Microfluidic devices, neuro-muscular models, and associated methods

Abstract

A device is disclosed that includes a body, a first channel formed and a second channel. The first channel and the second change have inlets from outside the device and outlets to outside the device. The device further incudes a first compartment formed within the body and a second compartment formed within the body. The device further includes a plurality of microchannels connecting the first compartment and the second compartment. The device further includes a plurality of interface regions. Each interface region of the plurality of interface regions connects the at least one first channel with the at least one second channel, the at least one second channel with the first compartment, or the at least one second channel with the second compartment.

Description

MICROFLUIDIC DEVICES, NEURO-MUSCULAR MODELS, AND ASSOCIATED METHODSFIELD OF THE INVENTION

[0001] The present invention relates generally to microfluidic devices, and more specifically, to microfluidic devices, models, and associated methods that provide for innervated and vascularized organ models.BACKGROUND OF THE INVENTION

[0002] Several disorders, such as neuromuscular diseases, are still incurable (e.g., ALS, Duchenne Muscular Dystrophy) and lack effective treatments. The main hindrance in drug development for these diseases is the lack of representative research models in which new and repurposed chemical formulations can be tested to predict their effects (e.g., therapeutic and toxicity) in humans. Animal models lack the human biology and physiology components to accurately mimic human diseases. Moreover, some diseases and / or their variations occur only in humans, which invalidates the use of animal models altogether. On the other hand, current in vitro cellular models of human diseases lack the complexity and maturation level to adequately mimic an organ / tissue behavior. Consequently, these fail to provide reliable data regarding a drug's therapeutic potential.

[0003] Accordingly, needs exist for a novel solution in which neuromuscular diseases can be simulated within a device that offers a convenient and reproducible test bed for drug discovery and preclinical testing, among other purposes.SUMMARY OF THE INVENTION

[0004] The term embodiment and like terms (e.g., implementation, configuration, aspect, example, and option) are intended to refer broadly to all of the subject matter of this disclosure and the claims below. Statements containing these terms should be understood not to limit the subject matter described herein or to limit the meaning or scope of the claims below. Embodiments of the present disclosure covered herein are defined by the claims below, not this summary. This summary is a high-level overview of various aspects of the disclosure and introduces some of the concepts that are further described in the Detailed Description section below. This summary is not intended to identify key or essential features of the claimed subject matter. This summary is also not intended to be used in isolation to determine the scope of the claimed subject matter. The subject matter should be understood by reference to appropriate portions of the entire specification of this disclosure, any or all drawings, and each claim.

[0005] According to certain aspects of the present disclosure, a device is disclosed. The device includes a body, at least one first channel formed within the body, and at least one second channel formed within the body. The at least one first channel has an inlet from outside the device and an outlet to outside the device. The at least onesecond channel has an inlet from outside the device and an outlet to outside the device. The device further includes a first compartment formed within the body and a second compartment formed within the body. The at least one second channel separates the first compartment and the second compartment from the at least one first channel. The device further includes a plurality of microchannels connecting the first compartment and the second compartment. The device further includes a plurality of interface regions. Each interface region of the plurality of interface regions connects the at least one first channel with the at least one second channel, the at least one second channel with the first compartment, or the at least one second channel with the second compartment.

[0006] According to certain further aspects, the first compartment has an open top. According to certain further aspects, the second compartment has an open top. According to certain further aspects, the plurality of microchannels are about 1 to 10 microns in diameter, about 900 microns to 6 millimeters (mm) in length, and about 120to 180 microns in height. According to some preferred aspects, the microchannels are about 8 microns in diameter, about 900 microns in length, and about 150 microns in height. Such sizes allow axons to extend into the plurality of microchannels and prevent neuron cell bodies from extending into the plurality of microchannels when neuronal tissue is within one of the compartments. According to certain further aspects, the at least one second channel includes two second channels, and the first compartment and the second compartment are between the two second channels in the body. The at least one first channel can include two first channels, and the two second channels are between the two first channels in the body. According to certain further aspects, the first compartment includes a plurality of grooves on a first surface. Each groove of the plurality of grooves can be less than about one micron wide and less than about one micron deep. For example, each groove can be from about 500 nanometers (nm) to 10 microns in width. The spacing (i.e., pitch) between adjacent grooves can be regular or variable and from about 500 nm to 10 microns in width. According to certain further aspects, the first compartment and the second compartment includes a plurality of grooves on a first surface. According to certain further aspects, each interface region of the plurality of interface regions is a pillar region formed of a plurality of pillars that allow material to pass between adjacent pillars. Each pillar of the plurality of pillars can have a top-down profile shaped as a rectangle or a trapezoid, such that the pillars can be shaped as a cuboid or a trapezoid prism. According to certain further aspects, the at least one first channel is about 50 microns to 1 mm in diameter, such as preferably about 400 microns in diameter. According to certain further aspects, the at least one second channel is about 50 microns to 2 mm in diameter, such as preferably about 800 microns in diameter. According to certain further aspects, the body is formed of polydimethylsiloxane (PDMS), glass, plastic (e.g., polycarbonate), or a polymeric substrate. The body can be transparent. According to certain further aspects, the body is bonded to a bottom substrate via a physical method or a chemical method. According to certain further aspects, the bottom substrate is formed of PDMS, glass, plastic, or a polymeric substrate. According to certain further aspects, the body includes a top substrate bonded to the bottom substrate. According to certain further aspects, the bottom substrate includes electrodes. According to certain further aspects, the first compartment, the second compartment, or both are more than one compartment such that the device includes three or more compartments.

[0007] According to certain additional aspects of the present disclosure, a human neuro-muscular model isdisclosed. The model includes the device disclosed above. The model further includes endothelial cells lining the at least one first channel disclosed above such that the at least one first channel is configured as at least one vascular channel. The model further includes aligned myotubes formed of myogenic progenitor cells within the first compartment disclosed above such that the first compartment is configured as a muscle compartment. The model further includes neuronal tissue within the second compartment disclosed above such that the second compartment is configured as a neuron compartment. According to certain further aspects, one or more of the at least one vascular channel, the muscle compartment, the neuron compartment, or a combination thereof model one or more neuromuscular disorders, one or more neurological disorders, one or more vascular disorders, one or more cancers, or a combination thereof. According to certain further aspects, the one or more neuromuscular disorders include amyotrophic lateral sclerosis, muscular dystrophy, myasthenia gravis, spina bifida, Charcot-Marie-Tooth disease, spinocerebellar ataxia, spinal muscular atrophy, Guillain-Barre Syndrome, Friedreich's Ataxia, myotonic dystrophy, Lambert-Eaton Myasthenic Syndrome, inclusion body myositis, one or more congenital myasthenic syndromes, or monomelic amyotrophy. According to certain further aspects, the one or more neurological disorders include Alzheimer's disease, Parkinson's disease, multiple sclerosis, neuropathy, Huntington's disease, Batten disease, Alpers' disease, Lewy Body disease, Gerstmann-Straussler-Scheinker disease, Kuru, or Leigh's disease. According to certain further aspects, the one or more vascular disorders include atherosclerosis, hemangioma, Kawasaki disease, arteritis, or fibromuscular dysplasia. According to certain further aspects, the one or more neuromuscular disorders, the one or more neurological disorders, the one or more vascular disorders, the one or more cancers, or the combination thereof are modeled by reproducing disease-associated mutations, exposure associated with disease-relevant physicochemical stimuli, adding cells and / or tissue that are associated with a disease, or a combination thereof. According to certain further aspects, the exposure associated with disease-relevant physicochemical stimuli includes exposure to toxins and / or chemicals, cytokine stimuli, metabolic changes, exposure to varying oxygen levels, exposure to at least one virus and / or at least one infection, serum or growth factor deprivation, radiation, or a combination thereof.

[0008] According to certain further aspects, one or more of the at least one first channel, the at least one second channel, the first compartment, and the second compartment are coated with one or more of poly-L-lysine, poly-D- lysine, poly-L-ornithine, fibronectin, vitronectin laminin, gelatin, fibrin, osteopontin, elastin, tenascin, keratin, silk, hyaluronic acid, alginate or a derivative thereof, natural or synthetic polysaccharide, proteoglycans, carbon nanotubes, polylactic acid, polyglycolic acid, a polyanhydride, a poly(lactide-co-glycolide) polymer, a polyamino acid, a poly(alkylene oxide), a polyethylene oxide), a poly(allylamine), poly(butylcyanoacrylate), poly (aspartic acid), a poly(acrylate), a polyester, polyhydroxybutyrate and poly-epsilon-caprolactone, a polyphosphazine, a poly(vinyl alcohol), a modified styrene polymer, polyurethane, poly(4-aminomethylstyrene), a pluronic polyol, a polyoxamer, a poly(uronic acid), a poly( vinylpyrrolidone), a polypyrrole, synthetic adhesion peptides (RGD and variations of these, IKVAV, or a combination thereof. According to certain further aspects, the neuronal tissue is one or more neurons, one or more neurospheres, or a combination thereof. According to certain further aspects, the neuron compartmentis coated with poly-L-lysine, poly-D-lysine, poly-L-ornithine, fibronectin, vitronectin laminin (all isoforms), gelatin, fibrin, osteopontin elastic, tenascin, keratin, silk, hyaluronic acid, alginate or a derivative thereof, natural or synthetic polysaccharide, proteoglycans, carbon nanotubes, polylactic acid, polyglycolic acid, a polyanhydride; a poly(lactide- co-glycolide) (PLGA) polymer, a polyamino acid, a poly(alkylene oxide), a polyethylene oxide), a poly(allylamine)(PAI\ / l), poly(butylcyanoacrylate) (PBCA), poly (aspartic acid), a poly(acrylate), a polyester, polyhydroxybutyrate and poly-epsilon-caprolactone, a polyphosphazine, a poly(vinyl alcohol), a modified styrene polymer, polyurethane, poly(4-aminomethylstyrene), a pluronic polyol, a polyoxamer, a poly(uronic acid), a poly(vinylpyrrolidone), a polypyrrole, synthetic adhesion peptides (RGD and variations of these, IKVAV and variations of these), or combinations thereof. According to certain further aspects, the one or more neurons, the one or more neurospheres, or the combination thereof contains primary human Schwann cells, stem-cell derived Schwann cells (e.g., embryonic stem cells or iPSCs), or Schwann cells directly converted from other cells such as fibroblasts. According to certain further aspects, the hiPSCs-derived Schwann cells can be from one or more patients with specific gene mutations. Alternatively, the mutations can be introduced by several methods, such as genetic manipulations (e.g., CRISPR and other methods). According to certain further aspects, the at least one second channel is filled with a hydrogel. According to certain further aspects, the hydrogel can contain collagen, fibrin, endothelial cells, pericytes, or a combination thereof. According to certain further aspects, the neuron compartment includes hydrogel. According to certain further aspects, one or more axons extend from the one or more neurons, the one or more neurospheres, or the combination thereof into the plurality of microchannels. According to certain further aspects, the one or more axons can form neuromuscular junctions with the aligned myotubes. According to certain further aspects, the one or more neurons, the one or more neurospheres, or the combination thereof can be optogenetically modified neurons to cause an electrical signal that travels through the one or more axons to the neuromuscular junctions and induce muscle fibers contraction when the one or more neurons, the one or more neurospheres, or the combination thereof are exposed to light of a predetermined wavelength and frequency. Accordingto certain further aspects, the model further includes an extracellular matrix material coating the muscle compartment. According to certain further aspects, the muscle fibers are anchored to the array of pillars. According to certain further aspects, the myotubes form a muscle sheet and the muscle sheet is anchored to the array of pillars. According to certain further aspects, the muscle sheet aligns with a plurality of grooves on a first surface of the first compartment. According to certain further aspects, the muscle sheet is one cell thick to allow for imaging of individual cells.

[0009] According to certain additional aspects of the present disclosure, a method of identifying a compound, a dosage of a compound, a dosing regime, or combination thereof, that effects activity of innervated muscle fibers is disclosed. The method includes making a first measurement of a biochemical, cellular, genetic, molecular, or morphological characteristic of neuronal cells, muscle cells, innervated muscle fibers, or combination thereof. The method further includes contacting the neuronal cells, muscle cells, innervated muscle fibers, or combination thereof with one or more amounts, for one or more dosing regimes, of a test compound, or a combination thereof. The method further includes making a second measurement of the biochemical, cellular, genetic, molecular, or morphologicalcharacteristic of the neuronal cells, muscle cells, or innervated muscle fibers, or combination thereof. The method further includes selecting the compound, the dosage of the compound, the dosing regime, or the combination thereof that effects neuronal activity, muscular activity, or a combination thereof if the compound improves or worsens the biochemical, genetic, cellular, molecular, or morphological characteristic of the neuronal cell, muscle cells, or innervated muscle fibers, or combination thereof.

[0010] According to certain further aspects, the method includes delivering the test compound directly through a first compartment, a second compartment, and / or a first channel of a device including the first compartment, the second compartment, and / or the first channel. According to certain further aspects, the first channel is vascularized and the test compound is perfused through the first channel. According to certain further aspects, the test compound is administered in a free form or as a payload of a delivery agent. According to certain further aspects, the method includes performing one or more assays on the neuronal cells, the muscle cells, the innervated muscle fibers, or the combination thereof. According to certain further aspects, the one or more assays include one or more assays on cell metabolic activity, mitochondrial function, oxidative stress, apoptosis, cell membrane activity.

[0011] According to certain additional aspects of the present disclosure, a method for identifying compounds that have pro / anti vascularization properties within organoids, tissue explants, or other assembly cells is disclosed. The method includes providing a device. The device includes a first channel formed of a hollow vascular tube; a second channel having a hydrogel; and a first compartment in fluid communication with one or both of the first channel and the second channel. The first compartment includes a target tissue embedded in a hydrogel containing endothelial cells and / or mural cells. The method further includes providing a compound in the first compartment and delivered through the first channel or second channel. The method further includes identifying and quantifying presence of vascular tubes within or around the target tissue using microscopic techniques. The method further includes determining that the compound is pro-vascularization when an amount of blood vessels at the target tissue is superior to control samples or determining that the compound is anti-vascularization when the amount of blood vessels at the target tissue is inferior to control samples.

[0012] According to certain additional aspects of the present disclosure, a method to identify one or more compounds that can have pro- or anti-innervation properties within organoids, tissue explants, and / or other assemblies of cells is disclosed. The method includes providing a device. The device includes a first channel that is vascularized, a second channel, a first compartment that has a neuronal population, and a second compartment that has a target tissue. The first compartment, the second compartment, or a combination thereof are in fluid communication with the first compartment, the second compartment, or a combination thereof. The method further includes providing a compound through the first channel, the second channel, the first compartment, and / or the second compartment. The method further includes assessing innervation by identifying and quantifying innervation contact points and nerve fibers in the target tissue by means of microscopic techniques.

[0013] According to certain additional aspects of the present disclosure, a method of detecting an effect on a biochemical, cellular, genetic, molecular, and / or morphological characteristic of a cancer is disclosed. The methodincludes providing a device. The device includes a first channel that is vascularized and contains one or more cancer cells in circulating or adherent form. The device further includes a second channel that is filled with a blank hydrogel or a hydrogel loaded with endothelial cells. The device further includes a first compartment or a second compartment that contains a cancer cell population that is dissociated or in spheroids. The first compartment, the second compartment, or a combination thereof are in fluid communication with the first channel, the second channel, or a combination thereof. The method further includes delivering a drug through the first channel, the first compartment, or the second compartment. The method further includes performing one or more microscopic and / or biochemical assays to detect an alteration in cancer cell proliferation, viability, migration, death / apoptosis, intravasation, and / or extravasation in the one or more cancer cells, the cancer cell population, or a combination thereof.

[0014] The above summary is not intended to represent each embodiment or every aspect of the present disclosure. Rather, the foregoing summary merely provides an example of some of the novel aspects and features set forth herein. The above features and advantages, and other features and advantages of the present disclosure, will be readily apparent from the following detailed description of representative embodiments and modes for carrying out the present invention, when taken in connection with the accompanying drawings and the appended claims. Additional aspects of the disclosure will be apparent to those of ordinary skill in the art in view of the detailed description of various embodiments, which is made with reference to the drawings, a brief description of which is provided below.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The disclosure, and its advantages and drawings, will be better understood from the following description of representative embodiments together with reference to the accompanying drawings. These drawings depict only representative embodiments and are therefore not to be considered as limitations on the scope of the various embodiments or claims.

[0016] FIG. 1 is cross-sectional plan view of a device that can be used as a neuro-muscular model, according to one application, according to aspects of the present disclosure.

[0017] FIGS. 2A and 2B are a perspective view and a top view of grooves formed on a surface of a muscle or neuron compartment, according to aspects of the present disclosure.

[0018] FIG. 3 is a detailed view of microchannels from FIG. 1, according to certain aspects of the present disclosure.

[0019] FIG. 4 is a top view of an interface region from FIG. 1, according to aspects of the present disclosure.

[0020] FIG. 5 is a detailed view of muscle tissue within the muscle compartment, according to certain aspects of the present disclosure.

[0021] FIG. 6 is a detailed view of a junction between a vascular channel and a wall channel, according to certain aspects of the present disclosure.

[0022] FIG. 7 is a detailed view of microchannels the microchannels of FIG. 1 with axons extending therethrough, according to certain aspects of the present disclosure.

[0023] FIG. 8 is a partial scanning electron micrograph depicting an overview of the design of a device, such as the device of FIG. 1, according to aspects of the present disclosure.

[0024] FIG. 9 is a micrograph showing formation of aligned and striated muscle fibers with peripheral nuclei from rat primary muscle cells in a muscle compartment of a device disclosed herein, according to aspects of the present disclosure. Cells were immunostained with alpha actinin (striations), and DAPI was used to visualize the cell nuclei.

[0025] FIGS. 10A and 10B are show anchored muscle fibers on micropillars of a device as disclosed herein, according to aspects of the present disclosure. The muscle fibers were immunostained with alpha actinin (FIG. 11A) are visibly anchored to the micropillars (FIG. 11B), as highlighted by the box.

[0026] FIGS. 11A and 11B show a front view and an isometric view, respectively, of formation of a vascular channel within a device as disclosed herein, according to aspects of the present disclosure. Endothelial cells (human vein endothelial cells, HUVEC) form a confluent, hollow and tight channel as visible by the 3D reconstruction of micrographs taken to CD31 immunostained cells.DETAILED DESCRIPTION

[0027] Various embodiments are described with reference to the attached figures, where like reference numerals are used throughout the figures to designate similar or equivalent elements. The figures are not necessarily drawn to scale and are provided merely to illustrate aspects and features of the present disclosure. Numerous specific details, relationships, and methods are set forth to provide a full understanding of certain aspects and features of the present disclosure, although one having ordinary skill in the relevant art will recognize that these aspects and features can be practiced without one or more of the specific details, with other relationships, or with other methods. In some instances, well-known structures or operations are not shown in detail for illustrative purposes. The various embodiments disclosed herein are not necessarily limited by the illustrated ordering of acts or events, as some acts may occur in different orders and / or concurrently with other acts or events. Furthermore, not all illustrated acts or events are necessarily required to implement certain aspects and features of the present disclosure.

[0028] For purposes of the present detailed description, unless specifically disclaimed, and where appropriate, the singular includes the plural and vice versa. The word "including" means "including without limitation." Moreover, words of approximation, such as "about," "almost," "substantially," "approximately," and the like, can be used herein to mean "at," "near," "nearly at," "within 3-5% of," "within acceptable manufacturing tolerances of," or any logical combination thereof. Similarly, terms "vertical" or "horizontal" are intended to additionally include "within 3-5% of" a vertical or horizontal orientation, respectively. Additionally, words of direction, such as "top,""bottom / ' "left," "right," "above," and "below" are intended to relate to the equivalent direction as depicted in a reference illustration; as understood contextually from the object(s) or element(s) being referenced, such as from a commonly used position for the object(s) or element(s); or as otherwise described herein.

[0029] The devices, models, and methods of the present disclosure provide for an in vitro platform that permits the realization of biological assays on a human organ model of muscle, such as skeletal muscle, smooth muscle, or cardiac muscle. However, according to additional implementations, the devices, models, and / or methods of the present disclosure can apply to other organs. In addition to the parenchyma, the models of the present disclosure also contain innervation and vascularization.

[0030] The devices of the present disclosure contain a unique design in which different compartments allow tissues to grow separately, under specific biochemical conditions (e.g., coating material, hydrogel support, culture medium, external cue actuations, etc.) and develop a biomimetic architecture. The interfaces between the different compartments also permit interactions among tissues, in particular, formation of neuromuscular junctions and vascularization of the target tissues for nutrient / drug delivery.

[0031] Specific aspects of devices disclosed herein provide an in vitro platform that is a model of the human neuromuscular system and recapitulates the native tissue architectural complexity and the biological / physiological interactions, both within a healthy and a pathological context. Aspects of devices disclosed herein specific to compartmentalization and control over tissue formation permit testing with reproducibility, convenience, and accuracy of different neuro-muscular aspects.

[0032] The models disclosed herein can be applied for several purposes, but a preferred application is the in vitro reproduction of human diseases, in particular neuromuscular disorders (but also neurological or others). One important neuromuscular disorder is Amyotrophic Lateral Sclerosis (ALS) because it is a disease with no cure and no effective treatment, which is in dire need of novel and accurate human research models. To reproduce ALS, hiPSCs cells from patients, which inherited specific gene mutations (e.g., SOD1, FUS mutations), can be used in the devices and models disclosed herein. This allows for the creation of diverse ALS phenotypes and reconstitutes ALS-related events, such as TDP-43 protein aggregation, NMJ degeneration, and muscle wasting. ALS-related gene mutations can also be introduced by several other techniques, such as, for example, CRISPR / Cas9 genome editing, transgene overexpression or knockdown, chemical induction of mutations, and gene targeting with recombinant DNA vectors.

[0033] By modelling ALS (or other diseases) within the devices of the present disclosure, the morphological (e.g., fiber striation, nuclei positioning, etc.), phenotypical (e.g., TDP-43 aggregation, etc.), and functional (e.g., muscle contraction speed, frequency, force, etc.) parameters, at single cell resolution, which are associated with the pathology, can all be measured. Furthermore, similar models of healthy tissue using the devices can serve as a benchmark for direct comparison.

[0034] The establishment of a disease model using the present devices allow for screening chemical compounds and detecting their therapeutic activity (e.g., recovery of muscle function) and toxicity effects (e.g., cell viability). Thus, one application of the present devices is drug discovery and preclinical testing of disease modifying drugs for specificpathologies, particularly neuromuscular disorders. However, the present disclosure is not limited to only these uses or their equivalents but extends to any other similar use of the devices.

[0035] Referring to FIG. 1, shown is a cross-sectional plan view of a device 100, according to aspects of the present disclosure. According to one implementation, and as further disclosed below, the device 100 provides for an in vitro platform that permits the realization of biological assays on a human organ model of muscle. The device 100 serves as the housing for inducing the reproducible formation of a miniature biomimetic and functional model of innervated / vascularized tissue. The device 100 includes a body 102. The body 102 can be formed of glass or a polymeric substrate, including, for example, polydimethylsiloxane (PDMS) or plastic. In preferred embodiments, the body 102 is transparent. However, the body 102 can also be opaque.

[0036] The device 100 further includes two first channels 104. However, although two first channels 104 are shown and described, the device 100 may have only one first channel 104. Each one of the first channels 104 includes an inlet 106 from outside the device 100 and an outlet 108 to outside the device 100. According to some implementations, each one of the first channels 104 is about 50 microns to 1 mm in diameter, such as preferably about 400 microns in diameter. However, although described as having a diameter, the first channels 104 can have cross-sections other than circular, such as square or rectangular.

[0037] The inlets 106 for both of the two first channels 104 can be on the same end of the device 100. Alternatively, the inlets 106 for the two first channels 104 can be on opposite ends of the device 100. This allows for cross-flow configurations relative to the two first channels 104.

[0038] As disclosed further below, the two first channels 104 can be modified such that the two first channels 104 are vascular channels. For example, the two first channels 104 can be modified to have endothelial cells line the inner surfaces thereof, similar vasculature found in vitro. Thus, for the purpose of this disclosure, the descriptions of first channel(s) and vasculature channel(s) are interchangeable when the first channels 104 are modified as vasculature channels.

[0039] According to some aspects, the two first channels 104 can include 1 to 1,000,000 cells. The cells can be endothelial cells, such as human or animal endothelial cells. Sources of human cells include, for example, Human Umbilical Vein Endothelial cells (HUVEC), Human Umbilical Artery Endothelial cells (HUAECs), Human Lung Microvascular Endothelial cells (HLMEC), Human adipose derived Endothelial cells, Human Aortic Endothelial cells (HAEC), Human Aortic Valve Endothelial cells, Human Coronary Artery Endothelial cells (HCAEC), Human Pulmonary Artery Endothelial cells, Human Cardiac Microvascular Endothelial cells, Human Dermal Microvasculr Endothelial cells, Human Iliac Artery Endothelial cells, Human Bladder microvascular endothelial cells, Human Brain microvascular endothelial cells, Human Placental Microvascular Endothelial Cells, Human Renal Glomerular Endothelial Cells (HRGECs), Human Liver Sinusoidal Endothelial Cells (LSECs).

[0040] The two first channels 104 can be perfused with cells, such as circulating cells, including, for example, erythrocytes, non-adherant immune cells (e.g., lymphocytes, marcophages, monocytes, granulocytes, dendritic cells), and tumor cells (circulating tumor cells). The two first channels 104 can also include chemical compoundssuch as small molecules or other drugs, nanoparticles or other nano / micro assemblies constructed from polymers, particles or lipids, with the intent to carry a load or modify the biological structure, culture medium or other liquid appropriate for cell culture, and / or liquids that emulate blood in terms of viscosity and / or composition.

[0041] The device 100 further includes two second channels 110. The two second channels 110 are between the two first channels 104. However, although two second channels 110 are shown and described, the device 100 may have only one second channel 110. Each one of the second channels 110 includes an inlet 112 from outside the device 100 and an outlet 114 to outside the device 100. According to some implementations, each one of the second channels 110 is about 50 microns to 2 mm in diameter, such as preferably about 800 microns in diameter. However, although the described as having a diameter, the second channels 110 can have cross-sections other than circular, such as square or rectangular.

[0042] The inlets 112 for both of the two second channels 110 can be on the same end of the device 100. Alternatively, the inlets 112 for the two second channels 110 can be on opposite ends of the device 100. This allows for cross-flow configurations relative to the two second channels 110.

[0043] Similarly, the inlets 112 for both of the two second channels 110 can be on the same end of the device 100 as the inlets 106 for both of the two first channels 104. Alternatively, the inlets 112 for both of the two second channels 110 can be on the opposite end of the device 100 as the inlets 106 for both of the two first channels 104. Alternatively, the inlets 112 for the two second channels 110 and the inlets 106 for the two first channels 104 can have any other arrangement, such as same ends on the same side or opposite ends on the same side.

[0044] According to some aspects, the two second channels 110 can include cells that are embedded in the material within the two second channels 110 (in the case of cell-laden hydrogels) or on top of these materials. The two second channels 110 can include endothelial cells as disclosed above for the two first channels 104. The two second channels 110 can further include supporting cells for blood vessels, such as pericytes, fibroblasts, smooth muscle cells, and mesenchymal stem cells. The two second channels 110 can further include glial cells of the central nervous system, such as astrocytes, microglia, oligodendrocytes, ependymal cells. The two second channels 110 can further include glial cells of the peripheral nerve system, such as Schwann cells and Satellite cells. The two second channels 110 can further include tendon cells, such as tenocytes (specialized fibroblasts).

[0045] As disclosed further below, the two second channels 110 can be filled with a hydrogel. In which case, the two second channels 110 can be considered wall channels because they can act like walls of organ environments. The hydrogel can be just the hydrogel (i.e., a blank hydrogel), or the hydrogel can contain other components, such as, for example, collagen (type I, II , III and IV), fibrin, poly-L-lysine, poly-D-lysine, poly-L-ornithine, fibronectin, vitronectin laminin (all isoforms), gelatin, fibrin, osteopontin, elastin, tenascin, keratin, silk, hyaluronic acid, alginate or a derivative thereof, natural or synthetic polysaccharide, proteoglycans, carbon nanotubes, polylactic acid, polyglycolic acid, a polyanhydride; a poly(lactide-co-glycolide) (PLGA) polymer, a polyamino acid, a poly(alkylene oxide), a polyethylene oxide), a poly(allylamine)(PAIVI), poly(butylcyanoacrylate) (PBCA), poly (aspartic acid), a poly(acrylate), a polyester, polyhydroxybutyrate and poly-epsilon-caprolactone, a polyphosphazine, a poly(vinylalcohol), a modified styrene polymer, polyurethane, poly(4-aminomethylstyrene), a pluronic polyol, a polyoxamer, a poly(uronic acid), a poly( vinylpyrrolidone), a polypyrrole, synthetic adhesion peptides (RGD and variations of these, IKVAV and variations of these), or a combination thereof. The hydrogel also can contain various cells, such as, for example, endothelial cells, pericytes (vascular applications) and fibroblasts, tenocytes and tenoblasts (muscle anchoring that is described below), etc. The hydrogel can contain appropriate tendon-specific cells, such as tenocytes and tenoblasts (e.g., of human and animal origin). Thus, for the purpose of this disclosure, the descriptions of second channel(s) 110 and wall channel(s) are interchangeable when the second channels are modified as wall channels.

[0046] The device 100 further includes a first compartment 116 and a second compartment 118, both of which are between the two second channels 110. However, in alternative implementations, the device 100 can include more than two compartments. The first compartment 116 is generally circular in shape. The second compartment 118 also is generally circular in shape. However, according to some implementations, the first compartment 116, the second compartment 118, and / or both can have other shapes, such as triangular, square, rectangular, etc.

[0047] 0ne or both of the first compartment 116 and the second compartment 118 can have an open top. The open top allows for tissue extraction (e.g., extraction of biological material, such as proteins or DNA, for protein / gene analysis); delivery of tissue and / or fluid (e.g., culture medium) directly into the compartments 116 and 118; and external manipulation and characterization, such as electrophysiological recording with patch clamping techniques. Alternatively, one or both of the first compartment 116 and the second compartment 118 can be closed.

[0048] As disclosed further below, the first compartment 116 can be modified such that the first compartment 116 is a muscle compartment. For example, the first compartment 116 can include aligned myotubes formed of myogenic progenitor cells. According to some implementations, the muscle compartment can be lined with an extracellular matrix material. According to some implementations, the muscle compartment can include a hydrogel made of a composition of materials such as fibrin, collagen, and more. Thus, for the purpose of this disclosure, the descriptions of first compartment 116 and the muscle compartment are interchangeable when the first compartment 116 is modified as a muscle compartment.

[0049] According to some aspects, the first compartment 116 can include muscle cells, such as myoblast, cardiac, skeletal, or smooth muscle cells. The cells can be human or animal origin. The human cells can be differentiated from induced pluripotent stems cells or embryonic stem cells. The first compartment can contain other non-muscle cells present in muscle tissue such as fibroblasts and neurons. The muscle cells can be in bundles or individualized. The muscles can be aligned or in a random position configuration. In some aspects, the cell number can be from 1 to 1,000,000 cells.

[0050] As disclosed further below, the second compartment 118 can be modified such that the second compartment 118 is a neuron compartment. For example, the second compartment 118 can include neuronal tissue. In which case, the second compartment 118 is configured as a neuron compartment. The neuronal tissue can be one or more neurons, one or more neurospheres, or a combination thereof. According to some implementations, the one or moreneurons, the one or more neurospheres, or the combination thereof can contain primary human Schwann cells, stemcell derived Schwann cells (e.g., embryonic stem cells or iPSCs), or Schwann cells directly converted from other cells such as fibroblasts. Specific examples include human pluripotent stem cells (hiPSCs)-derived Schwann cells (hiPSCs- SCs). For example, the hiPSCs-derived Schwann cells can be from one or more patients with specific gene mutations. Alternatively, certain mutations can be introduced artificially, such as with genetic manipulation techniques (e.g., CRISPR and other methods). The neuron compartment can also include hydrogel, as described herein. Thus, for the purpose of this disclosure, the descriptions of second compartment 118 and the neuron compartment are interchangeable when the second compartment 118 is modified as a neuron compartment.

[0051] According to some aspects, the neurons can be neuron stem cells or fully differentiated cells, wherein these can be motor neurons, sensory neurons or interneurons of human or animal origin. Human cells can be differentiated from induced pluripotent stems cells or embryonic stem cells. Neurons can be dissociated in single cells or formed as a neurosphere. The number of neurons can range from 1 to 1,000,000 neurons. Glial cells such as Schwann cells, satellite cells, oligondendrocytes, microglia, astrocytes, and ependymal cells can be also present, either dissociated or within the neurosphere. These cells can be of human or animal origin. Glial cell number can range from 1 to 1,000,000 cells.

[0052] Referring to FIG. 2, according to some implementations, the first compartment 116 can include a plurality of grooves 200 formed on the bottom surface 202. Each groove 200 can be less than about one micron wide and less than about one micron deep. The grooves can be made of glass or plastic materials and can include a specific biochemical coating. According to certain aspects, each groove can be from about 500 nanometers (nm) to 10 microns in width. The spacing (i.e., pitch) between adjacent grooves can be regular or variable and from about 500 nm to 10 microns in width. As discussed further below, the grooves 200 assist the muscle tissue forming the desired shape and configuration. The second compartment 118 can have the same groves 200 (i.e., the same process that forms the grooves 200 on the first compartment 116 can at the same time form the grooves 200 on the second compartment 118). According to some aspects, the grooves 20 can direct axonal elongation along the groove axis. Further, certain configurations of the device 100 can simultaneously contain grooves in the first compartment and the second compartment that have different orientations (e.g., perpendicular with each other).

[0053] The grooves 200 have an aligned configuration that is intended to direct cell organization in the same direction, thus imparting tissue anisotropy. The size of the grooves influences the number of connections that the cells establish with it. Small groove sizes (e.g., about 500 nm to 3 um) are thinner than the cell diameter, and thus one cell is in direct contact with multiple grooves. This is useful to obtain aligned tissue with high density. Larger grooves (e.g., about 3 to 10 um) do not permit cells to establish contact points with multiple grooves, thus restricting cell position to one groove only. This is useful to obtain aligned tissue with lower density and individualized (such as individual muscle fibers).

[0054] Referring back to FIG. 1, and also in reference to FIG. 3, connecting the first compartment 116 and the second compartment 118 are a plurality of microchannels 120. According to some aspects, the microchannels 120 are about200 microns to 6 mm in length, 1 to 10 microns in diameter, and 120 to 180 microns in diameter, although they can be shorter or longer. According to some preferred implementations, the microchannels 120 are 8 microns in diameter, 400 microns in length, and 150 microns in height. These dimensions with respect to length allow separate compartments but also allows axons to reach the other compartment quickly (e.g., within 1-2 days axons are able to span that distance). These dimensions with respect to diameter exclude cell bodies and allow only axons to go through. The number of microchannels 120 can control the number of axons that form, as disclosed below. According to some aspects, there can be 10 to 100 microchannels 120, and preferably 50 microchannels 120. This is to ensure that enough axons cross to the other compartment to have sufficient NMJs or other type of innervation points (e.g., when using other target organs such as organoids).

[0055] According to some implementations, the microchannels 120 are generally formed near the bottoms of the first compartment 116 and the second compartment 118, which aids axons in extending through the microchannels 120 from the bottom surface of the second compartment 118.

[0056] Referring back specifically to FIG. 1, the device 100 further includes a plurality of interface regions 122. Some of the interface regions 122 connect one of the two first channels 104 with one of the two second channels 110. Some of the interface regions 122 connect one of the two second channels 110 with the first compartment 116 (or other compartments, if present). Some of the interface regions 122 connect one of the two second channels 110 with the second compartment 118 (or other compartments, if present). The interface regions 122 allow material to pass through the associated elements to which the interface regions 122 join. Accordingly, the interface regions 122 may be formed of a membrane that selectively lets certain solids (e.g., cells, proteins, etc.) and fluids through but selectively prohibits other material.

[0057] Alternatively, and referring to FIG. 4, the interface regions 122 can be formed of a plurality of pillars 300 that allow material to pass therethrough. For example, spaces 302 exists between adjacent pillars 300, and these spaces 302 are sized to selectively allow material to pass therethrough and prohibit other material from passing therethrough. For example, the length of each side can be about 50 to 250 microns (in case of the trapezoid shape, the large side is typically 2 to 1.5x larger than the small side). Adjacent pillars 300 can be spaced about 10 to 50 microns apart. The pillars 300 can have various shapes. According to some implementations, and as shown in FIG. 3, each pillar 300 can have a top-down profile shaped as a rectangle or a trapezoid, such that the pillars 300 can be shaped as a cuboid or a trapezoid prism. According to a preferred aspect, the pillars 300 have a trapezoidal shape with the large sides facing the first channel 104 and the first / second (or more) compartments. In this way, the area of the second channel 110 is maximized, whereas the space between pillars 300 is minimized. This geometrical configuration is effective in trapping material within the second channel 110 (i.e., allowing the creation of a barrier), while permitting materia l / flu id / cel I crossing through the space between the pillars 300.

[0058] According to some embodiments, tendon-specific cells— such as tenocytes and tenoblasts (e.g., of human and animal origin)— and / or a tendon-mimicking material can attach to the pillars 300. The tendon-specific cells and / or tendon-mimicking material allow for anchoring muscle cells, fibers, and / or sheets within the first compartment 116to the pillars 300. This arrangement allows long-term tissue stabilization of the muscle cells, fibers, and / or sheets within the first compartment 116. A hydrogel can be introduced into the appropriate one or more of the two second channels 110 for providing and attaching the tendon-specific cells and / or the tendon-mimicking material to the desired pillars 300 (e.g., interface regions 122).

[0059] The device 100 can be formed via various techniques, such as a lithography process or by 3D printing. According to some implementations for the lithography process in particular, the device 100 can be formed by first fabricating a master mold via a 2-step lithography process. From this master mold, PDMS microfluidic parts can be produced by replica molding. These microfluidic parts can then be further bonded to a bottom support made of glass or a polymeric substrate (with or without nano / micro features). In a preferred embodiment, the body is made of a transparent material to allow for visual inspection of the elements of the devices. According to some implementations, the transparency allows for neuronal activity to be traced with imaging-based techniques, such as calcium imaging, as just one example. According to further implementations, the transparency allows for muscle contraction parameters (e.g., speed, force, etc.) to be recorded, such as by using a brightfield microscopy. Further, a semi-automated particle image velocimetry (PIV) analysis algorithm can be used to measure the parameters.

[0060] Muscle formation

[0061] Upon forming the device 100, the first compartment 116 can be modified into the muscle compartment by adding muscle tissue. The muscle tissue can be various different types of muscle tissue, such as skeletal, cardiac, smooth, and the like. The muscle compartment can also be pre-coated with an extracellular matrix-like material (e.g., Matrigel® by Corning Life Sciences, GelTrex™ by Thermo Fisher Scientific, etc.). According to some implementations, the muscle tissue can be formed by differentiating hiPSCs into myogenic progenitors cells that are added to the muscle compartment. However, alternatively, other types of cells, such as primary or stem-cell derived (e.g., embryonic stem cell-derived) of human or other animal origin (e.g., rat) cells can be used. The cells are allowed to grow and differentiate into myotubes through specific culture medium conditions (e.g., specific medium and hydrogel addition on top of the cells). The first compartment 116 can contain other non-muscle cells present in muscle tissue, such as, for example, fibroblasts and neurons. The muscle cells can be in one or more bundles, fibers, or sheets, or individualized. The muscle cells can range from 1 cell to 1,000,000 cells, for example. The muscles cells can be aligned or in a random position configuration. For example, the myotubes can further align because of the grooves 200 present in the body. Cell alignment occurs via an anisotropic contact guidance provided by the parallel grooves 200 on which the cells are cultured. Overtime, a muscle sheet of aligned and contractile myotubes is formed.

[0062] For example, and referring to FIG 5, the first compartment 116 is shown configured as the muscle compartment. The first compartment 116 includes a muscle sheet 500 of aligned and contractile myotubes. The muscle sheet 500 aligns with the grooves 200 (FIG. 2) in the first compartment 116. According to some implementations, the muscle sheet 500 can be one cell thick, which allows for imaging of the individual cells.

[0063] Neuronal tissue formation

[0064] Upon forming the device 100, the second compartment 118 can be modified into the neuron compartment by adding neuronal tissue. Neurons can be dissociated in single cells or formed as a neurosphere. The neuronal tissue can be one or more neurons, one or more neurospheres, or a combination thereof, such as peripheral neurons (e.g., sensory or motor), central nervous system (CNS) neurons, and the like. The number of neurons can range from 1 to 1,000,000 neurons. The neuronal tissue can be formed from human motor neurons differentiated from human induced pluripotent stem cells (hiPSCs) or embryonic stem cells. According to some implementations, neuronal tissue may or may not contain hiPSCs-derived Schwann cells (hiPSCs-SCs), depending if the neurons are intended to be myelinated. According to some aspects, the neurons can be neuron stem cells or fully differentiated cells, wherein these can be motor neurons, sensory neurons or interneurons of human or animal origin. Glial cells such as Schwann cells, satellite cells, oligondendrocytes, microglia, astrocytes, and ependymal cells can be also present, either dissociated or within the neurosphere. These cells can be of human or animal origin. Glial cell number can range from 1 to 1,000,000 cells.

[0065] According to some implementations, and as disclosed above, the neuronal tissue can be modified to express a light-sensitive protein, such as a blue light-sensitive protein (e.g., channel rhodopsin-2), which allows for light to trigger action potentials with light impulses. For example, the light-sensitive protein allows for spatiotemporal control via the light impulses. The light impulses can be provided to the device 100 according to various different approaches, such as having the device near a light emitting diode (LED) that can provide tunable LED firing patterns.

[0066] The neuron compartment can be coated with poly-L-lysine, poly-D-lysine, poly-L-ornithine, fibronectin, vitronectin laminin (all isorforms), collagen (type I, II , III and IV), gelatin, fibrin, osteopontin elastic, tenascin, keratin, silk, hyaluronic acid, alginate or a derivative thereof, natural or synthetic polysaccharide, proteoglycans, carbon nanotubes, polylactic acid, polyglycolic acid, a polyanhydride; a poly(lactide-co-glycolide) (PLGA) polymer, a polyamino acid, a poly(alkylene oxide), a polyethylene oxide), a poly(allylamine)(PAIVI), Poly(butylcyanoacrylate) (PBCA), Poly (aspartic acid), a poly(acrylate), a polyester, polyhydroxybutyrate and poly-epsilon-caprolactone, a polyphosphazine, a poly(vinyl alcohol), a modified styrene polymer, polyurethane, poly(4-aminomethylstyrene), a pluronic polyol, a polyoxamer, a poly(uronic acid), a poly(vinylpyrrolidone), a polypyrrole. Synthetic adhesion peptides (RGD and variations of these, IKVAV and variations of these)., or a combination thereof. The coating materials can partially imitate the native extracellular matrix and permit the adhesion of the neuron cells, as well as the extension of neurites through the compartment and microchannels. Further, in addition to the neuronal tissue, the neuron compartment can include a hydrogel (e.g., fibrin, collagen, etc., as described herein) to allow 3D neurite outgrowth.

[0067] Vascular tissue formation

[0068] Upon forming the device 100, the first channels 104 can be modified into the vascular channels by addingvascular tissue. The vascular tissue can be any type of vascular tissue, such as endothelial cells of any source (e.g., dermal, umbilical, lung, brain, etc.), mural cells (e.g., pericytes, fibroblasts, etc.), and the like. According to some implementations, the vascular tissue can be formed within the vascular channel by filling the wall channel 110 with a collagen / fibrin hydrogel or other materials. This hydrogel may be blank, thus acting as a simply a wall. Alternatively, the hydrogel can be embedded with endothelial cells, pericytes, fibroblasts, or other vascular-related cells, which produces three-dimensional microvessels within it. A collagen solution (e.g., 1 milligram per milliliter (mg / ml)) can be added to the vascular channel to coat it. However, other coatings or chemical / physical modifications can also be implemented in order to permit and enhance endothelial cell adhesion and proliferation After that, human endothelial cells (e.g., HUVECs, hiPSCs-ECs, HMVECs, etc.) can be added to the vascular channel. A seeding solution containing 0.5% gelatin within appropriate vascular culture medium can be added to the vascular channel to improve cell adhesion and retention within the channel. After that, the cells were cultured within an appropriate culture medium.

[0069] Based on the vascular channels having inlets and outlets, the vascular channels can be connected to a perfusion pump through tube connections at the inlets / outlets for delivering medium and subjecting cells therein to shear stress, simulating the physiological environment.

[0070] After forming the vascular channel, medium flow through the vascular channel and can contain one or more drugs, agents (e.g., nanoparticles), or other molecules of interest (e.g., growth factors) to be delivered to the other tissues. Medium flowing through the vascular channels can also contain cells (e.g., circulating tumor cells, red blood cells, immune cells, ...). The transparency of the device allows for the tissue vascularization to be evaluated with fluorescent microscopy. Based on the foregoing, the devices and models of the present disclosure allows for the formation of vascular tissue that resembles the native vascular architecture by depicting its multi-scale dimension.

[0071] Referring to FIG. 6, within the wall channels, microvessels 600 formed by the same endothelial cells and stabilized by human pericytes can form. These microvessels 600 can anastomose with the vascular channel to conduct perfused fluid from the vascular channel towards target tissue. Current technologies do not show this multiscale vascular dimension for targeted tissue perfusion and do not contain mural cells (e.g., pericytes or other mural cell types).

[0072] Neuromuscularjunction formation

[0073] 0nce the device 100 is prepared as discussed above, the device 100 acts as a human neuro-muscular model. Referring to FIG. 7, once the device 100 is modified as disclosed above with respect to at least the muscle compartment and the neuron compartment, axons 700 grow from the neuronal tissue (e.g., neurospheres) 702 and migrate into the muscle compartment 116 via the plurality of microchannnels 120. These microchannels 120 are specifically designed to only allow the axons 700 (unmyelinated or myelinated) to cross, which prevents neuronal cell bodies from crossing. This allows for the neuronal tissue 602 to develop in a biomimetic manner, with the axons 700 emanating from a soma and, optionally, further myelinated by hiPSCs-SCs. Specifically, myelin is an insulatinglayer, formed by SCs enwrapping of axons, which permits faster nerve conduction and is necessary for appropriate nerve and neuromuscular junction function. Myelin integrity is affected in several pathologies, such as multiple sclerosis. The devices and models of the present disclosure allow for neuronal tissue that is myelinated by human SCs. This results in the formation of a controlled number of myelinated neurites 700 that reach the muscle compartment and form mature neuromuscular junctions. Further, the NMJs are formed of three different cell types: stem cells, neurons, and muscle cells. As disclosed above, the number of axons 700 can be controlled by the number of microchannels 120 formed between the muscle compartment and the neuron compartment.

[0074] By stimulating optogenetically modified neurons with blue-light, as disclosed above, the neurons fire an electrical signal that travels through the axons to the NMJs and induces muscle fibers contraction. These contractions allow for on-demand external control of muscle activity to promote a superior maturation of the NMJ and muscle tissue. However, according to some implementations, besides optogenetic stimulation, the neurons can be stimulated with other means, such as electrodes or chemicals.

[0075] The devices, models, and methods disclosed herein have many technical advantages over the art. For example, they allow for determining a novel compound / therapeutic target for a clinical solution for currently uncured / untreated neuromuscular diseases, such as ALS. Moreover, the high cell resolution provided by the devices and models allows for the extraction of precise data regarding intracellular biochemical compositions and localization. This is achieved, in part, because the neuromuscular tissue is formed as a thin three dimensional layer that allows the visualization of muscle cells and neuromuscular junctions details with high resolution. The devices, models, and methods also allow for protocols to obtain mature motor neurons and muscle fibers that emulate the function and physiology of the neuromuscular junction at unprecedent levels. The devices and models are easy to construct and operate, and are able to generate identical replicas at each batch.

[0076] The devices, models, and methods allow for disease modeling by reproducing disease-associated mutations, such as by artificial genetic manipulations by gene knock in / gene knock out (e.g., with CRISPR) and direct use of cells from patients (e.g., iPSCs obtained from diseased patients that are subsequently differentiated). The devices, models, and methods allow for disease modeling by exposure to disease-relevant physicochemical stimuli, such as by toxins and chemicals (e.g., neuron exposure to beta-amyloid peptide in an Alzheimer model); cytokine stimuli (e.g., simulation of inflammation with pro-inflammatory cytokines); metabolic changes (e.g., glucose deprivation or overexposure (e.g. hyperglycaemic conditions to emulate diabetes)); oxygen levels (e.g., low oxygen levels to reproduce ischemia); virus and infections (e.g., viral introduction to study viral infection mechanism and virus pathogenesis); serum or growth factor deprivation (e.g., stress induction via removal of critical factors); and radiation exposure (e.g., model certain aspects of cancer related to ionizing radiation). The devices, models, and methods also allow for adding cells / tissue that is associated with a disease, such as cancer cell lines and patient derived ex vivo samples.

[0077] The devices, models, and methods allow for modeling various different diseases, such as for neuromuscular disorders (e.g., Amyotrophic Lateral Sclerosis (ALS), Muscular Dystrophy (e.g. Duchenne muscular dystrophy),Myasthenia Gravis, Spina bifida, Charcot-Marie-Tooth Disease (CMT), spinocerebellar ataxia, Spinal Muscular Atrophy (SMA), Guillain-Barre Syndrome, Friedreich's Ataxia, Myotonic Dystrophy, Lambert-Eaton Myasthenic Syndrome (LEMS), Inclusion Body Myositis (IBM), Congenital Myasthenic Syndromes (CMS), monomelic amyotrophy; Neurological disorders (e.g., Alzheimer's disease, Parkinson's disease, Multiple Sclerosis, Neuropathy, Huntington's disease, Batten Disease, Alpers' Disease, Lewy Body Disease, Gerstmann-Straussler-Scheinker Disease, Kuru, Leigh's Disease); vascular disorders (e.g., Atherosclerosis, Hemangioma, Kawasaki Disease, Arteritis, Fibromuscular Dysplasia (FMD)); and cancer (e.g., breast cancer, Schwannoma).

[0078] Assays can be conducted within the device or outside after tissue removal. Such assays include, for example, omics analysis (e.g., genomics, transcriptomics, metabolomics and proteomics); sample imaging with microscopic techniques of live and fixed cells (e.g., immunostained); electrophysiology measurements with use of external actuators (e.g., electrodes, needles, etc.) or chemical dies (ex. Calcium sensors); and toxicity assays, which are a series of experiments that assess the tissue's viability after drug exposure (e.g., cell metabolic activity; Mitochondrial function; Oxidative stress; Apoptosis; Cell membrane activity).

[0079] The devices can also provide for screening of angiogenic / anti-angiogenic compounds. For example, the devices can be used for a method to identify compounds with angiogenic or antiangiogenic properties with a device as disclosed herein. The first channel 104 can be a hollow vascular tube. The second channel 110 can be filled with a blank hydrogel (e.g., no cells). Compounds to be screened are added to the first and / or second compartment and / or perfused through the first channel. The assay can include identifying and quantifying sprouts from the vascular tube toward the second channel using microscopic techniques. A compound is considered pro angiogenic when induces higher formation of sprouts compared to a control sample. A compound is considered anti angiogenic when induces lower formation of sprouts compared to a control sample. The viability of tissue could be assayed, including cell metabolic activity, mitochondrial function, oxidative stress, apoptosis, cell membrane activity.

[0080] The devices can also provide for screening of pro / anti vascularization compounds to target organs / tissues. For example, the devices as disclosed herein can be used for a method to identify compounds that can have pro / anti vascularization properties within organoids, tissue explants or other assembly cells.

[0081] The device for this configuration consists of a first channel is composed of a hollow vascular tube, and a second channel is composed of a hydrogel with or without cells (cells of the second channel). A target tissue is placed at the first and / or second compartment. The target tissue can be embedded in a hydrogel containing endothelial cells and / or mural cells. A compound is added on the first / second compartments or delivered through the first channel or second channel. The vascularization is assessed by identifying and quantifying the presence of vascular tubes within / around the target tissue using microscopic techniques. This can be done in the device or outside, after tissue removal. A compound is considered to be pro vascularization when the amount of blood vessels at the target organ is superior to control samples. A compound is considered to be anti-vascularization when the amount of blood vessels at the target organ is inferior to control samples. Biochemical assays to determine the biochemical, cellular, genetic, molecular, or morphological characteristic of the tissues. Series of experiments thatassess the tissue's viability can include cell metabolic activity, mitochondrial function, oxidative stress, apoptosis, and cell membrane activity.

[0082] The devices can also provide for screening of pro / anti innervation compounds to target organs / tissues. For example, the devices as disclosed herein can be used for a method to identify compounds that can have pro / anti innervation properties within organoids, tissue explants or other assembly of cells. The device includes a first channel that can be vascularized, a first compartment that has a neuronal population, a second compartment has a target tissue, and a compound is added through the first / second channel or first / second compartment. Assays allow for innervation assessed by identifying and quantifying the innervation contact points and nerve fibers in the target tissue by means of microscopic techniques; ranges of methods to detect cell's biochemical, cellular, genetic, molecular, or morphological characteristic; functional measurements of neuron activity (e.g., electrophysiology) and toxicity assays for experiments that assess the tissue's viability, including cell metabolic activity, mitochondrial function, oxidative stress, apoptosis, and cell membrane activity.

[0083] The devices can also provide for a method to detect compounds that can have an effect on a cancer cell biochemical, cellular, genetic, molecular, or morphological characteristic. For example, the devices as disclosed herein can include a first channel is vascularized and can contain cancer cells in circulating or adherent form, a second channel is filled with a blank hydrogel or a hydrogel loaded with endothelial (and mural cells), and the first and / or second compartments contain a cancer cell population. The cancer cells can be dissociated or in spheroids. The assays can involve a drug being delivered through the first channel and / or first and second compartments. Microscopic and biochemical assays to detect an alteration in cancer cell proliferation, viability, migration, death / apoptosis, intravasation and extravasation.

[0084] Although the disclosed embodiments have been illustrated and described with respect to one or more implementations, equivalent alterations and modifications will occur or be known to others skilled in the art upon the reading and understanding of this specification and the annexed drawings. In addition, while a particular feature of the invention may have been disclosed with respect to only one of several implementations, such feature may be combined with one or more other features of the other implementations as may be desired and advantageous for any given or particular application.

[0085] While various embodiments of the present disclosure have been described above, it should be understood that they have been presented by way of example only, and not limitation. Numerous changes to the disclosed embodiments can be made in accordance with the disclosure herein, without departing from the spirit or scope of the disclosure. Thus, the breadth and scope of the present disclosure should not be limited by any of the above-described embodiments. Rather, the scope of the disclosure should be defined in accordance with the following claims and their equivalents.

Claims

CLAIMS1. A device comprising: a body; at least one first channel formed within the body, the at least one first channel having an inlet from outside the device and an outlet to outside the device; a first compartment formed within the body; a second compartment formed within the body; at least one second channel formed within the body and separating the first compartment and the second compartment from the at least one first channel, the at least one second channel having an inlet from outside the device and an outlet to outside the device; a plurality of microchannels connecting the first compartment and the second compartment; and a plurality of interface regions, each interface region of the plurality of interface regions connecting the at least one first channel with the at least one second channel, the at least one second channel with the first compartment, or the at least one second channel with the second compartment.

2. The device of claim 1, wherein the first compartment has an open top.

3. The device of claim 1, wherein the second compartment has an open top.

4. The device of claim 1, wherein the plurality of microchannels are about 1 to 10 microns in diameter.

5. The device of claim 1, wherein the at least one second channel includes two second channels, and the first compartment and the second compartment are between the two second channels in the body.

6. The device of claim 5, wherein the at least one first channel includes two first channels, and the two second channels are between the two first channels in the body.

7. The device of claim 1, wherein the first compartment includes a plurality of grooves on a first surface.

8. The device of claim 7, wherein each groove of the plurality of grooves is about 500 nanometers to 10 microns in wide and about 500 nanometers to 10 microns deep.

9. The device of claim 8, wherein a groove pitch of the plurality of grooves is about 500 nm to 10 pm.

10. The device of claim 1, wherein the first compartment and the second compartment includes a plurality of grooves on a first surface.

11. The device of claim 1, wherein each interface region of the plurality of interface regions is a pillar region formed of a plurality of pillars that allow material to pass between adjacent pillars.

12. The device of claim 11, wherein each pillar of the plurality of pillars is shaped as a trapezoid or a cuboid.

13. The device of claim 1, wherein the at least one first channel is about 50 microns to 1 mm in diameter.

14. The device of claim 1, wherein the at least one second channel is about 50 pm to 2 mm in diameter.

15. The device of claim 1, wherein the body is formed of polydimethylsiloxane (PDMS), glass, plastic, or apolymeric substrate.

16. The device of claim 15, wherein the body is transparent.

17. The device of claim 1, wherein the body is bonded to a bottom substrate via a physical method or a chemical method.

18. The device of claim 17, wherein the bottom substrate is formed of PDMS, glass, plastic, or a polymeric substrate.

19. The device of claim 17, wherein the bottom substrate includes electrodes.

20. The device of claim 1, wherein the first compartment, the second compartment, or both are more than one compartment such that the device includes three or more compartments.

21. A human neuro-muscular model comprising: the device of claim 1; endothelial cells lining the at least one first channel such that the at least one first channel is configured as at least one vascular channel; aligned myotubes formed of myogenic progenitor cells within the first compartment such that the first compartment is configured as a muscle compartment; and neuronal tissue within the second compartment such that the second compartment is configured as a neuron compartment.

22. The model of claim 21, wherein one or more of the at least one vascular channel, the muscle compartment, the neuron compartment, or a combination thereof model one or more neuromuscular disorders, one or more neurological disorders, one or more vascular disorders, one or more cancers, or a combination thereof.

23. The model of claim 22, wherein the one or more neuromuscular disorders include amyotrophic lateral sclerosis, muscular dystrophy, myasthenia gravis, spina bifida, Charcot-Marie-Tooth Disease, spinocerebellar ataxia, spinal muscular atrophy, Guillain-Barre Syndrome, Friedreich's Ataxia, myotonic dystrophy, Lambert- Eaton Myasthenic Syndrome, inclusion body myositis, one or more congenital myasthenic syndromes, or monomelic amyotrophy.

24. The model of claim 22, wherein the one or more neurological disorders include Alzheimer's disease, Parkinson's disease, multiple sclerosis, neuropathy, Huntington's disease, Batten disease, Alpers' disease, Lewy Body disease, Gerstmann-Straussler-Scheinker disease, Kuru, or Leigh's disease.

25. The model of claim 22, wherein the one or more vascular disorders include atherosclerosis, hemangioma, Kawasaki disease, arteritis, or fibromuscular dysplasia.

26. The model of claim 22, wherein the one or more neuromuscular disorders, the one or more neurological disorders, the one or more vascular disorders, the one or more cancers, or the combination thereof are modeled by reproducing disease-associated mutations, exposure associated with disease-relevantphysicochemical stimuli, adding cells and / or tissue that are associated with a disease, or a combination thereof.

27. The model of claim 26, wherein the exposure associated with disease-relevant physicochemical stimuli includes exposure to toxins and / or chemicals, cytokine stimuli, metabolic changes, exposure to varying oxygen levels, exposure to at least one virus and / or at least one infection, serum or growth factor deprivation, radiation, or a combination thereof.

28. The model of claim 21, wherein one or more of the at least one first channel, the at least one second channel, the first compartment, and the second compartment are coated with one or more of poly-L-lysine, poly-D-lysine, poly-L-ornithine, fibronectin, vitronectin laminin, gelatin, fibrin, osteopontin, elastin, tenascin, keratin, silk, hyaluronic acid, alginate or a derivative thereof, natural or synthetic polysaccharide, proteoglycans, carbon nanotubes, polylactic acid, polyglycolic acid, a polyanhydride, a poly(lactide-co- glycolide) polymer, a polyamino acid, a poly(alkylene oxide), a polyethylene oxide), a poly(allylamine), poly(butylcyanoacrylate), poly (aspartic acid), a poly(acrylate), a polyester, polyhydroxybutyrate and poly- epsilon-caprolactone, a polyphosphazine, a poly(vinyl alcohol), a modified styrene polymer, polyurethane, poly(4-aminomethylstyrene), a pluronic polyol, a polyoxamer, a poly(uronic acid), a poly(vinylpyrrolidone), a polypyrrole, synthetic adhesion peptides (RGD and variations of these, IKVAV, or a combination thereof.

29. The model of claim 21, wherein the neuronal tissue is formed from human motor neurons differentiated from human induced pluripotent stem cells (hiPSCs) or embryonic stem cells.

30. The model of claim 21, wherein the neuronal tissue is one or more neurons, one or more neurospheres, or a combination thereof.

31. The model of claim 21, wherein the one or more neurons, the one or more neurospheres, or the combination thereof contains primary human Schwann cells, stem-cell derived Schwann cells (e.g., embryonic stem cells or iPSCs), Schwann cells directly converted from other cells such as fibroblasts, or combinations thereof.

32. The model of claim 31, wherein the hiPSCs- the bottom substrate includes electrodes.

33. The model of claim 21, wherein the neuronal cells can contain one or more specific gene mutations.

34. The model of claim 21, wherein the at least one second channel is filled with a hydrogel.

35. The model of claim 34, wherein the hydrogel contains collagen, fibrin, endothelial cells, pericytes, or a combination thereof.

36. The model of claim 21, wherein the neuron compartment includes a hydrogel or a mixture of hydrogels or other materials.

37. The model of claim 21, wherein one or more axons extend from the one or more neurons, the one or more neurospheres, or the combination thereof into the plurality of microchannels.

38. The model of claim 37, wherein the one or more axons form neuromuscular junctions with the myotubes.

39. The model of claim 38, wherein the one or more neurons, the one or more neurospheres, or thecombination thereof are optogenetically modified neurons to cause an electrical signal that travels through the one or more axons to the neuromuscular junctions and induce muscle fibers contraction when the one or more neurons, the one or more neurospheres, or the combination thereof are exposed to light of a predetermined wavelength.

40. The model of claim 39, further comprising an array of pillars in the interface between the muscle compartment and the at least one second channel configured to anchor the aligned myotubes.

41. The model of claim 40, wherein the muscle fibers are anchored to the array of pillars.

42. The model of claim 40, wherein the myotubes form a muscle sheet and the sheet is anchored to the array of pillars.

43. The model of claim 42, wherein the muscle sheet aligns with a plurality of grooves on a first surface of the first compartment.

44. The model of claim 43, wherein the muscle sheet is one cell thick to allow for imaging of individual cells.

45. A method of identifying a compound, a dosage of a compound, a dosing regime, or combination thereof, that effects activity of innervated muscle fibers, the method comprising: making a first measurement of a biochemical, cellular, genetic, molecular, or morphological characteristic of neuronal cells, muscle cells, innervated muscle fibers, or combination thereof; contacting the neuronal cells, muscle cells, innervated muscle fibers, or combination thereof with one or more amounts, for one or more dosing regimes, of a test compound, or a combination thereof; making a second measurement of the biochemical, cellular, genetic, molecular, or morphological characteristic of the neuronal cells, muscle cells, or innervated muscle fibers, or combination thereof; and selecting the compound, the dosage of the compound, the dosing regime, or the combination thereof that effects neuronal activity, muscular activity, or a combination thereof if the compound improves or worsens the biochemical, genetic, cellular, molecular, or morphological characteristic of the neuronal cell, muscle cells, or innervated muscle fibers, or combination thereof.

46. The method of claim 45, further comprising delivering the test compound directly through a first compartment, a second compartment, and / or a first channel of a device including the first compartment, the second compartment, and / or the first channel.

47. The method of claim 46, wherein the first channel is vascularized and the test compound is perfused through the first channel.

48. The method of claim 46, wherein the test compound is administered in a free form or as a payload of a delivery agent.

49. The method of claim 45, further comprising performing one or more assays on the neuronal cells, the muscle cells, the innervated muscle fibers, or the combination thereof.

50. The method of claim 49, wherein the one or more assays include one or more assays on cell metabolic activity, mitochondrial function, oxidative stress, apoptosis, cell membrane activity.

51. A method for identifying compounds that have pro / anti vascularization properties within organoids, tissue explants, or other assembly cells, the method comprising: providing a device having: a first channel formed of a hollow vascular tube; and a second channel having a hydrogel; a first compartment in fluid communication with one or both of the first channel and the second channel, the first compartment including a target tissue embedded in a hydrogel containing endothelial cells and / or mural cells; providing a compound in the first compartment and delivered through the first channel or second channel; identifying and quantifying presence of vascular tubes within or around the target tissue using microscopic techniques; and determining that the compound is pro-vascularization when an amount of blood vessels at the target tissue is superior to control samples or determining that the compound is anti-vascularization when the amount of blood vessels at the target tissue is inferior to control samples.

52. A method to identify one or more compounds that can have pro- or anti-innervation properties within organoids, tissue explants, and / or other assemblies of cells, the method comprising: providing a device, the device including: a first channel that is vascularized; a second channel; a first compartment that has a neuronal population; and a second compartment that has a target tissue; wherein the first compartment, the second compartment, or a combination thereof are in fluid communication with the first compartment, the second compartment, or a combination thereof; providing a compound through the first channel, the second channel, the first compartment, and / or the second compartment; and assessing innervation by identifying and quantifying innervation contact points and nerve fibers in the target tissue by means of microscopic techniques.

53. A method of detecting an effect on a biochemical, cellular, genetic, molecular, and / or morphological characteristic of a cancer, the method comprising: providing a device, the device including:a first channel that is vascularized and contains one or more cancer cells in circulating or adherent form; a second channel that is filled with a blank hydrogel or a hydrogel loaded with endothelial cells; and a first compartment or a second compartment that contains a cancer cell population that is dissociated or in spheroids, the first compartment, the second compartment, or a combination thereof being in fluid communication with the first channel, the second channel, or a combination thereof; delivering a drug through the first channel, the first compartment, or the second compartment; and performing one or more microscopic and / or biochemical assays to detect an alteration in cancer cell proliferation, viability, migration, death / apoptosis, intravasation, and / or extravasation in the one or more cancer cells, the cancer cell population, or a combination thereof.

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