Tissue analysis systems, co-culture casting systems, and methods for functional interrogation of co-culture tissues

Abstract

Tissue analysis systems and co-culture casting systems include at least a multi-well co-culture casting plate and a muscle tissue casting plate. Methods of use include seeding a plurality of neuronal culture compartments of the multi-well co-culture casting plate with live neuronal material, seeding a plurality of muscle culture compartments of the muscle tissue casting plate with live muscle material, culturing, in the multi-well co-culture casting plate, a plurality of co-culture tissues that include a muscle cell culture cultured from the live muscle material and in situ neurospheres matured in the cell aggregation features from the live neuronal material, and causing an innervated portion of the live muscle material in each of the co-culture tissues to contract by stimulating the live neuronal material.

Description

TISSUE ANALYSIS SYSTEMS, CO-CULTURE CASTING SYSTEMS, AND METHODSFOR FUNCTIONAL INTERROGATION OF CO-CULTURE TISSUESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority to U.S. Provisional Application No. 63 / 508,551, filed June 16, 2023, and to U.S. Provisional Application No. 63 / 621 ,771 , filed January 17, 2024, the entire disclosures of which are hereby incorporated by reference.STATEMENT OF GOVERNMENT LICENSE RIGHTS

[0002] This invention was made with government support under TR004795 awarded by the National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD

[0003] This disclosure relates to the field of tissue engineering, for example the modeling of neuromuscular biology.BACKGROUND

[0004] Within the human body, the musculoskeletal skeletal system is responsible for a range of methods fundamentally important to life, from respiration to locomotion. Control over the musculature within the musculoskeletal system is exerted by the central nervous system through the neuromuscular junction and associated structures, termed the neuromuscular system. Consequently, diseases of the neuromuscular system lead to severe progressive diseases similar to those of the musculoskeletal system. Current models of the neuromuscular system rely upon animal models, which present significant limitations and fail to generate effective treatments which can be used in the clinic. Therefore, a model of the neuromuscular system based upon human cells, built in a controlled engineered environment, would enable research on the mechanisms of diseases, discovery of therapeutics, and toxicity screening of compounds.

[0005] To that end, previous work have attempted to develop models of the neuromuscular junction ("NMJ") through the culture of motor neurons and skeletal muscle cells in 2D and 3D devices. However, each known model presents limitations preventing widespread adoption in a profitable drug discovery pipeline. These limitations include non-scalable manufacturing technology, poor reproducibility between different laboratories, high levels of technicalexpertise to assemble, low throughput measurement technologies, low throughput seeding approaches, low biological mimicry and low percentages of innervation leading to narrow assay windows.

[0006] Specifically, initial models utilized simple 2D co-culture systems based within standard or lightly modified cultureware. These systems cannot replicate the 3D structure of the neuromuscular junction. Furthermore, 2D skeletal muscle cultures fail to generate a system able to allow examination of neuromuscular disease and physiology across time, a key feature for the study of progressive neuromuscular conditions. Finally, no compartmentalization of the cell types prevents unique stimulation of the cells without the use of cell modification or chemical stimulation reducing the range of applications and cells which these systems can be used for.

[0007] Microfluidic devices have been presented which allow communication of skeletal muscle cells and neurons while keeping cell bodies separate. However, these systems remain limited by the 2D environment, the limited timeframe of 2D muscle culture, and an inability to present a direct readout of skeletal muscle function.

[0008] Examples of 3D compartmentalized and non-compartmentalized devices have been presented. The non-compartmentalized systems suffer from the same limitations as 2D noncompartmentalized systems, with chemical stimulation likely to lead to cell death and optogenetic solutions requiring extensive genetic engineering precluding widespread adoption of these systems. The compartmentalized systems suffer from throughput limitations (e.g., due to measurement performed in series) and limited application (e.g., due to culturing of the neuronal spheroid component is carried out in a 2D environment).

[0009] None of the forgoing devices or methods show in situ generation of the neuronal components or guidance of axons beyond that exerted by muscle attraction. Therefore, the forgoing devices and methods cannot be utilized in a profitable drug discovery pipeline due to fundamental limitations on neuron survival rates and NMJ formation, as well as practical usability and repeatability limitations.SUMMARY

[0010] The present disclosure provides systems, devices, and methods enabling real-time high throughput readouts of muscle function in response to neural inputs in a 3D system allowing modeling of the neuromuscular system and associated diseases.

[0011] According to an aspect, The present disclosure provides methods of using a tissue analysis system comprising a co-culture casting system, the methods comprising: seeding a plurality of neuronal culture compartments of a multi-well co-culture casting plate with live neuronal material, wherein each neuronal culture compartment comprises 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, in the multi-well co-culture casting plate, a plurality of co-culture tissues, each of the co-culture tissues comprising a muscle cell culture cultured from the live muscle material and in situ neurospheres matured in the cell aggregation features from the live neuronal material; and causing an innervated portion of the live muscle material in each of the co-culture tissues to contract by stimulating the live neuronal material.

[0012] According to another aspect, the present disclosure provides tissue analysis systems and co-culture casting systems, comprising: a multi-well co-culture casting plate comprising a plurality of neuronal culture compartments each with a cell aggregation region formed at a bottom surface thereof, each cell aggregation region comprising a plurality of cell aggregation features; and a multi-well muscle tissue casting plate.

[0013] According to another aspect, the present disclosure provides methods of modelling a neuromuscular junction in a plurality of co-culture tissues, comprising: seeding a plurality of neuronal culture compartments of a multi-well plate with live neuronal material; casting a plurality of muscle cell cultures comprising live muscle material; culturing, in the multi-well plate, a plurality of co-culture tissues comprising the live muscle material and in situ neurospheres matured from the live neuronal material; and causing an innervated portion of the live muscle material in each of the co-culture tissues to contract by stimulating the live neuronal material.

[0014] According to another aspect, the present disclosure provides methods of modelling a plurality of neuromuscular junctions, comprising: seeding a plurality of neuronal culture compartments of a multi-well plate with live neuronal material by thawing cells comprising motor neurons directly in the plurality of neuronal culture compartments, wherein each neuronal culture compartment comprises cell aggregation features configured to aggregate the live neuronal material into cell aggregates; casting a plurality of muscle cell cultures comprising live muscle material; culturing, in the multi-well plate, a plurality of co-culture tissues comprising the live muscle material and in situ neurospheres matured from the liveneuronal material, in part by remodeling an interface between a linking hydrogel and a second hydrogel comprising the live muscle material; and contemporaneously causing an innervated portion of the live muscle material in each of the co-culture tissues to contract by stimulating the live neuronal material.

[0015] According to another aspect, the present disclosure provides methods of modelling a plurality of neuromuscular junctions, comprising: seeding a plurality of culture units of a multiwell plate with live neuronal material by thawing cells comprising motor neurons directly in a plurality of neuronal culture compartments, wherein each neuronal culture compartment comprises cell aggregation features configured to aggregate the live neuronal material into cell aggregates; casting a plurality of muscle cell cultures comprising live muscle material; culturing a plurality of co-culture tissues comprising the live muscle material and in situ neurospheres matured from the live neuronal material in the multi-well plate by communicating, in each of the culture units, the live neuronal material with the live muscle material along a communication channel; and contemporaneously causing an innervated portion of the live muscle material in each of the co-culture tissues to contract by stimulating the live neuronal material.

[0016] Advantageously, the devices and methods described herein enable in situ generation of neuronal components (single or multicellular), reducing the need to manipulate neuronal components following maturation. In another aspect, the systems enable active control of axon outgrowth in 3D tissues, leveraging principles of microfluidic flow, to ensure highly reproducible connections between neuronal and skeletal muscle components. In still another aspect, the systems enable real-time label -free measurement of skeletal muscle of large numbers (>24) of tissues in parallel, significantly improving over the throughput of any previously presented systems.

[0017] The devices and methods circumvent shortcomings of known systems and methods by enabling parallel functional measurements (e.g., using optical and / or magnetic sensing) and in situ spheroid creation, negating the need for individual spheroid handling. Additionally, the devices and methods circumvent throughput limitations and drive axonal guidance within a 3D hydrogel environment towards an engineered muscle tissue, better representing the process of neuromuscular junction development in vivo.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 illustrates a co-culture tissue formed according to methods of the present disclosure.

[0019] FIG. 2A illustrates a plan view of a multi-well co-culture casting plate according to an embodiment of the present disclosure.

[0020] FIG. 2B illustrates a detail plan view of one well of the casting plate of FIG. 2 A.

[0021] FIG. 2C illustrates a section view of the well of FIG. 2B.

[0022] FIG. 2D illustrates a perspective view of cell aggregation features of the well of FIG. 2B.

[0023] FIG. 3A illustrates a plan view of another multi-well co-culture casting plate according to an aspect of the present disclosure.

[0024] FIG. 3B illustrates a detail plan view of one well of the casting plate of FIG. 3 A.

[0025] FIG. 3C illustrates a first section view of the well of FIG. 3B.

[0026] FIG. 3D illustrates a second section view of the well of FIG. 3B.

[0027] FIG. 4A illustrates tissue analysis systems, methods of using tissue analysis systems and co-culture casting systems, and methods of modelling a plurality of neuromuscular junctions, according to an aspect of the present disclosure.

[0028] FIG. 4B illustrates exemplary methods of FIG. 4A for using a tissue analysis system and co-culture casting system.

[0029] FIG. 4C illustrates exemplary methods of FIG. 4A for modelling a plurality of neuromuscular junctions.

[0030] FIG. 4D illustrates additional exemplary methods of FIG. 4A for modelling a plurality of neuromuscular junctions.

[0031] FIG. 5 shows example images of cells formed according to the present disclosure.

[0032] FIG. 6 shows images of a muscle cell culture comprising live skeletal muscle material.

[0033] FIG. 7A shows a contraction force trace of a co-culture tissue formed according to the methods described herein.

[0034] FIG. 7B shows a force trace of a co-culture tissue formed according to the methods described herein.

[0035] FIG. 7C shows a graph of electrically evoked forces for tetanus response of a coculture tissue formed according to the methods described herein.

[0036] FIG. 7D shows of graph of electrically evoked forces for twitch response of a coculture tissue formed according to the methods described herein.

[0037] FIG. 8A shows a plot of active twitch forces of a co-culture tissue formed according to the methods described herein.

[0038] FIG. 8B shows a plot of time from contraction 10 to peak contraction of a co-culture tissue formed according to the methods described herein.

[0039] FIG. 8C shows a plot of time from contraction peak to relaxation of a co-culture tissue formed according to the methods described herein.

[0040] FIG. 9A is a trace showing response to blue light of co-culture tissues formed according to methods and devices of the present disclosure.

[0041] FIG. 9B plots the contraction force of the co-culture tissue of FIG. 9A in response to different blue light stimulation periods.

[0042] FIG. 9C plots the contraction force of the co-culture tissue of FIG. 9A from optical stimulation of the neuronal cell culture as a percentage the contraction force achieved by directly stimulating the muscle cell culture with field stimulation.

[0043] FIG. 9D plots the percentage capture of the contraction force of the co-culture tissue of FIG. 9A within 100ms of blue light stimulation.

[0044] FIG. 10A - FIG. 10C show contractile force traces of co-culture tissues formed according to the present disclosure before and after exposure to active Botulinum neurotoxin complex serotype A (BoNT-A).DETAILED DESCRIPTION

[0045] The devices and methods of the present disclosure allow for the in situ culture of cell types relevant to neuromuscular biology within devices which guide and control cell growth and cell-cell interactions while allowing real-time functional readouts of certain cell types (e.g., muscle cells co-cultured with neuronal cells). The devices and methods can be adapted to a variety of user interventions to create controlled experimental conditions for the study of musculoskeletal / neuromuscular disease / conditions, e.g., a neurotoxin potency assay.

[0046] As used herein, in situ means in the original place (e.g., original device) where the cells are originally formed. For example, neurospheres and neuronal cell cultures formed in a casting plate are in situ as long as those neurospheres or neuronal cell cultures remain unmoved from their situs of formation in the casting plate. Furthermore, cells remain in situ despitegrowth, extension, withdrawal, or other movement by such cells (e.g., axonal withdrawal). Furthermore, cells remain in situ within a device regardless of movement of the device itself. For example, neurospheres formed in a casting plate are in situ as long as said cells remain unmoved from their situs of formation in the casting plate, regardless of whether the casting plate is physically moved.

[0047] As used herein, to culture means to cultivate or grow one or more cell populations, e.g., in a hydrogel or other medium. Accordingly, to co-culture means to cultivate or grow two or more distinct cell populations in physical contact, e.g., through a hydrogel or other media. A co-culture tissue is a tissue comprising at least two distinct cell populations which have been co-cultured. Co-culture tissues may include, for example, two, three, four, five, or more distinct cell populations. In any embodiment, at least two of the cell populations may be biologically distinct (e.g., different cell types such as neuronal cells and muscle cells).

[0048] As used herein, the term hydrogel refers to a group of polymeric materials, the hydrophilic structure of which renders them capable of holding large amounts of water in their three-dimensional networks. Polymers from which hydrogels are made may be naturally- occurring, e.g., purified from a natural source, or synthetic, e.g., chemically-synthesized. The polymers may be water soluble or water insoluble. The ability of hydrogels to absorb water arises from hydrophilic functional groups attached to the polymeric backbone, while their resistance to dissolution arises from cross-links between network chains. The crosslinks may be chemical or physical; polymer crosslinks include covalent crosslinks, ionic crosslinks, hydrogen bonds, and / or hydrophobic interactions. Hydrogel-forming natural polymers 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 / synthesis methods. Hydrogels may be one, two- or multi-component systems comprising a three-dimensional network of polymer chains and water that fills the space between polymer macromolecules.

[0049] In some embodiments, hydrogel can include a network of polymer chains that are water-insoluble. Hydrogel can include a water-swollen and cross-linked polymeric network produced by a reaction of one or more monomers. Hydrogel can include polymeric material that exhibits the ability to swell and retain a significant fraction of water within its structure, but will not dissolve in water. Hydrogel can include a colloidal gel in which water is the dispersion medium. Their hydrophilic structure renders them capable of holding large amountsof water in their three-dimensional networks (e.g., hydrogels can be super absorbent and can contain 50%, 75%, 90%, 95% and over 99% water). Hydrogels can include natural and / or synthetic polymers. Hydrogels can possess a degree of flexibility very similar to natural tissue, due to their significant water content.

[0050] In a non-limiting example, a hydrogel comprises collagen. Various concentrations of collagen may be used. For example, a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1-10 mg / ml or more of collagen in an aqueous solution (such as cell culture medium, saline, or water) may be used. The collagen-containing hydrogel optionally also includes one or more of the following proteins: laminin, entactin, heparan sulfate proteoglycans, which are characterized by adhesive properties, as well as growth factors such as TGF-beta and EGF. An example of such a collagen-containing hydrogel includes MATRIGEL®. Thus in some embodiments, the hydrogel comprises MATRIGEL® (gelatinous protein mixture derived from mouse tumor cells) or an equivalent thereof. MATRIGEL® is the trade name for a gelatinous protein mixture secreted by Engelbreth-Holm- Swarm (EHS) mouse sarcoma cells that is produced and marketed by Corning Life Sciences. Thus “MATRIGEL®” may be substituted with a gelatinous protein mixture from another natural, synthetic, or commercial source. MATRIGEL® or a substitute of MATRIGEL® may be used, e.g., in a 1: 1, 1 :2, 1 :3, 1:4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 : 10 MATRIGEL®:collagen or collagemMATRIGEL® ratio.

[0051] In some embodiments, the hydrogel comprises an alginate or a derivative thereof, gelatin, collagen, agarose, a natural or synthetic polysaccharide, polylactic acid, polyglycolic acid, poly(lysine), a polyanhydride; a poly(lactide-co-glycolide) (PLGA) polymer, a polyamino acid, a poly(alkylene oxide), a polyethylene oxide), a poly(allylamine)(PAM), a poly(acrylate), a polyester, polyhydroxybutyrate and poly-epsilon-caprolactone, a polyphosphazine, a poly(vinyl alcohol), a modified styrene polymer, poly(4- aminomethyl styrene), a pluronic polyol, a polyoxamer, a poly(uronic acid), a poly(vinylpyrrolidone), and / or a copolymer comprising one or more of an alginate or a derivative thereof, gelatin, collagen, agarose, a natural or synthetic polysaccharide, polylactic acid, polyglycolic acid, poly(lysine), a polyanhydride; a poly(lactide-co-glycolide) (PLGA) polymer, a polyamino acid, a poly(alkylene oxide), a poly(ethylene oxide), a poly(allylamine)(PAM), a poly(acrylate), a polyester, polyhydroxybutyrate and poly-epsilon- caprolactone, a polyphosphazine, a poly(vinyl alcohol), a modified styrene polymer, poly(4-aminomethylstyrene), a pluronic polyol, a polyoxamer, a poly(uronic acid), and a poly(vinylpyrrolidone).

[0052] The presented devices and methods enable reproducible and robust co-culture of neurons, muscle and other neuromuscular related cell types to form ex vivo neuromuscular junctions. In particular, the devices and methods provide co-culture tissues comprising live neuronal material and live muscle material, which may be stimulated independently to assess NMJ function.

[0053] The live neuronal material may comprise, for example, cells, organoids, and / or tissues comprising neuronal cells, including, for example, stem cell derived cells, human and animal cell lines, primary human or animal bulk tissue explants or primary human or animal isolated cells. Representative live neuronal material includes, for example, iPSC derived motor neurons (e.g., using small molecule directed differentiation, with presence of motor neurons confirmed within the differentiated population using 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, microglial cells. These cells may contain mutations or disease characteristics relevant to human disease or may present a model of good health allowing the study of disease in comparison to healthy cultures. Live neuronal material may include any combination of the foregoing cell types, optionally in a medium (e.g., hydrogel) with other cell types. Optionally, the live neuronal material may express proteins acting as gated ion channels, e g., light gated ions channels including Channelrhodopsin -1 and -2. Expression of such proteins may be induced, e.g., by administering one or more promoters such as tetracyclene promoter or neuron-specific promoter. Advantageously, expression of such proteins may facilitate stimulation of the neurons. For example, in some embodiments, blue light sensitive channelrhodopsin expression by the live neuronal material facilitates blue light driven neuronal activation.

[0054] The presented devices provide one or more compartments in which single neuronal cells may be seeded and subsequently matured in a non-adherent environment promoting neurosphere formation. Subsequent addition of a biological / non-biological hydrogel provides an adherent substrate for neurite extension. The timing of hydrogel(s) addition, and thecomposition of the hydrogel(s) can be manipulated to best promote robust neurite extension. Neurites mature to axons with motile growth cones.

[0055] The live muscle material may comprise, for example, one or more of: cells, organoids, and / or tissues comprising muscle cells, including, for example, stem cell derived cells, human and animal cell lines, primary human or animal bulk tissue explants or primary human or animal isolated cells. Representative live muscle material includes, for example, iPSC derived myoblasts and primary human dermal fibroblasts (e.g., myoblasts characterized using desmin positivity, populations with >80% desmin positivity) myoblasts, skeletal muscle fibers (e.g., oxidative, oxidative-glycolytic, glycolytic), cardiac muscle cells (e.g., cardiomyocytes), smooth muscle cells, and satellite cells. Live muscle material may include any combination of the foregoing cell types, optionally in a medium (e.g., hydrogel) with other cell types (e.g., stromal cells). Said live muscle material may be assembled into engineered skeletal muscle tissues (EMTs) such as disclosed in International Patent Publication No. WO2017 / 156455A1 to Sniadecki et al., herein incorporated by reference in its entirety.

[0056] The cell culture comprising live neuronal material is co-cultured with the cell culture comprising the live muscle material, forming a co-culture tissue. In particular, axons extending from the live neuronal material communicate with the live muscle material, e.g., through a hydrogel interface. The intervening space can then be crossed by the axons through extension allowing neuron-muscle interactions (i.e., innervated muscle), which will mature to neuromuscular junctions. In some embodiments, the interface between the live neuronal material and live muscle material is remodeled to facilitate axon extension, e.g., by treating the interface with a serum media. In some embodiments, hydrogel patterning and growth factor accumulation / gradients can guide growth cone motility through the device to position the axon tip close to the introduced muscle.

[0057] The methods and devices thus allow for the individual maturation of neurons and skeletal muscle to provide tissue primed for interaction (see, e.g., FIG. 1 and FIG. 6), a characteristic missing from previous neuromuscular co-culture systems.

[0058] FIG. 1 illustrates a representative NMJ co-culture tissue 102 according to an aspect of the present disclosure, i.e., formed according to the methods and / or utilizing one or more of the devices described herein (such as the device of FIG. 2A - FIG. 2D). The co-culture tissue 102 comprises live muscle material 104 (visible as translucent matter having an overall boneshape) extending between a plurality of tissue fixtures 106a, 106b. Each of the tissue fixtures106a, 106b may be, for example, a flexible post, hook, clamp or a relatively rigid post, hook, or clamp, e.g., as described in International Patent Publication No. WO2017 / 156455A1 to Sniadecki et al.

[0059] The co-culture tissue 102 also comprises live neuronal material 108 visible in FIG. 1 as discreet dark gray neurospheres extending throughout the co-culture tissue 102. As evident from FIG. 1, the neurospheres are distributed in a pattern throughout the co-culture tissue 102, rather than at random. Said distribution of neurospheres may be controlled by modulating aspects of the methods and devices described herein, e.g., using cell aggregation features of a casting plate. Advantageously, the controlled distribution of neurospheres within the coculture tissue 102 can facilitate modeling of the NMJ, as a single neurosphere (such as neurosphere 110) may control a finite volume of the underlying live muscle material 104.

[0060] The live muscle material 104 may contract spontaneously, under direct stimulation, and / or under the control of the live neuronal material 108, which in turn may be stimulated through electrical, optical, or other stimulation means. When the live muscle material 104 contracts, one or both of the tissue fixtures 106a, 106b deflects relative to its resting position. The deflection may be measured through optical, magnetic, or other measurement means and utilized to determine the contraction force of the co-culture tissue 102. The contraction force and other parameters of the co-culture tissue 102 may be analyzed over time to assess the function of the NMJ.

[0061] FIG. 2A - FIG. 2D illustrate a multi-well neurosphere casting plate 200 according to a representative embodiment of the present disclosure. The casting plate 200 facilitates the casting of a cell culture comprising live neuronal material (namely mature neurospheres), i.e., a neuronal cell culture.

[0062] In some embodiments, any casting plate described herein (including casting plate 200) is part of a consumable assembly comprising at least one of a muscle tissue casting plate, a maintenance plate, and / or a lid. In some embodiments, any casting plate described herein (including casting plate 200) is part of a system comprising at least one of a muscle tissue casting plate, a maintenance plate, an optical stimulation device (e.g., a stimulation lid), an electrical stimulation device, and / or an instrument configured to measure contraction of coculture tissues formed in the casting plate. In some embodiments, any casting plate described herein (including casting plate 200) is part of an assay, e.g., a neurotoxin assay.

[0063] The casting plate 200 includes a plurality of culture units 202a, culture unit 202b, ... n arrayed in a Society For Biomolecular Screening (SBS) - compatible layout, for example a 24, 48, 96 compartment layout. For example, the casting plate 200 may have exterior dimensions configured for compatibility with the instrument described in International Patent Publication No. WO2021 / 173887A1, which is herein incorporated by reference in its entirety. The casting plate 200 includes twenty-four neuronal culture compartments in a 4x6 array; however, this is representative and not limiting. In some embodiments, each culture unit 202a-n is identical; in other embodiments, one or more of the culture units 202a differs from one or more others, e.g., to facilitate different experiments.

[0064] The casting plate 200 may be utilized with the methods described herein or independently therefrom. For example, a co-culture tissue may be cultured directly in each culture unit 202a-n, e.g., by communicating a first cell culture comprising live neuronal material (a neuronal cell culture) with a second cell culture comprising live muscle material (a muscle cell culture, such as an EMT). The cell culture comprising live muscle material may form a lattice between a plurality of tissue fixtures (e.g., post, hooks, clamps, or the like as shown in FIG. 4A) and cultured within a second casting plate before transfer to the casting plate 200, wherein the two cell cultures are co-cultured.

[0065] In some embodiments, the casting plate 200 may be composed at least in part of a rigid thermoplastic to improve manufacturability and prevent absorption of molecules from solutions held within the culture unit.

[0066] Turning to FIG. 2B and FIG. 2C, one representative culture unit 202a will now be described. The features described with respect to culture unit 202a are present in each culture unit of the casting plate 200.

[0067] The culture unit 202a is formed as a blind well of the casting plate 200. A neuronal culture compartment is formed as a casting trench 204 in a bottom (blind) surface of the well and is configured to be seeded with live neuronal material. Any neuronal culture compartment described herein, in any embodiment, is configured to allow the maintenance of neuronal and supporting cells therein for extended periods.

[0068] The dimensions and shape of the casting trench 204 generally correspond to a portion of a muscle tissue cell culture (e.g., an EMT) with which the neuronal cell culture will be cocultured. In the representative embodiment shown, casting trench 204 has a length L of about10mm to about 20mm (e.g., 10mm to 15mm), a width W of about 2mm to about 10mm (e.g., 3mm to 5mm), and a depth D of about 2mm to about 10mm (e.g., 3mm to 5mm).

[0069] Furthermore, the casting trench 204, in the absence of additional biological / non- biological substrates, may be non-cell adherent, but bio-compatible. This could be achieved through the choice of culture unit material, permanent surface treatments, and / or short term pre-use treatments.

[0070] The neuronal culture compartment or casting trench 204 includes an optional cell aggregation region 206 provided on (e.g., formed in) at least a portion of the bottom (blind) surface of the casting trench 204. The cell aggregation region 206 encompasses one or more types of cell aggregation features configured to aggregate the live neuronal material into one or more cell aggregates, i.e., to facilitate maturation into neurospheres. In the representative embodiment shown, cell aggregation region 206 has a length 1' of about 5mm to about 15mm (e.g., 10mm to 15mm) and a width w' of about 2mm to about 10mm (e.g., 2mm to 5mm). In different embodiments, the cell aggregation region 206 may be positioned in different locations on the bottom surface of the respective well, e.g., for locational control of the neurospheres.

[0071] Each neurosphere formed in the cell aggregation region 206 may range from about lOum to about 500um in diameter, containing about 500 to about 100,000 individual cells per neurosphere, Each neuronal culture compartment may be capable of generating between about 1 and about 100 individual neurospheres.

[0072] The cell aggregation features drive maturation of neurospheres at locations within the neuronal cell culture which are predetermined to facilitate stimulation and / or measurement of the co-culture tissue. Such location specificity of the neurospheres enables precise experimental control. For example, the cell aggregation features may be positioned at a location(s) predetermined to receive optical and / or electrical stimulation. As another example, the cell aggregation features may be positioned at a location(s) that facilitates subsequent measurement of neurosphere function, e.g., simultaneous measurement of neurosphere function with that of the live muscle material.

[0073] The cell aggregation features generally include chemical, electrical, and / or mechanical features that drive formation of the cell aggregates. For example, in some embodiments, the cell aggregation features comprise 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 the cell aggregation region 206 is provided with a hydrophobicsurface treatment, formed of a hydrophobic material, and / or imparted with an electrostatic charge to aggregate cells at predetermined locations of the cell aggregation region 206 (e.g., in a single aggregate location or in a predetermined pattern of aggregates).

[0074] Independently of or in addition to cell aggregation surface properties, the cell aggregation region 206 may comprise feature geometry configured to drive cell aggregation. The feature geometry may comprise a plurality of closely-packed cavities or cells, each forming a nadir or low point. For example, each cavity may be formed as an inverted pyramid, inverted cone, inverted triangular prism, dome, dimple, valley, depression, trench, bowl, trough, depression, hollow, or similar feature having a polygonal or organic base shape forming at least one nadir or low point into which cells will aggregate under gravitational action. In some embodiments, all such cavities or cells have a common shape and / or dimensions (as shown in FIG. 2D). In other embodiments, the feature geometry may include differently sized and / or shaped cavities or cells.

[0075] In some embodiments, the feature geometry is selected and arranged to reduce, minimize, or eliminate interstices between adjacent cavities (which otherwise may permit formation of randomly-located neurospheres). For example, in some embodiments, the feature geometry comprises tessellating surface features, i.e., geometric features which cohesively repeat without interstices. Representative examples include geometries having a base with three, four, six, or eight sides, such as equilateral triangles, hexagons, octagons, and parallelograms. In other embodiments, the interstices between the closely-packed cavities may be provided with hydrophobicity, electrostatic charge, and / or other feature to prevent formation of randomly-located neurospheres thereon.

[0076] FIG. 2D illustrates representative and non-limiting cell aggregation features configured to drive aggregation of neuronal cells. The cell aggregation region 206 includes cell aggregation features 208 comprising feature geometry formed as tessellating surface features (in this embodiment tessellating cavities). Each tessellating cavity is an inverted pyramid forming a nadir 210 with a four-sided (parallelogram) base. Different embodiments include different tessellating and non-tessellating geometries.

[0077] In general, the feature geometry has dimensions (e.g., depth, edge length) from about lum to about 10 cm, thereby allowing for spatially controlled cell aggregation and confinement. In the representative embodiment shown, the cell aggregation region 206 includes about 50 to about 250 tessellating surface features and a surface feature density ofabout 2 to about 10 tessellating surface features per square mm (e.g., 2-5 features per square mm). As shown in FIG. 2D, each edge 212 has a length of about 0.25mm to about 2mm (e.g., 0.25mm to about 0.75mm). Each of the cell aggregation features 208 has a depth (e.g., a z-axis distance from the plane containing edges 212 to the respective nadir) of about 0.25mm to about 2mm (e.g., 0.25mm to about 0.75mm).

[0078] The neuronal culture compartments may include any combination of the above features (e.g., surface properties and feature geometry) allowing the in situ generation of neurospheres from single cells. The inventors of the instant application have surprisingly and unexpectedly found that by maintaining the neuronal cells in situ at the time and during the process of coculturing with muscle tissue, an effective and replicable 3D structure of neuromuscular junction can be produced.

[0079] In use, a co-culture tissue may be cultured directly in each casting trench 204, e.g., according to the methods described herein. For example, a co-culture tissue may be cast directly in the casting trench 204 by co-culturing the neuronal cell culture comprising live neuronal material with the muscle cell culture comprising 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 of the neuronal cell culture cross the interface into the muscle tissue, maturing into neuromuscular junctions indicative of innervated muscle tissue.

[0080] FIG. 3A - FIG. 3D illustrate aspects of a multi-well neurosphere casting plate 300 according to another representative embodiment of the present disclosure. Casting plate 300 advantageously enables long distance communication and axonal extension between the live neuronal material and live muscle material, which may increase the assay window for progressive neuromuscular diseases characterized by a withdrawal of axons from muscle.

[0081] Like the embodiment of FIG. 2A - FIG. 2D, the casting plate 300 includes a plurality of culture units 302a, b, ... n arrayed in an SBS-compliant layout. Casting plate 300 differs from the embodiment of FIG. 2A - FIG. 2D in that each culture unit comprises at least one distinct neuronal culture compartments in fluidic communication with a muscle culture compartment via a fluidic channel. Thus, a neuronal cell culture is cultured within each neuronal culture compartment and interfaces with a muscle cell culture (e.g., an EMT) disposed in the muscle culture compartment at an intermediate location along the fluidic channel.

[0082] FIG. 3A shows a plan view of a portion of casting plate 300. FIG. 3B shows a plan view of culture unit 302a within the casting plate 300, whereas FIG. 3C and FIG. 3D showperpendicular section views of the culture unit 302a. Because all culture units share the same geometry and features in casting plate 300, common reference numerals are utilized in FIG. 3A - FIG. 3D across different culture units. For ease of understanding, culture unit 302a is illustrated with an EMT 336 placed therein, although the EMT is not part of culture unit 302a.

[0083] Turning first to FIG. 3A, aspects of culture unit 302b which are common to all culture units of the casting plate 300 will now be introduced. Culture unit 302b comprises a plurality of neuronal culture compartments 314a, 314b configured to allow the maintenance of neuronal and supporting cells therein for extended periods. Neuronal culture compartments 314a, 314b are in fluidic communication with each other via a communication channel 318, e.g., a microfluidic channel having a diameter on the order of hundreds of micrometers (shown in greater detail in FIG. 3C). The illustrated embodiment includes two opposed neuronal culture compartments 314a, 314b; however, other embodiments may include a single neuronal culture compartment or greater than two neuronal culture compartments (e g., three, four, or five neuronal culture compartments distributed about the muscle culture compartment).

[0084] A muscle culture compartment 316 includes the spacious volume within the culture unit 302b disposed between the neuronal culture compartments 314a, 314b, in order to accommodate and maintain the health of a muscle tissue such as EMT 336. The muscle cell culture will generally have been cultured in a separate casting plate and then transferred into the muscle culture compartment 316.

[0085] Communication channel 318 is a fluidic channel extending between neuronal culture compartments 314a, 314b and features an aperture 334 through a wall thereof. In some embodiments, the communication channel 318 has a diameter on the order of hundreds of micrometers. In some embodiments, the communication channel 318 has a diameter not less than about fifty micrometers, e.g., to facilitate manufacturing. Advantageously, this The aperture 334 fluidically connects the communication channel 318 to the muscle culture compartment 316. Thus, each of the neuronal culture compartments 314a, 314b and muscle culture compartment 316 are in fluid communication via the communication channel 318.

[0086] Referring to FIG. 3B, aspects of culture unit 302a are described in greater detail. As shown in the detail inset, at least a portion of a bottom (blind) surface of one, some, or all neuronal culture compartments 314a, 314b may optionally be provided with a cell aggregation region 306 with any type and combination of cell aggregation features 308 previously described. For example, some embodiments of neuronal culture compartments 314a and 314binclude surface features and / or feature geometry, e.g., comprising tessellating cavities configured to gravitationally aggregate neuronal cells.

[0087] Electrical and optical isolation between the neuronal culture compartments 314a, 314b and muscle culture compartment 316 may be achieved through imposition of one or more electrically insulating and optically opaque walls therebetween (e.g., wall 326). The bulk electrical resistance between any two points within each respective compartment is substantially lower (e.g., at least three orders of magnitude lower) than the electrical resistance between two points crossing an interface between compartments (e.g., via the aperture 334); consequently, electrical stimulation targeting cells in one compartment (e.g., neurospheres) will have negligible influence on cells in the other (e.g., muscle tissue) and vice versa. As such, in some embodiments, each compartment is configured to accommodate at least two electrically conductive components, referred to henceforth as electrodes, that can be put in direct electrical communication with an external device such as a stimulation device.

[0088] The neuronal culture compartment may be stimulated to evoke neuronal action potentials. Non-exclusive exemplary stimulation modes include: chemical stimulation (e.g., Glutamate); field potential stimulation (e.g., Amplitude 10-400mA, Frequency l-50Hz, Pulse widths l-20ms); direct contact electrical stimulation (Amplitude 10-400mA, Frequency 1- 50Hz, Pulse widths l-20ms); and / or light stimulation through light sensitive ion channels (Power 5-40 mW / mm2, Frequency l-100Hz, Pulse Widths l-20ms). In some embodiments, these stimulations are isolated to / unique to the neuronal culture compartment(s) and / or the muscle culture compartment, thus enabling independent stimulation of the neuronal cell or muscle portions of the co-culture tissue.

[0089] Accordingly, culture unit 302a receives a plurality of electrodes in each of the neuronal culture compartments 314a, 314b and in the muscle culture compartment 316. For example, electrode 320a extends into neuronal culture compartment 314a (to stimulate neurospheres cultured therein) and electrode 320b extends into the muscle culture compartment 316 (to stimulate EMT 336). These electrodes may be integrated with the casting plate 300 or part of a stimulation device such as a stimulation lid.

[0090] FIG. 3C and FIG. 3D show perpendicular section views of culture unit 302a. As shown in FIG. 3C, the neuronal culture compartments 314a, 314b are separated from the muscle culture compartment 316 by the communication channel 318 or connecting compartment. Casting plate 300 may be composed of one or more components, e.g., an uppercomponent 328 and a lower component 330, bonded together by an interposing component 332. In some embodiments, the interposing component 332 comprises at least a portion of the communication channel 318.

[0091] In some embodiments, communication channel 318 forms an open-ended channel with upper and lower walls and side walls where each terminus of the communication channel 318 is in communication with either of the neuronal culture compartments 314a, 314b or muscle culture compartment 316. These upper and lower walls may be contiguous with the upper and lower components 328, 330 of the casting plate 300, whereas the side walls may be contiguous with the upper or lower components 328, 330 or formed by the interposing component 332 disposed therebetween.

[0092] The channel dimensions of the communication channel 318 are sufficiently small to prevent infiltration of neurospheres, ensuring separation of the neuronal cells from the muscle cells within the culture unit 302a while allowing limited fluidic communication via neuritic outgrowths in a manner that mimics interactions between these two tissue types in-vivo. This communication channel 318 may be filled with liquid or a hydrogel of biological or non- biological origin.

[0093] In some embodiments, the communication channel 318 is configured to promote neuronal guidance from the neuronal culture compartments 314a, 314b to the muscle culture compartment 316. As one representative example, this may be achieved through the application of chemoattractant growth factors in the muscle culture compartment 316 and not the neuronal culture compartments 314a, 314b, generating a chemoattractant gradient. Non-exclusive examples would include neuronal growth factor (NGF) or Netrin.

[0094] In another embodiment, flow features within the communication channel 318 induce areas of turbulent and laminar hydrogel flow in patterns that optimize hydrogel alignment, such that when filled with a hydrogel, flow of the hydrogel therethrough during seeding aligns hydrogel components along the axis of the communication channel 318, promoting axonal guidance as axons extend through this patterned substrate.

[0095] Further, in some embodiments, the communication channel 318 has dimensions making it suitable for applications utilizing evaporative pumping. An example of this application includes providing a hydrogel of appropriate viscosity to the neuronal culture compartments 314a, 314b and / or muscle culture compartment 316, followed by the addition of an aqueous cell culture medium to the neuronal culture compartments 314a, 314b but not themuscle culture compartment 316. Evaporation in the muscle culture compartment 316 draws solvent and solutes through the communication channel 318 via intermolecular forces acting between the evaporating solvent and bulk solute and solvent molecules. This flow can be used to manufacture microenvironments in-situ that provide favorable conditions for rapid, directed axon growth. Fluid shear within the hydrogel caused by moving solvent can pattern any hydrogel within the communication channel 318. Furthermore, evaporative pumping can accumulate chemoattractant proteins at the aperture 334, generating high concentration chemoattractant gradients, as these factors are left behind at the terminus of the communication channel 318 when the encompassing solvent evaporates.

[0096] Under such guidance conditions, axons may be induced to extend up to 1mm per day allowing long distance communication between compartments. Advantageously, the ability to direct axonal outgrowth to any specific location within the muscle culture compartment 316 allows neurons to interface with the muscle cell culture in a spatially controllable manner. Interface points can be adjusted with respect to their size and position as suits particular measurement approaches and muscle tissue geometries.

[0097] FIG. 3D shows a section view of culture unit 302a, wherein the section extends across the muscle culture compartment 316. Accordingly, the EMT 336 is suspended within the muscle culture compartment 316 between two tissue fixtures 322a, 322b, which may form part of a separate assembly that extends into the muscle culture compartment 316 for the purpose of placing the EMT 336 in contact with the communication channel 318. For example, in some embodiments, tissue fixture 322a is a flexible post, whereas tissue fixture 322b is a relatively rigid post.

[0098] In use, a hydrogel may be added to the communication channel 318 such that it forms a structure 324 (e.g., a domed cell culture structure or a hydrogel structure) elevated from the roof of the communication channel 318. This structure 324 directly contacts the EMT 336, thus forming an interface between the two cell cultures and permitting co-culturing. The muscle culture compartment 316 is designed in such a way that the communication channel 318 surrounds or abuts the EMT 336 to increase the probability that axons extending from the neuronal cell culture interface with the EMT 336. In a non-limiting example of this, the communication between compartments may be achieved through a bulk hydrogel surrounding the EMT 336 with neuronal components embedded.

[0099] In some embodiments, the muscle culture compartment 316 enables the non-invasive readout of muscle function, both spontaneous and evoked, through the motion of one or more of the tissue fixtures 322a, 322b. This motion may be recorded through optical tracking or the change in magnetic field caused by a magnet embedded in one or both of the tissue fixtures 322a, 322b. Accordingly, the muscle culture compartment 316 may be formed of a transparent material to enable optical tracking. Additionally or alternatively, the casting plate 300 may be configured to fit within an instrument for magnetically and / or optically measuring movement of at least one tissue fixture in each culture unit 302a...n.

[0100] In the illustrated embodiment, the tissue fixtures 322a, 322b comprise part of a separate muscle tissue casting assembly, which is inserted into the casting plate 300.

[0101] The devices described herein, including the casting plates, consumables, and assays, may form part of a product line of such devices. For example, the present disclosure includes product lines comprising at least two different casting plates, wherein each of the at least two different casting plates is a casting plate of the present disclosure. For example, the product line may comprise at least two casting plates having different cell aggregation region configurations. As another example, the product line may comprise at least two casting plates, wherein a first casting plate is configured according to any of the embodiments described with respect to FIG. 2A - FIG. 2D, and wherein a second casting plate is configured according to any of the embodiments described with respect to FIG. 3A - FIG. 3D. Such different configurations may be suitable for assaying different cell cultures characterized by different neurological diseases.

[0102] The present disclosure includes tissue analysis systems comprising co-culture casting systems, including a multi-well co-culture casting plate and a multi-well muscle tissue casting plate. The multi-well co-culture casting plate may be any embodiment described herein with respect to FIG. 2A - FIG. 3D.

[0103] Any casting plate or culture unit of the present disclosure may form part of a tissue analysis system and / or co-culture casting system, and may optionally be utilized as part of one or more methods for: a) using such a tissue analysis system or co-culture casting system and / or b) modeling neuromuscular junction(s), which will now be described. Advantageously, the following methods enable effective, high throughput, and repeatable NMJ modeling by forming co-culture tissues with in situ neurospheres. The following methods may be performed independently of the devices described herein.

[0104] FIG. 4A schematically illustrates representative methods 400 for a) using a tissue analysis system comprising a co-culture casting system that includes a multi-well co-culture casting plate and a muscle tissue casting plate and for b) modeling a plurality of neuromuscular junctions, which include methods of casting a co-culture tissue comprising in situ neurospheres. In embodiments where at least some of the live neuronal material contains mutations or disease characteristics, the methods 400 may be restated as, and / or may form part of, methods of modelling the disease. In embodiments where at least some of the live neuronal material is treated with a substance (e.g., a neurotoxin), the methods 400 may be restated as, and / or may form part of, methods of assaying the substance.

[0105] Methods 400 generally include four phases. In phase one 402, cell cultures comprising neurospheres matured from live neuronal material are cast (i.e., neuronal cell cultures). In phase two 404, cell cultures comprising live muscle material are cast (muscle cell cultures). Phase one 402 and phase two 404 may be performed contemporaneously by a common entity or different entities. In phase three 406, the muscle cell cultures are co-cultured with the neuronal cell cultures, thus forming co-culture tissues comprising in situ neurospheres and live muscle material. Phase one 402, phase two 404, and phase three 406 collectively constitute a method of forming co-culture tissues comprising in situ neurospheres and live muscle material. In phase four 408, the co-culture tissues are stimulated. For example, the live muscle material of each co-culture tissue is caused to contract by stimulating the neurospheres of that co-culture tissue using electrical, optical, and / or chemical stimulation.

[0106] The broken line boxes of phase one 402 and phase two 404 show and encompass elements of representative tissue analysis systems and co-culture casting systems including a multi-well co-culture casting plate shown in the broken line box of phase one 402 and a multiwell muscle tissue casting plate shown in the broken line box of phase two 404. In some embodiments, the multi -well co-culture casting plate is the casting plate 200 of FIG. 2A - FIG. 2D; in other embodiments, the multi-well co-culture casting plate is the casting plate 300 of FIG. 3A - FIG. 3D. For simplicity, an individual well of each of the multi-well co-culture casting plate and the multi-well muscle tissue casting plate is respectively shown in phase one 402 and phase two 404. Said tissue analysis systems may optionally comprise a multi-well maintenance plate, such as that shown in the broken line box of phase four 408.

[0107] Representative steps of each phase will now be described. The methods described herein expressly include any combination of the following features, performed in anyreasonable order consistent with this disclosure. Each different combination and order of features and steps constitute a different method 400 of the present disclosure.

[0108] Phase one 402 (casting neuronal cell cultures).

[0109] In step 410, neurospheres are cast into neuronal cell cultures by seeding live neuronal material comprising neuronal cells (e.g., motor neurons) into a plurality of neuronal culture compartments and allowing the live neuronal material to mature into neurospheres in the neuronal culture compartments. Step 410 may include any combination of the following features:• Administering a hydrogel upon the live neuronal material in the neuronal culture compartments. Representative hydrogels include, for example, Type I rat tail collagen (with a concentration of about 1 mg / mL) + about 5% MATRIGEL® or equivalent. Restated, adding a plurality of neuronal cells to a hydrogel to form a neuronal hydrogel.• Seeding the live neuronal material into a plurality of neurosphere casting trenches, e.g., of a multi-well neurosphere casting plate.• Seeding the live neuronal material into a plurality of neuronal culture compartments in each well of a multi-well neurosphere casting plate.• Controlling a location of cell aggregates of the live neuronal material. In some embodiments, controlling the location of the cell aggregates includes patterning the cell aggregates and / or spacing apart the cell aggregates. In some embodiments, controlling the location of the cell aggregates comprises seeding the live neuronal material into (e.g., only into) at least one specific region within the neuronal culture compartment. In some such embodiments, the at least one specific region may comprise one or more of a cell aggregation region, an end region of the neuronal culture compartment, a side region of the neuronal culture compartment, a central region of the neuronal culture compartment, or a tissue fixture region.• Not seeding the live neuronal material into at least one exclusion region within the neuronal culture compartment. In some such embodiments, the at least one exclusion region may comprise one or more of an end region of the neuronal culture compartment, a central region of the neuronal culture compartment, a side region of the neuronal culture compartment, or a tissue fixture region. Not seeding the live neuronal material in at least one exclusion region may facilitate analysis of the co-culture tissue, e.g., with calcium and / or optical imaging.• Aggregating the live neuronal material into cell aggregates. In some such embodiments, aggregating the live neuronal material into cell aggregates comprises seeding live neuronal material into a cell aggregation region comprising cell aggregation features, e.g., surface properties (such as hydrophobicity and / or electrostatic charge) and / or feature geometry (such as tessellating cavities).• Each neuronal culture compartment comprises a casting trench (see FIG. 2A - FIG. 2D).• Each neuronal culture compartment is one of a plurality of neuronal culture compartments of a culture unit in a well of a multi-well plate (see FIG. 3A - FIG. 3D).• Each neuronal culture compartment and / or each casting trench comprises at least one cell aggregation region comprising cell aggregation features as described in any embodiment herein, including one or more of surface properties or feature geometry that drive cell aggregation.• Thawing the live neuronal material from cryopreservation directly in the plurality of neuronal culture compartments, optionally without a Rho kinase inhibitor. Directly thawing the neurons in the neuronal culture compartments, where they mature into neurospheres, which in situ neurospheres are cultured into co-culture tissue, increases neuron survival rates by eliminating a transfer step, improving reproducibility and simplifying experiment planning.• After seeding the live neuronal material, waiting a maturation period (e.g., 1-14 days) during which neurons of the live neuronal material mature into neurospheres, and then administering a linking hydrogel upon the in situ neurospheres.• The live neuronal material includes neurons expressing one or more proteins acting as gated ion channels, e.g., light gated ions channels including Channelrhodopsin -1 and -2. In some such embodiments, expression of such proteins may be induced, e.g., by administering one or more promoters such as a tetracyclene promoter or a neuron-specific promoter.• The neuronal hydrogel comprises a total amount of the plurality of neuronal cells ranging from about 5% to about 75% (w / wt) of the neuronal hydrogel. In another aspect, the neuronal hydrogel comprises the plurality of neuronal cells in an amount selected from the group consisting of 5% (w / wt), 10% (w / wt), 15%, (w / wt), 20% (w / wt), 25% (w / wt), 30% (w / wt), 35% (w / wt), 40% (w / wt), 45% (w / wt), 50% (w / wt), 55% (w / wt), 60% (w / wt), 65% (w / wt), 70% (w / wt), and 75% (w / wt), and a range thereof, of the neuronal hydrogel.

[0110] Step 412 may form part of phase one 402 or phase two 404. In step 412, a linking hydrogel is administered in each neuronal culture compartment on the in situ neurospheres matured from the live neuronal material. Such linking hydrogel facilitates adhesion of the neurospheres to the muscle cell culture. In some embodiments, the linking hydrogel is a collagen type I hydrogel, e.g., with low proportion of basement membrane extract from murine Engelbreth-Holm-Swarm (EHS) tumors (such as MATRIGEL®), which allows for robust neurite extension and compaction of the gel to bring neurospheres close to the muscle cell culture. Step 412 may include any combination of the following features:• Administering the linking hydrogel on in situ neurospheres in a plurality of neurosphere casting trenches, or in a plurality of neuronal culture compartments in each well of a multiwell neurosphere casting plate.• Not removing the neurospheres from the neuronal culture compartments before administering the linking hydrogel.[OHl] Thus, step 410 and optionally step 412 provide cell cultures comprising live neuronal material, i.e., in situ neurospheres.

[0112] Phase two 404 (casting muscle cell culture).

[0113] Step 412 may optionally be performed as part of 404 as discussed above.

[0114] In step 414, live muscle material is cast into muscle cell cultures by seeding the live muscle material in a plurality of muscle casting wells of a separate casting plate (as shown in FIG. 4A).

[0115] Step 414 may include any one or more of the following features:• A plurality of tissue fixtures (e.g., plurality of pairs of posts) is cast in the live muscle material, such that the resulting muscle cell cultures each form a lattice between two or more of the tissue fixtures (as shown in FIG. 3D and FIG. 4A).• The live muscle material is cast in a medium, e.g., a hydrogel which may differ from the hydrogel in which the neuronal cell culture is cast and / or the linking hydrogel.• Adding a plurality of muscle cells to the second medium, e.g., a hydrogel.• The muscle cell culture is an engineered muscle tissue (EMT).• Adding a plurality of muscle cells to a hydrogel to form a hydrogel comprising muscle tissue.• The hydrogel comprising muscle cells comprises a total amount of the plurality of muscle cells ranging from about 5% to about 75% (wt / wt) of the hydrogel comprisingmuscle cells. In another aspect, the hydrogel comprising muscle cells comprises the plurality of muscle cells in an amount selected from the group consisting of 5% (w / wt), 10% (w / wt), 15%, (w / wt), 20% (w / wt), 25% (w / wt), 30% (w / wt), 35% (w / wt), 40% (w / wt), 45% (w / wt), 50% (w / wt), 55% (w / wt), 60% (w / wt), 65% (w / wt), 70% (w / wt), and 75% (w / wt), and a range thereof, of the hydrogel comprising muscle cells.

[0116] In optional step 416, the muscle cell cultures are transferred to a maintenance plate, e.g., while the neurospheres are cultured in the linking hydrogel (see step 412). In some embodiments, the live muscle material is transferred to the maintenance plate after it matures into a muscle tissue construct (e.g., a lattice or EMT).

[0117] In any embodiment, phase one 402 and phase two 404 are performed contemporaneously and / or coordinated to conclude concurrently. For example, in some embodiments, phase one 402 and phase two 404 are performed over a common number of days and / or to conclude on a common day.

[0118] Phase three 406 (form co-culture tissues).

[0119] In step 418, each of the muscle cell cultures is co-cultured with, and allowed to attach to, one or more of the in situ neuronal cell cultures, thereby forming a plurality of co-culture tissues, each comprising both in situ neurospheres and live muscle material, wherein at least a portion of the live muscle material is innervated. Restated, the neurospheres remain in situ at the time of attaching and co-culturing a medium comprising the mature muscle cell culture to a medium comprising the neurospheres.

[0120] In some embodiments, each of the muscle cell cultures is communicated with a respective in situ neuronal cell culture by administering a muscle hydrogel containing the live muscle material to the each neurosphere casting trench comprising the linking hydrogel (linking hydrogel), thereby incorporating the neurospheres into the hydrogel(s) in close proximity to the live muscle material. The muscle tissue effectively forms around the neurospheres and the muscle and neuron compartments are separated by the hydrogel(s).

[0121] In some embodiments, the co-culture tissues are formed in the plurality of neuronal culture compartments in which the live neuronal material was cast in phase one 402. For example, in some embodiments, a muscle cell culture is transferred from a separate casting plate into each of the neuronal culture compartments. In some such embodiments, each neuronal culture compartment comprises a casting trench (see FIG. 2A - FIG. 2D).

[0122] In some embodiments, each neuronal culture compartment is one of a plurality of neuronal culture compartments of a culture unit in a well of a multi-well plate. Each culture unit may include one or more neuronal culture compartments and at least one muscle culture compartment (see FIG. 3A - FIG. 3D). In some such embodiments, the muscle cell cultures are transferred from a separate casting plate into the plurality of muscle culture compartments of the plurality of culture units. In some such embodiments, the muscle cell cultures are communicated with one or more neuronal cell cultures along a communication channel within a culture unit, e.g., a communication channel extending from the neuronal culture compartment, e.g., at an aperture of the communication channel.

[0123] In any embodiment, the muscle cell cultures are co-cultured with the in situ neuronal cell cultures 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) prior to commencing phase four 408. The attachment period enables the muscle cell cultures to attach to the respective neuronal cell cultures, e.g., the outgrowth of axons from the neuronal cell cultures across the hydrogel interface to the muscle cell cultures.

[0124] In any embodiment, the muscle cell cultures are co-cultured with the in situ neuronal cell cultures through a communication channel as defined according to any embodiment herein.

[0125] In any embodiment, the muscle cell cultures are co-cultured with the in situ neuronal cell cultures while maintaining electrical and / or optical isolation therebetween. For example, in the casting plate 300 of FIG. 3 A - FIG. 3D, the walls separating the neuronal culture compartments and the muscle culture compartments provide effective electrical insulation.

[0126] In any embodiment, step 418 further comprises remodeling an interface between the neuronal cell culture and the muscle cell culture, e.g., between the linking hydrogel and a hydrogel comprising the live muscle material. This step may facilitate neurite extension across the interface. The interface may include a mechanical interface between the two hydrogels by virtue of different properties thereof, e.g., different stiffness values. In some embodiments, remodeling the interface comprises reducing a stiffness gradient between the linking hydrogel and the muscle hydrogel. In some embodiments, remodeling the interface between the linking hydrogel and the muscle hydrogel comprises treating the interface with a serum media, e.g., for a treatment period of at least 12 hours or at least 24 hours. In some embodiments, the serum media is a media comprising 10% animal serum such as Fetal Bovine Serum (FBS), which isadministered to the tissues by adding the serum containing media into the plurality of casting wells without removing the tissues after the linking hydrogel is set in step 412.

[0127] Phase four 408 (stimulate co-culture tissues).

[0128] In step 420, the co-culture tissues are stimulated. In particular, an innervated portion of the live muscle material in each co-culture tissue is caused to contract by stimulating the live neuronal material, e.g., with one or more of chemical stimulation, electrical stimulation, or optical stimulation.

[0129] In any embodiment, the co-culture tissues are stimulated contemporaneously (in parallel), e.g., using chemical, electrical, and / or optical stimulus for each co-culture tissue in a common multi-well plate.

[0130] In any embodiment, the co-culture tissues are recorded contemporaneously (in parallel), e.g., using one or more recording electrodes for each co-culture tissue in a common multi-well plate.

[0131] In any embodiment, the co-culture tissues are stimulated and recorded contemporaneously (in parallel), e.g., in a common multi -well plate. Restated, each co-culture tissue may be contemporaneously stimulated and recorded, and the plurality of co-culture tissues may be stimulated contemporaneously with each other and / or recorded contemporaneously with each other.

[0132] In any embodiment, the live muscle material of each co-culture tissue is contracted by stimulating only the live neuronal material of that co-culture tissue with one or more of chemical stimulation, electrical stimulation, or optical stimulation.

[0133] In any embodiment, the live muscle material of each co-culture tissue is contracted by stimulating the live muscle material and the respective live neuronal material independently with one or more of chemical stimulation, electrical stimulation, or optical stimulation.

[0134] In some embodiments, namely where the co-culture tissues are formed in neuronal culture compartments, i.e., the co-culture tissues each comprise in situ neurospheres, step 420 is performed in part by activating in situ neurospheres of each co-culture tissue.

[0135] In some embodiments, the live muscle material of the co-culture tissue is contracted by stimulating at least the live neuronal material of the co-culture tissue after transferring the coculture tissue to a maintenance plate.

[0136] Thus, FIG. 4A introduces numerous repeatable, scalable methods of modelling a plurality of neuromuscular junctions.

[0137] FIG. 4B schematically illustrates exemplary specific methods of using tissue analysis systems previously introduced with respect to FIG. 4A. Such methods may be performed, for example, using the casting plate 200 of FIG. 2A - FIG. 2D or the casting plate 300 of FIG. 3 A - FIG. 3D. The methods described below expressly include any combination of the following features, performed in any reasonable order consistent with this disclosure.

[0138] In step 424, method 422 seeds a plurality of neuronal culture compartments of the multi-well co-culture casting plate with live neuronal material, wherein each neuronal culture compartment comprises a plurality of cell aggregation features configured to aggregate the live neuronal material into cell aggregates. In some embodiments, the cell aggregation features are disposed in a casting trench of the respective neuronal culture compartment. In some embodiments, the cell aggregation features comprise surface features (e.g., hydrophobicity and / or electrostatic charge) and / or feature geometry as described herein. For example, in some embodiments, the feature geometry comprises tessellating surface features such as tessellating cavities, wherein each tessellating cavity forms a nadir. In some embodiments, seeding the plurality of neuronal culture compartments with the live neuronal material comprises thawing cells comprising motor neurons directly in the plurality of neuronal culture compartments. In some embodiments, the multi-well co-culture casting plate comprises a plurality of culture units, wherein each culture unit comprises a muscle culture compartment in communication with at least one of the neuronal culture compartments via a communication channel.

[0139] In step 426, method 422 seeds a plurality of muscle culture compartments of the muscle tissue casting plate with live muscle material.

[0140] In step 428, method 422 cultures, in the multi-well co-culture casting plate, a plurality of co-culture tissues, each of the co-culture tissues comprising a muscle cell culture cultured from the live muscle material and in situ neurospheres matured in the cell aggregation features from the live neuronal material. In some embodiments, culturing the plurality of co-culture tissues comprises culturing at least one of the co-culture tissues in each neuronal culture compartment. In some embodiments, seeding the plurality of neuronal culture compartments with the live neuronal material (step 426) comprises administering, in each neuronal culture compartment, a linking hydrogel on the in situ neurospheres, and culturing each the plurality of co-culture tissues (step 428) comprises remodeling an interface between the linking hydrogeland a second hydrogel comprising the live muscle material, e.g., by treating the interface with a serum media.

[0141] In step 430, method 422 causes an innervated portion of the live muscle material in each of the co-culture tissues to contract by stimulating the live neuronal material. In some embodiments, step 430 comprises transferring the co-culture tissues to a maintenance plate prior to causing the innervated portion of the live muscle material in the co-culture tissues to contract by stimulating the live neuronal material. In some embodiments, step 430 comprises activating the in situ neurospheres with at least one of an optical, electrical, or chemical stimulus. In some embodiments, step 430 comprises contemporaneously causing the innervated portion of the live muscle material in each of the co-culture tissues to contract, optionally while contemporaneously recording a contraction of each of the co-culture tissues.

[0142] FIG. 4C schematically illustrates exemplary specific methods of modelling a plurality of neuromuscular junctions previously introduced with respect to FIG. 4A. Such methods may be performed, for example, using the casting plate 200 of FIG. 2A - FIG. 2D. The methods described below expressly include any combination of the following features, performed in any reasonable order consistent with this disclosure.

[0143] In step 432, method 448 seeds a plurality of neuronal culture compartments of a multiwell plate with live neuronal material by thawing cells comprising motor neurons directly in the plurality of neuronal culture compartments, wherein each neuronal culture compartment comprises cell aggregation features configured to aggregate the live neuronal material into cell aggregates. Optionally, the live neuronal material expresses at least one protein acting as a gated ion channel. The cell aggregation features may have any configuration previously introduced.

[0144] In step 434, method 448 seeds a plurality of muscle culture compartments of the muscle tissue casting plate with live muscle material and / or casts a plurality of muscle cell cultures comprising live muscle material. Alternatively, the muscle cell cultures may be received, e.g., from a third party that casts the muscle cell cultures.

[0145] In step 436, method 448 cultures, in the multi-well plate, a plurality of co-culture tissues comprising the live muscle material and in situ neurospheres matured from the live neuronal material, in part by remodeling an interface between a linking hydrogel and a second hydrogel comprising the live muscle material, e.g., by treating the interface with a serum media.

[0146] In step 438, method 448 contemporaneously causes an innervated portion of the live muscle material in each of the co-culture tissues to contract by stimulating the live neuronal material, e.g., with chemical, optical, and / or electrical stimuli. Optionally, causing the innervated portion of the live muscle material in each of the co-culture tissues to contract further comprises recording a contraction of each of the co-culture tissues contemporaneously with a stimulation thereof.

[0147] FIG. 4D schematically illustrates additional exemplary specific methods of modelling a plurality of neuromuscular junctions previously introduced with respect to FIG. 4A. Such methods may be performed, for example, using the casting plate 300 of FIG. 3 A - FIG. 3D. The methods described below expressly include any combination of the following features, performed in any reasonable order consistent with this disclosure.

[0148] In step 440, method 450 seeds a plurality of culture units of a multi-well plate with live neuronal material by thawing cells comprising motor neurons directly in a plurality of neuronal culture compartments, wherein each neuronal culture compartment comprises cell aggregation features configured to aggregate the live neuronal material into cell aggregates. Optionally, the live neuronal material expresses at least one protein acting as a gated ion channel. The cell aggregation features may have any configuration previously introduced.

[0149] In step 442, method 450 seeds a plurality of muscle culture compartments of the muscle tissue casting plate with live muscle material and / or casts a plurality of muscle cell cultures comprising live muscle material. Alternatively, the muscle cell cultures may be received, e g., from a third party that casts the muscle cell cultures.

[0150] In step 444, method 450 cultures a plurality of co-culture tissues comprising the live muscle material and in situ neurospheres matured from the live neuronal material in the multiwell plate by communicating, in each of the culture units, the live neuronal material with the live muscle material along a fluidic communication channel.

[0151] In step 446, method 450 contemporaneously causing an innervated portion of the live muscle material in each of the co-culture tissues to contract by stimulating the live neuronal material, e.g., with chemical, optical, and / or electrical stimuli. Optionally, causing the innervated portion of the live muscle material in each of the co-culture tissues to contract further comprises recording a contraction of each of the co-culture tissues contemporaneously with a stimulation thereof.

[0152] FIG. 5 - FIG. 9B demonstrate efficacy of modeling a neuromuscular junction according to the methods described herein.

[0153] FIG. 5 shows exemplary images of cells and tissues formed within devices of the present disclosure.

[0154] Image (A) shows in situ neuronal aggregates embedded in a cell aggregation region of a single well of a casting plate comprising feature geometry, i.e., tessellating cavities. The neuronal aggregates can be maintained to produce neurospheres. Scale bar 500um.

[0155] Image (B) shows exemplary neurite extension in a 3D matrix following in situ creation and maintenance of neurospheres in the casting plate of image (A) after 24 hours within adherent hydrogel, demonstrating the viability of the in situ neurosphere creation approaches described herein. Scale bar represents 500um.

[0156] Image (C) shows exemplary neurite extension within a casting plate comprising a neuronal culture compartment and a muscle culture compartment connected by a communication channel. In particular, Image (C) is a stitched image illustrating neurite growth from an iPSC-derived motor neuron neurosphere (white dotted circle) from the neuronal culture compartment, through the communication channel (white arrows), and making contact with an engineered muscle tissue on the right-hand side (white dotted border) in the muscle culture compartment. Neurites in the muscle chamber that are making contact with the muscle are highlighted by a white arrow head. Scale bar represents 500um.

[0157] FIG. 6 shows still frames from 20fps video taken during acetylcholine stimulation of a muscle cell culture comprising engineered skeletal muscle. The three still frames represent, from top to bottom: a resting state of the co-culture tissue before glutamate administration; maximal contraction of the co-culture tissue following administration of glutamate; and a relaxation state of the following maximum displacement. White dashed lines represent tissue length at rest and tissue length at maximal contraction. Scale bar represents 1mm. As shown, the live muscle material of the co-culture tissue contracts in response to stimulation of the live neuronal material therein, indicating viable NMJ formation.

[0158] FIG. 7A shows a contraction force trace of an NMJ co-culture tissue formed according to the methods described herein and comprising two-week old human iPSC engineered skeletal muscle with ImM and lOuM acetylcholine. In particular, skeletal muscle tissues comprised iPSC derived myoblasts and human dermal fibroblasts remixed at a ratio of 9: 1 were generated by suspending cells within a supporting hydrogel, at a density of 5xl06cells per mL ofhydrogel. The hydrogel comprised polymerized fibrinogen with 20% basement membrane extract from murine Engelbreth-Holm-Swarm (EHS) tumors. These tissues were matured for 14 days and exposed to Acetylcholine at 0.01 and 1 mM. The resulting contractile response was recorded though magnetic field tracking of a flexible magnetic post. Dashed black line represents moment of acetylcholine addition. Accordingly, the live muscle material of the coculture tissue is sensitive to acetylcholine stimulation.

[0159] FIG. 7B shows a force trace of an NMJ co-culture tissue (maintained in co-culture medium) formed according to the methods described herein and comprising two-week old motor neuron - skeletal muscle co-cultures, following stimulation with ImM glutamate. In particular, skeletal muscle tissues, comprising iPSC derived myoblasts and human dermal fibroblasts remixed at a ratio of 9: 1 were generated by suspending cells within a supporting hydrogel, at a density of 5xl06cells per mL of hydrogel. Co-culture tissues were generated as for the muscle tissues but included, in addition to myoblasts and fibroblasts, approx. 100 neurospheres at 1000 (Co-culture 1) or 250 (Co-culture 2) cells per neurosphere. Neurospheres were created in situ and included within tissues during tissue formation. Neurospheres comprised iPSC derived motor neurons 18 days post differentiation induction, which were then matured for 8 days before co-culture creation. The supporting hydrogel comprised polymerized fibrinogen with 20% basement membrane extract from murine Engelbreth-Holm-Swarm (EHS) tumors. These tissues were matured for 14 days and exposed to Glutamate at 0.1 mM. The resulting contractile response was recorded though magnetic field tracking of a flexible magnetic post. Dashed black line represents moment of glutamate addition. Accordingly, the live muscle material of the co-culture tissue is sensitive to glutamate stimulation of the live neuronal material.

[0160] FIG. 7C and FIG. 7D respectively show electrically evoked forces for tetanus response (100Hz) and twitch response (1Hz) of the co-culture tissue of FIG. 7B, stimulated at the given frequency with a biphasic pulse train with pulse widths of 5ms (5 high, 5 low) and an amplitude of 60mA.

[0161] FIG. 8A - FIG. 8C demonstrate that an NMJ co-culture tissue formed according to the methods described herein maintains the function of the live muscle material therein, in comparison to a muscle-only tissue. In particular, skeletal muscle only tissues, comprising iPSC derived myoblasts and human dermal fibroblasts remixed at a ratio of 9: 1 were generated by suspending cells within a supporting hydrogel, at a density of 7xl06cells per mL ofhydrogel. The hydrogel comprised polymerized fibrinogen with 20% basement membrane extract from murine Engelbreth-Holm-Swarm (EHS) tumors. NMJ co-culture tissues comprised skeletal muscle tissues with an additional neuronal component adhered to a mature skeletal muscle tissue. The neuronal component comprised approx. 100 neurospheres containing 2500 cells per neurosphere. These neurospheres comprised iPSC derived motor neurons 18 days post differentiation induction, which were then matured for 10 days before coculture creation. At Day 10 of skeletal muscle differentiation, neurospheres were suspended in a linking hydrogel (Type 1 collagen at 1 mg / mL plus 5% basement membrane extract from murine Engelbreth-Holm-Swarm (EHS) tumors) and skeletal muscle tissues introduced into the unset hydrogel within the neurosphere creation consumable. Following Day 10, all tissues were exposed to media containing 10% FBS for 24 hours, before being changed into a coculture media designed to support both muscle contractile function and neuronal axon extension. Functional readouts of skeletal muscle are displayed across time as an average from 30 twitch responses following 10 ms, 100 mA, 1 Hz biphasic stimulation pulses.

[0162] FIG. 8A shows a plot of active twitch forces of the foregoing co-culture tissue, in comparison to muscle-only tissue.

[0163] FIG. 8B shows a plot of time from contraction 10 to peak contraction of the co-culture tissue of FIG. 8A formed according to the methods described herein, in comparison to muscle- only tissue.

[0164] FIG. 8C shows a plot of time from contraction peak to relaxation of the co-culture tissue of FIG. 8A, in comparison to muscle-only tissue.

[0165] FIG. 9A shows a contractile force trace of an NMJ co-culture tissue formed as described above with respect to FIG. 8A - FIG. 8C, wherein the neuronal cell culture is stimulated with a 450 nm blue light (250 ms pulse train, 40 mW / mm2, 100 Hz, 80% duty cycle). The motor neurons of the co-culture tissue express Channelrhodopsin-2 (ChR2). Arrow heads indicate beginning of pulse trains. As shown, the live muscle material of the co-culture tissue contracts in response to blue light stimulation of the live neuronal material.

[0166] FIG. 9B - FIG. 9D display mean ± SD for n=4 tissues as described with respect to FIG. 9A for stimulation with blue light (250 ms pulse train, 40 mW / mm2, 100 Hz, 80% duty cycle) at 5, 2 and Is intervals.

[0167] FIG. 9B plots the contraction force of the NMJ co-culture tissue of FIG. 9A, i.e., peak height for blue light driven functional response of the muscle cell culture of the co-culture tissue.

[0168] FIG. 9C plots the contraction force of the NMJ co-culture tissue of FIG. 9A from optical stimulation of the neuronal cell culture as a percentage the contraction force achieved by directly stimulating the muscle cell culture with field stimulation, as a proxy for innervation density. Restated, FIG. 9C shows the blue light force expressed as a percentage compared to mean force produced by the same tissue under electrical stimulation (10 ms, 100 mA, 1 Hz biphasic stimulation pulses).

[0169] FIG. 9D plots the percentage capture of the contraction force of the NMJ co-culture tissue of FIG. 9A within 100ms of blue light stimulation. Restated, FIG. 9D shows the percentage of blue light stimulation pulses which elicit a peak within 100 ms following the end of the blue light stimulation pulse. As shown from FIG. 9A - FIG. 9D, direct NMJ activation of the co-culture tissues formed according to the methods described herein enables real time functional measurement of innervated tissues.

[0170] FIG. 10A - FIG. 10C show contractile force traces of an NMJ co-culture tissue formed as described above with respect to FIG. 8A - FIG. 8C. The co-culture tissues were exposed to active Botulinum neurotoxin complex serotype A (BoNT-A) at 10 pg toxin per tissue in maintenance culture media. Blue light driven contraction response was monitored across the following 8 hours in both treated and control groups.

[0171] FIG. 10A displays contractile force output (mean ± SD, n=4 tissues, of 10 contractions driven by blue light pulses, 250 ms pulse train, 40 mW / mm2, 100 Hz, 80% duty cycle) normalized to control values at each timepoint. As shown, treatment with BoNT-A caused complete loss of blue light driven contractile responses of the co-culture tissues.

[0172] FIG. 10B shows representative traces taken from individual co-culture tissues at 0 hours and 8 hours post treatment from groups displayed in FIG. 10A.

[0173] FIG. 10C shows representative force traces from electrical stimulation of the coculture tissues of FIG. 10A, post BoNT-A treatment. Following BoNT-A exposure, loss of functional response could be due to complete loss of muscle functional capacity; however, electrical stimulation (10 ms, 100 mA, 1 Hz biphasic stimulation pulses) of control and treated tissues showed no difference in underlying functional capacity, thereby confirming the specificity of blue light driven responses to the NMJs within the co-culture tissues.

[0174] The methods and devices described herein present a number of advantages in the modeling of neuromuscular disease. The first is the in situ generation of the neuronal component, i.e., neuronal aggregates, neurospheres, and neuronal cell cultures. Current models require manual handling of cells / cell aggregates / neurospheres, which is time consuming and technically challenging, resulting in reduced neuron survival, reproducibility, and scalability. The methods described herein further reduce variability through the optional direct thaw of single cells to the casting plate directly from resuscitation from cryopreservation, thereby allowing high-throughput batch control for experiments with high batch-batch sensitivity.

[0175] Secondly, communication of the live neuronal material with the live muscle material through a communication channel (e.g., the communication channel 318 of casting plate 300) allow long distance communication and axonal extension. Many neuromuscular diseases are progressive diseases characterized by a withdrawal of axons from muscle; this progressive instability has a short / small assay window on devices presented with short communication routes (limited by lack of guidance) and therefore are unlikely to be able to successfully model complex progressive diseases.

[0176] Thirdly, the devices and methods allow specific and independent stimulation of cell cultures (e.g., different cell compartments) allowing a comparison of two important metrics of neuromuscular junction performance: maximal direct evoked muscle function and innervation evoked muscle function. The latter can be achieved, for example, by directly stimulating the live muscle material with electrical, optical or chemical stimuli and measuring the contractile response of the muscle cell culture. The former can be achieved, for example, by stimulating the neuronal cell culture and measuring the contractile response of the muscle cell culture. The ratio of these measurements gives an estimation of innervation density, which is important in muscle function in health and disease.

[0177] In view of the foregoing, various inventive aspects disclosed herein may be characterized and claimed according to the following clauses:

[0178] 1. A method of using a tissue analysis system comprising a co-culture casting system, the method comprising: seeding a plurality of neuronal culture compartments of a multi-well co-culture casting plate with live neuronal material, wherein each neuronal culture compartment comprises 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, in the multi-well co-culturecasting plate, a plurality of co-culture tissues, each of the co-culture tissues comprising a muscle cell culture cultured from the live muscle material and in situ neurospheres matured in the cell aggregation features from the live neuronal material; and causing an innervated portion of the live muscle material in each of the co-culture tissues to contract by stimulating the live neuronal material.

[0179] 2 The method of clause 1, wherein seeding the plurality of neuronal culture compartments with the live neuronal material comprises thawing cells comprising motor neurons directly in the plurality of neuronal culture compartments.

[0180] 3. The method of clause 2, wherein culturing the plurality of co-culture tissues comprises culturing at least one of the co-culture tissues in each neuronal culture compartment.

[0181] 4 The method of clause 1, wherein the cell aggregation features comprise feature geometry.

[0182] 5. The method of clause 4, wherein the feature geometry comprises tessellating surface features.

[0183] 6. The method of clause 5, wherein the tessellating surface features comprise tessellating cavities, wherein each tessellating cavity forms a nadir.

[0184] 7 The method of clause 1, wherein the cell aggregation features comprise at least one of hydrophobicity or electrostatic charge.

[0185] 8. The method of clause 2 or clause 3, wherein seeding the plurality of neuronal culture compartments with the live neuronal material comprises administering, in each neuronal culture compartment, a linking hydrogel on the in situ neurospheres, wherein culturing each the plurality of co-culture tissues comprises remodeling an interface between the linking hydrogel and a second hydrogel comprising the live muscle material.

[0186] 9. The method of clause 8, wherein remodeling the interface comprises treating the interface with a serum media.

[0187] 10. The method of clause 2 or clause 3, wherein the cell aggregation features are disposed in a casting trench of the respective neuronal culture compartment.

[0188] 11. The method of clause 2, wherein culturing each of the co-culture tissues comprises communicating the live neuronal material with the live muscle material along a communication channel.

[0189] 12. The method of clause 11, wherein the multi-well co-culture casting plate comprises a plurality of culture units, wherein each culture unit comprises a muscle culture compartment in communication with at least one of the plurality of neuronal culture compartments via the communication channel.

[0190] 13. The method of clause 1, wherein causing an innervated portion of the live muscle material in each of the co-culture tissues to contract by stimulating the live neuronal material comprises transferring the co-culture tissues to a maintenance plate prior to causing the innervated portion of the live muscle material in the co-culture tissues to contract by stimulating the live neuronal material.

[0191] 14. The method of clause 1, wherein causing the innervated portion of the live muscle material in the co-culture tissue to contract comprises activating the in situ neurospheres with at least one of an optical, electrical, or chemical stimulus.

[0192] 15. The method of clause 2 or clause 3, wherein causing the innervated portion of the live muscle material in each of the co-culture tissues to contract comprises contemporaneously causing the innervated portion of the live muscle material in each of the co-culture tissues to contract.

[0193] 16. The method of clause 15, wherein causing the innervated portion of the live muscle material in each of the co-culture tissues to contract further comprises contemporaneously recording a contraction of each of the co-culture tissues.

[0194] 17. A tissue analysis system or co-culture casting system, comprising: a multi-well coculture casting plate comprising a plurality of neuronal culture compartments each with a cell aggregation region formed at a bottom surface thereof, each cell aggregation region comprising a plurality of cell aggregation features; and a multi-well muscle tissue casting plate.

[0195] 18. The tissue analysis system of clause 17, wherein the cell aggregation features comprise feature geometry.

[0196] 19. The tissue analysis system of clause 18, wherein the feature geometry comprises tessellating surface features.

[0197] 20. The tissue analysis system of clause 19, wherein the tessellating surface features comprise tessellating cavities, wherein each tessellating cavity forms a nadir.

[0198] 21. The tissue analysis system of clause 17, wherein the cell aggregation features comprise at least one of hydrophobicity or electrostatic charge.

[0199] 22. The tissue analysis system of clause 17, wherein each of the cell aggregation regions is disposed in a casting trench of the respective neuronal culture compartment.

[0200] 23. The tissue analysis system of clause 17, wherein the multi -well co-culture casting plate comprises a plurality of culture units, wherein each culture unit comprises a muscle culture compartment in communication with at least one of the neuronal culture compartments via a communication channel.

[0201] 24. A method of modelling a neuromuscular junction in a plurality of co-culture tissues, comprising: seeding a plurality of neuronal culture compartments of a multi-well plate with live neuronal material; casting a plurality of muscle cell cultures comprising live muscle material; culturing, in the multi-well plate, a plurality of co-culture tissues comprising the live muscle material and in situ neurospheres matured from the live neuronal material; and causing an innervated portion of the live muscle material in each of the co-culture tissues to contract by stimulating the live neuronal material.

[0202] 25. The method of clause 24, wherein seeding the plurality of neuronal culture compartments with the live neuronal material comprises thawing cells comprising motor neurons directly in the plurality of neuronal culture compartments.

[0203] 26. The method of clause 25, wherein culturing the plurality of co-culture tissues comprises culturing at least one of the co-culture tissues in each neuronal culture compartment.

[0204] 27. The method of clause 25 or clause 26, wherein each neuronal culture compartment comprises a plurality of cell aggregation features configured to aggregate the live neuronal material into cell aggregates.

[0205] 28. The method of clause 27, wherein the cell aggregation features comprise feature geometry configured to drive cell aggregation.

[0206] 29. The method of clause 28, wherein the feature geometry comprises tessellating surface features.

[0207] 30. The method of clause 29, wherein the tessellating surface features comprise tessellating cavities, wherein each tessellating cavity forms a nadir.

[0208] 31 The method of clause 27, wherein the cell aggregation features comprise at least one of hydrophobicity or electrostatic charge.

[0209] 32 The method of clause 25 or clause 26, wherein thawing cells comprising the motor neurons directly in the plurality of neuronal culture compartments comprises thawing the cells without a Rho kinase inhibitor.

[0210] 33. The method of clause 25 or clause 26, wherein seeding the plurality of neuronal culture compartments with the live neuronal material comprises administering, in each neuronal culture compartment, a linking hydrogel on the in situ neurospheres, wherein culturing each of the plurality of co-culture tissues comprises remodeling an interface between the linking hydrogel and a second hydrogel comprising the live muscle material.

[0211] 34. The method of clause 33, wherein remodeling the interface comprises treating the interface with a serum media.

[0212] 35. The method of clause 25 or clause 26, wherein the multi-well plate is a first casting plate and the plurality of neuronal culture compartments comprises casting trenches.

[0213] 36. The method of clause 25, wherein culturing the plurality of co-culture tissues comprises, for each of the co-culture tissues, communicating the live neuronal material with the live muscle material along a communication channel.

[0214] 37. The method of clause 36, wherein the multi-well plate comprises a plurality of culture units, wherein each culture unit comprises a muscle culture compartment in communication with at least one of the neuronal culture compartments via the communication channel.

[0215] 38 The method of clause 26, further comprising transferring the co-culture tissues to a maintenance plate prior to causing the innervated portion of the live muscle material in the coculture tissues to contract by stimulating the live neuronal material.

[0216] 39. The method of clause 25 or clause 26, wherein causing the innervated portion of the live muscle material in the co-culture tissue to contract by stimulating the live neuronal material comprises activating the in situ neurospheres.

[0217] 40. The method of clause 25 or clause 26, wherein causing the innervated portion of the live muscle material in each of the co-culture tissues to contract comprises contemporaneously causing the innervated portion of the live muscle material in each of the coculture tissues to contract.

[0218] 41. The method of clause 40, wherein causing the innervated portion of the live muscle material in each of the co-culture tissues to contract further comprises contemporaneously recording a contraction of each of the co-culture tissues.

[0219] 42. A method of modelling a plurality of neuromuscular junctions, comprising: seeding a plurality of neuronal culture compartments of a multi-well plate with live neuronal material by thawing cells comprising motor neurons directly in the plurality of neuronal culture compartments, wherein each neuronal culture compartment comprises cell aggregation features configured to aggregate the live neuronal material into cell aggregates; casting a plurality of muscle cell cultures comprising live muscle material; culturing, in the multi-well plate, a plurality of co-culture tissues comprising the live muscle material and in situ neurospheres matured from the live neuronal material, in part by remodeling an interface between a linking hydrogel and a second hydrogel comprising the live muscle material; and contemporaneously causing an innervated portion of the live muscle material in each of the co-culture tissues to contract by stimulating the live neuronal material.

[0220] 43. A method of modelling a plurality of neuromuscular junctions, comprising: seeding a plurality of culture units of a multi-well plate with live neuronal material by thawing cells comprising motor neurons directly in a plurality of neuronal culture compartments, wherein each neuronal culture compartment comprises cell aggregation features configured to aggregate the live neuronal material into cell aggregates; casting a plurality of muscle cell cultures comprising live muscle material; culturing a plurality of co-culture tissues comprising the live muscle material and in situ neurospheres matured from the live neuronal material in the multiwell plate by communicating, in each of the culture units, the live neuronal material with the live muscle material along a communication channel; and contemporaneously causing an innervated portion of the live muscle material in each of the co-culture tissues to contract by stimulating the live neuronal material.

[0221] Additionally the proposed methods and devices allow these outputs to be measured non-invasively across time, which enables observation of progressive degenerative diseases in a single tissue across long time periods, improving statistical power and significantly reducing cost for time course experiments.

[0222] Various changes can be made to the embodiments of the present disclosure as could be reasonably contemplated in view of the above-described description by any person skilled in the art. The following claims are presented as examples of embodiments of the present disclosure,but these claims should not be construed to limit other claims or other embodiments disclosed herein.

[0223] The detailed description set forth above in connection with the appended drawings, where like numerals reference like elements, are intended as a description of representative embodiments of the present disclosure and are not intended to represent the only embodiments. Each embodiment described m 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 the disclosure to the precise forms disclosed. Similarly, any steps described herein may be interchangeable with other steps, or combinations of steps, in order to achieve the same or substantially similar result. Further still, one or more features of any embodiment may be combined with one or more features of one or more embodiments to form additional embodiments, which are within the scope of the present disclosure.

[0224] Generally, the embodiments disclosed herein are non-limiting, and the inventors contemplate that other embodiments within the scope of this disclosure may include structures and functionalities from more than one specific embodiment shown in the FIGURES and described in the specification. It will be appreciated that variations and changes may be made by others, and equivalents employed, without departing from the spirit of the present disclosure. Accordingly, it is expressly intended that all such variations, changes, and equivalents fall within the spirit and scope of the present disclosure as claimed. For example, the present disclosure includes additional embodiments having combinations of any one or more features described above with respect to the representative embodiments.

[0225] In the foregoing description, specific details are set forth to provide a thorough understanding of representative embodiments of the present disclosure. It will be apparent to one skilled in the art, however, that the embodiments disclosed herein may be practiced without embodying all the specific details. In some instances, well-known process steps have not been described in detail in order not to unnecessarily obscure various aspects of the present disclosure.

[0226] The present application may include references to directions, such as “first,” "second," "vertical," "horizontal," "front," "rear," "left," "right," "top," and "bottom," “below,” “around,” etc. These references, and other similar references in the present application, are intended to assist in helping describe and understand the particular embodiment (such as when theembodiment is positioned for use) and are not intended to limit the present disclosure to these directions or locations.

[0227] The present application may also reference quantities and numbers. Unless specifically stated, such quantities and numbers are not to be considered restrictive, but exemplary of the possible quantities or numbers associated with the present application. Also in this regard, the present application may use the term "plurality" to reference a quantity or number. In this regard, the term "plurality" means any number that is more than one, for example, two, three, four, five, etc. The term "about," "approximately," etc., means plus or minus 5% of the stated value. The term "based upon" means "based at least partially upon." The term "between" includes the values recited in connection therewith. 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, z.e., any one of the following conditions satisfy all of the foregoing expressions: A; B; C; AB; AC; BC; ABC.

Claims

CLAIMSWhat is claimed is:

1. A method of using a tissue analysis system comprising a co-culture casting system, the method comprising: seeding a plurality of neuronal culture compartments of a multi-well co-culture casting plate with live neuronal material, wherein each neuronal culture compartment comprises 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, in the multi-well co-culture casting plate, a plurality of co-culture tissues, each of the co-culture tissues comprising a muscle cell culture cultured from the live muscle material and in situ neurospheres matured in the cell aggregation features from the live neuronal material; and causing an innervated portion of the live muscle material in each of the co-culture tissues to contract by stimulating the live neuronal material.

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

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

4. The method of claim 1, wherein the cell aggregation features comprise feature geometry.

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

6. The method of claim 5, wherein the tessellating surface features comprise tessellating cavities, wherein each tessellating cavity forms a nadir.

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

8. The method of claim 2 or claim 3, wherein seeding the plurality of neuronal culture compartments with the live neuronal material comprises administering, in each neuronal culture compartment, a linking hydrogel on the in situ neurospheres, wherein culturing each the plurality of co-culture tissues comprises remodeling an interface between the linking hydrogel and a second hydrogel comprising the live muscle material.

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

10. The method of claim 2 or claim 3, wherein the cell aggregation features are disposed in a casting trench of the respective neuronal culture compartment.

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

12. The method of claim 11, wherein the multi-well co-culture casting plate comprises a plurality of culture units, wherein each culture unit comprises a muscle culture compartment in communication with at least one of the plurality of neuronal culture compartments via the communication channel.

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

14. The method of claim 1, wherein causing the innervated portion of the live muscle material in the co-culture tissue to contract comprises activating the in situ neurospheres with at least one of an optical, electrical, or chemical stimulus.

15. The method of claim 2 or claim 3, wherein causing the innervated portion of the live muscle material in each of the co-culture tissues to contract comprises contemporaneouslycausing the innervated portion of the live muscle material in each of the co-culture tissues to contract.

16. The method of claim 15, wherein causing the innervated portion of the live muscle material in each of the co-culture tissues to contract further comprises contemporaneously recording a contraction of each of the co-culture tissues.

Images