Multilayer artificial myocardium

Abstract

This application describes a method for producing a multi-layered artificial myocardium. Specifically, the method involves (i) providing a liquid reconstitution mixture in a mold and (ii) culturing the mixture. The method is characterized by sequentially adding one or more additional liquid reconstitution mixtures to obtain a multi-layered artificial myocardium. The muscle ideally has the shape of a patch, pouch, or cylinder. Furthermore, the application relates to a multi-layered artificial myocardium made up of at least two layers, including collagen, cardiomyocytes, and non-cardiomyocytes. The multi-layered artificial myocardium is the basis for several in vitro and in vivo applications, such as the production of a multi-layered artificial myocardium for use in patients, e.g., for cardiac repair.

Description

[Background technology]

[0001] Background of the Invention Regenerating a failing heart remains a formidable challenge. A common cause of heart failure is heart attack. A heart attack, or myocardial infarction, occurs when arterial blood flow is blocked, resulting in hypoperfused tissue that is deprived of oxygen and nutrients. During a heart attack, millions, even up to a billion, cells rapidly die. This is the primary cause of cardiac performance decline and, clinically, heart failure. Because the heart is a largely postmitotic tissue, cardiac tissue cannot regenerate itself. This goal calls for the transplantation of artificial myocardium. Various artificial myocardial formats have been developed for applications in disease modeling, drug screening, and cardiac repair. We have published several applications of artificial human myocardial models for in vitro studies of cardiomyocyte maturation and hypertrophy (Tiburcy et al. 2017), disease modeling (Hanses et al., 2020), and drug screening (Mills et al. 2019), as well as for in vivo cardiac repair (Riegler et al. 2015).

[0002] One approach to support cardiomyocyte self-assembly into 3D tissues is the scaffold-containing approach, where the scaffold is primarily supported by either collagen or fibrin (Zimmermann et al. 2006, Tulloch et al. 2011, Soong et al. 2012, Zhang et al. 2013, Riegler et al. 2015, Weinberger et al. 2016, Tiburcy et al. 2017). Furthermore, non-myocytes are essential for creating 3D tissues. Similar to rodent models (Naito et al. 2006), these non-myocytes are primarily fibroblasts or stromal cells with fibroblastic activity (Kensah et al. 2013; Ronaldson-Bouchard et al. 2018; Tiburcy et al. 2017, Zhang et al. 2013).

[0003] In addition to cellular components and scaffolds, the highest degree of maturation is achieved with the support of biophysical stimuli, i.e., mechanical, electrical, or a combination of both (Ronaldson-Bouchard et al. 2018; Tiburcy et al. 2017). Mechanical loading is an absolute requirement for engineering highly mature myocardium.

[0004] To produce artificial myocardial tissue for treating patients with weakened cardiac muscle, the artificial myocardium must support the force of a beating human heart. The cardiac wall to be treated is typically 5–10 mm thick in patients (Kawel et al. 2012). Therefore, the graft ideally must reach this thickness and also be able to generate mechanical force to support cardiac wall function.

[0005] The main challenge in achieving such a thick artificial myocardium is to ensure a constant supply of oxygen and nutrients to the cells embedded in the artificial myocardium. Therefore, there is a need in the art for an artificial myocardium with a thickness suitable for supporting the human heart. The inventors have overcome this challenge through a repetitive and sequential casting process of the artificial myocardium. Summary of the Invention

[0006] To create a multilayered artificial myocardium (MEHM), a layer of condensed cell-containing hydrogel (reconstitution mixture) is coated with at least one other layer. This repetitive and sequential process ensures the formation of a thicker, stronger artificial myocardium, with at least one new layer integrating with the previous layer. In other words, the artificial myocardium grows in thickness through repeated and sequential layering. Furthermore, it is important that the artificial myocardium be perforated by mechanical supports and that poles pass through the artificial tissue to support tonic contractions and introduce channels for the supply of oxygen and nutrients throughout the artificial tissue.

[0007] One particular advantage of the methods disclosed herein is that cells within the reconstitution mixture / MEHM are always adequately supplied with nutrients and oxygen. Minimizing the total diffusion distance from the MEHM surface to the core of the MEHM during the manufacturing process produces a thick MEHM that can be used, for example, as an implant, while still being adequately supplied with oxygen and nutrients. This is achieved by a perforated MEHM design. The perforations are introduced, for example, by perforated poles, particularly those used for mechanical support (e.g., as shown in Figures 7 and 8). The poles can supply oxygen and / or nutrients to cells within the MEHM. The poles create "channels" through the reconstitution mixture / MEHM for supplying oxygen and / or nutrients and independently introduce mechanical load onto the MEHM.

[0008] To achieve such a thick MEHM, a reconstitution mixture containing cardiomyocytes, non-cardiomyocytes, and collagen is repeatedly and sequentially cast. The layers are integrated by repeatedly casting each layer on top of and / or underneath the previous layer. This is a key advantage of the disclosed method. By sequential casting, the layers condense by at least 20% of their initial volume before additional layers are added. For example, to avoid hypoxia and / or necrosis, the embedded cells are supplied with oxygen and nutrients by diffusion from the tissue surface. The reconstitution mixture is perforated by poles that span the tissue surface area. Therefore, the overall muscle thickness can be scaled as desired, taking into account diffusion and cell-specific oxygen and nutrient requirements.

[0009] In a first aspect, a method for producing a multi-layer artificial myocardium (MEHM) is provided, comprising the steps of: (i) providing a liquid reconstituted mixture in a mold, the reconstituted mixture being perforated by at least two poles; the reconstitution mixture comprising (a) collagen, (b) a cell mixture of cardiomyocytes and non-myocytes, and (c) an appropriate reconstitution medium, whereby the reconstitution mixture gels in the mold; (ii) incubating the mixture in the mold obtained by step (i) in an appropriate culture medium, whereby the reconstituted mixture condenses in the mold; (iii) a) adding a further liquid reconstitution mixture as defined in step (i) from above and / or below the condensed reconstitution mixture obtained by step (ii), wherein the further liquid reconstitution mixture gels, and then incubation is carried out under the same conditions as in step (ii), so that the further reconstitution mixture condenses in the mold; or b) transferring the concentrated reconstituted mixture obtained from step (ii) into a different mold, wherein said reconstituted mixture is perforated by at least two poles, and thereafter step (iii)a) is carried out in said different mold; This results in a multi-layer artificial myocardium (MEHM), Preferably, the MEHM is thickened by repeating step (iii)a) and / or step (iii)b) at least once, and (iv) optionally culturing the MEHM of step (iii) in said mold in a suitable maturation medium. wherein the MEHM is capable of contracting.

[0010] In another aspect, a multi-layered artificial myocardium (MEHM) obtained by the methods disclosed herein is contemplated.

[0011] Further described is a multi-layered artificial myocardium (MEHM) comprising (a) collagen and (b) a cellular mixture of cardiomyocytes and non-myocytes, wherein the MEHM comprises at least two layers.

[0012] Further disclosed is an artificial heart muscle (EHM), preferably an artificial heart patch, an artificial heart pouch, or an artificial heart cylinder, wherein the EHM comprises (a) collagen, and (b) a cellular mixture of cardiomyocytes and non-myocytes, and wherein the EHM has a thickness of at least about 0.6 mm.

[0013] Additionally, the present disclosure discloses the use of MEHM obtained by the methods disclosed herein or MEHM disclosed herein in the in vitro production of artificial human myocardium.

[0014] Finally, a multilayered EHM obtained by the methods disclosed herein or a MEHM as disclosed herein is described for use in medicine, particularly in heart failure. DETAILED DESCRIPTION OF THE INVENTION

[0015] Detailed Description of the Invention In a first aspect, a method for producing a multi-layer artificial myocardium (MEHM) is provided, comprising the steps of: (i) providing a liquid reconstituted mixture in a mold, the reconstituted mixture being perforated by at least two poles; the reconstitution mixture comprising (a) collagen, (b) a cell mixture of cardiomyocytes and non-myocytes, and (c) an appropriate reconstitution medium, and the reconstitution mixture is allowed to gel in a mold; (ii) incubating the mixture in the mold obtained by step (i) in an appropriate culture medium, whereby the reconstituted mixture condenses in the mold; (iii) a) adding a further liquid reconstitution mixture as defined in step (i) from above and / or below the condensed reconstitution mixture obtained by step (ii), wherein the further liquid reconstitution mixture gels, and then incubation is carried out under the same conditions as in step (ii), so that the further reconstitution mixture condenses in the mold; or b) transferring the concentrated reconstituted mixture obtained from step (ii) into a different mold, wherein said reconstituted mixture is perforated by at least two poles, and thereafter step (iii)a) is carried out in said different mold; This results in a multi-layer artificial myocardium (MEHM), Preferably, the MEHM is thickened by repeating step (iii)a) and / or step (iii)b) at least once, and (iv) optionally culturing the MEHM of step (iii) in the mold in a suitable maturation medium. wherein the MEHM is capable of contracting.

[0016] Generally, as used herein, an artificial myocardium (EHM) refers to an artificial myocardium containing cells and collagen that is capable of contracting. Furthermore, a multilayer artificial myocardium (MEHM) is created from at least two layers of artificial myocardium (EHM). To fabricate a multilayer artificial myocardium (MEHM), the layers are added sequentially. Sequential layering occurs, with each subsequent layer being added to the original (first) layer. The subsequent layer then integrates with the original (first) layer. This layering process may be repeated several times. In other words, the subsequent layer integrates with the previously added layer. As layering is performed, the MEHM thickens with each layer. An important advantage of this method is that a thicker MEHM is more stable and capable of sustaining greater forces than a single-layer EHM. Furthermore, another important feature of an MEHM is that it is capable of contracting, preferably as further defined below, similarly to natural cardiac myocardium. Ideally, such a multilayer artificial myocardium is suitable for use as a graft, such as a cardiac graft.

[0017] In one embodiment, the MEHM obtained by step (iii) and optionally step (iv) has contractile capacity, which can be assessed by visual inspection. Alternatively, MEHM contraction can be confirmed by the ability of the reconstituted mixture of step (i) to form a force-generating artificial myocardium (EHM) by performing steps (i), (ii), and optionally step (iv), wherein the artificial myocardium (EHM) is capable of generating a contractile force of at least 0.05 mN force of contraction (FOC) as measured under standard isometric conditions in Supplementary Figure 6C of Tiburcy et al. Circulation 135(19)1832-1847 (2017). More specifically, MEHM contraction can be further confirmed by performing steps (i), (ii), and (iv) to evaluate in parallel the ability of the reconstitution mixture of step (i) to form a loop-format, force-generating artificial human muscle, as described in Tiburcy et al. Circulation 135(19)1832-1847 (2017), where the loop-format artificial cardiac muscle generates a force of contraction (FOC) of at least 0.05 mN as measured in Supplementary Figure 6C of Tiburcy et al. Circulation 135(19)1832-1847 (2017). In other words, MEHM contraction can be confirmed by fabricating loop-format EHMs in parallel with the MEHMs. The advantage of such a parallel approach is that the formation of loop-format EHMs is well established (see, e.g., Tiburcy et al. 2017 and 2020) and can be performed in a 48-well format. Furthermore, when the force between two poles is measured, the force of contraction can be more conveniently ascertained in the loop format. Therefore, the quality of the reconstitution mixture and medium can be more easily assessed in the well-established loop format. Furthermore, a well-developed EHM in the loop format is expected to ensure that the MEHM is provided with comparable quality. Alternatively, the force of contraction (FOC) in the MEHM can also be measured directly.For example, Zimmermann et al. (2000) describe measuring FOC in tissue. Therefore, one skilled in the art can adapt the setup described above for measuring FOC in MEHM. In preferred embodiments, the MEHM produces a force of contraction (FOC) of at least 0.05 mN, more preferably at least 0.1 mN, more preferably at least 0.3 mN, more preferably at least 0.5 mN, more preferably at least 1 mN, more preferably at least 3 mN, even more preferably at least 5 mN, and most preferably at least 10 mN.

[0018] In another preferred embodiment, the thickness of each layer of the MEHM is at least about 0.1 mm, preferably at least about 0.15 mm, more preferably at least about 0.2 mm, more preferably at least about 0.25 mm, more preferably at least about 0.3 mm, more preferably at least about 0.4 mm, and most preferably at least about 0.5 mm. In a particularly preferred embodiment, the MEHM is about 0.2 mm to about 30 mm thick, preferably about 0.3 mm to about 30 mm, more preferably about 0.5 mm to about 30 mm, more preferably about 0.7 mm to about 25 mm, more preferably about 0.9 mm to about 23 mm, more preferably about 1.5 mm to about 20 mm, more preferably about 2 mm to about 18 mm, more preferably about 2.8 mm to about 15 mm, more preferably about 3.3 mm to about 13 mm, more preferably about 3.8 mm to about 12 mm, more preferably about 4.2 mm to about 11 mm, more preferably about 4.6 mm to about 10.5 mm, and most preferably about 5 mm to about 10 mm. As used herein, the term "approximately" when used with respect to the thickness of each layer of the multi-layer artificial myocardium of the present invention and the thickness of the artificial myocardium described herein means that the thickness may deviate from the respective numerical value by ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, or ±10%. This means, for example, that if the thickness of each layer is "approximately 0.3 mm," the thickness of this layer may be 0.27 mm (a deviation of -10%) to 0.33 mm (a deviation of +10%). Similarly, if the thickness of the artificial myocardium itself is approximately 0.8 mm, the thickness of the artificial myocardium may range from 0.72 mm to 0.88 mm.

[0019] Ideally, the MEHM will match the thickness of human myocardium, which is 5-12 mm thick in the non-contracted state. For example, Kawel et al. (2012) reported that the human heart wall is at most 12 mm thick at maximum relaxation. Nowosielski et al. (2009) reported that wall thickness typically increases by 60% during contraction. Thus, the human heart is estimated to have a maximum thickness of 20 mm. As a result, MEHM for use as a cardiac graft preferably has a thickness of 5-20 mm.

[0020] To the inventors' knowledge, this is the first thick EHM design in the art. A key advantage of the disclosed method is that the MEHM is created from at least two layers, and the at least two layers are integrated together. This repetitive, sequential layering ensures that the MEHM thickens with each layer while simultaneously minimizing the overall surface tissue diffusion distance throughout the MEHM. The desired surface tissue diffusion distance is ensured by the design and density of the perforation poles. Minimal or metabolically adequate surface tissue diffusion ensures that cells are constantly supplied with oxygen and nutrients. Without perforations, e.g., several centimeters deep, the reconstituted mixture or its multilayered tissue would exhibit rapid (tissue core) cell necrosis in the center of the reconstituted mixture due to insufficient oxygen and / or nutrients being supplied to cells in the tissue core.

[0021] In another embodiment, MEHM contraction can also be measured by video optics. For example, the fractional area change (FAC) of the MEHM can be measured. A method for assessing FAC has been previously described in Tiburcy et al. (2017). Briefly, the area of ​​the MEHM in a relaxed state is compared to the area of ​​the MEHM in a contracted state, and the change is reported as a percentage. In a particularly preferred embodiment, contraction is confirmed by measuring the fractional area change (FAC) of the MEHM using the method described in Tiburcy et al., Circulation 135(19)1832-1847 (2017), where the fractional area change (FAC) is at least 0.5% upon electrical stimulation, preferably at least 0.7%, more preferably at least 1%, more preferably at least 2%, more preferably at least 3%, and even more preferably at least 5%. In particularly preferred embodiments, the FAC is at most 20%, preferably at most 19%, more preferably at most 18%, even more preferably at most 17%, even more preferably at most 16%, and even more preferably at most 15%. Electrical stimulation can be applied by electric field or by point stimulation. An example of an electric field is shown in Example 4. Point stimulation using electrodes is also known to those skilled in the art. Generally, application of electric stimulation causes MEHM contraction. From Tiburcy et al. 2017, it can be deduced that an FAC of approximately 7% corresponds to an FOC of approximately 1 mN (Supplementary Figure 11). Based on this figure, those skilled in the art can correlate FOC and FAC as desired.

[0022] To produce such MEHMs, a liquid reconstitution mixture is dispensed into a mold. Generally, a "liquid" mixture is capable of flowing and filling the mold. Additionally, a medium can be dissolved into the liquid reconstitution mixture. As used herein, a "liquid" mixture is meant to contrast with a "gel-like" mixture, which is defined below, since a gel-like mixture cannot flow and cannot freely dissolve a medium into the gel-like mixture.

[0023] As used herein, a "reconstitution mixture" includes collagen, a cell mixture, and an appropriate reconstitution medium, where the cell mixture is composed of cardiomyocytes and non-myocytes. To tissue engineer an MEHM, one skilled in the art will readily understand that the reconstitution mixture forms the backbone of the MEHM, as the various components of the reconstitution mixture make up a significant portion of the artificial myocardium, i.e., reconstitute a significant portion of the myocardium.

[0024] Collagen is one of the components of the reconstitution mixture. Collagen is the major structural protein in the extracellular matrix found in various connective tissues throughout the body. Collagen is composed of amino acids that form elongated fibril triple helices, as is known to those skilled in the art. Furthermore, collagen supports artificial tissues, such as MEHM, providing structural support to cells from the outside. Those skilled in the art also know that collagen, such as "collagen solutions," can be purchased from various suppliers. For example, the reconstitution mixture may provide a final concentration of 0.5-3 mg / ml collagen, preferably 0.55-2.5 mg / ml collagen, more preferably 0.6-2.2 mg / ml collagen, 0.65-2 mg / ml collagen, more preferably 0.7-1.75 mg / ml collagen, more preferably 0.75-1.5 mg / ml collagen, more preferably 0.8-1.2 mg / ml collagen, even more preferably 0.85-1 mg / ml collagen, and most preferably about 0.9 mg / ml collagen. In a preferred embodiment, the collagen in step (i) is selected from the group consisting of type I collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type XII collagen, type XIII collagen, type XIV collagen, type XV collagen, and mixtures thereof. Those skilled in the art are aware that type I collagen is the most abundant collagen in the human body. In a particularly preferred embodiment, at least 90% of the collagen in the reconstituted mixture in step (i) is type I collagen. In particular, when the MEHM is used as a graft, the collagen in the reconstituted mixture in step (i) may be medical grade.

[0025] Of course, collagen can be obtained from various sources and suppliers. For example, collagen can be of bovine, equine, human, or marine origin. In a particularly preferred embodiment, collagen is of bovine origin. In a particularly preferred embodiment, the collagen used is free of porcine endogenous retroviruses and free of any transmissible spongiform encephalopathy. It is also contemplated that the collagen in the reconstituted mixture of step (i) further comprises one or more additional extracellular matrix components other than collagen. The term "extracellular matrix protein" refers to any extracellular matrix (ECM) protein known to those skilled in the art (Hynes and Naba (2012)). Furthermore, those skilled in the art are aware of the composition of extracellular matrices that can be used to prepare EHM, for example, from Mouw JK, et al. (2014). In particularly preferred embodiments, the one or more additional matrix components are selected from the group consisting of elastin, laminin, entactin, nidogen, proteoglycans (e.g., decorin), glycosaminoglycans (e.g., hyaluronic acid), and fibronectin. Furthermore, those skilled in the art are aware that commercially available synthetic mimetics of these extracellular proteins are also suitable for MEHM fabrication. For example, those skilled in the art are aware of publications such as O'Leary et al. (2011), which describe the self-assembly of collagen mimetic peptides from triple helices into nanofibers and hydrogels.

[0026] In a preferred embodiment, the cell mixture of step (i) is present in a reconstitution mixture of 1 to 26.5 x 10 6 cells / ml, preferably 2 to 13.2 x 10 6 cells / ml, more preferably 3 to 10 × 10 6 cells / ml, more preferably 3.5 to 9 × 10 6 cells / ml, more preferably 4-8 x 10 6 cells / ml, more preferably 4.3 to 7 × 10 6 cells / ml, more preferably 4.6 to 6 × 10 6 cells / ml, and even more preferably about 5×10 6This results in a final cell concentration of 1000 cells / ml. However, those skilled in the art can determine the appropriate concentration of cells in the reconstitution mixture. Those skilled in the art may be guided, for example, by Tiburcy et al. (2017) or Schlick et al. (2019) to determine the appropriate cell concentration and composition.

[0027] The cell mixture is composed of cardiomyocytes and non-muscle cells. Cardiomyocytes are muscle cells (myocytes) that make up the heart muscle (myocardium) in natural tissue. Each cardiomyocyte contains myofibrils, specialized organelles composed of long chains of sarcomeres—the basic contractile units of muscle cells. Those skilled in the art know that cardiomyocytes can be obtained commercially or by differentiation from pluripotent stem cells, e.g., induced pluripotent stem cells. In a particularly preferred embodiment, the cardiomyocytes are human cardiomyocytes. In another particularly preferred embodiment, the cardiomyocytes are derived from embryonic stem cells, which have not been produced using processes involving alteration of the genetic identity of human germline or the use of human embryos for industrial or commercial purposes. For example, the cardiomyocytes can be obtained from induced pluripotent stem cells, parthenogenetic stem cells, programmed somatic cells, or adult stem cells; preferably, the cardiomyocytes are derived from induced pluripotent stem cells. Those skilled in the art are familiar with induced pluripotent stem cells, parthenogenetic stem cells, or adult stem cells, as described in the art. Programmed somatic cells are directly reprogrammed into the desired cell type. This method avoids the need for induction of pluripotency and subsequent differentiation procedures (see, for example, Ieda et al. 2010, Song et al. 2012, Nam et al. 2013). In particular, those skilled in the art are familiar with various protocols in the literature describing the generation of cardiomyocytes by serum-free differentiation, as described in WO2015 / 040142, or other protocols such as those outlined in Burridge et al. (2012), "Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming," Cell Stem Cell. 2012 Jan 6;10(1):16-28.In addition, the chapter "Engineered Heart Muscle Models in Phenotypic Drug Screens of Zimmermann" in the book "Organotypic Models in Drug Development, Handbook of Experimental Pharmacology" (Springer, 2020) by Schafer-Korting_Stucchi Maria-Engler,_Landsiedel (87017223) summarizes various protocols for obtaining cardiomyocytes that can be used in the methods disclosed herein. It is also contemplated that the cardiomyocytes in these protocols may be non-human primate stem cell-derived cardiomyocytes, fetal cardiomyocytes, or neonatal cardiomyocytes. Those skilled in the art also know how to assess the identity of cardiomyocytes, for example, by flow cytometry or RNA sequencing. Using these methods, it is possible to confirm the expression of cardiomyocyte-specific markers such as ACTN2, TTN, RYR2, and / or troponin.

[0028] In one embodiment, the cell mixture of step (i) contains at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, and most preferably at least 50% cardiomyocytes. Those skilled in the art can determine the optimal ratio of cardiomyocytes to non-myocytes. Optimization of the optimal ratio is guided by the compaction of the EHM and, in particular, the ability of the EHM to contract. For example, Tiburcy et al. (2017) and Schlick et al. (2019) demonstrated that cardiomyocytes and non-myocytes, such as stromal cells, are required for the contraction and compaction of artificial human myocardium. Specifically, cardiomyocytes mediate contraction, while non-myocytes, such as stromal cells, mediate compaction. Furthermore, non-myocytes secrete extracellular matrix proteins, such as collagen, and / or bind to the extracellular matrix via integrins to support the three-dimensional organization of the EHM. In a preferred embodiment, contraction of the MEHM is induced by cardiomyocytes, thereby generating the contractile force of the MEHM.

[0029] As used herein, non-muscle cells refer to other cellular components of a cell mixture other than cardiomyocytes. Non-muscle cells are essential for the development of MEHM. For example, as demonstrated in Schlick et al. (2019), artificial human myocardium does not condense when non-muscle cells are not provided in the reconstitution mixture. In a preferred embodiment, the non-muscle cells are selected from one or more of the group consisting of stromal cells, endothelial cells, smooth muscle cells, and mesenchymal stem cells. Preferably, the non-muscle cells are stromal cells or endothelial cells. More preferably, the non-muscle cells are stromal cells. Even more preferably, the stromal cells are cardiac stromal cells. Even more preferably, the cardiac stromal cells have fibroblastic properties. Even more preferably, the cardiac stromal cells are fibroblasts. Typically, to support the formation of MEHM, non-muscle cells secrete extracellular matrix proteins and / or bind to the extracellular matrix via integrins. In other words, in a preferred embodiment, the non-muscle cells ideally condense the reconstituted material through cell-matrix interactions in step (ii). Therefore, any cell capable of biophysical cell-matrix interactions and secretion of extracellular matrix proteins is a suitable non-muscle cell. In particularly preferred embodiments, the non-muscle cells express CD90 as determined by flow cytometry, and preferably, the non-muscle cells express CD90, CD74, and CD44. As described above, ideally, the non-muscle cells secrete extracellular matrix proteins, particularly collagen, to facilitate cell-matrix interactions and / or express integrins, which condense the reconstitution mixture. Non-muscle cells can be obtained from a variety of sources. For example, non-muscle cells can be obtained from induced pluripotent stem cells, parthenogenetic stem cells, programmed somatic cells, adult stem cells, or mesenchymal stem cells; preferably, the non-muscle cells are derived from induced pluripotent stem cells. Furthermore, non-muscle cells can be obtained from embryonic stem cells, which have not been produced using processes involving alteration of the genetic identity of human germline cells or the use of human embryos for industrial or commercial purposes.In another preferred embodiment, the non-muscle cells are derived from a human subject, preferably from a patient, and even more preferably from a biopsy obtained from a patient for autologous or allogeneic administration of EHM. Autologous patient-derived non-muscle cells have the advantage that the cells are derived from the same patient and are therefore immunologically compatible with the patient. This is a particular advantage when MEHM is used as a transplant in a patient. Allogeneic cells also have the advantage that the cells are derived from the patient and therefore resemble the patient in need of an MEHM transplant. Numerous protocols are available to those skilled in the art for obtaining suitable non-muscle cells. For example, EP20188364.2 describes a serum-free method for obtaining cardiac stromal cells with fibroblastic characteristics. Furthermore, Witty et al. (2014), Iyer D, et al. (2015), Bao X, et al. (2016), or Bao X, et al. (2017) describe protocols for obtaining suitable non-muscle cells.

[0030] In a particularly preferred embodiment, the non-muscle cells of the cell mixture in step (i) are stromal cells, as described, for example, in EP20188364.2. Preferably, the stromal cells are human stromal cells. When the non-muscle cells are stromal cells, the cell mixture in step (i) may contain at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, and even more preferably at least 50% stromal cells. Those skilled in the art will understand that cardiomyocytes and non-muscle cells together constitute the cell mixture, i.e., 100%. Exemplary ratios of cardiomyocytes to non-muscle cells would be 50:50, 60:40, 70:30, 80:20, or 90:10, respectively. Of course, any intermediate ratio could also be selected. As mentioned above, several ratios have been tested and can be found in literature, such as Tiburcy et al. (2017) and Schlick et al. (2019).

[0031] Typically, the reconstitution medium comprises (a) a basal medium and (b) a serum-free supplement. The serum-free supplement may be included in the reconstitution medium, and thereby in the reconstitution mixture, culture medium, and / or maturation medium. In a preferred embodiment, the serum-free supplement can be formulated to provide a final concentration in (1) the reconstitution mixture, (2) the culture medium, and / or (3) the maturation medium. Of course, the concentrations and components can be selected independently for (1) the reconstitution mixture, (2) the culture medium, and / or (3) the maturation medium.

[0032] For example, a serum-free supplement can be formulated to provide a final concentration of any of the following components: 0.5 to 50 mg / ml albumin (preferably 1 to 40 mg / ml, more preferably 2 to 30 mg / ml, more preferably 3 to 20 mg / ml, more preferably 4 to 10 mg / ml, most preferably 4.5 to 7.5 mg / ml, for example, about 5 mg / ml); 1 to 100 μg / ml transferrin (preferably 2 to 90 μg / ml, more preferably 3 to 80 μg / ml, more preferably 4 to 70 μg / ml, more preferably 5 to 60 μg / ml, more preferably 6 to 50 μg / ml, more preferably 7 to 40 μg / ml, more preferably 8 to 30 μg / ml, more preferably 9 to 20 μg / ml, for example, about 10 μg / ml); 0.1-10 μg / ml ethanolamine (preferably 0.2-9 μg / ml, more preferably 0.3-8 μg / ml, even more preferably 0.4-7 μg / ml, even more preferably 0.5-6 μg / ml, more preferably 0.6-5 μg / ml, more preferably 0.7-4 μg / ml, more preferably 0.8-3 μg / ml, most preferably 1-2.5 μg / ml, e.g., about 2 μg / ml); 14.4 to 1446 nM selenium or a bioavailable salt thereof (preferably 40 to 700 nM, more preferably 70 to 300 nM, even more preferably 130 to 160 nM, and most preferably about 144.6 nM); 0.4-40 μg / ml L-carnitine HCl (preferably 0.5-30 μg / ml, more preferably 1-20 μg / ml, even more preferably 2-10 μg / ml, more preferably 3-5 μg / ml, most preferably about 4 μg / ml); 1-100 μg / ml fatty acid supplement (preferably 1.4-80 μg / ml, more preferably 1.8-40 μg / ml, even more preferably 2-24 μg / ml, more preferably 2.4-8 μg / ml, most preferably 3.2-6 μg / ml, e.g., about 4 μg / ml); and 0.0004 to 0.04 μg / ml triiodo-L-thyronine (T3) (preferably 0.0010 to 0.02 μg / ml, more preferably 0.0016 to 0.010 μg / ml, even more preferably 0.002 to 0.006 μg / ml, most preferably about 0.004 μg / ml).

[0033] The fatty acid supplement may include, for example, linoleic acid and / or linolenic acid.

[0034] For example, the bioavailable salt of selenium is sodium selenite, such that a final concentration of sodium selenite of 0.003-0.3 μg / ml (preferably 0.005-0.16 μg / ml, more preferably 0.010-0.1 μg / ml, even more preferably 0.02-0.05 μg / ml, and most preferably 0.03 μg / ml) is provided in the reconstitution mixture, culture medium, or reconstitution medium.

[0035] In another embodiment, the serum-free supplement may also contain one or more of vitamin A, D-galactose, progesterone, and putrescine. These components are beneficial to cell viability. The appropriate concentration of each component is known to those skilled in the art or can be easily determined by routine experimentation.

[0036] An example of a serum-free supplement may be prepared according to published protocols (see also Brewer et al. (1993)) or purchased commercially. For example, B27 minus insulin (Table 1) can be used. In a preferred embodiment, the serum-free supplement is provided by 0.2-20% (v / v) B27 minus insulin in the reconstitution mixture, culture medium, or reconstitution medium, either commercially available or preferably prepared according to Table 1. Preferably, the serum-free supplement is provided by 1-16% (v / v), more preferably 2-12% (v / v), more preferably 3-8% (v / v), even more preferably 3-8% (v / v), even more preferably 3.4-5% (v / v) B27 minus insulin, and most preferably about 4% (v / v) B27 minus insulin in the reconstitution mixture, culture medium, or reconstitution medium. Alternatively, B27 containing insulin may also be used. The optimal B27 composition can be determined empirically for each cell type used. Those skilled in the art know how to determine the optimal B27 composition and / or concentration. For example, Tiburcy et al. (2017) compared the use of B27 with the use of B27 minus insulin in Supplementary Figure IIIF, online-only data. Artificial muscles produced using B27 minus insulin exhibit greater force compared to muscles produced using B27. However, the figure demonstrates that B27 (including insulin) can be used to successfully create artificial human cardiac muscle to achieve good contractility.

[0037] Furthermore, the basal medium of the reconstitution medium, culture medium, and / or maturation medium may further contain ascorbic acid or a derivative thereof. For example, when ascorbic acid is contained in the basal medium of the reconstitution medium, culture medium, and / or maturation medium, a concentration of 10 to 1000 μM, preferably 50 to 400 μM, more preferably 100 to 300 μM, even more preferably 150 to 250 μM, and most preferably about 200 μM is used in the reconstitution medium, culture medium, and / or maturation medium. Even more preferred is ascorbic acid in the form of ascorbic acid-2-phosphate.

[0038] Additionally, the basal medium of the reconstitution medium, culture medium, and / or maturation medium may be RPMI, which further comprises pyruvate. Particularly preferred concentration ranges of pyruvate in RPMI are 0.1-10 mM pyruvate, more preferably 0.2-5 mM pyruvate, even more preferably 0.4-2.5 mM pyruvate, even more preferably 0.8-1.5 mM pyruvate, even more preferably 0.9-1.2 mM pyruvate, and most preferably about 1 mM pyruvate.

[0039] The basal medium of the reconstitution medium may be selected from, for example, Iscove's medium, RPMI, αMEM, and DMEM, or a mixture thereof. Preferably, the basal medium is selected from Iscove's medium, RPMI, αMEM, or a mixture thereof. More preferably, the basal medium is a mixture of Iscove's medium and RPMI. However, any suitable basal medium can be used in the method. Basal media are commercially available or can be prepared according to publicly available recipes, for example, according to publicly available recipes from the ATCC catalog. Generally, the media used herein combine appropriate amounts of glucose, lactate, and / or fatty acids as essential nutrients. Glucose, lactate, and / or fatty acids ensure adequate nutrient supply to the cells embedded in the reconstitution mixture.

[0040] If appropriate, amino acids may be added to the basal medium. If αMEM is used as the basal medium, the basal medium does not need to be supplemented with, for example, non-essential amino acids. Non-essential amino acids are commercially available as complex supplements. Such supplements include, for example, 750 mg / L glycine, 890 mg / L L-alanine, 1320 mg / L L-asparagine, 1330 mg / L L-aspartic acid, 1470 mg / L L-glutamic acid, 1150 mg / L L-proline, and 1050 mg / L L-serine.

[0041] In a particularly preferred embodiment, the reconstitution medium further comprises (c) 35 to 790 μM ascorbic acid, (d) 5 to 500 ng / ml IGF-1, (e) 0.3 to 26 ng / ml VEGF, (f) 0.5 to 53 ng / ml FGF-2, and (g) 0.5 to 10 ng / ml TGFβ 1. In an even more preferred embodiment, the reconstitution medium further comprises (c) 75 to 300 μM ascorbic acid, (d) 26 to 105 ng / ml IGF-1, (e) 1.3 to 5.2 ng / ml VEGF, (f) 2.6 to 10.5 ng / ml FGF-2, and (g) 1 to 6 ng / ml TGFβ 1. In an even more preferred embodiment, the medium further comprises (c) about 158 ​​uM ascorbic acid, (d) about 53 ng / ml IGF-1, (e) about 2.6 ng / ml VEGF, (f) about 5.3 ng / ml FGF-2, and about 3 ng / ml TGFβ1.

[0042] VEGF is VEGF 165 Those skilled in the art are aware of published protocols for suitable reconstitution media, such as Tiburcy et al. (2017) or Tiburcy et al. (2020), and those skilled in the art can identify similarly suitable protocols.

[0043] As used herein, the term "mold" can be freely selected and geometrically varied in shape. It is important that the mold retains the reconstitution mixture and that the reconstitution mixture gels within the mold. The mold may be custom-made for the intended purpose. For example, if the purpose is a human body implant, a custom-made mold may conform to the myocardial defect. If the purpose of the MEHM is testing, e.g., drug efficacy testing, a different shape may be used. Those skilled in the art can customize the mold shape according to their needs and purposes. For example, they may use a 3D printer to customize the mold shape. In a preferred embodiment, the mold is configured to facilitate the formation of a flat MEHM, a pouch-shaped MEHM, or a cylindrical MEHM. In a preferred embodiment, the mold is in the shape of a patch, pouch, or cylinder. It is even more preferred that the MEHM is in the shape of a patch, pouch, or cylinder. In an even more preferred embodiment, the patch is in the shape of a disk.

[0044] If the mold is configured to facilitate the formation of a planar MEHM, it may have a disk shape, preferably a circular or polygonal disk shape, and more preferably a hexagonal disk shape. Exemplary hexagonal disks are also shown in Figures 2 and 7 disclosed herein and used to form MEHMs as described in Example 2.

[0045] If the mold is configured to facilitate the formation of a pouch-shaped MEHM, it may have a pouch shape, preferably a spherical or ellipsoidal pouch shape. In a particularly preferred embodiment, the mold is formed by spherical or ellipsoidal inner and outer walls, and more preferably, the spherical or ellipsoidal inner wall is inflatable. An example of a pouch-shaped mold can be seen in FIG. 8A disclosed herein. Furthermore, WO 2008 / 058917 A1 and EP 2842581 A1 disclose methods for producing pouch-like artificial cardiac tissue as illustrated in FIG. 1 therein. Thus, those skilled in the art are generally aware of the creation of pouch-like artificial cardiac tissue. However, those skilled in the art are not aware of a multi-layer pouch-shaped EHM in which the reconstitution mixture (forming the MEHM) is perforated by at least two poles. As described above, for example, the mold may be 3D printed to adjust the radius of the pouch. After fabrication of the pouch form of the MEHM is complete, the pouch may be peeled from the mold and cut open if desired.

[0046] The mold may have a cylindrical shape, preferably a cylindrical mold, if it is configured to facilitate the formation of a cylindrical MEHM. In a particularly preferred embodiment, the cylindrical mold is formed by an inner and outer cylindrical wall. In another embodiment, the cylindrical mold is formed by a single cylindrical wall. The single cylindrical wall may be covered with the reconstitution mixture by centrifugation. An exemplary cylindrical mold is shown in FIG. 8B disclosed herein. As described above, the mold may be 3D printed to adjust the length or diameter of the cylinder, for example. An important advantage of cylindrical MEHMs is that large MEHMs can be produced using a relatively small space. Thus, several MEHMs can be produced in parallel. For example, the cylindrical MEHM may be cut open along its longitudinal axis prior to potential use in a patient.

[0047] The reconstitution mixture is perforated by at least two poles, as shown in exemplary Figures 2, 7, and 8. In one embodiment, the poles are flexible, allowing for the introduction of mechanical loads onto the MEHM. Generally, the poles serve at least two purposes: first, the poles penetrating the MEHM create channels that allow for a sufficient supply of oxygen and nutrients to the cells within the reconstitution mixture / MEHM; and second, the poles are important for the fabrication of the MEHM because they serve to provide physical stimulation for developing muscles. Those skilled in the art know that chemical as well as physical stimulation is important for the development of artificial muscles. In other words, the poles assist in the force generation of the MEHM, as muscle training is important for force generation in the MEHM. In one embodiment, the channels created by the perforated poles can allow for the perfusion of the MEHM with the culture medium of steps (ii) and (iii), and optionally the maturation medium and oxygen of step (iv), in steps (ii), (iii), and optionally (iv). In particularly preferred embodiments, perfusion of culture medium and / or maturation medium leads to adequate nutrient support of cardiomyocytes and non-myocytes during steps (ii), (iii), and / or optionally (iv).

[0048] In a preferred embodiment, the reconstitution mixture is perforated with at least two poles, and the perforated poles introduce channels into the reconstitution mixture / MEHM, thereby increasing the surface area of ​​the reconstitution mixture / MEHM. In an even more preferred embodiment, the reconstitution mixture / MEHM is perforated with at least two poles, increasing the surface area of ​​the MEHM and thereby controlling the diffusion distance of oxygen and nutrients across the MEHM. As further described below, this increase in surface area shortens the distance between cells and the surface of the reconstitution mixture / MEHM, on average, so that cells are adequately supplied with oxygen and / or nutrients, as may be confirmed by, for example, a hypoxia sensor (e.g., Hesse et al. 2014) or cell death (e.g., using known viability assay dyes). In a further preferred embodiment, the inter-pole distance is 0.1 to 10 mm, preferably 0.5 to 9 mm, more preferably 1 to 8 mm, even more preferably 2 to 7 mm, even more preferably 2.5 to 6 mm, even more preferably 3 to 5 mm, even more preferably 3.1 to 6 mm, even more preferably 3.2 to 5 mm, even more preferably 3.3 to 4 mm, even more preferably 3.4 to 3.7 mm, and most preferably about 3.5 mm. Figure 8 shows a highly preferred embodiment. The optimal inter-pole distance is particularly important for MEHMs; the MEHM may be thicker than the inter-pole distance, and then cells may have a distance to the next perforation pole that is shorter than the distance to the top or bottom surface.

[0049] In one embodiment, at least two poles perforate the reconstituted mixture from the bottom and / or top. Specifically, the reconstituted mixture may be perforated throughout its thickness. This means that the poles penetrate the reconstituted mixture. In a particularly preferred embodiment, at least two poles perforate the reconstituted mixture from the bottom, and the mold is disc-shaped. Furthermore, at least two poles penetrate the reconstituted mixture from the bottom of the mold. Even more preferably, the poles are permanently attached to the mold or base plate. For example, the poles may be firmly attached to the casting area of ​​the mold, or may be attached to a detachable base plate. The base plate can also be transferred to a different mold. An advantage of a base plate is that it can be detached, thereby allowing it to be transferred to a different mold if desired (see, for example, FIG. 8). For example, the poles may be an integral part of the mold as a permanently attached element, or they may be detachable elements. More preferably, the mold and at least two pole elements are 3D printed, and even more preferably, the mold with at least two poles is 3D printed as a single item. Even more preferably, the mold with at least two poles is 3D printed as a single item. 3D-printing has the advantage that molds such as discs can be custom-made and / or adapted. The base plate has the advantage that the MEHM is detachable and can be easily transported from the mold to the point of use, e.g., an operating room, in a suitable shipping container.

[0050] In another embodiment, at least two poles perforate the reconstituted mixture from above, and the mold is disk-shaped. In a particularly preferred embodiment, at least two poles are inserted into the liquid reconstituted mixture from above. The at least two poles may be inserted from above, for example, as a lattice comb.

[0051] In another embodiment, at least two poles are designed to pierce the reconstituted mixture from below, and the mold is designed to assist in forming a pouch. In a particularly preferred embodiment, the pouch is formed by an inner wall and an outer wall of a spherical or ellipsoidal shape. Even more preferably, at least two poles extend from the inner wall of the spherical or ellipsoidal shape, thereby piercing the reconstituted mixture. In a further preferred embodiment, at least two poles are permanently attached to the inner wall of the spherical or ellipsoidal shape.

[0052] In another embodiment, at least two poles perforate the reconstituted mixture from above, and the mold is designed to assist in forming a pouch. In a particularly preferred embodiment, the pouch is formed by an inner wall and an outer wall of a sphere or ellipsoid. In a further preferred embodiment, at least two poles extend from the outer wall of the sphere or ellipsoid, thereby perforating the reconstituted mixture. Even more preferably, at least two poles are permanently attached to the outer wall of the sphere or ellipsoid.

[0053] In another embodiment, at least two poles perforate the reconstituted mixture from below, and the mold is a cylindrical mold. In a preferred embodiment, the cylindrical mold is formed by a cylindrical inner wall and an outer wall. Even more preferably, at least two poles pass through the reconstituted mixture from the outer wall of the cylinder, thereby perforating the reconstituted mixture. In an even more preferred embodiment, the poles are permanently attached to the outer wall of the cylinder. In particular, the outer wall of the cylinder with at least two poles can be 3D printed as a single object. 3D printing has the advantage that any shape, such as a cylinder with at least two poles, can be produced and the size can be individually adjusted.

[0054] In another embodiment, at least two poles perforate the reconstituted mixture from above, and the mold is a cylindrical mold. In a preferred embodiment, the cylindrical mold is formed by an inner and outer cylindrical wall. In a more preferred embodiment, the at least two poles pass through the reconstituted mixture from the inner cylindrical wall. In an even more preferred embodiment, the poles are permanently attached to the inner cylindrical wall. In particular, the inner cylindrical wall with at least two poles may be 3D printed as a single object. 3D printing has the advantage that any shape, such as a cylinder with at least two poles, can be produced and the size can be individually adjusted. The mold and / or poles can be produced by any suitable engineering method, including 3D printing, injection molding, milling, or glassworking. In particular, the mold may be manufactured by injection molding, for example, by milling a PTFE block. In a highly preferred embodiment, the mold and / or poles are 3D printed.

[0055] The reconstituted mixture is cast into a mold, completely or partially filling the mold, i.e., the casting area. Those skilled in the art can determine the optimal volume depending on the size of the mold. Typically, one liquid reconstituted mixture is 1 mm to about 10 mm thick. Depending on the casting area and desired thickness, those skilled in the art can determine the optimal volume by simple multiplication. In a preferred embodiment, the volume of each liquid reconstituted mixture is 0.5 to 200 ml, preferably 1 to 150 ml, more preferably 1.5 to 100 ml, more preferably 2 to 50 ml, more preferably 2.5 to 40 ml, more preferably 3 to 30 ml, more preferably 3.5 to 25 ml, more preferably 4 to 20 ml, more preferably 4.5 to 15 ml, more preferably 5 to 11.5 ml, more preferably 6 to 9.5 ml, even more preferably 6.5 to 8.5 ml, and most preferably about 8 ml. An exemplary EHM using an 8 ml volume is shown in Figure 2 disclosed herein.

[0056] Molds can hold volumes ranging from 1 to 400 ml. The volume a mold can hold can be determined by multiplying the casting area by the potential height of the MEHM. For example, if the mold allows the formation of a circular disk, the mold's volume can be calculated by multiplying the casting area by the mold's height. An exemplary casting mold for producing hexagonal circular disks is shown in Figure 7A. If the mold is pouch-shaped, the mold's volume is the difference between the spherical or ellipsoidal volumes of the outer and inner walls. Similarly, if a cylindrical mold has outer and inner walls, the mold's volume can be determined by the difference in volume between the outer and inner walls of the cylinder. If a cylindrical mold only has one outer wall, it could theoretically hold the volume of the entire cylinder. In a preferred embodiment, the mold holds a volume of 2 to 300 ml, more preferably 3 to 200 ml, more preferably 4 to 100 ml, more preferably 5 to 80 ml, more preferably 6 to 60 ml, more preferably 6.5 to 50 ml, more preferably 7 to 40 ml, more preferably 7.5 to 30 ml, more preferably 8 to 23 ml, more preferably 13 to 19 ml, even more preferably 13 to 17 ml, and even more preferably about 15 ml. As mentioned above, the mold may be in the shape of a patch, a pouch, or a cylinder.

[0057] As described above, the mold has a casting area. The liquid reconstituted mixture is cast into the casting area. In other words, the size of the mold indicates the size of the casting area. Furthermore, those skilled in the art can determine the ideal casting area of ​​the mold according to their needs. For example, if it is desired that the MEHM be used as a graft, the mold may be custom-made, for example, using a 3D printer. In a preferred embodiment, the size of the mold is 1 to 400 cm. 2 , preferably 2 to 300 cm 2 , more preferably 3 to 200 cm 2 , more preferably 4 to 100 cm 2 , more preferably 5 to 80 cm 2, more preferably 6 to 60 cm 2 , more preferably 7 to 50 cm 2 , more preferably 8 to 40 cm 2 , more preferably 9 to 30 cm 2 , more preferably 10 to 23 cm 2 , more preferably 12 to 19 cm 2 , and even more preferably 13 to 17 cm 2 , and even more preferably about 16 cm 2 In a highly preferred embodiment, the mold is used to form a patch-shaped MEHM.

[0058] As described above, the reconstituted mixture is perforated by at least two poles. In a preferred embodiment, the reconstituted mixture is perforated by at least three poles. In an even more preferred embodiment, the poles are arranged in a lattice, more preferably, the poles are arranged in a triangular or rectangular lattice, and even more preferably, in a triangular lattice. An exemplary triangular lattice arrangement is shown, for example, in Figure 7 disclosed herein. However, one skilled in the art can identify other suitable lattice arrangements.

[0059] The appropriate number and diameter of the poles for perforating the reconstitution mixture depend on the size of the mold. The poles for perforating the reconstitution mixture serve two purposes: first, they ensure the formation of channels for sufficient oxygen and nutrient supply to the cells within the reconstitution mixture; and second, they support the training of developing muscles, i.e., they support the generation of muscle force. Those skilled in the art can determine the optimal number of poles depending on the size of the mold. Using simple experiments, those skilled in the art can determine whether cells are insufficiently supplied with oxygen and / or nutrients. For example, Hesse et al. (2014) described the use of hypoxia sensors in artificial tissues, such as artificial myocardium, to determine whether cells are hypoxic. Furthermore, the cells may die if they are not adequately supplied with oxygen and / or nutrients. This can also be confirmed by standard experiments using staining methods. In addition, those skilled in the art can determine the optimal number and diameter of the poles based on the contractility of the MEHM. As described above, shrinkage can be measured by FAC or FOC, for example, as described in Tiburcy et al. (2017) or Zimmermann et al. (2000). In preferred embodiments, the reconstituted mixture is perforated with at least 5 poles, more preferably at least 7 poles, more preferably at least 14 poles, even more preferably at least 20 poles, even more preferably at least 30 poles, even more preferably at least 37 poles, and most preferably at least 52 poles. Exemplary casting molds, such as those illustrated in Figures 7A and 7C herein, show 52 and 14 poles, respectively. To the best of the inventor's knowledge, there is no fixed upper limit to the number of poles. For example, the reconstituted mixture may be perforated with up to 2,000 poles, preferably up to 1,500 poles, and even more preferably up to 1,000 poles. Given the present disclosure, those skilled in the art can freely combine ranges of pole numbers.In another particularly preferred embodiment, the diameter of the poles is 0.5 to 3 mm, preferably 0.6 to 2.5 mm, more preferably 0.65 to 2 mm, even more preferably 0.7 to 1.7 mm, and even more preferably about 0.8 to 1.5 mm. Furthermore, as shown in Figure 7C, various diameters ranging from 0.8 to 1.1 mm have also been experimentally tested. In a particularly preferred embodiment, the reconstituted mixture may be perforated by at least seven poles, where the poles are arranged in a lattice pattern and the diameters of the poles form a gradient along one planar axis. An example of a diameter gradient is illustrated in Figure 7C disclosed herein. Furthermore, the diameter gradient can have the technical effect of producing a larger FAC measurement along the gradient than along poles with equal pole thickness. This finding is supported by experimental data such as that shown in Figure 7C.

[0060] For example, the casting mold depicted in Figure 7A contains 52 poles, each with a volume of 8 ml of liquid reconstitution mixture. One skilled in the art can also extrapolate the volume of the reconstitution mixture and the number of poles from this example.

[0061] In a preferred embodiment, each pole has a base region, which may be circular, rectangular, oval, triangular, or polygonal, and preferably the base region is circular. Circular base regions of poles are also illustrated, for example, in Figures 7 and 8 herein.

[0062] Advantageously, the poles create restoring forces to simulate the cardiac wall stresses of normal and / or infarcted hearts during the contraction cycle. A restoring force is a force that acts to move an object to an equilibrium position. As mentioned above, to introduce mechanical loads onto the MEHM, the poles are typically flexible. Therefore, the poles ideally have elastic properties (flexibility). Generally, the elastic properties of the poles are quantified by their modulus of elasticity, or Young's modulus, which is defined as the amount of stress required to achieve one unit of strain. For example, the higher the modulus, the less likely the material is to deform. Generally, the SI unit for this modulus is the pascal (Pa). For example, Rump et al. (2007) and Pislaru et al. (2014) describe the viscoelastic properties of normal and infarcted myocardium. An ideal artificial tissue would mimic the contractility of normal and / or infarcted myocardium (approximately 0.3-60 mN / mm). 2 ; Wiegerinck et al. 2009, Muleri et al. 1992) and viscoelastic (5–27 kPa; Rump et al. 2007) properties.

[0063] In particularly preferred embodiments, the restoring force of the pole ideally matches the wall tension of normal and / or diseased myocardium (10-200 kdyne / cm2 or 1-20 kPa; Fujita et al. 1993). Elastic properties can be defined by a working spring constant. In this case, the working spring constant can be evaluated at the point where the MEHM contacts the pole. Generally, the spring constant is defined as the amount of force required to deform the spring one unit of distance. The unit of measurement for spring constant is typically N / mm (Newtons per millimeter) or mN / mm (millinewtons per millimeter). In highly preferred embodiments, the elastic properties of the pole, defined by its elastic modulus, produce a restoring force of 0.5-50 mN / mm, preferably 0.7-40 mN / mm, more preferably 0.9-30 mN / mm, even more preferably 1-20 mN / mm, even more preferably 2-10 mN / mm, even more preferably 3-5 mN / mm, and most preferably about 4 mN / mm. In a particularly preferred embodiment, the restoring force of the pole corresponds to the wall tension of a normal and / or diseased human heart, and even more preferably, the wall tension is 1 to 20 kPa, the range also being disclosed in Fujita et al. (1993).

[0064] The elastic properties of the poles may exhibit different moduli of elasticity to establish biomechanical anisotropy, i.e., the moduli of elasticity exhibit variation between poles. In another embodiment, the elastic properties of the poles exhibit similar moduli of elasticity to establish biomechanical isotropy, i.e., the moduli of elasticity are uniform between poles.

[0065] In another embodiment, the pole is surrounded by a peripheral wall at the lower portion of the pole, the peripheral wall forming a ring-shaped ramp around the pole, the ring-shaped ramp tapering upward from the bottom. Exemplary dimensions of the peripheral wall are illustrated in FIG. 4B for a ring-shaped EHM. WO2017 / 207431A1 further describes such peripheral walls in detail. An advantage of a peripheral wall at the bottom of the pole is that the condensed reconstituted mixture moves upward along the ramp by compaction and / or muscle force generation. This upward sliding by compaction and / or muscle force generation facilitates the addition of additional reconstituted mixture from below, as described in step (iii) of the method.

[0066] In another embodiment, the pole tapers conically, with the pole having a maximum diameter at the bottom. Tapering the pole from bottom to top has the same advantages as the surrounding wall: the EHM can be peeled from the mold and slid upward to facilitate the addition of additional reconstitution mixture, particularly from the bottom.

[0067] Those skilled in the art are aware of appropriate conditions for conducting tissue culture experiments. In a preferred embodiment, steps (i), (ii), and / or optional (iv) are carried out at a temperature in the range of 36.4 to 37.6°C, preferably 36.6 to 37.4°C, preferably 36.8 to 37.2°C, and more preferably at about 37°C. In a further and further preferred embodiment, steps (i), (ii), and / or optional (iv) are carried out in a humidified cell culture incubator in the presence of 2 to 10% CO2, preferably 2.5 to 8% CO2, more preferably 3 to 7% CO2, even more preferably 3.5 to 6.5% CO2, even more preferably 4 to 6% CO2, and most preferably about 5% CO2. Generally, the culture may be carried out in ambient oxygen or an oxygen carrier, such as hemoglobin-based oxygen carriers (HBOCs) and perfluorocarbon-based oxygen carriers (PFOCs) (Iyer et al. 2007). In another preferred embodiment, steps (i), (ii), and / or (iv) are carried out in a humidified cell culture incubator in the presence of 5-40% O2, preferably 10-30% O2, more preferably 15-35% O2, even more preferably 17-33% O2, even more preferably 19-25% O2, and most preferably about 21% O2. Furthermore, those skilled in the art will know from common general knowledge that the above temperature and CO2 ranges are standard tissue culture conditions in the art. Furthermore, 21% O2 corresponds to the normal oxygen concentration in dry air.

[0068] In step (i), the liquid reconstitution mixture gels. When the reconstitution mixture gels, it is no longer liquid, i.e., non-liquid. In a preferred embodiment, gelation is characterized by the gel-like nature of the reconstitution mixture obtained by step (i). The gelation of the reconstitution mixture occurs due to the collagen in the reconstitution mixture. For example, if highly purified bovine dermal (skin) type I acid-solubilized collagen is used in the reconstitution mixture, the pH value of the reconstitution mixture is adjusted to a physiological pH, e.g., 7.0-8.0. Upon this pH shift, the collagen forms a gel. In practice, the ideal condition for collagen gel formation (3D-scaffold) is a pH of approximately 7. Biochemically, native collagen molecules are covalently cross-linked via hydroxylysine and lysine residues. Therefore, native collagen molecules can self-assemble into collagen fibrils and form hydrogels in vitro at 37°C and near neutral pH via cross-linking. The self-assembly gelation process can be initiated by a pH shift and occurs optimally at 37°C. The physicochemical properties of collagen hydrogels are affected by collagen concentration, polymerization pH, and ionic strength during crosslinking. This process is well known to those skilled in the art, and has been used to form 3D scaffolds (e.g., Tiburcy et al. 2017) for various tissue engineering applications. In particularly preferred embodiments, the reconstitution mixture has a pH of 7.0-7.8, preferably 7.2-7.6, more preferably 7.3-7.5, and most preferably about 7.4.

[0069] In another preferred embodiment, the gelation in step (i) is characterized by the culture medium surrounding the reconstituted mixture once added to the mold and not dissolving the reconstituted mixture. One skilled in the art can assess the gelation of the reconstituted mixture, for example, by testing whether the culture medium dissolves the reconstituted mixture after various time points. This test can also be performed in a laboratory tube in parallel with the production of the MEHM. Furthermore, the gelation in step (i) may be characterized by the reconstituted mixture being opaque, preferably as determined by visual inspection as shown in Figure 3B of Tiburcy M, et al. (2014).

[0070] In a highly preferred embodiment, step (i) is carried out for at least 15 minutes, preferably at most 24 hours, more preferably between 20 minutes and 15 hours, more preferably between 30 minutes and 8 hours, even more preferably between 45 minutes and 1.5 hours, and most preferably for about 1 hour.

[0071] In another embodiment, the culture medium in step (ii) comprises (a) basal medium, (b) serum-free supplement, (c) L-glutamine, (d) ascorbic acid, (e) IGF-1, (f) VEGF, and (g) TGFβ1. However, those skilled in the art can identify appropriate culture media. For example, Tiburcy et al. (2017), (2020) and Schlick et al. (2019) provide exemplary culture media suitable for culturing EHMs. Each of factors such as IGF-1, VEGF, and / or TGFβ1 can be replaced with factors with equal or similar effects. For example, TGFβ1 can be replaced with any other factor that stimulates non-muscle cells (especially stromal cells) to initiate compaction of the reconstituted mixture.

[0072] In a preferred embodiment, the basal medium of the culture medium is selected from Iscove's medium, αMEM, DMEM, and RPMI. Preferably, the basal medium is Iscove's medium or αMEM, and more preferably, the basal medium is Iscove's medium. However, any suitable basal medium can be used in the method. Basal media are commercially available or can be prepared according to publicly available recipes, for example, according to publicly available recipes from the ATCC catalog. Amino acids may be added to the basal medium as deemed appropriate. In another preferred embodiment, the culture medium may contain a serum-free supplement. The serum-free supplement may provide a final concentration in the culture medium as defined above. In another preferred embodiment, the culture medium in step (ii) may contain 0.4 to 10 mM L-glutamine, preferably 0.8 to 6 mM L-glutamine, more preferably 1.2 to 5 mM L-glutamine, more preferably 1.5 to 4 mM L-glutamine, more preferably 1.7 to 3 mM L-glutamine, and most preferably about 2 mM L-glutamine. In a highly preferred embodiment, the culture medium in step (ii) may contain 30 to 3000 μM ascorbic acid or a derivative thereof, preferably 100 to 1000 μM ascorbic acid or a derivative thereof, more preferably 180 to 500 μM ascorbic acid or a derivative thereof, more preferably 220 to 370 μM ascorbic acid or a derivative thereof, more preferably 270 to 330 μM ascorbic acid or a derivative thereof, and most preferably about 300 μM ascorbic acid or a derivative thereof. An example of a derivative of ascorbic acid is ascorbic acid-2-phosphate.

[0073] In another preferred embodiment, the culture medium in step (ii) contains 10 to 1000 ng / ml IGF1, preferably 50 to 500 ng / ml IGF1, more preferably 70 to 200 ng / ml IGF1, even more preferably 90 to 120 ng / ml IGF1, and most preferably about 100 ng / ml IGF1. In particular, the IGF1 may be human IGF1.

[0074] In another preferred embodiment, the culture medium in step (ii) contains 2.5 to 10 ng / ml VEGF, preferably 3 to 9 ng / ml VEGF, more preferably 3.5 to 8 ng / ml VEGF, more preferably 4 to 7 ng / ml VEGF, more preferably 4.5 to 6 ng / ml VEGF, and most preferably about 5 ng / ml VEGF. In particular, the VEGF may be human VEGF, and preferably the VEGF is VEGF 165 is.

[0075] In another preferred embodiment, the culture medium in step (ii) contains 5 to 20 ng / ml FGF-2, preferably 6 to 18 ng / ml FGF-2, more preferably 7 to 16 ng / ml FGF-2, more preferably 8 to 14 ng / ml, more preferably 9 to 12 ng / ml FGF-2, and most preferably about 10 ng / ml FGF-2. In particular, the FGF-2 may be human FGF-2.

[0076] In another preferred embodiment, the culture medium in step (ii) contains 2 to 8 ng / ml TGFβ1, preferably 3 to 7 ng / ml TGFβ1, more preferably 4 to 6 ng / ml TGFβ1, even more preferably 4.5 to 5.5 ng / ml TGFβ1, even more preferably about 5 ng / ml TGFβ1, and most preferably the TGFβ1 is human TGFβ1.

[0077] In another preferred embodiment, the culture medium in step (ii) contains about 750 mg / L glycine, about 890 mg / L L-alanine, about 1320 mg / L L-asparagine, about 1330 mg / L L-aspartic acid, about 1470 mg / L L-glutamic acid, about 1150 mg / L L-proline, and about 1050 mg / L L-serine.

[0078] During step (ii), the reconstituted mixture condenses. Step (ii) produces a condensed reconstituted mixture. In a preferred embodiment, the reconstituted mixture of step (ii) condenses by at least about 20%, preferably at least about 25%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, even more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, and even more preferably at least about 90% of the initial reconstituted mixture volume, preferably as determined by visual inspection and / or video optical analysis. The EHM volume can be assessed, for example, by planometry, assuming that the compaction of the EHM volume is symmetrical in all dimensions. In addition, the thickness can be accurately determined, for example, by ultrasound, laser interferometry, or optical coherence tomography. The EHM volume can be determined by a combination of area and thickness measurements by planometry. In a preferred embodiment, completion of step (ii) is confirmed by (a) fabricating a loop-format engineered heart muscle (EHM) in parallel with the MEHM by performing steps (i) and (ii), and (b) verifying the detachment of the loop-format engineered heart muscle (EHM) from the mold, as described in Figure 3C of Tiburcy M, et al., Collagen-based engineered heart muscle, Methods Mol Biol. (2014); 1181:167-76. Because the detachment is clearly visible, the detachment from the mold can be easily assessed by fabricating a loop-format EHM in parallel. For example, Figure 5C herein also shows that the loop-format EHM is clearly detached from the mold.In a further preferred embodiment, the completion of step (ii) can be further confirmed by preparing a loop-format EHM in parallel with the MEHM by following steps (i) and (ii), where the loop-format reconstituted mixture is condensed by at least 20%, preferably at least 25%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, and even more preferably at least 90% of the cross-sectional area of ​​the initial reconstituted mixture. The condensation (compactness) can be assessed by visual inspection and / or video optical analysis of the EHM. Figure 5C herein shows an example of optimal compaction of a loop-format EHM.

[0079] In a particularly preferred embodiment, step (ii) is carried out for at least 12 hours, preferably at most 15 days, more preferably 12 hours to 7 days, preferably 14 hours to 6 days, more preferably 16 hours to 5.5 days, more preferably 18 hours to 5 days, more preferably 20 hours to 4.5 days, even more preferably 22 hours to 4 days, even more preferably 23 hours to 3.5 days, and most preferably 24 hours to 3 days.

[0080] In step (iii), an additional reconstituted mixture is added to the reconstituted mixture of step (ii) from above and / or below. In a preferred embodiment, in step (iii)a), one additional reconstituted mixture is added to the top of the condensed reconstituted mixture of step (ii). In another preferred embodiment, in step (iii)a), one additional reconstituted mixture is added to the bottom of the condensed reconstituted mixture of step (ii). In another preferred embodiment, in step (iii)a), one additional reconstituted mixture is added to the condensed reconstituted mixture of step (ii) from above and below simultaneously. In an even more preferred embodiment, in step (iii)a), the additional reconstituted mixture coats the condensed reconstituted mixture of step (ii) from above and / or below. For example, the surface areas of the condensed reconstituted mixture of step (ii) and the additional reconstituted mixture of step (iii)a) are the same. For illustrative purposes, a five-layer EHM is fabricated by coating the condensed reconstitution mixture of step (ii) as shown in FIG. 3 , where the surface area of ​​the additional reconstitution mixture of step (iii)a) and the condensed reconstitution mixture of step (ii) are the same. In another example, in step (iii)a), the additional reconstitution mixture coats a limited area of ​​the condensed reconstitution mixture of step (ii) from the top or bottom. The surface area of ​​the additional reconstitution mixture of step (iii)a) may be smaller than the surface area of ​​the reconstitution mixture of step (ii). For illustrative purposes, FIG. 8C2 illustrates an inlet. An inlet can be placed above the condensed reconstitution mixture of step (ii) to supply a smaller liquid reconstitution mixture to the top of the condensed reconstitution mixture of step (ii). For example, the smaller surface area of ​​the reconstitution mixture of step (iii)a) can be tailored to a patient-specific defect or another desired shape. This has the advantage that a MEHM for use in cardiac repair can be strengthened in a specific, limited area compared to the remainder of the MEHM.

[0081] In another embodiment, the surface area of ​​the additional reconstituted mixture is larger than that of the condensed reconstituted mixture in step (ii). For example, in step (iii)b), the different mold has a larger casting area than the mold in step (i), and the additional reconstituted mixture coats at least the condensed reconstituted mixture in step (ii) from the top or bottom. Even more preferably, the additional reconstituted mixture coats the condensed reconstituted mixture in step (ii) from the top or bottom, filling the large casting area of ​​the different mold. For illustration, FIG. 8C1 illustrates a small-sized condensed reconstituted mixture being transferred to a mold with a large surface area. For example, the large surface area of ​​the different mold in step (iii)b) can be adjusted to a patient-specific defect. This has the advantage that the MEHM for use in cardiac repair can be tailored to specific needs.

[0082] Adding additional reconstitution mixture in step (iii) creates additional EHM layers. Thus, each additional reconstitution mixture increases the thickness of the EHM. The desired thickness can be determined by one skilled in the art. For example, the MEHM may be used as a graft for a human heart. Preferably, the human defect is imaged by state-of-the-art imaging methods, such as echocardiography, magnetic resonance imaging, or computed tomography, to determine the desired thickness and dimensions. In a preferred embodiment, step (iii)a) or (iii)b) is repeated at least two times, preferably at least three times, more preferably at least four times, more preferably at least five times, more preferably at least six times, more preferably at least seven times, more preferably at least eight times, even more preferably at least nine times, and most preferably at least ten times. In an even more preferred embodiment, step (iii)a) or (iii)b) is repeated 2 to 200 times, preferably 2 to 100 times, more preferably 2 to 80 times, more preferably 2 to 70 times, more preferably 2 to 60 times, more preferably 2 to 50 times, more preferably 2 to 40 times, more preferably 3 to 30 times, more preferably 3 to 25 times, more preferably 4 to 20 times, more preferably 4 to 15 times, more preferably 4 to 10 times, even more preferably 5 to 9 times, and most preferably 5 to 8 times. However, one skilled in the art can ascertain how many times step (iii) should be repeated or whether step (iii)a) or (iii)b) should be performed. The skilled artisan will be guided by the desired dimensions and shape, and will evaluate whether the layers should have the same surface area or whether each individual layer should be smaller or larger. For example, one skilled in the art may desire a cardiac graft. Therefore, the surface area of ​​each individual layer is customized to the size of the myocardial defect. Additionally, the thickness of the MEHM ideally matches that of healthy tissue. For example, one condensed reconstitution mixture may have a thickness of, for example, about 500 μm. To achieve a desired heart wall thickness of up to 10 mm (Kawel et al. (2012)), 20 layers of condensed reconstitution mixture may be stacked. In another example, the condensed reconstitution mixture may be thinner than 500 μm.Fifty layers of the reconstitution mixture may then need to be layered to achieve a typical heart wall thickness. One skilled in the art can ascertain how many layers of reconstitution mixture are needed to achieve an MEHM with a thickness typical of a patient's human heart and / or myocardial defect.

[0083] After step (iii) is completed, the EHM may be further matured in a maturation medium. Those skilled in the art can find exemplary compositions of maturation medium in, for example, Tiburcy et al. (2017) or Tiburcy et al. (2020). In a particularly preferred embodiment, the maturation medium for step (iv) is defined as the culture medium described above, except that TGFβ1 is omitted from the maturation medium compared to the culture medium. In an even more preferred embodiment, step (iv) is performed for 4 to 200 days, preferably 6 to 150 days, more preferably 8 to 120 days, more preferably 9 to 110 days, more preferably 10 to 90 days, more preferably 15 to 70 days, more preferably 20 to 50 days, and most preferably 28 to 42 days.

[0084] In a preferred embodiment, the MEHMs of step (iii) are cultured in an appropriate maturation medium in step (iv), and the MEHMs of step (iv) exhibit increased maturation as determined by increased abundance of cardiomyocyte sarcomeric proteins, such as alpha-sarcomeric actinin, myosin heavy chain protein, myosin light chain protein, and troponin, as determined by, for example, fluorescence microscopy, Western blotting, flow cytometry, or RNA sequencing after antibody labeling of one or several of the indicated proteins to determine the abundance of the respective protein-encoding transcripts, as described, for example, in Tiburcy et al. Circulation 135(19)1832-1847 (2017). Furthermore, the MEHM of step (iv) may exhibit increased force generation when compared to the MEHM of step (iii), wherein the increased force generation is confirmed by measuring fractional area change (FAC) using the method described in Tiburcy et al. Circulation 135(19)1832-1847 (2017), wherein the MEHM of step (iv) has a fractional area change (FAC) of at least 0.5% upon electrical stimulation, preferably at least 1%, more preferably at least 1.2%, more preferably at least 1%, more preferably at least 2%, more preferably at least 3%, and even more preferably at least 5%.

[0085] One particular advantage of the method disclosed herein is that the cells in the reconstituted mixture / MEHM are always adequately supplied with nutrients and oxygen. Ideally, the creation of channels through the perforated poles during the manufacturing process ensures a minimum acceptable surface tissue diffusion distance, thereby ensuring a sufficient supply of nutrients while producing a thick MEHM that can be used, for example, as an implant. For example, the inter-pole distance may be 3.5 mm, such that the diffusion distance, i.e., the distance from the pole (tissue surface) to the tissue core (the center between the two poles; see Figure 8 and further described above), is 1.75 mm. In other words, the diffusion distance from the tissue surface (pole or upper / lower surface) to the tissue core is optimally maintained. The diffusion distance can be adjusted by the number of perforated poles and the pole design. The optimal surface tissue diffusion distance can be confirmed by tissue hypoxia measurements (e.g., using the ODD-Luc hypoxia reporter described in Hesse AR, et al. (2014)) or cell viability assays (e.g., using known viability dyes). Viability dyes, also known as live / dead stains, are known in the art and can be purchased commercially from a variety of sources. For example, intracellular esterases can hydrolyze the dye in live cells to produce a hydrophilic, highly fluorescent compound, resulting in a fluorescent signal that can be measured in live cells, e.g., at Ex / Em = 485 / 530 nm. Dead cell dyes penetrate damaged cell membranes and exhibit a 40-fold increase in fluorescence upon binding to nucleic acids, resulting in bright red fluorescence (Ex / Em = 495 / 635 nm) in dead cells. In MEHM formulations, the surface-tissue distance is determined by the interpole distance of the perforated poles. An example of an appropriate interpole distance of 3.5 mm, resulting in a maximum surface-to-core diffusion distance of 1.75 mm, is illustrated in Figure 8. For illustrative purposes, selecting a 3.5 mm interpole distance results in a maximum surface-to-core diffusion distance of 1.75 mm, regardless of the thickness of the MEHM. Those skilled in the art can use the information provided herein to determine and adapt the optimal pole-to-pole distance in an MEHM as needed to ensure oxygen and nutrient support throughout an MEHM of desired dimensions and thickness.For example, when a monolayer EHM is prepared, e.g., by following steps (i), (ii), and (iv) and as shown on the right side of Figure 3C, 8 ml of liquid reconstitution mixture condenses to approximately 0.8 ml. Such a monolayer EHM may be 0.5 mm to 1 mm thick. Thus, the reconstitution mixture may condense by approximately 90% throughout the process. However, to sustain stress and support a beating heart, the thickness of an artificial cardiac tissue for use in the clinic is ideally at least 5 mm. As a thought experiment, to obtain an EHM with a thickness of at least 5 mm using a single reconstitution mixture, one skilled in the art would need to cast at least 80 ml of reconstitution mixture, which corresponds to a thickness of 50 mm (5 cm) of liquid reconstitution mixture. However, cells in the center of such a thick single reconstitution mixture would not survive such a process because they would die in the center of the reconstitution mixture due to hypoxia and nutrient deprivation, subsequently condensing the tissue. By introducing perforated poles, this limitation is circumvented as the desired optimal superficial tissue-core diffusion distance is established to ensure a sufficient supply of oxygen and nutrients throughout the MEHM.

[0086] Another important advantage of the method disclosed herein is that the layers are added sequentially. Thus, each additional reconstitution mixture is integrated with the condensed reconstitution mixture of step (ii). Because of this integration, the resulting MEHM is made up of several layers, but these layers are inseparable. In other words, the MEHM behaves like a single muscle as it is being formed, as each layer integrates to form a single entity. This is a particular advantage over the fused, layer-by-layer artificial cardiac myocardium disclosed in WO2007054286, Zimmermann et al. 2006, and Naito et al. 2006.

[0087] In a preferred embodiment, hypoxia is confirmed using a hypoxia reporter. In a particularly preferred embodiment, cardiomyocytes within the MEHM are well-oxygenated, preferably confirmed by the ODD-Luc hypoxia reporter as described in Hesse AR, et al. (2014). This embodiment is also supported by experimental evidence, as shown in Figure 3D. In the experiment, cardiomyocytes in the innermost layer of the MEHM and the monolayer EHM contain the ODD-Lux hypoxia reporter. As can be seen from the comparison in Figure 3D, the monolayer EHM actually exhibits greater hypoxia than the five-layer EHM. In a particularly preferred embodiment, the cardiomyocytes in the MEHM are adequately oxygenated, as determined by the ODD-Luc hypoxia reporter as described in Hesse AR et al. (2014), and preferably, sufficient oxygenation is ensured when the average relative luminescence of the cardiomyocytes of the MEHM, when obtained by following steps (i), (ii), and optionally step (iv) of the method, is at most 6 times, more preferably at most 5 times, more preferably at most 4 times, more preferably at most 3 times, more preferably at most 2.5 times, even more preferably at most 2 times, and even more preferably at most 1.5 times, the average relative luminescence of the cardiomyocytes of the monolayer EHM. In an even more preferred embodiment, the cardiomyocytes in the MEHM are well-oxygenated, as confirmed by the ODD-Luc hypoxia reporter as described in Hesse AR et al. (2014), and the average relative luminescence of cardiomyocytes in the MEHM is no greater than the average relative luminescence measured in cardiomyocytes in a monolayer EHM, as experimentally shown, for example, in Figure 3C.

[0088] In another aspect, a multi-layered artificial myocardium (MEHM) obtainable by the methods disclosed herein is also described. Also described is a multi-layered artificial myocardium (MEHM) obtainable by the methods disclosed herein.

[0089] In another aspect, a multilayered artificial myocardium (MEHM) is disclosed, wherein the MEHM comprises (a) collagen and (b) a cell mixture of cardiomyocytes and non-myocytes, and the EHM comprises at least two layers. The MEHM is preferably a patch, pouch, or cylinder. Even more preferably, the MEHM has a thickness of at least 0.2 mm. More preferably, the MEHM is fabricated by a repetitive, sequential layering process, resulting in 2 to 200 layers. More preferably, the layers are integrated with each other, thereby increasing the thickness of the MEHM. Those skilled in the art will recognize that the cell mixture requires a medium to provide nutrients to the cells. Even more preferably, each layer of the MEHM is derived from a reconstitution mixture, which comprises (a) collagen, (b) a cell mixture of cardiomyocytes and non-myocytes, and (c) an appropriate reconstitution medium.

[0090] In a preferred embodiment, a multi-layered artificial myocardium (MEHM) is described, wherein the MEHM is fabricated by a repetitive and sequential layering process, resulting in 2 to 200 layers, the layers being integral with one another, thereby increasing the thickness of the MEHM, and each layer is derived from a reconstitution mixture, each reconstitution mixture comprising: (a) collagen; (b) a cellular mixture of cardiomyocytes and non-myocytes; and (c) a suitable reconstitution medium.

[0091] Even more preferably, the MEHM is capable of contracting as measured by determining the fractional area change (FAC) of the MEHM using the method described in Tiburcy et al., Circulation 135(19)1832-1847 (2017). In even more preferred embodiments, the FAC is at least 0.5% upon electrical stimulation, preferably the FAC is at least 0.7%, more preferably at least 1%, more preferably at least 2%, more preferably at least 3%, and even more preferably at least 5%.

[0092] In highly preferred embodiments, MEHMs yield between 2 and 100 layers, preferably between 2 and 80 layers, more preferably between 2 and 70 layers, more preferably between 2 and 60 layers, more preferably between 2 and 50 layers, more preferably between 2 and 40 layers, more preferably between 3 and 30 layers, more preferably between 3 and 25 layers, and even more preferably between 4 and 20 layers. For example, 20 layers with an individual fully condensed thickness of 0.5 mm per reconstitution mixture may be a desirable setup for producing MEHMs for use as human grafts.

[0093] As a result of the at least two layers, the MEHM may be 0.2 mm to about 30 mm thick, preferably about 0.3 mm and up to about 30 mm, more preferably about 0.5 mm to about 30 mm, more preferably about 0.7 mm to about 25 mm, more preferably about 0.9 mm to about 20 mm, more preferably about 1 mm to about 17 mm, more preferably about 2.3 mm to about 15 mm, more preferably about 2.8 mm to about 14 mm, more preferably about 3.3 mm to about 13 mm, more preferably about 3.8 mm to about 12 mm, more preferably about 4.2 mm to about 11 mm, more preferably about 4.6 mm to about 10.5 mm, and most preferably about 5 mm to about 10 mm.

[0094] Even more preferably, the MEHM is not vascularized and / or is not under the control of the central nervous system. In a preferred embodiment, at least two poles perforate the MEHM with similar diffusion distances throughout the MEHM to ensure the supply of nutrients and oxygen to the cell mixture. For example, cardiomyocytes within the MEHM are adequately oxygenated, preferably as determined by the ODD-Luc hypoxia reporter as described in Hesse AR et al. (2014). In detail, sufficient oxygen can be supplied to cardiomyocytes in MEHM, and the oxygen supply can be confirmed by the ODD-Luc hypoxia reporter as described in Hesse AR et al. (2014), and preferably, sufficient oxygen supply can be ensured if the average relative luminescence of cardiomyocytes in MEHM is at most 6 times, more preferably at most 5 times, more preferably at most 4 times, more preferably at most 3 times, more preferably at most 2.5 times, even more preferably at most 2 times, and even more preferably at most 1.5 times the average relative luminescence of cardiomyocytes in monolayer EHM, which can be obtained by following step (i), step (ii), and optionally step (iv) of the method disclosed herein.

[0095] It is particularly preferred that the cardiomyocytes in the MEHM are well supplied with oxygen, as confirmed by the ODD-Luc hypoxia reporter as described in Hesse AR et al. (2014), and that the average relative luminescence of cardiomyocytes in the MEHM is not greater than the average relative luminescence measured in cardiomyocytes in the monolayer EHM.

[0096] In preferred embodiments, the MEHM is obtained by any embodiment or combination of embodiments of the methods disclosed herein. The MEHM disclosed herein can exhibit any of the characteristics as disclosed with respect to the methods disclosed herein.

[0097] In another aspect, the MEHM is obtained by performing steps (i)-(iii) of the method disclosed herein.

[0098] In a further aspect, an artificial myocardium, preferably an artificial cardiac patch, pouch, or cylinder, is disclosed, wherein the EHM comprises (a) collagen, and (b) a cell mixture of cardiomyocytes and non-myocytes, the EHM having a thickness of at least about 0.6 mm. Preferably, the EHM is a MEHM and comprises at least two layers.

[0099] In another aspect, the use of MEHM obtained by the methods disclosed herein or MEHM disclosed herein in an in vitro model for drug screening is also described. In particular, MEHM can be used in an in vitro model for drug toxicity screening or drug efficacy screening.

[0100] Furthermore, the use of MEHM obtained by the method disclosed herein or MEHM disclosed herein in the in vitro production of artificial human myocardium is also described. In a preferred embodiment, the MEHM has a three-dimensional shape that matches a patient-specific myocardial defect, and more preferably, the patient-specific myocardial defect is evaluated by MRI, ultrasound, computed tomography, positron emission tomography, and / or optical coherence tomography. Those skilled in the art are familiar with all of these methods, as they are standard techniques in the art. After evaluating the patient-specific three-dimensional myocardial defect, those skilled in the art can create MEHM with dimensions tailored to the purpose. For example, those skilled in the art can produce MEHM according to the thickness of the patient's myocardial wall, preferably according to the thickness of the patient's myocardial wall before the defect occurs.

[0101] The MEHM obtained by the methods disclosed herein or the MEHM disclosed herein may also be used as a research tool.

[0102] In a further aspect, the MEHM obtained by the method disclosed herein or the MEHM disclosed herein is also intended for use in medicine. In particular, the MEHM obtained by the method disclosed herein or the MEHM disclosed herein is intended for use in heart repair. For example, the MEHM can be a graft. The graft can be used to treat patients suffering from heart failure. In a preferred embodiment, the MEHM is permanently applied to the patient's heart. Such permanent application of the MEHM to the heart can be achieved using standard surgical procedures such as suturing, stapling, or gluing. For example, the MEHM can be suturing to the patient's heart.

[0103] Finally, a method of treating a subject suffering from heart failure is also disclosed, comprising administering to the patient's heart an MEHM obtained by the methods disclosed herein. In a preferred embodiment, the method comprises attaching the MEHM to the subject's heart, wherein the attaching preferably comprises suturing the MEHM onto the heart.

[0104] The present invention is further illustrated by the following aspects: 1. A method for producing a multi-layer artificial myocardium (MEHM), comprising the steps of: (i) providing a liquid reconstituted mixture in a mold, the reconstituted mixture being perforated by at least two poles; the reconstitution mixture comprising (a) collagen, (b) a cell mixture of cardiomyocytes and non-myocytes, and (c) an appropriate reconstitution medium, and the reconstitution mixture is allowed to gel in a mold; (ii) incubating the mixture in the mold obtained by step (i) in an appropriate culture medium, whereby the reconstituted mixture condenses in the mold; (iii) a) adding a further liquid reconstitution mixture as defined in step (i) from above and / or below the condensed reconstitution mixture obtained by step (ii), wherein the further liquid reconstitution mixture gels, and then incubation is carried out under the same conditions as in step (ii), so that the further reconstitution mixture condenses in the mold; or b) transferring the concentrated reconstituted mixture obtained from step (ii) into a different mold, wherein said reconstituted mixture is perforated by at least two poles, and thereafter step (iii)a) is carried out in said different mold; This results in a multi-layer artificial myocardium (MEHM), Preferably, the MEHM is thickened by repeating step (iii)a) and / or step (iii)b) at least once, and (iv) optionally culturing the MEHM of step (iii) in said mold in a suitable maturation medium. wherein the MEHM is capable of contracting. 2. The method of embodiment 1, wherein the ability of the MEHM to contract is assessed by visual inspection and / or by verifying the ability of the reconstitution mixture of step (i) to form a force-generating artificial myocardium by performing steps (i), (ii), and optionally step (iv), wherein the artificial myocardium is capable of generating a contractile force that has a force of contraction (FOC) of at least 0.05 mN as measured under standard isometric conditions in Supplementary Figure 6C of Tiburcy et al. Circulation 135(19)1832-1847 (2017). 3. The method of embodiment 2, wherein contraction of the MEHM is further confirmed by parallelly assessing the ability of the reconstitution mixture of step (i) to form a force-generating artificial human muscle in a loop format, as described in Tiburcy et al. Circulation 135(19)1832-1847 (2017), by performing steps (i), (ii), and optionally step (iv), and wherein the artificial myocardium in the loop format produces a force of contraction (FOC) of at least 0.05 mN as measured in Supplementary Figure 6C of Tiburcy et al. Circulation 135(19)1832-1847 (2017). 4. The method of any one of the preceding embodiments, wherein the MEHM produces a force of contraction (FOC) of at least 0.05 mN, more preferably at least 0.1 mN, more preferably at least 0.3 mN, more preferably at least 0.5 mN, more preferably at least 1 mN, more preferably at least 3 mN, even more preferably at least 5 mN, and most preferably at least 10 mN. 5. The method of any one of the previous embodiments, wherein each layer of the MEHM has a thickness of at least about 0.1 mm, at least about 0.15 mm, at least about 0.2 mm, at least about 0.25 mm, at least about 0.3 mm, at least about 0.4 mm, or at least about 0.5 mm. 6. The method of any one of the preceding aspects, wherein the MEHM is about 0.2 mm to about 30 mm thick, preferably about 0.3 mm to about 30 mm, preferably about 0.5 mm to about 30 mm thick, more preferably about 0.7 mm to about 25 mm, more preferably about 0.9 mm to about 20 mm, more preferably about 1.5 mm to about 17 mm, more preferably about 2 mm to about 15 mm, more preferably about 2.8 mm to about 14 mm, more preferably about 3.3 mm to about 13 mm, more preferably about 3.8 mm to about 12 mm, more preferably about 4.2 mm to about 11 mm, more preferably about 4.6 mm to about 10.5 mm, and most preferably about 5 mm to about 10 mm. 7. The method of any one of the preceding embodiments, wherein contraction of the MEHM is measured video-optically by fractional area change (FAC) measurements. 8. The method of any one of the preceding aspects, wherein the contraction is ascertained by measuring the fractional area change (FAC) of the MEHM using the method described in Tiburcy et al. Circulation 135(19)1832-1847 (2017), and wherein the fractional area change (FAC) is at least 0.5% upon electrical stimulation, preferably, the FAC is at least 0.7%, more preferably at least 1%, more preferably at least 2%, more preferably at least 3%, and even more preferably at least 5%. 9. The method of any one of the preceding aspects, wherein the reconstituted mixture has a pH of 7.0 to 7.8, preferably 7.2 to 7.6, more preferably 7.3 to 7.5, and most preferably the reconstituted mixture has a pH of about 7.4. 10. The method of any one of the preceding aspects, wherein the reconstitution mixture provides a final concentration of 0.5 to 3 mg / ml collagen, preferably 0.55 to 2.5 mg / ml collagen, more preferably 0.6 to 2.2 mg / ml collagen, more preferably 0.65 to 2 mg / ml collagen, more preferably 0.7 to 1.75 mg / ml collagen, more preferably 0.75 to 1.5 mg / ml collagen, more preferably 0.8 to 1.2 mg / ml collagen, even more preferably 0.85 to 1 mg / ml collagen, and most preferably about 0.9 mg / ml collagen. 11. The method of any one of the preceding aspects, wherein the collagen in step (i) is selected from the group consisting of collagen type I, collagen type III, collagen type IV, collagen type V, collagen type VI, collagen type XII, collagen type XIII, collagen type XIV, collagen type XV, and mixtures thereof. 12. The method of any one of the previous embodiments, wherein at least 90% of the collagen in the reconstituted mixture of step (i) is collagen type I. 13. The method of any one of the preceding aspects, wherein the collagen of the reconstituted mixture of step (i) is of medical grade. 14. The method of any one of the previous aspects, wherein the collagen of the reconstituted mixture of step (i) is bovine, equine, human, or marine collagen, preferably the collagen is bovine. 15. The method of any one of the preceding aspects, wherein the collagen in the mixture of step (i) further comprises one or more additional extracellular matrix components other than collagen, preferably wherein the one or more additional matrix components are selected from natural or synthetic extracellular matrix components, more preferably wherein the extracellular matrix component is selected from the group consisting of elastin, laminin, entactin, nidogen, proteoglycan, glycosaminoglycan, and fibronectin, or a synthetic mimetic thereof. 16. The cell mixture of step (i) is in a reconstitution mixture containing 1 to 26.5 × 10 6 cells / ml, preferably 2 to 13.2 x 10 6 cells / ml, more preferably 3 to 10 × 10 6 cells / ml, more preferably 3.5 to 9 × 10 6 cells / ml, more preferably 4-8 x 10 6 cells / ml, more preferably 4.3 to 7 × 10 6 cells / ml, more preferably 4.6 to 6 × 10 6 cells / ml, and even more preferably about 5×10 6 20. The method of any one of the preceding embodiments, resulting in a final cell concentration of cells / ml. 17. The method of any one of the preceding aspects, wherein the cardiomyocytes are human cardiomyocytes. 18. The method of any one of the preceding aspects, wherein the cardiomyocytes are derived from embryonic stem cells, and the cells have not been produced using a process involving modification of the genetic identity of human germline or using a process involving the use of human embryos for industrial or commercial purposes. 19. The method of any one of the preceding aspects, wherein the cardiomyocytes are derived from induced pluripotent stem cells, parthenogenetic stem cells, programmed somatic cells, or adult stem cells, preferably wherein the cardiomyocytes are derived from induced pluripotent stem cells. 20. The method of any one of the preceding aspects, wherein the cardiomyocytes are obtained by serum-free differentiation, preferably, the cardiomyocytes are obtained by following the protocol of WO2015 / 040142, or another protocol as outlined in Burridge et al. (2012), Production of de novo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming. Cell Stem Cell. 2012 Jan 6;10(1):16-28, or Zimmermann (2020). 21. The method of any one of the preceding aspects, wherein the cardiomyocytes are non-human primate stem cell-derived cardiomyocytes, fetal cardiomyocytes, or neonatal cardiomyocytes. 22. The method of any one of the preceding embodiments, wherein the cardiomyocytes express ACTN2, TTN, RYR2, and troponin as determined by flow cytometry or RNA sequencing. 23. The method of any one of the preceding aspects, wherein the cell mixture of step (i) comprises at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, and most preferably at least 50% cardiomyocytes. 24. The method of any one of the preceding embodiments, wherein contraction of the MEHM is caused by cardiomyocytes, thereby generating contractile force in the MEHM. 25. The method of any one of the preceding aspects, wherein the non-muscle cells are selected from one or more of the group consisting of stromal cells, endothelial cells, smooth muscle cells, and mesenchymal stem cells, preferably the non-muscle cells are stromal cells or endothelial cells, more preferably the non-muscle cells are stromal cells, even more preferably the stromal cells are cardiac stromal cells, even more preferably the cardiac stromal cells have fibroblastic properties, even more preferably the cardiac stromal cells are fibroblasts. 26. The method of any one of the preceding embodiments, wherein the non-muscle cells express CD90 as determined by flow cytometry, preferably the non-muscle cells express CD90, CD74, and CD44, and even more preferably the non-muscle cells secrete extracellular matrix proteins, particularly collagens, and / or express integrins to facilitate cell-matrix interactions, and the non-muscle cells condense the reconstitution mixture. 27. The method of any one of the preceding aspects, wherein the non-muscle cells are derived from embryonic stem cells, and the cells have not been produced using a process involving modification of the genetic identity of human germline or using a process involving the use of human embryos for industrial or commercial purposes. 28. The method of any one of the preceding aspects, wherein the non-muscle cells are derived from induced pluripotent stem cells, parthenogenetic stem cells, programmed somatic cells, adult stem cells, or mesenchymal stem cells, preferably, the non-muscle cells are derived from induced pluripotent stem cells. 29. The method of any one of the preceding aspects, wherein the non-muscle cells are obtained from a human subject, preferably obtained from a patient, and even more preferably derived from a biopsy obtained from a patient for autologous or allogeneic administration of MEHM. 30. The non-muscle cells are obtained by serum-free differentiation, preferably the non-muscle cells are derived from the cells described in EP20188364.2; Witty AD, Mihic A, Tam RY, et al., Generation of the epicardial lineage from human pluripotent stem cells, Nat Biotechnol. 2014;32(10):1026-1035; Iyer D, et al., Robust derivation of epicardium and its differentiated smooth muscle cell progeny from human pluripotent stem cells. Development, 2015 Apr 15;142(8):1528-41; Bao X, et al., Long-term self-renewing human epicardial cells generated from pluripotent stem cells under defined xeno-free conditions, Nat Biomed Eng. 2016;1; or Bao X, et al., Directed differentiation and long-term maintenance of epicardial cells derived from human pluripotent stem cells. 2017 Sep;12(9):1890-1900. 31. The method of any one of the previous aspects, wherein the non-muscle cells of the cell mixture of step (i) are stromal cells, preferably, the stromal cells are human stromal cells. 32. The method of embodiment 31, wherein the cell mixture of step (i) comprises at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, and even more preferably at least 50% stromal cells. 33. The method of any one of the preceding aspects, wherein non-muscle cells, preferably stromal cells, are capable of condensing the reconstitution mixture of step (ii). 34. The method of any one of the preceding embodiments, wherein the reconstituted medium comprises (a) a basal medium and (b) a serum-free supplement. 35. The method of embodiment 34, wherein the serum-free supplement provides a final concentration of 0.5-50 mg / ml albumin, 1-100 μg / ml transferrin, 0.1-10 μg / ml ethanolamine, 0.003-0.3 μg / ml sodium selenite, 0.4-40 μg / ml L-carnitine HCl, 0.1-10 μg / ml hydrocortisone, 0.05-5 μl / ml fatty acid supplement, and 0.0001-0.1 μg / ml triiodo-L-thyronine (T3). 36. The method of embodiment 34 or 35, wherein the serum-free supplement further comprises one or more ingredients selected from the group consisting of vitamin A, D-galactose, linoleic acid, linolenic acid, progesterone, and putrescine. 37. The method of any one of aspects 34-36, wherein the serum-free supplement is a B27 supplement minus insulin. 38. The method of any one of aspects 34 to 37, wherein the serum-free supplement is 2-6% (v / v) B27 supplement minus insulin, preferably, the serum-free supplement is 4% (v / v) B27 supplement minus insulin. 39. The method of any one of aspects 34 to 38, wherein the basal medium in step (i) is selected from Iscove's medium, RPMI, αMEM, and DMEM, or a mixture thereof; preferably, the basal medium is selected from Iscove's medium, RPMI, αMEM, or a mixture thereof; more preferably, the basal medium is a mixture of Iscove's medium and RPMI. 40. The reconstitution medium further contains (c) 35 to 790 uM ascorbic acid, (d) 5 to 500 ng / ml IGF-1, (e) 0.3 to 26 ng / ml VEGF, (f) 0.5 to 53 ng / ml FGF-2, and (g) 0.5 to 10 ng / ml TGFβ1; Preferably, the reconstitution medium further contains (c) 75 to 300 uM ascorbic acid, (d) 26 to 105 ng / ml IGF-1, (e) 1.3 to 5.2 ng / ml VEGF, (f) 2.6 to 10.5 ng / ml FGF-2, and (g) 1 to 6 ng / ml TGFβ1; More preferably, the method of any one of aspects 34 to 39, wherein the reconstitution medium further comprises (c) about 158 ​​uM ascorbic acid, (d) about 53 ng / ml IGF-1, (e) about 2.6 ng / ml VEGF, (f) about 5.3 ng / ml FGF-2, and about 3 ng / ml TGFβ1. 41. VEGF is VEGF 165 41. The method of embodiment 40, wherein 42. The method of any one of the previous embodiments, wherein the mold is configured to facilitate the formation of a planar-shaped EHM, a pouch-shaped EHM, or a cylindrical-shaped EHM. 43. The method of embodiment 42, wherein the mold is configured to facilitate the formation of a planar EHM, preferably the mold is in the shape of a disk, preferably the disk is in the shape of a circular or polygonal disk, more preferably the mold is in the shape of a hexagonal disk, and most preferably the disk has a patch-like shape; or the mold forms a confined three-dimensional space, preferably the three-dimensional space is in the shape of a circular or polygonal three-dimensional space, more preferably the mold is in the shape of a hexagonal three-dimensional space, and even more preferably the three-dimensional space has a patch-like shape. 44. The method of embodiment 42, wherein the mold is configured to facilitate the formation of a pouch-shaped EHM, preferably the mold is in the shape of a pouch, more preferably the mold is in the shape of a spherical or ellipsoidal pouch, even more preferably the mold is formed by spherical or ellipsoidal inner and outer walls, more preferably the spherical or ellipsoidal inner wall is inflatable. 45. The method of embodiment 42, wherein a mold is formed to facilitate the formation of a cylindrical EHM, preferably the mold is cylindrical in shape, more preferably the mold is a cylindrical mold, more preferably the cylindrical mold is formed by cylindrical inner and outer walls, or the cylindrical mold is formed by a single cylindrical wall, and the cylindrical mold is covered with the reconstituted mixture by centrifugation. 46. ​​The method of any one of the preceding embodiments, wherein the reconstituted mixture is perforated by at least two poles, and the poles are flexible so that a mechanical load can be introduced onto the MEHM. 47. The method of any one of the preceding aspects, wherein the pole allows for perfusion of the culture medium of steps (ii), (iii), and optionally the maturation medium and oxygen of step (iv) into the MEHM during steps (ii), (iii), and optionally (iv), and preferably the perfusion of culture medium and the perfusion of maturation medium leads to nutrient support for cardiomyocytes and non-myocytes during steps (ii), (iii), and / or optionally (iv). 48. The method of any one of the preceding embodiments, wherein at least two poles perforate the reconstituted mixture from the bottom or the top, preferably through its entire thickness. 49. The method of embodiment 48, wherein at least two poles perforate the reconstituted mixture from below, and wherein the mold is disk-shaped, preferably wherein at least two poles pass through the reconstituted mixture from the bottom of the mold, more preferably wherein the poles are permanently attached to the mold or base plate, more preferably wherein the poles are permanently attached to the casting area of ​​the mold or a removable base plate of the mold, and even more preferably wherein (a) the mold and at least two poles or (b) the removable base plate and at least two poles are 3D printed as a single entity. 50. The method of embodiment 48, wherein at least two poles perforate the reconstituted mixture from above, the mold is disk-shaped, and preferably, the at least two poles are inserted into the liquid reconstituted mixture from above. 51. The method of embodiment 48, wherein at least two poles perforate the reconstituted mixture from below, and wherein the mold is in the shape of a pouch, preferably wherein the pouch is formed by inner and outer walls of a sphere or ellipsoid, more preferably wherein the at least two poles extend from the inner wall of the sphere or ellipsoid, more preferably wherein the poles are permanently attached to the inner wall of the sphere or ellipsoid. 52. The method of embodiment 48, wherein at least two poles perforate the reconstituted mixture from the top, and wherein the mold is in the shape of a pouch, preferably wherein the pouch is formed by inner and outer spherical or ellipsoidal walls, more preferably wherein at least two poles extend from the outer spherical or ellipsoidal wall, and more preferably wherein the poles are permanently attached to the outer spherical or ellipsoidal wall. 53. The method of embodiment 48, wherein at least two poles perforate the reconstituted mixture from below, the mold is a cylindrical mold, the cylindrical mold being formed by a cylindrical inner wall and an outer wall, preferably, the at least two poles pass through the reconstituted mixture from the cylindrical outer wall, more preferably, the poles are permanently attached to the cylindrical outer wall, and even more preferably, the cylindrical outer wall with the at least two poles is 3D printed as a single object. 54. The method of embodiment 48, wherein at least two poles perforate the reconstituted mixture from the top, the mold is a cylindrical mold, the cylindrical mold is formed by a cylindrical inner wall and an outer wall, preferably the at least two poles pass through the reconstituted mixture from the cylindrical inner wall, more preferably the poles are permanently attached to the cylindrical inner wall, and even more preferably the cylindrical inner wall with the at least two poles is 3D printed as a single piece. 55. The method of any one of the preceding aspects, wherein each reconstituted mixture has a volume of 0.5 to 200 ml, preferably 1 to 150 ml, more preferably 1.5 to 100 ml, more preferably 2 to 50 ml, more preferably 2.5 to 40 ml, more preferably 3 to 30 ml, more preferably 3.5 to 25 ml, more preferably 4 to 20 ml, more preferably 4.5 to 15 ml, more preferably 5 to 11.5 ml, more preferably 6 to 9.5 ml, even more preferably 6.5 to 8.5, and most preferably about 8 ml. 56. The method of any one of the previous aspects, wherein the mold holds a volume of 1 to 400 ml, preferably 2 to 300 ml, more preferably 3 to 200 ml, more preferably 4 to 100 ml, more preferably 5 to 80 ml, more preferably 6 to 60 ml, more preferably 6.5 to 50 ml, more preferably 7 to 40 ml, more preferably 7.5 to 30 ml, more preferably 8 to 23 ml, more preferably 13 to 19 ml, even more preferably 13 to 17 ml, even more preferably about 15 ml, and most preferably, the mold is used to form an EHM that is in the shape of a patch, pouch, or cylinder. 57. Mold is 1-400cm 2 , preferably 2 to 300 cm 2 , more preferably 3 to 200 cm 2 , more preferably 4 to 100 cm 2 , more preferably 5 to 80 cm 2 , more preferably 6 to 60 cm 2 , more preferably 7 to 50 cm 2 , more preferably 8 to 40 cm 2 , more preferably 9 to 30 cm 2 , more preferably 10 to 23 cm 2 , more preferably 12 to 19 cm 2 , and even more preferably 13 to 17 cm 2 , and even more preferably about 16 cm 2 10. The method of any one of the preceding embodiments, wherein a mold is used to form a patch-shaped EHM, most preferably having a size of 10 mm. 58. The method of any one of the preceding embodiments, wherein the reconstituted mixture is perforated by at least three poles. 59. The method of any one of the preceding aspects, wherein the poles are arranged in a lattice, preferably the poles are arranged in a triangular or rectangular lattice, preferably a triangular lattice. 60. The method of any one of the preceding embodiments, wherein the reconstituted mixture is perforated by at least at least 5 poles, more preferably at least 7 poles, more preferably at least 14 poles, even more preferably at least 20 poles, even more preferably at least 30 poles, even more preferably at least 37 poles, most preferably at least 52 poles, and most preferably at least 61 poles. 61. The method of any one of the preceding aspects, wherein each pole has a base region, the base being circular, rectangular, oval, triangular, or polygonal, preferably the base region being circular. 62. The method of any one of the preceding aspects, wherein the poles have similar or different elastic properties. 63. The method of any one of the preceding aspects, wherein the elastic properties of the pole are defined by a working spring constant at the point where the MEHM is in contact with the pole, preferably having a restoring force of 0.5 to 50 mN / mm, more preferably 0.7 to 40 mN / mm, even more preferably 0.9 to 30 mN / mm, even more preferably 1 to 20 mN / mm, even more preferably 2 to 10 mN / mm, even more preferably 3 to 5 mN / mm, and most preferably about 4 mN / mm. 64. A method according to any one of the preceding aspects, wherein the elastic properties of the pole mimic the wall tension in a human heart, preferably the wall tension is between 1 and 20 kPa. 65. The method of any one of the preceding aspects, wherein the elastic properties of the pole exhibit varying elastic moduli to establish biomechanical anisotropy. 66. A method according to any one of aspects 1 to 64, wherein the elastic properties of the poles exhibit similar elastic moduli so as to establish biomechanical isotropy. 67. The method of any one of the preceding aspects, wherein the pole is surrounded by a perimeter wall at a lower portion of the pole, the perimeter wall forming a ring-shaped ramp around the pole, the ring-shaped ramp tapering upward from the bottom. 68. The method of any one of the preceding aspects, wherein the pole tapers conically, the pole having a maximum diameter at a lower portion. 69. The method of any one of the preceding embodiments, wherein the pole has a diameter of 0.5 to 3 mm, preferably 0.6 to 2.5 mm, more preferably 0.65 to 2 mm, even more preferably 0.7 to 1.7 mm, and even more preferably about 0.8 to 1.5 mm. 70. The method of embodiment 69, wherein the reconstituted mixture is perforated by at least seven poles, the poles arranged in a lattice shape, and the diameters of the poles form a gradient along one planar axis, as particularly illustrated in FIG. 7C, and preferably the FAC measurement value along the gradient is greater than the FAC measurement value along poles having equal pole thickness. 71. The method of any one of the preceding aspects, wherein steps (i), (ii), and / or optionally (iv) are carried out at a temperature in the range of 36.4 to 37.6°C, preferably in the range of 36.6 to 37.4°C, preferably in the range of 36.8 to 37.2°C, more preferably at about 37°C. 72. The method of any one of the preceding embodiments, wherein steps (i), (ii), and / or optionally (iv) are carried out in a humidified cell culture incubator in the presence of 2-10% CO2, preferably 2.5-8% CO2, more preferably 3-7% CO2, even more preferably 3.5-6.5% CO2, even more preferably 4-6% CO2, and most preferably about 5% CO2. 73. The method of any one of the preceding aspects, wherein steps (i), (ii), and / or optionally (iv) are carried out in a humidified cell culture incubator in the presence of 5-40% O2, preferably 10-30% O2, more preferably 15-35% O2, even more preferably 17-33% O2, even more preferably 19-25% O2, and most preferably about 21% O2. 74. The method of any one of the previous aspects, wherein the gelation in step (i) is characterized in that the reconstituted mixture obtained by step (i) is gel-like. 75. The method of any one of the preceding aspects, wherein the gelation in step (i) surrounds the reconstituted mixture when the culture medium is added to the mold and does not dissolve the reconstituted mixture. 76. The method of any one of the preceding aspects, wherein the gelation in step (i) is characterized by the reconstitution mixture being opaque, preferably as confirmed by visual inspection as shown in Figure 3B of Tiburcy M, Meyer T, Soong PL, Zimmermann WH. Collagen-based engineered heart muscle. Methods Mol Biol. 2014;1181:167-76. 77. The method of any one of the preceding embodiments, wherein step (i) is carried out for at least 15 minutes, preferably at most 24 hours, more preferably between 20 minutes and 15 hours, more preferably between 30 minutes and 8 hours, even more preferably between 45 minutes and 1.5 hours, and most preferably about 1 hour. 78. The method of any one of the preceding embodiments, wherein the culture medium in step (ii) comprises (a) a basal medium, (b) a serum-free supplement, (c) L-glutamine, (d) ascorbic acid, (e) IGF-1, (f) VEGF, and (g) TGFβ1. 79. The method of embodiment 78, wherein (a) the basal medium is selected from Iscove's medium, αMEM, DMEM, and RPMI, preferably, the basal medium is Iscove's medium or αMEM, more preferably, the basal medium is Iscove's medium. 80. The method of embodiment 78 or 79, wherein (b) the serum-free supplement is defined in any one of embodiments 35 to 38. 81. The method of any one of aspects 78 to 80, wherein the culture medium in step (ii) comprises 0.4 to 10 mM L-glutamine, preferably 0.8 to 6 mM L-glutamine, more preferably 1.2 to 5 mM L-glutamine, more preferably 1.5 to 4 mM L-glutamine, more preferably 1.7 to 3 mM L-glutamine, and most preferably about 2 mM L-glutamine. 82. The method of any one of aspects 78 to 81, wherein the culture medium in step (ii) comprises 30 to 3000 μM ascorbic acid or a derivative thereof, preferably 100 to 1000 μM ascorbic acid or a derivative thereof, more preferably 180 to 500 μM ascorbic acid or a derivative thereof, more preferably 220 to 370 μM ascorbic acid or a derivative thereof, more preferably 270 to 330 μM ascorbic acid or a derivative thereof, and most preferably about 300 μM ascorbic acid or a derivative thereof. 83. The method of embodiment 82, wherein the derivative of ascorbic acid is ascorbic acid-2-phosphate. 84. The method of any one of aspects 78 to 83, wherein the culture medium in step (ii) comprises 10 to 1000 ng / ml IGF1, preferably 50 to 500 ng / ml IGF1, more preferably 70 to 200 ng / ml IGF1, even more preferably 90 to 120 ng / ml IGF1, and most preferably about 100 ng / ml IGF1. 85. The method of embodiment 84, wherein the IGF1 is human IGF1. 86. The method of any one of aspects 78 to 85, wherein the culture medium in step (ii) comprises 2.5 to 10 ng / ml VEGF, preferably 3 to 9 ng / ml VEGF, more preferably 3.5 to 8 ng / ml VEGF, more preferably 4 to 7 ng / ml VEGF, more preferably 4.5 to 6 ng / ml VEGF, and most preferably about 5 ng / ml VEGF. 87. The VEGF is human VEGF, preferably VEGF 165 87. The method of embodiment 86, wherein 88. The method of any one of aspects 78 to 87, wherein the culture medium in step (ii) comprises 5 to 20 ng / ml FGF, preferably 6 to 18 ng / ml FGF, more preferably 7 to 16 ng / ml FGF, more preferably 8 to 14 ng / ml, more preferably 9 to 12 ng / ml FGF, and most preferably about 10 ng / ml FGF. 89. The method of any one of aspects 78 to 88, wherein the FGF-2 is human FGF-2. 90. The method of any one of aspects 78 to 89, wherein the culture medium in step (ii) comprises 2 to 8 ng / ml TGFβ1, preferably 3 to 7 ng / ml TGFβ1, more preferably 4 to 6 ng / ml TGFβ1, even more preferably 4.5 to 5.5 ng / ml TGFβ1, and even more preferably about 5 ng / ml TGFβ1, and most preferably, the TGFβ1 is human TGFβ1. 91. The method of any one of aspects 78-90, wherein the culture medium in step (ii) comprises about 750 mg / L glycine, about 890 mg / L L-alanine, about 1320 mg / L L-asparagine, about 1330 mg / L L-aspartic acid, about 1470 mg / L L-glutamic acid, about 1150 mg / L L-proline, and about 1050 mg / L L-serine. 92. The method of any one of the preceding embodiments, wherein the reconstituted mixture of step (ii) is condensed by at least about 20% of the initial reconstituted mixture volume, preferably at least about 25%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, and even more preferably at least about 90%, as determined by visual inspection or video optical analysis of the EHM volume. 93. The method of any one of the preceding embodiments, wherein completion of step (ii) is confirmed by fabricating a loop-format engineered heart muscle (EHM) in parallel with the MEHM by following steps (i) and (ii), and confirming release of the loop-format engineered heart muscle (EHM) from the mold after completion of step (ii), as described in Figure 3C of Tiburcy M, Meyer T, Soong PL, Zimmermann WH. Collagen-based engineered heart muscle. Methods Mol Biol. 2014;1181:167-176. 94. The method of any one of the preceding aspects, wherein step (ii) is carried out for at least 12 hours, preferably at most 15 days, more preferably between 12 hours and 7 days, preferably between 14 hours and 6 days, more preferably between 16 hours and 5.5 days, more preferably between 18 hours and 5 days, more preferably between 20 hours and 4.5 days, even more preferably between 22 hours and 4 days, even more preferably between 23 hours and 3.5 days, and most preferably between 24 hours and 3 days. 95. The method of any one of the previous embodiments, wherein in step (iii)a), one additional reconstituted mixture is added on top of the concentrated reconstituted mixture of step (ii). 96. The method of any one of aspects 1-94, wherein in step (iii)a), one additional reconstituted mixture is added from below the condensed reconstituted mixture of step (ii). 97. The method of any one of aspects 1 to 94, wherein in step (iii)a), one additional reconstituted mixture is added simultaneously from above and below to coat the condensed reconstituted mixture of step (ii). 98. The method of any one of the preceding aspects, wherein in step (iii)a), the further reconstituted mixture coats the condensed reconstituted mixture of step (ii) from above and / or below, and preferably the surface areas of the condensed reconstituted mixture of step (ii) and the further reconstituted mixture of step (iii)a) are the same. 99. The method of any one of aspects 1 to 97, wherein in step (iii)a), the further reconstituted mixture coats the limited area of ​​the condensed reconstituted mixture of step (ii) from above or below, and preferably the surface area of ​​the further reconstituted mixture of step (iii)a) is smaller than the surface area of ​​the reconstituted mixture of step (ii). 100. The method of any one of aspects 1 to 97, wherein in step (iii)b), the different mold has a larger casting area than the mold of step (i), and preferably, in step (iii)b), the further reconstituted mixture is coated at least from the top or bottom onto the condensed reconstituted mixture of step (ii), more preferably, in step (iii)b), the further reconstituted mixture is coated from the top or bottom onto the condensed reconstituted mixture of step (ii), filling the larger casting area of ​​the different mold, and more preferably, the surface area of ​​the further reconstituted mixture of step (iii)b) is larger than the surface area of ​​the condensed reconstituted mixture of step (ii). 101. The method of any one of the previous embodiments, wherein step (iii)a) or step (iii)b) is repeated at least 2 times, preferably at least 3 times, more preferably at least 4 times, more preferably at least 5 times, more preferably at least 6 times, more preferably at least 7 times, more preferably at least 8 times, even more preferably at least 9 times, and most preferably at least 10 times. 102. The method of any one of the preceding aspects, wherein step (iii)a) or step (iii)b) is repeated 2 to 200 times, preferably 2 to 100 times, more preferably 2 to 80 times, more preferably 2 to 70 times, more preferably 2 to 60 times, more preferably 2 to 50 times, more preferably 2 to 40 times, more preferably 3 to 30 times, more preferably 3 to 25 times, more preferably 4 to 20 times, more preferably 4 to 15 times, more preferably 4 to 10 times, even more preferably 5 to 9 times, and most preferably 5 to 8 times. 103. A method according to any one of the preceding aspects, wherein the maturation medium in step (iv) is defined as the culture medium according to any one of aspects 60 to 71 and 73, except that TGFβ1 is omitted from the maturation medium compared to the culture medium. 104. The method of any one of the preceding aspects, wherein step (iv) is carried out for 4 to 200 days, preferably 6 to 150 days, more preferably 8 to 120 days, more preferably 9 to 110 days, more preferably 10 to 90 days, more preferably 15 to 70 days, more preferably 20 to 50 days, and most preferably 28 to 42 days. 105. The method of any one of the preceding aspects, wherein in step (iv) the MEHMs of step (iii) are cultured in a suitable maturation medium, and the MEHMs of step (iv) exhibit increased force production compared to the MEHMs of step (iii), and the increased force production is confirmed by measuring fractional area change (FAC) using the method described in Tiburcy et al., Circulation 135(19)1832-1847 (2017), and the fractional area change (FAC) of the MEHMs of step (iv) is at least 0.5%, preferably at least 1%, more preferably at least 1.2%, more preferably at least 1%, more preferably at least 2%, more preferably at least 3%, and even more preferably at least 5% upon electrical stimulation. 106. The method of any one of the preceding embodiments, wherein cardiomyocytes within the MEHM are well oxygenated, preferably as determined by the ODD-Luc hypoxia reporter as described in Hesse AR, Levent E, Zieseniss A, Tiburcy M, Zimmermann WH, Katschinski DM: Lights on for HIF-1α: Genetically Enhanced Mouse Cardiomyocytes for Heart Tissue Imaging. Cell Physiol Biochem 2014;34:455-462. 107. The method of any one of the preceding aspects, wherein the cardiomyocytes in the MEHM are adequately oxygenated, and the oxygen supply is confirmed by the ODD-Luc hypoxia reporter as described in Hesse AR et al. (2014), and preferably sufficient oxygen supply is ensured when the average relative luminescence of the cardiomyocytes of the MEHM is at most 6-fold higher, more preferably at most 5-fold, more preferably at most 4-fold, more preferably at most 3-fold, more preferably at most 2.5-fold, even more preferably at most 2-fold, and even more preferably at most 1.5-fold higher than the average relative luminescence of the cardiomyocytes of the monolayer EHM obtained by following steps (i), (ii), and optionally step (iv) of the method. 108. The method of any one of the preceding aspects, wherein the cardiomyocytes in the MEHM are sufficiently oxygenated, the oxygen supply being confirmed by the ODD-Luc hypoxia reporter as described in Hesse AR et al. (2014), and the average relative luminescence of the cardiomyocytes in the MEHM is not greater than the average relative luminescence measured in the cardiomyocytes in the monolayer EHM. 109. A multi-layer artificial myocardium (MEHM) obtained by the method according to any one of aspects 1 to 108. 110. A multi-layer artificial myocardium (MEHM) obtainable by a method according to any one of aspects 1 to 108. 111. A multilayered artificial myocardium (MEHM), preferably an artificial heart patch, an artificial heart pouch, or an artificial heart cylinder, comprising at least two layers, comprising (a) collagen and (b) a cellular mixture of cardiomyocytes and non-myocytes. 112. The MEHM of embodiment 111, having a thickness of at least 0.2 mm. 113. The MEHM of embodiment 111 or 112, which is made by a repetitive and sequential layering process, resulting in 2 to 200 layers, and wherein the layers are integral with one another, thereby increasing the thickness of the MEHM. 114. Each layer originates from a reconstituted mixture; 114. The MEHM of any one of embodiments 111-113, wherein each reconstitution mixture comprises (a) collagen, (b) a cell mixture of cardiomyocytes and non-myocytes, and (c) an appropriate reconstitution medium, wherein the MEHM of any one of embodiments 111-113 is capable of contracting as measured by determining the fractional area change (FAC) of the MEHM using the method described in Tiburcy et al., Circulation 135(19)1832-1847 (2017). 115. The MEHM of any one of embodiments 111 to 114, which yields 2 to 100 layers, preferably 2 to 80 layers, more preferably 2 to 70 layers, more preferably 2 to 60 layers, more preferably 2 to 50 layers, more preferably 2 to 40 layers, more preferably 3 to 30 layers, more preferably 3 to 25 layers, and even more preferably 4 to 20 layers. 116. The MEHM of any one of aspects 111 to 115, wherein the FAC is at least 0.5% upon electrical stimulation, preferably, the FAC is at least 0.7%, more preferably at least 1%, more preferably at least 2%, more preferably at least 3%, and even more preferably at least 5%. 117. The MEHM of any one of aspects 111 to 116, having a thickness of 0.2 mm to about 30 mm, preferably about 0.3 mm and up to about 30 mm, more preferably about 0.5 mm to about 30 mm, more preferably about 0.7 mm to about 25 mm, more preferably about 0.9 mm to about 20 mm, more preferably about 1 mm to about 17 mm, more preferably about 2.3 mm to about 15 mm, more preferably about 2.8 mm to about 14 mm, more preferably about 3.3 mm to about 13 mm, more preferably about 3.8 mm to about 12 mm, more preferably about 4.2 mm to about 11 mm, more preferably about 4.6 mm to about 10.5 mm, and most preferably about 5 mm to about 10 mm. 118. The MEHM according to any one of aspects 111-117, which is not vascularized and / or not under the control of the central nervous system. 119. An MEHM according to any one of aspects 111 to 118, which is perforated by at least two poles with similar diffusion distances throughout the MEHM to ensure the supply of nutrients and oxygen to the cell mixture. 120. The MEHM of any one of embodiments 111 to 119, wherein cardiomyocytes within the MEHM are well-oxygenated, preferably as determined by the ODD-Luc hypoxia reporter as described in Hesse AR, Levent E, Zieseniss A, Tiburcy M, Zimmermann WH, Katschinski DM: Lights on for HIF-1α: Genetically Enhanced Mouse Cardiomyocytes for Heart Tissue Imaging. Cell Physiol Biochem 2014;34:455-462. 121. The MEHM according to any one of aspects 111 to 120, wherein the cardiomyocytes in the MEHM are adequately supplied with oxygen, and the oxygen supply is confirmed by the ODD-Luc hypoxia reporter as described in Hesse AR et al. (2014), and preferably, sufficient oxygen supply is ensured when the average relative luminescence of the cardiomyocytes of the MEHM is at most 6-fold higher, more preferably at most 5-fold higher, more preferably at most 4-fold higher, more preferably at most 3-fold higher, more preferably at most 2.5-fold higher, even more preferably at most 2-fold higher, and even more preferably at most 1.5-fold higher than the average relative luminescence of the cardiomyocytes of a monolayer EHM obtained by following step (i), step (ii), and optionally step (iv) of the method. 122. An MEHM described in any one of aspects 111 to 121, wherein the cardiomyocytes within the MEHM are sufficiently oxygenated, the oxygen supply being confirmed by the ODD-Luc hypoxia reporter as described in Hesse AR et al. (2014), and the average relative luminescence of the cardiomyocytes of the MEHM is not greater than the average relative luminescence measured in the cardiomyocytes of a monolayer EHM. 123. The MEHM of any one of embodiments 111 to 122, obtained by any one of the methods described in any one of embodiments 1 to 95. 124. A multi-layer artificial myocardium (MEHM) obtainable by carrying out steps (i) to (iii) of the method according to any one of aspects 1 to 102. 125. An artificial heart muscle (EHM), preferably an artificial heart patch, artificial heart pouch, or artificial heart cylinder, comprising (a) collagen, (b) a cellular mixture of cardiomyocytes and non-myocytes, and having a thickness of at least about 0.6 mm, preferably at least about, at least 0.61 mm, at least 0.62 mm, at least 0.63 mm, at least 0.64 mm, at least 0.65 mm, at least 0.66 mm, at least 0.67 mm, at least 0.68 mm, at least 0.69 mm, at least 0.7 mm, at least 0.8 mm, or at least 0.9 mm. 126. The EHM of embodiment 125, which is a multi-layer EHM and comprises at least two layers. 127. Use of an MEHM obtained by a method according to any one of embodiments 1 to 108 or an MEHM according to any one of embodiments 109 to 126 in an in vitro model for drug screening. 128. The use according to embodiment 127 in an in vitro model for drug toxicity screening or drug efficacy screening. 129. Use of an MEHM obtained by a method according to any one of aspects 1 to 108 or an MEHM according to any one of aspects 109 to 126 in the in vitro production of an artificial human myocardium, wherein preferably the MEHM has a three-dimensional shape that corresponds to a patient-specific myocardial defect, and more preferably the patient-specific myocardial defect is assessed by MRI, ultrasound, computed tomography, positron emission tomography, and / or optical coherence tomography. 130. Use of an MEHM obtained by a method according to any one of embodiments 1 to 108 or an MEHM according to any one of embodiments 109 to 126 as a research tool. 131. A multilayer EHM (MEHM) obtainable by a method according to any one of embodiments 1 to 108 or an MEHM according to any one of embodiments 109 to 126 for use in medicine. 132. A multilayered EHM (MEHM) obtainable by a method according to any one of aspects 1 to 108 or an MEHM according to any one of aspects 109 to 126, for use in cardiac repair, preferably for treating a patient suffering from heart failure. 133. The MEHM of embodiment 132, which is permanently applied to the patient's heart, preferably sutured to the patient's heart. 134. A method for treating a subject suffering from heart failure, comprising administering to the patient's heart an MEHM obtained by a method described in any one of embodiments 1 to 108 or an MEHM described in any one of embodiments 109 to 126. 135. The method of embodiment 134, comprising attaching the MEHM to the subject's heart, wherein the attaching preferably comprises suturing the MEHM onto the heart.

[0105] Of course, all embodiments disclosed herein can be applied alone or in combination with other embodiments. Furthermore, all embodiments disclosed herein apply to methods of making MEHM, as well as to MEHM and in vitro and in vivo uses of MEHM. [Brief explanation of the drawings]

[0106] [Figure 1]Schematic of the repeated lamination process for obtaining the multilayered artificial myocardium (MEHM) disclosed herein. Generally, the previous layer (condensed reconstitution mixture) and the newly added layer (reconstitution mixture) are integrated. Repeated lamination results in a thicker and stronger artificial myocardium. "Lamination on top" is achieved by adding new reconstitution mixture on top of the condensed reconstitution mixture. "Onion pattern" is achieved by immersing the first and subsequent stacks of integrated layers in newly added reconstitution mixture. "Lamination from bottom" is achieved by adding new reconstitution mixture from below to the condensed reconstitution mixture. This lamination can be repeated indefinitely, taking into account the requirements for oxygen and nutrient diffusion to the embedded cells. The supply of oxygen and nutrients is achieved by immersion in an oxygenated culture medium containing nutrients (especially glucose, lactate, and / or fatty acids) and by increasing the tissue surface area by design, i.e., by creating channels via perforated poles (not shown in the schematic; see Figures 2 and 3). The initial layer is indicated by a dotted line. The later layers are shown in black. [Figure 2] Scalability of Artificial Myocardium. Photographs show representative artificial myocardium formats we have used for disease modeling, drug screening, and cardiac repair. The volumes shown represent the initial volume of each artificial myocardium reconstitution mixture. In a layer-by-layer approach, to create the MEHMs disclosed herein, each reconstitution mixture can have these exemplary volumes. Mechanical loading is achieved by incorporating flexible poles into each tissue preparation. The shape and scale can be further adapted as needed. After the self-assembly process of a single-layer EHM is complete, the thickness (z-dimension) of the preparation shown is typically 0.5±0.1 mm. Repeated layering thickens and strengthens the MEHM as disclosed herein. Scale bar: 1 cm. [Figure 3]Scaling of EHM thickness through sequential / iterative layering and sufficient supply of oxygen and nutrients. A: A custom-designed casting mold with a stretching device (composed of 3D-printed flexible poles fixed at specified intervals to a rigid base plate) enables perforated EHM design and aims to facilitate tensile contraction of the developing EHM or MEHM against a specified resistance. B: Schematic of the perforated laminated patch design. C: Top and side views of a five-layer (left) and single-layer (right) EHM. The five-layer EHM is approximately 5-6 mm thick, while the single-layer EHM is approximately 1 mm thick. D: By utilizing the human ODD-Luc hypoxia reporter iPSC line to construct the middle layer of the five-layer construct (both the five-layer and monolayer EHM contain equal amounts of ODD-Luc cardiomyocytes; see Hesse et al. 2014 for the ODD-Lux hypoxia method), there was no evidence of elevated hypoxia in the significantly thicker five-layer design. The grayscale on the right indicates low and high ODD-Luc activity as evidence of hypoxia detection. The grayscale indicates the luminescence of the reporter cell line. The ODD-Luc signal intensity of the five-layer EHM (left) was lower compared to the background ODD-Luc signal of a single patch (right). In other words, the luminescence signal intensity of the monolayer EHM appears to be at least the same as that of the multilayer EHM, if not brighter. Thus, the five-layer EHM is not compromised by hypoxia. [Figure 4] Multiwell plate format for automated casting of loop format. A: Custom-made stretcher (with flexible poles fixed on the base plate) intended to support tonic contractions of loop format artificial cardiac myocardium, showing representative dimensions of pole dimensions and placement. B: Multiwell plate format for automated casting, parallel culture, and video-optical analysis of 48 loop format artificial cardiac myocardium, with a close-up of one well showing the casting cavity and flexible poles for maturation under defined mechanical loads (for further details, see Tiburcy et al. 2020). [Figure 5] EHM formation in loop format. A: Automated casting of EHM in a multiwell plate. B: One hour after casting, the EHM is in a non-liquid state (collagen gelation results in the opaque appearance of the EHM reconstitution mixture). C: 24 hours after casting, significant EHM compaction and suspension on a flexible pole for mechanical loading. D: EHM after complete compaction on day 18 of culture. The EHM is fully suspended on a flexible pole to impose bidirectional mechanical load and support tensile contraction under preload conditions. Scale bar: 5 mm. [Figure 6] A: Video-optical analysis of EHM contractions with or without stimulation. A: Screenshot of a custom-made video-optical analysis tool for functionally phenotyping EHMs by fractional area change (FAC) measurements. B: Device for electrical stimulation of the EHM to induce contractions at a defined rate. The electrical field stimulation intensity is increased (typically up to 2 V / cm) until the EHM is induced to contract at a defined beating rate. (C) Alternatively, local point stimulation can be applied with electrodes in contact with the EHM to induce synchronized EHM contractions for FAC analysis. [Figure 7]Multilayer EHM casting mold in patch format. A: A custom-designed casting mold with two recesses designed to hold the base plate and poles (bottom) and culture medium (top). The left recess with the flexible poles shows the condensed reconstitution mixture for illustrative purposes. Layers are stacked on top of, inserted below, or formulated around the first layer. B: Cross-section of a recess within the casting mold. Vertical perforated poles (gray and white) are shown, with the stacked EHM shown in black. C: Examples of pole designs with and without pole thickness gradients; note that the pole design (pole elastic properties, diameter, or shape) can control the uniform (no gradient; in this example, all poles have a diameter of 0.9 mm) and non-uniform (example of top-to-bottom gradient; pole diameters range from 1.1 mm to 0.7 mm in 0.1 mm increments) mechanical loads imposed on the EHM. D: Examples of EHMs producing isotropic (no gradient; pole diameters are the same) or anisotropic (vertical gradient; pole diameter gradient) contraction patterns. Contraction patterns were analyzed by comparing the FAC of the upper and lower parts of the EHM. When all pole diameters were the same, the upper and lower parts of the EHM exhibited similar contractions of approximately 1% FAC. In contrast, a vertical gradient increased the contractile force of the upper part to approximately 1.2% FAC (larger pole diameter) and decreased the contractile force of the lower part to approximately 0.7% FAC (smaller pole diameter). [Figure 8A]Figure 8: Multilayered artificial myocardium (MEHM) with configurable shapes. Examples of multilayered artificial myocardium (MEHM) designs. MEHMs can be produced in pouch (A), cylinder (B), and patch formats (C) with configurable thickness and xy dimensions (exemplified in patch format in C). In the MEHM pouch configuration (A), the MEHM is cast into a circular or spherical mold with a central element (either rigid or flexible due to the biomechanical load of the condensing EHM; similar to those described in Zimmermann WH, Yildirim Y, Eschenhagen T: Pouch-like engineered heart muscle tissue. WO2008058917 and Yildirim et al. (2007) Circulation). In the illustrated example, the pole-to-pole distance is 3.5 mm and the pole diameter is 1.5 mm, similar to the preferred embodiment described for the EHM patch (Figure 7). The size, shape, and interpole distance of the poles can be tailored as needed to create optimal loading and perforations for oxygen and nutrient delivery. In the case of a cylindrical shape (B), the poles can be fabricated to face inward or outward throughout the cylinder, preferably with the poles facing outward. Layering can be assisted by centrifugal forces (>2 g; "spin coating") induced by controlled rotation. (C) A heterogeneous ("patch-in-patch") multilayer strategy is demonstrated by placing a perforated patch assembly with perforated pole elements into a new mold to accommodate further layering (C1) or by using inserts to separate defined casting volumes within a larger casting mold design (C2). This allows for the freedom to design MEHMs with tunable thickness and xy dimensions to facilitate fabrication of patches tailored / individualized to the patient's heart. [Figure 8B] See legend to Figure 8A. [Figure 8C-1] See legend to Figure 8A. [Figure 8C-2] See legend to Figure 8A. [Example]

[0107] The following examples are intended to further illustrate, but not limit, the present invention. The examples illustrate the technical features, and the present invention also relates to combinations of the technical features presented in this section.

[0108] Example 1 Scalable production of engineered myocardium (EHM) in loop and patch formats As previously described by the inventors in Tiburcy et al. (2017) and Tiburcy et al. (2020), artificial human myocardium can be produced in loop format and as a patch. In particular, artificial cardiac patches are of paramount importance for in vivo cardiac repair applications. Figure 2 shows an overview of the various sizes and formats that have been previously produced and published (Tiburcy et al. (2017) and Tiburcy et al. (2020)).

[0109] Example 2 Scaling of EHM thickness by sequential and repetitive layering assisted by perforated poles that ensure mechanical loading and sufficient supply of oxygen and nutrients To generate cardiac muscle for treating heart failure patients, artificial cardiac muscle ideally needs to support the force of a beating human heart. The patient's cardiac wall thickness is typically 5-10 mm. Therefore, the implant ideally reaches this thickness and can be geometrically tailored to meet the specific patient's needs for mechanical support of the underperforming cardiac wall.

[0110] To fabricate thick (and reinforced) multilayered EHMs, we developed an iterative and sequential lamination method. Specifically, Figure 1 illustrates three different approaches to iterative and sequential lamination for thick artificial cardiac patches (note that perforated poles have been omitted for simplicity). In principle, the initial reconstitution mixture is compacted to a defined thickness and then coated with additional reconstitution mixture. Coating can be performed from the top, bottom, or both sides, as shown schematically in Figure 1. This process is repeated until the desired tissue thickness is achieved. Depending on the design of the casting mold, any shape and desired x-y dimensions can be created (individualized patch design). A prerequisite for the coating / lamination process is that the first and subsequent layers form a compact tissue. This compact tissue formation is achieved by iterative and sequential lamination of the individual reconstitution mixtures so that they merge with each other. Tissue compaction correlates with the type and content of stromal cells (Tiburcy et al. (2017); Schlick et al. (2019)). The type and content of stromal cells are selected so that the initial reconstitution mixture achieves 20% or greater compaction (Figures 5B-C exemplarily show EHM compaction in a loop format). This provides the structure and physical space for coating the condensing EHM with additional layers from the top and / or bottom, or with double-sided coating layers. We typically add a new layer 24 hours after casting the previous layer. These times can be shortened or extended as desired and verified experimentally. The desired tissue compaction process depends on the type and density of stromal cells, the overall cell composition, and the composition of the hydrogel and culture medium. The addition of an effective concentration of TGFb1 (e.g., as utilized for EHM formation in Tiburcy et al. (2017) and WO2017 / 207431) can accelerate the process. Additional reconstitution mixture may be added to the mold to coat the compacted multi-layered artificial myocardium.Using this method, the EHM can be thickened layer by layer, resulting in a multilayered EHM that can reach or even exceed the clinically desirable thickness of 5–10 mm. Because the human cell wall is 5–10 mm thick, cardiac patches with such thickness are expected to maintain sufficient mechanical stability to support heart wall function. Furthermore, the contractility of cardiomyocytes embedded within the EHM is expected to support native heart contractions.

[0111] Figure 3A illustrates a casting mold with a stretching device (composed of 3D-printed flexible poles fixed at specified intervals to a rigid, detachable base plate) that allows for perforated EHM design and facilitates tensile contraction of the developing patch against a defined resistance. Of course, the size of the casting mold and the number and size of the poles can be adapted. Figure 3B shows a schematic overview of the multilayer EHM disclosed herein. Figure 3C shows the fabricated five-layer EHM and a single-layer EHM next to each other. The bottom panel of Figure 3C provides a detailed comparison of the EHM thickness, demonstrating that the five-layer EHM is approximately 5–6 mm thick. Using the introduced perforated stack design, there is a constant diffusion distance as a result of the channels created by the perforated poles, and there is no constraint on the overall tissue thickness. Of course, the pole distance and size can be adapted to increase the number of channels and change the channel size and inter-channel distance.

[0112] A major challenge in attempting to achieve such thick, multilayered artificial myocardium (MEHM) is ensuring adequate oxygen and nutrient supply to cells embedded within the artificial myocardium. By using a perforated EHM design, we ensure sufficient oxygen and nutrient supply in any multilayer design. This is exemplified by our studies using the human ODD-Luc hypoxia reporter model (developed by us based on our previous experience with mouse models; Hesse et al. (2014)). Cardiomyocytes derived from the ODD-Luc human pluripotent stem cell line in monolayers and pentads (5–6 mm thick) showed no difference in ODD-Luc signal intensity above background (as reported in monolayered EHM). The channel diameter and density in the demonstrated EHM design were defined by the pole diameter (1.5 mm) and the circumferential distance between the poles (3.5 mm). Under these conditions (the effective maximum surface distance in the EHM is ≤1.75 mm), no hypoxia was observed. The perforated patch format is compatible with the vascular density of the human heart (2,000–5,000 capillaries / mm). 2 This contrasts with previous studies (<25 μm intercapillary distances). Therefore, the detection of hypoxia by cardiomyocytes, with oxygen supplied by diffusion over distances of >100 μm, was surprising and in contrast to theoretical assumptions (Radisic et al. (2005)). This discrepancy can be explained by the increased hypoxia tolerance of pluripotent stem cell-derived cardiomyocytes in the presence of nutrient-containing (glucose, lactate, and / or fatty acid)-containing culture medium.

[0113] The repeated and sequential layering, and in particular the perforated patch design, ensures that cells within the EHM are not compromised by hypoxia and receive sufficient nutrients. Another theoretical option for creating a 10 mm-thick EHM, for example, would be to directly cast a large volume of the reconstitution mixture in a single casting step. To achieve a 10 mm-thick EHM, the thickness of the reconstitution mixture would need to be approximately 100 mm. Cells within the mixture would need to be supplied with oxygen and nutrients over a distance of 1–10 centimeters, depending on the stage and extent of tissue compaction. Because the EHM is not vascularized, in centimeter-scale EHMs, embedded cells would be compromised by hypoxia-induced apoptosis and necrosis. To overcome this difficulty, we developed a method for repeated and sequential layering in a casting mold with perforated poles, as disclosed herein (Figures 3 and 7). This approach is advantageous over other approaches that do not guarantee sufficient oxygen and nutrient supply. Furthermore, it is essential to incorporate a mechanical support element into the penetration pole to assist in the tension contraction.

[0114] Materials and Methods for Example 2 Fabrication of 5-layer EHM: EHMs were constructed in custom-made casting molds (Figure 7) using the reconstitution mixture described in Tiburcy et al. (2017) and WO2015025030. After casting the reconstitution mixture (8 mL; see Figure 2), the EHM was observed to compact to less than 50% of its initial reconstitution volume within 3 days. After removing the culture medium, a second layer (composed of 6 mL of reconstitution mixture—the volume can be adjusted and depends on the mold design / surface area and application, and can be adapted depending on the EHM compaction) was pipetted into the casting mold to cover the compacted EHM from above and below. For example, 8 mL could be added, or a smaller volume, such as 2 mL, could be added to cover a limited area (see Example 5 for further illustration). After approximately 1 hour, gelation of the newly pipetted reconstitution mixture was complete, and culture medium was added to the mold. This process was repeated until five layers were obtained. The volume and timing of the reconstitution mixture for repeated deposition depend on the dimensions of the casting mold and the compaction of the EHM by stromal cells.

[0115] Preparation of hypoxia reporter strains The piggyBAC-ODD-Luc plasmid, containing firefly luciferase fused to the oxygen-dependent degradation domain (ODD) of HIF1α under the control of the chicken actin promoter (CAG), was cloned. Under normoxic conditions, the ODD domain is rapidly degraded by the ubiquitin-proteasome system. Under hypoxic conditions, the ODD domain is stabilized, resulting in measurable luminescence (Hesse et al., 2014). TC1133 iPSCs were electroporated with the piggyBAC-ODDLuc construct and a transposase vector. Forty-eight hours after electroporation, cells were selected with increasing concentrations of neomycin (250-500-750 μg / mL) for 7 days, after which colonies were manually picked and expanded.

[0116] Validation of hypoxia reporter strains Pluripotent stem cells expressing firefly luciferase fused to the oxygen-dependent degradation domain (ODD) of HIF1α under the control of the chicken actin promoter (CAG) were exposed to hypoxia (5% or 1% O2) and then lysed in 1X passive lysis buffer (Dual-Luciferase® Reporter Assay Systems, Promega) containing protease and phosphatase inhibitors (both from Roche). Cell-containing or cell-free (lysis buffer only) lysates were mixed with Luciferase Assay Reagent II (Dual-Luciferase® Reporter Assay Systems, Promega), and luminescence was measured using a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices). Mean ± SEM; n = 3. TIFF2023546466000001.tif22155

[0117] The luminescence signal from the stably integrated ODD-Luc reporter increased in response to low oxygen concentrations, demonstrating the utility of the ODD-Luc model for hypoxia detection in a human pluripotent stem cell model.

[0118] Imaging the bioluminescence of the hypoxia reporter EHM ODD-Luc EHMs were immersed in phosphate-buffered saline containing 1 mg / ml XenoLight D-luciferin (Perkin Elmer). Luminescence of the EHMs was imaged at 37°C using an IVIS Lumina III system (Perkin Elmer). Luminescence is displayed in grayscale in Figure 3D. Five-layer EHMs exhibited less luminescence than monolayer EHMs.

[0119] Example 3 Fabrication of loop-format EHM in multiwell plates to illustrate the coagulation process of artificial human myocardium Figure 5 shows a proof-of-principle schematic of the condensation (after 1 hour of gelation) and compaction (after 24 hours and 18 days) of the reconstitution mixture to form a force-generating loop-format EHM. For example, to support tonic contractions of artificial human myocardium, loop-format EHMs can be fabricated on a custom-made stretcher (with flexible poles fixed to a base plate) (Figure 4A). As previously disclosed in Tiburcy et al. (2020), 48 loop-format EHMs can be produced in parallel by using a defined multiwell plate (Figure 4B). This multiwell plate is further described in WO 2017 / 207431. This multiwell plate features a ring-channel mold in each mold. Each EHM is cast into the ring channel. During condensation and compaction of the reconstitution mixture, the artificial human myocardium detaches from the ring channel, moves upward, and tightly wraps around the two poles.

[0120] In the first step, the reconstitution mixture was dispensed into a multiwell plate mold as previously described in Tiburcy et al. 2017 or Tiburcy et al. 2020. When the reconstitution mixture was cast into the mold, it was a viscous, liquid, translucent solution. After 1 hour of incubation at 37°C in a humidified incubator containing 5% CO2, the artificial human myocardium became opaque as the reconstitution mixture solidified, primarily through cell-dependent gelation of the collagen hydrogel (see also Tiburcy et al. (2014) and Schlick et al. (2019)). After solidification (e.g., 1 hour, Figure 5B), culture medium supplying nutrients and growth factors was added to the mold (e.g., see Tiburcy et al. (2017) and Tiburcy et al. (2020) for exemplary medium compositions). For example, after 24 hours of culture, the loop detached from the mold, resulting in a visible space between the reconstitution mixture and the mold. Furthermore, after 24 hours, the reconstitution mixture detached from the ring channel and tightly wrapped around the two poles (Figure 5C). The process of EHM formation around the flexible poles is the action of stromal cell components and can be further facilitated by adding an effective concentration of TGFb1. After the artificial human myocardium was placed on the flexible poles, an artificial human myocardium maintenance medium without TGFb1 was used. Over the next 17 days, the mixture further condensed around the two poles, as illustrated in Figure 5D. A compact, contracting artificial human myocardium was formed. To simulate the natural tonic contraction cycle of the heart, the poles were designed to bend in response to EHM contraction and pull the EHM apart to a defined resting length during the EHM relaxation phase.

[0121] Example 4 Excitability of artificial myocardium (EHM) When cardiac myocytes are excited, they typically contract. To measure contraction within EHM, Tiburcy et al. (2017) described the fractional area change (FAC) of a patch during contraction. FAC is a video-optical measurement in which the surface area of ​​a patch is compared between a non-contracted and contracted state. Using this video-optical analysis tool, EHM phenotypes can be determined and compared. Figure 6A illustrates a custom-made video-optical analysis tool designed to facilitate measurements. Figure 6B shows the device for EHM electrical stimulation to induce contractions at a prescribed rate. The electrical field stimulation intensity (typically 2 V / cm) is adjusted until the EHM is induced to contract at a prescribed beating rate (typically controlled between 0.1 and 3 Hz). 2 Alternatively, local point stimulation with an electrode in contact with the EHM can be applied to induce EHM synchronous contractions at the desired beating rate for FAC analysis (Figure 6C).

[0122] FAC (Frequency Area Change) is measured by comparing the EHM surface area during video-optical imaging at peak systole (maximal contraction) and peak diastole (maximal relaxation). The change in surface area is recorded as a function of time.

[0123] Example 5 Various geometries for multilayer artificial myocardial design Generally, MEHMs can be fabricated in a variety of three-dimensional shapes. The general principle is a mold with perforated poles of uniform or non-uniform shape and dimensions to (i) impose mechanical resistance on the forming EHM and (ii) create perforated channels for nutrient and oxygen supply. In the EHM pouch configuration (as illustrated in Figure 8A), the EHM is cast into a circular or spherical mold with a central element (either rigid or flexible due to the biomechanical load of the condensing EHM; similar to those described in Zimmermann WH, Yildirim Y, Eschenhagen T: Pouch-like engineered heart muscle tissue. WO2008058917 and Yildirim et al. (2007) Circulation). In contrast to WO2008058917, perforated poles are created on top of the central element to ensure the creation of, for example, a >1 mm thick pouch in which muscle formation is controlled. In the illustrated example of Figure 8A, the pole-to-pole spacing is 3.5 mm and the pole diameter is 1.5 mm, similar to the preferred embodiment described for the EHM patch (Figure 7). The size, shape, and pole-to-pole spacing can be adapted as needed to create optimal loading and perforation for oxygen and nutrient delivery.

[0124] In a further example, the MEHM can also be cylindrical. In the case of a cylindrical shape (Figure 8B), the EHM can be fabricated with the poles facing inward or outward, allowing holes to be drilled into the reconstitution mixture. A key advantage of cylindrical EHMs is that many EHMs can be produced in parallel using a relatively small amount of space. After the cylindrical MEHM is produced, it can be cut open on one side to obtain patches or left as is to produce an MEHM cylinder / tube of desired dimensions. The MEHM dimensions can be scaled by freely adapting the circumference of the internal and / or external cylinder of the mold with the poles extending inward or outward / radially extending. For example, a mold in the shape of a cylinder with perforated poles is produced by prototyping 3D-printing to fit a container. The container then becomes the outer wall for producing the cylindrical EHM (e.g., a 50 ml polypropylene or glass tube). The container is filled with enough reconstitution mixture to coat the inside or outside of the cylinder. Layering can be assisted by centrifugal forces (>2g; "spin coating") induced by controlled rotation. Cylindrical / cylindrical EHMs fabricated in a longitudinally rotating cylindrical bioreactor must be performed at >2g. An inserted cylindrical stretcher has radially protruding poles to perforate the EHM. The centrifugal force applied to the EHM can be controlled by adjusting the rotation speed to mimic the increasing diastolic pressure during maturation. Furthermore, centrifugation ensures even spreading of the reconstitution mixture, ensuring a uniform EHM thickness. Even coating in a radially outward-splaying cylindrical design can be further improved by using an inner mold cylinder, which allows for even distribution of the reconstitution mixture throughout the spin-coating procedure. After coating and gelation, culture medium can be added to facilitate compaction. Layering can be repeated until the desired layer composition is achieved. Layers can be added on top of and / or below the previous reconstitution mixture.

[0125] Figure 8C shows further examples of MEHM designs. Figure 8C illustrates a heterogeneous ("patch-in-patch") multilayer strategy for creating MEHMs of varying thicknesses, as needed to produce personalized MEHMs, such as those for clinical use in cardiac repair. First, by placing a perforated patch assembly within a new mold to accommodate additional layering (C1), and second, by using inserts to separate defined casting volumes within a larger casting mold design (C2). This allows for the flexible design of MEHMs with (i) tunable thickness and (ii) xy dimensions to facilitate fabrication of patches tailored / individualized to the patient's heart. The described elements can be prepared by prototype 3D printing, injection molding, or other suitable engineering methods, as described in Tiburcy et al. (2017). The mold is typically produced from Teflon, PDMS, or agarose using milling or injection molding, while the perforated pole assembly is typically printed using 3D printing, for example, with a Connex350 (Stratasys) 3D printer, or by injection molding. For 3D printing, a combination of biocompatible MED610 polymer for the rigid components (e.g., the base plate in the case of a perforated patch design or the external mold elements in a pouch design) and TangoBlack polymer for the flexible elements such as the poles and cylinders can be applied.

[0126] table Table 1. Composition of serum-free supplement "B27 minus insulin" (50x concentration, liquid) 20 ml of "B27 minus insulin" per 500 ml of medium corresponds to 4% B27 minus insulin (v / v). TIFF2023546466000002.tif151163

[0127] List of References TIFF2023546466000003.tif239161TIFF2023546466000004.tif227161TIFF2023546466000005.tif229161TIFF2023546466000006.tif125161

Claims

1. 1. A method for producing a multi-layer artificial myocardium (MEHM), comprising: (i) providing a liquid reconstituted mixture into a mold, the reconstituted mixture is perforated by at least two poles; the reconstitution mixture comprising (a) collagen, (b) a cell mixture of cardiomyocytes and non-myocytes, and (c) an appropriate reconstitution medium, and the reconstitution mixture is allowed to gel within the mold; (ii) culturing the mixture in the mold obtained by step (i) in an appropriate culture medium, thereby condensing the reconstituted mixture in the mold; (iii) a) adding a further liquid reconstitution mixture as defined in step (i) from above and / or below the condensed reconstitution mixture obtained by step (ii), wherein the further liquid reconstitution mixture gels, and then incubation is carried out under the same conditions as in step (ii), so that the further reconstitution mixture condenses in the mold; or b) transferring the concentrated reconstituted mixture obtained from step (ii) into a different mold, wherein the reconstituted mixture is perforated by at least two poles, and thereafter step (iii)a) is carried out in the different mold; This results in a multi-layer artificial myocardium (MEHM), Preferably, the MEHM is thickened by repeating step (iii)a) and / or step (iii)b) at least once; and (iv) optionally culturing the MEHM of step (iii) in the mold in a suitable maturation medium. wherein the MEHM is capable of contracting.

2. 2. The method of claim 1, wherein the ability of the MEHM to contract is assessed by visual inspection and / or by verifying the ability of the reconstitution mixture of step (i) to form a force-generating artificial myocardium by performing steps (i), (ii), and optionally step (iv), wherein the artificial myocardium is capable of generating a contractile force that has a force of contraction (FOC) of at least 0.05 mN when measured under standard isometric conditions in Supplementary Figure 6C of Tiburcy et al. Circulation 135(19)1832-1847 (2017).

3. 3. The method of claim 1 or 2, wherein the contraction is confirmed by measuring the fractional area change (FAC) of the MEHM using the method described in Tiburcy et al. Circulation 135(19)1832-1847 (2017), and wherein the fractional area change (FAC) is at least 0.5% upon electrical stimulation, preferably the FAC is at least 0.7%, more preferably at least 1%, more preferably at least 2%, more preferably at least 3%, and even more preferably at least 5%.

4. 10. The method of claim 9, wherein the non-muscle cells are selected from one or more of the group consisting of stromal cells, endothelial cells, smooth muscle cells, and mesenchymal stem cells, preferably the non-muscle cells are stromal cells or endothelial cells, more preferably the non-muscle cells are stromal cells, even more preferably the stromal cells are cardiac stromal cells, even more preferably the cardiac stromal cells have fibroblastic properties, even more preferably the cardiac stromal cells are fibroblasts.

5. 10. The method of any one of the preceding claims, wherein the MEHM has the shape of a patch, pouch, or cylinder.

6. 10. The method according to any one of the preceding claims, characterized in that the reconstitution mixture is opaque when gelation in step (i) is confirmed, preferably by visual inspection as shown in Figure 3B of Tiburcy M, et al., Collagen-based engineered heart muscle, Methods Mol Biol. 2014;1181:167-76.

7. The method of any one of the preceding claims, wherein completion of step (ii) is confirmed by (a) performing steps (i) and (ii) to create a loop-format engineered heart muscle (EHM) in parallel with the MEHM, and (b) confirming that the loop-format engineered heart muscle (EHM) has been detached from the mold, as described in Figure 3C of Tiburcy M, et al., Collagen-based engineered heart muscle, Methods Mol Biol. 2014;1181:167-176.

8. 10. The method according to any one of the preceding claims, wherein step (iii)a) or step (iii)b) is repeated 2 to 200 times, preferably 2 to 100 times, more preferably 2 to 80 times, more preferably 2 to 70 times, more preferably 2 to 60 times, more preferably 2 to 50 times, more preferably 2 to 40 times, more preferably 3 to 30 times, more preferably 3 to 25 times, more preferably 4 to 20 times, more preferably 4 to 15 times, more preferably 4 to 10 times, even more preferably 5 to 9 times, and most preferably 5 to 8 times.

9. 10. The method of claim 1, wherein the cardiomyocytes in the MEHM are adequately oxygenated, preferably as determined by an ODD-Luc hypoxia reporter as described in Hesse AR, et al., Lights on for HIF-1α, Genetically Enhanced Mouse Cardiomyocytes for Heart Tissue Imaging, Cell Physiol Biochem, 2014;34:455-462, and wherein adequate oxygenation is ensured if the mean relative luminescence of the cardiomyocytes of the MEHM is at most 6-fold higher, more preferably at most 5-fold, more preferably at most 4-fold, more preferably at most 3-fold, more preferably at most 2.5-fold, even more preferably at most 2-fold, and even more preferably at most 1.5-fold higher than that of the monolayer EHM obtained by following steps (i), (ii), and optionally step (iv) of the method.

10. A multi-layered artificial myocardium (MEHM) obtainable by the method according to any one of claims 1 to 9.

11. A multilayered artificial myocardium (MEHM), preferably an artificial heart patch, an artificial heart pouch, or an artificial heart cylinder, comprising (a) collagen and (b) a cellular mixture of cardiomyocytes and non-muscle cells, and comprising at least two layers.

12. the MEHM is fabricated by a repetitive, sequential layer-by-layer process; the MEHM is made up of 2 to 200 layers; and the layers are integral with one another, thereby increasing the thickness of the MEHM; The MEHM of claim 11.

13. 13. The MEHM of claim 11 or 12, wherein the cardiomyocytes in the MEHM are adequately supplied with oxygen, as determined by the ODD-Luc hypoxia reporter as described in Hesse AR et al. (2014), and preferably when the average relative luminescence of the cardiomyocytes of the MEHM is at most 6-fold, more preferably at most 5-fold, more preferably at most 4-fold, more preferably at most 3-fold, more preferably at most 2.5-fold, even more preferably at most 2-fold, and even more preferably at most 1.5-fold higher than that of a monolayer EHM obtained by following steps (i), (ii), and optionally step (iv) of the method.

14. Use of an MEHM obtained by the method of any one of claims 1 to 9 or an MEHM of any one of claims 10 to 13 in the in vitro production of an artificial human myocardium, preferably wherein the MEHM has a three-dimensional shape that matches a patient-specific myocardial defect, more preferably wherein the patient-specific defect is assessed by MRI, ultrasound, computed tomography, positron emission tomography, and / or optical coherence tomography.

15. A multilayered EHM (MEHM) obtained by the method of any one of claims 1 to 9 or an MEHM of any one of claims 10 to 13 for use in cardiac repair, preferably for treating patients suffering from heart failure.

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