Artificial skeletal muscle tissue

Abstract

Embodiments described herein generally relate to three-dimensional skeletal muscle tissue comprising a hydrogel, a plurality of cells including skeletal muscle cells, and two or more anchors, at least one of which is a flexible anchor, characterized by one or more contractions in response to electrical and / or chemical stimuli.

Description

[Technical Field]

[0001] The present disclosure relates generally to artificial skeletal muscle tissue and uses thereof. [Background technology]

[0002] Skeletal muscle is one of the three major muscle types and is a form of striated muscle tissue that is under voluntary control of the somatic nervous system. Most skeletal muscles are attached to bones by bundles of collagen fibers known as tendons.

[0003] Skeletal muscle diseases encompass a variety of disorders, including those caused by genetic mutations, such as Duchenne muscular dystrophy (Yedigaryan L, Cells 2021), as well as functional impairments secondary to aging, inflammation, cancer (cachexia), trauma, and infection (Salucci et al., 2021; Rudroff et al., 2021). Recently, clinical studies have reported fatigue or muscle weakness as one of the long-term symptoms following COVID-19 infection (Huang C. et al., Lancet, 2021). Regardless of etiology, skeletal muscle disorders cause muscle degeneration and reduce patients' quality of life (Constantin-Teodosiu D et al., 2021). Unfortunately, to date, the underlying pathology of many skeletal muscle-related diseases has not been fully elucidated, resulting in limited treatment options. This clinical challenge is primarily due to the limited predictive value / predictive capabilities of currently available models, specifically 2D in vitro and animal models.

[0004] A major limitation of animal models is their inability to reliably recapitulate human physiology due to species differences, resulting in inaccurate predictions of drug response and poor clinical application (Hay T et al., 2014). Further disadvantages include the high cost and labor-intensive nature of animal experiments (Lowe DA, Alway SE, 2002; Sztretye ​​M et al., 2020). While in vitro 2D models of skeletal muscle are less labor-intensive than animal models, their limitations include the difficulty of maintaining cultures for long periods, resulting in immaturity characterized by a lack of cell alignment or tissue structure consistent with mature, native muscle tissue and a lack of mature contractile responses (Guo X et al., 2014; Cooper ST et al., 2004). Additionally, to date, no methodology exists for assessing 2D in vitro skeletal muscle contractility. The inability to fully recapitulate mature skeletal muscle organization and function in 2D inevitably limits their usefulness for new insights into muscle pathophysiology and drug discovery.

[0005] This clinical need prompts the development of new in vitro methods that may better model the complex pathophysiology of human skeletal muscle and thus facilitate the development of new therapeutic approaches. Summary of the Invention

[0006] FIELD OF THE INVENTION The embodiments described herein generally relate to three-dimensional (3D) artificial skeletal muscle tissue, methods of producing the tissue, and methods of using the tissue.

[0007] One aspect of the present disclosure relates to a three-dimensional skeletal muscle tissue comprising a hydrogel, a plurality of cells including skeletal muscle cells, and two or more anchors, wherein the skeletal muscle tissue is characterized by one or more contractions in response to electrical and / or chemical stimuli, and at least one of the anchors is a flexible anchor.

[0008] In some embodiments, the plurality of cells further comprises fibroblasts.

[0009] In some embodiments, the ratio of fibroblasts to skeletal muscle cells is about 1:5 to 1:50.

[0010] In some embodiments, the skeletal muscle cells comprise human skeletal muscle cells.

[0011] In some embodiments, the hydrogel comprises collagen or a collagen derivative, intestinal submucosa or a derivative thereof, cellulose or a cellulose derivative, proteoglycan, heparin sulfate, chondroitin sulfate, keratin sulfate, hyaluronic acid, elastin, fibronectin, laminin, fibrin, chitosan, alginate, Matrigel®, Geltrex®, agarose, decellularized extracellular matrix, polyethylene glycol or a derivative thereof, silicone or a derivative thereof, or a combination thereof. In some embodiments, the hydrogel comprises collagen or a collagen derivative. In some embodiments, the hydrogel comprises Matrigel®.

[0012] In some embodiments, the collagen comprises type I collagen, type III collagen, type IV collagen, type V collagen, type XI collagen, type XII collagen, or a combination thereof.

[0013] In some embodiments, at least a portion of the cells are encapsulated or embedded within the hydrogel.

[0014] In some embodiments, at least 50% of the cells are encapsulated or embedded within the hydrogel.

[0015] In some embodiments, the 3D skeletal muscle tissue further comprises fibrinogen and / or thrombin.

[0016] In some embodiments, the skeletal muscle tissue has about 30,000 to about 1,000,000 cells.

[0017] In some embodiments, the skeletal muscle tissue is about 0.1 mm 3 ~about 2.5mm 3 It has a volume of

[0018] In some embodiments, the skeletal muscle tissue comprises an A-band, an I-band, a Z-line, an M-line, an H-zone, or a combination thereof.

[0019] In some embodiments, the skeletal muscle tissue comprises striations, elongated nuclei, sarcomeres, or a combination thereof.

[0020] In some embodiments, the skeletal muscle tissue comprises acetylcholine receptors, slow-twitch fibers, fast-twitch fibers, or a combination thereof.

[0021] In some embodiments, the expression levels of genes associated with skeletal muscle tissue maturation (e.g., myosin isoform ratios), genes associated with calcium handling, and / or genes associated with sarcomeric proteins are substantially the same as those in native human skeletal muscle tissue. Upregulation of ablation-induced genes (e.g., ESRRG, NFATC2) and downregulation of developmental and fatigable contraction genes (e.g., MYH8, MYH1) can also be used as markers of mature skeletal muscle tissue.

[0022] In some embodiments, the one or more contractions generate a twitch force and / or a tetanic force.

[0023] In some embodiments, the chemical stimulus comprises acetylcholine, adenosine triphosphate, 4-chloro-m-cresol, or a combination thereof.

[0024] In some embodiments, the skeletal muscle tissue is characterized by transient changes in intracellular calcium concentration in response to electrical and / or chemical stimuli.

[0025] In some embodiments, the skeletal muscle tissue is characterized by shortened action potentials with significant hyperpolarization.

[0026] In some embodiments, the skeletal muscle tissue is held under tension between the anchors.

[0027] In some embodiments, the anchors can be adjusted to alter the tension in the skeletal muscle tissue.

[0028] In some embodiments, the flexible anchor is an elastic sensing element. In some embodiments, two or more anchors are elastic sensing elements.

[0029] In some embodiments, the flexible anchor comprises a synthetic polymer, a biopolymer, or a combination thereof.

[0030] In some embodiments, the polymer is degradable.

[0031] In some embodiments, the polymer is non-degradable.

[0032] In some embodiments, the flexible anchor comprises a polymer selected from the group consisting of polylactic acid, poly(lactic-co-glycolic acid), poly(caprolactone), polyglycolide, polylactic acid, polyhydroxobutyric acid, polyhydroxyalkanoic acid, chitosan, hyaluronic acid, poly(2-hydroxyethyl methacrylate), poly(ethylene glycol), poly(L-lactic acid) (PLA), poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PMMA), poly(glycerol sebacate), poly(octamethylene maleic (anhydride) citric acid) (POMaC), citric acid-free POMaC, poly(ε-caprolactone), polyurethane, silk, and combinations thereof.

[0033] In some embodiments, the polymer comprises POMaC.

[0034] In some embodiments, the flexible anchor has an elasticity of about 10 kPa to 0.8 MPa.

[0035] In some embodiments, at least one of the anchors is in the form of a polymer wire. In some embodiments, two or more anchors are in the form of polymer wires.

[0036] One aspect of the present disclosure relates to a tissue system comprising the 3D skeletal muscle tissue described herein and a bioreactor, the bioreactor comprising: a device having a well configured to grow 3D skeletal muscle tissue from cells seeded in the well, the well having a bottom; and two or more anchors positioned across the well such that a gap exists between the anchor and the bottom of the well, at least one of the anchors being flexible, the anchors configured to allow attachment of the 3D skeletal muscle tissue formed therebetween, thereby suspending the 3D skeletal muscle tissue above the bottom of the well, the flexible anchor configured to deform in response to contractile forces exerted on the flexible anchor by the three dimensional skeletal muscle tissue.

[0037] In some embodiments, the bioreactor further comprises at least two electrodes configured to apply electrical stimulation to the 3D skeletal muscle tissue of the bioreactor.

[0038] In some embodiments, the flexible anchor is an elastic sensing element. In some embodiments, two or more anchors are elastic sensing elements.

[0039] The above discussion regarding anchors (eg, elastic, polymer, etc.) is equally applicable to this embodiment.

[0040] In some embodiments, the bioreactor contains between 2 and 25 anchors per well.

[0041] In some embodiments, the bioreactor comprises a multi-well plate.

[0042] In some embodiments, the multiwell plate comprises 6 wells, 8 wells, 12 wells, 24 wells, 96 wells, 384 wells, or 1536 wells.

[0043] One aspect of the present disclosure relates to a method for measuring the effect of a test agent on contraction using the tissue system described herein, the method comprising: measuring a first value of a contractile property of 3D skeletal muscle tissue in a bioreactor before exposure to the test agent; contacting the 3D skeletal muscle tissue with the test agent for an incubation time under conditions sufficient for the test agent to modulate contraction; measuring a second value of the contractile property of the 3D skeletal muscle tissue after exposure to the test agent; and determining whether the test agent modulates contraction by comparing the first value to the second value.

[0044] In some embodiments, if there is a significant difference between the first value and the second value, the test agent modulates contraction.

[0045] In some embodiments, the test agent is selected from the group consisting of a small molecule, an antibody, an ion, a protein, a peptide, a lipid, DNA, RNA, a virus, a bacterium, a microparticle, a nanoparticle, a therapeutic agent, and a toxin.

[0046] In some embodiments, the incubation time is at least 7 days.

[0047] In some embodiments, the incubation time is at least 12 days.

[0048] One aspect of the present disclosure relates to a method for measuring the effect of a test agent on calcium transport using the tissue system described herein, the method comprising: measuring a first value of a calcium transport property of 3D skeletal muscle tissue in a bioreactor before exposure to the test agent; contacting the 3D skeletal muscle tissue with the test agent for an incubation time under conditions sufficient for the test agent to modulate calcium transport; measuring a second value of the calcium transport property of the 3D skeletal muscle tissue after exposure to the test agent; and comparing the first value to the second value to determine whether the test agent modulates calcium transport.

[0049] In some embodiments, measuring the first value or the second value comprises measuring a fluorescent signal of an intracellular calcium indicator within the 3D skeletal muscle tissue.

[0050] In some embodiments, the intracellular calcium indicator is selected from Fura-4F AM, Fura-2, Fluo-3, Fluo-4, Indo-1, Mag-Fura-5, and Mag-Fura-red.

[0051] In some embodiments, the test agent modulates calcium transients if there is a significant difference between the first calcium transient and the second calcium transient.

[0052] In some embodiments, the test agent is selected from the group consisting of a small molecule, an antibody, an ion, a protein, a peptide, a lipid, DNA, RNA, a virus, a bacterium, a microparticle, a nanoparticle, a therapeutic agent, and a toxin.

[0053] In some embodiments, the incubation time is at least 7 days.

[0054] In some embodiments, the incubation time is at least 12 days.

[0055] One aspect of the present disclosure relates to a method for stimulating myogenesis in vitro, the method comprising: (i) providing a hydrogel and a plurality of cells, including skeletal muscle cells, in a well of a bioreactor, the well having a bottom and two or more anchors positioned across the well such that a gap exists between the anchors and the bottom of the well, at least one of the anchors being a flexible anchor; and (ii) stimulating the skeletal muscle cells in the well with a series of electrical pulse trains separated by intervals over a period of time.

[0056] In some embodiments, each train has a duration of 0.5 to 4 seconds.

[0057] In some embodiments, each train has a duration of 0.5 to 2 seconds.

[0058] In some embodiments, each train has a frequency of 5-15 Hz.

[0059] In some embodiments, each train has a frequency of 8-12 Hz.

[0060] In some embodiments, each train has a duty cycle of 1-10%.

[0061] In some embodiments, the pulses in each train have a voltage between 3 and 10V.

[0062] In some embodiments, the interval is between 5 seconds and 60 minutes.

[0063] In some embodiments, the interval is between 5 seconds and 60 seconds.

[0064] In some embodiments, the period of time is between 10 minutes and 24 hours.

[0065] In some embodiments, the period of time is between 30 minutes and 2 hours.

[0066] In some embodiments, the method further comprises (iii) repeating step (ii) every 6 to 18 hours for a duration.

[0067] In some embodiments, step (ii) is repeated every 10 to 14 hours for a fixed duration.

[0068] In some embodiments, the duration is 1 to 6 weeks.

[0069] In some embodiments, the duration is 3 to 5 weeks.

[0070] In some embodiments, at least a portion of the cells are encapsulated or embedded within the hydrogel.

[0071] In some embodiments, the method comprises growing skeletal muscle cells in the wells in growth medium for 1-3 days.

[0072] In some embodiments, the method comprises differentiating the skeletal muscle cells in the wells in a differentiation medium for 3 to 50 days.

[0073] In some embodiments, the differentiation medium comprises glucose and insulin.

[0074] In some embodiments, the glucose concentration in the differentiation medium is 5 to 30 mM.

[0075] In some embodiments, the insulin concentration in the differentiation medium is between 0.5 nM and 10 nM.

[0076] In various embodiments, a method of stimulating myogenesis in vitro is provided, the method including: (i) providing a hydrogel and a plurality of cells, including skeletal muscle cells, into a well of a bioreactor, the well having a bottom and two or more anchors positioned across the well such that a gap exists between the anchors and the bottom of the well, at least one of the anchors being a flexible anchor; and (ii) stimulating the skeletal muscle cells in the well with a series of electrical pulse trains separated by intervals of 5 seconds to 60 minutes for 10 minutes to 24 hours, optionally with each train having a duration of 0.5 to 4 seconds and optionally with a frequency of 5 to 15 Hz.

[0077] In various embodiments, a method of stimulating myogenesis in vitro is provided, the method including: (i) providing a hydrogel and a plurality of cells, including skeletal muscle cells, into a well of a bioreactor, the well having a bottom and two or more anchors positioned across the well such that a gap exists between the anchors and the bottom of the well, at least one of the anchors being a flexible anchor; and (ii) stimulating the skeletal muscle cells in the well with a series of electrical pulse trains separated by intervals of 5 seconds to 60 seconds for 30 minutes to 2 hours, each train having a duration of 0.5 to 2 seconds, and optionally each train having a frequency of 8 to 12 Hz.

[0078] In various embodiments, a method of stimulating myogenesis in vitro is provided, the method including: (i) providing a hydrogel and a plurality of cells, including skeletal muscle cells, into a well of a bioreactor, the well having a bottom and at least two anchors positioned across the well such that a gap exists between the anchors and the bottom of the well, at least one of the anchors being a flexible anchor; (ii) stimulating the skeletal muscle cells in the well with a series of electrical pulse trains separated by intervals of 5 seconds to 60 minutes for 10 minutes to 24 hours, optionally wherein each train has a duration of 0.5 to 4 seconds and optionally wherein each train has a frequency of 5 to 15 Hz; and (iii) repeating step (ii) every 6 to 18 hours for 1 to 6 weeks.

[0079] In various embodiments, a method of stimulating myogenesis in vitro is provided, the method including: (i) providing a hydrogel and a plurality of cells, including skeletal muscle cells, into a well of a bioreactor, the well having a bottom and two or more anchors positioned across the well such that a gap exists between the anchors and the bottom of the well, at least one of the anchors being a flexible anchor; (ii) stimulating the skeletal muscle cells in the well with a series of electrical pulse trains separated by intervals of 5 seconds to 60 seconds for 30 minutes to 2 hours, each train having a duration of 0.5 to 2 seconds, and optionally, each train having a frequency of 8 to 12 Hz; and (iii) repeating step (ii) every 6 to 18 hours for 3 to 5 weeks.

[0080] Those skilled in the art will appreciate that the parameters and aspects of the stimulation protocol can be selected from any combination of the values ​​and ranges detailed above. In particular, those skilled in the art can combine values ​​or ranges for specific train durations, train frequencies, train duty cycles, pulse voltages, intervals, periods, whether step (iii) is included, the duration of step (iii), whether a growth step or a differentiation step is included, etc.

[0081] One aspect of the present disclosure relates to a method for stimulating myogenesis in vitro, the method comprising: (i) providing a hydrogel and a plurality of cells comprising skeletal muscle cells to a well of a bioreactor, the well having a bottom and two or more anchors positioned across the well such that a gap exists between the anchors and the bottom of the well, at least one of the anchors being a flexible anchor; and (ii) differentiating the skeletal muscle cells in the well in a differentiation medium comprising glucose and insulin.

[0082] In some embodiments, at least a portion of the cells are encapsulated or embedded within the hydrogel.

[0083] In some embodiments, the hydrogel and the plurality of cells are within a tissue system described herein.

[0084] In some embodiments, the method comprises growing skeletal muscle cells in the wells in growth medium for 1-3 days.

[0085] In some embodiments, the skeletal muscle cells are differentiated in differentiation medium for 3 to 50 days.

[0086] In some embodiments, the glucose concentration in the differentiation medium is 5 to 30 mM.

[0087] In some embodiments, the insulin concentration in the differentiation medium is between 0.5 nM and 10 nM.

[0088] Also provided is a method for stimulating myogenesis in vitro, the method comprising: (i) providing a hydrogel and a plurality of cells, including skeletal muscle cells, in a well of a bioreactor, the well having a bottom and two or more anchors positioned across the well such that a gap exists between the anchors and the bottom of the well, at least one of the anchors being a flexible anchor; (ii) growing the skeletal muscle cells in the well in a growth medium for 1 to 3 days; (iii) differentiating the skeletal muscle cells in the well in a differentiation medium comprising glucose and insulin for 3 to 50 days; and (iv) stimulating the skeletal muscle cells in the well with a series of electrical pulse trains separated by intervals over a period of time.

[0089] Also provided is a three-dimensional skeletal muscle tissue as described herein, which is obtainable by the method of stimulating myogenesis as described herein.

[0090] One aspect of the present disclosure relates to a method of monitoring the maturity of three-dimensional skeletal muscle tissue described herein, the method comprising (i) confirming the presence of myotubes in the tissue, (ii) confirming the presence of sarcomeres in myotubes in the tissue, (iii) measuring the fatigability of the tissue, and / or (iv) determining whether exercise-inducible genes (e.g., ESRRG, NFATC2) are upregulated and / or whether developmental and fatigable contractile genes (e.g., MYH8, MYH1) are downregulated. [Brief explanation of the drawings]

[0091] [Figure 1]

[0023] Figure 1 is a set of optical images taken on the Biowire™ II platform showing representative in vitro human skeletal tissue composed of PromoCell skeletal muscle cells (SkMCs) with or without cardiac fibroblasts (cFBs). The presence of fibroblasts did not significantly affect tissue compaction. Optical images were taken 12 days after cell seeding. [Figure 2]A series of representative contractility traces showing the active forces generated by the skeletal tissue. After 4 days of continuous electrical stimulation, the skeletal tissue no longer beat spontaneously (SPT) and could be captured at increasing frequencies (1 Hz, 2 Hz) when electrically stimulated. [Figure 3] 1 is a series of representative contractility traces showing the active forces generated by skeletal tissue over time. [Figure 4A] FIG. 1 is a schematic diagram showing the stimulation protocol used for skeletal tissue maturation in Example 1, where low frequency continuous stimulation was used. [Figure 4B] FIG. 1 is a schematic diagram showing the stimulation protocol used for skeletal tissue maturation in Example 1, which used high-frequency intermittent stimulation with a fixed interval between pulse trains. [Figure 4C] FIG. 1 is a schematic diagram showing the stimulation protocol used for skeletal tissue maturation in Example 1, which uses high-frequency intermittent stimulation with a fixed interval between pulse trains and increasing frequency of successive pulse trains. [Figure 5] The Biowire™ II platform supports the generation of engineered human skeletal muscle tissue. (A) Schematic representation of skeletal muscle tissue formation. A cell suspension in a hydrogel is seeded onto the Biowire™ II platform. Over time, tissue compaction occurs, resulting in the tissue suspended between two polymer wires (i). Skeletal muscle tissue was maintained in growth medium for 2 days and then switched to differentiation medium for 5 weeks. On day 7 of differentiation, a subset of skeletal muscle tissue was mobilized by electric field stimulation (ii). Contractile force was measured noninvasively by optically tracking the deflection of the polymer wire under electric field stimulation (iii, iv). (B) Representative immunofluorescence images of skeletal muscle tissue composed of multiple myofibers stained with F-actin (green) and DAPI (blue) (i), the distribution of myofibers within a 30 mm Z-stack (ii), and quantification of sarcomere length of myofibers acquired with a SoRa (super-resolution) confocal system (iii). (n=8 myotubes) [Figure 6]Increasing the time of differentiation culture promotes myotube formation and modulates developmental genes and contractility. (A) Confocal analysis of skeletal muscle tissue immunostained for F-actin (i) and relative quantification of myotube diameter (ii) demonstrates an increase in myotube size over the maturation period. (B) Expression of key developmental genes, myogenin, fetal, and neonatal MyHC, is modulated over maturation time. From D21, skeletal muscle tissue also expresses adult MyHC genes. (C) Skeletal muscle tissue generates tetanic force already at day 7 of differentiation (i). Tetanic force to 100 Hz electrical stimulation increases over maturation time (ii). (E) Representative traces of skeletal muscle tissue showing increased force with increasing electrical stimulation frequency and maturation time. Data represent mean ± SEM (n = 20). **p < 0.005. [Figure 7] Tissue exercise results in enhanced myotube formation, improved force, modulation of contractile exercise parameters, improved fatigability, and modulation of MyHC expression. Confocal analysis of skeletal muscle tissue immunostained for F-actin (i) and relative quantification of myotube diameter (ii) demonstrate increased myotube size in D21-stimulated tissue compared to time-matched unstimulated tissue. (B) Stimulated tissue exhibits increased peak amplitude (i), rise slope (ii), and rise time (iii) at both 1 Hz and 100 Hz, with a decrease in decay time at 100 Hz over time (iv). Data represent mean ± SEM (n = 20). (C) Fatigueability is measured as a 50% force drop during 10 min of repeated submaximal tetanic stimulation (i). Exercise enhances tissue fatigue resistance over the maturation period (ii). Data represent mean ± SEM (n = 9). (D) Exercise induces modulation of adult MyHC expression, specifically, the transition from IIx MyHC to IIa MyHC. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001. [Figure 8]These results demonstrate that engineered human skeletal tissue responds appropriately to chronic treatment with clenbuterol and dexamethasone. (A) Chronic clenbuterol treatment increases the force generated by stimulated tissue (Ai) and shortens the rise time (ii) compared to baseline and PBS controls. (B) Chronic dexamethasone treatment decreases the force generated by both stimulated and unstimulated tissue. (C) Chronic clenbuterol treatment somewhat mitigates the effects of dexamethasone in stimulated tissue. Statistics refer to unpaired t-tests between stimulated tissue treated with dexamethasone and stimulated tissue treated with both dexamethasone and clenbuterol. Data represent mean ± SEM. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001. [Figure 9] Figure 1 shows the results of exercise of skeletal muscle tissue with improved force. Representative traces of contractility of skeletal muscle tissue stimulated for 2 weeks versus time-matched unstimulated skeletal muscle tissue show that greater force was generated by stimulated tissue at each stimulation frequency. [Figure 10] Figure 1 shows kinetic parameters of skeletal muscle tissue chronically exposed to clenbuterol and dexamethasone. Clenbuterol treatment does not affect decay (relaxation) time in either stimulated or unstimulated tissue compared to untreated controls (A). Two weeks of dexamethasone treatment shortens decay time in unstimulated tissue but does not affect rise time in either stimulated or unstimulated tissue (Bi, ii). [Figure 11] Exercised skeletal tissue expresses key proteins for insulin-stimulated glucose transport, specifically the insulin receptor (InsR) and GLUT4, demonstrating a significant increase in glucose uptake in response to insulin. (A) Representative Western blotting of skeletal tissue showing expression of the insulin receptor (InsRbeta) and GLUT4. β-actin was used as a protein equivalent load. (B) Representative glucose uptake (+ / - insulin-stimulated) of skeletal tissue in NbActiv4 differentiation medium or customized medium. [Figure 12A] We show that long-term exercise (electrical stimulation) alters the expression of exercise-induced genes and muscle fiber contractile genes. RNA sequencing analysis revealed that when stimulated tissue was compared with unstimulated tissue, the expression of exercise-induced genes (e.g., ESRRG, NFATC2) was increased, while the expression of developmental and fatigue-induced contractile genes (e.g., MYH8, MYH1) was decreased. [Figure 12B] Chronic exercise (electrical stimulation) alters the expression of exercise-induced and muscle fiber contractile genes. GO analysis highlights several differentially regulated pathways affected by stimulation. [Figure 13A] Chronic dexamethasone treatment alters gene expression in engineered skeletal tissue. RNAseq analysis highlights the upregulation of glucocorticoid-inducible genes, including the atrogen TRIM63 and FBXO32, in dexamethasone-treated tissue. [Figure 13B] We show that chronic dexamethasone treatment alters gene expression in engineered skeletal tissue. GO analysis highlights several differentially regulated pathways that were altered by dexamethasone treatment. DETAILED DESCRIPTION OF THE INVENTION

[0092] One aspect of the present disclosure relates to a three-dimensional skeletal muscle tissue comprising a hydrogel, a plurality of cells including skeletal muscle cells, and two or more anchors, wherein the skeletal muscle tissue is characterized by one or more contractions in response to electrical and / or chemical stimuli, and at least one of the anchors is a flexible anchor.

[0093] In some embodiments, the electrical stimulus that causes one or more contractions can be about 0.1 to 100 Hz.

[0094] In some embodiments, the chemical stimulus that causes one or more contractions comprises acetylcholine, adenosine triphosphate, 4-chloro-m-cresol, or a combination thereof.

[0095] In some embodiments, the plurality of cells may further comprise fibroblasts. In some embodiments, the ratio of the number of fibroblasts to the number of skeletal muscle cells may be about 1:3 or less, about 1:3.5 or less, about 1:4 or less, about 1:4.5 or less, about 1:5 or less, about 1:5.5 or less, or about 1:6 or less. In some embodiments, the ratio of the number of fibroblasts to the number of skeletal muscle cells may be at least about 1:70, at least about 1:65, at least about 1:60, at least about 1:55, at least about 1:50, at least about 1:45, or at least about 1:40.

[0096] Combinations of the above ranges for the ratio of fibroblasts to skeletal muscle cells are also possible (e.g., at least about 1:70 to about 1:3 or at least about 1:60 to about 1:4.5), including all values ​​and ranges therebetween. For example, the ratio of fibroblasts to skeletal muscle cells can be about 1:50 to 1:5.

[0097] In some embodiments, the plurality of cells may further comprise myoblasts. The plurality of cells may further comprise other cell types, such as endothelial cells, immune cells, neurons, etc.

[0098] The cells can be derived from any animal, including humans or non-human animals. In some embodiments, the cells are human cells. In some embodiments, the cells are non-human cells, such as rat cells or mouse cells. Thus, skeletal muscle cells can be derived from humans and / or non-human animals, fibroblasts can be derived from humans and / or non-human animals, myoblasts can be derived from humans and / or non-human animals, endothelial cells can be derived from humans and / or non-human animals, and immune cells can be derived from humans and / or non-human animals.

[0099] Depending on the animal from which the cells are derived, the skeletal muscle tissue produced therefrom can exhibit the same or substantially the same properties as animal skeletal muscle tissue, such as human skeletal muscle tissue, mouse skeletal muscle tissue, or rat skeletal muscle tissue.

[0100] In some embodiments, the cells are derived from stem cells. The term "stem cells" includes embryonic stem cells ("ESCs"), fetal stem cells ("FSCs"), and adult (or somatic) stem cells ("SSCs"). In terms of potential, stem cells can be totipotent (also known as omnipotent) (stem cells that can differentiate into embryonic and extraembryonic cell types), pluripotent stem cells (capable of differentiating into almost any cell type), multipotent stem cells (capable of differentiating into many cell types), oligopotent stem cells (capable of differentiating into only a few cell types), or unipotent (capable of generating only one cell type). Stem cells can be commercially available, obtained / isolated directly from patients, or obtained / isolated from other suitable sources. Thus, skeletal muscle cells can be derived from pluripotent stem cells, fibroblasts can be derived from pluripotent stem cells, myoblasts can be derived from pluripotent stem cells, endothelial cells can be derived from pluripotent stem cells, and / or immune cells can be derived from pluripotent stem cells. In various embodiments, the skeletal muscle cells can be derived from induced pluripotent stem cells (iPSCs), the fibroblasts can be derived from iPSCs, the myoblasts can be derived from iPSCs, the endothelial cells can be derived from iPSCs, and / or the immune cells can be derived from iPSCs. In various embodiments, the skeletal muscle cells can be derived from human iPSCs, the fibroblasts can be derived from human iPSCs, the myoblasts can be derived from human iPSCs, the endothelial cells can be derived from human iPSCs, and / or the immune cells can be derived from human iPSCs.

[0101] Combinations of the above cell types are also possible (e.g., human iPSC-derived skeletal muscle cells and non-human animal-derived fibroblasts).

[0102] In some embodiments, the cells are wild-type healthy cells. In some embodiments, the cells are diseased cells (e.g., have one or more genetic mutations that lead to a diseased phenotype). In some embodiments, the cells have been pretreated with a compound or drug (e.g., the cells have been incubated with a drug for a period of time).

[0103] The hydrogel can comprise collagen or a collagen derivative, intestinal submucosa or a derivative thereof, cellulose or a cellulose derivative, proteoglycan, heparin sulfate, chondroitin sulfate, keratin sulfate, hyaluronic acid, elastin, fibronectin, thrombin, laminin, fibrin, chitosan, alginate, Matrigel®, Geltrex®, agarose, decellularized extracellular matrix, polyethylene glycol or a derivative thereof, silicone or a derivative thereof, or a combination thereof. In some embodiments, the hydrogel can comprise Matrigel® or Geltrex®. In some embodiments, the hydrogel can comprise Matrigel®.

[0104] In some embodiments, the hydrogel may comprise at least about 1% by weight collagen or collagen derivative, at least about 2% by weight collagen or collagen derivative, at least about 3% by weight collagen or collagen derivative, at least about 4% by weight collagen or collagen derivative, at least about 5% by weight collagen or collagen derivative, at least about 6% by weight collagen or collagen derivative, at least about 7% by weight collagen or collagen derivative, at least about 8% by weight collagen or collagen derivative, at least about 9% by weight collagen or collagen derivative, or at least about 10% by weight collagen or collagen derivative.

[0105] In some embodiments, the hydrogel may contain about 50% or less by weight of collagen or collagen derivatives, about 45% or less by weight of collagen or collagen derivatives, about 40% or less by weight of collagen or collagen derivatives, about 35% or less by weight of collagen or collagen derivatives, about 30% or less by weight of collagen or collagen derivatives, about 25% or less by weight of collagen or collagen derivatives, about 20% or less by weight of collagen or collagen derivatives, or about 15% or less by weight of collagen or collagen derivatives.

[0106] Combinations of the above ranges for the weight ratio of collagen or collagen derivative in the hydrogel are also possible (e.g., at least about 1 wt % to about 50 wt % or at least about 5 wt % to about 40 wt %), including all values ​​and ranges therebetween.

[0107] In some embodiments, the collagen comprises type I collagen, type III collagen, type IV collagen, type V collagen, type XI collagen, type XII collagen, or a combination thereof.

[0108] In some embodiments, at least a portion of the cells are encapsulated or embedded within the hydrogel. In some embodiments, at least about 40% of the cells are encapsulated or embedded within the hydrogel. In some embodiments, at least about 45% of the cells are encapsulated or embedded within the hydrogel. In some embodiments, at least about 50% of the cells are encapsulated or embedded within the hydrogel. In some embodiments, at least about 55% of the cells are encapsulated or embedded within the hydrogel. In some embodiments, at least about 60% of the cells are encapsulated or embedded within the hydrogel. In some embodiments, at least about 65% of the cells are encapsulated or embedded within the hydrogel. In some embodiments, at least about 70% of the cells are encapsulated or embedded within the hydrogel.

[0109] In some embodiments, about 100% of the cells are encapsulated or embedded within the hydrogel. In some embodiments, about 99% or less of the cells are encapsulated or embedded within the hydrogel. In some embodiments, about 95% or less of the cells are encapsulated or embedded within the hydrogel. In some embodiments, about 90% or less of the cells are encapsulated or embedded within the hydrogel. In some embodiments, about 85% or less of the cells are encapsulated or embedded within the hydrogel. In some embodiments, about 80% or less of the cells are encapsulated or embedded within the hydrogel.

[0110] Combinations of the above ranges for the percentage of cells encapsulated or embedded in the hydrogel are also possible (e.g., at least about 40% to about 100%, or at least about 50% to about 99% or less), including all values ​​and ranges therebetween.

[0111] In some embodiments, the 3D skeletal muscle tissue may further comprise fibrinogen and / or thrombin. The fibrinogen may comprise human and / or non-human fibrinogen. In some embodiments, the thrombin may comprise human and / or non-human thrombin.

[0112] In some embodiments, the volume of the 3D skeletal muscle tissue is at least about 0.1 mm 3 , at least about 0.5 mm 3 , at least about 1 mm 3 , at least about 1.5 mm 3 , at least about 2 mm 3 , or at least about 2.5 mm 3 It could be.

[0113] In some embodiments, the volume of the 3D skeletal muscle tissue is about 10 mm 3 Below, approximately 9mm 3 Below, approximately 8mm 3 Below, approximately 7mm 3 Below, approximately 6mm 3 Below, about 5mm 3 Below, approximately 4mm 3Below, approximately 3mm 3 Less than or equal to 2.5 mm 3 It can be the following:

[0114] A combination of the above ranges for the volume of 3D skeletal muscle tissue (e.g., at least about 0.1 mm 3 ~about 10mm 3 Less than or equal to about 0.1 mm 3 ~about 2.5mm 3 ) are also possible, including all values ​​and ranges therebetween.

[0115] The number of cells in the 3D skeletal muscle tissue varies depending on the volume of the tissue. In some embodiments, the number of cells can be at least about 20,000, at least about 25,000, at least about 30,000, at least about 35,000, at least about 40,000, at least about 45,000, or at least about 50,000. In some embodiments, the number of cells can be about 5,000,000 or less, about 2,000,000 or less, about 1,000,000 or less, about 900,000 or less, 800,000 or less, about 700,000 or less, 600,000 or less, about 500,000 or less, 400,000 or less, about 300,000 or less, 200,000 or less, or 100,000 or less.

[0116] Combinations of the above ranges for the number of cells in the tissue are also possible (eg, at least about 20,000 to a maximum of about 5,000,000, or at least about 30,000 to a maximum of about 1,000,000), including all values ​​and ranges therebetween.

[0117] The 3D skeletal muscle tissue can be characterized by morphology, structural organization, elasticity, extensibility, gene expression, protein expression, excitability, contractility, calcium translocation, electrophysiology, biochemical signaling, or a combination thereof. The 3D skeletal muscle tissue can have one or more characteristics substantially identical to healthy, native human skeletal muscle tissue. In some embodiments, the 3D skeletal muscle tissue can fully reproduce the structure and function of native skeletal muscle tissue, such as human skeletal muscle tissue.

[0118] In some embodiments, the 3D skeletal muscle tissue may include A-bands, I-bands, Z-lines, M-lines, H-zones, T-tubules, or combinations thereof.

[0119] In some embodiments, the 3D skeletal muscle tissue can include striations, elongated nuclei, sarcomeres, or a combination thereof. In some embodiments, the 3D skeletal muscle tissue can include acetylcholine receptors, slow-twitch fibers, fast-twitch fibers, or a combination thereof. Slow-twitch and fast-twitch fibers in skeletal muscle tissue are described, for example, in C. Handschin, et al., "External Physical and Biochemical Stimulation to Enhance Skeletal Muscle Bioengineering," Adv. Drug Deliv. Rev. 2015, 82-83, 168-175; D. Pette and G. Vrbova, "The Contribution of Neuromuscular Stimulation in Elucidating Muscle Plasticity Revisited," Eur. J. Transl. Myol. 2017, 27, 33-39, the contents of each of which are incorporated herein by reference.

[0120] In some embodiments, the expression levels of maturation-associated genes (e.g., myosin isoform ratio), genes associated with calcium handling, genes associated with sarcomeric proteins (e.g., dystrophin, myosin heavy chain, alpha actinin), exercise-induced genes (e.g., ESRRG, NFATC2), developmental and / or fatigable contraction genes (e.g., MYH8, MYH1) in the 3D skeletal muscle tissue are substantially the same as in native human skeletal muscle tissue.

[0121] In some embodiments, the one or more contractions generate a twitch force and / or a tetanic force.

[0122] In some embodiments, the 3D skeletal muscle tissue may be characterized by transient changes in intracellular calcium concentration in response to electrical and / or chemical stimuli. The transient changes in intracellular calcium concentration result in depolarization of muscle fibers, resulting in muscle fiber contractility. In some embodiments, the electrical stimulation is between about 0.1 and 100 Hz. In some embodiments, the chemical stimulation comprises acetylcholine, adenosine triphosphate, 4-chloro-m-cresol, or a combination thereof.

[0123] In some embodiments, the electrophysiological characteristic of the 3D skeletal muscle tissue is a shortened action potential with a pronounced hyperpolarization.

[0124] The anchors can be placed in any suitable position relative to the tissue, as long as the tissue can be attached to the anchors. In some embodiments, the anchors are placed at first and second ends (i.e., distal and proximal ends, or "opposite ends") of the tissue. Advantageously, the anchors allow for measurement of tissue contractile forces due to the flexibility of the flexible anchors.

[0125] The term "attached" is intended to cover embodiments in which the anchor is at least partially embedded within tissue or is fixedly or loosely connected to the surface of tissue.

[0126] In some embodiments, the skeletal muscle tissue is held under tension between the anchors. The inventors have advantageously discovered that holding the tissue under tension during myogenesis improves the formation of skeletal muscle tissue.

[0127] In some embodiments, one or more anchors (e.g., flexible anchors) can be adjusted to change the tension of the skeletal muscle tissue. One or more properties of the anchor (or anchors) can be adjusted. For example, the stiffness of the anchor (or anchors) can be adjusted (e.g., the stiffness of a flexible anchor can be adjusted) to change the tension of the skeletal muscle tissue. The tunability of the anchor point material is an advantage over platforms known in the prior art, which typically use rigid PDMS molds.

[0128] Skeletal muscle tissue is not limited to having two such anchors, but may include two or more, such as 2-30 anchors, e.g., 2-25, 2-20, 2-15, or 2-10 anchors. In some embodiments, skeletal muscle tissue may include 2, 3, 4, 5, 6, 7, 8, 9, or more anchors. Any number of anchors can be attached to skeletal muscle tissue, as long as there is the ability to form connective tissue around and between at least a portion of each anchor.

[0129] In some embodiments, the flexible anchor is an elastic sensing element. If there are multiple flexible anchors, any number or all of the flexible anchors can be elastic sensing elements.

[0130] The skeletal muscle tissue is not limited to having one flexible anchor, but may include multiple flexible anchors, such as 1-30 flexible anchors, e.g., 1-25, 1-20, 1-15, or 1-10 flexible anchors. In some embodiments, the skeletal muscle tissue may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or more flexible anchors. Any number of the flexible anchors may be elasticity-sensing elements.

[0131] The anchors can have an orientation that is perpendicular or substantially perpendicular to the longitudinal axis of the skeletal muscle tissue. The anchors can have an orientation that is parallel or substantially parallel to the longitudinal axis of the skeletal muscle tissue. The anchors can have an orientation that is oblique or substantially oblique to the longitudinal axis of the skeletal muscle tissue.

[0132] In some embodiments, the flexible anchor comprises a synthetic polymer, a biopolymer, or a combination thereof. The polymer may be biodegradable or non-biodegradable.

[0133] In some embodiments, the flexible anchor comprises a polymer having a Young's modulus in the range of 10 kPa to 800 kPa. For example, the Young's modulus of the polymer can be in the range of 20 kPa to 700 kPa, 20 kPa to 600 kPa, 20 kPa to 500 kPa, 50 kPa to 500 kPa, or 100 kPa to 500 kPa. In some embodiments, the polymer can have a Young's modulus of about 150 kPa, about 200 kPa, about 250 kPa, about 300 kPa, about 350 kPa, about 400 kPa, about 450 kPa, about 500 kPa, or about 550 kPa.

[0134] In some embodiments, flexible anchors can include polymers whose mechanical properties can be tuned by controlling the polymerization using different crosslinking energies. Tunability can also be controlled by the mixing ratio of the polymer units during the polymerization reaction.

[0135] In some embodiments, the polymer can be at least one of polylactic acid, poly(lactic-co-glycolic acid), poly(caprolactone), polyglycolide, polylactic acid, polyhydroxobutyric acid, polyhydroxyalkanoic acid, chitosan, hyaluronic acid, hydrogel, poly(2-hydroxyethyl methacrylate), poly(ethylene glycol), poly(L-lactic acid) (PLA), poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PMMA), poly(glycerol sebacate), poly(octamethylene maleic anhydride citric acid) (POMaC), citric acid-free POMaC, poly(ε-caprolactone), polyurethane, silk, nanoengineered materials, copolymers, blended polymers, or combinations thereof. In some embodiments, the flexible anchor comprises POMaC.

[0136] The shape, thickness, length, orientation, and surface topography of the flexible anchors can be varied in any number of suitable ways, so long as the flexible anchors are capable of deforming, bending, or otherwise changing shape in response to the contractile action or activity of the tissue connected therebetween, and such deformation, bending, or other shape change can be reliably measured. In some embodiments, the flexible anchors are in the form of a wire, such as, for example, a polymer wire.

[0137] The above discussion regarding flexible anchors is equally applicable to two or more anchors, for example, the two or more anchors may include POMaC and / or may be in the form of a polymer wire.

[0138] In some embodiments, the anchor is porous, allowing for the delivery of nutrients and growth factors to the skeletal muscle tissue. In some embodiments, the anchor is non-absorbable.

[0139] In some embodiments, the anchors are fluorescent. In some embodiments, the flexible anchors are fluorescent. This allows for monitoring the contractile properties of the tissue using fluorescence techniques.

[0140] One aspect of the present disclosure relates to 3D diseased skeletal muscle tissue comprising a plurality of diseased skeletal muscle cells. Compared to healthy skeletal muscle tissue, the 3D diseased skeletal muscle tissue can be characterized by reduced force-generating capacity, altered calcium handling, altered electrophysiology, reduced proliferation and differentiation capacity, altered extracellular matrix material (ECM), or a combination thereof. In some embodiments, when the 3D diseased skeletal muscle tissue is stimulated by electrical and / or chemical stimuli, the tissue does not contract or contracts to a significantly reduced extent compared to healthy skeletal muscle tissue.

[0141] Muscle cells attach and bind to the ECM. Furthermore, the ECM can provide a suitable and permissive environment for muscle development and function. Alterations in the ECM can be indicative of pathologies in skeletal muscle tissue. See K. Grzelkowska-Kowalczyk, "The Importance of Extracellular Matrix in Skeletal Muscle Development and Function," in F. Travascio (ed.) "Composition and Function of the Extracellular Matrix in the Human Body," IntechOpen 2016, the contents of which are incorporated herein by reference.

[0142] In some embodiments, the tissue of interest can be treated with an agent known in the art to cause cellular damage (e.g., a toxin, mutagen, radiation, infectious agent, or chemical) to induce damage to the tissue. In some embodiments, standard recombinant techniques can be used to modify the tissue of interest and induce a pathology. For example, the technique of homologous recombination can be used to insert a gene into a cell or to "knock out" gene expression of a gene of interest. For a review of homologous recombination, see Lewin, B., Genes V, Oxford University Press, New York, 1994, pp. 968-997; and Capecchi, M., (1989) Science 244:1288-1292; Capecchi, M., (1989) Trends Genet. 5(3):70-76. In some embodiments, the tissue of interest is damaged as a result of an inherited genetic defect, which can be a single gene defect or a multifactorial defect.

[0143] The 3D diseased skeletal muscle tissue can be used in disease models. For example, the disease models can mimic one or more muscle diseases, such as Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, myotonic dystrophy, congenital fiber type imbalance, myosinopathy, Pompe disease, obesity and type 2 diabetes, muscle inactivity, aging / sarcopenia, heart failure, and chronic obstructive pulmonary disease. See J. Talbot and L. Maves, "Skeletal muscle fiber type: using insights from muscle developmental biology to dissect targets for susceptibility and resistance to muscle disease," Wiley Interdiscip Rev Dev Biol. 2016, 5, 518-534, the contents of which are incorporated herein by reference.

[0144] One aspect of the present disclosure relates to a tissue system including the 3D skeletal muscle tissue described herein and a bioreactor. The bioreactor includes a device having a well configured to grow 3D skeletal muscle tissue from cells seeded therein, the well having a bottom, and two or more anchors positioned across the well such that a gap exists between the anchors and the bottom of the well. A gap also exists between the bottom of the well and the tissue suspended by the two or more anchors. At least one anchor is a flexible anchor.

[0145] The inventors have demonstrated that skeletal muscle tissue generated in a bioreactor is superior to in vitro muscle tissue generated by prior art methods. In particular, the generated muscles are morphologically similar to in vivo skeletal muscle tissue and exhibit a higher twitch-to-tetanic ratio. This platform also has the significant advantage that muscle growth, differentiation, motility, and contractility assessment can all be performed within the same platform, eliminating the need to transfer tissue to another location at any step.

[0146] The shape of the wells is not particularly limited and can be square, rectangular, circular, oval, oblong, triangular, or any combination of shapes. Other dimensions of the wells can also be varied in any suitable manner. For example, the depth of the wells, the height of the wells, the length of the wells, and the overall volume of the wells can be varied in any suitable manner.

[0147] For example, the length, height, or width of the well can be about 0.1 to 1 mm, about 0.2 to 2 mm, about 0.3 to 3 mm, about 0.4 to 4 mm, about 0.5 to 5 mm, about 0.6 to 6 mm, about 0.7 to 7 mm, about 0.8 to 8 mm, about 0.9 to 9 mm, about 1 to 10 mm, about 1 to 100 mm, or about 10 to 100 mm.

[0148] The well can be characterized by a longitudinal axis, which can be along the length of the well.

[0149] In some embodiments, the wells may be disposed within cell culture wells on a multi-well plate. In some embodiments, the multi-well plate may include multiple wells, such as 6 wells, 8 wells, 12 wells, 24 wells, 48 ​​wells, 96 wells, 384 wells, or 1536 wells.

[0150] The two or more anchors function as anchor points for tissue formed therebetween, the anchors are configured to allow attachment of the 3D skeletal muscle tissue formed therebetween, thereby suspending the 3D skeletal muscle tissue above the bottom of the well, and the flexible anchors are configured to deform in response to contractile forces exerted by the 3D skeletal muscle tissue on the flexible anchors.

[0151] The bioreactor is not limited to two anchors per well, but may include more than two, such as 2-30 anchors per well, e.g., 2-25, 2-20, 2-15, or 2-10 anchors per well. In some embodiments, the bioreactor may include 2, 3, 4, 5, 6, 7, 8, 9, or more anchors per well. Any number of anchors can be provided per well, as long as tissue can form around each anchor and bond between them to suspend the tissue above the bottom of the well.

[0152] The bioreactor is not limited to having one flexible anchor per well, but may include multiple flexible anchors, such as 1-30 flexible anchors per well, e.g., 1-25, 1-20, 1-15, or 1-10 flexible anchors per well. In some embodiments, the bioreactor may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or more flexible anchors per well. Any number of the flexible anchors may be elastic sensing elements.

[0153] The anchors can have an orientation that is perpendicular or substantially perpendicular to the longitudinal axis of the well, and the tissue can be aligned in the same direction or substantially in the same direction as the longitudinal axis of the well.

[0154] The anchors can have an orientation that is parallel or substantially parallel to the longitudinal axis of the well.

[0155] The anchors can have an oblique or substantially oblique orientation relative to the longitudinal axis of the well.

[0156] The above discussion regarding anchors and flexible anchors is equally applicable to this aspect.

[0157] Cells can be seeded into the hydrogel to generate skeletal muscle tissue, and the above discussion regarding cells and hydrogels is equally applicable to this embodiment.

[0158] Neurons can be seeded in a location separate from the skeletal muscle tissue within the bioreactor, and the neurons can then grow and / or extend through the channels to contact the skeletal muscle tissue.

[0159] In some embodiments, the ratio of the number of neurons to the number of skeletal muscle cells can be about 1:3 or less, about 1:3.5 or less, about 1:4 or less, about 1:4.5 or less, about 1:5 or less, about 1:5.5 or less, or about 1:6 or less. In some embodiments, the ratio of the number of neurons to the number of skeletal muscle cells can be at least about 1:70, at least about 1:65, at least about 1:60, at least about 1:55, at least about 1:50, at least about 1:45, or at least about 1:40.

[0160] Combinations of the above ranges for the ratio of neurons to skeletal muscle cells are also possible (e.g., at least about 1:70 to less than about 1:3, at least about 1:60 to less than about 1:4.5), including all values ​​and ranges therebetween. For example, the ratio of neurons to skeletal muscle cells can be about 1:50 to 1:5.

[0161] The bioreactor may further include electrodes configured to provide electrical stimulation to skeletal muscle tissue. Each electrode may include conductive carbon, gold, platinum, palladium, stainless steel, tin, tungsten, titanium, or combinations thereof. Further examples of conductive materials for tissue stimulation are described in Merrill et al., "Electrical Stimulation of Excitable Tissue: Design of Efficacious and Safe Protocols," J. of Neuroscience Methods 2005, 141, 171-198; Tandon et al., "Characterization of Electrical Stimulation Electrodes for Cardiac Tissue Engineering," Proceedings of the 28th IEEE, pages 845-848, the contents of each of which are incorporated herein by reference.

[0162] Further features of the bioreactor are described in US20160282338, the entire contents of which are incorporated by reference.

[0163] Typically, to generate mature 3D skeletal muscle tissue, cells encapsulated or embedded in a hydrogel are electrically stimulated according to a stimulation protocol, also known as an "exercise regimen."

[0164] In one embodiment, the stimulation protocol may include a low-frequency pulse train or trains over an extended duration. See FIG. 4A, which is a schematic diagram illustrating an example of such a stimulation protocol. The pulse trains may be continuous (i.e., a single pulse train with no intervals between them) or intermittent (i.e., multiple pulse trains separated by intervals without stimulation).

[0165] In some embodiments, the low frequency can be at least about 0.1 Hz, at least about 0.2 Hz, at least about 0.3 Hz, at least about 0.4 Hz, at least about 0.5 Hz, or at least about 0.6 Hz, at least about 0.7 Hz, at least about 0.8 Hz, or at least about 0.9 Hz, at least about 1 Hz, at least about 1.5 Hz, at least about 2 Hz, at least about 2.5 Hz, or at least about 3 Hz. In some embodiments, the low frequency can be about 5 Hz or less, about 4.5 Hz or less, about 4 Hz or less, about 3.5 Hz or less, about 3 Hz or less, about 2.5 Hz or less, about 2 Hz or less, about 1.5 Hz or less, or about 1 Hz or less.

[0166] Combinations of the above ranges of low frequencies are also possible (e.g., at least about 0.1 Hz to about 1 Hz or less, or at least about 0.5 Hz to about 2 Hz or less), including all values ​​and ranges therebetween. In some embodiments, the low frequency is about 0.1 Hz, about 0.2 Hz, about 0.3 Hz, about 0.4 Hz, about 0.5 Hz, about 0.6 Hz, about 0.7 Hz, about 0.8 Hz, about 0.9 Hz, or about 1.0 Hz.

[0167] In some embodiments, the extended duration can be at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 10 hours, at least about 20 hours, at least about 40 hours, or at least about 1 week. In some embodiments, the extended duration can be about 3 weeks or less, about 2.5 weeks or less, about 2 weeks or less, about 1.5 weeks or less, or about 1 week or less, about 3 days or less, or about 24 hours or less.

[0168] Combinations of the above ranges for extended duration are also possible (eg, at least about 1 hour to no more than about 3 weeks, or at least about 5 hours to no more than about 2 weeks), including all values ​​and ranges therebetween.

[0169] In another embodiment, the stimulation protocol may include high-frequency pulse train(s) of extended duration, which may be continuous (i.e., a single pulse train with no intervals between them) or intermittent (i.e., multiple pulse trains separated by intervals without stimulation).

[0170] In some embodiments, the stimulation protocol may include multiple intermittent high-frequency pulse trains (i.e., separated by intervals without electrical stimulation). Compared to mature 3D cardiac tissue, generating mature 3D skeletal muscle tissue may require high-frequency pulses (e.g., at least about 5 Hz). Without wishing to be bound by theory, the high-frequency requirement is due to the ability of the skeletal tissue to generate sustained contracture through repeated high-frequency pulses. Repeated sustained contractures result in maturation of the skeletal tissue. See FIG. 4B, which is a schematic diagram illustrating an example of such a stimulation protocol.

[0171] In some embodiments, the high frequency can be at least about 5 Hz, at least about 10 Hz, at least about 15 Hz, at least about 20 Hz, at least about 25 Hz, at least about 30 Hz, at least about 35 Hz, or at least about 40 Hz. In some embodiments, the high frequency can be about 100 Hz or less, about 95 Hz or less, about 90 Hz or less, about 85 Hz or less, about 80 Hz or less, about 75 Hz or less, about 70 Hz or less, about 65 Hz or less, or about 60 Hz or less.

[0172] Combinations of the above ranges of high frequencies are also possible (eg, at least about 5 Hz to about 100 Hz or less, or at least about 10 Hz to about 60 Hz or less), including all values ​​and ranges therebetween.

[0173] In some embodiments, the duration of each pulse train can be at least about 1 second, at least about 10 seconds, at least about 30 seconds, at least about 60 seconds, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, or at least about 60 minutes. In some embodiments, the duration of each pulse train can be about 3 hours or less, about 2.5 hours or less, about 2 hours or less, about 1.5 hours or less, or about 1 hour or less.

[0174] Combinations of the above ranges for the duration of each pulse train are also possible (eg, at least about 1 second to less than or equal to about 3 hours, or at least about 30 seconds to less than or equal to about 1 hour), including all values ​​and ranges therebetween.

[0175] In some embodiments, the extended duration can be at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 5 hours, at least about 10 hours, at least about 12 hours, at least about 15 hours, at least about 18 hours, at least about 20 hours, or at least about 24 hours. In some embodiments, the extended duration is 4 weeks or less, 3 weeks or less, 2 weeks or less, 1 week or less, 72 hours or less, 48 ​​hours or less, 24 hours or less, 20 hours or less, 18 hours or less, 15 hours or less, 12 hours or less, 10 hours or less, 5 hours or less, 3 hours or less, 2 hours or less, or 1 hour or less.

[0176] Combinations of the above ranges for extended duration are also possible (eg, at least about 5 minutes to up to about 4 weeks), including all values ​​and ranges therebetween.

[0177] In some embodiments, the stimulation protocol can include about 1-60 high-frequency pulse trains per hour, e.g., about 1-55, about 1-50, about 1-45, about 1-40, or about 10-60 high-frequency pulse trains per hour.

[0178] In yet another embodiment, a stimulation protocol can include a first frequency pulse train of a first duration and a second frequency pulse train of a second duration, where the first frequency increases at a first ramp rate to the second frequency. See FIG. 4C for a schematic diagram illustrating an example of such a stimulation protocol. In some embodiments, a stimulation protocol can include a series of first frequency pulse trains over a first duration and a series of second frequency pulse trains over a second duration, where the first frequency increases at a first ramp rate to the second frequency.

[0179] In some embodiments, the first frequency can be at least about 0.1 Hz, at least about 0.2 Hz, at least about 0.3 Hz, at least about 0.4 Hz, at least about 0.5 Hz, or at least about 0.6 Hz, at least about 0.7 Hz, at least about 0.8 Hz, or at least about 0.9 Hz, at least about 1 Hz, at least about 1.5 Hz, at least about 2 Hz, at least about 2.5 Hz, or at least about 3 Hz. In some embodiments, the first frequency can be about 5 Hz or less, about 4.5 Hz or less, about 4 Hz or less, about 3.5 Hz or less, about 3 Hz or less, about 2.5 Hz or less, about 2 Hz or less, about 1.5 Hz or less, or about 1 Hz or less.

[0180] Combinations of the above ranges of the first frequency are also possible (e.g., at least about 0.1 Hz to about 1 Hz or less, or at least about 0.5 Hz to about 2 Hz or less), including all values ​​and ranges therebetween. In some embodiments, the first frequency is about 0.1 Hz, about 0.2 Hz, about 0.3 Hz, about 0.4 Hz, about 0.5 Hz, about 0.6 Hz, about 0.7 Hz, about 0.8 Hz, about 0.9 Hz, or about 1.0 Hz.

[0181] In some embodiments, the first duration can be at least about 1 second, at least about 10 seconds, at least about 30 seconds, at least about 60 seconds, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, or at least about 60 minutes. In some embodiments, the first duration can be about 3 hours or less, about 2.5 hours or less, about 2 hours or less, about 1.5 hours or less, or about 1 hour or less.

[0182] Combinations of the above ranges for the first duration are also possible (e.g., at least about 1 second to not more than about 3 hours, or at least about 30 seconds to not more than about 1 hour), including all values ​​and ranges therebetween. In some embodiments, the first duration is about 1 hour.

[0183] In some embodiments, the second frequency can be at least about 10 Hz, at least about 15 Hz, at least about 20 Hz, at least about 25 Hz, at least about 30 Hz, at least about 35 Hz, or at least about 40 Hz. In some embodiments, the second frequency can be about 100 Hz or less, about 95 Hz or less, about 90 Hz or less, about 85 Hz or less, about 80 Hz or less, about 75 Hz or less, about 70 Hz or less, about 65 Hz or less, or about 60 Hz or less.

[0184] Combinations of the above ranges of the first frequency are also possible (eg, at least about 10 Hz to about 100 Hz or less, or at least about 10 Hz to about 60 Hz or less), including all values ​​and ranges therebetween.

[0185] In some embodiments, the second duration can be at least about 1 second, at least about 10 seconds, at least about 30 seconds, at least about 60 seconds, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, or at least about 60 minutes. In some embodiments, the second duration can be about 3 hours or less, about 2.5 hours or less, about 2 hours or less, about 1.5 hours or less, or about 1 hour or less.

[0186] Combinations of the above ranges for the second duration are also possible (e.g., at least about 1 second to not more than about 3 hours, or at least about 30 seconds to not more than about 1 hour), including all values ​​and ranges therebetween. In some embodiments, the second duration is about 1 hour.

[0187] In some embodiments, the first ramp rate can be at least about 0.1 Hz / hour.

[0188] In some embodiments, the stimulation protocol may further include a third frequency pulse train over a third duration. In some embodiments, the stimulation protocol may further include a series of third frequency pulse trains over a third duration. The third frequency may be less than or greater than the second frequency. The stimulation protocol may further include a fourth frequency pulse train over a fourth duration, a fifth frequency pulse train over a fifth duration, etc. The stimulation protocol may further include a series of fourth frequency pulse trains over a fourth duration, a series of fifth frequency pulse trains over a fifth duration, etc.

[0189] Any of the above stimulation protocols may be applied to cells once a day, twice a day, three times a day, or four or more times a day.

[0190] In some embodiments, electrical stimulation is terminated once a specific phenotype (e.g., slow-twitch or fast-twitch fibers) is achieved, and analysis can be performed based on the expression of markers (e.g., type II myosin heavy chain, which is predominantly expressed in fast-twitch fibers, and type I myosin heavy chain, which is predominantly expressed in slow-twitch fibers). Contractility and calcium translocation parameters also allow for characterization of slow-twitch and fast-twitch fibers. In some embodiments, electrical stimulation is terminated once a specific phenotype (e.g., the presence of myotubes and / or sarcomeres, which can be analyzed microscopically) is achieved.

[0191] In some embodiments, electrical stimulation is terminated once one or more measurable parameters are achieved, for example, a minimum tetanus-to-baseline ratio of greater than 2 or a minimum tetanus force of 50 μN.

[0192] Another aspect of the present disclosure relates to a method for stimulating myogenesis in vitro, the method comprising: (i) providing a hydrogel and a plurality of cells comprising skeletal muscle cells into a well of a bioreactor, the well having a bottom and two or more anchors positioned across the well such that a gap exists between the anchors and the bottom of the well, at least one of the anchors being a flexible anchor; and (ii) stimulating the skeletal muscle cells over a period of time with a series of electrical pulse trains separated by intervals.

[0193] The above discussion regarding the hydrogel, the plurality of cells, the anchor, and the bioreactor is equally applicable to this embodiment.

[0194] In some embodiments, the period of time is at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 5 hours, at least 10 hours, at least 12 hours, at least 15 hours, at least 18 hours, at least 20 hours, or at least 24 hours. In some embodiments, the period of time is 24 hours or less, 20 hours or less, 18 hours or less, 15 hours or less, 12 hours or less, 10 hours or less, 5 hours or less, 3 hours or less, 2 hours or less, or 1 hour or less.

[0195] In some embodiments, the period of time is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, at least 28 days, at least 35 days, or at least 42 days. In some embodiments, the period of time is 8 weeks or less, 7 weeks or less, 6 weeks or less, 5 weeks or less, 4 weeks or less, 3 weeks or less, 2 weeks or less, or 1 week or less.

[0196] In some embodiments, the period of time is between 10 minutes and 24 hours, hi some embodiments, the period of time is between 30 minutes and 2 hours.

[0197] Combinations of the above ranges of fixed time periods are also possible (eg, at least 5 minutes to about 1 week or less), including all values ​​and ranges therebetween.

[0198] The series of electrical pulse trains can be any multiple consecutive electrical pulse trains, hi some embodiments, the electrical pulse train is a train of biphasic square wave pulses.

[0199] In some embodiments, the duration of each pulse train can be at least about 0.1 seconds, at least about 0.2 seconds, at least about 0.3 seconds, at least about 0.4 seconds, at least about 0.5 seconds, at least about 0.6 seconds, at least about 0.7 seconds, at least about 0.8 seconds, at least about 0.9 seconds, at least about 1 second, at least about 2 seconds, at least about 3 seconds, at least about 4 seconds, at least about 5 seconds, at least about 6 seconds, at least about 7 seconds, at least about 8 seconds, at least about 9 seconds, at least about 10 seconds, at least about 15 seconds, at least about 20 seconds, at least about 25 seconds, at least about 30 seconds, at least about 1 minute, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, or at least about 30 minutes. In some embodiments, the duration of each pulse train can be about 3 hours or less, about 2.5 hours or less, about 2 hours or less, about 1.5 hours or less, about 1 hour or less, about 30 minutes or less, about 15 minutes or less, about 10 minutes or less, about 5 minutes or less, about 1 minute or less, about 30 seconds or less, about 25 seconds or less, about 20 seconds or less, about 15 seconds or less, about 10 seconds or less, or about 5 seconds or less.

[0200] Combinations of the above ranges for the duration of each pulse train are also possible (e.g., at least about 0.1 seconds to not more than about 3 hours), including all values ​​and ranges therebetween. In various embodiments, the duration of each pulse train is 0.5 to 4 seconds. In various embodiments, the duration of each pulse train is 0.5 to 2 seconds. The pulse trains can all have the same duration (e.g., all pulse trains in a series have a duration of 1 second) or can have different durations (e.g., the first pulse train in a series has a duration of 0.8 seconds, the second pulse train has a duration of 1.2 seconds, etc.).

[0201] In some embodiments, the pulse train has a low frequency and has a pulse repetition frequency of at least about 0.1 Hz, at least about 0.2 Hz, at least about 0.3 Hz, at least about 0.4 Hz, at least about 0.5 Hz, or at least about 0.6 Hz, at least about 0.7 Hz, at least about 0.8 Hz, or at least about 0.9 Hz, at least about 1 Hz, at least about 1.5 Hz, at least about 2 Hz, at least about 2.5 Hz, or at least about 3 Hz. In some embodiments, the pulse repetition frequency is about 5 Hz or less, about 4.5 Hz or less, about 4 Hz or less, about 3.5 Hz or less, about 3 Hz or less, about 2.5 Hz or less, about 2 Hz or less, about 1.5 Hz or less, or about 1 Hz or less.

[0202] In some embodiments, the pulse train is high frequency and has a pulse repetition frequency of at least about 5 Hz, at least about 6 Hz, at least about 7 Hz, at least about 8 Hz, at least about 9 Hz, at least about 10 Hz, at least about 12 Hz, at least about 15 Hz, at least about 20 Hz, at least about 25 Hz, at least about 30 Hz, at least about 35 Hz, or at least about 40 Hz. In some embodiments, the pulse repetition frequency is about 100 Hz or less, about 95 Hz or less, about 90 Hz or less, about 85 Hz or less, about 80 Hz or less, about 75 Hz or less, about 70 Hz or less, about 65 Hz or less, about 60 Hz or less, about 55 Hz or less, about 50 Hz or less, about 45 Hz or less, about 40 Hz or less, about 35 Hz or less, about 30 Hz or less, about 25 Hz or less, or about 20 Hz or less.

[0203] Combinations of the above ranges of pulse repetition frequencies are also possible (e.g., at least about 5 Hz to about 100 Hz or less), including all values ​​and ranges therebetween. Combinations of the above ranges and values ​​for the low-frequency pulse train and the high-frequency pulse train are also possible (e.g., a pulse repetition frequency of about 1 Hz for the first pulse train and a pulse repetition frequency of about 10 Hz for the second pulse train). In some embodiments, each train has a pulse repetition frequency of 5-15 Hz. In some embodiments, each train has a pulse repetition frequency of 8-12 Hz.

[0204] In some embodiments, each train has a duty cycle of 0.5-20%. In some embodiments, each train has a duty cycle of 1-10%. In some embodiments, each train has a duty cycle of 2-5%. In some embodiments, each train has a duty cycle of 4%.

[0205] In some embodiments, each pulse in the pulse train has a pulse width of at least about 1 millisecond, at least about 2 milliseconds, at least about 3 milliseconds, at least about 4 milliseconds, at least about 5 milliseconds, at least about 10 milliseconds, at least about 15 milliseconds, at least about 20 milliseconds, at least about 25 milliseconds, at least about 30 milliseconds, at least about 40 milliseconds, or at least about 50 milliseconds. In some embodiments, each pulse in the pulse train has a pulse width of about 50 milliseconds or less, about 40 milliseconds or less, about 30 milliseconds or less, about 25 milliseconds or less, about 20 milliseconds or less, about 15 milliseconds or less, or about 10 milliseconds or less.

[0206] Combinations of the above ranges for pulse width are also possible (e.g., at least about 1 millisecond to not more than about 50 milliseconds, or at least about 2 milliseconds to not more than about 10 milliseconds), including all values ​​and ranges therebetween. In some embodiments, the pulse width is at least about 1 millisecond to not more than about 10 milliseconds. In some embodiments, the pulse width is about 4 milliseconds.

[0207] In some embodiments, the pulses in each train have a pulse amplitude of at least 3 V, at least 4 V, at least 5 V, at least 6 V, at least 7 V, at least 8 V, at least 9 V, or at least 10 V. In some embodiments, the pulses in each train have a pulse amplitude of 50 V or less, 45 V or less, 40 V or less, 35 V or less, 30 V or less, 25 V or less, 20 V or less, 15 V or less, or 10 V or less.

[0208] Combinations of the above ranges of pulse amplitudes are also possible (e.g., at least about 3V to no more than 45V), including all values ​​and ranges therebetween. In some embodiments, the pulses in each train have a voltage between 3 and 10V.

[0209] In some embodiments, the interval between pulse trains is at least 3 seconds, at least 4 seconds, at least 5 seconds, at least 6 seconds, at least 7 seconds, at least 8 seconds, at least 9 seconds, at least 10 seconds, at least 15 seconds, at least 20 seconds, at least 30 seconds, at least 45 seconds, at least 60 seconds, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 45 minutes, or at least 60 minutes. In some embodiments, the interval between pulse trains is 120 minutes or less, 90 minutes or less, 60 minutes or less, 45 minutes or less, 30 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, 4 minutes or less, 3 minutes or less, 2 minutes or less, or 1 minute or less.

[0210] Combinations of the above ranges for the interval are also possible (e.g., at least about 3 seconds to up to 120 minutes), including all values ​​and ranges therebetween. In some embodiments, the interval is 5 seconds to 60 minutes. In some embodiments, the interval is 5 minutes to 60 minutes. In some embodiments, the interval is 10 seconds.

[0211] In some embodiments, the method comprises applying step (ii) to the cells once a day, twice a day, three times a day, or four or more times a day.

[0212] In some embodiments, the method comprises repeating step (ii) every 6 to 18 hours for a period of time. In some embodiments, the method comprises repeating step (ii) every 8 to 16 hours for a period of time. In some embodiments, the method comprises repeating step (ii) every 10 to 14 hours for a period of time. In some embodiments, the method comprises repeating step (ii) every 11 to 13 hours for a period of time. In some embodiments, the method comprises repeating step (ii) every 12 hours for a period of time.

[0213] In some embodiments, the duration is at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 5 weeks, In some embodiments, the duration is 8 weeks or less, 7 weeks or less, 6 weeks or less, 5 weeks or less, 4 weeks or less, or 3 weeks or less.

[0214] Combinations of the above ranges for duration are also possible (e.g., from 5 days to 8 weeks, inclusive), including all values ​​and ranges therebetween. In some embodiments, the duration is 1 to 6 weeks. In some embodiments, the duration is 3 to 5 weeks. In some embodiments, the duration is 4 weeks.

[0215] In some embodiments, electrical stimulation is terminated once a specific phenotype (e.g., slow-twitch or fast-twitch fibers) is achieved, and analysis can be performed based on the expression of markers (e.g., type II myosin heavy chain, which is predominantly expressed in fast-twitch fibers, and type I myosin heavy chain, which is predominantly expressed in slow-twitch fibers). Contractility and calcium translocation parameters also allow for characterization of slow-twitch and fast-twitch fibers. In some embodiments, electrical stimulation is terminated once a specific phenotype (e.g., the presence of myotubes and / or sarcomeres, which can be analyzed microscopically) is achieved.

[0216] In some embodiments, electrical stimulation is terminated once one or more measurable parameters are achieved, for example, a minimum tetanus-to-baseline ratio of greater than 2 or a minimum tetanus force of 50 μN.

[0217] In some embodiments, the method includes growing skeletal muscle cells in the well in growth medium for at least 1 day, at least 2 days, or at least 3 days. In some embodiments, the method includes growing skeletal muscle cells in the well in growth medium for no more than 5 days, no more than 4 days, no more than 3 days, or no more than 2 days. This method advantageously allows for cell growth, tissue movement, and muscle contraction measurement in the same well without moving or dissociating the tissue.

[0218] Combinations of the above ranges for the growth step are also possible (e.g., at least 1 day to no more than 5 days), including all values ​​and ranges therebetween. In some embodiments, the method includes growing the skeletal muscle cells in Growth Medium for 1 to 3 days. In some embodiments, the method includes growing the skeletal muscle cells in Growth Medium for 2 days.

[0219] The growth step may be an initial step that occurs before the stimulation step (i.e., before step (ii)). There may also be other steps (e.g., a differentiation step) between the growth and stimulation steps.

[0220] The growth medium can be any suitable culture medium that allows cell proliferation. For example, the growth medium can be skeletal muscle cell growth medium (Promocell or Sigma-Aldrich). Typically, the growth medium contains serum.

[0221] In some embodiments, the method comprises differentiating skeletal muscle cells in a well in differentiation medium for at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, at least 28 days, at least 35 days, at least 42 days, or at least 49 days. In some embodiments, the method comprises differentiating skeletal muscle cells in differentiation medium for up to 100 days, up to 90 days, up to 80 days, up to 70 days, up to 60 days, up to 50 days, or up to 40 days. This method advantageously allows cells to be grown and differentiated in the same well where tissue is moved and muscle contraction is measured, without moving and / or separating the tissue.

[0222] Combinations of the above ranges for the differentiation step are also possible (e.g., at least 3 days to up to 100 days), including all values ​​and ranges therebetween. In some embodiments, the method involves differentiating the skeletal muscle cells in differentiation medium for 3 to 50 days. In some embodiments, the method involves differentiating the skeletal muscle cells in differentiation medium for 20 to 40 days.

[0223] The differentiation step may be an initial step that occurs before the stimulation step (i.e., before step (ii)). If a growth step is present, the differentiation step may occur between the growth step and the stimulation step.

[0224] The differentiation medium can be any suitable culture medium that allows cells to differentiate into mature skeletal muscle cells. For example, the differentiation medium can be NbActiv4 (BrainBitsLLC.com). In some embodiments, the differentiation medium contains glucose. In some embodiments, the glucose concentration in the differentiation medium is 5-30 mM.

[0225] In some embodiments, the differentiation medium contains insulin. In some embodiments, the insulin concentration in the differentiation medium is 0.5 nM to 10 nM. In some embodiments, the insulin concentration in the differentiation medium is 1 to 8 nM. In some embodiments, the insulin concentration in the differentiation medium is 3 to 5 nM.

[0226] In some embodiments, the differentiation medium contains glucose and insulin, and the glucose concentration in the differentiation medium is 5 to 30 mM, and the insulin concentration in the differentiation medium is 0.5 nM to 10 nM.

[0227] Another aspect of the present disclosure relates to a method for stimulating myogenesis in vitro, the method comprising: (i) providing a hydrogel and a plurality of cells comprising skeletal muscle cells in a well of a bioreactor, the well having a bottom and two or more anchors positioned across the well such that a gap exists between the anchors and the bottom of the well, at least one of the anchors being a flexible anchor; and (ii) differentiating the skeletal muscle cells in the well in a differentiation medium comprising glucose and insulin.

[0228] The above descriptions regarding the hydrogel, the plurality of cells, the anchor, and the bioreactor are equally applicable to this aspect. The above descriptions regarding the differentiation step and differentiation medium are equally applicable to this aspect.

[0229] In some embodiments, the method includes growing the skeletal muscle cells in the wells in a growth medium for at least 1 day, at least 2 days, or at least 3 days, hi some embodiments, the method includes growing the skeletal muscle cells in the wells in a growth medium for no more than 5 days, no more than 4 days, no more than 3 days, or no more than 2 days.

[0230] Combinations of the above ranges for the growth step are also possible (e.g., at least 1 day to no more than 5 days), including all values ​​and ranges therebetween. The above discussion of growth steps and growth media is equally applicable to this embodiment.

[0231] Also provided is a method for stimulating myogenesis in vitro, the method comprising: (i) providing a hydrogel and a plurality of cells, including skeletal muscle cells, in a well of a bioreactor, the well having a bottom and two or more anchors positioned across the well such that a gap exists between the anchors and the bottom of the well, at least one of the anchors being a flexible anchor; (ii) growing the skeletal muscle cells in the well in a growth medium for 1 to 3 days; (iii) differentiating the skeletal muscle cells in the well in a differentiation medium comprising glucose and insulin for 3 to 50 days; and (iv) stimulating the skeletal muscle cells in the well with a series of electrical pulse trains separated by intervals over a period of time.

[0232] The above discussion regarding the hydrogel, the plurality of cells, the anchor, the bioreactor, the growth step, the differentiation step, and the stimulation step is equally applicable to this aspect.

[0233] In some embodiments, the tissue systems described herein may be used to measure the effect on the contractility of skeletal muscle tissue formed therein as a result of exposure of a subject to a test agent.

[0234] In some embodiments, the tissue system may be used to (a) test the efficacy and safety (including toxicity) of test agents (e.g., experimental pharmacological agents), (b) define the pharmacokinetics and / or pharmacodynamics of pharmacological agents, (c) characterize the properties and therapeutic effects of pharmacological agents on a subject, (d) screen experimental pharmacological agents, and / or (e) provide transplantable engineered tissue for use in regenerative medicine to treat damaged and / or diseased tissue.

[0235] Accordingly, one aspect of the present disclosure provides a method for measuring the effect of a test agent on contraction using the tissue system described herein, the method comprising: measuring a first value of a contractile property of 3D skeletal muscle tissue in a bioreactor before exposure to the test agent; contacting the 3D skeletal muscle tissue with the test agent for an incubation time under conditions sufficient for the test agent to modulate contraction; measuring a second value of the contractile property of the 3D skeletal muscle tissue after exposure to the test agent; and determining whether the test agent modulates contraction by comparing the first value with the second value.

[0236] The contractile property can be contractile force, twitch force, tetanic force, time to tetanus, rate of tetanic force onset, rate of tetanic force decay, rate of relaxation from tetanus, or a combination thereof.

[0237] In some embodiments, the test agent modulates contraction when there is a significant difference between the first and second values, e.g., at least about a 10% difference, at least about a 15% difference, at least about a 20% difference, at least about a 25% difference, at least about a 30% difference, at least about a 35% difference, at least about a 40% difference, at least about a 45% difference, or at least about a 50% difference.

[0238] In some embodiments, measuring the contractile force comprises measuring an amount of movement imposed by the 3D skeletal muscle tissue on the flexible anchor from a first position to a second position. In some embodiments, measuring the contractile force comprises measuring an amount of movement imposed by the 3D skeletal muscle tissue on two or more anchors from a first position to a second position.

[0239] In some embodiments, the test agent modulates the contractile force when there is a significant difference between the first contractile force and the second contractile force, e.g., when there is a difference of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%.

[0240] In some embodiments, contractile force can be measured by optical microscopy, as disclosed in US20160282338.

[0241] Another aspect of the present disclosure relates to a method for measuring the effect of a test agent on calcium transport using the tissue system described herein, the method comprising: measuring a first value of a calcium transport property of 3D skeletal muscle tissue in a bioreactor before exposure to the test agent; contacting the 3D skeletal muscle tissue with the test agent for an incubation time under conditions sufficient for the test agent to modulate calcium transport; measuring a second value of the calcium transport property of the 3D skeletal muscle tissue after exposure to the test agent; and comparing the first value to the second value to determine whether the test agent modulates calcium transport.

[0242] The calcium translocation property can be the magnitude of calcium translocation, the time constant of calcium translocation, the rate of calcium translocation, or a combination thereof.

[0243] In some embodiments, the test agent modulates calcium transients when there is a significant difference between the first and second values, e.g., at least about a 10% difference, at least about a 15% difference, at least about a 20% difference, at least about a 25% difference, at least about a 30% difference, at least about a 35% difference, at least about a 40% difference, at least about a 45% difference, or at least about a 50% difference.

[0244] In some embodiments, measuring the calcium transport properties comprises measuring the fluorescent signal of an intracellular calcium indicator within the 3D skeletal muscle tissue.

[0245] In some embodiments, the intracellular calcium indicator is selected from Fura-4F AM, Fura-2, Fluo-3, Fluo-4, Indo-1, Mag-Fura-5, and Mag-Fura-red.

[0246] Another aspect of the present disclosure relates to a method of evaluating the safety of a test agent using the tissue system described herein, the method comprising: (a) contacting skeletal muscle tissue with the test agent; (b) measuring an effect on one or more physiological parameters indicative of safety; and (c) comparing the physiological parameters in (b) with the same physiological parameters measured from a control bioreactor not exposed to the test agent, wherein a statistically significant change in the physiological parameters in (b) compared to the same physiological parameters measured from the control bioreactor indicates that the test agent lacks safety.

[0247] Undesirable toxic effects caused by administration of a test agent can be screened for in several ways. The tissue system described herein can be used to determine the toxic dosimetry range of a test agent. The effect of increasing concentrations (doses) of the test agent on skeletal muscle tissue can be monitored to detect toxicity. If a toxic effect is observed, it is due to the concentration of test agent / cell cm. 2 By calculating the toxic concentration according to the distribution of cells within skeletal muscle tissue, one skilled in the art can extrapolate to biological systems and estimate the toxic dose in subjects of various weights and developmental stages.

[0248] The tissue systems described herein can also be used to evaluate the efficacy of test agents. Efficacy can be detected by measuring individual parameters associated with repair, strengthening, improvement, and / or regeneration in disease models involving diseased skeletal muscle tissue. Pathological conditions can be induced or can be the result of pre-existing conditions in the tissue donor, including conditions associated with genetic abnormalities. Either induced or pre-existing conditions can constitute debilitating conditions resulting from prior drug exposure. Test agents can be analyzed for efficacy in the disease models of the present disclosure.

[0249] Using the methods of the present invention, various doses of individual test agents and combinations of test agents can be screened in panels of tissues with diverse genetic backgrounds to determine the pharmacogenetic efficacy profile of the test agent, e.g., multiple doses of a test agent or combinations of a test agent can be screened for efficacy, or lack thereof, specific to one or more genetic backgrounds.

[0250] Typically, the test agent can be incubated with skeletal muscle tissue at a dose range predicted to be therapeutically effective for a duration sufficient to produce an effect (e.g., a metabolic effect or an effect indicating toxicity or efficacy). Incubation times range from about 1 minute to 24 hours, but can be extended to several days or weeks as needed. Incubation conditions typically include standard culture conditions known in the art, such as an incubation temperature of about 37°C and a culture medium compatible with skeletal muscle tissue.

[0251] In some embodiments, the test agent is selected from the group consisting of a small molecule, an antibody, an ion, a protein, a peptide, a lipid, DNA, RNA, a virus, a bacterium, a microparticle, a nanoparticle, a therapeutic agent, and a toxin.

[0252] Examples of test drugs include opioid analgesics, anti-inflammatory drugs such as antihistamines and nonsteroidal anti-inflammatory drugs (NSAIDs), diuretics such as carbonic anhydrase inhibitors, loop diuretics, hyperdiuretics, thiazides and thiazide-like drugs, and potassium-sparing diuretics, drugs that affect the kidneys and cardiovascular system such as angiotensin-converting enzyme (ACE) inhibitors, cardiac drugs such as organic nitrates, calcium channel blockers, sympatholytics, vasodilators, and beta-adrenergic receptor antagonists. These include, but are not limited to, agonists and antagonists, alpha adrenergic receptor agonists and antagonists, cardiac glycosides, antiarrhythmic drugs, drugs that affect hyperlipoproteinemia, such as 3-hydroxymethylglutaryl-coenzyme A (HMG-CoA) inhibitors, antitumor agents, such as alkylating agents, antimetabolites, natural products, antibiotics, and other drugs, immunomodulators, antidiabetic agents, and antibacterial agents, such as antibacterial, antiviral, antifungal, antiprotozoal, and antiparasitic agents.

[0253] In some embodiments, the incubation time is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 14 days, at least 21 days, or at least 28 days. In some embodiments, the incubation time is 100 days or less, 90 days or less, 80 days or less, 70 days or less, 60 days or less, 50 days or less, 40 days or less, 30 days or less, or 20 days or less.

[0254] Combinations of the above ranges for incubation time are also possible (e.g., at least 1 day to up to 100 days), including all values ​​and ranges therebetween. In some embodiments, the incubation time is at least 7 days. In some embodiments, the incubation time is at least 12 days.

[0255] Another aspect of the present disclosure relates to a method of monitoring the maturity of 3D skeletal muscle tissue described herein, comprising (i) confirming the presence of myotubes in the tissue, (ii) confirming the presence of sarcomeres in myotubes in the tissue, (iii) measuring the fatigability of the tissue, and / or (iv) determining whether exercise-induced genes (e.g., ESRRG, NFATC2) are upregulated and / or whether developmental and fatigable contractile genes (e.g., MYH8, MYH1) are downregulated.

[0256] To confirm the presence of myotubes in tissue, the tissue must be stained (e.g., F-actin immunostaining) and observed using a microscope such as a confocal microscope. Confirmation of the presence of myotubes in tissue may involve evaluation of gene expression or protein content. Confirmation of the presence of myotubes in tissue may involve the use of laser diffraction.

[0257] Confirming the presence of sarcomeres in myotubes within a tissue may involve staining the tissue (e.g., F-actin and / or troponin immunostaining) and observing the tissue using a microscope, such as a confocal microscope.

[0258] Tissue fatigability can be measured using any suitable method of measuring loss of force production over time. In some embodiments, an isometric fatigue method is used (e.g., inducing tetanus and measuring fatigue over a period of time, such as 30 seconds). In some embodiments, a chronic fatigue method is used (e.g., multiple stimulated contractions over an extended period of time). As a non-limiting example, fatigability can be measured by measuring loss of force production during 10 minutes of repeated submaximal tetanic stimulation (80 Hz, 0.4 seconds duration) that resulted in a 50% force decrease at time zero. Those skilled in the art will recognize that different frequencies and durations can be used.

[0259] Also provided is a three-dimensional skeletal muscle tissue as described herein, which is obtainable by the method of stimulating myogenesis as described herein.

[0260] While the present invention has been described in conjunction with various embodiments and examples, it is not intended that the present invention be limited to such embodiments or examples. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those skilled in the art.

[0261] While various inventive embodiments have been described and illustrated herein, those skilled in the art will readily conceive of various other means and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein, and each such variation and / or modification is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application or applications utilizing the teachings of the present invention. Those skilled in the art will recognize numerous equivalents to the specific inventive embodiments described herein. Accordingly, the above-described embodiments are provided by way of example only, and it should be understood that, within the scope of the appended claims and their equivalents, the inventive embodiments may be practiced otherwise than as specifically described and claimed. The inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and / or methods is within the scope of the present disclosure, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0262] All definitions defined and used herein should be understood to supersede dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0263] The indefinite articles "a" and "an," as used in the specification and claims, unless expressly indicated to the contrary, should be understood to mean "at least one." All ranges cited herein are inclusive.

[0264] As used throughout this specification and claims, the terms "substantially," "approximately," and "about" generally mean plus or minus 10% of the stated value, e.g., about 100 includes 90-110.

[0265] The term "and / or," as used in the specification and claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" of the elements so conjoined. Other elements, whether related or unrelated to the elements specifically identified, can optionally be present other than the elements specifically identified by the "and / or" clause. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language such as "comprising," can, in one embodiment, refer to A only (optionally including elements other than B); in another embodiment, refer to B only (optionally including elements other than A); in yet another embodiment, refer to both A and B (optionally including other elements); and so forth.

[0266] As used in this specification and the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, meaning that at least one of a number or list of elements is included, and more than one element is included, optionally including additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of" or "only any one of," or "consisting of," as used in the claims, mean that only one element of a number or list of elements is included. Generally, the term "or" as used herein will be interpreted as indicating exclusive alternatives (i.e., "either / or, but not both") only when preceded by terms indicating exclusivity, such as "either," "one of," "only one of," or "only one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0267] As used in this specification and claims, the phrase "at least one" in reference to a list of one or more elements should be understood to refer to at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each element specifically listed in the list of elements, and not necessarily excluding combinations of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to the elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") refers to, in one embodiment, at least one (optionally, multiple) A's, with no B's present (and optionally including elements other than B); in another embodiment, at least one (optionally, multiple) B's, with no A's present (and optionally including elements other than A's); and in yet another embodiment, at least one (optionally, multiple) A's and at least one (optionally, multiple) B's.

[0268] In the claims and the above specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "consisting of," and the like, are understood to be open-ended, i.e., to mean including, but not limited to. Only the transitional phrases "consisting of" and "essentially consisting of" shall be closed or semi-closed transitional phrases, respectively, as defined in U.S. Patent Office Manual of Patent Examining Procedure Section 2111.03.

[0269] As used in this specification and claims, the term "substantially the same" refers to a first value being within 10% of a second value. For example, if A is substantially the same as B and B is 100, the value of A can range from 90 to 110. If A is substantially the same as B and B is 200, the value of A can range from 180 to 220.

[0270] As used herein, the term "POMaC" refers to poly(octamethylene maleic anhydride) citric acid) (POMaC) or a POMaC prepolymer comprising a mixture of 1,8-octanediol, citric acid, and maleic anhydride. See Tran et al., "Synthesis and characterization of a biodegradable elastomer featuring a dual crosslinking mechanism," Soft Matter, Jan 1, 2010;6(11):2449-2461, the entire contents of which are incorporated herein by reference.

[0271] As used herein, the term "test agent" refers to any substance being evaluated for its ability to diagnose, cure, mitigate, treat, or prevent disease in a subject, or any substance intended to alter the structure or function of a subject's body. In embodiments, the test agent may be a "drug," as that term is defined in Section 321(g)(1) of the Federal Food, Drug, and Cosmetic Act. Test agents may also be referred to as "pharmacological agents," including, but not limited to, chemical compounds, biologicals, proteins, peptides, antibodies, nucleic acids, lipids, polysaccharides, supplements, diagnostic agents, and immunomodulators.

[0272] As used herein, the term "toxicity" is defined as an undesirable effect (including unnecessary or overly exaggerated pharmacological effects) on human cells or tissues caused by a test agent, or a test agent used in combination with other pharmaceutical agents. A similar term used in this context is "adverse reaction."

[0273] As used herein, the term "non-human" refers to all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, mice, rats, etc.

[0274] Terms such as “first,” “second,” and the like may be used herein to describe various elements, components, regions, layers, and / or sections, but it will be understood that these elements, components, regions, layers, and / or sections are not limited by these terms. These terms are used only to distinguish one element, component, region, layer, or section from another element, component, region, layer, or section. Thus, a first element, component, region, layer, or section described below may be referred to as a second element, component, region, layer, or section without departing from the teachings of the example embodiments. Spatially relative terms such as “below,” “bottom,” “lower,” “above,” and “top” may be used herein for convenience of description to describe the relationship of one element or feature to another element(s) or feature(s), as shown in the figures. It will be understood that spatially relative terms are intended to encompass various orientations of the device during use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures were turned over, elements described as "below" or "beneath" other elements or features would then be positioned "above" the other elements or features. Thus, the exemplary term "below" can encompass both an orientation of above and below. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.

[0275] As used herein, the term "hydrogel" refers to a physically or chemically crosslinked polymer network that can absorb large amounts of water and is commonly used to form tissue engineering scaffolds. These can be classified into different categories depending on various parameters, such as the manufacturing method, loading, and mechanical and structural properties. See S. Van Vlierberghe et al., "Biopolymer-Based Hydrogels as Scaffolds for Tissue Engineering Applications: A Review," Biomacromolecules, 2011, 12(5), pp. 1387-1408, the contents of which are incorporated herein by reference.

[0276] As used herein, the term "pulse" refers to a single electrical stimulation event in which the applied voltage or current varies from a steady-state baseline, with each pulse generally consisting of a single positive phase and a single negative phase. The term "waveform" is used to describe the shape of an individual pulse. Each electrical pulse may include either a monophasic or biphasic waveform, which may be, for example, asymmetric, symmetric, square, sinusoidal, etc. A pulse is defined by its pulse width (i.e., the time elapsed between the rising and falling edges of the pulse) and / or pulse amplitude.

[0277] As used herein, the term "pulse train" refers to a series of individual pulses. A pulse train may be defined by the number of pulses in the train and / or the duration of the train (i.e., the length of time between the first and last pulses in the pulse train). A pulse train may be defined by the pulse repetition frequency (the number of pulses in a specified unit of time, in Hz) and / or the pulse interval (the duration between corresponding points in successive pulses) and / or the duty cycle (the fraction of one period that the signal is active; a period is the time it takes for the signal to complete an on and off cycle; duty cycle = (pulse width x 100%) / total period of the signal).

[0278] As used herein, the term "spacing" refers to the duration and distance between successive pulse trains. The spacing between pulse trains may also be referred to as a relaxation period.

[0279] As used herein, the term "myogenesis" refers to the formation of skeletal muscle tissue.

[0280] As used herein, the term "encapsulated or embedded within a hydrogel" refers to cells that are immobilized within the surrounding hydrogel. This term is intended to cover embodiments in which all cells are completely contained and surrounded by the hydrogel, as well as embodiments in which some cells are completely contained and surrounded by the hydrogel and some cells penetrate and / or are located on the surface of the hydrogel.

[0281] The term "artificial" as used herein with respect to skeletal tissue refers to tissue that is grown and differentiated outside the body, and does not include skeletal tissue that is grown and / or matured inside the body (e.g., does not include muscle tissue removed from a living organism and embedded in a hydrogel). "Artificial" may be used interchangeably with "in vitro."

[0282] As used herein, the term "anchor" refers to an element that serves as an anchor point and allows attachment of the formed 3D skeletal muscle tissue between them, thereby suspending the 3D skeletal muscle tissue (e.g., above the bottom of the well).

[0283] As used herein, the term "flexible anchor" refers to an anchor that can bend or flex without breaking. A flexible anchor can deform in response to contractile forces exerted by 3D skeletal muscle tissue.

[0284] Unless otherwise specified, the claims should not be construed as limited to the described order or elements. Those skilled in the art should recognize that various changes in form and detail can be made without departing from the spirit and scope of the appended claims. All embodiments that come within the spirit and scope of the following claims and equivalents thereof are claimed. [Example]

[0285] Example 1 Human skeletal muscle cells (SkMCs) (catalog number C-12530) were purchased from PromoCell at passage 2. Cells were maintained in growth medium (catalog numbers C23260 and C-39365) and expanded according to the manufacturer's instructions. Cells from passages 3 to 5 were used for experiments. After detachment with trypsin / EDTA solution, SkMCs were embedded in a hydrogel with the following composition: rat tail collagen 3 mg / mL, human fibrinogen 8.2 mg / mL, Matrigel® 1:9, and thrombin 50 units / mL (1 / 25 of the total volume). In some experiments, collagen was not used. Tissues with viable SkMCs at a cell density of 30,000 to 100,000 were expected. In some experiments, human cardiac fibroblasts were added at a ratio of 1:10. SkMCs were maintained in growth medium for 2 days, then in differentiation medium (PromoCell catalog number C-39366) for 1 week, and then maintained in growth medium until the end of the study.

[0286] SkMC tissue compaction occurred adequately 24 h after seeding and progressed gradually over 2–3 days. The presence of fibroblasts did not significantly affect tissue compaction (Figure 1).

[0287] One week after tissue seeding, external electrical stimulation was applied. To test the hypothesis that electrical stimulation plays an important role in the maturation and functionality of skeletal tissue, some tissue was left unstimulated as a control. Stimulation started at 1 Hz and increased by 0.3 Hz per day. Medium changes occurred every two days.

[0288] The first appearance of contractions in response to external stimulation occurred after 4 days of stimulation. No contractions were observed in unstimulated tissue (Figure 2).

[0289] SkMC tissue contractility was analyzed over time up to day 13. No increase in active force was observed over time (Figure 3).

[0290] Example 2. introduction Engineered 3D human skeletal muscle models are a promising approach to overcome the limitations of 2D in vitro systems and animal models (Broer T et al., 2020; Khodabukus A, 2021). Engineered 3D models can recapitulate complex tissue architectures by providing structural and mechanical features typical of native tissues. Embedding cells in hydrogels that can fix them in three dimensions creates cell-extramatrix (ECM) interactions that are important for outside-in biochemical signaling. The ECM not only provides a favorable environment for muscle development and function, but is also dynamic and may play an important role in the progression of conditions such as diabetes, muscular dystrophy, and muscle aging (Grzelkowska-Kowalczyk K, 2016). Furthermore, it has been demonstrated that embedding myoblasts in hydrogels anchored between two adhesion points can induce myotube alignment by recapitulating the mechanical features present in native skeletal muscle, and mechanical tension and alignment can promote sarcomere maturation (Jalal S et al., 2021). Perhaps more importantly, these advantages of engineered 3D skeletal tissue models allow for the promotion of physiologically relevant contractile phenotypes. While various 3D skeletal muscle models have been proposed over the past decade ( Jalal S et al., 2021 ; Cho S, Jang J, 2021 ), no current model fully recapitulates all aspects of complex skeletal muscle physiology.

[0291] Here, we used the Biowire™ II platform to generate engineered 3D human skeletal muscle tissue. The Biowire™ II platform consists of two elastic polymer wires that suspend the tissue, recreating the mechanical tension typical of skeletal muscle's native environment (Figure 5Ai). The elastic polymer wires are flexible (and fluorescent), allowing for measurement of contractile force. We demonstrated the differentiation of human myoblasts into myotubes and increased twitch and tetanic forces. Seven days after seeding the tissue onto the Biowire™ II platform, electric field stimulation was applied to a subset of the tissue. Electrical stimulation improved myotube alignment and fatigue resistance compared to unstimulated tissue. Electrical stimulation also induced a switch from fast to slow myosin heavy chain (MyHC) isoforms, a change commonly reported in vivo with exercise training (Demirel HA et al., 1999; Short KR et al., 2005). Furthermore, the 3D skeletal muscle tissue responded predictably to compounds with known mechanisms of action. Together, these results demonstrate an engineered 3D human skeletal muscle model that recapitulates key features of human muscle physiology, which can be used for drug discovery and disease modeling applications.

[0292] result Engineered human skeletal tissue on the Biowire™ II platform.

[0293] Human skeletal muscle tissue was generated by encapsulating primary human skeletal muscle cells (skMDCs) in a hydrogel composed of Matrigel and fibrin, as described in the Materials and Methods section, and then seeding the cell suspension onto a Biowire™ II platform. To mimic skeletal muscle physiology, fibroblasts were added at a 1:20 ratio to provide structural support through ECM protein synthesis (Chapman MA et al., 2016) and stimulate myogenesis (Mackey AL et al., 2017). Under these experimental conditions, myoblasts and fibroblasts self-organized and gradually remodeled the hydrogel after 24 hours to form tissue suspended between two polymer wires (Figure 5Ai). The skeletal tissue was maintained within the platform for 2 days before differentiation was initiated and then monitored for an additional 33 days. On day 7 of differentiation, the skeletal tissue was divided into groups; one group received an exercise regimen consisting of intermittent external electrical stimulation, while the other group was maintained as a control culture without stimulation (Figure 5Aii).

[0294] Confocal analysis of skeletal muscle tissue immunostained for F-actin demonstrated that at the end of the maturation protocol, the skeletal tissue was composed of multiple myotubes (Figure 5Bi), which were distributed in three dimensions (Figure 5Bii). One of the distinctive properties of skeletal muscle in vivo is the presence of sarcomeres, which are responsible for muscle force generation. Super-resolution images acquired using the SoRa spinning disk confocal system (see Materials and Methods) enabled visualization and quantification of sarcomeres within myotubes. The sarcomere length, measured as the distance between the Z-lines, was 2.34 ± 0.09 µm (Figure 5Biii), which is similar to the sarcomere length of approximately 2.4 µm reported for whole rat muscle (Moo et al., 2016; 2018).

[0295] Effect of differentiation time and Biowire™ II platform on the structure and function of engineered skeletal tissue.

[0296] Skeletal tissue was cultured in differentiation medium and structurally analyzed over time. F-actin staining revealed enhanced myotube formation over time (Figure 6Ai). Quantification of myotube diameter revealed a statistically significant increase in diameter at day 21 compared to day 7 (8.22 ± 0.90 μm at day 7 and 11.76 ± 0.3 μm at day 21). At day 35, we observed no further increase in myotube diameter compared to day 21 (Figure 6Aii). Furthermore, combining the Biowire™ II platform with differentiation-time-regulated developmental genes, embryonic genes myogenin and embryonic (Myh3) MyHC, increased in expression from day 7 to day 21 and decreased in expression by day 35. Neonatal (Myh8) MyHC expression increased as a function of differentiation time. Interestingly, the expression of adult MyHC isoforms (Myh1, Myh2, and Myh7) increased in engineered skeletal tissue starting at day 21 (Figure 6B). The Biowire™ II platform allows for the measurement of contractility parameters by imaging the deflection of the polymer wire as a function of time, as previously reported (Figure 5iii, iv) (Feric et al., 2019). A key characteristic of skeletal muscle is its ability to generate tetanus, which consists of the muscle's ability to sustain a contraction triggered by the firing of action potentials at a very high rate by innervating motor neurons. We mimicked this high-speed stimulation in vitro by applying electrical stimulation at frequencies ≤100 Hz. We calculated the amplitude, rise time (time to amplitude), rise (contraction) slope, and decay (relaxation) time to 50% of 1 Hz twitch and tetanus contractions generated using high frequencies (Figure 5iv). After 7 days of differentiation, skeletal tissue was already able to generate tetanic force with high-frequency stimulation (24.8 ± 10.3 μN at 100 Hz and 3.9 ± 1.6 μN at 1 Hz) (Figure 6Ci). When monitored longitudinally, the skeletal tissue generated a constant force at 1 Hz stimulation throughout the entire observation period (up to 35 days). However, when stimulated at 100 Hz, the tissue increased its tetanic force during the first 3 weeks (days 7–21) and then maintained the same amount of force for the remaining 2 weeks of observation (Figure 6Cii).Similar to 100 Hz, skeletal tissue exhibited a time-dependent increase in twitch amplitude at 5 Hz, 10 Hz, and 20 Hz, as shown in the representative traces in Figure 6D.

[0297] Exercise regulates skeletal tissue morphology, gene expression, contractility, and fatigability.

[0298] On day 7 of tissue differentiation, we applied a 4-week intermittent electrical stimulation protocol. F-actin immunostaining demonstrated that exercise promoted myotube formation (Figure 7Ai), and we observed a statistically significant increase in muscle fiber diameter in exercised tissue (2-week stimulated tissue) at day 21 compared to unexercised (unstimulated) tissue (Figure 7Aii). We also observed enhanced contractility with exercise. Notably, after 1 week of stimulation, exercised skeletal tissue showed a significant increase in twitch amplitude and ascending (contraction) slope at both 1 Hz and 100 Hz stimulation compared to unstimulated tissue. Although the force generated by stimulated tissue reached a plateau after 2 weeks of stimulation and subsequently decreased, the force generated by stimulated tissue was significantly higher than that of unstimulated tissue at all time points (Figure 7Bi,ii, Figure 9A, Figure 9B). The ascending (contraction) time also increased with exercise, but only at later time points (Figure 7Biii). Finally, exercised skeletal tissue showed a 50% decrease in decay (relaxation) time when stimulated at 100 Hz compared to unstimulated skeletal tissue (Figure 7Biv). Taken together, these data demonstrate that the electrical stimulation regimen applied to exercise skeletal tissue resulted in a dramatic modulation of skeletal contractility parameters.

[0299] Skeletal muscle fatigue, the loss of force in response to contractile activity, occurs both physiologically and in pathological conditions. Under physiological conditions, exercise training can improve muscle fatigue (Hunter SK, 2018). To examine whether our exercise program could mimic physiological function, we subjected the skeletal tissue to repeated submaximal tetanic stimulation for 10 minutes on day 7, leading to a fatigue state (measured as a 50% force loss). We then applied the same fatigue protocol throughout the observation period and measured fatigue weekly until day 35. Figure 7C shows that fatigue resistance of skeletal tissue improved by 3–4 weeks after the start of the exercise program compared to unexercised tissue.

[0300] To investigate the molecular profile underlying the functional changes mediated by electrical stimulation, the major MyHC isoforms known to be involved in skeletal contractility were analyzed. Interestingly, unstimulated tissue expressed more type IIx (fast-twitch) MyHC than stimulated tissue, whereas exercised tissue expressed more type IIa (slow-twitch) MyHC than unstimulated tissue (Figure 7D). A transition from type IIx (fast-twitch) to type IIa (slow-twitch) MyHC isoforms is commonly observed in vivo following exercise training (Demirel HA et al., 1999; Short KR et al., 2005).

[0301] Skeletal tissue exhibits the expected contractile response to chronic treatment with known compounds.

[0302] We investigated the response capacity of engineered skeletal muscle tissue to compounds with known mechanisms of action and the contribution of exercise training to these pharmacological responses. Clenbuterol is a β2 agonist that has been reported to increase skeletal muscle mass in some animal models (Zeman et al., 1998) and can partially prevent or reverse muscle loss in experimental models of muscle atrophy (Lynch and Ryall, 2008). Furthermore, it has been demonstrated to reduce fat mass in young, healthy individuals (Hostrup M, Onslev J, 2021). However, its potential as an anti-wasting and anti-obesity drug remains controversial. Indeed, clenbuterol was banned in the United States due to concerns about skeletal and cardiac toxicity (Burniston JG et al., 2002; Burniston JG et al., 2005; Barry AR, Graham MM, 2013). Skeletal tissues subjected to 2 weeks of exercise and time-matched unstimulated tissues were chronically treated with 0.1 μM clenbuterol for 2 weeks. This dose has been demonstrated to improve contractility in several in vitro skeletal muscle models (Madden L. et al., 2015). Data show that after 1 or 2 weeks of treatment at all stimulation frequencies (1 Hz, 5 Hz, 10 Hz, 20 Hz, and 100 Hz), clenbuterol treatment significantly increased contractile amplitude in stimulated tissues compared to controls (tissues treated with PBS vehicle). In contrast, chronic clenbuterol administration did not affect contractility in unstimulated tissues (Figure 8Ai). Quantification of kinetic parameters revealed that rise (contraction) time was dramatically reduced in stimulated tissues but was unaffected in unstimulated tissues compared to untreated controls (Figure 8Aii). However, decay (relaxation) time was unaffected in either stimulated or unstimulated tissues compared to untreated controls (Figure 10A).

[0303] Dexamethasone, like other glucocorticoids, is widely used to treat neuromuscular diseases; however, long-term side effects, such as muscle atrophy, are well known (Schaecke H. et al., 2002; Schakman O. et al., 2013; Gupta A. et al., 2013). One week of dexamethasone treatment dramatically reduced the force generated in both stimulated and unstimulated tissue. High-frequency stimulation force was further reduced after two weeks of dexamethasone treatment (Figure 8B). Regarding contractile kinetic parameters, two weeks of dexamethasone treatment dramatically reduced the decay time by 50% in unstimulated tissue (Figure 10Bi), but did not affect the rise time in either stimulated or unstimulated tissue (Figure 10Bii). Finally, tissues were treated for two weeks with dexamethasone alone or a combination of dexamethasone and clenbuterol. Figure 8C shows that clenbuterol mitigated some of the effects of dexamethasone on stimulated tissue, as demonstrated by comparing the peak amplitudes of tissue exposed to clenbuterol and dexamethasone with those of tissue exposed to dexamethasone alone. Specifically, after 2 weeks of treatment, the mean peak amplitudes relative to vehicle for stimulated tissue exposed to clenbuterol and dexamethasone compared with those of stimulated tissue exposed to dexamethasone alone were 0.052 vs. 0.0064, 0.024 vs. 0.006, 0.023 vs. 0.005, and 0.014 vs. 0.004 for stimulations at 5 Hz, 10 Hz, 20 Hz, and 100 Hz, respectively. Thus, as shown in Figure 8A, we confirmed that clenbuterol had no effect on unstimulated skeletal tissue.

[0304] Discussion Many skeletal muscle disorders remain untreated due to a lack of knowledge about skeletal muscle pathophysiology and disease progression, as well as a lack of predictive models for drug development, resulting in treatments that are often symptomatic (Stephenson D et al., 2022).

[0305] Herein, we provide a platform and protocol that enables the generation of engineered 3D human skeletal muscle tissue. We first optimized a tissue engineering protocol that allows the fusion of human myoblasts into myotubes over time in culture. Myogenesis involves a series of events, including stem and progenitor cell commitment, proliferation, differentiation, and fusion, leading to the generation of multinucleated skeletal muscle fibers (Bentzinger CF et al., 2012). These events are tightly controlled by the expression of developmental genes. Myogenin is a transcription factor that controls muscle cell fusion during development and is specifically expressed during terminal myoblast differentiation in embryonic development (Andres V and Walsh K, 1999; Guo K et al., 1995; Zhang P et al., 1999). Developing mammalian skeletal muscle is also characterized by the expression of specific myosin isoforms, including fetal and neonatal myosin heavy chains, which are contractile proteins that are transiently expressed during development. After birth, developmental myosin isoforms disappear and are replaced by adult myosin isoforms (fast and slow myosins) (Schiaffino et al., 2015). We found that longer differentiation times promote myotube formation, as evidenced by an increase in myotube diameter as a function of time cultured in differentiation medium. After 5 weeks of differentiation, skeletal muscle tissue consisted of three-dimensionally distributed myotubes, exhibiting sarcomere organization and sarcomere length similar to in vivo reports (Moo et al., 2016; 2018). Furthermore, increased differentiation time modulates developmental genes. In particular, the early (embryonic) developmental genes, myogenin and embryonic myosin heavy chain (MYHC3), increased from days 7 to 21 of differentiation, then decreased at later time points (day 35). The neonatal gene, myosin heavy chain (MYH8), gradually increased in a time-dependent manner, whereas expression of adult myosins (MYHC1, MYHC2, MYHC7) increased at differentiation day 21. These data demonstrate that by recapitulating physiology, the Biowire™ II platform and differentiation protocol generated skeletal muscle tissue with physiologically relevant morphology and developmental gene expression.

[0306] Skeletal muscle contractions are triggered by action potentials initiated by motor neurons and then propagating along the muscle cell membrane. To characterize the contractility of engineered skeletal muscle tissue, we applied external electrical stimulation mimicking innervation. A key characteristic of skeletal muscle is its ability to generate tetanic contractions (Sweeney HL, Hammers DW, 2018), which consist of the muscle's ability to sustain contractions triggered by high-frequency action potentials. Compared to twitch contractions (triggered by low-frequency action potentials), tetanic contractions are more physiologically relevant. Our engineered skeletal muscle tissue exhibited fully fused tetanus in response to 100 Hz electrical stimulation by day 7. Furthermore, tetanic force increased with increasing stimulation frequency and maturation time.

[0307] Next, we exercised engineered 3D skeletal muscle tissue by applying chronic, intermittent external electrical stimulation. Chronic stimulation promoted myotube formation and enhanced force compared to unstimulated tissue. Specifically, after 2 weeks of electrical stimulation, the 3D skeletal muscle tissue generated twitch and tetanic forces that were four and eight times greater, respectively, than those generated by time-matched unstimulated tissue. This increase in force in response to electrical stimulation is consistent with previous observations in skeletal mouse and human models (Huang YC et al., 2006; Ito A et al., 2014; Khodabukus A et al., 2019). However, our engineered skeletal tissue outperformed known models, demonstrating increases in maximum twitch and tetanic forces.

[0308] We observed that while both twitch and tetanic forces of stimulated 3D skeletal muscle tissue rapidly increased after only one week of stimulation, force values ​​began to decrease by the fourth week of stimulation. The decrease in force after four weeks of stimulation is likely due to the contraction of some myotubes over time. Exercise increased the uptake time during tetanic contractions and decreased half-relaxation time compared to unstimulated tissue, as well as twitch amplitude and the mathematically related uptake slope. Exercise also produced tissue with enhanced fatigue resistance compared to unstimulated tissue. Interestingly, as reported in in vivo endurance exercise training studies, improved force and fatigue resistance correlate with a shift from type IIx (fast myosin isoform) to type IIa (slow myosin isoform) myosin heavy chains (Demirel HA et al., 1999; Short KR et al., 2005). Overall, the applied exercise training resulted in engineered skeletal muscle tissue that exhibited contractile behavior similar to that seen in vivo. Furthermore, the expression of slow myosin isoforms, coupled with a delayed rise time and increased fatigue resistance in stimulated tissue, indicates that our stimulation protocol tuned myotubes toward a slow fiber phenotype. Thus, the present invention offers the advantage of being able to tailor 3D skeletal muscle tissue toward different muscle fiber phenotypes by mimicking different neural firing patterns using alternative electrical stimulation regimes (Huang YC et al., 2006). Similarly, the present invention enables the modeling of disease states by adjusting stimulation protocols. Some skeletal diseases preferentially affect specific fiber types; for example, in Duchenne muscular dystrophy, type II fibers degenerate first, while type I fibers are only affected later in the disease (Webster C. et al., 1988; Pedemonte M. et al., 1999). Furthermore, modulating the proportion of skeletal muscle fiber types is a promising therapeutic approach for many muscle diseases.These results demonstrate that skeletal tissue generated using the methods developed herein can recapitulate various skeletal fiber types and proportions in vitro, thereby 1) providing new insights into genetic and molecular pathways and 2) providing a novel drug discovery platform. Furthermore, long-term exercise has been shown to result in altered expression of exercise-induced genes and muscle fiber contractile genes. RNA-seq analysis revealed elevated expression of exercise-induced genes (e.g., ESRRG, NFATC2) and decreased expression of developmental and fatigable contractile genes (e.g., MYH8, MYH1) when comparing stimulated and unstimulated tissue (see Figure 12A). GO analysis also revealed several differentially regulated pathways affected by stimulation (see Figure 12B).

[0309] One hope of engineered 3D skeletal muscle systems is to obtain a more predictive model for drug screening than currently available models, reducing the high rate of compound failure that leads to clinical trials (Broer T. et al., 2020). To test the model's pharmacological predictability, the inventors exposed the 3D skeletal tissue to compounds with known mechanisms of action and confirmed that in vitro responses mirrored those predicted in vivo. Chronic β2-adrenergic stimulation is known to affect only slow-twitch muscle fibers in vivo, causing a transition from slow-twitch to fast-twitch muscle fiber type. This transition is associated with a positive inotropic effect. In particular, chronic treatment of animal models with β2-agonists results in increased twitch and tetanic force (Dodd SL et al., 1996; Zhang KM et al., 1996), maximal shortening velocity (Dodd SL et al., 1996), and a reduction in time to peak tension (Zhang KM et al., 1996). Recently, the ability of β2 agonists to enhance muscle strength has also been demonstrated in humans (Hostrup M et al., 2015). Herein, we demonstrate that exercised skeletal muscle tissue responds appropriately to chronic clenbuterol treatment. Specifically, two weeks of clenbuterol treatment increased twitch amplitude in exercised (stimulated) tissue but had no effect on the contractility of unstimulated tissue. We further confirmed the accuracy of our model predicting clenbuterol response by analyzing contraction velocity. Treatment of exercised skeletal tissue with clenbuterol resulted in faster contraction velocity than stimulated controls, suggesting that clenbuterol has the effect of inducing a transition from slow to fast fiber types.

[0310] Two weeks of exposure to dexamethasone resulted in a dramatic decrease (up to 90%) in both twitch and tetanic forces in engineered skeletal tissue. Glucocorticoid-induced muscle dysfunction, including muscle weakness, is widely recognized and is thought to be due to atrophy (Shin YS et al., 2000; Schakman O. et al., 2013). Furthermore, by simultaneously treating tissue with clenbuterol and dexamethasone, we demonstrated that clenbuterol mitigated some of the effects of dexamethasone on stimulated tissue. Taken together, these data demonstrate that our skeletal model can be used to predict pharmacological responses.

[0311] Two weeks of exposure to altered gene expression in engineered skeletal tissue. Specifically, RNA-seq analysis shows upregulation of glucocorticoid-induced genes, including the atrogen TRIM63 and FBXO32, in dexamethasone-treated tissue (see Figure 13A). GO analysis also shows that several distinct regulatory pathways are altered by dexamethasone treatment (see Figure 13B).

[0312] Materials and Methods Engineered human skeletal tissue formation Primary human skeletal muscle cells (skMDCs) (Cook Myosite, Pittsburgh, PA) and dermal fibroblasts (Lonza, Allendale, NJ) were maintained in 2D culture according to the manufacturer's recommendations. To form skeletal tissue, cells were embedded in a hydrogel containing 5 mg / mL fibrinogen, 1:5 Matrigel, and 25 U / mL thrombin (1 / 25 of the total volume) and seeded onto a Biowire™ II platform, consisting of polystyrene microwells containing parallel poly(octamethylene maleic anhydride) citrate (POMaC) wires (Feric NT et al., 2019). Each tissue consisted of 90,000 skMDCs and 4,500 dermal fibroblasts. The tissues were stored in growth medium (Cook Myosite) for 2 days and then in NbActiv4 differentiation medium (Brainbits, Springfield, IL). The medium was changed twice weekly. On day 7 of differentiation, some tissues were subjected to intermittent external electrical stimulation with a 1-second, 10 Hz pulse train (each pulse lasting 4 ms) twice daily for 10 seconds each for 1 hour in a custom chamber equipped with parallel carbon electrodes (Feric NT et al., 2019).

[0313] Immunofluorescence analysis of engineered human skeletal tissue Skeletal tissue was fixed in paraformaldehyde for 30 minutes and then permeabilized with Triton 0.1% for an additional 30 minutes at room temperature (RT). After washing with PBS, tissue was incubated with Alexa Fluor 488 Phalloidin (ThermoFisher Scientific, Waltham, MA) diluted 1:400 in PBS for 1 hour at room temperature. After washing with PBS, nuclei were counterstained with DAPI. Images were acquired using a CSU-W1 spinning disk confocal and analyzed using NIS-Elements software (Nikon Instruments Inc, Edgewood, New York). Super-resolution images were acquired using a CSU-W1 SoRa spinning disk confocal system (Nikon Instruments Inc, Edgewood, New York).

[0314] Measuring the contractility of engineered human skeletal tissue Contractility of skeletal tissue was measured by tracking the deflection of POMaC wires as a function of time, as previously described (Zaho et al., 2019). The tissue was transferred to a custom chamber equipped with parallel carbon electrodes for external electric field stimulation within an environmental chamber at 37°C and 5% CO2. The wires were illuminated using 350 nm excitation. Videos were acquired using a Zyla 4.2 sCMOS camera (Andor, South Windsor, CT) equipped with a 470 nm emission filter using NIS-Elements software. Videos were analyzed using a custom Matlab program to measure contractile force. Twitch contractions and varying degrees of tetanic contraction were obtained by stimulating the tissue for 6 seconds at 1 Hz, 5 Hz, 10 Hz, 20 Hz, and 100 Hz. Fatigue was measured as the loss of force generation during 10 minutes of repeated submaximal tetanic stimulation (80 Hz, 0.4 second duration) that resulted in a 50% force reduction at time zero.

[0315] Drug testing of engineered human skeletal tissue Two-week stimulated tissues and time-matched unstimulated tissues were treated with 0.1 μM clenbuterol and 10 μM dexamethasone (Sigma Aldrich, St. Louis, MO). Clenbuterol stock solution was prepared in PBS according to the manufacturer's recommendations. Dexamethasone stock solution was prepared in ethanol. Compounds and relative controls in experimental and control tissues were exchanged every two days during a 14-day complete medium exchange. Tissues were analyzed weekly for contractility, as described above. Contractility for each tissue was first normalized to its relative baseline and then to the vehicle control group.

[0316] Statistics Analysis was by one-way ANOVA followed by a post-hoc test (Bonferroni ad hoc test). Student's t-test was performed for paired observations. A value of p<0.05 was considered statistically significant. Statistical analysis was performed using GraphPad PRISM 8.0 software. Unless otherwise stated, all experiments were independently repeated at least three times.

[0317] Example 3, Skeletal muscle is highly metabolically active. In skeletal muscle, insulin binding to the insulin receptor (IR) initiates a signaling cascade involving the insulin receptor substrate-1 / phosphoinositide 3-kinase / Akt (IRS-1 / PI3K / Akt) pathway, leading to translocation of insulin-sensitive glucose transporter protein 4 (GLUT4) to the plasma membrane and facilitating glucose diffusion into the cell. Impairment of the signaling events leading to insulin-stimulated glucose uptake leads to insulin resistance and the development of type 2 diabetes. The causes of insulin resistance are numerous, and the underlying mechanisms are multifactorial. The development of 3D models of insulin resistance that closely mimic human physiology may elucidate novel mechanisms controlling skeletal muscle glucose uptake. Identifying the mechanisms leading to insulin resistance is essential for more effective treatment of type 2 diabetes.

[0318] Skeletal tissues were maintained in growth medium for 2 days, then in NbActiv4 differentiation medium or customized medium (Neurobasal medium + B27 supplement without insulin + 20 mM glucose + 6.9 nM insulin). Tissues were maintained in differentiation medium for 7 days without electrical stimulation, followed by chronic electrical stimulation for 7–10 days.

[0319] Figure 11A shows representative Western blots of tissues at day 7 and day 14 showing the expression of insulin receptor (InsRbeta) and GLUT4. These data demonstrate that exercised skeletal tissues expressed proteins important for insulin-stimulated glucose transport, specifically InsR and GLUT4.

[0320] Figure 11B shows representative glucose uptake in skeletal tissue in NbActiv4 medium or customized medium, with or without insulin stimulation. These data demonstrate that exercised skeletal tissue significantly increased glucose uptake in response to insulin.

Claims

1. A three-dimensional skeletal muscle tissue comprising a hydrogel, a plurality of cells including skeletal muscle cells, and two or more anchors, wherein the skeletal muscle tissue is characterized by one or more contractions in response to electrical and / or chemical stimuli, and at least one of the anchors is a flexible anchor.

2. The three-dimensional skeletal muscle tissue of claim 1 , wherein the plurality of cells further comprises fibroblasts.

3. The three-dimensional skeletal muscle tissue of claim 2, wherein the ratio of said fibroblasts to said skeletal muscle cells is about 1:5 to 1:

50.

4. The three-dimensional skeletal muscle tissue of any one of claims 1 to 3, wherein the skeletal muscle cells comprise human skeletal muscle cells.

5. 5. The three-dimensional skeletal muscle tissue of any one of claims 1 to 4, wherein the hydrogel comprises collagen or a collagen derivative, intestinal submucosa or a derivative thereof, cellulose or a cellulose derivative, proteoglycan, heparin sulfate, chondroitin sulfate, keratin sulfate, hyaluronic acid, elastin, fibronectin, thrombin, laminin, fibrin, chitosan, alginate, Matrigel®, Geltrex, agarose, decellularized extracellular matrix, polyethylene glycol or a derivative thereof, silicone or a derivative thereof, or a combination thereof.

6. The three-dimensional skeletal muscle tissue of claim 5 , wherein the hydrogel comprises collagen or a collagen derivative.

7. 7. The three-dimensional skeletal muscle tissue of claim 6, wherein the collagen comprises type I collagen, type III collagen, type IV collagen, type V collagen, type XI collagen, type XII collagen, or a combination thereof.

8. 6. The three-dimensional skeletal muscle tissue of claim 5, wherein the hydrogel comprises Matrigel® or Geltrex®.

9. The three-dimensional skeletal muscle tissue of any one of claims 1 to 8, wherein at least a portion of the cells are encapsulated or embedded within the hydrogel.

10. 10. The three-dimensional skeletal muscle tissue of claim 9, wherein at least 50% of the cells are encapsulated or embedded within the hydrogel.

11. The three-dimensional skeletal muscle tissue of any one of claims 1 to 10, further comprising fibrinogen and / or thrombin.

12. The three-dimensional skeletal muscle tissue of any one of claims 1 to 11, having from about 30,000 to about 1,000,000 cells.

13. Approximately 0.1mm ３ ~Approx. 2.5mm ３ The three-dimensional skeletal muscle tissue of any one of claims 1 to 12, having a volume of

14. The three-dimensional skeletal muscle tissue of any one of claims 1 to 13, further comprising A-bands, I-bands, Z-lines, M-lines, H-zones, or combinations thereof.

15. The three-dimensional skeletal muscle tissue of any one of claims 1 to 14, further comprising striations, elongated nuclei, sarcomeres, or a combination thereof.

16. The three-dimensional skeletal muscle tissue of any one of claims 1 to 15, further comprising acetylcholine receptors, slow muscle fibers, fast muscle fibers, or a combination thereof.

17. 17. The three-dimensional skeletal muscle tissue of any one of claims 1 to 16, wherein the expression levels of maturation-associated genes, calcium handling-associated genes, and / or sarcomeric protein-associated genes are substantially the same as the expression levels in native human skeletal muscle tissue.

18. The three-dimensional skeletal muscle tissue of any one of claims 1 to 17, wherein the one or more contractions generate a twitch force and / or a tetanic force.

19. The three-dimensional skeletal muscle tissue of any one of claims 1 to 18, wherein the chemical stimulus comprises acetylcholine, adenosine triphosphate, 4-chloro-m-cresol, or a combination thereof.

20. 20. The three-dimensional skeletal muscle tissue of any one of claims 1 to 19, characterized in that intracellular calcium concentration changes temporarily in response to electrical and / or chemical stimuli.

21. 21. The three-dimensional skeletal muscle tissue of any one of claims 1 to 20, characterized by shortened action potentials with pronounced hyperpolarization.

22. The three-dimensional skeletal muscle tissue of any one of claims 1 to 21, wherein the skeletal muscle tissue is held under tension between the anchors.

23. The three-dimensional skeletal muscle tissue of any one of claims 1 to 22, wherein the anchors can be adjusted to change the tension in the skeletal muscle tissue.

24. The three-dimensional skeletal muscle tissue of any one of claims 1 to 23, wherein the flexible anchor is an elastic sensing element.

25. 25. The three-dimensional skeletal muscle tissue of claim 24, wherein the flexible anchor comprises a synthetic polymer, a biopolymer, or a combination thereof.

26. 26. The three-dimensional skeletal muscle tissue of claim 25, wherein the polymer is degradable.

27. 26. The three-dimensional skeletal muscle tissue of claim 25, wherein the polymer is non-degradable.

28. 28. The three-dimensional skeletal muscle tissue of any one of claims 24-27, wherein the flexible anchor comprises a polymer selected from the group consisting of polylactic acid, poly(lactic-co-glycolic acid), poly(caprolactone), polyglycolide, polylactic acid, polyhydroxobutyric acid, polyhydroxyalkanoic acid, chitosan, hyaluronic acid, poly(2-hydroxyethyl methacrylate), poly(ethylene glycol), poly(L-lactic acid) (PLA), poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PMMA), poly(glycerol sebacate), poly(octamethylene maleic (anhydride) citric acid) (POMaC), citric acid-free POMaC, poly(ε-caprolactone), polyurethane, silk, and combinations thereof.

29. 30. The three-dimensional skeletal muscle tissue of claim 28, wherein the polymer comprises POMaC.

30. The three-dimensional skeletal muscle tissue of any one of claims 24 to 29, wherein the flexible anchor has an elasticity of approximately 10 kPa to 0.8 MPa.

31. The three-dimensional skeletal muscle tissue of any one of claims 1 to 30, wherein at least one of the anchors is in the form of a polymer wire, and optionally, two or more of the anchors are in the form of the polymer wire.

32. A tissue system comprising the three-dimensional skeletal muscle tissue of any one of claims 1 to 31 and a bioreactor, wherein the bioreactor: a device having a well configured to grow the three-dimensional skeletal muscle tissue from cells seeded in the well, the well having a bottom; and two or more anchors positioned across the well such that a gap exists between the anchors and a bottom of the well, at least one of the anchors being a flexible anchor, the anchors being configured to allow attachment of the three-dimensional skeletal muscle tissue formed therebetween, thereby suspending the three-dimensional skeletal muscle tissue above the bottom of the well, the flexible anchor being configured to deform in response to a contractile force exerted by the three-dimensional skeletal muscle tissue on the flexible anchor.

33. 33. The tissue system of claim 32, wherein the bioreactor further comprises at least two electrodes configured to apply electrical stimulation to the three-dimensional skeletal muscle tissue of the bioreactor.

34. 34. The tissue system of claim 32 or claim 33, wherein the flexible anchor is an elastic sensing element, and optionally the two or more anchors are elastic sensing elements.

35. 35. The tissue system of claim 34, wherein the flexible anchor comprises a synthetic polymer, a biopolymer, or a combination thereof.

36. 36. The tissue system of claim 35, wherein the polymer is degradable.

37. 36. The tissue system of claim 35, wherein the polymer is non-degradable.

38. 38. The tissue system of any one of claims 34-37, wherein the flexible anchor comprises a polymer selected from the group consisting of polylactic acid, poly(lactic-co-glycolic acid), poly(caprolactone), polyglycolide, polylactic acid, polyhydroxobutyric acid, polyhydroxyalkanoic acid, chitosan, hyaluronic acid, poly(2-hydroxyethyl methacrylate), poly(ethylene glycol), poly(L-lactic acid) (PLA), poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PMMA), poly(glycerol sebacate), poly(octamethylene maleic (anhydride) citric acid) (POMaC), citric acid-free POMaC, poly(ε-caprolactone), polyurethane, silk, and combinations thereof.

39. 39. The tissue system of claim 38, wherein the polymer comprises POMaC.

40. The tissue system of any one of claims 34 to 39, wherein the flexible anchor has an elasticity of about 10 kPa to 0.8 MPa.

41. The tissue system of any one of claims 32 to 40, wherein the two or more anchors are in the form of polymer wires.

42. 41. The tissue system of any one of claims 32 to 40, wherein the bioreactor comprises 2 to 25 anchors per well.

43. The tissue system of any one of claims 32 to 42, wherein the bioreactor comprises a multi-well plate.

44. 44. The tissue system of claim 43, wherein the multiwell plate comprises 6 wells, 8 wells, 12 wells, 24 wells, 96 wells, 384 wells, or 1536 wells.

45. A method for measuring the effect of a test agent on contraction using the tissue system of any one of claims 32 to 44, comprising: measuring a first value of a contractile property of the three-dimensional skeletal muscle tissue in the bioreactor prior to exposure to the test agent; contacting the three-dimensional skeletal muscle tissue with the test agent for an incubation period under conditions sufficient for the test agent to modulate the contraction; measuring a second value of the contractile property of the three-dimensional skeletal muscle tissue after exposure to the test agent; and determining whether the test agent modulates the contraction by comparing the first value with the second value.

46. 46. ​​The method of claim 45, wherein the test agent modulates the contraction if there is a significant difference between the first value and the second value.

47. 47. The method of claim 45 or claim 46, wherein the test agent is selected from the group consisting of a small molecule, an antibody, an ion, a protein, a peptide, a lipid, DNA, RNA, a virus, a bacterium, a microparticle, a nanoparticle, a therapeutic agent, and a toxin.

48. 48. The method of any one of claims 45 to 47, wherein the incubation time is at least 7 days.

49. 49. The method of claim 48, wherein the incubation time is at least 12 days.

50. A method for measuring the effect of a test agent on calcium transport using the tissue system of any one of claims 32 to 44, comprising: measuring a first value of a calcium transport property of the three-dimensional skeletal muscle tissue in the bioreactor prior to exposure to the test agent; contacting the three-dimensional skeletal muscle tissue with the test agent for an incubation period under conditions sufficient for the test agent to modulate the calcium transient; measuring a second value of the calcium transport property of the three-dimensional skeletal muscle tissue after exposure to the test agent; comparing the first value to the second value to determine whether the test agent modulates the calcium transport.

51. 51. The method of claim 50, wherein measuring the first value or the second value comprises measuring a fluorescent signal of an intracellular calcium indicator within the three-dimensional skeletal muscle tissue.

52. 52. The method of claim 51, wherein the intracellular calcium indicator is selected from Fura-4F AM, Fura-2, Fluo-3, Fluo-4, Indo-1, Mag-Fura-5, and Mag-Fura-red.

53. 53. The method of any one of claims 50 to 52, wherein the test agent modulates the calcium transient if there is a significant difference between the first calcium transient and the second calcium transient.

54. 54. The method of any one of claims 50 to 53, wherein the test agent is selected from the group consisting of a small molecule, an antibody, an ion, a protein, a peptide, a lipid, DNA, RNA, a virus, a bacterium, a microparticle, a nanoparticle, a therapeutic agent, and a toxin.

55. 55. The method of any one of claims 50 to 54, wherein the incubation time is at least 7 days.

56. 56. The method of claim 55, wherein the incubation time is at least 12 days.

57. 1. A method for stimulating myogenesis in vitro, comprising: (i) providing a hydrogel and a plurality of cells, including skeletal muscle cells, in a well of a bioreactor, the well having a bottom, two or more anchors positioned across the well such that a gap exists between the anchors and the bottom of the well, and at least one of the anchors is a flexible anchor; (ii) stimulating the skeletal muscle cells in the well with a series of electrical pulse trains separated by intervals over a period of time.

58. 58. The method of claim 57, wherein each train has a duration of between 0.5 and 4 seconds.

59. 59. The method of claim 58, wherein each train has a duration of between 0.5 and 2 seconds.

60. 60. A method according to any one of claims 57 to 59, wherein each train has a frequency of 5 to 15 Hz.

61. 61. The method of claim 60, wherein each train has a frequency between 8 and 12 Hz.

62. A method according to any one of claims 57 to 61, wherein each train has a duty cycle of 1 to 10%.

63. 63. A method according to any one of claims 57 to 62, wherein the pulses in each train have a voltage of between 3 and 10V.

64. 64. The method of any one of claims 57 to 63, wherein the interval is between 5 seconds and 60 minutes.

65. 65. The method of claim 64, wherein the interval is between 5 seconds and 60 seconds.

66. 66. The method of any one of claims 57 to 65, wherein the period of time is between 10 minutes and 24 hours.

67. 67. The method of claim 66, wherein the period of time is between 30 minutes and 2 hours.

68. 68. The method of any one of claims 57 to 67, further comprising: (iii) repeating step (ii) every 6 to 18 hours for a fixed duration.

69. 69. The method of claim 68, wherein step (ii) is repeated every 10 to 14 hours for a fixed duration.

70. 70. The method of claim 68 or claim 69, wherein the duration is from 1 to 6 weeks.

71. 71. The method of claim 70, wherein the duration is 3 to 5 weeks.

72. 72. The method of any one of claims 57 to 71, wherein at least a portion of the cells are encapsulated or embedded within the hydrogel.

73. 73. The method of any one of claims 57 to 72, comprising growing the skeletal muscle cells in the wells in growth medium for 1 to 3 days.

74. 74. The method of any one of claims 57 to 73, comprising differentiating the skeletal muscle cells in the well in a differentiation medium for 3 to 50 days.

75. 75. The method of any one of claims 57 to 74, wherein the differentiation medium comprises glucose and insulin.

76. 76. The method of claim 75, wherein the glucose concentration in the differentiation medium is 5 to 30 mM.

77. 77. The method of claim 75 or 76, wherein the insulin concentration in the differentiation medium is 0.5 nM to 10 nM.

78. 1. A method for stimulating myogenesis in vitro, comprising: (i) providing a hydrogel and a plurality of cells, including skeletal muscle cells, in a well of a bioreactor, the well having a bottom, two or more anchors positioned across the well such that a gap exists between the anchors and the bottom of the well, and at least one of the anchors is a flexible anchor; (ii) differentiating the skeletal muscle cells in the well in a differentiation medium containing glucose and insulin.

79. 79. The method of claim 78, wherein at least a portion of the cells are encapsulated or embedded within the hydrogel.

80. The method of claim 78 or 79, wherein the hydrogel and the plurality of cells are in a tissue system according to any one of claims 32 to 44.

81. 81. The method of any one of claims 78 to 80, comprising growing the skeletal muscle cells in the wells in growth medium for 1 to 3 days.

82. 82. The method of any one of claims 78 to 81, wherein the skeletal muscle cells are differentiated in the differentiation medium for 3 to 50 days.

83. 83. The method of any one of claims 78 to 82, wherein the glucose concentration in the differentiation medium is 5 to 30 mM.

84. 84. The method of any one of claims 78 to 83, wherein the insulin concentration in the differentiation medium is between 0.5 nM and 10 nM.

85. 1. A method for stimulating myogenesis in vitro, comprising: (i) providing a hydrogel and a plurality of cells, including skeletal muscle cells, in a well of a bioreactor, the well having a bottom, two or more anchors positioned across the well such that a gap exists between the anchors and the bottom of the well, and at least one of the anchors is a flexible anchor; (ii) growing the skeletal muscle cells in the wells in growth medium for 1 to 3 days; (iii) differentiating the skeletal muscle cells in the wells for 3 to 50 days in a differentiation medium containing glucose and insulin; (iv) stimulating the skeletal muscle cells in the well with a series of electrical pulse trains separated by intervals over a period of time.

86. The three-dimensional skeletal muscle tissue of any one of claims 1 to 31, wherein the three-dimensional skeletal muscle tissue is obtained by a method according to any one of claims 57 to 85.

87. A method for monitoring the maturation of three-dimensional skeletal muscle tissue according to any one of claims 1 to 31, comprising: (i) confirming the presence of myotubes in said tissue; (ii) confirming the presence of sarcomeres in myotubes within said tissue; and / or (iii) measuring the fatigability of said tissue; and / or (iv) determining whether exercise-induced genes (e.g., ESRRG, NFATC2) are upregulated and / or whether developmental and fatigable contraction genes (e.g., MYH8, MYH1) are downregulated.

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