Artificial skeletal muscle tissue

Abstract

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

Description

ARTIFICIAL SKELETAL MUSCLE TISSUETechnical Field

[0001] The present disclosure relates generally to artificial skeletal muscle tissues and use thereof.Background

[0002] As one of three major muscle types, skeletal muscle is a form of striated muscle tissue, which is under the 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 include a spectrum of disorders that include genetic mutations such as Duchenne Muscular Dystrophy (Yedigaryan L, Cells 2021), and dysfunctions secondary to aging, inflammation, cancer (cachexia), injury, infections (Salucci et al, 2021; Rudroff et al., 2021). More recently, clinical studies have reported fatigue or muscle weakness as one of the longterm symptoms of post-COVID-19 infection (Huang C. et al., Lancet, 2021). Regardless of the etiology, skeletal muscle disorders result in muscle degeneration and impairment of the patient’s life quality (Constantin-Teodosiu D et al., 2021). Unfortunately, to date the underlying pathology of many skeletal muscle-related diseases is not fully understood, limiting treatment options. This clinical problem is mainly due to the limited predictive value / capability of the models currently available, specifically 2D in vitro and animal models.

[0004] The main limitation of animal models is the inability to reliably recapitulate human physiology because of species differences, resulting in inaccurate drug response predictions and unsuccessful clinical translation (Hay T al., 2014). Additional disadvantages include the high cost and intensive labor requirements for animal studies (Lowe DA, Alway SE, 2002; Sztretye M et al., 2020). In vitro 2D models of skeletal muscle are less labor-intensive than animal models, however, their limitations include a difficulty to maintain cultures in the long-term resulting in cellular immaturity characterized by a lack of cellular alignment or tissue architecture consistent with mature native musculature, and a lack of a mature contractile response (Guo X et al., 2014; Cooper ST et al., 2004). Additionally, to date methodologies to assess 2D in vitro skeletal muscle contractility do not exist. The inability to fully recapitulate the organization and function of mature skeletal muscle in 2D inevitably limits their utility for new insights in muscle pathophysiology anddrug discovery.

[0005] This clinical need prompts the development of novel in vitro methods that are better able to model the complex pathophysiology of human skeletal muscle and in turn advance the development of new treatments.Summary

[0006] Embodiments described herein relate generally to three-dimensional (3D) artificial skeletal muscle tissues, methods of producing the tissues, and methods of using the tissues.

[0007] One aspect of the present disclosure relates to a 3D skeletal muscle tissue comprising a hydrogel, a plurality of cells that includes skeletal muscle cells, and two or more anchors, wherein the skeletal muscle tissue is characterized by one or more contractions in response to an electrical and / or chemical stimulation, and wherein 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 fibroblasts and the skeletal muscle cells are at a ratio of between about 1 :5 and 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, a 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 inside the hydrogel.

[0014] In some embodiments, at least 50% of the cells are encapsulated or embedded inside 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 has a volume of about 0.1 mm3to about 2.5 mm3.

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

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

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

[0021] In some embodiments, expression levels of genes related to maturation of skeletal muscle tissue (e.g., myosin isoforms ratio), genes related to calcium handling, and / or genes related to sarcomeric proteins are substantially the same as those in a native human skeletal muscle tissue. Upregulation of excise-induced genes (e.g. ESRRG, NFATC2) and downregulation developmental and fatigable contractile genes (e.g., MYH8, MYH1) can also be used as a marker 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 stimulation comprises acetylcholine, adenosine triphosphate, 4-chloro-m-cresol, or a combination.

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

[0025] In some embodiments, the skeletal muscle tissue is characterized by a shortened action potential with prominent 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 modify the tension of the skeletal muscle tissue.

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

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

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

[0031] In some embodiments, the polymer is nondegradable.

[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, polylactide, polyhydroxobutyrate, polyhydroxyalcanoic acid, chitosan, hyaluronic acid, poly(2- hydroxyethyl-methacrylate), poly(ethylene glycol), poly(L-lactide) (PLA), poly(dimethysiloxane) (PDMS), poly(methylmethacrylate) (PMMA), poly(glycerol sebacate), poly(octamethylene maleate (anhydride) citrate) (POMaC), POMaC without citric acid, poly(s-caprolactone), polyurethane, silk, and a combination thereof.

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

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

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

[0036] One aspect of the present disclosure relates to a tissue system comprising a 3D skeletal muscle tissue described herein and a bioreactor, wherein the bioreactor comprises: a device having a well configured for growing the 3D skeletal muscle tissue from cells seeded therein, wherein the well has a bottom; and two or more anchors disposed across the well such that there is a gap between the anchors and the bottom of the well, wherein at least one of the anchors is a flexible anchor, wherein the anchors are configured to permit attachment of the 3D skeletal muscle tissue formed therebetween, thereby suspending the 3D skeletal muscle tissue above the bottom of the well, and wherein the flexible anchor is configured to deform in response to a contractile force 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 an 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, the two or more anchors are elastic sensing elements.

[0039] The description above in relation to the anchors (e.g., elasticity, polymers, etc.) is equally applicable to this aspect.

[0040] In some embodiments, the bioreactor comprises 2 to 25 anchors per well.

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

[0042] In some embodiments, the multi-well 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 an effect of a test agent on contraction using the tissue system described herein, comprising: measuring a first value of a contraction characteristic of the 3D skeletal muscle tissue in the 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 the contraction; measuring a second value of the contraction characteristic of the 3D 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.

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

[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, bacteria, 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 an effect of a test agent on a calcium transient using the tissue system described herein, comprising: measuring a first value of a calcium transient characteristic of the 3D skeletal muscle tissue in the 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 the calcium transient; measuring a second value of the calcium transient characteristic of the 3D skeletal muscle tissue after exposure to the test agent; and determining whether the test agent modulates the calcium transient by comparing the first value with the second value.

[0049] In some embodiments, measuring the first value or second value comprises measuring a fluorescence signal of an intracellular calcium indicator in 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, and Indo-1, Mag-Fura-5, and Mag-Fura-red.

[0051] In some embodiments, the test agent modulates the calcium transient when there is asignificant 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, bacteria, 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 of stimulating myogenesis in vitro, comprising: (i) providing a hydrogel and a plurality of cells that includes skeletal muscle cells to a well of a bioreactor, wherein the well has a bottom and two or more anchors are disposed across the well such that there is a gap between the anchors and the bottom of the well, and wherein at least one of the anchors is a flexible anchor; and (ii) stimulating the skeletal muscle cells in the well over a period of time with a series of electrical pulse trains separated by intervals.

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

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

[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 between 1 and 10%.

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

[0062] In some embodiments, the intervals are 5 s to 60 m.

[0063] In some embodiments, the intervals are 5 s to 60 s.

[0064] In some embodiments, the period of time is 10 m to 24 h.

[0065] In some embodiments, the period of time is 30 m to 2 h.

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

[0067] In some embodiments, step (ii) is repeated every 10 h to 14 h for a 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 inside the hydrogel.

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

[0072] In some embodiments, the method comprises the step of differentiating the skeletal muscle cells in the well in a differentiation medium for 3-50 days.

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

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

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

[0076] In various embodiments, there is provided a method of stimulating myogenesis in vitro, comprising: (i) providing a hydrogel and a plurality of cells that includes skeletal muscle cells to a well of a bioreactor, wherein the well has a bottom and two or more anchors are disposed across the well such that there is a gap between the anchors and the bottom of the well, and wherein at least one of the anchors is a flexible anchor; and (ii) stimulating the skeletal muscle cells in the well over 10 m to 24 h with a series of electrical pulse trains separated by intervals of 5 s to 60 m, optionally wherein each train has a 0.5 to 4 s duration, optionally wherein each train has a frequency of 5-15 Hz.

[0077] In various embodiments, there is provided a method of stimulating myogenesis in vitro, comprising: (i) providing a hydrogel and a plurality of cells that includes skeletal muscle cells to a well of a bioreactor, wherein the well has a bottom and two or more anchors are disposed across the well such that there is a gap between the anchors and the bottom of the well, and wherein at least one of the anchors is a flexible anchor; and (ii) stimulating the skeletal muscle cells in the well over 30 m to 2 h with a series of electrical pulse trains separated by intervals of 5 s to 60 s, optionally wherein each train has a 0.5 to 2 s duration, optionally wherein each train has a frequency of 8-12 Hz.

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

[0079] In various embodiments, there is provided a method of stimulating myogenesis in vitro, comprising: (i) providing a hydrogel and a plurality of cells that includes skeletal muscle cells to a well of a bioreactor, wherein the well has a bottom and two or more anchors are disposed across the well such that there is a gap between the anchors and the bottom of the well, and wherein at least one of the anchors is a flexible anchor; (ii) stimulating the skeletal muscle cells in the well over 30 m to 2 h with a series of electrical pulse trains separated by intervals of 5 s to 60 s, optionally wherein each train has a 0.5 to 2 s duration, optionally wherein each train has a frequency of 8-12 Hz; and (iii) repeating step (ii) every 6 h to 18 h for 3 to 5 weeks.

[0080] It will be appreciated by the skilled person that the parameters and aspects of the stimulation protocol may be selected in any combination from the values and ranges detailed above. In particular, the skilled person may combine the value or range of any particular train duration, train frequency, train duty cycle, pulse voltage, interval, period of time, whether step (iii) is included, the duration of step (iii), whether a growth step or differentiation step is included etc.

[0081] One aspect of the present disclosure relates to a method of stimulating myogenesis in vitro, comprising: (i) providing a hydrogel and a plurality of cells that includes skeletal muscle cells to a well of a bioreactor, wherein the well has a bottom and two or more anchors are disposed across the well such that there is a gap between the anchors and the bottom of the well, and wherein at least one of the anchors is 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 inside the hydrogel.

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

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

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

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

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

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

[0089] There is also provided the three-dimensional skeletal muscle tissue as described herein, wherein the three-dimensional skeletal muscle tissue has been obtained by a method of stimulating myogenesis as described herein.

[0090] One aspect of the present disclosure relates to a method of monitoring the maturity of the three-dimensional 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 fatiguability of the tissue; and / or (iv) determining whether exercise- induced genes (e.g. ESRRG, NFATC2) are upregulated and / or developmental and fatigable contractile genes (e.g., MYH8, MYH1) are downregulated.Brief Description of the Drawings

[0091] FIG. l is a set of optical images showing a representative in vitro human skeletal tissue, pictured in the Biowire™ II platform, composed of PromoCell skeletal muscle cells (SkMCs) with or without cardiac fibroblasts (cFBs). The presence of fibroblasts did not significantly affect tissue compaction. The optical images were acquired 12 days after cell seeding.

[0092] FIG. 2 is a series of representative contractility traces showing the active force generated by skeletal tissues. After 4 days of continuous electrical stimulation, skeletal tissues were not spontaneously beating (SPT) and when electrically stimulated were able to capture at increasing frequencies (1Hz, 2Hz).

[0093] FIG. 3 is a series of representative contractility traces showing the active force generated overtime by the skeletal tissues.

[0094] FIGs. 4A-4C are schematic diagrams showing the stimulation protocol used for maturation of skeletal tissue in Example 1 using (FIG. 4A) low frequency continuous stimulation,(FIG. 4B) high frequency intermittent stimulation with fixed intervals between pulse trains, and (FIG. 4C) high frequency intermittent stimulation with fixed intervals between pulse trains and increasing frequency of successive pulse trains.

[0095] FIG. 5 shows the Biowire™II platform supporting the generation of engineered human skeletal muscle tissues. (A) Diagrammatic representation of skeletal muscle tissue formation. Cell suspension in a hydrogel is seeded in the Biowire™II platform. Tissue compaction occurs over time, resulting in tissues suspended in between 2 polymer wires (i). Skeletal muscle tissues were maintained for 2 days in growth media then switched to differentiation media for 5 weeks. At day 7 of differentiation, a subset of skeletal tissues was exercised by electrical field stimulation (ii). Contractile forces are measured non-invasively by optically tracking the deflection of the polymer wire under electrical field stimulation (iii, iv). (B) Representative immunofluorescence images of skeletal muscle tissues composed of several myofibers stained with F-actin (green) and DAPI (blue) (i), myofiber distribution in a 30 mm z stack (ii), and quantification of sarcomere length of myofibers acquired with SoRa (super resolution) confocal system (iii). (n=8 myotubes).

[0096] FIG. 6 shows increasing time in a differentiation culture enhances myotube formation, modulates developmental genes and contractility. (A) Confocal analysis of skeletal muscle tissues immune-stained with F-Actin (i) and relative quantification of myotube diameter (ii), showing increasing myotube size over maturation period. (B) Expression of key developmental genes, myogenin, embryonic and neonatal MyHC, is modulated over maturation time. Starting from D21 skeletal muscle tissues express also adult MyHC genes. (C) Skeletal muscle tissues generate tetanic forces already at day 7 of differentiation (i). Tetanic forces in response to 100Hz electrical stimulation increase over maturation time (ii). (E) Representative traces of skeletal muscle tissues showing increasing forces at increasing electrical stimulation frequencies and over increasing maturation periods. Data represent mean ± SEM (n=20). **p<0.005.

[0097] FIG. 7 shows exercise results in tissues with enhanced myotube formation, improved force, modulation of kinetic parameters of contractility, improved fatigability and with MyHC modulations. Confocal analysis of skeletal muscle tissues immunostained with F-Actin (i) and relative quantification of myotube diameter (ii), showing increased myotube size in D21 stimulated tissues as compared to time-matched unstimulated tissues. (B) Stimulated tissues show increased peak amplitude (i), rise slope (ii), rise time (iii) at both 1Hz and 100Hz, decreased decay time at 100Hz overtime (iv). Data represent mean ± SEM (n=20). (C) Fatigability is measured as 50%force reduction during a 10 minute-repeat submaximal tetanic stimulus (i). Exercise results in tissues with increased fatigue resistance over maturation periods (ii). Data represent mean ± SEM (n=9). (D) Exercise results in the modulation of adult MyHC expression, specifically in a IIx to Ila MyHC shift. *p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001.

[0098] FIG. 8 shows engineered human skeletal tissues respond properly to clenbuterol and dexamethasone chronic treatment. (A) Chronic clenbuterol treatment increases forces produced by stimulated tissues as compared to baseline and to PBS controls (Ai) and decreases rise time (ii). (B) Chronic dexamethasone treatment decreases forces produced by both stimulated and unstimulated tissues. (C) Chronic clenbuterol treatment mitigates some dexamethasone effects in stimulated tissues. Statistic refers to unpaired t test between stimulated tissues treated with dexamethasone and stimulated tissues treated with both dexamethasone and clenbuterol. Data represent mean ± SEM. *p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001.

[0099] FIG. 9 shows exercise results in skeletal muscle tissues with improved force. Representative traces of contractility of 2 week- stimulated and time-matched unstimulated skeletal muscle tissues, showing higher forces generated by stimulated tissues at each stimulation frequency.

[0100] FIG. 10 shows kinetic parameters of skeletal muscle tissues chronically exposed to clenbuterol and dexamethasone. Clenbuterol treatment does not affect decay (relaxation) time, in either stimulated or unstimulated tissues, as compared to untreated controls (A). 2 week- dexamethasone treatment decreases decay time in unstimulated tissues, without affecting rise time in either stimulated or unstimulated tissues (Bi, ii).

[0101] FIG. 11 shows exercised skeletal tissues express key proteins for insulin-stimulated glucose transport, specifically insulin receptor (InsR) and GLUT4, and exhibit a significant increase in glucose uptake in response to insulin. (A) Representative western blotting of skeletal tissues showing expression of insulin receptor (InsRbeta) and GLUT4. P-actin has been used as protein equal loading. (B) Representative glucose uptake of skeletal tissues in NbActiv4 differentiation media or customized media, + / - insulin stimulation.

[0102] FIGs. 12A and 12B show that chronic exercise (electrical stimulation) results in altered expression of exercise-induced genes and myofiber contractile genes. A) RNAseq analysis identified upregulated exercise-induced genes (e.g. ESRRG, NFATC2) and downregulated developmental and fatigable contractile genes (e.g., MYH8, MYH1) in stimulatedvs. unstimulated tissues. B) GO analysis highlights several differentially regulated pathways impacted by stimulation.

[0103] FIGs. 13 A and 13B show that chronic dexamethasone treatment results in altered gene expression in engineered skeletal tissues. A) RNAseq analysis highlighting upregulation of glucocorticoid-induced genes, including the atrogenes TRIM63 and FBXO32 in dexamethasone- treated tissues. B) GO analysis highlights several differentially regulated pathways altered by dexamethasone treatment.Detailed Description

[0104] One aspect of the present disclosure relates to a 3D skeletal muscle tissue comprising a hydrogel, a plurality of cells that includes skeletal muscle cells, and two or more anchors, wherein the skeletal muscle tissue is characterized by one or more contractions in response to an electrical and / or chemical stimulation, and wherein at least one of the anchors is a flexible anchor.

[0105] In some embodiments, the electrical stimulation to elicit the one or more contractions can be about 0.1-100 Hz.

[0106] In some embodiments, the chemical stimulation to elicit the one or more contractions comprises acetylcholine, adenosine triphosphate, 4-Chloro-m-cresol, or a combination thereof.

[0107] In some embodiments, the plurality of cells can further include fibroblasts. In some embodiments, the number of fibroblasts and the number of skeletal muscle cells can be at a ratio of no more than about 1 :3, no more than about 1 :3.5, no more than about 1 :4, no more than about 1 :4.5, no more than about 1 :5, no more than about 1 :5.5, or no more than about 1 :6. In some embodiments, the number of fibroblasts and the number of skeletal muscle cells can be at a ratio of 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.

[0108] Combinations of the above-referenced ranges for the ratio of the fibroblasts over the skeletal muscle cells are also possible (e.g., at least about 1 :70 to no more than about 1 :3, or at least about 1 :60 to no more than about 1 :4.5), inclusive of all values and ranges therebetween. For example, the ratio of the fibroblasts over the skeletal muscle cells can be between about 1 :50 and 1 :5.

[0109] In some embodiments, the plurality of cells can further include myoblasts. The pluralityof cells can further include other cell types, including endothelial cells, immune cells, and neurons.

[0110] The cells can be derived from any animal including human 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 and mouse cells. Accordingly, the skeletal muscle cells can be derived from a human and / or a non-human animal; the fibroblasts can be derived from a human and / or a non- human animal; the myoblasts can be derived from a human and / or a non-human animal; the endothelial cells can be derived from a human and / or a non-human animal; and the immune cells can be derived from a human and / or a non-human animal.

[0111] Depending on the animal from which the cells are derived, the skeletal muscle tissue produced therefrom can exhibit the same or substantially the same characteristics as a skeletal muscle tissue of the animal, such as a human skeletal muscle tissue, a mouse skeletal muscle tissue, or a rat skeletal muscle tissue.

[0112] 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”). The stem cells, in terms of potency potential, can be totipotent (a.k.a omnipotent) (stem cells that can differentiate into embryonic and extra-embryonic cell types), pluripotent stem cells (can differentiate into nearly all cells), multipotent stem cells (can differentiate into a number of cell types), oligopotent stem cells (can differentiate into only a few cell types), or unipotent (can produce only one cell type). Stem cells can be obtained commercially, or obtained / isolated directly from patients, or from any other suitable source. Accordingly, the skeletal muscle cells can be derived from pluripotent stem cells; the fibroblasts can be derived from pluripotent stem cells; the myoblasts can be derived from pluripotent stem cells; the endothelial cells can be derived from pluripotent stem cells; and / or the immune cells can be derived from pluripotent stem cells. In various embodiments, the skeletal muscle cells can be derived from induced pluripotent stem cells (iPSC); the fibroblasts can be derived from iPSC; the myoblasts can be derived from iPSC; the endothelial cells can be derived from iPSC; and / or the immune cells can be derived from iPSC. In various embodiments, the skeletal muscle cells can be derived from human iPSC; the fibroblasts can be derived from human iPSC; the myoblasts can be derived from human iPSC; the endothelial cells can be derived from human iPSC; and / or the immune cells can be derived from human iPSC.

[0113] Combinations of the above-referenced cell types are also possible (e.g., skeletal muscle cells derived from human iPSC and fibroblasts derived from a non-human animal).

[0114] 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 leading a disease phenotype). In some embodiments, the cells have been pre-treated with a compound or agent (e.g., the cells have been incubated for a period of time with a drug).

[0115] The hydrogel can include collagen or a collagen derivative, intestinal submucosa or a derivative thereof, cellulose or a cellulose derivative, a 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 include Matrigel® or Geltrex. In some embodiments, the hydrogel can include Matrigel®.

[0116] In some embodiments, the hydrogel can include at least about 1 wt% collagen or a collagen derivative, at least about 2 wt% collagen or a collagen derivative, at least about 3 wt% collagen or a collagen derivative, at least about 4 wt% collagen or a collagen derivative, at least about 5 wt% collagen or a collagen derivative, at least about 6 wt% collagen or a collagen derivative, at least about 7 wt% collagen or a collagen derivative, at least about 8 wt% collagen or a collagen derivative, at least about 9 wt% collagen or a collagen derivative, or at least about 10 wt% collagen or a collagen derivative.

[0117] In some embodiments, the hydrogel can include no more than about 50 wt% collagen or a collagen derivative, no more than about 45 wt% collagen or a collagen derivative, no more than about 40 wt% collagen or a collagen derivative, no more than about 35 wt% collagen or a collagen derivative, no more than about 30 wt% collagen or a collagen derivative, no more than about 25 wt% collagen or a collagen derivative, no more than about 20 wt% collagen or a collagen derivative, or no more than about 15 wt% collagen or a collagen derivative.

[0118] Combinations of the above-referenced ranges for the weight ratio of collagen or a collagen derivative in the hydrogel are also possible (e.g., at least about 1 wt% to no more than about 50 wt%, or at least about 5 wt% to no more than about 40 wt%), inclusive of all values and ranges therebetween.

[0119] 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.

[0120] In some embodiments, at least a portion of the cells are encapsulated or embeddedinside the hydrogel. In some embodiments, at least about 40% of the cells are encapsulated or embedded inside the hydrogel. In some embodiments, at least about 45% of the cells are encapsulated or embedded inside the hydrogel. In some embodiments, at least about 50% of the cells are encapsulated or embedded inside the hydrogel. In some embodiments, at least about 55% of the cells are encapsulated or embedded inside the hydrogel. In some embodiments, at least about 60% of the cells are encapsulated or embedded inside the hydrogel. In some embodiments, at least about 65% of the cells are encapsulated or embedded inside the hydrogel. In some embodiments, at least about 70% of the cells are encapsulated or embedded inside the hydrogel.

[0121] In some embodiments, about 100% of the cells are encapsulated or embedded inside the hydrogel. In some embodiments, no more than about 99% of the cells are encapsulated or embedded inside the hydrogel. In some embodiments, no more than about 95% of the cells are encapsulated or embedded inside the hydrogel. In some embodiments, no more than about 90% of the cells are encapsulated or embedded inside the hydrogel. In some embodiments, no more than about 85% of the cells are encapsulated or embedded inside the hydrogel. In some embodiments, no more than about 80% of the cells are encapsulated or embedded inside the hydrogel.

[0122] Combinations of the above-referenced 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 no more than about 99%), inclusive of all values and ranges therebetween.

[0123] In some embodiments, the 3D skeletal muscle tissue can further include fibrinogen and / or thrombin. The fibrinogen can include human and / or non-human fibrinogen. In some embodiments, the thrombin can include human and / or non-human thrombin.

[0124] In some embodiments, the volume of the 3D skeletal muscle tissue can be at least about 0.1 mm3, at least about 0.5 mm3, at least about 1 mm3, at least about 1.5 mm3, at least about 2 mm3, or at least about 2.5 mm3.

[0125] In some embodiments, the volume of the 3D skeletal muscle tissue can be no more than about 10 mm3, no more than about 9 mm3, no more than about 8 mm3, no more than about 7 mm3, no more than about 6 mm3, no more than about 5 mm3, no more than about 4 mm3, no more than about 3 mm3, or no more than about 2.5 mm3.

[0126] Combinations of the above-referenced ranges for the volume of the 3D skeletal muscle tissue are also possible (e.g., at least about 0.1 mm3to no more than about 10 mm3, or at least about 0.1 mm3to no more than about 2.5 mm3), inclusive of all values and ranges therebetween.

[0127] The number of cells in the 3D skeletal muscle tissue depends 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, at least about 50,000. In some embodiments, the number of cells can be no more than about 5,000,000, no more than about 2,000,000, no more than about 1,000,000, no more than about 900,000, no more than 800,000, no more than about 700,000, no more than 600,000, no more than about 500,000, no more than 400,000, no more than about 300,000, no more than 200,000, or no more than 100,000.

[0128] Combinations of the above-referenced ranges for the number of cells in the tissue are also possible (e.g., at least about 20,000 to no more than about 5,000,000, or at least about 30,000 to no more than about 1,000,000), inclusive of all values and ranges therebetween.

[0129] The 3D skeletal muscle tissue can be characterized by morphology, structural organization, elasticity, extensibility, gene expression, protein expression, excitability, contractility, calcium transients, electrophysiology, biochemical signaling, or a combination thereof. The 3D skeletal muscle tissue can have one or more features substantially the same as a healthy native human skeletal muscle tissue. In some embodiments, the 3D skeletal muscle tissue can fully recapitulate the organization and function of native skeletal muscle tissues, e.g., human skeletal muscle tissues.

[0130] In some embodiments, the 3D skeletal muscle tissue can include an A band, an I band, a Z line, a M line, a H zone, a T tubule, or a combination thereof.

[0131] In some embodiments, the 3D skeletal muscle tissue can include cross-striations, elongated nuclei, sarcomeres, or a combination thereof. In some embodiments, the 3D skeletal muscle tissue can include an acetylcholine receptor, slow twitch fibers, fast twitch fibers, or a combination thereof. Slow twitch fibers and fast twitch fibers in skeletal muscle tissues are disclosed 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.

[0132] In some embodiments, expression levels of genes related to maturation (e.g., myosin isoforms ratio), genes related to calcium handling, genes related to sarcomeric proteins (e.g.,dystrophin, myosin heavy chain, or alpha actinin), exercise-induced genes (e.g. ESRRG, NFATC2) and developmental and / or fatigable contractile genes (e.g., MYH8, MYH1) in the 3D skeletal muscle tissue are substantially the same as those in a native human skeletal muscle tissue.

[0133] In some embodiments, the one or more contractions generate a twitch force and / or a tetanic force.

[0134] In some embodiments, the 3D skeletal muscle tissue can be characterized by a transient change in intracellular calcium concentration in response to an electrical and / or chemical stimulation. The transient changes in intracellular calcium concentration can be a consequence of sarcolemmal depolarization, resulting in myofiber contractility. In some embodiments, the electrical stimulation can be about 0.1-100 Hz. In some embodiments, the chemical stimulation comprises acetylcholine, adenosine triphosphate, 4-chloro-m-cresol, or a combination thereof.

[0135] In some embodiments, the electrophysiology of the 3D skeletal muscle tissue can be characterized by a shortened action potential with prominent hyperpolarization.

[0136] The anchors may be positioned at any suitable points in relation to the tissue provided that the tissue is attached to the anchors. In some embodiments, the anchors are positioned at a first end and a second end of the tissue (i.e., a distal end and a proximal end; or “opposing ends”). Advantageously, the anchors allow for measurement of contractile force of the tissue by virtue of the flexibility of the flexible anchor.

[0137] The term “attached” is intended to cover embodiments where the anchors are at least partially embedded within the tissue, or are fixedly or loosely connected to the surface of the tissue.

[0138] In some embodiments, the skeletal muscle tissue is held under tension between the anchors. The inventors have advantageously found that holding the tissue under tension during myogenesis results in improved formation of skeletal muscle tissue.

[0139] In some embodiments, one or more of the anchors (e.g., the flexible anchor) can be adjusted to modify the tension of the skeletal muscle tissue. One or more properties of the anchor (or anchors) may be adjusted. For example, the stiffness of the anchor (or anchors) may be adjusted to modify the tension of the skeletal muscle tissue (e.g., the stiffness of the flexible anchor may be adjusted). The tuneability of the material of the anchor points is an advantage over platforms known in the prior art, which typically use rigid PDMS molds.

[0140] The skeletal muscle tissue is not limited to having two such anchors, but may include more than two, such as 2-30 anchors, e.g., 2-25, 2-20, 2-15, or 2-10 anchors. In some embodiments,the skeletal muscle tissue can include 2, 3, 4, 5, 6, 7, 8, 9, or more anchors. Any number of anchors may be attached to the skeletal muscle tissue so long as there is the ability to form a tissue that forms around at least part of each of the anchors and becomes joined therebetween.

[0141] In some embodiments, the flexible anchor is an elastic sensing element. Where there is more than one flexible anchor, any number, or all, of the flexible anchors may be elastic sensing elements.

[0142] The skeletal muscle tissue is not limited to having one flexible anchor, but may include more than one, 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 can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or more flexible anchors. Any number of the flexible anchors may be elastic sensing elements.

[0143] 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 diagonal or substantially diagonal to the longitudinal axis of the skeletal muscle tissue.

[0144] In some embodiments, the flexible anchor comprises a synthetic polymer, a biologic polymer, or a combination thereof. The polymer can be biodegradable or non-biodegradable.

[0145] 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 polymer can have a Young’s modulus 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.

[0146] In some embodiments, the flexible anchor can comprise a polymer whose mechanical properties are tunable by controlling the polymerization using different crosslinking energy. Tunability can also be controlled by the ratio of the mixtures of polymer units during the polymerization reaction.

[0147] In some embodiments, the polymer can be at least one of polylactic acid, poly(lactic- co-glycolic) acid, poly(caprolactone), polyglycolide, polylactide, polyhydroxobutyrate, polyhydroxyalcanoic acid, chitosan, hyaluronic acid, a hydrogel, poly(2-hydroxyethyl- methacrylate), poly(ethylene glycol), poly(L-lactide) (PLA), poly(dimethysiloxane) (PDMS),poly(methylmethacrylate) (PMMA), poly(glycerol sebacate), poly(octam ethylene maleate (anhydride) citrate) (POMaC), POMaC without citric acid, poly(s-caprolactone), polyurethane, silk, a nanofabricated material, a co-polymer, a blended polymer, or a combination thereof. In some embodiments, the flexible anchor comprises POMaC.

[0148] The shape, thickness, length, orientation, and surface topographical properties of the flexible anchor can vary any number of suitable ways so long as the flexible anchor is capable of deforming, bending, or otherwise changing shape in response to the contractile action or activity of the tissue connected therebetween, and that such deforming, bending, or otherwise shape changing can be reliably measured. In some embodiments, the flexible anchor is in the form of a wire, e.g., a polymer wire.

[0149] The above description in relation to the flexible anchor may be equally applicable to the two or more anchors. For example, the two or more anchors may comprise POMaC, and / or may be in the form of polymer wires.

[0150] In some embodiments, the anchors are porous, thereby permitting delivery of nutrients and growth factors to the skeletal muscle tissue. In some embodiments, the anchors are nonabsorbent.

[0151] In some embodiments, the anchors are fluorescent. In some embodiments, the flexible anchor is fluorescent. This allows the monitoring of contractile properties of the tissue using fluorescent techniques.

[0152] One aspect of the present disclosure relates to a 3D diseased skeletal muscle tissue that includes a plurality of diseased skeletal muscle cells. As compared to healthy skeletal muscle tissues, the 3D disease skeletal muscle tissue can be characterized by reduced force generationcapacity, alteration in calcium handling, alteration in electrophysiology, reduced proliferation and differentiation ability, alteration in extracellular matrix material (ECM), or a combination thereof. In some embodiments, when the 3D diseased skeletal muscle tissue is excited by an electrical and / or chemical stimulation, the tissue does not contract or contracts at a significantly reduced amount as compared to a healthy skeletal muscle tissue.

[0153] Muscle cells adhere to and connect with the ECM. In addition, the ECM can provide an appropriate and permissive environment for muscle development and functioning. Alternations in the ECM can signal a disease state in the skeletal muscle tissue. See K. Grzelkowska- Kowalczyk, “The Importance of Extracellular Matrix in Skeletal Muscle Development andFunction” in “Composition and Function of the Extracellular Matrix in the Human Body,” edited by F. Travascio, IntechOpen 2016, the contents of which are incorporated herein by reference.

[0154] In some embodiments, tissues of interest can be treated with agents known in the art to cause cellular damage (e.g., toxins, mutagens, radiation, infectious agents, or chemical agents), inducing injury in the tissue. In some embodiments, tissues of interest can be altered using standard recombinant techniques to induce a disease state. For example, techniques of homologous recombination can be used to insert a transgene into a cell, or "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 injured as a result of an inherited genetic defect, which can be a single gene defect or a multifactorial defect.

[0155] The 3D diseased skeletal muscle tissue can be used in a disease model. For example, the disease model can mimic one or more muscle diseases such as Duchenne muscular dystrophy, Facioscapulohumeral muscular dystrophy, myotonic dystrophy, congenital fiber type disproportion, myosinopathies, 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.

[0156] One aspect of the present disclosure relates to a tissue system comprising a 3D skeletal muscle tissue described herein and a bioreactor. The bioreactor comprises: a device having a well configured for growing the 3D skeletal muscle tissue from cells seeded therein, wherein the well has a bottom; and two or more anchors disposed across the well such that there is a gap between the anchors and the bottom of the well. There is also a gap between the bottom of the well and the tissue suspended on the two or more anchors. At least one of the anchors is a flexible anchor.

[0157] The inventors have demonstrated that skeletal muscle tissues generated within their bioreactor are superior to ex vivo muscle tissues generated by prior art methods. In particular, the generated muscle is more morphologically similar to in vivo skeletal muscle tissue and has a greater ratio between twitch and tetanus. The platform also provides the significant advantage that the muscle can be grown, differentiated, exercised and assessed for contractility all within the sameplatform, without the need to move the tissue to a different setting for any of the steps.

[0158] The shape of the well is not limited in any particular manner and can be square, rectangular, circular, oval, oblong, triangular, or any combination of shapes. The other dimensions of the well also may vary in any suitable manner. For example, the depth of the well, height of the well, and length of the well, and the overall volume of the well may be varied in any suitable way.

[0159] For example, the length, height, or width of the well can be about 0.1-1 mm, about 0.2- 2 mm, about 0.3-3 mm, about 0.4-4 mm, about 0.5-5 mm, about 0.6-6 mm, about 0.7-7 mm, about 0.8-8 mm, about 0.9-9 mm, about 1-10 mm, about l-100mm, or about 10-100mm.

[0160] The well can be characterized by a longitudinal axis. The longitudinal axis can be along the length of the well.

[0161] In some embodiments, the well can be positioned inside a cell culture well on a multiwell plate. In some embodiments, the multi-well plate can include a plurality of wells, such as 6 wells, 8 wells, 12 wells, 24 wells, 48 wells, 96 wells, 384 wells, or 1536 wells.

[0162] The two or more anchors function as anchor points for a tissue formed therebetween. The anchors are configured to permit 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 anchor is configured to deform in response to a contractile force exerted on the flexible anchor by the 3D skeletal muscle tissue.

[0163] The bioreactor is not limited to having 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 can include 2, 3, 4, 5, 6, 7, 8, 9, or more anchors per well. Any number of anchors per well may be provided so long as there is the ability to form a tissue that forms around each of the anchors and becomes joined therebetween such that the tissue is suspended above the bottom of the well.

[0164] The bioreactor is not limited to having one flexible anchor per well, but may include more than one, 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 can 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.

[0165] The anchors can have an orientation that is perpendicular or substantially perpendicular to the longitudinal axis of the well. The tissue can be aligned in the same or substantially the samedirection as the longitudinal axis of the well.

[0166] The anchors can have an orientation that is parallel or substantially parallel to the longitudinal axis of the well.

[0167] The anchors can have an orientation that is diagonal or substantially diagonal to the longitudinal axis of the well.

[0168] The above description in relation to the anchors and the flexible anchor is equally applicable to this aspect.

[0169]

[0170] To produce the skeletal muscle tissue, the cells can be seeded in a hydrogel. The above description in relation to the cells and the hydrogel is equally applicable to this aspect.

[0171] Neurons can be seeded in a location in the bioreactor different from the skeletal muscle tissue. The neurons can then grow and / or extend via a channel to contact the skeletal muscle tissue.

[0172] In some embodiments, the number of neurons and the number of skeletal muscle cells can be at a ratio of no more than about 1 :3, no more than about 1 :3.5, no more than about 1 :4, no more than about 1 :4.5, no more than about 1 :5, no more than about 1 :5.5, or no more than about 1 :6. In some embodiments, the number of neurons and the number of skeletal muscle cells can be at a ratio of 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.

[0173] Combinations of the above-referenced ranges for the ratio of the neurons over the 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), inclusive of all values and ranges therebetween. For example, the ratio of the neurons over the skeletal muscle cells can be between about 1 :50 and 1 :5.

[0174] The bioreactors can further include electrodes configured to provide electrical stimulations to the skeletal muscle tissue. Each electrode can include conductive carbon, gold, platinum, palladium, stainless steel, tin, tungsten, titanium, or a combination thereof. Further examples of conductive materials for tissue stimulation can be found at 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 28thIEEE, pages 845-848, the contents of each of which are incorporated herein by reference.

[0175] Additional features of the bioreactor can be found at US20160282338, the contents ofwhich are incorporated by reference in their entireties.

[0176] In general, to produce mature 3D skeletal muscle tissues, the cells encapsulated or embedded in the hydrogel are subject to electrical stimulations in accordance with a stimulation protocol. The stimulation protocol may also be referred to as an “exercise regime”.

[0177] In one aspect, the stimulation protocol can include a low frequency train or trains of pulses for an extended duration. See FIG. 4A, which is a schematic diagram illustrating an example of such stimulation protocol. The train of pulses may be continuous (i.e., a single train of pulses with no intervals), or intermittent (i.e., a plurality of trains of pulses separated by intervals of no stimulation).

[0178] 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 no more than about 5 Hz, no more than about 4.5 Hz, no more than about 4 Hz, no more than about 3.5 Hz, no more than about 3 Hz, no more than about 2.5 Hz, no more than about 2 Hz, no more than about 1.5 Hz, or no more than about 1 Hz.

[0179] Combinations of the above-referenced ranges for the low frequency are also possible (e.g., at least about 0.1 Hz to no more than about 1 Hz, or at least about 0.5 Hz to no more than about 2 Hz), inclusive of 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.

[0180] 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 one week. In some embodiments, the extended duration can be no more than about 3 weeks, no more than about 2.5 weeks, no more than about 2 weeks, no more than about 1.5 weeks, or more than about one week, no more than about 3 days, or no more than about 24 hours.

[0181] Combinations of the above-referenced ranges for the extended duration are also possible (e.g., 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), inclusive of all values and ranges therebetween.

[0182] In another aspect, the stimulation protocol can include a high frequency train or trainsof pulses for an extended duration. The train of pulses may be continuous (i.e., a single train of pulses with no intervals), or intermittent (i.e., a plurality of trains of pulses separated by intervals of no stimulation).

[0183] In some embodiments, the stimulation protocol can include a plurality of intermittent high frequency pulse trains (i.e., separated by intervals of no electrical stimulation). As compared to mature 3D cardiac tissues, high frequency pulses (e.g., at least about 5 Hz) may be required to produce mature 3D skeletal muscle tissues. Without wishing to be bound by theory, the high frequency requirement is because of the ability of skeletal tissue to generate sustained contractures under repeat high frequency pulses. Repeat sustained contractures results in maturation of skeletal tissue. See FIG. 4B, which is a schematic diagram illustrating an example of such stimulation protocol.

[0184] 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 no more than about 100 Hz, no more than about 95 Hz, no more than about 90 Hz, no more than about 85 Hz, no more than about 80 Hz, no more than about 75 Hz, no more than about 70 Hz, no more than about 65 Hz, or no more than about 60 Hz.

[0185] Combinations of the above-referenced ranges for the high frequency are also possible (e.g., at least about 5 Hz to no more than about 100 Hz, or at least about 10 Hz to no more than about 60 Hz), inclusive of all values and ranges therebetween.

[0186] In some embodiments, the duration for each pulse train can be at least about one 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, at least about 60 minutes. In some embodiments, the duration for each pulse train can be no more than about 3 hours, no more than about 2.5 hours, no more than about 2 hours, no more than about 1.5 hours, or no more than about 1 hour.

[0187] Combinations of the above-referenced ranges for the duration for each pulse train are also possible (e.g., at least about one second to no more than about 3 hours, or at least about 30 seconds to no more than about 1 hour), inclusive of all values and ranges therebetween.

[0188] 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 no more than 4 weeks, no more than 3 weeks, no more than 2 weeks, no more than 1 week, no more than 72 hours, no more than 48 hours, no more than 24 hours, no more than 20 hours, no more than 18 hours, no more than 15 hours, no more than 12 hours, no more than 10 hours, no more than 5 hours, no more than 3 hours, no more than 2 hours, or no more than 1 hour.

[0189] Combinations of the above-referenced ranges for the extended duration are also possible (e.g., at least about 5 minutes to no more than about 4 weeks), inclusive of all values and ranges therebetween.

[0190] 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.

[0191] In yet another aspect, the stimulation protocol can include a first frequency pulse train for a first duration, a second frequency pulse train for a second duration, wherein the first frequency is increased to the second frequency at a first ramp rate. See FIG. 4C, which is a schematic diagram illustrating an example of such stimulation protocol. In some embodiments, the stimulation protocol can include a series of first frequency pulse trains for a first duration, a series of second frequency pulse trains for a second duration, wherein the first frequency is increased to the second frequency at a first ramp rate.

[0192] 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 no more than about 5 Hz, no more than about 4.5 Hz, no more than about 4 Hz, no more than about 3.5 Hz, no more than about 3 Hz, no more than about 2.5 Hz, no more than about 2 Hz, no more than about 1.5 Hz, or no more than about 1 Hz.

[0193] Combinations of the above-referenced ranges for the first frequency are also possible (e.g., at least about 0.1 Hz to no more than about 1 Hz, or at least about 0.5 Hz to no more than about 2 Hz), inclusive of 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.

[0194] In some embodiments, the first duration can be at least about one 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, at least about 60 minutes. In some embodiments, the first duration can be no more than about 3 hours, no more than about 2.5 hours, no more than about 2 hours, no more than about 1.5 hours, or no more than about 1 hour.

[0195] Combinations of the above-referenced ranges for the first duration are also possible (e.g., at least about one second to no more than about 3 hours, or at least about 30 seconds to no more than about 1 hour), inclusive of all values and ranges therebetween. In some embodiments, the first duration is about 1 hour.

[0196] 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 no more than about 100 Hz, no more than about 95 Hz, no more than about 90 Hz, no more than about 85 Hz, no more than about 80 Hz, no more than about 75 Hz, no more than about 70 Hz, no more than about 65 Hz, or no more than about 60 Hz.

[0197] Combinations of the above-referenced ranges for the second frequency are also possible (e.g., at least about 10 Hz to no more than about 100 Hz, or at least about 10 Hz to no more than about 60 Hz), inclusive of all values and ranges therebetween.

[0198] In some embodiments, the second duration can be at least about one 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, at least about 60 minutes. In some embodiments, the second duration can be no more than about 3 hours, no more than about2.5 hours, no more than about 2 hours, no more than about 1.5 hours, or no more than about 1 hour.

[0199] Combinations of the above-referenced ranges for the second duration are also possible (e.g., at least about one second to no more than about 3 hours, or at least about 30 seconds to no more than about 1 hour), inclusive of all values and ranges therebetween. In some embodiments, the second duration is about 1 hour.

[0200] In some embodiments, the first ramp rate can be at least about 0.1 Hz / hour.

[0201] In some embodiments, the stimulation protocol can further include a third frequency pulse train for a third duration. In some embodiments, the stimulation protocol can further include a series of third frequency pulse trains for a third duration. The third frequency can be either smaller or greater than the second frequency. The stimulation protocol can further include a fourth frequency pulse train for a fourth duration, a fifth frequency pulse train for a fifth duration, etc. The stimulation protocol can further include a series of fourth frequency pulse trains for a fourth duration, a series of fifth frequency pulse trains for a fifth duration, etc.

[0202] Any one of the above stimulation protocols can be applied to the cells once a day, twice a day, three times a day, or more than three times a day.

[0203] In some embodiments, the electrical stimulation is terminated when a certain phenotype is achieved, e.g., slow twitch fibers or fast twitch fibers, which can be analyzed based on the expression of markers (type II myosin heavy chain mainly expressed in fast twitch fibers and type I myosin heavy chain mainly expressed in slow twitch fibers). Parameters of contractility and calcium transients can also allow the characterization of slow twitch fibers and fast twitch fibers. In some embodiments, the electrical stimulation is terminated when a certain phenotype is achieved, e.g., the presence of myotubes and / or sarcomeres, which can be analyzed by microscopy.

[0204] In some embodiments, the electrical stimulation is terminated when one or more measurable parameters are achieved, e.g., a minimum tetanus to baseline ratio of greater than 2, or a minimum tetanus force of 50 pN.

[0205] Another aspect of the present disclosure relates to a method of stimulating myogenesis in vitro, comprising: (i) providing a hydrogel and a plurality of cells that includes skeletal muscle cells to a well of a bioreactor, wherein the well has a bottom and two or more anchors are disposed across the well such that there is a gap between the anchors and the bottom of the well, and wherein at least one of the anchors is a flexible anchor; and (ii) stimulating the skeletal muscle cells over aperiod of time with a series of electrical pulse trains separated by intervals.

[0206] The description above in relation to the hydrogel, the plurality of cells, the anchors, and the bioreactor is equally applicable to this aspect.

[0207] 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 no more than 24 hours, no more than 20 hours, no more than 18 hours, no more than 15 hours, no more than 12 hours, no more than 10 hours, no more than 5 hours, no more than 3 hours, no more than 2 hours, or no more than 1 hour.

[0208] 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, at least 42 days. In some embodiments, the period of time is no more than 8 weeks, no more then 7 weeks, no more than 6 weeks, no more than 5 weeks, no more than 4 weeks, no more than 3 weeks, no more than 2 weeks, no more than 1 week.

[0209] In some embodiments, the period of time is 10 minutes to 24 hours. In some embodiments, the period of time is 30 minutes to 2 hours.

[0210] Combinations of the above-referenced ranges for the period of time are also possible (e.g., at least 5 minutes to no more than about 1 week), inclusive of all values and ranges therebetween.

[0211] The series of electrical pulse trains may be any suitable plurality of electrical pulse trains in succession. In some embodiments, the electrical pulse trains are trains of biphasic square wave pulses.

[0212] In some embodiments, the duration of each train of pulses 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 15seconds, 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 train of pulses can be no more than about 3 hours, no more than about 2.5 hours, no more than about 2 hours, no more than about 1.5 hours, no more than about 1 hour, no more than about 30 minutes, no more than about 15 minutes, no more than about 10 minutes, no more than about 5 minutes, no more than about 1 minute, no more than about 30 seconds, no more than about 25 seconds, no more than about 20 seconds, no more than about 15 seconds, no more than about 10 seconds, or no more than about 5 seconds.

[0213] Combinations of the above-referenced ranges for the duration for each train of pulses are also possible (e.g., at least about 0.1 seconds to no more than about 3 hours), inclusive of all values and ranges therebetween. In various embodiments, the duration of each train of pulses can be 0.5 to 4 seconds. In various embodiments, the duration of each train of pulses can be 0.5 s to 2 seconds. The pulse trains may all have the same duration (e.g., all of the pulse trains in the series have a duration of 1 second) or may have different durations (e.g., a first pulse train in the series has a duration of 0.8 s, a second pulse train in the series has a duration of 1.2 s, etc.).

[0214] In some embodiments, the pulse trains have a low frequency and have 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 no more than about 5 Hz, no more than about 4.5 Hz, no more than about 4 Hz, no more than about 3.5 Hz, no more than about 3 Hz, no more than about 2.5 Hz, no more than about 2 Hz, no more than about 1.5 Hz, or no more than about 1 Hz.

[0215] In some embodiments, the pulse trains have a high frequency and have 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 no more than about 100 Hz, no more than about 95 Hz, no more than about 90 Hz, no more than about 85 Hz, no more than about 80 Hz, no more than about 75 Hz, no more than about 70 Hz, no more than about 65 Hz, no morethan about 60 Hz, no more than about 55 Hz, no more than about 50 Hz, no more than about 45 Hz, no more than about 40 Hz, no more than about 35 Hz, no more than about 30 Hz, no more than about 25 Hz, or no more than about 20 Hz.

[0216] Combinations of the above-referenced ranges for the pulse repetition frequency are also possible (e.g., at least about 5 Hz to no more than about 100 Hz), inclusive of all values and ranges therebetween. Combinations of the above-referenced ranges and values for the low frequency and high frequency pulse trains are also possible (e.g., a first pulse train has a pulse repetition frequency of about 1 Hz, and a second pulse train has a pulse repetition frequency of about 10 Hz). In various 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.

[0217] In some embodiments, each train has a duty cycle of between 0.5 and 20%. In some embodiments, each train has a duty cycle of between 1 and 10%. In some embodiments, each train has a duty cycle of between 2 and 5%. In some embodiments, each train has a duty cycle of 4%.

[0218] In some embodiments, each pulse within the pulse train has a pulse width of at least about 1 ms, at least about 2 ms, at least about 3 ms, at least about 4 ms, at least about 5 ms, at least about 10 ms, at least about 15 ms, at least about 20 ms, at least about 25 ms, at least about 30 ms, at least about 40 ms, or at least about 50 ms. In some embodiments, each pulse within the pulse train has a pulse width of no more than about 50 ms, no more than about 40 ms, no more than about 30 ms, no more than about 25 ms, no more than about 20 ms, no more than about 15 ms, or no more than about 10 ms.

[0219] Combinations of the above-referenced ranges for the pulse width are also possible (e.g., at least about 1 ms to no more than about 50 ms, or at least about 2 ms to no more than about 10 ms), inclusive of all values and ranges therebetween. In some embodiments, the pulse width is at least about 1 ms to no more than about 10 ms. In some embodiments, the pulse width is about 4 ms.

[0220] In some embodiments, the pulses within 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 within each train have a pulse amplitude of no more than 50 V, no more than 45 V, no more than 40 V, no more than 35 V, no more than 30 V, no more than 25 V, no more than 20 V, no more than 15 V, or no more than 10 V.

[0221] Combinations of the above-referenced ranges for the pulse amplitude are also possible(e.g., at least about 3 V to no more than 45 V), inclusive of all values and ranges therebetween. In some embodiments, the pulses in each train have a voltage of 3-10 V.

[0222] In some embodiments, the intervals between the pulse trains are 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 least9 seconds, at least 10 seconds, at least 15 seconds, at least 20 seconds, at least 30 seconds, at least 45 seconds, at least 60 s, 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 intervals between the pulse trains are no more than 120 minutes, no more than 90 minutes, no more than 60 minutes, no more than 45 minutes, no more than 30 minutes, no more than 15 minutes, no more than 10 minutes, no more than 5 minutes, no more than 4 minutes, no more than 3 minutes, no more than 2 minutes, or no more than 1 minute.

[0223] Combinations of the above-referenced ranges for the intervals are also possible (e.g., at least about 3 seconds to no more than 120 minutes), inclusive of all values and ranges therebetween. In some embodiments, the intervals are 5 seconds to 60 minutes. In some embodiments, the intervals are 5 seconds to 60 seconds. In some embodiments, the intervals are10 seconds.

[0224] In some embodiments, the method comprises applying step (ii) to the cells once a day, twice a day, three times a day, or more than three times a day.

[0225] In some embodiments, the method comprises repeating step (ii) every 6 hours to 18 hours for a duration. In some embodiments, the method comprises repeating step (ii) every 8 hours to 16 hours for a duration. In some embodiments, the method comprises repeating step (ii) every 10 hours to 14 hours for a duration. In some embodiments, the method comprises repeating step (ii) every 11 to 13 hours for a duration. In some embodiments, the method comprises repeating step (ii) every 12 hours for a duration.

[0226] 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 no more than 8 weeks, no more than 7 weeks, no more than 6 weeks, no more than 5 weeks, no more than 4 weeks, or no more than 3 weeks.

[0227] Combinations of the above-referenced ranges for the duration are also possible (e.g., at least 5 days to no more than 8 weeks), inclusive of 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. Insome embodiments, the duration is about 4 weeks.

[0228] In some embodiments, the electrical stimulation is terminated when a certain phenotype is achieved, e.g., slow twitch fibers or fast twitch fibers, which can be analyzed based on the expression of markers (type II myosin heavy chain mainly expressed in fast twitch fibers and type I myosin heavy chain mainly expressed in slow twitch fibers). Parameters of contractility and calcium transients can also allow the characterization of slow twitch fibers and fast twitch fibers. In some embodiments, the electrical stimulation is terminated when a certain phenotype is achieved, e.g., the presence of myotubes and / or sarcomeres, which can be analyzed by microscopy.

[0229] In some embodiments, the electrical stimulation is terminated when one or more measurable parameters are achieved, e.g., a minimum tetanus to baseline ratio of greater than 2, or a minimum tetanus force of 50 pN.

[0230] In some embodiments, the method comprises the step of growing the skeletal muscle cells in the well in a growth medium for at least 1 day, at least 2 days, or at least 3 days. In some embodiments, the method comprises the step of growing the skeletal muscle cells in the well 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. The method advantageously allows the cells to be grown, the tissue to be exercised, and muscle contractions to be measured in the same well, without having the move and / or detach the tissue.

[0231] Combinations of the above-referenced ranges for the growth step are also possible (e.g., at least 1 day to no more than 5 days), inclusive of all values and ranges therebetween. In some embodiments, the method comprises the step of growing the skeletal muscle cells in a growth medium for 1-3 days. In some embodiments, the method comprises the step of growing the skeletal muscle cells in a growth medium for 2 days.

[0232] The growth step may be an initial step that occurs prior to the stimulation step (i.e., prior to step (ii)). There may be other steps in between the growth step and the stimulation step (e.g., a differentiation step).

[0233] The growth medium can be any suitable culture medium that enables the cells to proliferate. For example, the growth medium may be skeletal muscle cell growth medium (Promocell or Sigma-Aldrich). Typically, the growth medium comprises serum.

[0234] In some embodiments, the method comprises the step of differentiating the skeletal muscle cells in the well in a differentiation medium for at least 3 days, at least 4 days, at least 5days, 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 the step of differentiating the skeletal muscle cells in a differentiation medium for no more than 100 days, no more than 90 days, no more than 80 days, no more than 70 days, no more than 60 days, no more than 50 days, or no more than 40 days. The method advantageously allows the cells to be grown and differentiated in the same well in which the tissue is exercised, and muscle contractions measured, without having the move and / or detach the tissue.

[0235] Combinations of the above-referenced ranges for the differentiation step are also possible (e.g., at least 3 day to no more than 100 days), inclusive of all values and ranges therebetween. In some embodiments, the method comprises the step of differentiating the skeletal muscle cells in a differentiation medium for 3-50 days. In some embodiments, the method comprises the step of differentiating the skeletal muscle cells in a differentiation medium for 20- 40 days.

[0236] The differentiation step may be an initial step that occurs prior to the stimulation step (i.e., prior to step (ii)). When a growth step is present, the differentiation step may occur between the growth step and the stimulation step.

[0237] The differentiation medium can be any suitable culture medium that enables the cells to differentiate into mature skeletal muscle cells. For example, the differentiation medium may be NbActiv4 (BrainBitsLLC.com). In some embodiments, the differentiation medium comprises glucose. In some embodiments, the concentration of glucose in the differentiation medium is 5-30 mM.

[0238] In some embodiments, the differentiation medium comprises insulin. In some embodiments, the concentration of insulin in the differentiation medium is 0.5 nM-10 nM. In some embodiments, the concentration of insulin in the differentiation medium is 1-8 nM. In some embodiments, the concentration of insulin in the differentiation medium is 3-5 nM

[0239] In some embodiments, the differentiation medium comprises glucose and insulin. In some embodiments, the concentration of glucose in the differentiation medium is 5-30 mM and the concentration of insulin in the differentiation medium is 0.5 nM-10 nM.

[0240] Another aspect of the present disclosure relates to a method of stimulating myogenesis in vitro, comprising: (i) providing a hydrogel and a plurality of cells that includes skeletal muscle cells to a well of a bioreactor, wherein the well has a bottom and two or more anchors are disposedacross the well such that there is a gap between the anchors and the bottom of the well, and wherein at least one of the anchors is a flexible anchor; and (ii) differentiating the skeletal muscle cells in the well in a differentiation medium comprising glucose and insulin.

[0241] The description above in relation to the hydrogel, the plurality of cells, the anchors, and the bioreactor is equally applicable to this aspect. The description above in relation to the differentiation step and the differentiation medium is also equally applicable to this aspect.

[0242] In some embodiments, the method comprises the step of growing the skeletal muscle cells in the well in a growth medium for at least 1 day, at least 2 days, or at least 3 days. In some embodiments, the method comprises the step of growing the skeletal muscle cells in the well 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.

[0243] Combinations of the above-referenced ranges for the growth step are also possible (e.g., at least 1 day to no more than 5 days), inclusive of all values and ranges therebetween. The description above in relation to the growth step and the growth medium is equally applicable to this aspect.

[0244] There is also provided a method of stimulating myogenesis in vitro, comprising: (i) providing a hydrogel and a plurality of cells that includes skeletal muscle cells to a well of a bioreactor, wherein the well has a bottom and two or more anchors are disposed across the well such that there is a gap between the anchors and the bottom of the well, and wherein at least one of the anchors is a flexible anchor; (ii) growing the skeletal muscle cells in the well in a growth medium for 1-3 days; (iii) differentiating the skeletal muscle cells in the well in a differentiation medium comprising glucose and insulin for 3-50 days; and (iv) stimulating the skeletal muscle cells in the well over a period of time with a series of electrical pulse trains separated by intervals.

[0245] The description above in relation to the hydrogel, the plurality of cells, the anchors, the bioreactor, the growth step, the differentiation step and the stimulation step is equally applicable to this aspect.

[0246] In some embodiments, the tissue system described herein can be used for measuring the effect on contractility of the skeletal muscle tissue formed therein resulting from exposure to a test agent of interest.

[0247] In some embodiments, the tissue system can be used for (a) testing of the efficacy and safety (including toxicity) of a test agent (e.g., an experimental pharmacologic agent), (b) definingthe pharmacokinetics and / or pharmacodynamics of a pharmacologic agent, (c) characterizing the properties and therapeutic effects of a pharmacologic agent on a subject, (d) screening an experimental pharmacologic agents, and / or (e) providing implantable engineered tissues for use in regenerative medicine for treating damaged and / or diseased tissues.

[0248] Accordingly, one aspect of the present disclosure provides a method for measuring an effect of a test agent on contraction using the tissue system described herein, comprising: measuring a first value of a contraction characteristic of the 3D skeletal muscle tissue in the 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 the contraction; measuring a second value of the contraction characteristic of the 3D 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.

[0249] The contraction characteristic can be a contractile force, a twitch force, a tetanic force, time to tetanus, rate of tetanic force generation, rate of tetanic force rundown, rate of relaxation from tetanic force, or a combination thereof.

[0250] In some embodiments, the test agent modulates the contraction when there is a significant difference between the first value and the second value, e.g., a difference of at least about 10%, a difference of at least about 15%, a difference of at least about 20%, a difference of at least about 25%, a difference of at least about 30%, a difference of at least about 35%, a difference of at least about 40%, a difference of at least about 45%, or a difference of at least about 50%.

[0251] In some embodiments, the measurement of a 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, the measurement of a contractile force comprises measuring an amount of movement imposed by the 3D skeletal muscle tissue on the two or more anchors from a first position to a second position.

[0252] 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., a difference of at least about 10%, a difference of at least about 15%, a difference of at least about 20%, a difference of at least about 25%, a difference of at least about 30%, a difference of at least about 35%, a difference of at least about 40%, a difference of at least about 45%, or a differenceof at least about 50%.

[0253] In some embodiments, the contractile force can be measured by optical microscopy as disclosed by US20160282338.

[0254] Another aspect of the present disclosure relates to a method for measuring an effect of a test agent on a calcium transient using the tissue system described herein, comprising: measuring a first value of a calcium transient characteristic of the 3D skeletal muscle tissue in the 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 the calcium transient; measuring a second value of the calcium transient characteristic of the 3D skeletal muscle tissue after exposure to the test agent; and determining whether the test agent modulates the calcium transient by comparing the first value with the second value.

[0255] The calcium transient characteristic can be magnitude of calcium transient, time constant of calcium transient, rate of calcium transient, or a combination thereof.

[0256] In some embodiments, the test agent modulates the calcium transient when there is a significant difference between the first value and the second value, e.g., a difference of at least about 10%, a difference of at least about 15%, a difference of at least about 20%, a difference of at least about 25%, a difference of at least about 30%, a difference of at least about 35%, a difference of at least about 40%, a difference of at least about 45%, or a difference of at least about 50%.

[0257] In some embodiments, the measurement of a calcium transient characteristic comprises measuring a fluorescence signal of an intracellular calcium indicator in the 3D skeletal muscle tissue.

[0258] In some embodiments, the intracellular calcium indicator is selected from Fura-4F AM, Fura-2, Fluo-3, Fluo-4, and Indo-1, Mag-Fura-5, and Mag-Fura-red.

[0259] Another aspect of the present disclosure relates to a method for evaluating the safety of a test agent using the tissue system described herein, comprising: (a) contacting the skeletal muscle tissue with the test agent; (b) measuring the effect on one or more physiological parameters indicative of safety; (c) comparing the physiological parameters in (b) to 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) as compared to the same physiological parameters measured from the control bioreactor indicates that the test agent lacks safety.

[0260] The undesired effects of toxicity caused by administration of a test agent can be screened in several ways. The tissue system described herein can be used to determine the range of toxic dosimetry of a test agent. The effect of increasing concentrations of the test agent (i.e., dose) on skeletal muscle tissue can be monitored to detect toxicity. A toxic effect, when observed, can be equated with a measurement of test agent concentration / cells cm2. By calculating the toxic concentration according to the distribution of cells in the skeletal muscle tissue, one of skill in the art can extrapolate to the living system, to estimate toxic doses in subjects of various weights and stages in development.

[0261] The tissue system described herein can also be used to evaluate a test agent's efficacy. Efficacy can be detected by measuring individual parameters associated with the repair, enhancement, improvement and / or regeneration of a disease model comprising a diseased skeletal muscle tissue. The diseased state can be induced or can be the result of a pre-existing condition in the tissue donor, including conditions relating to inherited genetic abnormalities. Either the induced or pre-existing condition can comprise a weakened state resulting from a previous drug exposure. Test agents can be analyzed for efficacy in disease models of the present disclosure.

[0262] Using methods of the invention, various doses of individual test agents and combinations of test agents can be screened in panels comprised of tissues having diverse genetic backgrounds to determine the pharmacogenetic efficacy profile of the test agents. For example, multiple doses of, or combinations with, test agents will be screened for efficacy, or the lack thereof, specific to one or more genetic backgrounds.

[0263] In general, test agents can be incubated with the skeletal muscle tissue in a dosage range estimated to be therapeutic and for a duration sufficient to produce an effect (e.g., metabolic effects or effects indicating to toxicity or efficacy). The incubation time can range from about one minute to 24 hours, or can be extended as necessary for several days or even weeks. The incubation conditions typically involve standard culture conditions known in the art, including culture temperatures of about 37 degrees Celsius, and culture mediums compatible with the skeletal muscle tissue.

[0264] 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, bacteria, a microparticle, a nanoparticle, a therapeutic agent, and a toxin.

[0265] Examples of test agents include, but are not limited to, opioid analgesics, anti-inflammatory drugs such as antihistamines and non-steroidal anti-inflammatory drugs (NSAIDs), diuretics such as carbonic anhydrase inhibitors, loop diuretics, high-ceiling diuretics, thiazide and thiazide-like agents, and potassium-sparing diuretics, agents that impinge on the renal and cardiovascular systems such as angiotensin converting enzyme (ACE) inhibitors, cardiac drugs such as organic nitrates, calcium channel blockers, sympatholytic agents, vasodilators, P- adrenergic receptor agonists and antagonists, a-adrenergic receptor agonists and antagonists, cardiac glycosides, anti-arrhythmic drugs, agents that affect hyperlipoproteinemias such as 3- hydroxymethylglutaryl-coenzyme A (HMG-CoA) inhibitors, anti -neoplastic agents such as alkylating agents, antimetabolites, natural products, antibiotics, and other drugs, immunomodulators, anti-diabetic agents, and anti-microbial agents such as antibacterial agents, antiviral agents, antifungal agents, antiprotozoal agents, and antihelminthic agents.

[0266] 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 no more than 100 days, no more than 90 days, no more than 80 days, no more than 70 days, no more than 60 days, no more than 50 days, no more than 40 days, no more than 30 days, or no more than 20 days.

[0267] Combinations of the above-referenced ranges for the incubation time are also possible (e.g., at least 1 day to no more than 100 days), inclusive of 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.

[0268] Another aspect of the present disclosure relates to a method of monitoring the maturity of the 3D skeletal muscle tissue as 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 fatiguability of the tissue; and / or (iv) determining whether exercise-induced genes (e.g. ESRRG, NFATC2) are upregulated and / or developmental and fatigable contractile genes (e.g., MYH8, MYH1) are downregulated.

[0269]

[0270] Confirming the presence of myotubes in the tissue may involve staining the tissue (e.g., F-Actin immunostaining) and observing the tissue using microscopy, such as confocal microscopy. Confirming the presence of myotubes in the tissue may involve assessing gene expression orprotein content. Confirming the presence of myotubes in the tissue may involve using laser diffraction.

[0271] Confirming the presence of sarcomeres in myotubes in the tissue may involve staining the tissue (e.g., F-Actin and / or troponin immunostaining) and observing the tissue using microscopy, such as confocal microscopy.

[0272] The fatigability of the tissue may be measured using any suitable method which measures the loss of force generation over time. In some embodiments, an isometric fatigue method is used (e.g., tetanus is induced and fatigue is measured over a time period, such as 30 s). In some embodiments, a chronic fatigue method is used (e.g., multiple stimulated contractions over a longer period of time). As a non-limiting example, fatiguability may be measured by measuring the loss of force generation during a 10 minute-repeat submaximal tetanic stimulus (80Hz, 0.4s duration) that resulted in 50% force reduction at time zero. The skilled person will recognise that different frequencies and durations may be used.

[0273] There is also provided the three-dimensional skeletal muscle tissue as described herein, wherein the three-dimensional skeletal muscle tissue has been obtained by a method of stimulating myogenesis as described herein.

[0274] While the present teachings have been described in conjunction with various embodiments and examples, it is not intended that the present teachings 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 of skill in the art.

[0275] While various inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications 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 upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims andequivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0276] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0277] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” Any ranges cited herein are inclusive.

[0278] The terms “substantially”, “approximately,” and “about” used throughout this Specification and the claims generally mean plus or minus 10% of the value stated, e.g., about 100 would include 90 to 110.

[0279] The phrase “and / or,” as used herein in the specification and in the 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 fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” may refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0280] As used herein in the specification and in 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” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, whenused in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0281] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those 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”) may refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0282] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0283] As used herein in the specification and in the claims, the term “substantially the same” refers to a first value that is within 10% of a second value. For example, if A is substantially the same as B, and B is 100, A can have a value ranging from 90 to 110. If A is substantially the same as B, and B is 200, A can have a value ranging from 180 to 220.

[0284] As used herein, the term "POMaC" refers to poly(octam ethylene maleate (anhydride) citrate) (POMaC) or the POMaC prepolymer which comprises a mixture of 1,8-octandiol, citrateacid, and maleic anhydride. Reference can be made to Tran et al., "Synthesis and characterization of a biodegradable elastomer featuring a dual crosslinking mechanism," Soft Matter, Jan 1, 2010; 6(11): 2449-2461, which is incorporated herein by reference in its entirety.

[0285] As used herein, the term "test agent" is any substance that is evaluated for its ability to diagnose, cure, mitigate, treat, or prevent disease in a subject, or is intended to alter the structure or function of the body of a subject. A test agent in an embodiment can be a "drug" as that term is defined under the Food Drug and Cosmetic Act, Section 321(g)(1). Test agents can include, but are not limited to, chemical compounds, biologic agents, proteins, peptides, antibodies, nucleic acids, lipids, polysaccharides, supplements, diagnostic agents and immune modulators and may also be referred to as "pharmacologic agents."

[0286] As used herein, the term "toxicity" is defined as any unwanted effect on human cells or tissue caused by a test agent, or test agent used in combination with other pharmaceuticals, including unwanted or overly exaggerated pharmacological effects. An analogous term used in this context is "adverse reaction."

[0287] As used herein, the term “non-human” refers to all vertebrates, e.g., mammals and nonmammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, mice, rats, and the like.

[0288] It will be understood that, although the terms "first", "second", etc. may be used herein to describe various elements, components, regions, layers and / or sections, these elements, components, regions, layers and / or sections should not be limited by these terms. These terms are only used 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 discussed below could be termed a second element, component, region, layer or section without departing from the teachings of example embodiments. Spatially relative terms, such as "beneath," "below," "lower," "above," "upper" and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is turned over, elements described as "below" or "beneath" other elements or features would then be oriented "above" the other elements or features. Thus, the exemplary term "below" can encompass both an orientation of above and below. Thedevice may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.

[0289] As used herein, the term “hydrogel” refers to a physically or chemically cross-linker polymer network that is able to absorb large amounts of water and is a common material for forming tissue engineering scaffolds. They can be classified into different categories depending on various parameters including the preparation method, the charge, and the mechanical and structural characteristics. Reference can be made to S. Van Vlierberghe et al., “Biopolymer-Based Hydrogels As Scaffolds for Tissue Engineering Applications: A Review,” Biomacromolecules, 2011, 12(5), pp. 1387-1408, which is incorporated herein by reference.

[0290] As used herein, the term “pulse” refers to a single electrical stimulation event in which an applied electrical voltage or current changes from a steady state baseline, each pulse generally consisting of a single positive going phase and a single negative going phase. The term waveform” is used to represent the shape of an individual pulse. Each of the electrical pulses may comprise either a monophasic or biphasic waveform, which may be, for example, asymmetric, symmetric, square, sinusoidal, and the like. A pulse may be defined by pulse width (i.e., the elapsed time between the leading and trailing edges of the pulse) and / or pulse amplitude.

[0291] As used herein, the term “pulse train” refers to a series of individual pulses. The pulse train may be defined by the number of pulses in the train and / or the train duration (i.e., the length of time between the first and last pulses in the pulse train). The pulse train may be defined by the pulse repetition frequency (i.e., the number of pulses in a specified unit of time; Hz) and / or the pulse spacing (the duration between corresponding points of successive pulses) and / or the duty cycle (i.e., the fraction of one period in which a signal is active, the period being the time it takes for a signal to complete an on-and-off cycle; duty cycle = (pulse width x 100%) / total period of the signal.).

[0292] As used herein the term “interval” refers the duration of and spacing between successive pulse trains. The interval between pulse trains may also be referred to as a relaxation period.

[0293] As used herein, the term “myogenesis” refers to the formation of skeletal muscular tissue.

[0294] As used herein, the term “encapsulated or embedded inside the hydrogel” refers to the cells being fixed within the surrounding hydrogel. This term is intended to cover embodimentswhere all of the cells are fully contained and enclosed by the hydrogel, and also embodiments where some of the cells are fully contained and enclosed by the hydrogel, and some of the cells are breaching the surface and / or positioned on the surface of the hydrogel.

[0295] As used herein in relation to the skeletal tissue, the term “artificial” refers to the tissue being grown and differentiated in vitro, and does not include skeletal tissues that grew and / or matured in vivo (e.g., a muscle tissue removed from a living organism and embedded in a hydrogel is not included). “Artificial” may be used interchangeably with “ex vivo”.

[0296] As used herein, the term “anchors” refers to elements that function as anchor points and permit attachment of the 3D skeletal muscle formed therebetween, thereby suspending the 3D skeletal muscle tissue (e.g., above the bottom of a well).

[0297] As used herein the term “flexible anchor” refers to an anchor which is capable of being flexed or bent without breaking. The flexible anchor is able to deform in response to a contractile force exerted on it by the 3D skeletal muscle tissue.

[0298] The claims should not be read as limited to the described order or elements unless stated to that effect. It should be understood that various changes in form and detail may be made by one of ordinary skill in the art 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 thereto are claimed.ExamplesExample 1.

[0299] Human Skeletal Muscle Cells (SkMCs) (catalog no. C-12530) were purchased from PromoCell at passage 2. Cells were maintained in culture in growth medium (catalog no. C23260, C-39365) and expanded by following the manufacturing instructions. Cells between passage 3 and passage 5 were used for the experiments. After detachment with trypsin / EDTA solution, SkMCs were embedded in a hydrogel with the following composition: rat tail collagen 3mg / mL, human fibrinogen 8.2 mg / mL, Matrigel® 1 :9, 50 unit / ml thrombin 1 / 25 of total volume. In some experiments, collagen was not used. Tissues with a cell density between 30,000 and 100,000 viable SkMCs were envisioned. In some experiments, human cardiac fibroblasts were added in a ratio 1 : 10. SkMCs were maintained 2 days in growth medium, followed by 1 week in differentiation medium (catalog no. C-39366 from PromoCell) and successively maintained in growth medium until the end of the study.

[0300] SkMC tissue compaction occurred properly, starting 24 hours after seeding and gradually proceeding overtime for 2-3 days. The presence of fibroblasts did not significantly affect tissue compaction (FIG. 1).

[0301] After 1 week of tissue seeding, external electrical stimulation was applied. Some tissues were kept unstimulated, as control, to test the hypothesis that the electrical stimulation plays a key role in skeletal tissue maturation and therefor functionality. The stimulation was started at 1Hz with a 0.3Hz increase / day. Media changed occurred every 2 days.

[0302] The first appearance of contraction in response to external stimulation occurred after 4 days of stimulation. No contraction was observed in unstimulated tissues (FIG. 2).

[0303] SkMC tissue contractility was analyzed overtime until day 13. No increase in active force was observed overtime (FIG. 3).Example 2.

[0304] Introduction

[0305] Engineered 3D human skeletal muscle models represent a promising approach to overcoming the limitations of 2D in vitro systems and animal models (Broer T et al., 2020; Khodabukus A, 2021). Engineered 3D models can recreate complex tissue architectures by providing structural and mechanical features typical of native tissues. By embedding cells in hydrogel, wherein cells can tri-dimensionally anchor, cell-extracellular matrix (ECM) interactions are formed that are critical for outside-in biochemical signaling. The ECM not only provides a permissive environment for muscle development and function but are dynamic and can play a critical role in the progression of conditions such as diabetes, muscular dystrophies and muscle aging (Grzelkowska-Kowalczyk K, 2016). Additionally, it has been demonstrated that embedding myoblasts in a hydrogel anchored between two attachment points can induce myotube alignment by recreating mechanical features present in the 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 can promote a physiologically relevant contractile phenotype. In the last decade, a variety of 3D skeletal muscle models have been proposed (Jalal S et al., 2021; Cho S, Jang J, 2021), however, none of the current models fully recapitulate all aspects of the complex skeletal muscle physiology.

[0306] Here the inventors generated engineered 3D human skeletal muscle tissues using the Biowire™II platform. The Biowire™II platform consists of two elastic polymer wires between which tissues are suspended, recreating the mechanical tension typical of the native skeletal muscle environment (FIG. 5 Ai). The elastic polymer wires allow for measurement of contractile force by virtue of their flexibility (and also fluorescence). The inventors demonstrated differentiation of human myoblasts into myotubes and increased twitch and tetanic force. Seven days after the tissues were seeded in the Biowire™II platform, electrical field stimulation was applied to a subset of the tissues. The electrical stimulation improved myotube alignment and resistance to fatigue, as compared to unstimulated tissues. Electrical stimulation also induced the switch from fast myosin heavy chain (MyHC) isoforms to slow MyHC isoforms, a transformation commonly reported in vivo with exercise training (Demirel HA et al., 1999; Short KR et al., 2005). Additionally, the 3D skeletal muscle tissues responded as expected to compounds with known mechanisms of action. Taken together these results demonstrate an engineered 3D human skeletal muscle model thatrecapitulates important hallmarks of human muscle physiology that can be used in drug discovery and disease modeling applications.

[0307] Results

[0308] Engineering human skeletal tissues in the Biowire™!! platform.

[0309] Human skeletal muscle tissues were generated by encapsulating primary human skeletal muscle cells skMDCs in a hydrogel composed of Matrigel and fibrin and then seeding the cell suspension into the Biowire™!! platform, as described in Material and Methods. To mimic skeletal muscle physiology, fibroblasts were added in a 1 :20 ratio to both provide structural support through ECM protein synthesis (Chapman MA et al., 2016), and stimulate myogenesis (Mackey AL et al., 2017). In these experimental conditions, myoblasts and fibroblasts self-organized and gradually remodeled the hydrogel to form a tissue suspended between two polymer wires after 24 hours (FIG. 5Ai). Skeletal tissues were cultured for 2 days in the platform before differentiation was initiated and then monitored for another 33 days. On day 7 of differentiation, the skeletal tissues were divided into groups wherein one group was subjected to an exercise regime consisting of intermittent external electrical stimulation, and the other group was maintained as an unstimulated control culture (FIG. 5 Aii).

[0310] Confocal analysis of skeletal muscle tissues immunostained with F-actin demonstrated that at the end of the maturation protocol the skeletal tissues were composed of several myotubes (FIG. 5Bi) and the myotubes were tridimensionally distributed (FIG. 5Bii). One defining property of skeletal muscle in vivo is the presence of sarcomeres, responsible for force production by muscles. Super-resolution images acquired by using a SoRa spinning disk confocal system (see Material and Methods) allowed for visualization and quantification of sarcomeres in the myotubes. The sarcomere length, measured as the distance between Z-lines, was 2.34 ± 0.09pm (FIG. 5Biii), similar to the sarcomere length of approximately 2.4 pm reported for rat whole muscle (Moo et al., 2016; 2018).

[0311] Effect of differentiation time and the Biowire™!! platform on engineered skeletal tissue structure and function.

[0312] Skeletal tissues were analyzed structurally over time in culture in a differentiation media. F-actin staining revealed enhanced myotube formation over time (FIG. 6Ai). Quantification of myotube diameter resulted in a statistical increase in diameter at day 21 as compared to day 7 (8.22 ± 0.90 pm at day 7; 11.76 ± 0.3 pm at day 21). At day 35, the inventorsdid not observe a further increase in myotube diameter relative to day 21 (FIG. 6Aii). Additionally, the combination of the Biowire™II platform and differentiation time modulated developmental genes. The embryonic genes, myogenin and embryonic (Myh3) MyHC, had increased expression from day 7 to 21 and decreased expression by day 35. The neonatal (Myh8) MyHC expression increased as a function of differentiation time. Interestingly, starting from day 21 engineered skeletal tissues had increased expression of the adult MyHC isoforms (Myhl, Myh2, Myh7) (FIG. 6B). The Biowire™II platform allows for measuring parameters of contractility by imaging the deflection of a polymer wire as a function of time (FIG 5iii, iv), as previously reported (Feric et al., 2019). An important hallmark of skeletal muscles is the ability to generate tetanus, consisting in the muscle ability to sustain a contraction evoked by the emission of action potentials at a very high rate by the innervating motor neuron. The inventors mimicked this high-rate stimulus in vitro by applying an electrical stimulus at <100 Hz. The inventors calculated the amplitude, rise time (time to amplitude), rise (contraction) slope and decay (relaxation) time to 50% of 1 Hz twitches and tetanus generated using high frequencies (FIG. 5iv). After 7 days of differentiation, skeletal tissues were already able to generate tetanic forces upon high frequency stimulation (at 100Hz, 24.8 ± 10.3 pN; at 1Hz, 3.9 ± 1.6 pN) (FIG. 6Ci). When monitored over time, skeletal tissues generated constant forces at 1 Hz stimulation for the entire period of observation (up to 35 days). However, when stimulated at 100 Hz, tissues generated increasing tetanic forces in the first 3 weeks (day 7 to 21), and subsequently they maintained the same amount of forces for the remaining 2 weeks of observation (FIG. 6Cii). Similar to 100 Hz, skeletal tissues showed a time-dependent increase in twitch amplitude at 5 Hz, 10 Hz and 20 Hz, as demonstrated by the representative traces in FIG. 6D.

[0313] Exercise modulates skeletal tissue morphology, gene expression, contractility and fatigability.

[0314] At day 7 of tissue differentiation, the inventors applied a 4-week intermittent electrical stimulation protocol. F-actin immunostaining demonstrated that exercise enhanced myotube formation (FIG. 7Ai), as the inventors observed a statistical increase in myofiber diameter of day 21 -exercised tissues (2-week-stimulated tissues) as compared to time-matched non-exercised (unstimulated) tissues (FIG. 7Aii). The inventors also observed enhanced contractility with exercise. Specifically, after 1 week of stimulation, exercised skeletal tissues displayed a significant increase in twitch amplitude and rise (contraction) slope both at 1 Hz and 100 Hz stimulationrelative to unstimulated tissues. Although forces produced by stimulated tissues reached a plateau after 2 weeks of stimulation and subsequently decreased, the force generated by the stimulated tissues was significantly higher than the unstimulated tissues at all time points (FIG. 7Bi, ii, FIG. 9A, FIG. 9B). Rise (contraction) time was similarly increased by exercise, however only in the later time points (FIG. 7Biii). Finally, exercised skeletal tissues showed a decreased decay (relaxation) time to 50% when stimulated at 100 Hz as compared to unstimulated skeletal tissues (FIG. 7Biv). All together these data demonstrate that the electrical stimulation regime applied to exercise the skeletal tissues resulted in dramatic modulations of the skeletal contractility parameters.

[0315] Skeletal muscle fatigability is the loss of force in response to contractile activity, which occurs both physiologically and in diseased states. In physiological situations, exercise training can improve muscle fatigability (Hunter SK, 2018). To investigate if the inventors’ exercise regime was able to recapitulate the physiology, the inventors allowed skeletal tissues to reach fatigability, measured by 50% force reduction, by applying a 10 minute repeated submaximal tetanic stimulus to skeletal tissues on day 7. The inventors then measured fatigability weekly up to day 35, by applying the same fatigability protocol for the entire period of observation. FIG. 7C shows that by 3 to 4 weeks of the exercise regime, skeletal tissues had improved fatigue resistance, as compared to non-exercised tissues.

[0316] To investigate the molecular profile underlying the functional changes mediated by the electrical stimulation, key MyHC isoforms, known to be involved in skeletal contractility, were analyzed. Interestingly, unstimulated tissues expressed more type IIx (fast twitch) MyHC than stimulated tissues, while exercised tissues expressed more type Ila (slow twitch) MyHC than unstimulated tissues (FIG. 7D). The shift from the IIx (fast twitch) to Ila (slow twitch) MyHC isoform is commonly observed in vivo with exercise training (Demirel HA et al., 1999; Short KR et al., 2005).

[0317] Skeletal tissues display expected contractility responses to chronic treatments with known compounds.

[0318] The inventors investigated the ability of their engineered skeletal muscle tissues to respond to compounds with known mechanisms of action, as well as the contribution of exercise training to these pharmacological responses. Clenbuterol is a P2 agonist, reported to increase skeletal muscle mass in some animal models (Zeman et al., 1998) and able to partially prevent orrestore muscle loss in experimental models of muscle wasting (Lynch and Ryall, 2008). Additionally, 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. In fact, clenbuterol was banned in the USA because of skeletal and cardiac toxicity- related concerns (Burniston JG et al., 2002; Bumiston JG et al., 2005; Barry AR, Graham MM, 2013). Skeletal tissues exercised for two weeks and time matched unstimulated tissues were chronically treated with 0.1 pM clenbuterol for 2 weeks. This dose was demonstrated to improve contractility in some in vitro skeletal muscle models (Madden L. et al., 2015). The data demonstrates that treatment with clenbuterol resulted in a significant increase in twitch amplitude in stimulated tissues as compared to controls (PBS vehicle-treated tissues) after 1 week or 2 weeks of treatment at all stimulation frequencies (1 Hz, 5 Hz, 10 Hz, 20 Hz, 100 Hz). On the contrary, chronic clenbuterol treatment did not affect contractility in unstimulated tissues (FIG. 8Ai). Quantification of the kinetic parameters revealed that rise (contraction) time was dramatically decreased in stimulated tissues, but unaffected in unstimulated tissues, as compared to untreated controls (FIG. 8Aii). Decay (relaxation) time, however, was not affected in either stimulated or unstimulated tissues, as compared to untreated controls (FIG. 10A).

[0319] Dexamethasone, like other glucocorticoids, is widely used in the treatment of neuromuscular diseases, although long-term side effects, such as muscle atrophy, are widely known (Schacke H. et al., 2002; Schakman O. et al., 2013; Gupta A. et al., 2013). A one week dexamethasone treatment dramatically decreased the force produced by both stimulated and unstimulated tissues. At high frequency stimulation forces were further decreased after 2 weeks of dexamethasone treatment (FIG. 8B). Regarding kinetic parameters of contractility, 2 week- dexamethasone treatment resulted in a dramatic decrease in decay time to 50% in unstimulated tissues (FIG. lOBi), without affecting rise time in either stimulated or unstimulated tissues (FIG. lOBii). Finally, we treated tissues with dexamethasone alone or in combination with clenbuterol for 2 weeks. FIG. 8C shows that clenbuterol mitigated some of the effects exerted by dexamethasone on stimulated tissues, as demonstrated by comparing peak amplitudes of tissues exposed to clenbuterol and dexamethasone to peak amplitudes of tissues exposed only to dexamethasone. Specifically, after 2 week-treatment, the mean peak amplitude relative to vehicle of stimulated tissues exposed to clenbuterol and dexamethasone as compared to mean peak amplitude of stimulated tissues exposed only to dexamethasone was the following: 0.052 vs.0.0064, 0.024 vs. 0.006, 0.023 vs. 0.005, 0.014 vs. 0.004 respectively, when stimulated at 5 Hz, 10 Hz, 20 Hz and 10 0Hz. Additionally, as demonstrated in FIG. 8 A, the inventors confirmed that clenbuterol did not have any effect on unstimulated skeletal tissues.

[0320] Discussion

[0321] Many skeletal muscle disorders remain untreated due to lack of knowledge of the skeletal muscle pathophysiology and disease progression, together with a lack of predictive models for drug development. Consequently, treatments are often only palliative (Stephenson D et al., 2022).

[0322] Here the inventors have provided a platform and protocol that allows the generation of engineered 3D human skeletal muscle tissues. The inventors first optimized a tissue engineering protocol that allowed for the fusion of human myoblasts into myotubes over time in culture. Myogenesis involves a sequence of events that include stem and progenitor cell commitment, proliferation, differentiation, and fusion to give rise to multinucleated skeletal myofibers (Bentzinger C.F. et al., 2012). These events are strictly controlled by the expression of developmental genes. Myogenin is a transcription factor that regulates myocyte fusion during development, specifically it is 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 embryonic and neonatal myosin heavy chains, that are contractile proteins transiently expressed during development. After birth, developmental myosin isoforms disappear and are replaced with adult myosin isoforms (fast and slow myosins) (Schiaffino et al., 2015). The inventors determined that increased differentiation time enhanced myotube formation, as myotube diameter increased as a function of time cultured in a differentiation media. After 5 weeks of differentiation, skeletal muscle tissues were composed of tridimensional distributed myotubes that displayed sarcomeric organization and sarcomeric lengths similar to in vivo reports (Moo et al., 2016; 2018). Additionally, increasing differentiation time modulated developmental genes. Specifically, early (embryonic) developmental genes, myogenin and embryonic myosin heavy chain (MYHC3), increased from day 7 to day 21 of differentiation then subsequently decreased at later time points (day 35). The neonatal gene, myosin heavy chain (MYH8), progressively increased in a time-dependent manner, whereas expression of adult myosins (MYHC1, MYHC2, MYHC7) increased on day 21 of differentiation. By recapitulating the physiology, these datademonstrate that the Biowire™!! platform and the differentiation protocol resulted in skeletal muscle tissues with physiologically relevant morphology and developmental gene expression.

[0323] Skeletal muscle contraction is triggered by an action potential initiated by motor neurons that subsequently propagates along the muscle cell plasma membrane. To characterize their engineered skeletal muscle tissues in terms of contractility, the inventors applied an external electrical stimulation that mimics innervation. An important hallmark of skeletal muscle is the ability to generate tetanus (Sweeney HL, Hammers DW, 2018), which consists in the muscle’s ability to sustain a contraction caused by action potentials occurring at high rate. As compared to twitch (evoked by low frequency action potentials), tetanus is more physiological relevant. The inventors engineered skeletal muscle tissues displayed completely fused tetanus in response to 100 Hz electrical stimulation on day 7. Moreover, tetanic forces increased in response to higher stimulation frequencies and to increasing maturation time.

[0324] The inventors then exercised engineered 3D skeletal muscle tissues by applying a chronic intermittent external electrical stimulation. Chronic stimulation resulted in enhanced myotube formation, as well as force when compared to unstimulated tissues. Specifically, after 2 weeks of electrical stimulation, 3D skeletal muscle tissues generated twitch and tetanic forces 4- fold and 8-fold greater respectively than forces generated by time-matched unstimulated tissues. Increased force in response to electrical stimulation is consistent with previous observations in skeletal murine and human models (Huang YC et al., 2006; Ito A et al., 2014; Khodabukus A et al., 2019). However, the inventors’ engineered skeletal tissues were superior to known models, displaying the greatest twitch and tetanic fold increase in force.

[0325] In terms of both twitch and tetanic forces in their stimulated 3D skeletal muscle tissues, the inventors observed that while forces increased rapidly, after only 1 week of stimulation by week 4 of stimulation force values started to decrease. The decrease in force after 4 weeks of stimulation could be ascribed to the contraction of some myotubes over time. Similar to the twitch amplitude and the mathematically related rise (contraction) slope, exercise increased the rise (contraction) time and decreased the half relaxation time during tetanic contractions relative to unstimulated tissues. Exercise also resulted in tissues with enhanced fatigue resistance as compared to unstimulated tissues. Interestingly, enhanced force and fatigue resistance are correlated with a type IIx (fast myosin isoform) to Ila (slow myosin isoform) myosin heavy chain shift as reported in in vivo endurance exercise training studies (Demirel HA et al., 1999; Short KRet al., 2005). Overall, the applied exercise training resulted in engineered skeletal muscle tissues with contractility behavior like that seen in vivo. Furthermore, the expression of slow myosin isoforms, together with slower rise time and increased fatigue resistance in stimulated tissues indicate that the inventors’ stimulation protocol tuned myotubes towards a slow-fiber phenotype. As such, the present invention provides the advantage of being able to tune 3D skeletal muscle tissues towards different muscle fiber phenotypes by mimicking different neural firing patterns using alternative electrical stimulation regimes (Huang YC et al., 2006). Similarly, the present invention allows for the modelling of disease states by adjusting the stimulation protocol. Some skeletal diseases preferentially affect specific fiber types, for instance, in Duchenne Muscular Dystrophy, type II fibers are the first ones to degenerate, conversely type I fibers are affected only in a late stage of the disease (Webster C. et al., 1988; Pedemonte M. et al., 1999). Additionally, 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 reproduce different skeletal fiber types and proportions in vitro and thereby 1) provide new insights into genetic and molecular pathways and 2) offer a new drug discovery platform. In addition, chronic exercise has been shown to result in altered expression of exercise- induced genes and myofiber contractile genes. RNAseq analysis identified upregulated exercise- induced genes (e.g. ESRRG, NFATC2) and downregulated developmental and fatigable contractile genes (e.g., MYH8, MYH1) in stimulated vs. unstimulated tissues (see Figure 12A). GO analysis also highlights several differentially regulated pathways impacted by stimulation (see Figure 12B).

[0326] One of the expectations of engineering 3D skeletal muscle systems is to obtain more predictive models for drug screening than the models currently available, and reduce, in turn, the high-rate failure of compounds that never reach clinical trials (Broer T. et al., 2020). To test the pharmacological predictivity of their model, the inventors exposed 3D skeletal tissues to compounds with known mechanism of actions, to ensure that the in vitro responses were as expected in vivo. Chronic P2- adrenergic stimulation is known to affect only slow twitch fibers in vivo and induce a slow-to-fast fiber-type shift. This transition is associated with a positive inotropic effect. Specifically, chronic treatments of animal models with P2 agonists result in increased twitch and tetanic forces (Dodd SL et al., 1996; Zhang KM et al., 1996), maximal shortening velocity (Dodd SL et al., 1996) and shortened time-to-peak tension (Zhang KM et al.,1996). Recently the ability of P2 agonists to enhance muscle force has been demonstrated in humans as well (Hostrup M et al., 2015). Here, the inventors demonstrated that their exercised skeletal muscle tissues properly respond to chronic clenbuterol treatment. Specifically, 2 weeks of clenbuterol treatment resulted in increased twitch amplitude in exercised (stimulated) tissues, without affecting contractility in unstimulated tissues. The inventors further confirm the accuracy of their model to predict clenbuterol response, by analyzing the contraction speed. Exercised skeletal tissues treated with clenbuterol showed a faster contraction speed than stimulated controls, suggesting the effect of clenbuterol of inducing a slow-to-fast fiber-type shift.

[0327] A two-week exposure to dexamethasone resulted in a dramatic decrease (up to 90%) in both twitch and tetanic forces in engineered skeletal tissues. Glucocorticoid-induced muscle dysfunctions such as muscle weakness are widely recognized, with mechanisms ascribed to atrophy (Shin YS et al., 2000; Schakman O. et al., 2013). Additionally, by simultaneously treating tissues with clenbuterol and dexamethasone, the inventors showed that clenbuterol mitigates some of the effects exerted by dexamethasone on stimulated tissues. All together these data demonstrate that the inventors’ skeletal model can be used to predict pharmacological responses.

[0328] The two-week exposure to altered gene expression in engineered skeletal tissues. In particular, RNAseq analysis shows upregulation of glucocorticoid-induced genes, including the atrogenes TRIM63 and FBXO32 in dexamethasone-treated tissues (see Figure 13 A). GO analysis also shows that several differentially regulated pathways are altered by dexamethasone treatment (see Figure 13B).

[0329] Materials and Methods

[0330] Engineered Human Skeletal Tissue Formation

[0331] Primary human skeletal muscle cells skMDC (Cook Myosite, Pittsburgh, PA) and dermal fibroblasts (Lonza, Allendale, NJ) were maintained in 2D culture following the manufacturing recommendations. For skeletal tissue formation, cells were embedded in hydrogel composed of fibrinogen 5mg / mL, matrigel 1 :5 and 25 U / ml thrombin 1 / 25 of total volume and seeded in the Biowire™!! platform, consisting of polystyrene microwells containing parallel poly (octamethylene maleate (anhydride) citrate) (POMaC) wires (Feric NT et al., 2019). Each tissue consisted of 90000 skMDC and 4500 dermal fibroblasts. Tissues were kept 2 days in growth media (Cook Myosite) and subsequently in NbActiv4 differentiation media (Brainbits, Springfield, IL). Media was changed twice / week. At day 7 of differentiation, some tissues were subjected tointermittent external electrical stimulation in custom chambers containing parallel carbon electrodes (Feric NT et al., 2019) with a 1 second, 10 Hz pulse train (each pulse having a 4 ms duration) every 10 seconds for 1 hour, twice per day.

[0332] Immunofluorescence Analysis of Engineered Human Skeletal Tissues

[0333] Skeletal tissues were fixed in paraformaldehyde 30 minutes and subsequently permeabilized with Triton 0.1% for additional 30 minutes at room temperature (RT). After washing with PBS, tissues were incubated with Alexa Fluor 488 Phalloidin (ThermoFisher Scientific, Waltham, MA), diluted 1 :400 in PBS, 1 hour at RT. 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).

[0334] Contractility Measurements of Engineered Human Skeletal Tissues

[0335] Skeletal tissue contractility was measured by tracking the deflection of the POMaC wires as a function of time, as previously described (Zaho et al., 2019). Tissues were transferred to a custom chamber containing parallel carbon electrodes to provide external field stimulation in 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) with a 470 nm emission filter using NIS-Elements software. To measure contractile force, the videos were analyzed using a custom Matlab program. Twitch and different degrees of tetanus were acquired by stimulating the tissues at 1Hz, 5Hz, 10Hz, 20Hz and 100Hz for 6 seconds. Fatigability was measured as loss of force generation during a 10 minute-repeat submaximal tetanic stimulus (80Hz, 0.4s duration) that resulted in 50% force reduction at time zero.

[0336] Drug Testing of Engineered Human Skeletal Tissues

[0337] 2 week-stimulated tissues and time-matched unstimulated tissues were treated with 0.1 pM clenbuterol and lOpM dexamethasone (Sigma Aldrich, St. Louis, MO). Clenbuterol stock solution was prepared in PBS, following the manufacturing recommendation. Dexamethasone stock solution was prepared in ethanol. Compounds and relative controls, in experimental and control tissues respectively, were replaced every 2 days during full media change for 14 days. Tissues were analyzed weekly for contractility, as described above. Each tissue’s contractility was first normalized to relative baseline, subsequently to vehicle controls.

[0338] Statistics

[0339] Analysis by one-way ANOVA was followed by post-hoc testing (Bonferroni’s ad hoc test). Student 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. All experiments were independently replicated at least three times unless otherwise noted.Example 3.

[0340] Skeletal muscle is highly metabolically active. In skeletal muscle, binding of insulin to the insulin receptor (IR) initiates a signaling cascade that involves insulin receptor substrate- 1 / phosphoinositide 3-kinase / Akt (IRS-1 / PI3K / Akt) pathway and results in the translocation of the insulin-sensitive glucose transporter protein 4 (GLUT4) to the plasma membrane, which leads to facilitated diffusion of glucose into the cell. Impairment in signaling events guiding insulin- stimulated glucose uptake leads to development of insulin resistance and type 2 diabetes. Causes of insulin resistance are numerous and the mechanisms are multifactorial. The development of 3D models of insulin resistance that closely mimic the human physiology may unravel novel mechanisms regulating skeletal muscle glucose uptake. Identifying mechanisms leading to insulin resistance will be essential for a more effective treatment of type-2 diabetes.

[0341] Skeletal tissues were kept in growth media for 2 days, and subsequently kept in Nb Activ4 differentiation media or customized media (Neurobasal media+B27 supplement without insulin + glucose 20mM + insulin 6.9nM). The tissues were kept in the differentiation media for 7 days without electrical stimulation, followed by 7-10 days with chronic electrical stimulation.

[0342] Figure 11 A shows representative western blotting of day 7 tissues versus day 14 tissues showing expression of insulin receptor (InsRbeta) and GLUT4. These data demonstrate that exercised skeletal tissues expressed key proteins for insulin-stimulated glucose transport, specifically InsR and GLUT4.

[0343] Figure 1 IB shows representative glucose uptake of skeletal tissues in NbActiv4 media or customized media, with or without insulin stimulation. These data demonstrate that exercised skeletal tissues exhibited a significant increase in glucose uptake in response to insulin.

Claims

What is claimed is:

1. A three-dimensional skeletal muscle tissue comprising a hydrogel, a plurality of cells that includes skeletal muscle cells, and two or more anchors, wherein the skeletal muscle tissue is characterized by one or more contractions in response to an electrical and / or chemical stimulation, and wherein 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 fibroblasts and the skeletal muscle cells are at a ratio of between about 1 :5 and 1 :50.

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

5. The three-dimensional skeletal muscle tissue of any one of claims 1-4, wherein the hydrogel comprises collagen or a collagen derivative, intestinal submucosa or a derivative thereof, cellulose or a cellulose derivative, a 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. 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. 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 1-8, wherein at least a portion of the cells are encapsulated or embedded inside the hydrogel.

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

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

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

13. The three-dimensional skeletal muscle tissue of any one of claims 1-12, having a volume of about 0.1 mm3to about 2.5 mm3.

14. The three-dimensional skeletal muscle tissue of any one of claims 1-13, further comprising an A band, an I band, a Z line, a M line, a H zone, or a combination thereof.

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

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

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

18. The three-dimensional skeletal muscle tissue of any one of claims 1-17, 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 1-18, wherein the chemical stimulation comprises acetylcholine, adenosine triphosphate, 4-Chloro-m-cresol, or a combination.

20. The three-dimensional skeletal muscle tissue of any one of claims 1-19, characterized by a transient change in intracellular calcium concentration in response to an electrical and / or chemical stimulation.

21. The three-dimensional skeletal muscle tissue of any one of claims 1-20, characterized by a shortened action potential with prominent hyperpolarization.

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

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

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

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

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

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

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, polylactide, polyhydroxobutyrate, polyhydroxyalcanoic acid, chitosan, hyaluronic acid, poly(2-hydroxyethyl- methacrylate), poly(ethylene glycol), poly(L-lactide) (PLA), poly(dimethysiloxane) (PDMS), poly(methylmethacrylate) (PMMA), poly(glycerol sebacate), poly(octamethylene maleate (anhydride) citrate) (POMaC), POMaC without citric acid, poly(s-caprolactone), polyurethane, silk, and a combination thereof.

29. 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 24-29, wherein the flexible anchor has an elasticity from about 10 kPa to 0.8 MPa.

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

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

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

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

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

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

37. The tissue system of claim 35, wherein the polymer is nondegradable.

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, polylactide, polyhydroxobutyrate, polyhydroxyalcanoic acid, chitosan, hyaluronic acid, poly(2-hydroxyethyl-methacrylate), polyethylene glycol), poly(L- lactide) (PLA), poly(dimethysiloxane) (PDMS), poly(methylmethacrylate) (PMMA), poly(glycerol sebacate), poly(octamethylene maleate (anhydride) citrate) (POMaC), POMaC without citric acid, poly(s-caprolactone), polyurethane, silk, and a combination thereof.

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

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

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

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

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

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

45. A method for measuring an effect of a test agent on contraction using the tissue system of any one of claims 32-44, comprising: measuring a first value of a contraction characteristic of the three-dimensional skeletal muscle tissue in the bioreactor before exposure to the test agent; contacting the three-dimensional skeletal muscle tissue with the test agent for an incubation time under conditions sufficient for the test agent to modulate the contraction; measuring a second value of the contraction characteristic 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. The method of claim 45, wherein the test agent modulates the contraction when there is a significant difference between the first value and the second value.

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, bacteria, a microparticle, a nanoparticle, a therapeutic agent, and a toxin.

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

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

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

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

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

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

54. The method of any one of claims 50-53, 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, bacteria, a microparticle, a nanoparticle, a therapeutic agent, and a toxin.

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

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

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

58. The method of claim 57, wherein each train has a duration of 0.5 to 4 s duration.

59. The method of claim 58, wherein each train has a 0.5 s to 2 s duration.

60. The method of any one of claims 57-59, wherein each train has a frequency of 5-15 Hz.

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

62. The method of any one of claims 57-61, wherein each train has a duty cycle of between 1 and 10%.

63. The method of any one of claims 57-62, wherein the pulses in each train have a voltage of 3-10 V.

64. The method of any one of claims 57-63, wherein the intervals are 5 s to 60 m.

65. The method of claim 64, wherein the intervals are 5 s to 60 s.

66. The method of any one of claims 57-65, wherein the period of time is 10 m to 24 h.

67. The method of claim 66, wherein the period of time is 30 m to 2 h.

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

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

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

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

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

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

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

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

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

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

78. A method of stimulating myogenesis in vitro, comprising:(i) providing a hydrogel and a plurality of cells that includes skeletal muscle cells to a well of a bioreactor, wherein the well has a bottom and two or more anchors are disposed across the well such that there is a gap between the anchors and the bottom of the well, and wherein at least one of the anchors is a flexible anchor; and(ii) differentiating the skeletal muscle cells in the well in a differentiation medium comprising glucose and insulin.

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

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

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

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

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

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

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

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

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

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