Production of engineered skeletal muscle in a bioreactor

Abstract

This invention is in the field of skeletal muscle engineering. The present invention relates to a method for producing an engineered skeletal muscle tissue from pluripotent stem cells, comprising five defined steps, thereby producing contractile engineered skeletal muscle tissue, and the method is characterized in that the steps (i)-(iii) are carried out in a bioreactor under suitable conditions, and wherein the method does not include the use of a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma. Furthermore, an engineered skeletal muscle tissue obtainable by a method disclosed herein is also disclosed wherein said engineered skeletal muscle tissue generates a contraction force of at least 0.5 millinewtons (mN) upon a stimulus from 1 Hz to 150 Hz, wherein said engineered skeletal muscle tissue is serum-free, and wherein said engineered skeletal muscle tissue does not comprise a transgene. Medical and non-medical uses of the engineered skeletal muscle tissue are also described.

Description

[0001] Production of engineered skeletal muscle in a bioreactor

[0002] Background of the invention

[0003] This invention is in the field of skeletal muscle engineering which is increasingly being exploited in transforming industrial applications. One of the most important applications will be the mass production of lab-grown meat to reduce the use of animals and enable sufficient meat production according to demand and reduce the environmental burden associated with conventional meat production. In addition, the use of human muscle is being sought in drug development (for muscle diseases, but also for metabolic diseases such as insulin resista nce / diabetes) and regenerative as well as reparative medicine.

[0004] Traditionally, meat production has relied on the slaughter of animals to obtain their skeletal muscle tissue. However, raising animals is not only expensive but also produces a lot of climate-damaging carbon dioxide, so engineered meat, i.e. engineered skeletal muscle, is one way to overcome these drawbacks. In addition, skeletal muscle in the human body enables breathing, posture and movement. However, major injuries often do not heal properly and can leave permanent damage. In addition, myopathic diseases such as Duchenne muscular dystrophy cause skeletal muscle wasting, which could be treated with engineered skeletal muscles that can repair or regenerate. It follows that there is a need in the art to obtain engineered skeletal muscle, for example for meat production or as a therapy for disease and injury.

[0005] The theory of tissue engineering is to generate the required cell types and differentiate them in an engineered environment to produce an in w / o-like tissue. The greatest challenge is to start with stem cells and differentiate them into differentiated skeletal muscle tissue. In tissue engineering, the extracellular environment plays an important role because developmentally relevant information must be provided to the differentiating cells. For example, dissociation, such as passaging of cells, disrupts cell-cell connectivity, geometric cell positioning and cell- extracellular matrix connectivity. This environment needs to be re-established during tissue engineering (Zimmermann et al. 2004, Tiburcy et al. 2017, Shahriyari et al., 2022, Shahriyari et al., 2023). Furthermore, differentiated skeletal muscle tissue consists not only of skeletal muscle fibers, but also of stromal / connective tissue cells, in particular satellite cells, which are formed in response to their environment and chemical stimuli.

[0006] Those skilled in the art will be aware of various tissue engineering methods related to skeletal muscle cells using 2D cell cultures, small animal models or muscle tissue extracted from small animals (Beldjilali-Labro et al. (2018) and Khodabukus et al. (2018)). For example, Chai et al. (2016) describe the production of muscle fibers in a 2D method. In addition, the inventors previously described the generation of skeletal muscle in WO2021 / 074126 Al using a serum- free method. However, the described method still had drawbacks. For example, the method was standardly performed on Matrigel, which is not a defined substance. The method described in WO2021 / 074126 Al requires a surface coating such as Matrigel to work. Furthermore, due to the undefined nature of Matrigel, such a protocol is not as reproducible as the protocol according to the present invention.

[0007] Moreover, the differentiation of stem cells into skeletal muscle tissue requires not only chemical but also physical stimuli. Thus, a specific combination of chemical and physical stimuli, in addition to surface topography and structural composition, appears to reliably generate functional muscle tissue in vitro (Liao et al. (2008), Pavesi et al. (2015)). The development of a robust, defined and reproducible differentiation and maturation protocol is a very important step to enable the production of skeletal muscle tissue.

[0008] Numerous approaches have been described to generate skeletal muscle in vitro. In most cases, myogenic cells capable of forming multi nucleated muscle cells are combined with a matrix designed to provide some of the cues that the extracellular matrix of muscle would provide. Notably, the groups that have generated muscle with relevant force production have relied on a hydrogel approach, where myogenic cells are embedded in a matrix consisting of collagen or fibrin. These approaches require the use of additional exogenous extracellular matrix (Matrigel®) to allow the formation of multinucleated, well-developed muscle cells, a prerequisite for generating twitch force. However, Matrigel® is an ill-defined extracellular matrix exudate derived from Engelbrecht Holm Swarm mouse tumours, which is incompatible with potential industrial applications. Efforts are being made to replace Matrigel® in the generation of organoids, but this has proved extremely difficult for skeletal muscle (Kozlowski et al. 2021). Skeletal muscle tissue (Shahriyari et al. 2022) and organoids (Shahriyari et al. 2023) previously described by the inventors rely on the addition of hydrogel (such as collagen or fibrin) and or in a preferred embodiment are prepared with additional contain Matrigel® supplementation. In these disclosed approaches, none of the steps is carried out in a bioreactor.

[0009] Therefore, there is a need in the art to obtain improved engineered skeletal muscle without the use of an undefined reconstitution and / or coating substances such as hydrogels and in particular Matrigel. In addition, there is a need in the art for a scalable production to produce the required biomass for food and medical applications comprised by muscle cells and related extracellular matrix.

[0010] Summary of the Invention The present invention describes methods for producing an engineered skeletal muscle tissue from pluripotent stem cells which is serum-free, and the different chemical substances and their concentrations as well as the physical stimulation are defined. This invention enables the utilization of spheroids to produce (i) migratory muscle cells as well as (ii) the cell-autonomous extracellular matrix to finally generate tissue engineered muscle with relevant force production. The technology is the basis for a defined and highly scalable muscle generation process. This approach eliminates the need for exogenous extracellular matrix such as Matrigel, with a poorly defined and variable composition. In other words, the methods do not include the use of a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma (Matrigel). This is because the method is at least partially carried out in a bioreactor. In addition, in steps (i) to (iii), the method does not require the addition of further hydrogels such as collagen or fibrin, because the myomatrix spheroid produce their own extracellular matrix (ECM) environment. The resultant myomatrix aggregates can be further processed to produce skeletal muscle of a desired size and geometry using, e.g., cast molding, fusion, stacking, or 3D printing approaches.

[0011] The present invention relates to a method for producing an engineered skeletal muscle tissue from pluripotent stem cells, comprising the steps of

[0012] (i) forming two or more pluripotent stem cell aggregates from pluripotent stem cells;

[0013] (ii) inducing mesoderm differentiation of the pluripotent stem cell aggregates;

[0014] (iii) inducing the myogenic specification thereby obtaining two or more myomatrix spheroids;

[0015] (iv) expanding and maturing the two or more myomatrix spheroids into myomatrix spheroids containing skeletal myoblasts and migratory muscle progenitors;

[0016] (v) maturing myomatrix spheroids containing skeletal myoblasts and migratory muscle progenitors into myomatrix spheroids containing skeletal myotubes; thereby producing contractile engineered skeletal muscle tissue, the method is characterized in that the steps (i)-(iii) are carried out in a bioreactor under suitable conditions; and wherein the method does not include the use of a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma, as further defined in the claims.

[0017] An engineered skeletal muscle tissue obtainable by a method disclosed herein is also disclosed wherein said engineered skeletal muscle tissue generates a contraction force of at least 0.5 millinewtons (mN) upon a stimulus from 1 Hz to 150 Hz, wherein said engineered skeletal muscle tissue is serum-free, and wherein said engineered skeletal muscle tissue does not comprise a transgene, as further defined in the claims.

[0018] Figures

[0019] Figure 1. Schematic of the bioreactor- based differentiation protocol utilizing production of myomatrix spheroids

[0020] Differentiation protocol for bioreactor-based directed differentiation of pluripotent stem cells (PSCs) into myomatrix spheroids containing skeletal myoblasts and satellite-like cells. The first step corresponds to formation of PSCs aggregates that undergo mesodermal induction (step 2) and myogenic specification (step 3), differentiated into 3D myomatrix spheroids that can further undergo myogenic expansion and maturation, e.g., either in suspension in a bioreactor or in suitable molds with or without hydrogel support. The timeline and the addition of factors is purely exemplary.

[0021] Figure 2. Development of myomatrix spheroids.

[0022] Morphology of pluripotent stem cell aggregates during differentiation into myomatrix spheroids. Myomatrix spheroids were produced form a transgenic iPSC line with stable expression of a muscle reporter. Muscle cells that express sarcomeric a-actinin (ACTN2), representing the specific and matured muscle biomass, are labelled by the fluorescent reporter protein citrine (green). Scale bar: 500 pm.

[0023] Figure 3. Fusion of myomatrix spheroids in a collagen 1 hydrogel.

[0024] Myomatrix spheroids differentiated for 13 days were cast into a collagen 1 hydrogel and cultured in expansion medium. 24 hrs after seeding cells started to migrate out of the spheroids (Day 22: arrows). Aggregate structure is lost and spheroids immerse into a confluent tissue by day 7. Scale bar: 500 pm

[0025] Figure 4. Characterization of migrating cells emanating from the myomatrix spheroids.

[0026] Immunostaining of myomatrix spheroids in collagen 1 hydrogel after 7 days of culture. Staining for muscle migrating muscle progenitors (nuclear staining of PAX7) as well as differentiating muscle (ACTN2) cells emanating from the myomatrix spheroids core (nuclei-rich region labelled by asterisks). Scale bar: 100 pm.

[0027] Figure 5. RNA expression indicates formation of muscle and non-muscle cell populations in myomatrix spheroids.

[0028] RNA counts measured by nCounter™ platform. Data indicates loss of pluripotency (POU5F1) followed by induction of paraxial mesoderm (TBX6). Induction of somite markers (PAX3) and migratory muscle progenitors (LBX1) is paralleled by induction on non-myocyte markers (PRRX1, FAP). Myomatrix spheroids were cultured in stirred-tank bioreactors until day 21 (n=3) followed by harvest for muscle generation. Some aggregates were continued in suspension culture in low-attachment 6 well plates at 37°C on a rocking plate (65 rpm) for day 30 and day 37 samples.

[0029] Figure 6. Macroscopic fusion of myomatrix spheroids into skeletal muscle tissue rings.

[0030] (A) Casting of myomatrix spheroids in a ring-shaped mold in a collagen 1-hydrogel without Matrigel. (B) Fusion of myomatrix spheroids in collagen 1 hydrogel after 8 days of culture. (C) Myomatrix spheroids-derived muscle tissue ring on a metal holder for mechanical loading. (D) Skeletal muscle ring after maturation at day 51 of culture. Green fluorescence from muscle reporter (ACTN2-Citrine) confirms muscle-containing tissue. Scale bar: 500 pm.

[0031] Figure 7. Function of myomatrix spheroid-derived engineered skeletal muscle.

[0032] Muscle twitch tension was measured by isometric force recordings in Tyrode's solution in ther- mostatted organ baths at 37°C. Electrical field stimulation was performed at 0.5 Hz to generate single twitches or at 100 Hz for 4 sec to generate a tetanic contraction ("tetanus").

[0033] Figure 8. Morphology of myomatrix spheroids-derived engineered skeletal muscle

[0034] Immunostaining of a-actinin (green), Collagen VI (magenta), nuclei (blue). Scale bar: 50 pm.

[0035] Figure 9. Contractile function of engineered skeletal muscle from myomatrix spheroids at different stages of development.

[0036] Twitch tension of engineered skeletal muscle made from myomatrix spheroids at day 17 (n=l), day 21 (n=3), and day 30 (n=4) of differentiation without addition of Matrigel. For comparison engineered skeletal muscle tissue made according to PCT / EP2020 / 078738 (WO2021 / 074126 Al) with (n=17) or without Matrigel (n=4) are shown. Contractile function was assessed at day 56 of differentiation. This experiment provides a direct comparison illustrating the technical advance over the previous application WO'126.

[0037] Figure 10. Contractile function of engineered skeletal muscle from myomatrix spheroids with or without exogenous matrix.

[0038] Myomatrix spheroids at day 21 of differentiation were cast-molded into chambers in a collagen I-hydrogel (+collagen, n=3) or without a hydrogel (-collagen, n=5) to form engineered skeletal muscle. Contractile function was measured by optical analysis of movement of two flexible poles upon electrical stimulation with 40 Hz, 20 ms bipolar pulses, 20 V. Contraction curves of individual ESM show comparable development of tetanic twitch tension demonstrating that the myomatrix of bioreactor-derived spheroids is sufficient to allow fusion into a force-generating muscle.

[0039] Detailed description

[0040] The present disclosure relates to a method for producing an engineered skeletal muscle tissue from pluripotent stem cells, comprising the steps of

[0041] (i) forming two or more pluripotent stem cell aggregates from pluripotent stem cells;

[0042] (ii) inducing mesoderm differentiation of the pluripotent stem cell aggregates;

[0043] (iii) inducing the myogenic specification thereby obtaining two or more myomatrix spheroids;

[0044] (iv) expanding and maturing the two or more myomatrix spheroids into myomatrix spheroids containing skeletal myoblasts and migratory muscle progenitors;

[0045] (v) maturing myomatrix spheroids containing skeletal myoblasts and migratory muscle progenitors into myomatrix spheroids containing skeletal myotubes; thereby producing contractile engineered skeletal muscle tissue, the method is characterized in that the steps (i)-(iii) are carried out in a bioreactor under suitable conditions; and wherein the method does not include the use of a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma, as defined in the claims.

[0046] The skilled person is generally aware of the general differentiation steps required for the differentiation from pluripotent stem cells to an engineered skeletal muscle tissue (see e.g. PCT / EP2020 / 078738 published as WO2021 / 074126 Al). Said differentiation is induced by defined factors / inhibitors. In comparison to PCT / EP2020 / 078738, step (i) is added, which is carried out in a bioreactor. The addition of this step has the advantage that the cells are capable of forming an initial three-dimensional network on which the three-dimensional skeletal muscle can be built on. Furthermore, it is important that said additional step (i) is carried out in a bioreactor. Moreover, compared to PCT / EP2020 / 078738 (WO2021 / 074126 Al), steps (ii) and (iii) are also carried out in a bioreactor according to the present invention. This is further advantageous as the growth environment of the cells and spheroids can be tightly controlled as to for example nutrient and oxygen supply, metabolite clearance, and pH, which cannot be achieved in monolayer cultures on plates. According to the present invention, bioreactors are the only viable way to achieve sufficient biomass for application in meat industry and regenerative medicine. In addition, culturing in a bioreactor provides tight control over the selforganization and differentiation of PSCs into muscle cells and myomatrix spheroids. According to the invention at least steps (i) to (iii) of the method are carried out in a bioreactor.

[0047] It is contemplated that steps (iv) and (v) are also carried out in a bioreactor. The conditions in the bioreactor in steps (iv) and (v) may be individually selected from the conditions disclosed herein for steps (i) to (iii). It is further contemplated that the bioreactor may be a stirred tank bioreactor. Alternatively, steps (iv) and (v) could be carried out in a casting mold. Possible casting molds are for example described in WO2022 / 084429. The method may be further characterized in that steps (iv) and (v) be carried under mechanical conditioning for myomatrix spheroid fusion to the desired scale. For example, the two or more myomatrix spheroids obtained by step (iii) are further embedded in an extracellular matrix hydrogel after completion step (iii). The skilled person is generally aware of hydrogels used for tissue engineering. Ideally, the extracellular matrix hydrogel is selected from a collagen hydrogel, a laminin hydrogel, a hyaluronic acid hydrogel, a gelatin hydrogel, a fibrin / fibrinogen hydrogel, a poly lactic acid hydrogel, an alginate hydrogel, a methylcellulose hydrogel, a polyethylene glycol hydrogel, a hydrogel derived from decellularized tissues, a matrix from spider silk, decellularized extracellular matrix from human or animal organ systems, aliginates, xanthan gum, vegetable binders or combinations thereof. In a preferred embodiment, the extracellular matrix hydrogel is a collagen hydrogel. The skilled person is aware that various extracellular matrix hydrogels are commercially available such as from Gibco, Sigma-Aldrich, or LLC Collagen solutions. Collagen I used in the Examples presented herein was obtained from LLC Collagen solutions (catalog number FS22024).

[0048] After completion of step (iii) of the method, the pluripotent stem cells have formed myomatrix spheroids. Specifically, step (iii) may be completed when the myomatrix spheroids express the mRNA of PRRX1 (Paired related homeobox 1) and / or FAP (Fibroblast activation protein-o). Ideally, the myomatrix spheroids express the mRNA of PRRX1 and FAP. Figure 5 provides exemplary data of the expression of PRRX1 and FAP reported as RPKM (Reads Per Kilobase per Million mapped reads). Furthermore, the myomatrix spheroids may comprise a mesenchymal myomatrix-producing non-myocyte cell population. Ideally, said mesenchymal myomatrixproducing non-myocyte cell population comprises stromal cells, Fibre / Adipogenic Progenitors (FAP)-cells, satellite cells and / or neurons. It is preferred that said mesenchymal myomatrixproducing non-myocyte cell population comprises stromal cells, FAP-cells, satellite cells and neurons. The mRNA of PRRX1 and / or FAP may be indicative of the presence of a mesenchymal myomatrix-producing non-myocyte cell population.

[0049] Figure 1 provides a schematic overview of the method steps. Ideally, step (iii) is completed after at least 17 days counted from the start of the method. It is also contemplated that step (iii) is completed after at least 18 days, preferably after at least 19 days, more preferably after at least 20 days, and more preferably after at least 21 days. In some embodiments, step (iii) is completed after at most 28 days, preferably at most 25 days, more preferably at most 22 days, and even more preferably at most 21 days. The upper and lower day limits may be freely combined. For example, step (iii) may completed from 19-25 days counted from the start of the method.

[0050] It is described that the collagen may be type I collagen. As illustrated in the Example Section, collagen is particularly suitable. Said type I collage may be of bovine origin, porcine origin, human origin or marine origin. Said collagen may also be produced by recombinant expression. Especially, the collagen may be of bovine origin.

[0051] As known in the art, different tissues have different degrees of stiffness, i.e., the ratio between stress and strain, which is also called Young's modulus and presented as E and generally expressed in Pascals (Pa) and ranging from 200 Pa in bone marrow to 20 GPa in bone (see e.g., Akhmanova M, et al. (2015)). Extracellular matrix is typically present with similar stiffness, but this can vary depending on its preparation. Typical ECM hydrogel preparations exhibit a stiffness from 10 Pa to 20 kPa. Said stiffness may be determined by rheological measurement. It is preferred that the extracellular matrix hydrogel may have a stiffness from 15 Pa to 100 kPa, more preferably from 200 Pa to 12 kPa, even more preferably from 500 Pa to 8 kPa, even more preferably from 1 kPa to 6 kPa, even more preferably from 2 kPa to 4 kPa.

[0052] The myomatrix spheroids generated by completion of steps (i)-(iii) may be embedded into an engineered form in the presence of extracellular matrix hydrogel as defined above. The engineered form may have the form of a ring, ribbon, strand, patch, pouch, or cylinder. Alternatively, the individual engineered skeletal muscle tissues may be fused. This allows the generation of thicker and tissues, which may be advantageous, in particular for the production of artificial meat. Preferably, the embedded myomatrix spheroids are embedded in the extracellular matrix hydrogel which are casted into a casting mold or which are 3D printed. In preferred embodiments, the myomatrix spheroids generated by completion of steps (i)-(iii) may be casted into a casting mold. Said casting may be with or without a hydrogel to support the self-assembly into a desired form (see e.g. Figure 10 herein for experimental support). The desired form may have the shape of a ring, ribbon, strand, patch, pouch, or cylinder. Alternatively, the individual engineered skeletal muscle tissues may be fused. This allows the generation of thicker tissues, which may be advantageous, in particular for the production of artificial meat.

[0053] The myomatrix spheroids after completion of step (iii) may be casted with or without supporting hydrogel into molds of desired geometry and size to facilitate the self-assembly as ring, ribbon, strand, patch, pouch, or cylinder structures or allow fusion of multiple layers into desired geometries.

[0054] Alternatively, the myomatrix spheroids after completion of step (iii) may be 3D printed with or without supporting hydrogel and / or support material into a desired geometry and size to facilitate the self-assembly, e.g., as a ring, ribbon, strand, patch, pouch, or cylinder structures.

[0055] With regard to step (ii), said step may be carried out by culturing the pluripotent stem cell aggregates in a basal medium comprising an effective amount of (a) FGF2, (b) a GSK3 inhibitor, (c) one or more, preferably one or two, SMAD inhibitor(s), and (d) a serum-free additive comprising transferrin, insulin, progesterone, putrescine and selenium or a bioavailable salt thereof. It is even more preferred that the basal medium comprises (c) one SMAD inhibitor. Exemplary media used are described in detail in the Example section. Specifically, an exemplary (c) serum-free additive is provided in Table 2 herein.

[0056] Furthermore, step (iii) may be carried out by culturing the cell aggregates obtained in step (ii) in a basal medium comprising an effective amount of (a) a gamma-secretase / NOTCH inhibitor, (b) FGF2, and (c) a serum-free additive as defined in the preceding paragraph, followed by continuing the cultivation in the medium with the addition of an effective amount of (d) HGF, followed by culturing the cells in a basal medium comprising an effective amount of (a) a gamma secretase / NOTCH inhibitor, (b) HGF, (c) a serum-free additive as defined in the preceding paragraph, and (d) knockout serum replacement (KSR) thereby obtaining two or more myomatrix spheroids. The skilled person is generally aware of KSR. Exemplary ingredients and concentrations are provided in Table 6 herein.

[0057] The myomatrix spheroids obtained after step (iii) may be aggregates of cells comprising somite progenitor cells and myomatrix proteins. Conducting step (iii) in a bioreactor enables a closer emulation of the physiological conditions governing myogenic specification in vivo. Unlike previously reported 2D-based myogenic specification protocol, wherein cells are uniformly exposed to differentiation factors, myogenic specification in a bioreactor exposes cells within aggregates to varying concentrations of nutrients and factors based on their localization within the aggregate. This process likely facilitates intracellular signalling, which further supports myogenic specification into different cell types. For example, the somite progenitor cells may express the marker(s) selected from the group of PAX3, LBX1, and MET. Ideally, all three markers are expressed. In addition, the myomatrix proteins may be COL6A1-3 and / or FN1. Preferably, both myomatrix proteins are expressed. The person skilled in the art is aware of the methods by which expression of the markers can be assessed, such as Western Blot, RNA- seq, RT-PCR, immunofluorescence confocal microscopy. The aggregates obtained from step (iii) are referred hereinto as myomatrix spheroids.

[0058] Exemplary markers after each step of the method are as follows, wherein one or more of these markers is ideally expressed:

[0059] In particular, the myomatrix spheroids obtained after step (iii) may have a size from 100 pm to 400 pm, preferably from 120 to 300 pm, more preferably from 150 to 250 pm, even more preferably from 160 to 220 pm and the most preferably of about 200 pm.

[0060] Regarding step (iv), said step may be carried out by culturing said myomatrix spheroids in a basal medium comprising an effective amount of (a) HGF, (b) a serum-free additive as defined above, and (c) knockout serum replacement (KSR).

[0061] With regard to step (v), said step may be carried out by culturing the myomatrix spheroids containing skeletal myoblasts and migratory muscle progenitors obtained in step (iv) under mechanical or electrical stimulation, preferably mechanical stimulation, in a basal medium, comprising an effective amount of (a) a serum-free additive as defined above, and (b) an additional serum-free additive comprising transferrin, ethanolamine, selenium or a bioavailable salt thereof, L-carnitine, fatty acid additive, and triiodo-L-thyronine (T3), preferably wherein the additional serum-free additive additionally comprises albumin.

[0062] It is preferred that the myomatrix spheroids containing skeletal myoblasts and migratory muscle progenitors obtained in step (iv) further contain stromal cells, FAP-cells, satellite cells and neuronal cells.

[0063] Upon completion of the method steps, the engineered skeletal muscle tissue ideally generates a contraction force of at least 0.5 millinewtons (mN) upon a stimulus from 1 Hz to 150 Hz. It is preferred that said contraction force is at least 0.6 mN, more preferably at least 0.7 mN, more preferably at least 0.8 mN, more preferably at least 0.9 mN, and most preferably at least 1 mN. It is further preferred that said stimulus is from 5 Hz to 140 Hz, more preferably from 10 Hz to 130 Hz, more preferably from 20 Hz to 125 Hz, more preferably from 40 Hz to 120 Hz, more preferably from 60 Hz to 115 Hz, more preferably from 80 Hz to 110 Hz, more preferably from 90 Hz to 105 Hz, and most preferably about 100 Hz. In particular, the engineered skeletal muscle tissue may generate a contraction force of at least 0.5 millinewtons (mN) upon a stimulus of about 100Hz.

[0064] The pluripotent stem cells are ideally of animal or human origin. For example, the pluripotent stem cells may be of porcine origin, bovine origin, caprine origin, equine origin, ovine origin, ruminant origin, avian origin, fish origin or primate origin. Furthermore, the pluripotent stem cells may be selected from induced pluripotent stem cells, parthenogenetic stem cells, and pluripotent cells produced via chemical reprogramming or non-human embryonic stem cells. It is preferred that the pluripotent stem cells are induced pluripotent stem cells or parthenogenetic stem cells. The pluripotent stem cells are not human embryonic stem cells.

[0065] As disclosed above, the steps (i)-(iii) are carried out in a bioreactor. Said bioreactor is ideally a stirred-tank type of bioreactor, a rocker bioreactor, an air-lift bioreactor, a fixed-bed bioreactor, a continuous bioreactor, or a fed-batch bioreactor. It is preferred that the bioreactor is a stirred-tank type of bioreactor.

[0066] The basal medium in step (i), step (ii), step (iii), step (iv) and / or in step (v) may be selected from DMEM, DMEM / F12, RPMI, IMDM, alphaMEM, Medium 199, Hams F-10, Hams F-12. The skilled person is generally aware of these media and also knows that these media are commercially available. It is preferred that the basal medium is DMEM. The basal medium may be supplemented with pyruvate and / or non-essential amino acids. Furthermore, the basal medium may comprise from 1 to 4.5 g / l glucose. An exemplary composition of a preferred medium including specific concentrations is disclosed in Table 4 herein. Ideally, step (i) is carried out in the presence of ROCK inhibitor. The skilled person is generally aware of ROCK inhibitors and that said ROCK inhibitors are commercially available. Exemplary ROCK inhibitor of which one may be selected from are Y27632, H-1152P, thiazovivin, fasudil, hydroxyfasudil, GSK429286A and RKI1447. It is preferred that the ROCK inhibitor is selected from the group consisting of Y27632, H-1152P, thiazovivin, fasudil and Hydroxyfasudil. For example, the ROCK inhibitor is selected from the group consisting of Y27632 and H-1152P. Ideally, the ROCK inhibitor is Y27632. If the ROCK inhibitor is Y27632, the ROCK inhibitor is used at a concentration of 0.5-20 pM, preferably 1-16 pM, more preferably 2-12 pM, more preferably 4-11 pM, more preferably 7-10 pM, and most preferably at a concentration of about 10 pM. In such a scenario the stem cell medium is ideally iPS-Brew XF.

[0067] At step (i) the cells may be seeded into the bioreactor at concentration from 1.3 x 105cells / ml to 5.4 x 105cells / ml, more preferably from 2.5 x 105cells / ml to 4 x 105cells / ml, most preferably at around 3.75xl05 / ml.

[0068] Step (i) is ideally completed, when the aggregates reach size of at least from 100 pm to 400 pm, preferably from 120 to 300 pm, more preferably from 150 to 250 pm, even more preferably from 160 to 220 pm and the most preferably about 200 pm. The aggregates obtained after step (i) ideally express one or more pluripotency marker(s). Said one or more pluripotency marker(s) may be selected from the group consisting of OCT4, NANOG, TRA1-60, and SSEA5. It is preferred that all of these pluripotency markers are expressed. The skilled person is aware of methods to detect the expression of pluripotency markers. For example, the expression of marker(s) is / are detectable by flow-cytometry, by immunostaining methods such as Western blot, by immunostaining methods combined with fluorescence microscopy, by light microscopy, by PCR, RNA-amplification, by RNA-fish or by RNA-seq techniques. It is preferred that the markers are detected by flow-cytometry. Flow-cytometry has the advantage that it can be determined which percentage of the population of cells is expressing a marker. Ideally, the one or more pluripotency marker(s) is / are expressed by at least 75% of cells in a population, which, for example can be determined by flow cytometry. It is more preferred that at least 80% of cells in the population express one or more pluripotency marker. Alternatively at least 85% of cells in the population or at least 90% of cells in the population express one or more pluripotency marker. For example, 90% of the cells in the aggregates obtained after step (i) may express OCT4, NANOG, TRA1-60, and SSEA5.

[0069] Step (i) may carried out for 72 to 288 hours. An exemplary timeline is also shown in Figure 1. It is preferred that step (i) is carried out for 80 to 270 hours, in particular for 90 to 255 hours, more preferably for 96 to 230 hours, and even more preferably for 120 to 220 hours. As shown in the Example section, step (i) is ideally carried out for about 216 hours.

[0070] Scalable production in bioreactors is achieved by pluripotent stem cell aggregation without the need for microcarrier systems. By adjusting the size if iPSC aggregates through mechanical shear (stirring speed) and equal distribution of nutrients and oxygen is ensured. In some embodiment, the temperature in the bioreactor is from 34 to 39 °C. It is preferred that the temperature in the bioreactor is from 36 °C to 39°C, preferably from 36.5 to 38 °C and most preferably at around 37 °C. It is preferred that the temperature in the bioreactor is from 34 to 37 °C for step (i), (ii) and / or (iii), which can be individually selected. In some embodiments, the temperature may be adjusted to correspond to the preferable temperature of organism of origin. For example, for cultivation of fish cells the temperature in bioreactor may be from 20 to 30 °C. In particular, the aeration in the bioreactor may be from 20 to 26%, preferably from 21 to 25 %, preferably from 22 to 24 %, more preferably from 23 to 24%. It is most preferred that the aeration in the bioreactor is around 23.8%. Ideally, the aeration is continuously measured. It is preferred that the aeration in the bioreactor is from 20 to 26% for step (i), (ii) and / or (iii), which can be individually selected. In some embodiments, the bioreactor is a stirred-tank type of bioreactor. Such bioreactors have a stirring rate. For example, the stirring rate within said bioreactor may be from 90 to 130 rpm. It is preferred that the stirring rate is from 100 to 120 rpm. In some preferred embodiments, the stirring rate is around 110 rpm. It is preferred that the stirring rate in the bioreactor is from 90 to 130 rpm for step (i), (ii) and / or (iii), which can be individually selected. Ideally, the stirring rate is at around 110 rpm to 130 rpm for step (i) of the method. In preferred embodiments the stirring rate is from 90 rpm to 110 rpm in step (ii) and / or step (iii). It has been found that the disclosed stirring rates are advantageous over higher or lower stirring rates for the development of skeletal muscle tissue. It is further preferred that the bioreactor is a stirred-tank type of bioreactor having downward stirring. Furthermore, it was found that downward stirring is particularly advantageous for the development of skeletal muscle tissue. In preferred embodiment, the stirred-tank type of bioreactor with a stirrer set at an angle from 35° to 55°, preferably from 37.5° to 52.5 °, more preferably from 40° to 50°, even more preferably from 42.5° to 47.5° and most preferably at around 45°. It is preferred that the stirrer set at the angle from 35° to 55° in the bioreactor is for step (i), (ii) and / or (iii), which can be individually selected.

[0071] The pH value of the medium may be controlled by the bioreactor. Ideally the pH is controlled by a CO2 supply. If necessary, a base, such as NaOH, can be added to adjust the pH. The pH- value in the medium may be from 6.6 to 7.6, preferably from 6.7 to 7.6, more preferably from 6.8 to 7.6, more preferably from 6.9 to 7.5, more preferably from 7.0 to 7.5 and even more preferably about 7.4. It is preferred that the pH value is 6.6 to 7.6 for step (i), (ii) and / or (iii), which can be individually selected.

[0072] The bioreactor may be configured to have a cell culture volume of between about 2 mL - 20,000 L. Preferred bioreactors may have a volume of from 250 mL to 2000L. An exemplary bioreactor suitable for the method of the present invention is a Sartorius BIOSTAT™ B-DCUII Triple. Such bioreactors are available from volumes of 250 mL to 2000L. The advantage of such stirred-tank bioreactors is the seamless scalability for commercial production. In some embodiments, such as application in edible meat production, the cell culture volume in the preferred bioreactor is from about 10 L to about 2,000 L. In some embodiments such as medical and research applications, the preferred cell culture volume in the bioreactor is from 10 to 200 L. This may be less or more depending on the specific application - production at the point-of-consumption or point-of-care requirement vs mass production of food or medicines.

[0073] With regard to the bioreactor conditions, Step (ii) and / or step (iii) may be carried out under the same conditions as step (i). The conditions as described here may be individually selected for step (ii) and (iii). In other words, the conditions of step (i), (ii) and (iii) may be different for each step. Alternatively, the conditions in step (ii) and (iii) may be individually the same as in step (i).

[0074] In step (ii), the GSK3 inhibitor may be selected from the group consisting of CHIR99021, CHIR98014, SB216763, TWS119, tideglusib, SB415286, 6-bromoindirubin-3-oxime and a valproate salt. The skilled person is generally aware of GSK3 inhibitors and that such GSK3 inhibitors are commercially available. It is preferred that the GSK3 inhibitor is CHIR99021. In some embodiments, the SMAD inhibitor is selected from the group consisting of LDN193189, K02288, LDN214117, ML347, LDN212854, DMH1, SB431542, SB505124, A83-01 or a combination thereof in step (ii). The skilled person is generally aware of SMAD inhibitors and that such SMAD inhibitors are commercially available. It is preferred that one or two SMAD inhibitors are selected from the group listed above. It is even more preferred that one of the two SMAD inhibitors is LDN193189. Ideally, the one SMAD inhibitor is LDN193189, which for example has also been used in the Example section.

[0075] In step (ii) the effective amount of FGF2 is 1-15 ng / ml, preferably 2.5-14 ng / ml, more preferably 5-13 ng / ml, even more preferably 7.5-12.5 ng / ml, even more preferably 8-12 ng / ml, even more preferably 9-11 ng / ml, and most preferably about 10 ng / ml; and / or the serum-free additive provides in the medium a final concentration of 50-500 pg / ml transferrin, 1-20 pg / ml insulin, 0.001-0.1 pg / ml progesterone, 5-50 pg / ml putrescine and 6-600 nM selenium or a bioavailable salt thereof, in particular sodium selenite, and / or the GSK3 inhibitor is CHIR99021, and the effective amount is 1-20 pM, preferably 2-19 pM, more preferably 3-18 pM, even more preferably 4-17 pM, even more preferably 5-16 pM, even more preferably 6-15 pM, even more preferably 7-14 pM, even more preferably 7.5-13 pM, even more preferably 8-12 pM, even more preferably 9-11 pM, and most preferably about 10 pM; and / or the SMAD inhibitor is LDN193189, and the effective amount is 0.05-5 pM, preferably 0.1-2.5 pM, more preferably 0.2-1 pM, even more preferably 0.25-5 0.8 pM, even more preferably 0.3-0.75 pM, even more preferably 0.35-0.7 pM, even more preferably 0.4-0.6 pM, even more preferably 0.45-0.55 pM, and most preferably about 0.5 pM.

[0076] The method of any preceding embodiments, wherein the step (ii) is completed when the cells express one or both markers TBX6 and CDX2. The skilled person is generally aware that he / she can detect the expression of TBX6 and / or CDX2, for example by flow cytometry. As mentioned above, the advantage of flow cytometry is that the skilled person can detect, which percentage of the population is expressing a specific marker. Alternatively, the expression of the marker(s) is / are detectable by immunostaining methods such as Western blot, by immunostaining methods combined with fluorescence microscopy or light microscopy, by PCR, by RNA-amplification, by RNA-fish or by RNA-seq techniques. Ideally, at least 75% of cells in the population after completion of step (ii) express one or both marker(s) (TBX6 and CDX2). It is preferred that at least 80% of cells in the population, more preferably at least 85% of cells in the population, and most preferably at least 90% of cells in the population express TBX6 and / or CDX2. Ideally, both markers are expressed.

[0077] The step (ii) may carried out for 24 to 132 hours. For example, step (ii) may be carried out for 48 to 120 hours. In particular, step (ii) may be carried out for 60 to 114 hours or, preferably, for 72 to 108 hours, or more preferably for 84 to 102 hours. As shown in Figure 1, it is most preferred that step (ii) is carried out for about 96 hours.

[0078] In step (ii) the medium may comprise a serum-free additive. Said serum-free additive may be 0.1-10% (v / v) N2 additive. The skilled person is aware of N2 additive and that it is commercially available. Table 2 herein provides an exemplary composition of N2 additive. It is preferred that the serum-free additive may be 0.3-7.5% (v / v) N2 additive, more preferably 0.5- 5% (v / v) N2 additive, more preferably 0.75%-2% (v / v) N2 additive, more preferably 0.9%- 1.2% (v / v) N2 additive, and most preferably about 1% (v / v) N2 additive. The advantage of a serum-free additive is that it is fully defined so that the method is defined, robust and reproducible. In some embodiments, the basal medium in step (ii), step (iii), step (iv), and / or in step (v) is selected from DMEM, DMEM / F12, RPMI, IMDM, alphaMEM, Medium 199, Hams F-10, Hams F- 12. The medium may be individually selected for each step or may be the same in these steps. The skilled person is aware that these media are commercially available. Ideally, the basal medium is DMEM. Table 4 shows the composition of DMEM, which can be used. Furthermore, the basal medium may be supplemented with pyruvate and / or non-essential amino acids. Table 3 provides the ingredients and concentration of exemplary non-essential amino acids. Furthermore, the basal medium may comprise from 1 to 4.5 g / l glucose.

[0079] The cultivation in step (iii) may be carried out in the presence of (a) a gamma-secre- tase / NOTCH inhibitor, (b) FGF2, (c) the serum-free additive, and (d) HGF for 36 to 60 hours, preferably for 42 to 54 hours, and even more preferably for about 48 hours; followed by the cultivation which may be carried out in the presence of (a) a gamma-secre- tase / NOTCH inhibitor, (b) HGF, (c) the serum-free additive, and (d) knockout serum replacement (KSR) for 72 to 120 hours, preferably for 76 to 114 hours, more preferably for 84 to 108 hours, even more preferably for 90 to 102 hours, and most preferably for about 96 hours.

[0080] For example, the gamma-secretase / NOTCH inhibitor used in step (iii) could be selected from DAPT, RO4929097, semagacestat (LY450139), avagacestat (BMS-708163), BMS-299897, dibenzazepine (YO-01027), LY411575, IMR-1, and L685458. The preferable gamma-secretase / NOTCH inhibitor is DAPT. The person skilled in the art is aware of other the gamma- secretase / NOTCH inhibitor that may be used and that they are commercially available from various suppliers. The gamma-secretase / NOTCH inhibitor used in the Example section was purchased from TOCRIS (Cat. No. 2634).

[0081] In step (iii), the effective amount of FGF2 may be 15-30 ng / ml, preferably 17.5-25 ng / ml, more preferably 18-22 ng / ml, even more preferably 19-21 ng / ml, and most preferably about 20 ng / ml. The skilled person is capable of determining an effective amount of FGF2. The skilled person is generally aware that the effective amount of an inhibitor, a cell signalling protein, or receptor / enzyme agonist varies with the availability and biological activity of the respective substance.

[0082] The effective amount of HGF used in the step (iii) may be 1-15 ng / ml, preferably 2.5-14 ng / ml, more preferably 5-13 ng / ml, even more preferably 7.5-12.5 ng / ml, even more preferably 8-12 ng / ml, even more preferably 9-11 ng / ml, and most preferably about 10 ng / ml. As noted above, the person skilled in the art is generally aware that the effective amount or concentration depends on availability and biological activity of the particular substance. Furthermore, the skilled person is general capable of determining an effective amount of HGF.

[0083] If the gamma-secretase / NOTCH inhibitor is DAPT in step (iii), the effective amount may be 1- 20 pM, preferably 2-19 pM, more preferably 3-18 pM, even more preferably 4-17 pM, even more preferably 5-16 pM, even more preferably 6-15 pM, even more preferably 7-14 pM, even more preferably 7.5-13 pM, even more preferably 8-12 pM, even more preferably 9-11 pM, and most preferably about 10 pM. As noted above, the person skilled in the art is generally aware that the effective amount or concentration depends on availability and biological activity of the particular substance. Furthermore, the skilled person is general capable of determining an effective amount of the gamma-secretase / NOTCH inhibitor.

[0084] The "knockout serum replacement" (KSR) used herein refers to the mixture of ascorbic acid, insulin, transferrin, and albumin, each present at an effective concentration. In a preferred embodiment, KSR also includes an effective concentration of selenium or its bioavailable salt, along with glutathione and trace elements. In a further preferred embodiment, KSR comprises the combination of substances listed in Table 6. In the most preferred embodiment, KSR comprises the components outlined in Table 6 at the specified concentrations. The 'knockout serum replacement' (KSR) is known to the person skilled in the art, who is aware that KSR is readily accessible commercially from various suppliers (for example, Gibco). Ideally, the KSR is used in an amount of 5-20% (v / v), preferably 6-17.5% (v / v), more preferably 7-15% (v / v), more preferably 8%-12% (v / v), more preferably 9%-l 1% (v / v), and most preferably about 10% (v / v) KSR. In the most preferred embodiment the KSR is used in the presence of a reducing agent such as beta-mercaptoethanol and / or alpha-thioglycerol. It is preferred that the KSR is used in step (iii). It is preferred that the KSR is used in step (iv). It is even more preferred that the KSR is used in steps (iii) and (iv).

[0085] Ideally, step (iv) is carried out by culturing said myomatrix spheroids in a basal medium comprising an effective amount of (a) HGF, (b) a serum-free additive as defined herein, and (c) knockout serum replacement (KSR). In general the skilled person is capable to determine an effective amount. For example, the effective amount of HGF used in step (iv) may be 1-15 ng / ml, preferably 2.5-14 ng / ml, more preferably 5-13 ng / ml, even more preferably 7.5-12.5 ng / ml, even more preferably 8-12 ng / ml, even more preferably 9-11 ng / ml, and most preferably about 10 ng / ml.

[0086] In some embodiments, the cells after step (iv) comprise a first and a second population of cells. In particular, step (iv) may be completed when the cells within myomatrix spheroids express at least one marker selected from a group of PAX3, LBX1, MET, corresponding to the first population of cells and at least two markers selected from the group of PRRX1, FAP, EBF2, COL6A1-3 and FN1, corresponding to the second population of cells. In strongly preferred embodiments, all of these markers are expressed after completion of step (iv).

[0087] In the preferred embodiment, 40 % to 60% of the cells in the myomatrix spheroids after completion of step (iv) express PAX3, LBX1, and / or MET corresponding to the first population of cells. Ideally, 40 % to 60% of the cells in the myomatrix spheroids after completion of step (iv) express PRRX1, FAP, EBF2, COL6A1-3 and / or FN1 corresponding to the second population of cells. In preferred embodiment, 45 % to 55% of the cells in the myomatrix spheroids after completion of step (iv) express PAX3, LBX1, and / or MET; and / or 45 % to 55% of the cells in the myomatrix spheroids after completion of step (iv) express PRRX1, FAP, EBF2, COL6A1-3 and / or FN1. It is even more preferred that around 50% of the cells in the myomatrix spheroids after completion of step (iv) express PAX3, LBX1, and / or MET; and / or wherein around 50% of the cells in the myomatrix spheroids after completion of step (iv) express PRRX1, FAP, EBF2, COL6A1-3 and / or FN1. In preferred embodiments, 75% of cells in the first population of cells express PAX3, LBX1, and / or MET, preferably at least 80% of cells in the first population of cells, more preferably at least 85% of cells in the first population of cells, and most preferably at least 90% of cells in the first population of cells. It is also preferred that 75% of cells in the second population of cells express PRRX1, FAP, EBF2, COL6A1-3 and / or FN1, preferably at least 80% of cells in the second population of cells, more preferably at least 85% of cells in the second population of cells, and most preferably at least 90% of cells in the second population of cells. The person skilled in the art is aware of the methods used for assessing expression of these markers. For example, the skilled person may use flow-cytometry, immunostaining methods such as Western blot, immunostaining methods combined with fluorescence microscopy or polarized light microscopy, PCR, RNA-amplification, RNA-FISH or RNA-seq techniques. The most preferred method for detecting expression of the markers is flow-cytometry. By using flow cytometry, the skilled person is able to quantify the percentage of cells expressing the markers.

[0088] In preferred embodiments, the medium of step (v) comprises an additional serum-free additive. Said additional serum-free additive may comprise 1-100 pg / ml transferrin, 0.1-10 pg / ml ethanolamine, 17.4-1744 nM selenium or a bioavailable salt thereof, in particular sodium selenite, 0.4-40 pg / ml L-carnitine, 0.05-5 pl / ml fatty acid additive, 0.0001-0.1 pg / ml triiodo-L- thyronine (T3). It is preferred that the additional serum-free additive additionally provides a final concentration of 0.5-50 mg / ml albumin in the medium. Ideally, the additional serum-free additive is B27. The skilled person is aware of B27. In particular, the serum-free additive in step (v) may be at a concentration of 0.1-10% (v / v) B27, preferably 0.5-8% (v / v), more preferably 1-6% (v / v), even more preferably 1.5-4% (v / v), and most preferably about 2% (v / v) B27. For example, the composition of B27 is listed in the Table 5 herein. The skilled person can find further information in Brewer et al. (1993).

[0089] In step (v), the myomatrix spheroids may be cultured under a stimulation. In preferred embodiment, said myomatrix spheroids may be cultured in a dedicated casting mold or may be 3D printed in a suitable environment. It is preferred that said myomatrix spheroids are cultured with or without support material such as a hydrogel (see e.g. Figure 10 providing experimental evidence). Said stimulation may be mechanical or electrical. Preferably, the mechanical stimulation is a static tension, a dynamic stimulation, or an auxotonic stimulation. An example of a static tension is an isometric muscle contraction, in which the muscle undergoes only a change in tension and no change in length. Therefore, the muscle does not shorten as a result of isometric muscle contraction. In an auxotonic contraction, both the muscle length and the muscle tension change. Dynamic strain can occur, for example, when the muscle is suspended on flexible supports to promote auxotonic contraction. The most preferable mechanical stimulation is a static tension. Mechanical stimulation, such as static tension, can be applied using a stretching device, which is commonly used in the art.

[0090] Alternatively, the myomatrix spheroids in step (v) may be cultured under an electrical stimulation. Preferably, the electrical stimulation is applied as field or point stimulation, more preferably wherein the electrical stimulation is applied at above a contraction inducing voltage threshold, even more preferably wherein the contraction inducing voltage threshold is determined by visual or video-optic inspection of muscle tissue contraction upon an electrical stimulus. The skilled person is generally capable of determining a contraction inducing voltage threshold upon examination of the muscle tissue contraction in response to an electrical stimulus. Alternatively and less preferably, cellular depolarisation to induce contraction can also be induced by light stimulation. Optogenetic proteins that can be stimulated by specific wavelengths are known in the art and consist of, for example, channelorhodopsin, chrimson.

[0091] It is preferred that the method does not comprise a skeletal myoblast enrichment step. The skeletal myoblast enrichment is commonly referred to the step, wherein non-differentiated cells are removed from the population by means of, e.g., flow cytometry or other sorting methods. For example, the method does not comprise a skeletal myoblast enrichment by cell selection or a skeletal myoblast enrichment by anti body- based cell selection.

[0092] The engineered skeletal muscle tissue obtainable according to the methods disclosed herein can be used for in vitro assay for studying effects of chemical substances, for example, drugs or nutrients. Therefore, the engineered skeletal muscle tissue can be used in pharmacological testing of safety and / or efficacy of chemical compounds, or other interventions, such as physical stimuli.

[0093] These assays may present advantages over in vivo testing, as in vitro studies are more cost- effective and time-efficient. Despite being carried out in vitro, they provide a more physiologically relevant conditions than 2D-cell cultures.

[0094] An example of such application is in vitro method for testing the efficacy of a drug candidate on a skeletal muscle tissue, comprising the steps of

[0095] (a) generating an engineered skeletal muscle tissue using any of the methods of the invention,

[0096] (b) optionally inflicting damage on the engineered skeletal muscle tissue, and

[0097] (c) contacting the engineered skeletal muscle tissue from step (a) or (b) with a drug candidate; preferably wherein the method further comprises determining the contraction force and / or structure of the engineered skeletal muscle tissue and / or metabolic function and / or molecular parameters and / or protein biochemical parameters before and / or after step (c).

[0098] The contraction force of the skeletal muscle tissue may be measured by the contraction experiments described herein. Additionally, the muscle tissue could be examined microscopically by light microscopy experiments, also including fluorescence microscopy, as described herein. The metabolic function of the cells can be assessed by measuring, for example, oxygen consumption, extracellular acid generation rate or glycolysis. The metabolic parameters can be measured, for example, by Seahorse Metabolic Flux Analyzer, which is known to those skilled in the art. Expression of molecular markers in response to the treatment can be assessed by transcriptome analysis methods, such as RNA sequencing or RT-PCR. Change in proteome composition in response to the treatment may be assessed by mass spectrometry or antibodybased methods. For example, common biomarkers (proteins or RNA) related to skeletal muscle could assessed. For example, creatine kinase (also known as creatine kinase CK, CPK, or creatine phosphokinase) and L-lactate dehydrogenase (LDH) could be relevant biomarkers used in such assay.

[0099] Drug candidates comprise pharmacological drug candidates such as drug candidates comprising small molecule compounds and protein-based or nucleic acid-based molecules. Additionally, gene therapeutic drug candidates could be tested, that involve the modification of genome by introducing corresponding nucleic acids or protein-nucleic acid combinations. Moreover, drug candidates may consist of endogenous substances produced by the body, allowing for the evaluation of hormonal or hormone-like signalling effects. Notable examples of such signalling substances include myokines like myostatin, follistatin, irisin, visfatin, and myonectin.

[0100] Similarly, the engineered skeletal muscle obtainable by a method as disclosed herein can be used in in vitro methods for testing the toxicity of a substance on an engineered skeletal muscle tissue, comprising the steps of:

[0101] (a) generating an engineered skeletal muscle tissue using any of the methods of embodiments of the invention,

[0102] (b) contacting the engineered skeletal muscle tissue from step (a) with a substance to be tested.

[0103] It is preferred that said method further comprises determining contraction force and / or engineered skeletal muscle tissue structure and / or metabolic function and / or molecular parameters and / or protein biochemical parameters before and / or after step (b).

[0104] The methods to determine contraction force, metabolic function, molecular parameters and protein biochemical parameters discussed above equally apply herein.

[0105] In vitro method comprising usage of an engineered skeletal muscle generated according to the invention can be used for testing effect of nutrients and dietary supplements. Such method may comprise the following steps:

[0106] (a) generating an engineered skeletal muscle tissue using any of the methods the invention,

[0107] (b) contacting the engineered skeletal muscle tissue from step (a) with a nutrient or dietary supplement to be tested,

[0108] It is preferred that such a method further comprises determining contraction force and / or engineered skeletal muscle tissue structure and / or metabolic function and / or molecular parameters and / or protein biochemical parameters before and / or after step (b).

[0109] Similarly, the methods to determine contraction force, metabolic function, molecular parameters and protein biochemical parameters discussed above equally apply herein.

[0110] An engineered skeletal muscle obtainable by any of the described methods is disclosed. Furthermore, an engineered skeletal muscle obtained by any of the described methods is disclosed herein. In preferred embodiments, said engineered skeletal muscle does not comprise blood vessels and / or innervation by central nervous system. The engineered skeletal muscle tissue according to the invention ideally generates a contraction force of at least 0.5 millinewtons (mN) upon a stimulus from 1 Hz to 150 Hz, preferably at least 0.6 mN, more preferably at least 0.7 mN, more preferably at least 0.8 mN, more preferably at least 0.9 mN, and most preferably at least 1 mN. For example, the stimulus is from 5 Hz to 140 Hz, more preferably from 10 Hz to 130 Hz, more preferably from 20 Hz to 125 Hz, more preferably from 40 Hz to 120 Hz, more preferably from 60 Hz to 115 Hz, more preferably from 80 Hz to 110 Hz, more preferably from 90 Hz to 105 Hz, and most preferably about 100 Hz. In a preferred embodiment, the engineered skeletal muscle tissue according to the invention generates a contraction force of at least 0.5 millinewtons (mN) upon a stimulus of about 100 Hz.

[0111] The generated force of the engineered skeletal muscle tissue may also be measured using a surrogate in, e.g., in ring format, in case the shape and form or the engineered skeletal muscle tissue does not allow for a suitable contraction force measurement. For example, surrogate of the engineered skeletal muscle tissue may be generated in ring-shape in order to measure the contraction force, while the engineered skeletal muscle tissue may be in an unsuitable format to measure the contraction force, e.g. in steak format.

[0112] It is preferred that said engineered skeletal muscle tissue is serum-free. This has the advantage that the engineered skeletal muscle tissue is generated by a defined protocol. Furthermore, a serum-free muscle tissue is free of unknown factors. Ideally, the engineered skeletal muscle tissue is Matrigel-free. This has the advantage that the muscle is fully defined and can be produced in a more reducible fashion. In particular, said engineered skeletal muscle tissue may not comprise a transgene. This has the advantage that the cells in the muscle tissue have differentiated based on the physical and chemical cues, e.g. by being in a bioreactor. In such an approach, the natural environment of differentiating cells is modelled so that the obtained tissue is as close as possible to the natural skeletal muscle tissue.

[0113] Ideally, said engineered skeletal muscle tissue has not undergone genetic editing. Preferably such genetic editing may be CRISPR-Cas editing, TALEN editing or LoxP editing. For example, such genetic editing is CRISPR-Cas editing.

[0114] A engineered skeletal muscle tissue according to the invention for use in treating skeletal muscle disease is also disclosed. This muscle disease may be a hereditary or a non-hereditary muscle disease. This muscle disease may be a tendon disease. It is preferred that hereditary muscle disease is selected from Duchenne muscular dystrophy, Becker muscular dystrophy, Emery-Dreifuss muscular dystrophy, and congenital muscular dystrophies with collagen VI de- ficiency. Furthermore, the non-hereditary muscle disease may be selected from a drug induced, a mechanical inflicted, a blast injury inflicted, a thermally inflicted, a radiation inflicted, a metabolically induced and a osmotically induced disease.

[0115] Moreover, a engineered skeletal muscle tissue for use in treating a matrix-driven congenital muscular dystrophy is disclosed. Said the matrix-driven congenital muscular dystrophy may be Collagen VI muscle dystrophy or Collagen IV muscle dystrophy.

[0116] In addition, an engineered skeletal muscle tissue for use in treating a hereditary or a and non- hereditary muscle disease is disclosed and wherein the engineered skeletal muscle is ideally a muscle replacement, a muscle augmentation. Alternatively, the engineered skeletal muscle tissue may be a muscle supplementation. It is preferred that the engineered skeletal muscle releases trophic, protective, and / or regenerative factors. Such an engineered skeletal muscle has the advantage that the natural skeletal muscle is supported.

[0117] Furthermore, the use of the engineered skeletal muscle tissue as cultured meat is disclosed. One advantage of the invention is that the cultured meat can be cultured to have a desired shape and size. For example, a typical steak or filet shape can be cultured. Specifically, the cultured meat can have said desired shape by casting the cells after step (iii) into a mould having a specified shape or 3D printing technologies may be used. In preferred embodiments, said cultured meat is edible cultured meat. Such edible cultured meat may additionally comprise recombinant protein (for example myoglobin, myosin, actin, or hemoglobin), extracted protein (for example from fungus, yeast, insects, or algae), plant-based protein (for example from soybean, pea, wheat, starch), fibers or thickeners (for example alginate, locust bean gum, xanthan gum, guar gum, carrageenan, cellulose, chitosan, decellularized plants) or combinations thereof. Ideally, the engineered skeletal muscle tissue provides artificial meat for consumption by humans without the need to sacrifice animals. Plant based protein may include soy bean, wheat, pea or a combination thereof. This production method has the advantage that it eliminates or at least drastically reduces the need to raise and farm animals for food. This is also expected to reduce the agriculture-related pollution of the environment and undesirable land-use. Alternatively, the engineered skeletal muscle tissue may be mixed with meat from animals. This has the advantage that less meat from animals is required but the natural taste of meat is retained as much as possible. Furthermore, the engineered skeletal muscle tissue according to the invention may be mixed with fungi--based meat alternatives. Such a fungi-based meat alternative may be a mycelium meat alternative. A further advantage of cultured meat is that it can be produced without the use of antibiotics, which is not the case in conventional meat production using farm animals. Hence, cultured meat is expected to reduce antibiotic resistance and may even reduce zoonotic disease outbreaks. Another advantage of cultured meat is that it can be controlled to be pathogen-free. This includes to absence of bacteria, fungus, virus, and prions.

[0118] A method for producing myomatrix spheroids from pluripotent stem cells, comprising the steps of

[0119] (i) forming two or more pluripotent stem cell aggregates from pluripotent stem cells;

[0120] (ii) inducing mesoderm differentiation of the pluripotent stem cell aggregates by culturing pluripotent stem cell aggregates in a basal medium comprising an effective amount of (a) FGF2, (b) a GSK3 inhibitor, (c) one or more SMAD inhibitors, and (d) a serum-free additive comprising transferrin, insulin, progesterone, putrescine and selenium or a bioavailable salt thereof;

[0121] (iii) inducing the myogenic specification by culturing the cell aggregates obtained in step (ii) in a basal medium comprising an effective amount of (a) a gamma-secre- tase / NOTCH inhibitor, (b) FGF2, and (c) a serum-free additive as in (ii), followed by continuing the cultivation in the medium with the addition of an effective amount of (d) HGF, followed by culturing the cells in a basal medium comprising an effective amount of (a) a gamma secretase / NOTCH inhibitor, (b) HGF, (c) a serum-free additive as in (ii), and (d) knockout serum replacement (KSR); wherein steps (i)-(iii) are carried out in bioreactor; and wherein the method is free of a solubilized basement membrane preparation extracted from the Engelbreth-Holm- Swarm mouse sarcoma.

[0122] Ideally, the obtained myomatrix spheroids express the myomatrix spheroid markers. Exemplary markers are COL6A1-3 and FNl.As already detailed above, the expression of myomatrix spheroid marker(s) may detectable by flow-cytometry; or by immunostaining methods such as Western blot; or by immunostaining methods combined with fluorescence microscopy or polarized light microscopy; or by PCT, RNA-amplification, RNA-fish or RNA-seq techniques; preferably by flow-cytometry. In some embodiment, the cells in the myomatrix spheroids express at least one marker selected from a group of PAX3, LBX1, MET, corresponding to a first population of cells; and at least two markers selected from the group of PRRX1, FAP, EBF2, COL6A1-3 and FN1, corresponding to a second population of cells. In strongly preferred embodiments, all said markers are expressed in the myomatrix spheroids. Ideally, 40 % to 60% of the cells in the myomatrix spheroids express PAX3, LBX1, and / or MET corresponding to the first population of cells. Furthermore, 40 % to 60% of the cells in the myomatrix spheroids may express PRRX1, FAP, EBF2, COL6A1-3 and / or FN1 corresponding to the second population of cells. In preferred embodiments, 45 % to 55% of the cells in the myomatrix spheroids express PAX3, LBX1, and / or MET. In preferred embodiments, 45 % to 55% of the cells in the myomatrix spheroids express PRRX1, FAP, EBF2, COL6A1-3 and / or FN1. It is more preferred that around 50% of the cells in the myomatrix spheroids express PAX3, LBX1, and / or MET; and / or wherein around 50% of the cells in the myomatrix spheroids express PRRX1, FAP, EBF2, COL6A1-3 and / or FN1.

[0123] The obtained myomatrix spheroids according to said method may be used in methods of production of engineered skeletal muscle tissue such as edible meat.

[0124] A method for producing engineered skeletal muscle tissue from two or more myomatrix spheroids comprising the following steps is also disclosed:

[0125] (I) expanding and maturing the two or more myomatrix spheroids into myomatrix spheroids containing skeletal myoblasts and migratory muscle progenitors by culturing said myomatrix spheroids in a basal medium comprising an effective amount of (a) HGF, (b) a serum-free additive comprising transferrin, insulin, progesterone, putrescine and selenium or a bioavailable salt thereof, and (c) knockout serum replacement (KSR);

[0126] (II) maturing myomatrix spheroids containing skeletal myoblasts and migratory muscle progenitors into myomatrix spheroids containing skeletal myotubes by culturing the cells obtained in step (I) under mechanical stimulation in a basal medium, comprising an effective amount of (a) a serum-free additive as in step (I), and (b) an additional serum-free additive comprising transferrin, ethanolamine, selenium or a bioavailable salt thereof, L-carnitine, fatty acid additive, and triiodo-L-thyro- nine (T3), preferably wherein the additional serum-free additive additionally comprises albumin.

[0127] Said method is free of a solubilized basement membrane preparation extracted from the En- gelbreth-Holm-Swarm mouse sarcoma. By carrying out steps (I) and (II) an engineered contractile skeletal muscle tissue is produced.

[0128] When performing said method, the two or more myomatrix spheroids may be obtained or are obtainable by a method according to the present disclosure.

[0129] The invention is described in more detail in the following embodiments:

[0130] 1. A method for producing an engineered skeletal muscle tissue from pluripotent stem cells, comprising the steps of (i) forming two or more pluripotent stem cell aggregates from pluripotent stem cells;

[0131] (ii) inducing mesoderm differentiation of the pluripotent stem cell aggregates;

[0132] (iii) inducing the myogenic specification thereby obtaining two or more myomatrix spheroids;

[0133] (iv) expanding and maturing the two or more myomatrix spheroids into myomatrix spheroids containing skeletal myoblasts and migratory muscle progenitors;

[0134] (v) maturing myomatrix spheroids containing skeletal myoblasts and migratory muscle progenitors into myomatrix spheroids containing skeletal myotubes; thereby producing contractile engineered skeletal muscle tissue, the method is characterized in that the steps (i)-(iii) are carried out in a bioreactor under suitable conditions; and wherein the method does not include the use of a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma. The method of the embodiment 1, wherein the two or more myomatrix spheroids obtained by step (iii) are further embedded in an extracellular matrix hydrogel after completion step (iii), preferably wherein the embedded myomatrix spheroids embedded in the extracellular matrix hydrogel are casted into a mold or are 3D printed; or wherein steps (iv) or (v) are carried out in a bioreactor under suitable conditions. The method of embodiment 2, wherein the extracellular matrix hydrogel is selected from a collagen hydrogel, a laminin hydrogel, a hyaluronic acid hydrogel, a gelatin hydrogel, a fibrin / fibri nogen hydrogel, a poly lactic acid hydrogel, an alginate hydrogel, a methylcellulose hydrogel, a polyethylene glycol hydrogel, a hydrogel derived from decel- I ularized tissues, a matrix from spider silk, decellularized extracellular matrix from human or animal organ systems, aliginates, xanthan gum, vegetable binders or combinations thereof; preferably wherein the extracellular matrix hydrogel is a collagen hydrogel. The method of any of the embodiments 2-3, wherein step (iii) is completed when the myomatrix spheroids express the mRNA of PRRX1 and / or FAP, preferably wherein the myomatrix spheroids comprise a mesenchymal myomatrix-producing non-myocyte cell population, more preferably wherein said mesenchymal myomatrix-producing non-myocyte cell population comprises stromal cells, FAP-cells, satellite cells and neurons. The method of any of the embodiments 2-4, wherein step (iii) is completed after at least 17 days counted from the start of the method, preferably after at least 18 days, more preferably after at least 19 days, more preferably after at least 20 days more preferably after at least 21 days; and / or wherein step (iii) is completed after at most 28 days, preferably at most 25 days, more preferably at most 22 days, and even more preferably at most 21 days. The method of any of the embodiments 2-5, wherein the collagen is type I collagen, more preferably of bovine origin, porcine origin, human origin or marine origin, in particular collagen of bovine origin. The method of any of the embodiments 2-6, wherein the extracellular matrix hydrogel has a stiffness from 15 Pa to 100 kPa, more preferably from 200 Pa to 12 kPa, even more preferably from 500 Pa to 8 kPa, even more preferably from 1 kPa to 6 kPa, even more preferably from 2 kPa to 4 kPa; preferably wherein the stiffness is determined by rheological measurement. The method of any preceding embodiments, wherein the myomatrix spheroids generated by completion of steps (i)-(iii) are embedded into an engineered form in the presence of extracellular matrix hydrogel as defined in embodiments 2-7. The method of any of the embodiment 8, wherein the engineered form has the form of a ring, ribbon, strand, patch, pouch, or cylinder, wherein optionally individual engineered skeletal muscle tissues are fused. The method of any of the preceding embodiments, wherein step (ii) is carried out by culturing the pluripotent stem cell aggregates in a basal medium comprising an effective amount of (a) FGF2, (b) a GSK3 inhibitor, (c) one or more, preferably one or two, SMAD inhibitor(s), and (d) a serum-free additive comprising transferrin, insulin, progesterone, putrescine and selenium or a bioavailable salt thereof. The method of any of the preceding embodiments, wherein step (iii) is carried out by culturing the cell aggregates obtained in step (ii) in a basal medium comprising an effective amount of (a) a gamma-secretase / NOTCH inhibitor, (b) FGF2, and (c) a serum- free additive as defined in embodiment 10, followed by continuing the cultivation in the medium with the addition of an effective amount of (d) HGF, followed by culturing the cells in a basal medium comprising an effective amount of (a) a gamma secre- tase / NOTCH inhibitor, (b) HGF, (c) a serum-free additive as defined in embodiment 10, and (d) knockout serum replacement (KSR) thereby obtaining two or more myomatrix spheroids. The method of any of the preceding embodiments, wherein step (iv) is carried out by culturing said myomatrix spheroids in a basal medium comprising an effective amount of (a) HGF, (b) a serum-free additive as defined in embodiment 10, and (c) knockout serum replacement (KSR). The method of any of the preceding embodiments, wherein step (v) is carried out by culturing the myomatrix spheroids containing skeletal myoblasts and migratory muscle progenitors, preferably further containing stromal cells, FAP-cells, satellite cells and neuronal cells, obtained in step (iv) under mechanical or electrical stimulation, preferably mechanical stimulation, in a basal medium, comprising an effective amount of (a) a serum-free additive as in embodiment 10, and (b) an additional serum-free additive comprising transferrin, ethanolamine, selenium or a bioavailable salt thereof, L-carnitine, fatty acid additive, and triiodo-L-thyronine (T3), preferably wherein the additional serum- free additive additionally comprises albumin. The method of any of the preceding embodiments, wherein the myomatrix spheroids obtained after step (iii) are aggregates of cells comprising somite progenitor cells and myomatrix proteins; preferably wherein the somite progenitor cells express the marker(s) selected from the group of PAX3, LBX1, and MET; preferably wherein the myomatrix proteins are COL6A1-3 and FN1. The method of any of the preceding embodiments, wherein myomatrix spheroids obtained after step (iii) have a size from 100 pm to 400 pm, preferably from 120 to 300 pm, more preferably from 150 to 250 pm, even more preferably from 160 to 220 pm and the most preferably about 200 pm. The method of any preceding embodiments, wherein the engineered skeletal muscle tissue generates a contraction force of at least 0.5 millinewtons (mN) upon a stimulus from 1 Hz to 150 Hz, preferably at least 0.6 mN, more preferably at least 0.7 mN, more preferably at least 0.8 mN, more preferably at least 0.9 mN, and most preferably at least 1 mN; preferably wherein the stimulus is from 5 Hz to 140 Hz, more preferably from 10 Hz to 130 Hz, more preferably from 20 Hz to 125 Hz, more preferably from 40 Hz to 120 Hz, more preferably from 60 Hz to 115 Hz, more preferably from 80 Hz to 110 Hz , more preferably from 90 Hz to 105 Hz, and most preferably about 100 Hz. 17. The method of any preceding embodiments, wherein the pluripotent stem cells are of animal or human origin, preferably of porcine origin, bovine origin, caprine origin, equine origin, ovine origin, ruminant origin, avian origin, fish origin or primate origin, in particular wherein the pluripotent stem cells are animal or human pluripotent stem cells;

[0135] 18. The method of any preceding embodiments, wherein the pluripotent stem cells are selected from induced pluripotent stem cells, parthenogenetic stem cells, and pluripotent cells produced via chemical reprogramming or non-human embryonic stem cells, in particular wherein the pluripotent stem cells are induced pluripotent stem cells or parthenogenetic stem cells.

[0136] 19. The method of any preceding embodiments, wherein the steps (i)-(iii) are carried out in a stirred-tank type of bioreactor, a rocker bioreactor, an air-lift bioreactor, a fixed-bed bioreactor, a continuous bioreactor, or a fed-batch bioreactor, most preferably in a stirred-tank type of bioreactor.

[0137] 20. The method of any preceding embodiments, wherein the basal medium in step (i), step (ii), step (iii), step (iv) and / or in step (v) is selected from DMEM, DMEM / F12, RPMI, IMDM, alphaMEM, Medium 199, Hams F-10, Hams F-12, wherein the basal medium is preferably DMEM, in particular wherein the basal medium is supplemented with pyruvate and / or non-essential amino acids, even more preferably wherein the basal medium comprises from 1 to 4.5 g / l glucose.

[0138] 21. The method of any preceding embodiments, wherein step (i) is carried out in the presence of ROCK inhibitor.

[0139] 22. The method of the embodiment 21, wherein the ROCK inhibitor is selected from the group consisting of Y27632, H-1152P, thiazovivin, fasudil, hydroxyfasudil, GSK429286A and RKI1447, preferably the ROCK inhibitor is selected from the group consisting of Y27632, H-1152P, thiazovivin, fasudil and hydroxyfasudil, more preferably the ROCK inhibitor is selected from the group consisting of Y27632 and H-1152P, particularly preferably wherein the ROCK inhibitor is Y27632.

[0140] 23. The method of the embodiment 21 or 22, wherein the ROCK inhibitor is Y27632 and is used at a concentration of 0.5-20 pM, preferably 1-16 pM, more preferably 2-12 pM, more preferably 4-11 pM, more preferably 7-10 pM, and most preferably at a concentration of about 10 pM; and / or wherein the stem cell medium is iPS-Brew XF. The method of any preceding embodiments, wherein at step (i) the cells are seeded into the bioreactor at concentration from 1.3 x 105cells / ml to 5.4 x 105cells / ml, more preferably from 2.5 x 105cells / ml to 4 x 105cells / ml, most preferably at 3.75xl05 / ml. The method of any of the preceding embodiments, wherein the step (i) is completed, when the aggregates reach size of at least from 100 pm to 400 pm, preferably from 120 to 300 pm, more preferably from 150 to 250 pm, even more preferably from 160 to 220 pm and the most preferably about 200 pm; and / or wherein the aggregates express one or more pluripotency marker(s). The method of embodiment 25, wherein the one or more pluripotency marker(s) is / are selected from the group consisting of OCT4, NANOG, TRA1-60, and SSEA5. The method of embodiment 26, wherein the expression of marker(s) is / are detectable by flow-cytometry; or by immunostaining methods such as Western blot; by immunostaining methods combined with fluorescence microscopy or light microscopy; or by PCR, RNA-amplification, RNA-fish or RNA-seq techniques; preferably by flow-cytom- etry. The method of the embodiments any of embodiments 25-27, wherein the pluripotency marker(s) is / are expressed by at least 75% of cells in a population, more preferably by at least 80% of cells in the population, even more preferably by at least 85% of cells in the population, and most preferably by at least 90% of cells in the population. The method of any of the preceding embodiments, wherein the step (i) is carried out for 72 to 288 hours, preferably for 80 to 270 hours, more preferably for 90 to 255 hours, even more preferably for 96 to 230 hours, more preferably for 120 to 220 hours, and most preferably for about 216 hours. The method of any preceding embodiments, wherein the step (i) is carried out in a bioreactor at a temperature from 34 to 37 °C, preferably at 37 °C; with aeration from 21 to 25%, preferably 23.8%; upon stirring from 90 to 130 rpm, more preferably from 100 to 120 rpm. The method of any one of the preceding embodiments, wherein in step (ii), the GSK3 inhibitor is selected from the group consisting of CHIR99021, CHIR98014, SB216763, TWS119, tideglusib, SB415286, 6-bromoindirubin-3-oxime and a valproate salt, preferably wherein the GSK3 inhibitor is CHIR99021; and / or wherein in step (ii) the SMAD inhibitor is selected from the group consisting of LDN193189, K02288, LDN214117, ML347, LDN212854, DMH1, SB431542, SB505124, A83-01 or a combination thereof, preferably wherein one or two SMAD inhibitor are selected from said group, more preferably wherein one of the two SMAD inhibitors is LDN193189, and even more preferably wherein the one SMAD inhibitor is LDN193189.

[0141] 32. The method of any one of the preceding embodiments, wherein in step (ii) the effective amount of FGF2 is 1-15 ng / ml, preferably 2.5-14 ng / ml, more preferably 5-13 ng / ml, even more preferably 7.5-12.5 ng / ml, even more preferably 8-12 ng / ml, even more preferably 9-11 ng / ml, and most preferably about 10 ng / ml; and / or the serum-free additive provides in the medium a final concentration of 50-500 pg / ml transferrin, 1-20 pg / ml insulin, 0.001-0.1 pg / ml progesterone, 5-50 pg / ml putrescine and 6-600 nM selenium or a bioavailable salt thereof, in particular sodium selenite, and / or the GSK3 inhibitor is CHIR99021, and the effective amount is 1-20 pM, preferably 2-19 pM, more preferably 3-18 pM, even more preferably 4-17 pM, even more preferably 5-16 pM, even more preferably 6-15 pM, even more preferably 7-14 pM, even more preferably 7.5-13 pM, even more preferably 8-12 pM, even more preferably 9-11 pM, and most preferably about 10 pM; and / or the SMAD inhibitor is LDN193189, and the effective amount is 0.05- 5 pM, preferably 0.1-2.5 pM, more preferably 0.2-1 pM, even more preferably 0.25-5 0.8 pM, even more preferably 0.3-0.75 pM, even more preferably 0.35-0.7 pM, even more preferably 0.4-0.6 pM, even more preferably 0.45-0.55 pM, and most preferably about 0.5 pM.

[0142] 33. The method of any preceding embodiments, wherein the step (ii) is completed when the cells express one or both markers TBX6 and CDX2.

[0143] 34. The method of embodiment 33, wherein the expression of the marker(s) is / are detectable by flow-cytometry; or by immunostaining methods such as Western blot; or by immunostaining methods combined with fluorescence microscopy or light microscopy; or by PCR, RNA-amplification, RNA-fish or RNA-seq techniques; preferably by flow-cytom- etry.

[0144] 35. The method of embodiments 33 or 34, wherein at least 75% of cells in the population express the marker(s) defined in embodiment 33, more preferably at least 80% of cells in the population, even more preferably at least 85% of cells in the population, and most preferably at least 90% of cells in the population.

[0145] 36. The method of any preceding embodiments, wherein the step (ii) is carried out for 24 to 132 hours, preferably for 48 to 120 hours, more preferably for 60 to 114 hours, even more preferably for 72 to 108 hours, more preferably for 84 to 102 hours, and most preferably for about 96 hours. The method of any of the preceding embodiments, wherein the serum-free additive in step (ii) is 0.1-10% (v / v) N2 additive, preferably 0.3-7.5% (v / v) N2 additive, more preferably 0.5-5% (v / v) N2 additive, more preferably 0.75%-2% (v / v) N2 additive, more preferably 0.9%-1.2% (v / v) N2 additive, and most preferably about 1% (v / v) N2 additive. The method of any of the preceding embodiments, wherein the basal medium in step (ii), step (iii), step (iv), and / or in step (v) is selected from DMEM, DMEM / F12, RPMI, IMDM, alphaMEM, Medium 199, Hams F-10, Hams F-12, wherein the basal medium is preferably DMEM, in particular wherein the basal medium is supplemented with pyruvate and / or non-essential amino acids, even more preferably wherein the basal medium comprises from 1 to 4.5 g / l glucose. The method of any of the preceding embodiments, wherein in step (iii), the cultivation is carried out in the presence of (a) a gamma-secretase / NOTCH inhibitor, (b) FGF2, and (c) the serum-free additive for 36 to 60 hours, preferably for 42 to 54 hours, and most preferably for about 48 hours; followed by the cultivation carried out in the presence of (a) a gamma-secretase / NOTCH inhibitor, (b) HGF, (c) the serum-free additive, and (d) knockout serum replacement (KSR) for 72 to 120 hours, preferably for 76 to 114 hours, more preferably for 84 to 108 hours, even more preferably for 90 to 102 hours, and most preferably for about 96 hours. The method of any one of the preceding embodiments, wherein in step (iii), the gamma- secretase / NOTCH inhibitor is selected from the group DAPT, RO4929097, semagacestat (LY450139), avagacestat (BMS-708163), BMS-299897, dibenzazepine (YO-01027), LY411575, IMR-1, and L685458, preferably wherein the gamma-secretase / NOTCH inhibitor is DAPT. The method of embodiment 39 or 40, wherein in step (iii), the effective amount of FGF2 is 15-30 ng / ml, preferably 17.5-25 ng / ml, more preferably 18-22 ng / ml, even more preferably 19-21 ng / ml, and most preferably about 20 ng / ml; and / or the effective amount of HGF is 1-15 ng / ml, preferably 2.5-14 ng / ml, more preferably 5- 13 ng / ml, even more preferably 7.5-12.5 ng / ml, even more preferably 8-12 ng / ml, even more preferably 9-11 ng / ml, and most preferably about 10 ng / ml; and / or the gamma-secretase / NOTCH inhibitor is DAPT, and the effective amount is 1-20 |jM, preferably 2-19 pM, more preferably 3-18 pM, even more preferably 4-17 pM, even more preferably 5-16 pM, even more preferably 6-15 pM, even more preferably 7-14 pM, even more preferably 7.5-13 pM, even more preferably 8-12 pM, even more preferably 9-11 pM, and most preferably about 10 pM; and / or the KSR is used in an amount of 5-20% (v / v), preferably 6-17.5% (v / v), more preferably 7-15% (v / v), more preferably 8%-12% (v / v), more preferably 9%-l 1% (v / v), and most preferably about 10% (v / v) KSR; in particular wherein the KSR is used in the presence of a reducing agent such as beta-mercaptoethanol and / or alpha-thioglycerol. The method of embodiment 12, wherein in step (iv) the effective amount of HGF is 1-15 ng / ml, preferably 2.5-14 ng / ml, more preferably 5-13 ng / ml, even more preferably 7.5- 12.5 ng / ml, even more preferably 8-12 ng / ml, even more preferably 9-11 ng / ml, and most preferably about 10 ng / ml; and / or the KSR is used in an amount of 5-20% (v / v), preferably 6-17.5% (v / v), more preferably 7-15% (v / v), more preferably 8%-12% (v / v), more preferably 9%-l 1% (v / v), and most preferably about 10% (v / v) KSR; in particular wherein the KSR is used in the presence of a reducing agent such as beta-mercaptoethanol and / or alpha-thioglycerol. The method of any of the preceding embodiments, wherein the step (iv) is completed when the cells express at least one marker selected from a group of PAX3, LBX1, MET, corresponding to a first population of cells; and at least two markers selected from the group of PRRX1, FAP, EBF2, COL6A1-3 and FN1, corresponding to a second population of cells; preferably when all markers are expressed in the myomatrix spheroids after completion of step (iv). The method of the embodiment 43, wherein 40 % to 60% of the cells in the myomatrix spheroids after completion of step (iv) express PAX3, LBX1, and / or MET corresponding to the first population of cells; and / or wherein 40 % to 60% of the cells in the myomatrix spheroids after completion of step (iv) express PRRX1, FAP, EBF2, COL6A1-3 and / or FN1 corresponding to the second population of cells; preferably wherein 45 % to 55% of the cells in the myomatrix spheroids after completion of step (iv) express PAX3, LBX1, and / or MET; and / or wherein 45 % to 55% of the cells in the myomatrix spheroids after completion of step (iv) express PRRX1, FAP, EBF2, COL6A1-3 and / or FN1; more preferably wherein around 50% of the cells in the myomatrix spheroids after completion of step (iv) express PAX3, LBX1, and / or MET; and / or wherein around 50% of the cells in the myomatrix spheroids after completion of step (iv) express PRRX1, FAP, EBF2, COL6A1- 3 and / or FN1.

[0146] 45. The method of the embodiments 43 or 44, wherein at least 75% of cells in the first and / or the second population of cells express the marker(s) defined in embodiment 43, preferably at least 80% of cells in the first and / or the second population of cells, more preferably at least 85% of cells in the first and / or the second population of cells, and most preferably at least 90% of cells in the first and / or the second population of cells .

[0147] 46. The method of any of embodiments 43-45, wherein the expression of the marker(s) is / are detectable by flow-cytometry; or by immunostaining methods such as Western blot; or by immunostaining methods combined with fluorescence microscopy or polarized light microscopy; or by PCR, RNA-amplification, RNA-fish or RNA-seq techniques; preferably by flow-cytometry.

[0148] 47. The method of any of the preceding embodiments, wherein in step (v) the additional serum-free additive provides in the medium a final concentration of 1-100 pg / ml transferrin, 0.1-10 pg / ml ethanolamine, 17.4-1744 nM selenium or a bioavailable salt thereof, in particular sodium selenite, 0.4-40 pg / ml L-carnitine, 0.05-5 pl / ml fatty acid additive, 0.0001-0.1 pg / ml triiodo-L-thyronine (T3), preferably wherein the additional serum-free additive additionally provides in the medium a final concentration of 0.5-50 mg / ml albumin.

[0149] 48. The method of any of the preceding embodiments, wherein the additional serum-free additive in step (v) is 0.1-10% (v / v) B27, preferably 0.5-8% (v / v), more preferably 1- 6% (v / v), even more preferably 1.5-4% (v / v), and most preferably about 2% (v / v) B27.

[0150] 49. The method of any of the preceding embodiments, wherein in step (v), the myomatrix spheroids are cultured under a stimulation and wherein said stimulation is mechanical, preferably wherein the mechanical stimulation is a static tension, a dynamic stimulation, or an auxotonic stimulation, and more preferably wherein the mechanical stimulation is a static tension.

[0151] 50. The method of any of embodiments 1-48, wherein in step (v), the myomatrix spheroids are cultured under a stimulation and wherein said stimulation is electrical, preferably, wherein the electrical stimulation is applied as field or point stimulation, more preferably wherein the electrical stimulation is applied at above a contraction inducing voltage threshold, even more preferably wherein the contraction inducing voltage threshold is determined by visual or video-optic inspection of muscle tissue contraction upon an electrical stimulus. The method of any of the preceding embodiments, wherein the method does not comprise a skeletal myoblast enrichment step, preferably not an enrichment step by cell selection, more preferably not an enrichment step by anti body- based cell selection. An in vitro method for testing the efficacy of a drug candidate on an engineered skeletal muscle tissue, comprising the steps of

[0152] (a) generating an engineered skeletal muscle tissue using any of the methods of embodiments 1-51,

[0153] (b) optionally inflicting damage on the engineered skeletal muscle tissue, and

[0154] (c) contacting the engineered skeletal muscle tissue from step (a) or (b) with a drug candidate; preferably wherein the method further comprises determining the contraction force and / or structure of the engineered skeletal muscle tissue and / or metabolic function and / or molecular parameters and / or protein biochemical parameters before and / or after step (c). An in vitro method for testing the toxicity of a substance on an engineered skeletal muscle tissue, comprising the steps of

[0155] (a) generating an engineered skeletal muscle tissue using any of the methods of embodiments 1-51,

[0156] (b) contacting the engineered skeletal muscle tissue from step (a) with a substance to be tested, preferably wherein the method further comprises determining contraction force and / or engineered skeletal muscle tissue structure and / or metabolic function and / or molecular parameters and / or protein biochemical parameters before and / or after step (b). An in vitro method for testing the effect of nutrients and dietary supplements on engineered skeletal muscle tissue performance, comprising the steps of

[0157] (a) generating an engineered skeletal muscle tissue using any of the methods of embodiments 1-51,

[0158] (b) contacting the engineered skeletal muscle tissue from step (a) with a nutrient or dietary supplement to be tested, preferably wherein the method further comprises determining contraction force and / or engineered skeletal muscle tissue structure and / or metabolic function and / or molecular parameters and / or protein biochemical parameters before and / or after step (b).

[0159] 55. An engineered skeletal muscle tissue obtainable by the method of any of embodiments 1 to 51.

[0160] 56. The engineered skeletal muscle tissue of embodiment 55, wherein said engineered skeletal muscle tissue does not comprise blood vessels.

[0161] 57. The engineered skeletal muscle tissue of embodiments 55 or 56, wherein said engineered skeletal muscle tissue does not comprise innervation by the central nervous system.

[0162] 58. The engineered skeletal muscle tissue of any of embodiments 55-57, wherein said engineered skeletal muscle tissue generates a contraction force of at least 0.5 millinewtons (mN) upon a stimulus from 1 Hz to 150 Hz, preferably at least 0.6 mN, more preferably at least 0.7 mN, more preferably at least 0.8 mN, more preferably at least 0.9 mN, and most preferably at least 1 mN; preferably wherein the stimulus is from 5 Hz to 140 Hz, more preferably from 10 Hz to 130 Hz, more preferably from 20 Hz to 125 Hz, more preferably from 40 Hz to 120 Hz, more preferably from 60 Hz to 115 Hz, more preferably from 80 Hz to 110 Hz , more preferably from 90 Hz to 105 Hz, and most preferably about 100 Hz.

[0163] 59. The engineered skeletal muscle tissue of any of embodiments 55-58, wherein said engineered skeletal muscle tissue is serum-free.

[0164] 60. The engineered skeletal muscle tissue of any of embodiments 55-59, wherein said engineered skeletal muscle tissue does not comprise a transgene.

[0165] 61. The engineered skeletal muscle tissue of any of embodiments 55-60, wherein said engineered skeletal muscle tissue has not undergone genetic editing, preferably by CRISPR- Cas editing, TALEN editing or LoxP editing, and more preferably CRISPR-Cas editing.

[0166] 62. The engineered skeletal muscle tissue obtainable by or obtained by a method according to any of embodiments 1-51 or the engineered skeletal muscle tissue according to any of embodiments 55-61 for use in treating skeletal muscle disease, in particular a hereditary or a non-hereditary muscle disease and / or tendon disease, preferably wherein the hereditary muscle disease is selected from Duchenne muscular dystrophy, Becker muscular dystrophy, Emery-Dreifuss muscular dystrophy, and congenital muscular dystrophies with collagen VI deficiency; preferably wherein the non-hereditary muscle disease is selected from a drug induced, a mechanical inflicted, a thermally inflicted, a radiation inflicted, a metabolically induced and a osmotically induced disease. The engineered skeletal muscle tissue obtainable by a method according to any of embodiments 1-51 or the engineered skeletal muscle tissue according to any of embodiments 55-61 for use in treating a matrix-driven congenital muscular dystrophy, preferably wherein the matrix-driven congenital muscular dystrophy is Collagen VI muscle dystrophy or Collagen IV muscle dystrophy. The engineered skeletal muscle tissue obtainable by a method according to any of embodiments 1-51 or the engineered skeletal muscle tissue according to any of embodiments 55-61 for use in treating a hereditary or a non-hereditary muscle disease, wherein the engineered skeletal muscle is a muscle replacement, a muscle augmentation, or a muscle supplementation and / or wherein the engineered skeletal muscle releases trophic, protective, and / or regenerative factors. Use of the engineered skeletal muscle tissue obtainable by a method according to any of embodiments 1-51 or the engineered skeletal muscle tissue according to any of embodiments 55-61 as cultured meat, preferably as edible cultured meat, more preferably wherein the cultured meat additionally comprises recombinant protein, extracted protein, plant based protein, fibers or combinations thereof. A method for producing myomatrix spheroids from pluripotent stem cells, comprising the steps of

[0167] (i) forming two or more pluripotent stem cell aggregates from pluripotent stem cells;

[0168] (ii) inducing mesoderm differentiation of the pluripotent stem cell aggregates by culturing pluripotent stem cell aggregates in a basal medium comprising an effective amount of (a) FGF2, (b) a GSK3 inhibitor, (c) one or more SMAD inhibitors, and (d) a serum-free additive comprising transferrin, insulin, progesterone, putrescine and selenium or a bioavailable salt thereof;

[0169] (iii) inducing the myogenic specification by culturing the cell aggregates obtained in step (ii) in a basal medium comprising an effective amount of (a) a gamma-secre- tase / NOTCH inhibitor, (b) FGF2, and (c) a serum-free additive as in (ii), followed by continuing the cultivation in the medium with the addition of an effective amount of (d) HGF, followed by culturing the cells in a basal medium comprising an effective amount of (a) a gamma secretase / NOTCH inhibitor, (b) HGF, (c) a serum-free additive as in (ii), and (d) knockout serum replacement (KSR); wherein steps (i)-(iii) are carried out in bioreactor; wherein the method is free of a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma. The method of embodiment 66, wherein myomatrix spheroids express the myomatrix spheroid markers, preferably wherein the myomatrix spheroid markers are COL6A1-3 and FN1. The method of embodiments 66 or 67, wherein the expression of myomatrix spheroid marker(s) is / are detectable by flow-cytometry; or by immunostaining methods such as Western blot; or by immunostaining methods combined with fluorescence microscopy or polarized light microscopy; or by PCT, RNA-amplification, RNA-fish or RNA-seq techniques; preferably by flow-cytometry. The method of any of the embodiments 66-68, the cells in the myomatrix spheroids express at least one marker selected from a group of PAX3, LBX1, MET, corresponding to a first population of cells; and at least two markers selected from the group of PRRX1, FAP, EBF2, COL6A1-3 and FN1, corresponding to a second population of cells; preferably when all markers are expressed in the myomatrix spheroids. The method of embodiment 69, wherein 40 % to 60% of the cells in the myomatrix spheroids express PAX3, LBX1, and / or MET corresponding to the first population of cells; and / or wherein 40 % to 60% of the cells in the myomatrix spheroids express PRRX1, FAP, EBF2, COL6A1-3 and / or FN1 corresponding to the second population of cells; preferably wherein 45 % to 55% of the cells in the myomatrix spheroids express PAX3, LBX1, and / or MET; and / or wherein 45 % to 55% of the cells in the myomatrix spheroids express PRRX1, FAP, EBF2, COL6A1-3 and / or FN1; more preferably wherein around 50% of the cells in the myomatrix spheroids express PAX3, LBX1, and / or MET; and / or wherein around 50% of the cells in the myomatrix spheroids express PRRX1, FAP, EBF2, COL6A1- 3 and / or FN1. The method of any of embodiments 66-70, wherein myomatrix spheroids are used in methods of production of engineered skeletal muscle tissue. A method for producing engineered skeletal muscle tissue from two or more myomatrix spheroids comprising the steps of

[0170] (I) expanding and maturing the two or more myomatrix spheroids into myomatrix spheroids containing skeletal myoblasts and migratory muscle progenitors by culturing said myomatrix spheroids in a basal medium comprising an effective amount of (a) HGF, (b) a serum-free additive comprising transferrin, insulin, progesterone, putrescine and selenium or a bioavailable salt thereof, and (c) knockout serum replacement (KSR);

[0171] (II) maturing myomatrix spheroids containing skeletal myoblasts and migratory muscle progenitors into myomatrix spheroids containing skeletal myotubes by culturing the cells obtained in step (I) under mechanical stimulation in a basal medium, comprising an effective amount of (a) a serum-free additive as in step (I), and (b) an additional serum-free additive comprising transferrin, ethanolamine, selenium or a bioavailable salt thereof, L-carnitine, fatty acid additive, and triiodo-L-thyro- nine (T3), preferably wherein the additional serum-free additive additionally comprises albumin; wherein the method is free of a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma; thereby producing engineered contractile skeletal muscle tissue.

[0172] 73. The method of embodiment 72, wherein the two or more myomatrix spheroids are obtained by a method according to embodiments 66-71.

[0173] EXAMPLES

[0174] The following examples are intended to further illustrate the invention, without confining its scope. The examples describe technical features, and the invention also relates to combinations of the technical features presented in this section. Methods and materials that were used in examples are described after the examples.

[0175] Example 1 - Directed differentiation of human pluripotent stem cells (hPSCs) into myomatrix spheroids in a bioreactor

[0176] The inventors developed a method for the bioreactor-based directed differentiation of induced pluripotent stem into 3D myomatrix spheroids. The method described here is transgene- and serum-free and is carried out in a bioreactor. In this method, hPSCs (human pluripotent stem cells) form aggregates that undergo treatment with a specific temporal sequence of agents (small molecules and inhibitors and stimulators) to induce the differentiation of human pluripotent stem cells into myomatrix spheroids (steps 1-3, as shown in Figure 1). The myomatrix spheroids then undergo myogenic expansion (step 4) and myogenic maturation (step 5). These steps could be carried out in bioreactor or myomatrix spheroids could be casted into hydrogels. Alternatively, myomatrix spheroids could be casted into molds without presence of hydrogel. In this scenario, sufficient stabilization of the cells is needs to be ensured, e.g. by the production of extracellular matrix of the cells of the myomatrix spheroids.

[0177] The schematic of the bioreactor-based differentiation protocol is outlined in Figure 1, and it shows the sequence of the different agents added to the medium. In addition, Figure 1 shows the stages of differentiation into skeletal myoblasts / myotubes and satellite cells i.e., the induction of mesoderm differentiation, the induction of myogenic specification, the (myogenic) expansion and maturation into skeletal myoblasts and satellite cells, and the maturation into skeletal myotubes and satellite cells. Myomatrix spheroids are obtained by completion of stages 1-3 and further expanded (step 4) and matured (step 5).

[0178] The human induced pluripotent stem cell (iPSC) line TCI 133 wild type (Baghbaderani et al., 2015; Master Cell Bank Lot#: 50-001-21) and a transgenic reporter line, in which the C-termi- nal part of the sarcomeric o-actinin gene (ACTN2) found in the sarcomeres of muscle cells was tagged with a citrine fluorochrome (TC1133-ACTN2-Citrine), were used for the experiments in the Example section. Alternatively, other pluripotent stem cells, not embryonic stem cells, can be used. The transgenic reporter cell line was used to visualize sarcomeres and therefore confirm successful differentiation into muscle tissue.

[0179] To perform the method, human pluripotent stem cells (TCI 133 and TC1133-ACTN2-Citrine) were seeded into a bioreactor at 3.75xlOE5 / ml density (500 ml, Sartorius BIOSTAT™ B-DCUII Triple) in StemMACS iPSC Brew medium (Miltenyi Biotec) under 60 % perfusion. The perfusion is a bioreactor cell culture process in which cells are retained in the bioreactor while ensuring a constant flow of fresh medium. All bioreactors steps in this protocol are carried out in a bioreactor under 60 % perfusion (0.6 working volumes in 24 hrs, or 180 ml in case of a working volume of 300 ml). This equates to a medium exchange rate of 7,5 ml / h. Constant medium exchange was enabled by a spin filter that retains cell aggregates while removing used medium. The exchange rate can be measured by either weighing fresh medium supply through the feed tube or weighing spent medium removed through the waste tube. Before seeding, the cell concentration was measured with Nucleocounter NC-200 Type 900-0201. The temperature of the culture medium was maintained at 37°C. Dissolved oxygen concentration (DO) was held at 23.8% and monitored using an oxygen sensor (Hamilton; Oxyferm FDA VP 120). pH was continuously monitored and maintained at around 7.4. The bioreactor was equipped with a stirrer set at a 45° angle. For the first two days of cultivation, stirring was set at 120 rpm in a downward direction, followed by 100 rpm downward stirring thereafter.

[0180] The iPSC aggregates were allowed to form for 5 days. The iPSC aggregates were then passaged using Versene (Thermo Scientific) and reseeded (0.5-1X10E6 cells / ml) in the bioreactor for 4 days in basal medium (iPSC Brew Medium) containing 10 |jM Y-27632 inhibitor. Step 1 was considered as completed when aggregates reached the size of 100-200 pm and expressed pluripotency markers (OCT4, NANOG, TRA1-60, SSEA5), which was typically achieved on around day 8. By culturing hPSC in bioreactor iPSC Brew Medium and in iPSC Brew Medium in the presence of 10 pM Y-27632 inhibitor, iPSC aggregates were obtained suitable for use in the next step of the bioreactor-based differentiation protocol.

[0181] On the day 9, the media was changed to Brew-N2-FCL, containing FGF-2 and CHIR99021 and LDN193189 (i.e. FCL) inhibitors, the next day the media were changed to N2-FCL and aggregates were cultivated for an additional day (see Table 2 for composition of N2)

[0182] BrewN2-FCL: 25% Brew complete medium, DMEM containing 1 g / l glucose and L-alanyl-L- glutamine (GlutaMAX™, see Table 4 for composition of DMEM) supplemented with pyruvate (Gibco), 1% serum-free additive N-2 (lOOx) (Thermo Scientific), 1% non-essential amino acids (lOOx) (MEM-NEAA, Invitrogen), 10 ng / ml recombinant bFGF (Peprotech), 10 pM CHIR-99021 (Stemgent), 0.5 pM LDN193189 (Stemgent).

[0183] N2-FCL: DMEM containing 1 g / l glucose and L-alanyl-L-glutamine (GlutaMAX™) supplemented with pyruvate (Gibco), 1% Pen / Strep (Invitrogen), 1% serum-free additive N-2 (lOOx) (Thermo Scientific), 1% non-essential amino acids (lOOx) (MEM-NEAA, Invitrogen), 10 ng / ml recombinant bFGF (Peprotech), 10 pM CHIR-99021 (Stemgent), 0.5 pM LDN193189 (Stemgent).

[0184] The above described step corresponds to step 2, i.e., mesodermal induction, as shown in Figure 1. The completion of this step is characterized by aggregates expressing paraxial mesoderm markers, such as TBX6 and CDX2.

[0185] On the day 12, the aggregates were washed with DMEM and the media was changed to N2- FD and left to cultivate until day 14, wherein the media was changed to N2-FHD, containing FGF-2, HGF and DAPT.

[0186] N2-FD medium: DMEM with 1 g / l glucose and L-alanyl-L-glutamine (GlutaMAX™) supplemented with pyruvate (Gibco), 1% Pen / Strep (Invitrogen), 1% serum-free additive N-2 (lOOx) (Thermo Scientific), 1% non-essential amino acids (lOOx) (MEM-NEAA, Invitrogen), 20 ng / ml recombinant bFGF (Peprotech), 10 uM DAPT (TOCRIS).

[0187] N2-FHD medium: DMEM with 1 g / l glucose and L-alanyl-L-glutamine (GlutaMAX™) supplemented with pyruvate (Gibco), 1% Pen / Strep (Invitrogen), 1% serum-free additive N-2 (lOOx) (Thermo Scientific), 1% non-essential amino acids (lOOx) (MEM-NEAA, Invitrogen), 20 ng / ml recombinant bFGF (Peprotech), 10 pM DAPT (TOCRIS),

[0188] 10 ng / ml recombinant HGF (Peprotech).

[0189] Starting from day 16, the media was changed to N2-HKD, which contains HGF, KSR and DAPT. N2-HKD medium: DMEM with 1 g / l glucose and L-alanyl-L-glutamine (GlutaMAX™) supplemented with pyruvate (Gibco), 1% Pen / Strep (Invitrogen), 1% serum-free additive N-2 (lOOx) (Thermo Scientific), 1% non-essential amino acids (lOOx) (MEM-NEAA, Invitrogen), 0.1 mM 2-mercaptoethanol (Invitrogen), 10 pM DAPT (TOCRIS), 10 ng / ml recombinant HGF (Pepro- tech), 10% knockout serum replacement (Life Technologies; see Table 6 for composition of knockout serum replacement, KSR).

[0190] The aggregates were cultivated for approximately 3 more days until the expression of somite progenitor cells (PAX3, LBX1, MET), somite non-myocytes (PRRX1, FAP, EBF2) and myomatrix (COL6A1-3, FN1) was detected. Aggregates expressing above-mentioned markers were obtained by sequential culturing of aggregated in N2-FD, N2-FHD and N2-HKD media as outlined above and termed myomatrix spheroids.

[0191] Step 4 involved myogene specification of myomatrix spheroids in expansion medium (N2-HK medium) containing HGF and KSR, which continued until expression of migratory muscle progenitors was detected (PAX7, LBX1, MYODI), approximately for 7 days.

[0192] N2-30 HK medium: DMEM with 1 g / l glucose and L-alanyl-L-glutamine (GlutaMAX™) supplemented with pyruvate (Gibco), 1% Pen / Strep (Invitrogen), 1% serum-free additive N-2 (lOOx) (Thermo Scientific), 1% non-essential amino acids (lOOx) (MEM-NEAA, Invitrogen), 0.1 mM 2-mercaptoethanol (Invitrogen), 10 ng / ml recombinant HGF (Peprotech), 10% knockout serum replacement (Life Technologies).

[0193] Samples were collected throughout the differentiation protocol imaged using confocal microscopy. Figure 2 shows microscopic images of aggregates formed in Step 1 (day 9), Step 2 (day 16), step 3 (day 21, myomatrix spheroids), after step 4 (day 30) and matured myomatrix spheroids (day 37). Starting with the round, homogeneous iPSC aggregates before applying the differentiation protocol (day 9, Figure 2), the aggregates became more elongated around day 16 and started to develop two distinct halves, which became very apparent on day 21: One half was noticeably darker, while the other was of a bright color. From day 21, aggregates usually increased in size the most. On day 30 and 37 of differentiation, the TC1133-ACTN2- Citrine aggregates showed fluorescence signal, confirming the presence of actinin found in the sarcomeres in the muscle (Figure 2, day 37), which indicates that the differentiation protocol can be applied to iPSC aggregates derived from a bioreactor to obtain myomatrix spheroids.

[0194] The markers that indicate completion of each step of differentiation protocol are listed in Table

[0195] 1. The aggregate size was measured by microscopic imaging. The expression of the maker proteins could be detected by antibody staining on by detection of corresponding mRNA by RNA-seq of RT-PCR.

[0196] Table 1

[0197] Example 2: Production of Engineered Skeletal Muscle tissue from myomatrix spheroids

[0198] The ability of myomatrix spheroids obtained in Example 1 (from steps 1-3) to fuse and form engineered skeletal muscle tissue was tested.

[0199] The myomatrix spheroids differentiated for 22 days (steps 1-3) were casted into a collagen 1 hydrogel and cultured in expansion medium. 24 hrs after seeding cells into the hydrogel, the cell migration out of the myomatrix spheroid was detected (see Figure 3, migrating cells are shown with arrows at day 22) and by day 7 of maturation (day 27 in total), the aggregate structure was lost and myomatrix spheroids immerse into a confluent tissue.

[0200] Therefore, the myomatrix spheroids obtained showed migration of cells out of the aggregates leading to immersion of myomatrix spheroids into a compact tissue (Figure 2, day 27). Most importantly, this process did not require the addition of extracellular matrix (Matrigel) due to myomatrix spheroids containing both myogenic progenitor and mesenchymal cells, the source of myogenic extracellular matrix. The migrating population largely consisted of LBX1+, MET+, and PAX7+ myogenic progenitor cells which were specifically generated by the differentiation protocol of Example 1.

[0201] Specifically, immunostaining of spheroids in collagen 1 hydrogel after 7 days of culture in hydrogel (day 27 from the start of the protocol) showed that PAX7-positively stained migrating muscle progenitors and well as differentiating muscle cells (ACTN2-positive) were emanating from the core of myomatrix spheroids (nuclei-rich region labelled by asterisks on Figure 4).

[0202] The myomatrix spheroids began expressing PRRX1 and FAP around day 18 of the protocol, with significant levels of expression attained by day 21 (Figure 5). Consequently, day 21 serves as an optimal timepoint for utilizing myomatrix spheroids in myogenic expansion and fusion into muscle tissues, whether casted in molds or in 2D layers, with or without presence of hydrogels.

[0203] Upon embedding myomatrix spheroids into collagen I hydrogel, the spheroids fused into a compact muscle tissue that can be cultured under mechanical load. Myomatrix spheroids from day 21 of differentiation protocol (Example 1, Steps 1-3) were cased in a ring-shaped mold in a collagen 1-hydrogel without addition of Matrigel. After 8 days of culture (on day 30), myomatrix spheroids fused into the mold (Figure 6). On the day 42, the resulting ring mold containing fused myomatrix spheroids was then started to be cultured under mechanical load, i.e. stretching (Figure 6). After 30 more days of culture (day 72), the muscle-containing tissue was formed which was confirmed by expression of ACTN2-Citrine (Figure 6, day 72 shows fluorescent signal of Citrine-labelled ACTN2).

[0204] Example 3 - Functional and morphological characterization of myomatrix-derived engineered skeletal muscle tissue

[0205] The skeletal muscle tissue in the form of a ring was tested in organ baths (Fdhr Medical Instruments) containing Tyrode's solution (e.g., in mmol / L: 120 NaCI, 1 MgCI2, 1.8 CaCI2, 5.4 KCI, 22.6 NaHCO3, 4.2 NaH2PO4, 5.6 glucose, and 0.56 ascorbate) at 37°C and continuous gassing with 5% CO2and 95% O2. The engineered skeletal muscle tissue is mechanically stretched, and the maximum force amplitude (force of contraction =FOC) is typically measured at electric field stimulation frequencies in the range of 1-100 Hz (4 ms rectangular pulses; 200 mA).

[0206] The results of the contraction experiments are shown in Figure 7 show representative contraction force curves of the engineered skeletal muscle tissue at different stimulus frequencies. At a stimulation of 0.5 Hz (top panel), four single contractions were recorded with a single duration of approximately 0.6 seconds; at a stimulation of 100 Hz (bottom graph), a fully developed tetanus was detected.

[0207] The contraction experiments illustrate that the engineered skeletal muscle tissue derived from myomatrix spheroids generates force upon electrical stimulation. The tested skeletal muscle tissues showed reproducible contraction frequency and contraction force in response to stimulation frequencies between 1 Hz and 100 Hz, and the contraction and relaxation times after a single stimulus were approximately 0.18 and 0.34 seconds respectively (time to 90% con- traction / relaxation).

[0208] Myomatrix spheroid-derived EMS was stained with antibodies against actinin, Collagen VI and nuclei. Presence of essential components of muscle extracellular matrix (collagen VI) indicated that the extracellular matrix is endogenous and cell-derived and can support generation larger muscle constructs without supplementation with exogenous matrix proteins (Figure 8).

[0209] Example 4 - Comparison of contractile function of engineered skeletal muscle tissue obtained in a bioreactor compared to engineered skeletal muscle obtained previously

[0210] The twitch tension of the engineered skeletal muscle tissue obtained in a bioreactor was compared to engineered skeletal muscle generated according the previously published differentiation protocol (PCT / EP2020 / 078738 published as WO2021 / 074126 Al) carried out with and without Matrigel (for 56 days) (Figure 9).

[0211] The engineered skeletal muscle tissue according to PCT / EP2020 / 078738 differentiated without Matrigel showed low twitch tension (below 0.025 mM) while engineered skeletal muscle prepared from myomatrix spheroids differentiated without Matrigel showed sufficient twitch tension of (around 1.2 mM at day 47 and around 0.9 mM at day 56).

[0212] This experiment demonstrates that engineered skeletal muscle derived from myomatrix spheroids without addition of Matrigel, exhibits superior contractile functionality compared to engineered skeletal muscle generated according to PCT / EP2020 / 078738 without Matrigel. It also demonstrates that between day 17 and day 21 of myomatrix spheroid differentiation the essential properties to enable contractile muscle tissue formation develop. This coincides with the development of migratory muscle progenitor cells (LBX1+) and muscle non-myocytes (FAP+, PRRX1+; Figure 5). These findings underline the advantages of preparing engineered skeletal muscle using myomatrix spheroids using a bioreactor, as it eliminates the need for using an undefined substance like Matrigel. Without using a bioreactor for at least steps 1-3, the omission of a substance like Matrigel would not have been possible. Hence, the present methods is an improvement for the generation of a defined, robust and reproducible production of engineered skeletal muscle, which is contractile. Example 5 - Generation of contractile muscle from myomatrix spheroids without collagen hydrogel

[0213] This experiment tested if bioreactor-derived myomatrix spheroids generate contractile muscle tissue without the addition of exogenous hydrogel matrices like collagen. This requires a suitable culture vessel to assemble myomatrix spheroids in close proximity to fuse with each other. In this experiment a custom-made muscle tissue culture mold made from PMMA was used to assemble myomatrix aggregates inside a collagen hydrogel or without a collagen hydrogel (Hofemeier et al. Elife 2021). Identical volumes of day 21 aggregates (15 pl) were cast into the wells in a total volume of 25 pl supplemented with a collagen hydrogel (+collagen condition) or additional medium (-collagen condition). Aggregates were left to settle in the wells for 90 min. For 7 days the forming muscle tissue received expansion medium (Figure 1, step 4) followed by 3 weeks of myogenic maturation (Figure 1, step 5). Contractile function was measured by optical analysis of flexible poles that were sit inside the custom-made culture mold and around which the muscle tissue forms. Individual contraction traces of muscle tissues are displayed in Figure 10. Compared to muscle tissue made with a collagen hydrogel (-(-collagen), tissue that was formed purely from aggregates (-collagen) showed comparable contractions with a similar twitch tension (~1.8 mN).

[0214] This experiment underscores the finding that cell-autonomous matrix (myomatrix) from muscle spheroids is sufficient to grow a functional muscle. Muscle generation requires migration and fusion of myoblasts which will only take place if supportive extracellular matrix is provided. Cell-autonomous matrix may be exploited via the myomatrix spheroid approach to enable muscle generation without adding any exogenous matrix.

[0215] Materials and methods

[0216] Bioreactor cultivation

[0217] Two human induced pluripotent stem cell (iPSC) line TC1133 wild-type (Baghbaderani et al., 2015; Master Cell Bank Lot#: 50-001-21) and transgenic reporter line with sarcomeric o-actinin gene (ACTN20) tagged with Citrine fluorochrome (TC1133-ACTN2-Citrine).

[0218] The hPSC were seeded into 500 ml Sartorius BIOSTAT™ B-DCUII Triple bioreactor with 320 ml of media at 3.75xlOE5 / ml density and cultivated with 60% of perfusion of media per day (meaning that 60% of the media volume was exchanged with fresh media during a given day). Other parameters used for cultivation: 37°C, pH 7.4, dOz 23.8%, 100-120 rpm downstirr, with pH and oxygen levels being continuously monitored using pH sensor (Hamilton; Easyferm Plus VP 120) and oxygen sensor (Hamilton; Oxyferm FDA VP 120). The conditions were further developed based on WO 2021 / 116362 Al.

[0219] Immunostaining and confocal imaging

[0220] Aggregates for imaging were collected and fixed using 4% RotiTM-Histofix (Cat.-No. P087.4, Roth) for at least one day at 4°C and washed with DPBS (Cat.-No. 14190-094, Thermo Scientific) before collecting them with a 1000 pl tip in 2.0 ml tubes containing 1 ml blocking solution (500 ml 1 x DPBS (-CaCI2-MgCI2), 27 ml FBS, 5 g BSA, 2.6 ml TritonX-100) and then placed for at least 1 h at 4°C on a shaker (~ 450 rpm). Blocking solution was aspirated and exchanged for 500 pl of antibodies diluted in the blocking solution. Tubes were placed in a rack on a shaker overnight at 4 °C and then washed three times with 1 ml each: first two times with 4 °C (cold) DPBS, the last time with cold blocking solution. Secondary antibodies (including Hoechst-33342) were added in a volume of 500 pl blocking buffer and were incubated overnight at 4 °C, covered in aluminum foil. Aggregates were washed three times again, the first time in cold blocking solution, then two times in cold DPBS. Approximately 100 pl of the aggregates in DPBS were collected with a 1000 pl tip and placed into a slide with preformed dips (Cat.-No. 1LN7.1, Roth). DPBS was aspirated using a 100 pl tip and 100 pl of Fluoromount- GTM(Cat.-No. 0100-01, SouthernBiotech) was added and sealed with a cover slip. Aggregates were left at least overnight at RT before imaging with confocal microscopy (LSM710, Zeiss). Fluorescent imaging of TC1133-ACTN2-Citrine aggregates was done using the CQ1 confocal imaging cytometer (Tokogawa) with excitation and emission wavelengths AEx= 516 nm and AEm=529 nm.

[0221] RNA Extraction

[0222] For the RNA extraction, cell lysates embedded in Trizol reagent (Thermo Fisher) were homogenized by vortexing. For every 1 ml of Trizol reagent, 200 pl of chloroform was added (Appli- Chem). Reagent tubes were tightly closed and inverted five times followed by 5 min incubation at room temperature. Samples were then centrifuged at 10,000-12,000 x g for 15 minutes. The aqueous phase containing RNA was transferred to fresh reagent tubes, followed by the addition of 500 pl of isopropanol (Roth) to precipitate the RNA. The reagent tubes were vor- texed, allowed to stand at room temperature for 10 min, and then centrifuged at 12,000 x g for an additional 10 min. The supernatant was removed and 1 ml of 70% EtOH / diethyl pyrocarbonate (DEPC) H2O was added to wash the pellet. After gently tapping the reagent tube to dissolve and wash the pellet, the samples were centrifuged one more time at 12,000 x g for 5 minutes, and the supernatant was removed. The pellets were left open for 5-10 minutes until the remaining liquid had evaporated, and the RNA was resuspended in DEPC H2O. The RNA concentration was quantified using the Nanodrop spectrophotometer (Thermo Fisher Scientific).

[0223] Quantitative RNA transcript analyses

[0224] Total RNA was isolated from myomatrix spheroids and engineered skeletal muscle using Trizol reagent (Thermo Fisher Scientific) as described in previous section.

[0225] According to the manufacturer's instructions, 1 pg of the RNA sample was treated with DNase I (Roche), and then the sample was reverse transcribed into complementary DNA (cDNA) using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative PCR was performed using the Fast SYBR Green Master Mix (Thermo Fisher Scientific) and the AB7900 HT Fast Real-Time PCR System (Applied Biosystems). Alternatively, transcriptome analysis was performed by RNA counting using the nCounter platform (Nanostring).

[0226] Isometric force measurements

[0227] The contractile function of engineered skeletal muscle tissue was measured under isometric conditions in an organ bath filled with gassed (5% CO2 / 95% 02) Tyrode solution (containing in mmol / L): 120 NaCI, 1 MgCI2, 0.2 CaCI2, 5.4 KCI, 22.6 NaHCO3, 4.2 NaH2PO4, 5.6 glucose, and 0.56 ascorbate) at 37°C. To verify the force-length relationship - while ENGINEERED SKELETAL MUSCLEs were electrically stimulated at 0.5 or 1 Hz with 5 ms rectangular pulses of 200 mA - muscle length was increased by mechanical stretching in intervals of 125 pm, until the maximum contraction force was observed.

[0228] At the length of maximum force generation, tetanic contraction force was assessed under defined stimulation frequencies (4-second stimulation at 10, 20, 40, 60, 80, and 100 Hz).

[0229] Cast molding of myomatrix aggregates without hydrogel

[0230] To generate muscle from myomatrix aggregates with or without an exogenous hydrogel, muscle tissue culture molds were applied that were milled from polymethyl methacrylate (PMMA) as described in Hofemeier et al. Elife 2021 and disclosed in PCT / EP2021 / 062976 published as WO / 2021 / 229097. The PMMA molds consist of 2 parts: the bottom part of the PMMA chamber contains the ellipsoid culture wells (diameters: 3.5 mm, 6.5 mm) and is glued onto a microscopy cover glass (VWR, Radnor, USA) using PDMS (Sylgard 170 silicone, Sigma, St. Louis, USA) which is cured for 24 hr at room temperature. The upper part represents the PMMA lid, which extends two 16 mm long ellipsoid posts per well (diameters: 0.68 mm, 1.3 mm) with a distance of 3.3 mm between each opposing posts. Prior to use, the molds were sterilized using 70% ethanol and the wells were coated with a Poloxamere solution in ddH2O over night at 4°C (5% Pluronic F-127, Sigma, St. Louis, USA) to render the surface non-adhesive. Identical vol- umes of day 21 aggregates (15 |_il) were cast into the wells in a total volume of 25 pl supplemented with a collagen hydrogel (+collagen condition) or additional medium (-collagen condition). Aggregates were left to settle in the wells for at least 90 min. For 7 days the forming muscle tissue received expansion medium according to (PCT / EP2020 / 078738 published as WO2021 / 074126 Al) followed by 3 weeks of myogenic maturation.

[0231] Optical measurements of muscle tissue function

[0232] Muscle function was measured by deflection of ellipsoid posts integrated into the lid of a custom-made PMMA chamber (Hofemeier et al. Elife 2021). For post-deflection analysis, the top part of the post was imaged with high-resolution microscopy (Olympus, CKX53SF) and a time series of individual pictures was obtained. Utilizing a custom written program in Matlab (Mathworks, Natick, USA), the strong contrast of the post compared to the surrounding tissue was chosen for movement tracking. A line of pixels located at the center of the post of each image was defined to identify the outline of the post by calculating the gradient of pixel intensities along this line. Subpixel resolution was achieved by the fit of a Gaussian function to these peaks and determining the central position. Subsequently, the pixel values were converted into units of length using the pre-calibrated pixel sizes. Analysis of the entire time stack in that manner gave rise to a time-dependent displacement signal for the post edges. Finally, the twitch tension was determined by multiplication of the post displacement by the spring constant of the post (39.2 ± 0.8 pN / pm; Hofemeier et al. Elife 2021).

[0233] Table 2: Composition of the serum-free additive N-2 in lOOx effective concentration (liquid form), i.e. 1% (v / v) corresponds to a single (lx) effective concentration

[0234] Table 3: Composition of the non-essential amino acids in lOOx effective concentration (lOOx)

[0235] Table 4: DMEM, low glucose at 1 g / l, GlutaMAX™ supplemented with pyruvate (Gibco, catalogue number: 10567014) Table 5: Composition of the additional serum-free B27 additive at 50X effective concentration (liquid form)

[0236] :,o ml of the 50X B27 additive per 500 ml of medium corresponds to 2% (v / v) Table 6: Composition of Knockout Serum Replacement (KSR)

[0237]

[0238] Table 7

[0239] List of References

[0240] WO2021 / 074126 Al

[0241] WO 2021 / 116362 Al

[0242] WO2022 / 084429

[0243] Akhmanova M, Osidak E, Domogatsky S, Rodin S, Domogatskaya A. Physical, Spatial, and Molecular Aspects of Extracellular Matrix of In Vivo Niches and Artificial Scaffolds Relevant to Stem Cells Research. Stem Cells Int. 2015;2015: 167025.

[0244] Baghbaderani BA, Tian X, Neo BH, Burkall A, Dimezzo T, Sierra G, Zeng X, Warren K, Kovarcik DP, Fellner T, Rao MS. cGMP-Manufactured Human Induced Pluripotent Stem Cells Are Available for Pre-clinical and Clinical Applications. Stem Cell Reports. 5(4): 647-59.

[0245] Beldjilali-Labro M, Garcia Garcia A, Farhat F, Bedoui F, Grosset J, Dufresne M, Legallais C. Biomaterials in Tendon and Skeletal Muscle Tissue Engineering: Current Trends and Challenges. Materials 2018 (11) 1116

[0246] Brewer, G. J., Torricelli, J. R., Evege, E. K. and Price, P. J. (1993), Optimized survival of hippocampal neurons in B27-supplemented neurobasal™, a new serum-free medium combination. J. Neurosci. Res., 35: 567-576. doi: 10.1002 / jnr.490350513

[0247] Hofemeier AD, Limon T, Muenker TM, Wallmeyer B, Jurado A, Afshar ME, Ebrahimi M, Tsukanov R, Oleksiievets N, Enderlein J, Gilbert PM, Betz T. Global and local tension measurements in biomimetic skeletal muscle tissues reveals early mechanical homeostasis. Elife. 2021 Jan 18;10:e60145. doi: 10.7554 / eLife.60145.

[0248] Kozlowski MT, Crook CJ, Ku HT. Towards organoid culture without Matrigel. Commun Biol. 2021 Dec 10;4(l): 1387. doi: 10.1038 / s42003-021-02910-8.

[0249] Khodabukus A, Prabhu N, Wang J, Bursae N. In Vitro Tissue-Engineered Skeletal Muscle Models for Studying Muscle Physiology and Disease. Adv. Healthcare Mater. 2018 (7) 1701498

[0250] Liao IC, Liu JB, Bursae N, Leong KW. Effect of Electromechanical Stimulation on the Maturation of Myotubes on Aligned Electrospun Fibers. Cell Mol Bioeng. 2008 Sep 1; 1(2-3): 133-145.

[0251] Pavesi A, Adriani G, Rasponi M, Zervantonakis IK, Fiore GB, Kamm RD. Controlled electromechanical cell stimulation on-a-chip. Sci Rep. 2015 Jul 2;5: 11800. Shahriyari M, Islam MR, Sakib MS, Rinn M, Rika A, Kruger D, Kaurani L, Gisa V, Winterhoff M, Anandakumar H, Shomroni O, Schmidt M, Salinas G, Unger A, Linke WA, Zschuntzsch J, Schmidt J, Bassel-Duby R, Olson EN, Fischer A, Zimmermann WH, Tiburcy M (2022) Engineered skeletal muscle recapitulates human muscle development, regeneration, and dystrophy. J Cachexia Sarcopenia Muscle. 13:3106-3121.

[0252] Shahriyari M, Rinn M, Hofemeier AD, Babych A, Zimmermann WH, Tiburcy M (2023) Protocol to develop force-generating human skeletal muscle organoids. STAR Protoc. 5(1): 102794.

[0253] Tiburcy M, Hudson JE, Balfanz P, Schlick S, Meyer T, Chang Liao ML, Levent E, Raad F, Zeidler S, Wingender E, Riegler J, Wang M, Gold JD, Kehat I, Wettwer E, Ravens U, Dierickx P, van Laake LW, Goumans MJ, Khadjeh S, Toischer K, Hasenfuss G, Couture LA, Unger A, Linke WA, Araki T, Neel B, Keller G, Gepstein L, Wu JC, Zimmermann WH. Defined Engineered Human Myocardium With Advanced Maturation for Applications in Heart Failure Modeling and Repair. Circulation. 2017 May 9; 135(19): 1832-1847.

[0254] Zimmermann WH, Melnychenko I, Eschenhagen T. Engineered Heart Tissue for Regeneration of Diseased Hearts. Biomaterials (2004) 25: 1639-1647

Claims

Claims1. A method for producing an engineered skeletal muscle tissue from pluripotent stem cells, comprising the steps of(i) forming two or more pluripotent stem cell aggregates from pluripotent stem cells;(ii) inducing mesoderm differentiation of the pluripotent stem cell aggregates;(iii) inducing the myogenic specification thereby obtaining two or more myomatrix spheroids;(iv) expanding and maturing the two or more myomatrix spheroids into myomatrix spheroids containing skeletal myoblasts and migratory muscle progenitors;(v) maturing myomatrix spheroids containing skeletal myoblasts and migratory muscle progenitors into myomatrix spheroids containing skeletal myotubes; thereby producing contractile engineered skeletal muscle tissue, the method is characterized in that the steps (i)-(iii) are carried out in a bioreactor under suitable conditions; and wherein the method does not include the use of a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma.

2. The method of the claim 1, wherein the two or more myomatrix spheroids obtained by step (iii) are further embedded in an extracellular matrix hydrogel after completion of step (iii), preferably wherein the extracellular matrix hydrogel is a collagen hydrogel.

3. The method of claim 2, wherein the extracellular matrix hydrogel has a stiffness from 15 Pa to 100 kPa, more preferably from 200 Pa to 12 kPa, even more preferably from 500 Pa to 8 kPa, even more preferably from 1 kPa to 6 kPa, even more preferably from 2 kPa to 4 kPa; preferably wherein the stiffness is determined by rheological measurement.

4. The method of any of the preceding claims, wherein step (ii) is carried out by culturing the pluripotent stem cell aggregates in a basal medium comprising an effective amount of (a) FGF2, (b) a GSK3 inhibitor, (c) one or more, preferably one or two, SMAD inhibitors), and (d) a serum-free additive comprising transferrin, insulin, progesterone, putrescine and selenium or a bioavailable salt thereof.

5. The method of any of the preceding claims, wherein step (iii) is carried out by culturing the cell aggregates obtained in step (ii) in a basal medium comprising an effective amount of (a) a gamma-secretase / NOTCH inhibitor, (b) FGF2, and (c) a serum-free additive as defined in claim 4, followed by continuing the cultivation in the medium with the addition of an effective amount of (d) HGF, followed by culturing the cells in a basal medium comprising an effective amount of (a) a gamma secretase / NOTCH inhibitor, (b) HGF, (c) a serum-free additive as defined in claim 4, and (d) knockout serum replacement (KSR) thereby obtaining two or more myomatrix spheroids.

6. The method of any of the preceding claims, wherein step (iv) is carried out by culturing said myomatrix spheroids in a basal medium comprising an effective amount of (a) HGF, (b) a serum-free additive as defined in claim 4, and (c) knockout serum replacement (KSR).

7. The method of any of the preceding claims, wherein step (v) is carried out by culturing the myomatrix spheroids containing skeletal myoblasts and migratory muscle progenitors obtained in step (iv) under mechanical stimulation in a basal medium, comprising an effective amount of (a) a serum-free additive as in claim 4, and (b) an additional serum- free additive comprising transferrin, ethanolamine, selenium or a bioavailable salt thereof, L-carnitine, fatty acid additive, and triiodo-L-thyronine (T3), preferably wherein the additional serum-free additive additionally comprises albumin.

8. The method of any preceding claims, wherein the engineered skeletal muscle tissue generates a contraction force of at least 0.5 millinewtons (mN) upon a stimulus from 1 Hz to 150 Hz, preferably at least 0.6 mN, more preferably at least 0.7 mN, more preferably at least 0.8 mN, more preferably at least 0.9 mN, and most preferably at least 1 mN; preferably wherein the stimulus is from 5 Hz to 140 Hz, more preferably from 10 Hz to 130 Hz, more preferably from 20 Hz to 125 Hz, more preferably from 40 Hz to 120 Hz, more preferably from 60 Hz to 115 Hz, more preferably from 80 Hz to 110 Hz , more preferably from 90 Hz to 105 Hz, and most preferably about 100 Hz.

9. The method of any preceding claims, wherein the steps (i)-(iii) are carried out in a stirred-tank type of bioreactor or in a rocker bioreactor, or in an air-lift bioreactor, or in a fixed-bed bioreactor, or in a continuous bioreactor, or in a fed-batch bioreactor, most preferably in a stirred-tank type of bioreactor.

10. The method of any of the preceding claims, wherein the method does not comprise a skeletal myoblast enrichment step, preferably not an enrichment step by cell selection, more preferably not an enrichment step by anti body- based cell selection.

11. An engineered skeletal muscle tissue obtainable by the method of any of claims 1-10, wherein said engineered skeletal muscle tissue generates a contraction force of at least 0.5 millinewtons (mN) upon a stimulus from 1 Hz to 150 Hz, wherein said engineered skeletal muscle tissue is serum-free, and wherein said engineered skeletal muscle tissue does not comprise a transgene.

12. The engineered skeletal muscle tissue obtainable by a method according to any of claims 1-10 or the engineered skeletal muscle tissue according to claim 11 for use in treating skeletal muscle injuries, hereditary and non-hereditary myopathies, tendon injuries, and / or a matrix-driven congenital muscular dystrophy, preferably wherein the matrix- driven congenital muscular dystrophy a Collagen VI muscle dystrophy or Duchenne Muscular Dystrophy.

13. Use of the engineered skeletal muscle tissue obtainable by a method according to any of claims 1-10 or the engineered skeletal muscle tissue according to claim 11 as cultured meat, preferably as edible cultured meat, more preferably wherein the cultured meat additionally comprises recombinant protein, extracted protein, plant-based protein, fibers, or combinations thereof.

14. A method for producing myomatrix spheroids from pluripotent stem cells, comprising the steps of(i) forming two or more pluripotent stem cell aggregates from pluripotent stem cells;(ii) inducing mesoderm differentiation of the pluripotent stem cell aggregates by culturing pluripotent stem cell aggregates in a basal medium comprising an effective amount of (a) FGF2, (b) a GSK3 inhibitor, (c) one or more SMAD inhibitors, and (d) a serum-free additive comprising transferrin, insulin, progesterone, putrescine and selenium or a bioavailable salt thereof;(iii) inducing the myogenic specification by culturing the cell aggregates obtained in step (ii) in a basal medium comprising an effective amount of (a) a gamma-secre- tase / NOTCH inhibitor, (b) FGF2, and (c) a serum-free additive as in (ii), followed by continuing the cultivation in the medium with the addition of an effective amount of (d) HGF, followed by culturing the cells in a basal medium comprising an effectiveamount of (a) a gamma secretase / NOTCH inhibitor, (b) HGF, (c) a serum-free additive as in (ii), and (d) knockout serum replacement (KSR); wherein steps (i)-(iii) are carried out in a bioreactor; wherein the method is free of a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma.

15. A method for producing engineered skeletal muscle tissue from two or more myomatrix spheroids comprising the steps of(I) expanding and maturing the two or more myomatrix spheroids into myomatrix spheroids containing skeletal myoblasts and migratory muscle progenitors by culturing said myomatrix spheroids in a basal medium comprising an effective amount of (a) HGF, (b) a serum-free additive comprising transferrin, insulin, progesterone, putrescine and selenium or a bioavailable salt thereof, and (c) knockout serum replacement (KSR);(II) maturing myomatrix spheroids containing skeletal myoblasts and migratory muscle progenitors into myomatrix spheroids containing skeletal myotubes by culturing the cells obtained in step (I) under mechanical stimulation in a basal medium, comprising an effective amount of (a) a serum-free additive as in step (I), and (b) an additional serum-free additive comprising transferrin, ethanolamine, selenium or a bioavailable salt thereof, L-carnitine, fatty acid additive, and triiodo-L-thyro- nine (T3), preferably wherein the additional serum-free additive additionally comprises albumin; wherein the method is free of a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma; thereby producing engineered contractile skeletal muscle tissue.

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