimensional co-culture systems based on standard or slightly modified culture dishes. These systems cannot replicate the three-dimensional structure of the neuromuscular junction. Furthermore, two-dimensional skeletal muscle cultures cannot generate systems that allow for the examination of neuromuscular diseases and physiology over time, a key feature for studying progressive neuromuscular conditions. Finally, the lack of compartmentalization for cell types limits the ability to provide unique stimulation to cells without the use of cell modification or chemical stimulation, thus reducing the applicability of these systems and the cell types that can be used.

[0008] Microfluidic devices have been proposed that allow skeletal muscle cells and neurons to communicate while maintaining cell body separation. However, these systems remain limited by the two-dimensional environment, the finite time frame for two-dimensional muscle culture, and the inability to provide direct readout of skeletal muscle function.

[0009] Examples of three-dimensional compartmentalized and non-compartmentalized devices have been proposed. Non-compartmentalized systems suffer from the same limitations as two-dimensional non-compartmentalized systems: chemical stimulation can lead to cell death, and optogenetic solutions require extensive genetic engineering, thus hindering the widespread adoption of these systems. Compartmentalized systems suffer from throughput limitations (e.g., because measurements are performed serially) and limited applications (e.g., because the culture of neuronal spheroid components is carried out in a two-dimensional environment).

[0010] None of the aforementioned devices or methods demonstrated in situ generation of neuronal components or axonal guidance beyond muscle attraction. Therefore, due to fundamental limitations in neuronal survival and NMJ formation, as well as limitations in practical availability and reproducibility, the aforementioned devices and methods cannot be used for profitable drug discovery pathways. Summary of the Invention

[0011] This disclosure provides systems, apparatus, and methods that enable real-time, high-throughput reading of muscle function in response to neural input in a three-dimensional system, thereby allowing for modeling of the neuromuscular system and related diseases.

[0012] According to one aspect, this disclosure provides methods for using a tissue analysis system including a co-culture casting system, the methods comprising: seeding a plurality of neuronal culture compartments of a porous co-culture casting plate with live neuronal material, wherein each neuronal culture compartment contains a plurality of cell aggregation features configured to aggregate the live neuronal material into cell aggregates; seeding a plurality of muscle culture compartments of a muscle tissue casting plate with live muscle material; culturing a plurality of co-cultured tissues in the porous co-culture casting plate, each co-cultured tissue comprising a muscle cell culture cultured from the live muscle material and an in situ neurosphere formed from the live neuronal material maturing in the cell aggregation features; and stimulating the live neuronal material to cause contraction of the innervated portion of the live muscle material in each co-cultured tissue.

[0013] According to another aspect, this disclosure provides a tissue analysis system and a co-culture casting system, comprising: a porous co-culture casting plate including a plurality of neuron culture chambers, each neuron culture chamber having a cell aggregation region formed on its bottom surface, each cell aggregation region containing a plurality of cell aggregation features; and a porous muscle tissue casting plate.

[0014] According to another aspect, this disclosure provides a method for modeling neuromuscular junctions in multiple co-cultured tissues, comprising: seeding multiple neuronal culture compartments of a multiwell plate with live neuronal material; casting multiple muscle cell cultures containing live muscle material; culturing multiple co-cultured tissues in the multiwell plate, the co-cultured tissues containing live muscle material and in situ neurospheres matured from the live neuronal material; and stimulating the live neuronal material to cause contraction of the innervated portion of the live muscle material in each co-cultured tissue.

[0015] According to another aspect, this disclosure provides a method for modeling multiple neuromuscular junctions, comprising: seeding multiple neuronal culture chambers of a multi-well plate with live neuronal material by directly thawing cells containing motor neurons in multiple neuronal culture chambers, wherein each neuronal culture chamber contains cell aggregation features configured to aggregate the live neuronal material into cell aggregates; casting multiple muscle cell cultures containing live muscle material; culturing multiple co-culture tissues in the multi-well plate, the co-culture tissues containing live muscle material and in situ neurospheres matured from the live neuronal material, partially achieved by remodeling the interface between a connecting hydrogel and a second hydrogel containing the live muscle material; and simultaneously stimulating the live neuronal material to cause the innervated portion of the live muscle material in each co-culture tissue to contract.

[0016] According to another aspect, this disclosure provides a method for modeling multiple neuromuscular junctions, comprising: seeding multiple culture units of a multiwell plate with live neuronal material by directly thawing cells containing motor neurons in multiple neuronal culture compartments, wherein each neuronal culture compartment contains cell aggregation features configured to aggregate the live neuronal material into cell aggregates; casting multiple muscle cell cultures containing live muscle material; culturing multiple co-culture tissues in the multiwell plate by connecting the live neuronal material and the live muscle material along a communication channel in each culture unit, the co-culture tissues comprising live muscle material and in situ neurospheres matured from the live neuronal material; and simultaneously stimulating the live neuronal material to cause contraction of the innervated portion of the live muscle material in each co-culture tissue.

[0017] Advantageously, the apparatus and methods described herein enable in-situ generation of neuronal components (single-cell or multi-celled), thereby reducing the need to manipulate neuronal components after maturation. In another aspect, these systems utilize microfluidic flow principles, enabling active control of axonal growth in three-dimensional tissues to ensure highly reproducible connections between neurons and skeletal muscle components. Furthermore, these systems enable parallel, real-time, label-free measurement of skeletal muscle in large numbers (≥24 tissues), significantly improving the throughput of any previously proposed system.

[0018] These devices and methods circumvent the drawbacks of known systems and methods by enabling parallel functional measurements (e.g., using optical and / or magnetic induction) and in-situ sphere creation, thereby eliminating the need for individual sphere handling. Furthermore, these devices and methods overcome throughput limitations and drive axonal guidance toward engineered muscle tissue within a three-dimensional hydrogel environment, better representing the process of neuromuscular junction development in vivo. Attached Figure Description

[0019] Figure 1 The co-culture tissue formed according to the method of this disclosure is shown.

[0020] Figure 2A A plan view of a porous co-grown casting plate according to one embodiment of the present disclosure is shown.

[0021] Figure 2B Showing Figure 2A Detailed plan view of a hole in a cast plate.

[0022] Figure 2C Showing Figure 2B Cross-sectional view of the hole.

[0023] Figure 2D Showing Figure 2B A three-dimensional diagram of the cell aggregation characteristics of the pores.

[0024] Figure 3A A plan view of another porous co-culture cast plate according to one aspect of this disclosure is shown.

[0025] Figure 3B Showing Figure 3A Detailed plan view of a hole in a cast plate.

[0026] Figure 3C Showing Figure 3B First cross-sectional view of the hole.

[0027] Figure 3D Showing Figure 3B Second cross-sectional view of the hole.

[0028] Figure 4A A tissue analysis system according to one aspect of this disclosure, a method using the tissue analysis system and a co-cultivation casting system, and a method for modeling multiple neuromuscular junctions are demonstrated.

[0029] Figure 4B Demonstrated the use of microstructure analysis systems and co-cultivation casting systems Figure 4A Exemplary method.

[0030] Figure 4C This demonstrates a method for modeling multiple neuromuscular junctions. Figure 4A Exemplary method.

[0031] Figure 4D This demonstrates a method for modeling multiple neuromuscular junctions. Figure 4A Additional exemplary methods are provided.

[0032] Figure 5 An example image of a cell formed according to this disclosure is shown.

[0033] Figure 6 An image of a muscle cell culture containing live skeletal muscle material is shown.

[0034] Figure 7A A diagram showing the contractile force trajectory of a co-cultured tissue formed according to the method described herein is presented.

[0035] Figure 7B A force trajectory diagram of the co-cultured tissue formed according to the method described herein is shown.

[0036] Figure 7C Electro-induced force maps of the tetanic response of co-cultured tissues formed according to the method described herein are shown.

[0037] Figure 7D Electro-evoked force maps of twitching responses in co-cultured tissues formed according to the methods described herein are shown.

[0038] Figure 8A A graph showing the active twitching force of co-cultured tissues formed according to the method described herein is presented.

[0039] Figure 8B A graph showing the time from shrinkage 10 to peak shrinkage of co-culture tissue formed according to the method described herein is presented.

[0040] Figure 8C A graph showing the time from peak contraction to relaxation of co-cultured tissues formed according to the method described herein is presented.

[0041] Figure 9A This is a trajectory diagram showing the response of a co-cultured tissue formed according to the method and apparatus of this disclosure to blue light.

[0042] Figure 9B Drawn Figure 9A Co-cultured tissues showed contractile forces in response to different blue light stimulation periods.

[0043] Figure 9C Drawn Figure 9A The percentage of contractile force generated by co-cultured tissues through optical stimulation of neuronal cell cultures compared to the contractile force achieved by direct electric field stimulation of muscle cell cultures.

[0044] Figure 9D Drawn Figure 9APercentage of contractile force captured by co-cultured tissue within 100 ms of blue light stimulation.

[0045] Figures 10A to 10C The diagram shows the contractile force trajectory of co-cultured tissue formed according to this disclosure before and after exposure to active botulinum neurotoxin complex serum type A (BoNT-A). Detailed Implementation

[0046] The devices and methods disclosed herein allow for the in situ culture of cell types relevant to neuromuscular biology within the device, guiding and controlling cell growth and intercellular interactions, while also allowing for real-time functional readouts of certain cell types, such as muscle cells co-cultured with neurons. These devices and methods can be adapted to a variety of user interventions to create controlled experimental conditions for studying musculoskeletal / neuromuscular diseases / conditions, such as neurotoxic potency assays.

[0047] As used herein, in situ means the original location where the cells initially formed (e.g., the original apparatus). For example, neurospheres and neuronal cell cultures formed in a casting plate are in situ as long as they remain in their formation location within the casting plate without being moved. Furthermore, the cells remain in situ despite growth, elongation, retraction, or other movements (e.g., axonal retraction). Additionally, the cells remain in situ within the apparatus regardless of whether the apparatus itself is moved. For example, neurospheres formed in a casting plate are in situ as long as the cells remain in their formation location within the casting plate without being moved, regardless of whether the casting plate is physically moved.

[0048] As used herein, “culture” means to cultivate or grow one or more cell populations, for example, in a hydrogel or other culture medium. Therefore, “co-culture” means to cultivate or grow two or more distinct cell populations in physical contact, for example, via a hydrogel or other culture medium. A co-cultured tissue is a tissue containing at least two distinct cell populations that have been co-cultured. A co-cultured tissue may include, for example, two, three, four, five, or more distinct cell populations. In any embodiment, the at least two cell populations may be biologically distinct (e.g., different cell types, such as neurons and muscle cells).

[0049] As used herein, the term hydrogel refers to a group of polymeric materials whose hydrophilic structure enables them to retain a large amount of water within their three-dimensional network. The polymers constituting a hydrogel can be naturally occurring, for example, purified from a natural source, or synthetic, for example, chemically synthesized. The polymer can be water-soluble or water-insoluble. The hydrogel's ability to absorb water derives from hydrophilic functional groups attached to the polymer backbone, while its resistance to dissolution derives from cross-linking between the network chains. Cross-linking can be chemical or physical; polymer cross-linking includes covalent cross-linking, ionic cross-linking, hydrogen bonding, and / or hydrophobic interactions. Natural polymers that form hydrogels include purified proteins (such as collagen and gelatin) and purified polysaccharides (such as starch, alginate, and agarose). Synthetic polymers that form hydrogels are prepared using chemical polymerization / synthetic methods. Hydrogels can be single-component, two-component, or multi-component systems comprising a three-dimensional network of polymer chains and water filling the spaces between the polymer macromolecules.

[0050] In some embodiments, the hydrogel may comprise a network of water-insoluble polymer chains. The hydrogel may comprise a water-swollen and cross-linked polymer network resulting from the reaction of one or more monomers. The hydrogel may comprise polymeric materials that exhibit swelling capacity and retain a significant amount of water within their structure but are not soluble in water. The hydrogel may comprise a colloidal gel in which water is the dispersion medium. Their hydrophilic structure enables them to retain a large amount of water in their three-dimensional network (e.g., the hydrogel may be superabsorbent and may contain 50%, 75%, 90%, 95%, and more than 99% water). The hydrogel may comprise natural and / or synthetic polymers. Due to their significant water content, the hydrogel may have a degree of flexibility very similar to natural tissues.

[0051] In a non-limiting example, the hydrogel contains collagen. Various concentrations of collagen can be used. For example, concentrations of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1 to 10 mg / ml or more of collagen in an aqueous solution (such as cell culture medium, saline, or water) can be used. The collagen-containing hydrogel optionally also includes one or more of the following proteins: laminin, nestin, heparan sulfate proteoglycan (characterized by adhesive properties), and growth factors (such as TGF-β and EGF). An example of such a collagen-containing hydrogel is MATRIGEL. ® Therefore, in some embodiments, the hydrogel contains MATRIGEL. ® (A gelatinous protein mixture derived from mouse tumor cells) or its equivalent. MATRIGEL ®MATRIGEL is a trade name for a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, manufactured and marketed by Corning Life Sciences. Therefore, "MATRIGEL" ® "It can be replaced with a gelatinous protein mixture from another natural, synthetic, or commercial source. MATRIGEL can be used." ® Or MATRIGEL ® Alternatives, such as MATRIGEL in ratios of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10. ® Collagen or collagen protein: MATRIGEL ® Proportion.

[0052] In some embodiments, the hydrogel comprises alginate or derivatives thereof, gelatin, collagen, agarose, natural or synthetic polysaccharides, polylactic acid, polyglycolic acid, poly(lysine), polyanhydride; poly(lactide-co-glycolic acid) (PLGA) polymer, polyamino acids, poly(oxyethylene), poly(ethylene oxide), poly(allylamine) (PAM), poly(acrylate), polyester, polyhydroxybutyrate and poly-ε-caprolactone, polyphosphazene, poly(vinyl alcohol), modified styrene polymer, poly(4-aminomethylstyrene), Pranic polyol, poloxamer, poly(uronic acid), poly(vinylpyrrolidone), and... / or a copolymer containing one or more of alginate or its derivatives, gelatin, collagen, agarose, natural or synthetic polysaccharides, polylactic acid, polyglycolic acid, poly(lysine), and polyanhydride; poly(lactide-co-glycolic acid) (PLGA) polymers, polyamino acids, poly(oxyethylene), poly(ethylene oxide), poly(allylamine) (PAM), poly(acrylate), polyesters, polyhydroxybutyrate and poly-ε-caprolactone, polyphosphazene, poly(vinyl alcohol), modified styrene polymers, poly(4-aminomethylstyrene), Pranic polyol, poloxamer, poly(uronic acid), and poly(vinylpyrrolidone).

[0053] The proposed devices and methods enable reproducible and robust co-culturing of neurons, muscle, and other neuromuscular-related cell types to form ex vivo neuromuscular junctions (NMJs). Specifically, these devices and methods provide co-cultured tissues comprising live neuronal material and live muscle material, which can be independently stimulated to assess NMJ function.

[0054] Live neuronal materials can comprise, for example, cells, organoids, and / or tissues containing neurons, including, for example, stem cell-derived cells, human and animal cell lines, primary human or animal bulk tissue explants, or isolated cells from primary humans or animals. Representative live neuronal materials include, for example, iPSC-derived motor neurons (e.g., those differentiated using small molecules and confirmed to contain motor neurons in the differentiated population via the transcription factor ISL-1 / 2), sensory neurons, motor neurons, interneurons, pyramidal cells, Purkinje cells, basket cells, chandelier cells, olfactory receptor neurons, dopaminergic neurons, serotonergic neurons, cholinergic neurons, GABAergic neurons, projection neurons, local circuit neurons, Schwann cells, oligodendrocytes, nociceptors, mirror neurons, hypothalamic neurons, astrocytes, and microglia. These cells may contain mutations or disease features associated with human diseases, or may present a good model of health, thus allowing for disease studies by comparison with healthy cultures. The living neuronal material may comprise any combination of the aforementioned cell types, optionally in a culture medium (e.g., a hydrogel) containing other cell types. Optionally, the living neuronal material may express proteins that act as gated ion channels, such as light-gated ion channels, including rhodopsin-1 and -2. Expression of such proteins can be induced, for example, by application of one or more promoters, such as tetracycline promoters or neuron-specific promoters. Advantageously, expression of such proteins can promote neuronal stimulation. For example, in some embodiments, blue light-sensitive rhodopsin expression in the living neuronal material promotes blue light-driven neuronal activation.

[0055] The proposed device provides one or more compartments in which single neurons can be seeded and subsequently matured in a non-adhesive environment that promotes neurosphere formation. A biological / non-biological hydrogel is then added to provide an adhesive matrix for neurite extension. The timing of hydrogel addition and the composition of the hydrogel can be manipulated to optimally promote robust neurite extension. The neurite matures into an axon with a motile growth cone.

[0056] Living muscle materials may comprise, for example, one or more of the following: cells containing muscle cells, organoids, and / or tissues, including, for example, stem cell-derived cells, human and animal cell lines, primary human or animal explants, or isolated cells from primary humans or animals. Representative living muscle materials include, for example, iPSC-derived myoblasts and primary human dermal fibroblasts (e.g., myoblasts characterized using desmin positivity, with a population desmin positivity rate >80%), myoblasts, skeletal muscle fibers (e.g., oxidative, oxidative-glycolytic, glycolytic), cardiomyocytes (e.g., cardiac muscle cells), smooth muscle cells, and satellite cells. Living muscle materials may comprise any combination of the aforementioned cell types, optionally in a culture medium (e.g., a hydrogel) containing other cell types (e.g., stromal cells). The living muscle materials may be assembled into engineered skeletal muscle tissue (EMT), such as those disclosed by Sniadecki et al. in International Patent Publication No. WO2017 / 156455A1, the entire contents of which are incorporated herein by reference.

[0057] Cell cultures containing living neuronal material are co-cultured with cell cultures containing living muscle material to form co-cultured tissues. Specifically, axons extending from the living neuronal material communicate with the living muscle material, for example, through a hydrogel interface. The axons can then extend through the intermediate space, allowing neuron-muscle interaction (i.e., nerve-innervated muscle), which will mature into a neuromuscular junction. In some embodiments, the interface between the living neuronal material and the living muscle material is remodeled to promote axonal extension, for example, by treating the interface with a serum culture medium. In some embodiments, hydrogel patterning and growth factor accumulation / gradients can guide growth cone movement through the device to position the axonal tip near the introduced muscle.

[0058] Therefore, these methods and devices allow for the individual maturation of neurons and skeletal muscle to provide tissues ready for interaction (see, for example, Figure 1 and Figure 6 This is a feature that was missing from previous neuromuscular co-culture systems.

[0059] Figure 1 A representative NMJ co-culture tissue 102 according to one aspect of this disclosure is shown, namely, according to the methods described herein and / or utilizing one or more devices described herein (such as, Figures 2A to 2DThe co-cultured tissue 102 comprises a living muscle material 104 (visible as a translucent substance having an integral bone shape) extending between a plurality of tissue fixation members 106a, 106b. Each tissue fixation member 106a, 106b may be, for example, a flexible column, hook, clamp or a relatively rigid column, hook or clamp, as described, for example, as described by Sniadecki et al. in International Patent Publication No. WO2017 / 156455A1.

[0060] The co-cultured tissue 102 also contains living neuronal material 108, in Figure 1 This is visible as discrete dark gray neurospheres extending throughout the co-cultured tissue 102. From... Figure 1 It is evident that the neurospheres are patterned throughout the co-cultured tissue 102, rather than randomly. The distribution of these neurospheres can be controlled by adjusting aspects of the methods and apparatus described herein, for example, using the cell aggregation characteristics of the casting plate. Advantageously, the controlled distribution of neurospheres within the co-cultured tissue 102 facilitates NMJ modeling because individual neurospheres (such as neurosphere 110) can control the limited volume of the underlying living muscle material 104.

[0061] The living muscle material 104 can contract spontaneously, under direct stimulation, and / or under the control of the living neuron material 108, which in turn can be stimulated by electrical, optical, or other means. When the living muscle material 104 contracts, one or both of the tissue fixation elements 106a and 106b deflect relative to their rest position. This deflection can be measured by optical, magnetic, or other means and is used to determine the contractile force of the co-cultured tissue 102. The contractile force and other parameters of the co-cultured tissue 102 can be analyzed over time to assess the function of the NMJ.

[0062] Figures 2A to 2D A porous neurosphere casting plate 200 according to a representative embodiment of this disclosure is shown. The casting plate 200 facilitates the casting of cell cultures containing living neuronal material (i.e., mature neurospheres), i.e., neuronal cell cultures.

[0063] In some embodiments, any of the casting plates described herein (including casting plate 200) is part of a consumable assembly that includes at least one of a muscle tissue casting plate, a maintenance plate, and / or a cover plate. In some embodiments, any of the casting plates described herein (including casting plate 200) is part of a system that includes at least one of a muscle tissue casting plate, a maintenance plate, an optical stimulation device (e.g., a stimulation cover), an electrical stimulation device, and / or an instrument configured to measure the contraction of co-cultured tissue formed in the casting plate. In some embodiments, any of the casting plates described herein (including casting plate 200) is part of an assay (e.g., a neurotoxin assay).

[0064] The casting plate 200 includes multiple culture units 202a, 202b, ... n, arranged in a layout conforming to the Society for Biomolecular Screening (SBS) standards, such as 24, 48, or 96 compartment layouts. For example, the external dimensions of the casting plate 200 can be configured for compatibility with the instrument described in International Patent Publication No. WO2021 / 173887A1, the entire contents of which are incorporated herein by reference. The casting plate 200 includes twenty-four neuron culture compartments arranged in a 4×6 array; however, this is representative and not limiting. In some embodiments, each culture unit 202a-n is identical; in other embodiments, one or more culture units 202a differ from one or more other culture units, for example, to facilitate different experiments.

[0065] The casting plate 200 can be used with or independently of the methods described herein. For example, co-cultured tissues can be cultured directly in each culture unit 202a-n, for instance, by connecting a first cell culture containing live neuronal material (neuronal cell culture) to a second cell culture containing live muscle material (muscle cell culture, such as EMT). The cell culture containing live muscle material can be placed in multiple tissue fixation devices (e.g., columns, hooks, clamps, etc., such as... Figure 4A A grid is formed between the cells (as shown), and the two cell cultures are co-cultured in a second casting plate before being transferred to a casting plate 200.

[0066] In some embodiments, the cast plate 200 may be at least partially composed of rigid thermoplastic to improve manufacturability and prevent the absorption of molecules held in the solution within the culture unit.

[0067] Go to Figure 2B and Figure 2C A representative culture unit 202a will now be described. The features described regarding culture unit 202a exist in each culture unit of the cast plate 200.

[0068] Culture unit 202a is formed as a blind hole in cast plate 200. Neuron culture chambers are formed as casting grooves 204 in the bottom (blind) surface of the holes and are configured to seed live neuronal material. Any neuron culture chamber described herein, in any embodiment, is configured to allow for the long-term maintenance of neurons and supporting cells therein.

[0069] The size and shape of the casting groove 204 typically correspond to a portion of a muscle tissue cell culture (e.g., EMT) with which the neuronal cell culture will be co-cultured. In the representative embodiment shown, the casting groove 204 has a length L of about 10 mm to about 20 mm (e.g., 10 mm to 15 mm), a width W of about 2 mm to about 10 mm (e.g., 3 mm to 5 mm), and a depth D of about 2 mm to about 10 mm (e.g., 3 mm to 5 mm).

[0070] Furthermore, in the absence of an additional biological / non-biological matrix, the casting trench 204 may exhibit non-cell adhesion but biocompatibility. This can be achieved through the selection of culture unit materials, permanent surface treatment, and / or short-term pretreatment.

[0071] The neuron culture chamber or casting groove 204 includes an optional cell aggregation region 206 disposed on at least a portion of the bottom (blind) surface of the casting groove 204. The cell aggregation region 206 includes one or more types of cell aggregation features configured to aggregate live neuronal material into one or more cell aggregates, i.e., to promote maturation into neurospheres. In the representative embodiment shown, the cell aggregation region 206 has a length l' of about 5 mm to about 15 mm (e.g., 10 mm to 15 mm) and a width w' of about 2 mm to about 10 mm (e.g., 2 mm to 5 mm). In different embodiments, the cell aggregation region 206 may be positioned at different locations on the bottom surface of the corresponding opening, for example, for neurosphere positioning control.

[0072] Each neurosphere formed in cell aggregation region 206 can range in diameter from about 10 μm to about 500 μm, and each neurosphere contains about 500 to about 100,000 individual cells. Each neuron culture compartment can generate between about 1 and about 100 individual neurospheres.

[0073] Cell aggregation features drive the maturation of neurosphere locations within neuronal cell cultures; these locations are pre-defined to facilitate stimulation and / or measurement of the co-cultured tissue. This location specificity of the neurosphere enables precise experimental control. For example, cell aggregation features can be positioned at predetermined locations to receive optical and / or electrical stimulation. As another example, cell aggregation features can be positioned to facilitate subsequent measurement of neurosphere function (e.g., simultaneously measuring neurosphere function with the function of living muscle material).

[0074] Cell aggregation features typically include chemical, electrical, and / or mechanical features that drive cell aggregate formation. For example, in some embodiments, cell aggregation features include one or more surface properties to drive cell aggregation, such as at least one of hydrophobicity or electrostatic charge. As another example, in some embodiments, the surface of cell aggregation region 206 is provided with a hydrophobic surface treatment, formed of a hydrophobic material, and / or endowed with an electrostatic charge to aggregate cells at predetermined locations in cell aggregation region 206 (e.g., at a single aggregate location or in an aggregate of a predetermined pattern).

[0075] Independent of or attached to the surface properties of cell aggregation, cell aggregation region 206 may include a characteristic geometry configured to drive cell aggregation. This characteristic geometry may include a plurality of closely packed cavities or units, each cavity or unit forming a lowest point or low point. For example, each cavity may be formed as an inverted pyramid, inverted cone, inverted triangular prism, dome, pit, valley, depression, groove, bowl, trough, depression, cavity, or similar feature having a polygonal or organic substrate shape, thereby forming at least a lowest point or low point to which cells will aggregate under gravity. In some embodiments, all such cavities or units have a common shape and / or size (e.g., Figure 2D (As shown). In other embodiments, the feature geometry may include cavities or units of different sizes and / or shapes.

[0076] In some embodiments, the feature geometry is selected and arranged to reduce, minimize, or eliminate gaps between adjacent cavities (which could otherwise allow the formation of randomly positioned neural spheres). For example, in some embodiments, the feature geometry includes tessellation surface features, i.e., geometric features that repeat coherently without gaps. Representative examples include geometries with triangular, quadrilateral, hexagonal, or octagonal bases (such as equilateral triangles, hexagons, octagons, and parallelograms). In other embodiments, the gaps between closely packed cavities may be provided with hydrophobic, electrostatic, and / or other features to prevent the formation of randomly positioned neural spheres thereon.

[0077] Figure 2D Representative and non-limiting cell aggregation features configured to drive neuronal cell aggregation are shown. Cell aggregation region 206 includes cell aggregation features 208, which include characteristic geometries formed as mosaic surface features (mosaic cavities in this embodiment). Each mosaic cavity is an inverted pyramid, forming a lowest point 210 with a quadrilateral (parallelogram) base. Different embodiments include different mosaic and non-mosaic geometries.

[0078] Generally, the feature geometry has dimensions (e.g., depth, side length) of about 1 μm to about 10 cm, thereby allowing spatially controlled cell aggregation and confinement. In the representative embodiment shown, the cell aggregation region 206 includes about 50 to about 250 mosaic surface features, and a surface feature density of about 2 to about 10 mosaic surface features per square millimeter (e.g., 2 to 5 features per square millimeter). Figure 2D As shown, each edge 212 has a length of about 0.25 mm to about 2 mm (e.g., 0.25 mm to about 0.75 mm). Each cell aggregation feature 208 has a depth of about 0.25 mm to about 2 mm (e.g., 0.25 mm to about 0.75 mm) (e.g., the z-axis distance from the plane containing edge 212 to the corresponding lowest point).

[0079] Neuron culture compartments can include any combination of the aforementioned features (e.g., surface properties and characteristic geometry), allowing for the in-situ generation of neurospheres from single cells. The inventors of this application have surprisingly discovered that by maintaining neuronal cells in situ during and throughout co-culturing with muscle tissue, efficient and reproducible three-dimensional structures of neuromuscular junctions can be generated.

[0080] In use, co-cultured tissues can be directly cultured in each casting groove 204, for example, according to the methods described herein. For instance, co-cultured tissues can be cast directly in the casting groove 204 by co-culturing a neuronal cell culture containing live neuronal material with a muscle cell culture containing live muscle material. In some examples, the two cell cultures share a hydrogel interface having an area corresponding to the cell aggregation region 206. Axons from the neuronal cell culture cross this interface into the muscle tissue, maturing into neuromuscular junctions that indicate nerve-innervated muscle tissue.

[0081] Figures 3A to 3D Various aspects of a porous neurosphere casting plate 300 according to another representative embodiment of this disclosure are shown. The casting plate 300 advantageously enables long-distance communication and axonal extension between living neuronal material and living muscle material, which can increase the measurement window for progressive neuromuscular diseases characterized by axonal retraction from muscle.

[0082] and Figures 2A to 2D Similar to the implementation scheme, the cast plate 300 includes multiple culture units 302a, b, ... n, which are arranged in a layout conforming to the SBS standard. The cast plate 300 and... Figures 2A to 2DThe difference in the implementation scheme is that each culture unit includes at least one distinct neuron culture chamber, which is fluidly connected to a muscle culture chamber via a fluid channel. Therefore, neuronal cell cultures are cultured in each neuron culture chamber and interfaced with a muscle cell culture (e.g., EMT) disposed in the muscle culture chamber at a midpoint along the fluid channel.

[0083] Figure 3A A plan view of a portion of the casting plate 300 is shown. Figure 3B A plan view of the culture unit 302a within the casting plate 300 is shown, while Figure 3C and Figure 3D A vertical cross-sectional view of culture unit 302a is shown. Since all culture units in the cast plate 300 share the same geometry and features, therefore... Figures 3A to 3D The different culture units use the same reference numerals. For ease of understanding, culture unit 302a is shown as containing EMT 336, although EMT is not part of culture unit 302a.

[0084] First turn Figure 3A The aspects of culture unit 302b, which are common to all culture units in cast plate 300, will now be described. Culture unit 302b includes multiple neuron culture chambers 314a, 314b, which are configured to allow for the long-term maintenance of neurons and supporting cells within them. The neuron culture chambers 314a, 314b are fluidly connected to each other via connecting channels 318, wherein the connecting channels are, for example, microfluidic channels with diameters on the order of hundreds of micrometers (in... Figure 3C (Shown in more detail below). The illustrated embodiment includes two opposing neuron culture chambers 314a, 314b; however, other embodiments may include a single neuron culture chamber or more than two neuron culture chambers (e.g., three, four, or five neuron culture chambers distributed around the muscle culture chamber).

[0085] The muscle culture chamber 316 includes a spacious volume within the culture unit 302b disposed between the neuron culture chambers 314a, 314b to accommodate and maintain the health of muscle tissue, such as EMT 336. Muscle cell cultures are typically cultured in separate casting plates and then transferred to the muscle culture chamber 316.

[0086] The connecting channel 318 is a fluid channel extending between the neuron culture chambers 314a and 314b, and has an opening 334 in its wall. In some embodiments, the connecting channel 318 has a diameter on the order of several hundred micrometers. In some embodiments, the diameter of the connecting channel 318 is not less than about fifty micrometers, for example, to facilitate manufacturing. Advantageously, this opening 334 fluidly connects the connecting channel 318 to the muscle culture chamber 316. Thus, each of the neuron culture chambers 314a and 314b is in fluid communication with the muscle culture chamber 316 via the connecting channel 318.

[0087] See Figure 3B Aspects of culture unit 302a are described in more detail. As shown in the detailed illustrations, at least a portion of the bottom (blind) surface of one, some, or all of the neuron culture chambers 314a, 314b may optionally be provided with a cell aggregation region 306 having any type and combination of the previously described cell aggregation features 308. For example, some embodiments of neuron culture chambers 314a and 314b include surface features and / or feature geometries, such as including mosaic cavities configured to aggregate neuronal cells by gravity.

[0088] Electrical and optical isolation between neuron culture chambers 314a, 314b and muscle culture chamber 316 can be achieved by providing one or more electrically insulating and optically opaque walls (e.g., wall 326) between them. Within each respective chamber, the volume resistance between any two points is much lower (e.g., at least three orders of magnitude lower) than the resistance between two points across the interface between chambers (e.g., via opening 334). Therefore, electrical stimulation of a cell (e.g., neurosphere) in one chamber will have a negligible effect on a cell (e.g., muscle tissue) in another chamber, and vice versa. Thus, in some embodiments, each chamber is configured to house at least two conductive components, hereinafter referred to as electrodes, which can be directly electrically connected to an external device, such as a stimulation device.

[0089] Neuronal culture compartments can be stimulated to induce neuronal action potentials. Non-exclusive exemplary stimulation modalities include: chemical stimulation (e.g., glutamate); field potential stimulation (e.g., amplitude 10 to 400 mA, frequency 1 to 50 Hz, pulse width 1 to 20 ms); direct contact electrical stimulation (amplitude 10 to 400 mA, frequency 1 to 50 Hz, pulse width 1 to 20 ms); and / or photostimulation via photosensitive ion channels (power 5 to 40 mW / mm²). 2(Frequency 1 to 100 Hz, pulse width 1 to 20 ms). In some implementations, these stimuli are isolated and / or uniquely applied to neuronal culture chambers and / or muscle culture chambers, thereby enabling independent stimulation of neuronal cells or muscle portions of the co-cultured tissue.

[0090] Therefore, culture unit 302a receives multiple electrodes in each of the neuron culture chambers 314a, 314b and in the muscle culture chamber 316. For example, electrode 320a extends into neuron culture chamber 314a (to stimulate the neurospheres cultured therein), and electrode 320b extends into muscle culture chamber 316 (to stimulate the EMT 336). These electrodes may be integrated with the casting plate 300 or as part of a stimulation device such as a stimulation cap.

[0091] Figure 3C and Figure 3D A vertical cross-sectional view of culture unit 302a is shown. (See figure) Figure 3C As shown, neuron culture chambers 314a and 314b are separated from muscle culture chamber 316 via a connecting channel 318 or connecting chamber. The casting plate 300 may consist of one or more components, such as an upper component 328 and a lower component 330, which are joined together by an intermediary component 332. In some embodiments, the intermediary component 332 includes at least a portion of the connecting channel 318.

[0092] In some embodiments, the connecting channel 318 forms an open channel having upper and lower walls and side walls, wherein each end of the connecting channel 318 communicates with either of the neuron culture chambers 314a, 314b or the muscle culture chamber 316. These upper and lower walls may be continuous with the upper and lower components 328, 330 of the casting plate 300, while the side walls may be continuous with the upper or lower components 328, 330, or formed by an intervening component 332 disposed therebetween.

[0093] The communication channel 318 is small enough to prevent neurosphere infiltration, thereby ensuring the separation of neurons from muscle cells within culture unit 302a, while allowing limited fluid connectivity via neurite growth in a manner that mimics the in vivo interaction of these two tissue types. This communication channel 318 can be filled with liquid or hydrogel of biological or non-biological origin.

[0094] In some implementations, the connectivity channel 318 is configured to facilitate neuronal guidance from neuronal culture chambers 314a, 314b to muscle culture chamber 316. As a representative example, this can be achieved by applying chemokines in muscle culture chamber 316, rather than in neuronal culture chambers 314a, 314b, thereby generating a chemokine gradient. Non-exclusive examples would include neuronal growth factor (NGF) or Netrin.

[0095] In another embodiment, the flow characteristics within the connecting channel 318 induce turbulent and laminar hydrogel flow in a region whose pattern optimizes the hydrogel alignment such that when the hydrogel is filled, the hydrogel flows through the channel during inoculation, causing the hydrogel components to align along the axis of the connecting channel 318, thereby promoting axon guidance as the axons extend through this patterned matrix.

[0096] Additionally, in some embodiments, the size of the connecting channel 318 makes it suitable for applications utilizing evaporative pumping. One example of such an application involves supplying a hydrogel of appropriate viscosity to neuronal culture chambers 314a, 314b and / or muscle culture chamber 316, followed by the addition of aqueous cell culture medium to the neuronal culture chambers 314a, 314b, but not the muscle culture chamber 316. Evaporation in the muscle culture chamber 316 attracts solvent and solute through the connecting channel 318 via intermolecular forces acting between the evaporating solvent and the host solute and solvent molecules. This flow can be used to create microenvironments in situ, providing favorable conditions for rapid, directional axonal growth. The fluid shear within the hydrogel caused by the moving solvent can pattern any hydrogel within the connecting channel 318. Furthermore, evaporative pumping can accumulate chemokines at the opening 334, thereby generating a high concentration gradient of chemokines, as these factors remain at the ends of the connecting channel 318 when the contained solvent evaporates.

[0097] Under such guided conditions, axons can be induced to extend at a rate of up to 1 mm per day, thus allowing long-distance communication between compartments. Advantageously, the ability to guide axonal growth to any specific location within the muscle culture compartment 316 allows neurons to connect to the muscle cell culture interface in a spatially controlled manner. The size and location of the interface points can be adjusted to suit specific measurement methods and muscle tissue geometries.

[0098] Figure 3DA cross-sectional view of culture unit 302a is shown, extending through muscle culture chamber 316. Thus, the EMT 336 is suspended between two tissue fixation members 322a, 322b within the muscle culture chamber 316. These fixation members may form part of a separate assembly extending into the muscle culture chamber 316 to contact the EMT 336 with the communication channel 318. For example, in some embodiments, tissue fixation member 322a is a flexible column, while tissue fixation member 322b is a relatively rigid column.

[0099] In use, hydrogel can be added to the connecting channel 318 to form structure 324 (e.g., a dome-shaped cell culture structure or a hydrogel structure) that rises from the top of the connecting channel 318. This structure 324 directly contacts the EMT 336, thereby forming an interface between the two cell cultures and allowing co-culture. The muscle culture compartment 316 is designed such that the connecting channel 318 surrounds or adjoins the EMT 336 to increase the probability of axons extending from the neuronal cell culture connecting to the interface of the EMT 336. In a non-limiting example of this design, communication between compartments can be achieved by surrounding the EMT 336 and embedding neuronal components within a bulk of hydrogel.

[0100] In some embodiments, the muscle culture chamber 316 enables non-invasive reading of muscle function, including spontaneous and induced function, by the movement of one or more tissue fixation elements 322a, 322b. This movement can be recorded by optical tracking or by changes in the magnetic field induced by magnets embedded in one or both tissue fixation elements 322a, 322b. Therefore, the muscle culture chamber 316 can be formed of a transparent material to enable optical tracking. Additionally or alternatively, the cast plate 300 can be configured to be adapted to an instrument that measures the movement of at least one tissue fixation element in each culture unit 302a...n magnetically and / or optically.

[0101] In the illustrated embodiment, tissue fixation members 322a and 322b form part of a separate muscle tissue casting assembly that is inserted into the casting plate 300.

[0102] The apparatus described herein, including cast plates, consumables, and assays, can form part of a product line of such apparatuses. For example, this disclosure includes a product line comprising at least two different cast plates, each of the at least two different cast plates being a cast plate of this disclosure. For example, the product line may include at least two cast plates with different cell aggregation region configurations. As another example, the product line may include at least two cast plates, wherein the first cast plate is configured according to… Figures 2A to 2D Any implementation configuration described, and wherein the second casting plate is configured according to the relevant provisions. Figures 3A to 3DAny implementation configuration described. Such different configurations may be suitable for assaying different cell cultures characterized by different neurological diseases.

[0103] This disclosure includes a tissue analysis system comprising a co-culture casting system, which includes a porous co-culture casting plate and a porous muscle tissue casting plate. The porous co-culture casting plate can be described herein as... Figures 2A to 3D Any implementation scheme described.

[0104] Any casting plate or culture unit of this disclosure can be incorporated into a tissue analysis system and / or a co-culture casting system, and can optionally be used as part of one or more methods for: a) using such a tissue analysis system or co-culture casting system, and / or b) modeling the neuromuscular junction, which will now be described. Advantageously, the following methods achieve efficient, high-throughput, and reproducible NMJ modeling by forming co-cultured tissue with in situ neurospheres. The following methods can be performed independently of the apparatus described herein.

[0105] Figure 4A Representative methods 400 are schematically illustrated for a) using a tissue analysis system including a co-culture casting system comprising a porous co-culture casting plate and a muscle tissue casting plate, and for b) modeling multiple neuromuscular junctions, including methods for casting co-culture tissue containing in situ neurospheres. In embodiments where at least some of the living neuronal material contains mutational or disease characteristics, method 400 may be restated as a disease modeling method and / or may form part of a disease modeling method. In embodiments where at least some of the living neuronal material is treated with a substance (e.g., a neurotoxin), method 400 may be restated as a method for determining the substance and / or may form part of a method for determining the substance.

[0106] Method 400 typically comprises four stages. In the first stage 402, a cell culture containing neurospheres matured from living neuronal material is cast (i.e., a neuronal cell culture). In the second stage 404, a cell culture containing living muscle material is cast (a muscle cell culture). The first stage 402 and the second stage 404 can be performed by a common entity or by different entities simultaneously. In the third stage 406, the muscle cell culture is co-cultured with the neuronal cell culture to form a co-cultured tissue containing in situ neurospheres and living muscle material. The first stage 402, the second stage 404, and the third stage 406 together constitute a method for forming a co-cultured tissue containing in situ neurospheres and living muscle material. In the fourth stage 408, the co-cultured tissue is stimulated. For example, by stimulating the neurospheres of the co-cultured tissue using electrical, optical, and / or chemical stimuli, the living muscle material in each co-cultured tissue is induced to contract.

[0107] The dashed boxes in stages 402 and 404 show and include elements of a representative tissue analysis system and a co-culture casting system, including a porous co-culture casting plate shown in the dashed box of stage 402 and a porous muscle tissue casting plate shown in the dashed box of stage 404. In some embodiments, the porous co-culture casting plate is... Figures 2A to 2D The casting plate 200; in other embodiments, the porous co-cultivation casting plate is Figures 3A to 3D The casting plate 300. For simplicity, individual wells of each of the porous co-culture casting plate and the porous muscle tissue casting plate are shown in the first stage 402 and the second stage 404, respectively. The tissue analysis system may optionally include a porous support plate, such as the one shown in the dashed box in the fourth stage 408.

[0108] Representative steps for each stage will now be described. The methods described herein explicitly include any combination of the following features, performed in any reasonable order consistent with this disclosure. Each different combination and order of features and steps constitutes a different method 400 of this disclosure.

[0109] Phase 1 402 (casting neuronal cell cultures).

[0110] In step 410, neurospheres are cast into neuronal cell cultures by seeding live neuronal material containing neuronal cells (e.g., motor neurons) into multiple neuronal culture chambers and allowing the live neuronal material to mature into neurospheres within the neuronal culture chambers. Step 410 may include any combination of the following features:

[0111] • Apply hydrogels to live neuronal material in the neuronal culture compartment. Representative hydrogels include, for example, type I rat tail collagen (approximately 1 mg / mL) + approximately 5% MATRIGEL. ® Or equivalent. In other words, multiple neurons are added to the hydrogel to form a neuronal hydrogel.

[0112] • Inoculate living neuron material into multiple neurosphere casting grooves, such as casting grooves of a porous neurosphere casting plate.

[0113] • Live neuron material was seeded into multiple neuron culture compartments in each well of a porous neurosphere casting plate.

[0114] • Controlling the location of cell aggregates of live neuron material. In some embodiments, controlling the location of cell aggregates includes patterning the cell aggregates and / or spaced them apart. In some embodiments, controlling the location of cell aggregates includes seeding the live neuron material into (e.g., only into) at least one specific region within a neuron culture chamber. In some such embodiments, at least one specific region may include one or more of the following: a cell aggregate region, an end region of a neuron culture chamber, a lateral region of a neuron culture chamber, a central region of a neuron culture chamber, or a tissue fixation region.

[0115] • Live neuronal material is not seeded into at least one exclusion region within the neuronal culture chamber. In some such embodiments, the at least one exclusion region may include one or more regions of the terminal region, central region, lateral region, or tissue fixation region of the neuronal culture chamber. Not seeding live neuronal material into at least one exclusion region can facilitate the analysis of the co-cultured tissue, for example, by calcium and / or optical imaging.

[0116] • Aggregating live neuron material into cell aggregates. In some such embodiments, aggregating live neuron material into cell aggregates includes seeding the live neuron material into a cell aggregate region containing cell aggregation features, such as surface properties (e.g., hydrophobicity and / or electrostatic charge) and / or characteristic geometry (e.g., embedded cavities).

[0117] • Each neuron culture compartment includes a casting groove (see Figures 2A to 2D ).

[0118] • Each neuron culture compartment is one of several neuron culture compartments in a culture unit within a well of a multi-well plate (see [link]). Figures 3A to 3D ).

[0119] Each neuron culture chamber and / or each casting groove includes at least one cell aggregation region containing cell aggregation features as described in any embodiment herein, including one or more surface properties or feature geometries that drive cell aggregation.

[0120] • Live neuronal material was directly thawed from cryopreservation in multiple neuronal culture chambers, optionally without the use of Rho kinase inhibitors. Neurons were directly thawed in the neuronal culture chambers, where they matured into neurospheres. These in situ neurospheres were then cultured into co-culture tissues. This process improved neuronal survival by eliminating the transfer step, thereby improving reproducibility and simplifying experimental planning.

[0121] • After inoculating the live neuronal material, wait for a maturation period (e.g., 1 to 14 days) during which the neurons of the live neuronal material mature into neurospheres, and then apply a connecting hydrogel to the in situ neurospheres.

[0122] • Living neuronal materials include neurons that express one or more proteins that act as gated ion channels (e.g., light-gated ion channels, including channels rhodopsin-1 and -2). In some such embodiments, the expression of such proteins can be induced, for example, by administering one or more promoters, such as tetracycline promoters or neuron-specific promoters.

[0123] The total number of neurons contained in the neuronal hydrogel ranges from approximately 5% to approximately 75% (by weight) of the neuronal hydrogel. In another aspect, the amount of neurons contained in the neuronal hydrogel is selected from the group consisting of: 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, and 75% (by weight) of the neuronal hydrogel, and ranges thereof.

[0124] Step 412 may form part of either the first stage 402 or the second stage 404. In step 412, in each neuronal culture compartment, a connecting hydrogel is applied to an in situ neurosphere matured from live neuronal material. This connecting hydrogel promotes adhesion of the neurosphere to the muscle cell culture. In some embodiments, the connecting hydrogel is a type I collagen hydrogel, for example, one with a low proportion of mouse Engelbreth–Holm–Swarm (EHS) tumor basement membrane extract (such as MATRIGEL). ® This allows for robust neurite extension and hydrogel compaction to bring the neurosphere close to the muscle cell culture. Step 412 may include any combination of the following features:

[0125] • Apply connecting hydrogels to in situ neurospheres in multiple neurosphere casting grooves, or to in situ neurospheres in multiple neuron culture compartments in each well of a porous neurosphere casting plate.

[0126] • Do not remove the neurospheres from the neuron culture compartment before applying the connecting hydrogel.

[0127] Therefore, steps 410 and optional step 412 provide cell cultures containing living neuronal material (i.e., in situ neurospheres).

[0128] Phase 2 404 (casting muscle cell culture).

[0129] Step 412 may optionally be performed as part of a 404 error, as discussed above.

[0130] In step 414, the live muscle material is inoculated onto a separate casting plate (such as...) Figure 4A Live muscle material is cast into muscle cell cultures through multiple muscle casting holes (as shown).

[0131] Step 414 may include any one or more of the following features:

[0132] • Cast multiple tissue fixation elements (e.g., multiple pairs of columns) into living muscle material, such that the resulting muscle cell cultures each form a mesh between two or more tissue fixation elements (e.g., Figure 3D and Figure 4A (As shown).

[0133] • Live muscle material is cast in a culture medium, such as a hydrogel, which may be different from the hydrogel used to cast neuronal cell cultures and / or the connecting hydrogel.

[0134] • Add multiple muscle cells to a second culture medium (e.g., hydrogel).

[0135] Muscle cell culture is engineered muscle tissue (EMT).

[0136] • Add multiple muscle cells to a hydrogel to form a hydrogel containing muscle tissue.

[0137] The total amount of muscle cells contained in the hydrogel containing muscle cells ranges from approximately 5% to approximately 75% (by weight) of the hydrogel containing muscle cells. Alternatively, the amount of muscle cells contained in the hydrogel containing muscle cells is selected from the group consisting of: 5% (by weight), 10% (by weight), 15% (by weight), 20% (by weight), 25% (by weight), 30% (by weight), 35% (by weight), 40% (by weight), 45% (by weight), 50% (by weight), 55% (by weight), 60% (by weight), 65% (by weight), 70% (by weight), and 75% (by weight), and ranges thereof, of the hydrogel containing muscle cells.

[0138] In optional step 416, the muscle cell culture is transferred to a maintenance plate, for example, when neurospheres are cultured in a connecting hydrogel (see step 412). In some embodiments, the live muscle material is transferred to the maintenance plate after maturing into a muscle tissue structure (e.g., a mesh or EMT).

[0139] In any implementation, Phase 1 402 and Phase 2 404 are executed concurrently and / or coordinated to terminate simultaneously. For example, in some implementations, Phase 1 402 and Phase 2 404 are executed over a common number of days and / or terminate on the same day.

[0140] Phase 3, 406 (Forming a co-cultivation organization).

[0141] In step 418, each muscle cell culture is co-cultured with one or more in situ neuronal cell cultures and allowed to attach to the latter, thereby forming multiple co-cultured tissues, each containing an in situ neurosphere and live muscle material, wherein at least a portion of the live muscle material is innervated by nerves. In other words, when a culture medium containing mature muscle cell cultures is attached and co-cultured with a culture medium containing neurospheres, the neurospheres remain in situ.

[0142] In some implementations, each muscle cell culture is incorporated into the hydrogel by applying a muscle hydrogel containing live muscle material to each neurosphere casting groove containing a connecting hydrogel, thereby communicating with the corresponding in situ neuronal cell culture. This brings the neurosphere into close proximity to the live muscle material. Muscle tissue effectively forms around the neurosphere, and muscle compartments and neuronal compartments are separated by the hydrogel.

[0143] In some embodiments, co-cultured tissue is formed in multiple neuron culture compartments, in which live neuron material is cast in the first stage 402. For example, in some embodiments, muscle cell cultures are transferred from individual casting plates to each neuron culture compartment. In some such embodiments, each neuron culture compartment includes a casting groove (see [link to documentation]). Figures 2A to 2D ).

[0144] In some implementations, each neuron culture compartment is one of multiple neuron culture compartments in a culture unit within a well of a multi-well plate. Each culture unit may include one or more neuron culture compartments and at least one muscle culture compartment (see [link to relevant documentation]). Figures 3A to 3D In some such embodiments, muscle cell cultures are transferred from a single casting plate to multiple muscle culture compartments within multiple culture units. In some such embodiments, the muscle cell cultures are in communication with one or more neuronal cell cultures within the culture unit along a communication channel (e.g., a communication channel extending from a neuronal culture compartment), for example, at the opening of the communication channel.

[0145] In any embodiment, the muscle cell culture is co-cultured with the orthotopic neuronal cell culture for an attachment period of at least about one hour to at least about seventy-two hours (e.g., at least about forty-eight hours), and then the fourth phase 408 begins. The attachment period enables the muscle cell culture to attach to the corresponding neuronal cell culture, for example, the axonal growth of the neuronal cell culture reaches the muscle cell culture through the hydrogel interface.

[0146] In any embodiment, muscle cell cultures are co-cultured with in situ neuronal cell cultures via communication channels as defined in any embodiment herein.

[0147] In any implementation, muscle cell cultures are co-cultured with orthotopic neuronal cell cultures while maintaining electrical and / or optical isolation between them. For example, in Figures 3A to 3D In the cast plate 300, the walls separating the neuron culture chamber and the muscle culture chamber provide effective electrical insulation.

[0148] In any embodiment, step 418 further includes reshaping the interface between the neuronal cell culture and the muscle cell culture, for example, the interface between the connecting hydrogel and the hydrogel containing living muscle material. This step can facilitate the extension of neurites across the interface. This interface may include a mechanical interface between them due to the different properties of the two hydrogels (e.g., different stiffness values). In some embodiments, reshaping this interface includes reducing the stiffness gradient between the connecting hydrogel and the muscle hydrogel. In some embodiments, reshaping the interface between the connecting hydrogel and the muscle hydrogel includes treating the interface with a serum culture medium, for example, for a treatment period of at least 12 hours or at least 24 hours. In some embodiments, the serum culture medium is a medium containing 10% animal serum (such as fetal bovine serum (FBS)), and the tissue is applied by adding the serum-containing medium to a plurality of casting wells without removing the tissue after the connecting hydrogel has solidified in step 412.

[0149] Phase 408 (Stimulation co-culture tissue).

[0150] In step 420, the co-cultured tissues are stimulated. Specifically, the innervated portion of the live neuronal material in each co-cultured tissue is induced to contract by stimulating the live neuronal material, for example, by one or more of chemical, electrical, or optical stimulation.

[0151] In any implementation, co-cultured tissues are stimulated simultaneously (in parallel), for example, by using chemical stimulation, electrical stimulation, and / or light stimulation on each co-cultured tissue in a common multiwell plate.

[0152] In any implementation, co-cultured tissues are recorded simultaneously (in parallel), for example, using one or more recording electrodes for each co-cultured tissue in a common multiwell plate.

[0153] In any implementation, co-cultured tissues are stimulated and recorded simultaneously (in parallel), for example, in a co-well plate. In other words, each co-cultured tissue can be stimulated and recorded simultaneously, and multiple co-cultured tissues can be stimulated and / or recorded simultaneously with each other.

[0154] In any implementation, the live muscle material of each co-cultured tissue contracts by stimulating the live neuronal material of that co-cultured tissue with one or more of the following: chemical stimulation, electrical stimulation, or light stimulation.

[0155] In any implementation, the live muscle material of each co-cultured tissue contracts independently by stimulating the live muscle material and the corresponding live neuronal material with one or more of chemical, electrical, or optical stimuli.

[0156] In some implementations, where the co-cultured tissues are formed in neuronal culture compartments, i.e., each co-cultured tissue contains an in situ neurosphere, step 420 is performed by activating the in situ neurosphere of each co-cultured tissue.

[0157] In some implementations, after the co-cultured tissue is transferred to a maintenance plate, the live muscle material of the co-cultured tissue is induced to contract by stimulating at least the live neuronal material of the co-cultured tissue.

[0158] therefore, Figure 4A Many repeatable and scalable methods for modeling multiple neuromuscular junctions have been introduced.

[0159] Figure 4B It schematically shows the previous information about Figure 4A The present invention describes an exemplary specific method for using an organizational analysis system. Such a method may, for example, use... Figures 2A to 2D 200 or cast plate Figures 3A to 3D The method is performed using a cast plate 300. The method described below explicitly includes any combination of the following features, performed in any reasonable order consistent with this disclosure.

[0160] In step 424, method 422 seedes multiple neuronal culture chambers of a porous co-culture casting plate with live neuronal material, wherein each neuronal culture chamber contains multiple cell aggregation features configured to aggregate the live neuronal material into cell aggregates. In some embodiments, these cell aggregation features are disposed in casting grooves of the respective neuronal culture chambers. In some embodiments, these cell aggregation features include surface features (e.g., hydrophobicity and / or electrostatic charge) and / or feature geometries as described herein. For example, in some embodiments, the feature geometries include mosaic surface features, such as mosaic cavities, wherein each mosaic cavity forms a lowest point. In some embodiments, seeding multiple neuronal culture chambers with live neuronal material includes directly thawing cells containing motor neurons in the multiple neuronal culture chambers. In some embodiments, the porous co-culture casting plate includes multiple culture units, wherein each culture unit includes a muscle culture chamber connected to at least one neuronal culture chamber via a communication channel.

[0161] In step 426, method 422 inoculates multiple muscle culture compartments of a muscle tissue casting plate with live muscle material.

[0162] In step 428, method 422 cultivates multiple co-culture tissues in a porous co-culture casting plate, each co-culture tissue comprising a muscle cell culture cultured from live muscle material and an in situ neurosphere matured from live neuronal material in a cell aggregation feature. In some embodiments, cultivating multiple co-culture tissues includes cultivating at least one co-culture tissue in each neuronal culture compartment. In some embodiments, seeding multiple neuronal culture compartments with live neuronal material (step 426) includes applying a connecting hydrogel to the in situ neurosphere in each neuronal culture compartment, and cultivating each co-culture tissue (step 428) includes reshaping the interface between the connecting hydrogel and a second hydrogel containing live muscle material, for example, by treating the interface with serum culture medium.

[0163] In step 430, method 422 causes the innervated portion of the live muscle material in each co-cultured tissue to contract by stimulating the live neuronal material. In some embodiments, step 430 includes transferring the co-cultured tissue to a maintenance plate before causing the innervated portion of the live muscle material in the co-cultured tissue to contract by stimulating the live neuronal material. In some embodiments, step 430 includes activating the in situ neurosphere using at least one of light stimulation, electrical stimulation, or chemical stimulation. In some embodiments, step 430 includes simultaneously causing the innervated portion of the live muscle material in each co-cultured tissue to contract, optionally recording the contraction of each co-cultured tissue simultaneously.

[0164] Figure 4CIt schematically shows the previous information about Figure 4A This section introduces several exemplary specific modeling methods for neuromuscular junctions. Such methods can, for example, use... Figures 2A to 2D The method is performed on a cast plate 200. The method described below explicitly includes any combination of the following features, performed in any reasonable order consistent with this disclosure.

[0165] In step 432, method 448 seedes a multi-well plate with live neuronal material by directly thawing cells containing motor neurons in multiple neuronal culture chambers, wherein each neuronal culture chamber contains a cell aggregation feature configured to aggregate the live neuronal material into cell aggregates. Optionally, the live neuronal material expresses at least one protein that acts as a gated ion channel. These cell aggregation features may have any of the previously described configurations.

[0166] In step 434, method 448 inoculates multiple muscle culture compartments of the muscle tissue casting plate with live muscle material and / or casts multiple muscle cell cultures containing live muscle material. Alternatively, muscle cell cultures may be received, for example, from a third party that casts the muscle cell cultures.

[0167] In step 436, method 448 cultivates multiple co-cultured tissues in a multi-well plate, the co-cultured tissues comprising live muscle material and in situ neurospheres matured from live neuronal material, partly by remodeling the interface between the connecting hydrogel and a second hydrogel containing live muscle material (e.g., by treating the interface with serum culture medium).

[0168] In step 438, method 448 involves stimulating the living neuronal material, for example with chemical stimulation, light stimulation, and / or electrical stimulation, while simultaneously causing the innervated portion of the living muscle material in each co-cultured tissue to contract. Optionally, causing the innervated portion of the living muscle material in each co-cultured tissue to contract also includes recording the contraction while stimulating each co-cultured tissue.

[0169] Figure 4D It schematically shows the previous information about Figure 4A Additional exemplary specific modeling methods for several neuromuscular junctions are introduced. Such methods can, for example, use... Figures 3A to 3D The method is performed using a cast plate 300. The method described below explicitly includes any combination of the following features, performed in any reasonable order consistent with this disclosure.

[0170] In step 440, method 450 seedes multiple culture units of a multiwell plate with live neuronal material by directly thawing cells containing motor neurons in multiple neuronal culture compartments, wherein each neuronal culture compartment contains a cell aggregation feature configured to aggregate the live neuronal material into cell aggregates. Optionally, the live neuronal material expresses at least one protein that acts as a gated ion channel. These cell aggregation features may have any of the previously described configurations.

[0171] In step 442, method 450 inoculates multiple muscle culture compartments of a muscle tissue casting plate with live muscle material and / or casts multiple muscle cell cultures containing live muscle material. Alternatively, muscle cell cultures may be received, for example, from a third party that casts the muscle cell cultures.

[0172] In step 444, method 450 cultivates multiple co-cultured tissues in a multi-well plate by connecting live neuronal material and live muscle material along a fluid communication channel in each culture unit. The co-cultured tissues contain live muscle material and in situ neurospheres matured from live neuronal material.

[0173] In step 446, method 450 stimulates the living neuronal material, for example with chemical stimulation, light stimulation, and / or electrical stimulation, while simultaneously causing the innervated portion of the living muscle material in each co-cultured tissue to contract. Optionally, causing the innervated portion of the living muscle material in each co-cultured tissue to contract also includes recording the contraction while stimulating each co-cultured tissue.

[0174] Figures 5 to 9B This demonstrates the effectiveness of modeling the neuromuscular junction using the method described in this paper.

[0175] Figure 5 Exemplary images of cells and tissues formed within the apparatus of this disclosure are shown.

[0176] Image (A) shows in situ neuronal aggregates embedded in a single pore of a cast plate containing a characteristic geometry (i.e., mosaic cavity). These neuronal aggregates can be maintained to generate neurospheres. Scale bar: 500 μm.

[0177] Image (B) illustrates exemplary neurite extension in a three-dimensional matrix within an adhesive hydrogel 24 hours after in situ creation and maintenance of the neurosphere in the cast plate of Image (A), demonstrating the feasibility of the in situ neurosphere creation method described herein. Scale bar represents 500 μm.

[0178] Image (C) illustrates an exemplary neurite extension within a cast plate comprising a neuronal culture chamber and a muscle culture chamber connected by a connecting channel. Specifically, image (C) is a stitched image showing a neurite emanating from an iPSC-derived motor neuron globus (white dashed circle) in the neuronal culture chamber, growing through the connecting channel (white arrow), and contacting engineered muscle tissue on the right side of the muscle culture chamber (white dashed border). The neurite in contact with the muscle in the muscle chamber is highlighted with a white arrowhead. The scale bar represents 500 μm.

[0179] Figure 6 Still frames from a 20fps video taken during acetylcholine stimulation of a muscle cell culture containing engineered skeletal muscle are shown. The three still frames, from top to bottom, represent: the quiescent state of the co-cultured tissue before glutamate application; the maximally contracted state of the co-cultured tissue after glutamate application; and the relaxed state after subsequent maximal displacement. The white dashed lines represent the tissue length at rest and at maximal contraction. The scale bar represents 1 mm. As shown, the live muscle material in the co-cultured tissue contracts in response to stimulation of the live neuronal material within it, indicating the formation of a living NMJ.

[0180] Figure 7A The contractile force trajectory of an NMJ co-culture tissue formed according to the method described herein is shown. This tissue comprises human iPSC-engineered skeletal muscle cultured for two weeks and treated with 1 mM and 10 μM acetylcholine. Specifically, the skeletal muscle tissue comprises a 9:1 remixture of iPSC-derived myoblasts and human dermal fibroblasts, generated by suspending the cells in a supporting hydrogel at a cell density of 5 × 10⁶ cells / mL hydrogel. 6 Cells. The hydrogel contained polymeric fibrinogen and 20% mouse Engelbreth–Holm–Swarm (EHS) tumor basement membrane extract. These tissues matured for 14 days and were then exposed to 0.01 mM and 1 mM acetylcholine. The resulting contractile responses were recorded by tracking a flexible magnetic column using a magnetic field. The black dashed line represents the moment acetylcholine was added. Thus, the live muscle material from the co-cultured tissues was sensitive to acetylcholine stimulation.

[0181] Figure 7B The force trajectory diagram of an NMJ co-culture tissue (maintained in a co-culture medium) formed according to the method described herein, comprising motor neuron-skeletal muscle co-culture cultured for two weeks and recorded after stimulation with 1 mM glutamate, is shown. Specifically, the skeletal muscle tissue comprises iPSC-derived myoblasts and human dermal fibroblasts remixed in a 9:1 ratio, generated by suspending the cells in a supporting hydrogel at a cell density of 5 × 10⁶ cells / mL hydrogel. 6The co-cultured tissues were generated in the same manner as muscle tissue, but contained approximately 100 neurospheres in addition to myoblasts and fibroblasts, each containing 1000 cells (co-culture 1) or 250 cells (co-culture 2). The neurospheres were created in situ and contained within the tissue during tissue formation. The neurospheres contained iPSC-derived motor neurons 18 days after differentiation induction, which were then further matured for 8 days before co-culture creation. The supporting hydrogel contained polymeric fibrinogen and 20% mouse Engelbreth–Holm–Swarm (EHS) tumor basement membrane extract. These tissues matured for 14 days and were then exposed to 0.1 mM glutamate. The resulting contractile responses were recorded by tracking a flexible magnetic column using a magnetic field. The black dashed line represents the moment of glutamate addition. Therefore, the live muscle material of the co-cultured tissues was sensitive to glutamate stimulation of the live neuronal material.

[0182] Figure 7C and Figure 7D They are shown respectively Figure 7B The co-cultured tissues were electrically evoked in tetanic (100 Hz) and twitching (1 Hz) responses using biphasic pulse trains with a pulse width of 5 ms (5 high, 5 low) and an amplitude of 60 mA at a given frequency.

[0183] Figures 8A to 8C This study demonstrated that NMJ co-cultured tissues formed according to the method described herein retained the function of the live muscle material within them, compared to pure muscle tissue. Specifically, the pure skeletal muscle tissue comprised a 9:1 remixture of iPSC-derived myoblasts and human dermal fibroblasts, generated by suspending the cells in a supporting hydrogel at a cell density of 7 × 10⁶ cells / mL hydrogel. 6The hydrogel contained polymeric fibrinogen and 20% mouse Engelbreth–Holm–Swarm (EHS) tumor basement membrane extract. The NMJ co-culture tissue contained skeletal muscle tissue and additional neuronal components attached to the mature skeletal muscle tissue. The neuronal components contained approximately 100 neurospheres, each containing 2500 cells. These neurospheres contained iPSC-derived motor neurons 18 days after differentiation induction, which then matured for another 10 days before co-culture creation. On day 10 of skeletal muscle differentiation, the neurospheres were suspended in a connecting hydrogel (1 mg / mL type I collagen plus 5% mouse Engelbreth–Holm–Swarm (EHS) tumor basement membrane extract), and the skeletal muscle tissue was introduced into the unsolidified hydrogel within the neurosphere creation consumable. After day 10, all tissues were exposed to a medium containing 10% FBS for 24 hours, then replaced with a co-medium designed to support muscle contraction and neuronal axonal extension. Functional readings of skeletal muscle over time are shown as the average of 30 twitching responses recorded after a 10 ms, 100 mA, 1 Hz biphasic stimulation pulse.

[0184] Figure 8A The diagram shows a comparison of the active twitching force of the aforementioned co-cultured tissue with that of pure muscle tissue.

[0185] Figure 8B The method described herein is shown. Figure 8A A comparison of the time from contraction 10 to peak contraction in co-cultured tissue with that of pure muscle tissue.

[0186] Figure 8C It shows Figure 8A A comparison of the time from peak contraction to relaxation in co-cultured tissue with that of pure muscle tissue.

[0187] Figure 9A As shown above regarding Figures 8A to 8C The contractile force trajectory diagram of the formed NMJ co-cultured tissue, wherein the neuronal cell culture was treated with 450nm blue light (250ms pulse train, 40mW / mm). 2 Stimulation was applied at 100 Hz (80% duty cycle). The motor neurons in the co-cultured tissue expressed the rhodopsin-2 (ChR2) channel. The arrowhead indicates the start of the pulse train. As shown in the figure, the live muscle material in the co-cultured tissue contracted in response to blue light stimulation of the live neuronal material.

[0188] Figures 9B to 9D The mean ± SD of n=4 tissues is shown, as shown regarding Figure 9A The procedure involved stimulation with blue light (250ms pulse train, 40mW / mm2, 100Hz, 80% duty cycle) at intervals of 5 seconds, 2 seconds, and 1 second.

[0189] Figure 9B Drawn Figure 9A The contractile force of NMJ co-cultured tissues, i.e., the peak height of the blue light-driven functional response of muscle cell cultures in co-cultured tissues.

[0190] Figure 9C Drawn Figure 9A The percentage of contractile force generated by optical stimulation of neuronal cell cultures in NMJ co-culture tissues compared to the contractile force achieved by direct electric field stimulation of muscle cell cultures is used as a representative of nerve innervation density. In other words, Figure 9C The blue light stimulation force is shown as a percentage, compared to the average force produced by the same tissue under electrical stimulation (10 ms, 100 mA, 1 Hz biphasic stimulation pulse).

[0191] Figure 9D Drawn Figure 9A The percentage of contractile force capture in NMJ co-cultured tissues within 100 ms of blue light stimulation. In other words, Figure 9D This shows the percentage of blue light stimulation pulses that triggered the peak within 100 ms after the end of the blue light stimulation pulse. (Example) Figures 9A to 9D As shown, direct NMJ activation in co-cultured tissues formed according to the method described in this paper enables real-time functional measurements of neurally innervated tissues.

[0192] Figures 10A to 10C As shown above regarding Figures 8A to 8C The contractile force trajectory of the formed NMJ co-cultured tissues was plotted. The co-cultured tissues were exposed to active botulinum neurotoxin complex serum type A (BoNT-A) at a dose of 10 µg toxin per tissue in maintenance medium. Blue light-driven contractile responses in the treated and control groups were monitored over the next 8 hours.

[0193] Figure 10A The contractile force output (mean ± SD, n = 4 tissues, 10 blue light pulse-driven contractions, 250 ms pulse train, 40 mW / mm², 100 Hz, 80% duty cycle) is shown, normalized to control values ​​at each time point. As shown in the figure, treatment with BoNT-A resulted in the complete loss of the blue light-driven contractile response in the co-cultured tissues.

[0194] Figure 10B It shows from Figure 10A The images show representative trajectory plots obtained from a single co-culture tissue at 0 and 8 hours post-treatment for the groups shown.

[0195] Figure 10C This shows the effect after BoNT-A processing. Figure 10ARepresentative force trajectory diagrams obtained from electrical stimulation of co-cultured tissues. The loss of functional response after BoNT-A exposure may be due to complete loss of muscle function; however, electrical stimulation (10 ms, 100 mA, 1 Hz biphasic stimulation pulses) of treated and control tissues showed no difference in basic functional capacity, thus confirming the specificity of the blue light-driven response to NMJ in co-cultured tissues.

[0196] The methods and apparatus described in this paper offer several advantages for modeling neuromuscular diseases. Firstly, there is the in-situ generation of neuronal components (i.e., neuronal aggregates, neurospheres, and neuronal cell cultures). Current models require manual processing of cells / cell aggregates / neurospheres, which is both time-consuming and technically challenging, leading to reduced neuronal survival, reproducibility, and scalability. The methods described in this paper further reduce variability by optionally thawing single cells directly from cryopreservation onto casting plates, thereby allowing for high-throughput batch control for experiments with high batch sensitivity.

[0197] Secondly, the living neuronal material is connected to the living muscle material via a connecting channel (e.g., connecting channel 318 of the casting plate 300), allowing for long-distance communication and axonal extension. Many neuromuscular diseases are progressive, characterized by axonal retraction from the muscle; this progressive instability has a short / small measurement window on devices with short connecting paths (limited by a lack of guidance), making it unlikely to successfully simulate complex progressive diseases.

[0198] Third, these devices and methods allow for specific and independent stimulation of cell cultures (e.g., different cell compartments), thereby enabling comparison of two important indicators of neuromuscular junction performance: maximum directly induced muscle function and innervation-induced muscle function. The latter can be achieved, for example, by directly stimulating living muscle material with electrical, light, or chemical stimulation and measuring the contractile response of the muscle cell culture. The former can be achieved, for example, by stimulating neuronal cell cultures and measuring the contractile response of the muscle cell cultures. The ratio of these measurements provides an estimate of innervation density, which is important for muscle function in both healthy and diseased states.

[0199] In view of the foregoing, the various inventive aspects disclosed herein may be characterized and claimed under the following terms:

[0200] 1. A method of using a tissue analysis system including a co-culture casting system, the method comprising: seeding a plurality of neuronal culture compartments of a porous co-culture casting plate with live neuronal material, wherein each neuronal culture compartment includes a plurality of cell aggregation features configured to aggregate the live neuronal material into cell aggregates; seeding a plurality of muscle culture compartments of a muscle tissue casting plate with live muscle material; culturing a plurality of co-cultured tissues in the porous co-culture casting plate, each co-cultured tissue comprising a muscle cell culture cultured from the live muscle material and an in situ neurosphere matured from the live neuronal material in the cell aggregation features; and stimulating the live neuronal material to cause contraction of the innervated portion of the live muscle material in each co-cultured tissue.

[0201] 2. The method according to Clause 1, wherein seeding the plurality of neuronal culture chambers with the live neuron material comprises directly thawing the cells containing motor neurons in the plurality of neuronal culture chambers.

[0202] 3. The method according to Clause 2, wherein culturing the plurality of co-cultured tissues comprises culturing at least one of the co-cultured tissues in each neuron culture chamber.

[0203] 4. The method according to Clause 1, wherein the cell aggregation feature includes a characteristic geometry.

[0204] 5. The method according to Clause 4, wherein the feature geometry includes tessellated surface features.

[0205] 6. The method according to Clause 5, wherein the tiling surface feature includes tiling cavities, wherein each tiling cavity forms a lowest point.

[0206] 7. The method according to Clause 1, wherein the cell aggregation characteristic includes at least one of hydrophobicity or electrostatic charge.

[0207] 8. The method according to Clause 2 or Clause 3, wherein seeding the plurality of neuronal culture chambers with the live neuron material includes applying a connecting hydrogel on the in situ neurosphere in each neuronal culture chamber, wherein culturing each of the plurality of co-cultured tissues includes remodeling the interface between the connecting hydrogel and a second hydrogel containing the live muscle material.

[0208] 9. The method according to Clause 8, wherein reshaping the interface comprises treating the interface with a serum culture medium.

[0209] 10. The method according to Clause 2 or Clause 3, wherein the cell aggregation feature is disposed in the casting groove of the corresponding neuron culture chamber.

[0210] 11. The method according to Clause 2, wherein culturing each of the co-cultured tissues comprises communicating the living neuronal material with the living muscle material along a communication channel.

[0211] 12. The method according to Clause 11, wherein the porous co-culture casting plate comprises a plurality of culture units, wherein each culture unit comprises a muscle culture chamber connected via the communication channel to at least one neuron culture chamber among the plurality of neuron culture chambers.

[0212] 13. The method according to Clause 1, wherein causing the innervated portion of the live muscle material in each of the co-cultured tissues to contract by stimulating the live neuronal material comprises transferring the co-cultured tissues to a maintenance plate prior to causing the innervated portion of the live muscle material in the co-cultured tissues to contract by stimulating the live neuronal material.

[0213] 14. The method according to Clause 1, wherein contracting the innervated portion of the living muscle material in the co-cultured tissue comprises activating the in situ neurosphere using at least one of light stimulation, electrical stimulation, or chemical stimulation.

[0214] 15. The method according to Clause 2 or Clause 3, wherein contracting the innervated portion of the live muscle material in each of the co-cultured tissues comprises simultaneously contracting the innervated portion of the live muscle material in each of the co-cultured tissues.

[0215] 16. The method according to Clause 15, wherein causing the innervated portion of the living muscle material in each of the co-cultured tissues to contract further comprises simultaneously recording the contraction of each of the co-cultured tissues.

[0216] 17. A tissue analysis system or co-culture casting system, comprising: a porous co-culture casting plate, the porous co-culture casting plate including a plurality of neuron culture compartments, each neuron culture compartment having a cell aggregation region formed on its bottom surface, each cell aggregation region including a plurality of cell aggregation features; and a porous muscle tissue casting plate.

[0217] 18. The tissue analysis system according to Clause 17, wherein the cell aggregation feature includes a characteristic geometry.

[0218] 19. The tissue analysis system according to Clause 18, wherein the feature geometry includes tessellated surface features.

[0219] 20. The tissue analysis system according to Clause 19, wherein the mosaic surface feature includes mosaic cavities, wherein each mosaic cavity forms a lowest point.

[0220] 21. The tissue analysis system according to Clause 17, wherein the cell aggregation characteristic includes at least one of hydrophobicity or electrostatic charge.

[0221] 22. The tissue analysis system according to Clause 17, wherein each of the said cell aggregation regions is disposed in a casting groove in the corresponding neuron culture chamber.

[0222] 23. The tissue analysis system according to Clause 17, wherein the porous co-culture casting plate comprises a plurality of culture units, wherein each culture unit comprises a muscle culture chamber and at least one of the neuron culture chambers connected via a communication channel.

[0223] 24. A method for modeling a neuromuscular junction in multiple co-cultured tissues, comprising: seeding multiple neuronal culture compartments of a multiwell plate with live neuronal material; casting multiple muscle cell cultures containing live muscle material; culturing multiple co-cultured tissues in the multiwell plate, the co-cultured tissues comprising the live muscle material and in situ neurospheres matured from the live neuronal material; and stimulating the live neuronal material to cause contraction of the innervated portion of the live muscle material in each of the co-cultured tissues.

[0224] 25. The method according to Clause 24, wherein seeding the plurality of neuronal culture chambers with the live neuron material comprises directly thawing cells containing motor neurons in the plurality of neuronal culture chambers.

[0225] 26. The method according to Clause 25, wherein culturing the plurality of co-cultured tissues comprises culturing at least one of the co-cultured tissues in each neuron culture chamber.

[0226] 27. The method according to Clause 25 or Clause 26, wherein each neuron culture compartment comprises a plurality of cell aggregation features configured to aggregate the live neuron material into cell aggregates.

[0227] 28. The method according to Clause 27, wherein the cell aggregation feature includes a feature geometry configured to drive cell aggregation.

[0228] 29. The method according to Clause 28, wherein the feature geometry includes tessellated surface features.

[0229] 30. The method according to Clause 29, wherein the tessellation surface feature includes tessellation cavities, wherein each tessellation cavity forms a lowest point.

[0230] 31. The method according to Clause 27, wherein the cell aggregation characteristic includes at least one of hydrophobicity or electrostatic charge.

[0231] 32. The method according to Clause 25 or Clause 26, wherein directly thawing the cells containing the motor neurons in the plurality of neuronal culture compartments includes thawing the cells without a Rho kinase inhibitor.

[0232] 33. The method according to Clause 25 or Clause 26, wherein seeding the plurality of neuronal culture chambers with the live neuron material includes applying a connecting hydrogel on the in situ neurosphere in each neuronal culture chamber, wherein culturing each of the plurality of co-cultured tissues includes remodeling the interface between the connecting hydrogel and a second hydrogel containing the live muscle material.

[0233] 34. The method according to clause 33, wherein reshaping the interface comprises treating the interface with a serum culture medium.

[0234] 35. The method according to Clause 25 or Clause 26, wherein the porous plate is a first cast plate and the plurality of neuron culture chambers include casting grooves.

[0235] 36. The method according to Clause 25, wherein culturing the plurality of co-cultured tissues comprises, for each of the co-cultured tissues, communicating the living neuronal material with the living muscle material along a communication channel.

[0236] 37. The method according to Clause 36, wherein the multi-well plate comprises a plurality of culture units, wherein each culture unit comprises a muscle culture chamber and at least one neuron culture chamber connected via the communication channel.

[0237] 38. The method according to Clause 26 further comprises transferring the co-cultured tissue to a maintenance plate before stimulating the innervated portion of the live muscle material in the co-cultured tissue to contract by means of stimulating the live neuronal material.

[0238] 39. The method according to Clause 25 or Clause 26, wherein stimulating the living neuronal material to cause contraction of the innervated portion of the living muscle material in the co-cultured tissue includes activating the in situ neurosphere.

[0239] 40. The method according to Clause 25 or Clause 26, wherein contracting the innervated portion of the live muscle material in each of the co-cultured tissues comprises simultaneously contracting the innervated portion of the live muscle material in each of the co-cultured tissues.

[0240] 41. The method according to Clause 40, wherein causing the innervated portion of the living muscle material in each of the co-cultured tissues to contract further comprises simultaneously recording the contraction of each of the co-cultured tissues.

[0241] 42. A method for modeling multiple neuromuscular junctions, comprising: seeding a plurality of neuronal culture chambers of a multiwell plate with live neuronal material by directly thawing cells containing motor neurons in the plurality of neuronal culture chambers, wherein each neuronal culture chamber contains a cell aggregation feature configured to aggregate the live neuronal material into cell aggregates; casting a plurality of muscle cell cultures containing live muscle material; culturing a plurality of co-culture tissues in the multiwell plate, the co-culture tissues comprising the live muscle material and in situ neurospheres matured from the live neuronal material, partially achieved by remodeling the interface between a connecting hydrogel and a second hydrogel containing the live muscle material; and simultaneously stimulating the live neuronal material to cause contraction of the innervated portion of the live muscle material in each of the co-culture tissues.

[0242] 43. A method for modeling multiple neuromuscular junctions, comprising: seeding multiple culture units of a multiwell plate with live neuronal material by directly thawing cells containing motor neurons in multiple neuronal culture compartments, wherein each neuronal culture compartment contains a cell aggregation feature configured to aggregate the live neuronal material into cell aggregates; casting multiple muscle cell cultures containing live muscle material; culturing multiple co-culture tissues in the multiwell plate by communicating the live neuronal material and the live muscle material along a communication channel in each of the culture units, the co-culture tissues comprising the live muscle material and in situ neurospheres matured from the live neuronal material; and simultaneously stimulating the live neuronal material to cause contraction of the innervated portion of the live muscle material in each of the co-culture tissues.

[0243] Furthermore, the proposed methods and apparatus allow for non-invasive measurement of these outputs over time, enabling long-term observation of progressive degenerative diseases in a single tissue, thereby improving statistical power and significantly reducing the cost of time-process experiments.

[0244] Those skilled in the art will reasonably envision various modifications that can be made to the embodiments of this disclosure in light of the foregoing description. The following claims are presented as examples of embodiments of this disclosure, but they should not be construed as limiting the other claims or other embodiments disclosed herein.

[0245] The detailed description above, taken in conjunction with the accompanying drawings (where like reference numerals denote like elements), is intended as a description of representative embodiments of this disclosure and not as representing the only embodiments. Each embodiment described in this disclosure is provided as an example or illustration and should not be construed as preferred or advantageous over other embodiments. The illustrative embodiments provided herein are not intended to be exhaustive or to limit this disclosure to the precise form disclosed. Similarly, any step described herein may be interchanged with other steps or combinations of steps to achieve the same or substantially similar results. Furthermore, one or more features of any embodiment may be combined with one or more features of one or more embodiments to form additional embodiments within the scope of this disclosure.

[0246] Generally, the embodiments disclosed herein are non-limiting, and the inventors envision other embodiments within the scope of this disclosure that may include the structure and functionality of more than one specific embodiment shown in the drawings and described in the specification. It should be understood that variations and modifications may be made by others, and equivalents may be employed, without departing from the spirit of this disclosure. Therefore, it is expressly intended that all such variations, modifications, and equivalents fall within the spirit and scope of the claimed disclosure. For example, this disclosure includes additional embodiments having combinations of any one or more features described above with respect to representative embodiments.

[0247] In the foregoing description, specific details have been set forth to provide a thorough understanding of representative embodiments of this disclosure. It will be apparent to those skilled in the art that the embodiments disclosed herein can be practiced without showing all the specific details. In some cases, well-known process steps have not been described in detail to avoid unnecessarily obscuring various aspects of this disclosure.

[0248] This application may include references to directions, such as “first,” “second,” “vertical,” “horizontal,” “front,” “back,” “left,” “right,” “top,” “bottom,” “below,” “around,” etc. These and other similar references in this application are intended to aid in the description and understanding of particular embodiments (such as when embodiments are positioned for use) and are not intended to limit this disclosure to these directions or locations.

[0249] This application may also refer to quantities and quantities. Unless otherwise specified, these quantities and quantities should not be considered limiting, but rather examples of possible quantities or quantities associated with this application. Moreover, in this regard, this application may use the term "multiple" to refer to a quantity or quantity. In this regard, the term "multiple" means any quantity more than one, such as two, three, four, five, etc. The terms "about," "approximately," etc., refer to adding or subtracting 5% of the stated value. The term "based on" means "at least partially based on." The term "between" includes the stated value associated with it. The expressions "at least one of A, B, or C"; "at least one of A, B, and C"; and "at least one of A, B, and / or C" have the same meaning, i.e., any one of the following conditions satisfies all the foregoing expressions: A; B; C; AB; AC; BC; ABC.

Claims

1. A method using a microstructure analysis system including a co-cultivation casting system, the method comprising: Multiple neuron culture compartments of a porous co-culture casting plate are seeded with live neuron material, wherein each neuron culture compartment contains multiple cell aggregation features configured to aggregate the live neuron material into cell aggregates. Multiple muscle culture compartments were created by inoculating live muscle material into a muscle tissue casting plate. Multiple co-culture tissues are cultured in the porous co-culture casting plate, each co-culture tissue comprising a muscle cell culture from the live muscle material and an in situ neurosphere formed from the live neuronal material maturing in the cell aggregation feature; as well as By stimulating the living neuronal material, the nerve-innervated portion of the living muscle material in each of the co-cultured tissues contracts.

2. The method of claim 1, wherein seeding the plurality of neuronal culture chambers with the live neuron material comprises directly thawing the cells containing motor neurons in the plurality of neuronal culture chambers.

3. The method of claim 2, wherein culturing the plurality of co-cultured tissues comprises culturing at least one of the co-cultured tissues in each neuron culture chamber.

4. The method of claim 1, wherein the cell aggregation feature includes a characteristic geometry.

5. The method of claim 4, wherein the feature geometry comprises tessellated surface features.

6. The method of claim 5, wherein the tiling surface feature includes tiling cavities, wherein each tiling cavity forms a lowest point.

7. The method of claim 1, wherein the cell aggregation characteristic includes at least one of hydrophobicity or electrostatic charge.

8. The method according to claim 2 or claim 3, The seeding of the plurality of neuron culture chambers with the live neuron material includes, in each neuron culture chamber, the application of a connecting hydrogel to the in situ neurosphere. Cultivating each of the plurality of co-cultured tissues includes reshaping the interface between the connecting hydrogel and the second hydrogel containing the living muscle material.

9. The method of claim 8, wherein remodeling the interface comprises treating the interface with a serum culture medium.

10. The method according to claim 2 or claim 3, wherein the cell aggregation feature is disposed in the casting groove of the corresponding neuron culture chamber.

11. The method of claim 2, wherein culturing each of the co-cultured tissues comprises communicating the living neuronal material with the living muscle material along a communication channel.

12. The method of claim 11, wherein the porous co-culture casting plate comprises a plurality of culture units, wherein each culture unit comprises a muscle culture chamber connected via the communication channel and at least one neuron culture chamber among the plurality of neuron culture chambers.

13. The method of claim 1, wherein causing the innervated portion of the live muscle material in each of the co-cultured tissues to contract by stimulating the live neuronal material comprises transferring the co-cultured tissues to a maintenance plate prior to causing the innervated portion of the live muscle material in the co-cultured tissues to contract by stimulating the live neuronal material.

14. The method of claim 1, wherein contracting the innervated portion of the living muscle material in the co-cultured tissue comprises activating the in situ neurosphere using at least one of light stimulation, electrical stimulation, or chemical stimulation.

15. The method of claim 2 or claim 3, wherein contracting the nerve-innervated portion of the live muscle material in each of the co-cultured tissues comprises simultaneously contracting the nerve-innervated portion of the live muscle material in each of the co-cultured tissues.

16. The method of claim 15, wherein causing the innervated portion of the living muscle material in each of the co-cultured tissues to contract further comprises simultaneously recording the contraction of each of the co-cultured tissues.

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