Multilayer engineered myocardium

Abstract

The present application describes a method for manufacturing a multilayer engineered myocardium. In particular, the method involves (i) providing a liquid reconstituted mixture in a mold, and (ii) culturing the mixture. The method is characterized by the sequential addition of one or more additional liquid reconstituted mixtures to obtain a multilayer engineered myocardium. The muscle ideally has the form of a patch, a pocket or a cylinder. Furthermore, the present application relates to a multilayer engineered myocardium derived from at least 2 layers, comprising collagen, cardiomyocytes and non-muscle cells. The multilayer engineered myocardium lays the foundation for several in vitro and in vivo applications, for example the production of a multilayer engineered myocardium for a patient, for example for cardiac repair.

Description

[0001] Background of the Invention Regenerating a failing heart remains a significant challenge. A common cause of heart failure is a heart attack. A heart attack, or myocardial infarction, is caused by obstruction of arterial blood flow, leading to insufficient tissue perfusion and deprivation of oxygen and nutrients. During a heart attack, millions, even billions, of cells rapidly die, a major cause of decreased cardiac function, clinically termed heart failure. Because the heart is primarily post-mitotic tissue, it cannot regenerate on its own. Implanting engineered heart muscle is a goal pursued in this field. Different engineered heart muscle formats have been developed for disease modeling, drug screening, and cardiac repair. The inventors and others have published several applications of engineered human heart muscle models for in vitro studies of cardiomyocyte maturation and hypertrophy (Tiburcy et al., 2017), disease modeling (Hanses et al., 2020), drug screening (Mills et al., 2019), and in vivo cardiac repair (Riegler et al., 2015).

[0002] One approach to support the self-assembly of cardiomyocytes into 3D tissues is the scaffold-based approach, where the scaffold is primarily supported by 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 crucial for the generation of three-dimensional tissues. Similar to rodent models (Naito et al. 2006), these non-myocytes are primarily fibroblasts or stromal cells with fibroblast activity (Kensah et al. 2013; Ronaldson-Bouchard et al. 2018; Tiburcy et al. 2017, Zhang et al. 2013).

[0003] Beyond cellular components and scaffolds, the highest level of maturation has been achieved with the support of biophysical stimulation, namely mechanical stimulation, electrical stimulation, or a combination of both (Ronaldson-Bouchard et al., 2018; Tiburcy et al., 2017). For the engineering of highly mature myocardium, mechanical loading is an absolute requirement.

[0004] In order to produce artificial myocardial tissue for the treatment of patients with myocardial weakness, engineered myocardium needs to provide the force to support the beating of a human heart. The thickness of the heart wall to be treated in patients is typically 5-10 mm (Kawel et al., 2012), meaning that the implant must ideally achieve this thickness and must also be able to generate mechanical forces to support the function of the heart wall.

[0005] A key challenge in achieving engineered cardiac muscle of such thickness is ensuring that the cells embedded within it receive adequate oxygen and nutrients at all times. Therefore, engineered cardiac muscle of sufficient thickness to support the human heart is required in this field. The inventors have overcome this challenge through a repetitive and sequential casting process for engineered cardiac muscle. Summary of the Invention

[0006] To create multilayer engineered heart muscle (MEHM), a dense layer (reconstitution mixture) of cellular hydrogel is coated with at least one other layer. This repetitive and sequential process ensures the formation of thickened and strengthened engineered heart muscle, in which at least one new layer fuses with the previous layer. In other words, the thickness of the engineered heart muscle is increased through repeated and sequential layering. Furthermore, it is important that the engineered heart muscle is penetrated by a mechanical support, where poles extend through the engineered tissue to support auxotonic contraction and introduce channels for oxygen and nutrient supply throughout the engineered tissue.

[0007] A particular advantage of the method disclosed herein is that the cells within the reconstructed mixture / MEHM receive adequate nutrition and oxygen at all times. This adequate oxygen and nutrient supply is achieved by minimizing the total diffusion distance from the MEHM surface to the MEHM core (center) throughout the manufacturing process, while still producing a thickened MEHM (e.g., suitable for use as an implant). This is accomplished through a perforated MEHM design. For example, perforations are introduced by perforating poles, particularly for mechanical support (e.g., perforated poles). Figure 7 and Figure 8(As shown). These rods are capable of supplying oxygen and / or nutrients to the cells in the MEHM. The rods create “channels” by remodeling the mixture / MEHM for supplying oxygen and / or nutrients and independently introduce mechanical loads to the MEHM.

[0008] To obtain MEHM of this thickness, a reconstructed mixture comprising cardiomyocytes, non-cardiomyocytes, and collagen is repeatedly and sequentially cast. A key advantage of this method is that the layers merge by repeatedly casting each layer on top of and / or from the bottom of the previous layer. Through sequential casting, each layer has condensed by at least 20% of its original volume before adding another layer. The embedded cells obtain oxygen and nutrients from the tissue surface through diffusion, avoiding, for example, hypoxia and / or necrosis. The reconstructed mixture is perforated with rods, which expands the tissue's surface area. Therefore, the total thickness of the muscle can be scaled as needed, taking into account both diffusion and the cell-specific oxygen and nutrient requirements.

[0009] In a first aspect, a method for manufacturing multilayer engineered myocardium (MEHM) is disclosed, wherein the method includes the following steps:

[0010] (i) A liquid reconstitution mixture is provided in a mold, wherein the reconstitution mixture is perforated by at least two rods. The reconstitution mixture comprises (a) collagen, (b) a mixture of cardiomyocytes and non-cardiomyocytes, and (c) a suitable reconstitution medium, wherein the reconstitution mixture undergoes gelation in the mold.

[0011] (ii) The mixture obtained in step (i) in the mold is cultured in a suitable culture medium, thereby compacting the reconstituted mixture in the mold;

[0012] (iii) a) Adding a further liquid reconstituted mixture as defined in step (i) from top and / or bottom to the densified reconstituted mixture obtained in step (ii), whereby the further liquid reconstituted mixture gels, and then incubating under the same conditions as in step (ii), whereby the further reconstituted mixture densifies in a mold; or b) The compacted reconstituted mixture obtained in step (ii) is transferred to different molds, wherein the reconstituted mixture is perforated by at least two rods, and then step (iii)a) is performed in the different molds; This allows for the creation of multilayer engineered myocardium (MEHM). Preferably, the MEHM is thickened by repeating step (iii) a) and / or step (iii) b) at least once, and

[0013] (iv) Optionally, the MEHM from step (iii) in the mold is cultured in a suitable maturating medium.

[0014] The MEHM mentioned therein is capable of shrinking.

[0015] On the other hand, a multilayer engineered myocardium (MEHM) was conceived using the method disclosed herein.

[0016] In addition, multilayer engineered myocardium (MEHM) is described, wherein the MEHM comprises (a) a mixture of collagen and (b) cardiomyocytes and non-myocellular cells, and wherein the MEHM comprises at least 2 layers.

[0017] In addition, engineered heart muscle (EHM) is disclosed, preferably an engineered heart patch, an engineered heart pouch, or an engineered heart cylinder, wherein the EHM comprises (a) collagen, (b) a mixture of cardiomyocytes and non-myocellular cells, and wherein the thickness of the EHM is at least about 0.6 mm.

[0018] Furthermore, this invention discloses the use of MEHM obtained by the methods disclosed herein, or MEHM as disclosed herein, in the in vitro fabrication of engineered human myocardium.

[0019] Finally, the use of multilayer EHMs or MEHMs obtained by the methods disclosed herein in medicine, particularly for heart failure, is described. Detailed Implementation

[0020] In a first aspect, a method for manufacturing multilayer engineered myocardium (MEHM) is disclosed, wherein the method includes the following steps: (i) A liquid reconstitution mixture is provided in a mold, wherein the reconstitution mixture is perforated by at least two rods. The reconstitution mixture comprises (a) collagen, (b) a mixture of cardiomyocytes and non-cardiomyocytes, and (c) a suitable reconstitution medium, wherein the reconstitution mixture gels in the mold. (ii) The mixture obtained in step (i) in the mold is cultured in a suitable culture medium, thereby densifying the reconstituted mixture in the mold; (iii) a) Adding a further liquid reconstituted mixture as defined in step (i) from top and / or bottom to the densified reconstituted mixture obtained in step (ii), whereby the further liquid reconstituted mixture gels, and then incubating under the same conditions as in step (ii), whereby the further reconstituted mixture densifies in a mold; or b) The densified reconstituted mixture obtained in step (ii) is transferred to different molds, wherein the reconstituted mixture is perforated by at least two rods, and then step (iii) a) is performed in the different molds; This allows for the creation of multilayer engineered myocardium (MEHM). Preferably, the MEHM is thickened by repeating step (iii) a) and / or step (iii) b) at least once, and (iv) Optionally, the MEHM from step (iii) in the mold is cultured in a suitable maturation medium. The MEHM mentioned therein is capable of shrinking.

[0021] Generally, the engineered heart muscle (EHM) used in this article refers to an artificial heart muscle containing cells and collagen that is contractile. Furthermore, multilayer engineered heart muscle (MEHM) is derived from at least two layers of engineered heart muscle (EHM). To create a multilayer engineered heart muscle (MEHM), layers are added sequentially. During sequential layering, subsequent layers are added on top of the original (first) layer. The subsequent layer then fuses with the original (first) layer. This layering process may be repeated several times. In other words, a subsequent layer fuses with a previously added layer. During layering, the MEHM thickens layer by layer. The main advantage of this method is that a thickened MEHM is more stable than a single-layer EHM and can withstand greater forces. Furthermore, another key feature of MEHM is its contractile ability, similar to natural heart muscle, preferably as further defined below. Ideally, this multilayer engineered heart muscle is suitable for use as an implant, such as a cardiac implant.

[0022] In one implementation, the MEHM obtained by step (iii) and optional step (iv) has contractile capacity, which can be assessed by visual inspection. Alternatively, the contractile capacity of the MEHM can be determined by the ability of the reconstructed mixture of step (i) to form a force-generating engineered myocardium (EHM) by performing steps (i), (ii), and optional (iv), and wherein the engineered myocardium (EHM) is capable of generating a contractile force of at least 0.05 mN (force of contraction, FOC), as in the additional section of Tiburcy et al. Circulation 135(19)1832-1847 (2017). Figure 6 The contraction of the MEHM can be further determined by evaluating in parallel the ability of the reconstructed mixture of step (i) to form a loop-format of force-generating engineered human muscle by performing steps (i), (ii), and (iv), as described in Tiburcy et al., Circulation 135(19)1832-1847 (2017), and wherein the loop-format engineered myocardium generates a contractile force (FOC) of at least 0.05 mN, as described in the supplement to Tiburcy et al., Circulation 135(19)1832-1847 (2017). Figure 6The shrinkage of the MEHM is measured in C. In other words, the shrinkage of the MEHM can be determined by generating an EHM in a ring-shaped form parallel to the MEHM. The advantage of this parallel method is that the formation of the ring-shaped EHM is well established (e.g., see Tiburcy et al., 2017 and 2020) and can also be performed in a 48-well format. Furthermore, the shrinkage force can be more easily determined in the ring-shaped form because the force between the two rods is measured. Therefore, the quality of the reconstituted mixture and the medium can be more easily evaluated in a well-established ring-shaped form. In addition, it is expected that a well-developed ring-shaped EHM can also ensure that the quality of the provided MEHM is the same. Alternatively, the shrinkage force (FOC) in the MEHM can also be measured directly. For example, Zimmermann et al. (2000) described the measurement of FOC in tissue. Therefore, the technician can also adjust the settings to measure the FOC in the MEHM. In a preferred embodiment, the contractile force (FOC) generated by the MEHM is 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.

[0023] 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 thickness of the MEHM is from about 0.2 mm to about 30 mm, preferably from about 0.3 mm to about 30 mm, preferably from about 0.5 mm to about 30 mm, more preferably from about 0.7 mm to about 25 mm, more preferably from about 0.9 mm to about 23 mm, more preferably from about 1.5 mm to about 20 mm, more preferably from about 2 mm to about 18 mm, more preferably from about 2.8 mm to about 15 mm, more preferably from about 3.3 mm to about 13 mm, more preferably from about 3.8 mm to about 12 mm, more preferably from about 4.2 mm to about 11 mm, more preferably from about 4.6 mm to about 10.5 mm, and most preferably from about 5 mm to about 10 mm. When referring to both the thickness of a single layer of the multilayer engineered myocardium of the present invention and the thickness of the engineered myocardium described herein, the term "about" means that the thickness may deviate from the corresponding value by ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, or ±10%. This means, for example, if the thickness of a single layer is "about 0.3 mm", then the thickness of that layer ranges from 0.27 mm (with a deviation of -10%) to 0.33 mm (with a deviation of +10%). Similarly, if the engineered myocardium itself has a thickness of about 0.8 mm, then the engineered myocardium includes a thickness of 0.72 mm to 0.88 mm.

[0024] Ideally, MEHMs should match the thickness of human myocardium, ranging from 5 mm to 12 mm in a non-systolic state. For example, Kawel et al. (2012) stated that the human heart wall is at most 12 mm thick during maximal diastole. Nowosielski et al. (2009) stated that the heart wall thickness typically increases by 60% during systole. Therefore, the maximum thickness of the human heart is estimated to be 20 mm. Thus, MEHMs used as cardiac implants preferably have a thickness between 5 mm and 20 mm.

[0025] To the inventors' knowledge, this is the first thickened EHM design in the art. A key advantage of the disclosed method is that the MEHM originates from at least two layers fused together. Through this continuous and repeated lamination, the MEHM thickens layer by layer while ensuring minimal total surface-to-tissue diffusion distance throughout the MEHM. The design and density of the perforating poles ensure the desired surface-to-tissue diffusion distance. Minimal or metabolically adequate surface-to-tissue diffusion ensures that cells consistently receive oxygen and nutrient supply. Reconstructed mixtures without, for example, perforations of several centimeters or their multilayered tissue will exhibit rapid (tissue core) necrosis of cells in the central part of the reconstructed mixture, as these cells will not be supplied with sufficient oxygen and / or nutrients.

[0026] In another embodiment, the contraction of the MEHM can also be measured video-optically. For example, the fractional area change (FAC) of the MEHM can be measured. The method for evaluating FAC has been described by Tiburcy et al. (2017). In short, the area of ​​the MEHM in the diastolic and contracted states is compared, and the change is reported as a percentage. In a particularly preferred embodiment, the contraction is determined 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) during electrical stimulation is at least 0.5%, 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 a particularly preferred embodiment, 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 achieved via an electric field or through 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, electrical stimulation leads to MEHM contraction. From the 2017 study by Tiburcy et al., it can be deduced that approximately 7% FAC corresponds to approximately 1 mN FOC (attached Figure 11). Based on this figure, those skilled in the art can correlate FOC and FAC if desired.

[0027] To manufacture this MEHM, a liquid remodeling mixture is provided in the mold. Generally, the "liquid" mixture is capable of flowing and filling the mold. Furthermore, a medium can dissolve into the liquid remodeling mixture. As used herein, "liquid" mixture refers to a mixture that is distinctly different from a "gel-like" mixture, because gel-like mixtures are not flowable and the medium cannot freely dissolve into them, as defined below.

[0028] As used herein, a “reconstitution mixture” includes collagen, a cell mixture, and a suitable reconstitution medium, wherein the cell mixture consists of cardiomyocytes and non-myomyocytes. Those skilled in the art will readily understand that the reconstitution mixture forms the basis of MEHM, because the different components of the reconstitution mixture construct key parts of engineered cardiomyocytes, i.e., reconstruct key parts of cardiomyocytes to engineer MEHM tissue.

[0029] Collagen is one of the components of the reconstructed mixture. Collagen is a major structural protein in the extracellular matrix of various connective tissues in the body. It is known to those skilled in the art that collagen is composed of amino acids forming triple helices of elongated fibrils. Furthermore, collagen supports engineered tissues, such as MEHM, and provides external support to cellular structures. Those skilled in the art also know that collagen can be purchased from various suppliers, such as Collagen Solutions. For example, the reconstituted mixture can provide collagen at a final concentration between 0.5 mg / ml and 3 mg / ml, preferably between 0.55 mg / ml and 2.5 mg / ml, more preferably between 0.6 mg / ml and 2.2 mg / ml, between 0.65 mg / ml and 2 mg / ml, more preferably between 0.7 mg / ml and 1.75 mg / ml, more preferably between 0.75 mg / ml and 1.5 mg / ml, more preferably between 0.8 mg / ml and 1.2 mg / ml, even more preferably between 0.85 mg / ml and 1 mg / ml, and most preferably about 0.9 mg / ml. In a preferred embodiment, the collagen in step (i) is selected from the following: 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 know 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 of step (i) is type I collagen. Specifically, if MEHM is used as an implant, the collagen in the reconstituted mixture of step (i) may be medical grade.

[0030] Of course, collagen can come from different sources and different suppliers. For example, collagen can be bovine, equine, human, or marine. In a particularly preferred embodiment, the collagen is bovine. In a particularly preferred embodiment, the collagen used is free of porcine endogenous retroviruses and any infectious spongiform encephalopathy. It is also anticipated that the collagen in the reconstituted mixture of step (i) will also contain one or more additional extracellular matrix components in addition to collagen. The term “extracellular matrix protein” refers to any extracellular matrix (ECM) protein known to a person skilled in the art (Hynes and Naba (2012)). Furthermore, those skilled in the art are familiar with the composition of the extracellular matrix, which can be used to generate EHM, for example, Mouw JK et al. (2014). In a particularly preferred embodiment, the one or more additional matrix components are selected from the following: elastin, laminin, entactin, nidogen, proteoglycans (e.g., core proteoglycans), glycosaminoglycans (e.g., hyaluronic acid), and fibronectin. Furthermore, those skilled in the art know that there are commercially available synthetic mimics of the extracellular proteins, and that these synthetic mimics are also suitable for manufacturing MEHMs. For example, those skilled in the art are familiar with literature such as O'Leary et al. (2011), which describes the self-assembly of collagen mimic peptides from triple helices to nanofibers and hydrogels.

[0031] In a preferred embodiment, the cell mixture in step (i) provides a final cell concentration of 1 × 10⁻⁶ in the reconstituted mixture. 6 -26.5×10 6 cells / ml, preferably 2×10⁻⁶ cells / ml 6 -13.2×10 6 Cells / ml, more preferably 3×10⁻⁶ cells / ml 6 -10×10 6 Cells / ml, more preferably 3.5 × 10⁻⁶ cells / ml 6 -9×10 6 cells / ml, more preferably 4×10⁻⁶ cells / ml 6 -8×10 6 Cells / ml, more preferably 4.3 × 10⁻⁶ cells / ml. 6 -7×10 6 Cells / ml, more preferably 4.6 × 10⁶ cells / ml. 6 -6×10 6 Cells / ml, more preferably about 5×10⁻⁶ cells / ml. 6Cells / ml. However, technicians are able to determine the appropriate cell concentration in the reconstituted mixture. For example, technicians can determine the appropriate cell concentration and composition with guidance from Tiburcy et al. (2017) or Schlick et al. (2019).

[0032] The cellular mixture consists of cardiomyocytes and non-myocellular cells. Cardiomyocytes are muscle cells (myocytes) that make up the cardiac muscle (heart muscle) in natural tissues. Each cardiomyocyte contains myofibrils (myofibrils are 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 commercially available or obtained by differentiation from pluripotent stem cells, such as 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, wherein the cells are not produced using methods involving modification of human germline genetic characteristics or processes involving the use of human embryos for industrial or commercial purposes. For example, cardiomyocytes can be 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. Those skilled in the art know induced pluripotent stem cells, parthenogenetic stem cells, or adult stem cells as described in the art. Programmed somatic cells are directly reprogrammed to the desired cell type. This approach avoids the induction of pluripotency and subsequent differentiation (e.g., see Ieda et al., 2010, Song et al., 2012, Nam et al., 2013). In particular, those skilled in the art are aware of various protocols in the literature describing cardiomyocyte production, such as via serum-free differentiation as described in WO2015 / 040142, or alternative protocols, such as those described in Burridge et al. (2012), Production of denovo cardiomyocytes: human pluripotent stem cell differentiation and direct reprogramming, Cell Stem Cell. 2012 Jan 6;10(1):16-28. Furthermore, Zimmermann's work in Schäfer-Korting_Stucchi Maria-Engler,_Landsiedel (87017223) Organotypic Models in Drug Development, Handbook of Experimental Pharmacology (Springer, 2020) Engineered Heart Muscle Models in Phenotypic Drug ScreensThis chapter summarizes various protocols for obtaining cardiomyocytes that can be used with the methods disclosed herein. These protocols also take into account that the cardiomyocytes may be derived from non-human primate stem cells, fetal, or neonatal cardiomyocytes. Those skilled in the art are also aware of methods for assessing cardiomyocyte identity, such as flow cytometry or RNA sequencing. For example, these methods allow for the determination of the expression of markers ACTN2, TTN, RYR2, and / or troponin, which are characteristic of cardiomyocytes.

[0033] In one embodiment, the cell mixture in 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. Those skilled in the art can determine the optimal ratio of cardiomyocytes to non-myomyocytes. Optimization of the optimal ratio will be guided by the compaction of the EHM, particularly its contractile capacity. For example, Tiburcy et al. (2017) and Schlick et al. (2019) demonstrated that non-myomyocytes, such as cardiomyocytes and stromal cells, are essential for the contraction and compaction of engineered human myocardium. Specifically, cardiomyocytes induce contraction, while non-myomyocytes, such as stromal cells, induce compaction. Furthermore, non-myomyocytes secrete extracellular matrix proteins, such as collagen, to support the three-dimensional organization of the EHM and / or bind to the extracellular matrix via integrins. In a preferred embodiment, the contraction of the MEHM is induced by cardiomyocytes, thereby causing the development of contractile force in the MEHM.

[0034] As used herein, non-muscular cells are the cellular components of a cellular mixture other than cardiomyocytes. Non-muscular cells are essential for the development of MEHMs. For example, as demonstrated by Schlick et al. (2019), engineered human cardiomyocytes do not compact when non-muscular cells are not provided in the reconstitution mixture. In a preferred embodiment, the non-muscular cells are selected from one or more of the following: stromal cells, endothelial cells, smooth muscle cells, and mesenchymal stem cells, preferably stromal cells or endothelial cells, more preferably stromal cells, even more preferably cardiac stromal cells, even more preferably fibroblastic cells, and even more preferably fibroblasts. Typically, non-muscular cells secrete extracellular matrix proteins and / or bind to the extracellular matrix via integrins to support MEHM formation. In other words, in a preferred embodiment, the non-muscular cells are ideally capable of compacting the reconstitution through cell-matrix interactions in step (ii). Therefore, any cell capable of performing biophysical cell-matrix interactions and secreting extracellular matrix proteins is a suitable non-muscle cell. In a particularly preferred embodiment, as determined by flow cytometry, the non-muscle cell expresses CD90, preferably CD90, CD74, and CD44. As described above, the non-muscle cell ideally secretes extracellular matrix proteins, particularly collagen, and / or expresses integrins to promote cell-matrix interactions, wherein the non-muscle cell densifies and remodels the mixture. Non-muscle cells can be obtained from a variety of sources. For example, non-muscle cells can be derived from induced pluripotent stem cells, parthenogenetic stem cells, programmed somatic cells, adult stem cells, or mesenchymal stem cells, preferably wherein the non-muscle cells are derived from induced pluripotent stem cells. Furthermore, non-muscle cells can be derived from embryonic stem cells, wherein the cells are not produced using methods involving modification of human germline genetic characteristics or processes involving the use of human embryos for industrial or commercial purposes. In another preferred embodiment, the non-muscle cells are derived from biopsy tissue obtained from a human individual, preferably from a patient, and more preferably from a patient for autologous or allogeneic administration of EHM. The advantage of autologous patient-derived non-muscle cells is that the cells are obtained from the same patient, and therefore these cells are immunologically compatible with the patient. This is a particular advantage when MEHM is used as a patient implant. Allogeneic cells also have the advantage of being patient-derived, and therefore these cells are similar to the cells of the patient requiring the MEHM implant. Technicians can use a large number of protocols to obtain suitable non-muscle cells. For example, EP20188364.2 describes a serum-free method for obtaining cardiac stromal cells with fibroblast characteristics.In addition, Witty et al. (2014), Iyer D et al. (2015), Bao X et al. (2016), or Bao X et al. (2017) described protocols for obtaining suitable non-myocellular cells.

[0035] In a particularly preferred embodiment, the non-myocellular cells in the cell mixture of step (i) are stromal cells, as described, for example, in EP20188364.2, preferably human stromal cells. When the non-myocellular cells are stromal cells, the cell mixture of 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-myocellular cells together constitute the cell mixture, i.e., 100%. Exemplary ratios of cardiomyocytes and non-myocellular cells would be 50:50, 60:40, 70:30, 80:20, or 90:10, respectively. Of course, any intermediate ratio can also be chosen. As mentioned above, various ratios have been tested and can be found in the literature, for example, Tiburcy et al. (2017) and Schlick et al. (2019).

[0036] 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, thereby in the reconstitution mixture, culture medium, and / or maturation medium. In a preferred embodiment, the serum-free supplement may be formulated to provide a final concentration in (1) the reconstitution mixture, (2) the culturing medium, and / or (3) the maturation medium. Of course, the concentration and composition may be independently selected for use in (1) the reconstitution mixture, (2) the culture medium, or (3) the maturation medium.

[0037] For example, serum-free supplements can be formulated to provide a final concentration of the following component in any of (1)-(3): 0.5-50 mg / ml (preferably 1-40 mg / ml, more preferably 2-30 mg / ml, more preferably 3-20 mg / ml, more preferably 4-10 mg / ml, most preferably 4.5-7.5 mg / ml, for example about 5 mg / ml) of albumin;

[0038] Transferrin at 1-100 μg / ml (preferably 2-90 μg / ml, more preferably 3-80 μg / ml, more preferably 4-70 μg / ml, more preferably 5-60 μg / ml, more preferably 6-50 μg / ml, more preferably 7-40 μg / ml, more preferably 8-30 μg / ml, more preferably 9-20 μg / ml, for example about 10 μg / ml);

[0039] Ethanolamine in the range of 0.1-10 μg / ml (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, and most preferably 1-2.5 μg / ml, for example about 2 μg / ml);

[0040] 14.4-1446 nM selenium or its bioavailable salt, (preferably 40-700 nM, more preferably 70-300 nM, even more preferably 130-160 nM, most preferably about 144.6 nM);

[0041] L-carnitine hydrochloride in the form of 0.4-40 μg / ml (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, and most preferably about 4 μg / ml).

[0042] Fatty acid supplements of 1-100 μg / ml (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, for example about 4 μg / ml); and

[0043] Triiodo-L-thyroxine (T3) in the range of 0.0004-0.04 μg / ml (preferably 0.0010-0.02 μg / ml, more preferably 0.0016-0.010 μg / ml, even more preferably 0.002-0.006 μg / ml, and most preferably about 0.004 μg / ml).

[0044] For example, fatty acid supplements may include linoleic acid and / or alpha-linolenic acid.

[0045] For example, the bioavailable salt of selenium is sodium selenite, so that the final concentration of sodium selenite provided in the reconstitution mixture, culture medium or reconstitution medium is 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).

[0046] 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 concentrations of the individual components are known to those skilled in the art or can be readily determined through routine experiments.

[0047] One example of a serum-free supplement can be prepared according to published protocols (see also Brewer et al. (1993)) or can be purchased commercially. For example, insulin-free B27 (B27 minus insulin) can be used (Table 1). In a preferred embodiment, the serum-free supplement is provided by 0.2-20% (v / v) of insulin-free B27 in a reconstitution mixture, culturing medium, or reconstitution medium, preferably 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) of insulin-free B27, and most preferably about 4% (v / v) of insulin-free B27 in a reconstitution mixture, culturing medium, or reconstitution medium, as commercially available or preferably prepared according to Table 1. Alternatively, insulin-containing B27 can also be used. The optimal B27 composition can be determined experimentally for each cell type used. Those skilled in the art know how to determine the optimal B27 composition and / or its concentration. For example, Tiburcy et al. (2017) compared the use of B27 and insulin-free B27 in Figure IIIF, which is available online only. Engineered muscle produced with insulin-free B27 exhibited higher force than muscle produced with B27. However, the figure also shows that B27 (with insulin) can be used to successfully generate engineered human cardiac muscle to achieve good contractility.

[0048] Furthermore, the basal medium of the reconstructed medium, culture medium, and / or maturation medium may additionally contain ascorbic acid or its derivatives. For example, when ascorbic acid is included in the basal medium of the reconstructed medium, culture medium, and / or maturation medium, a concentration of 10-1000 μM, preferably 50-400 μM, more preferably 100-300 μM, even more preferably 150-250 μM, and most preferably about 200 μM is provided in the reconstructed mixture, culture medium, and / or maturation medium. Even more preferred is ascorbic acid present in the form of ascorbate-2-phosphate.

[0049] Furthermore, the basal medium for the reconstructed medium, culture medium, and / or maturation medium can be RPMI medium, which also contains pyruvate. A particularly preferred concentration range for pyruvate in RPMI is 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.

[0050] The basal medium for the reconstitution medium can be, for example, selected from Iscove medium, RPMI, αMEM, and DMEM, or mixtures thereof, preferably selected from Iscove medium, RPMI, αMEM, or mixtures thereof, more preferably a mixture of Iscove medium and RPMI. However, any suitable basal medium can be used in this method. The basal medium is commercially available or can be produced according to publicly available formulations, for example, from the ATCC product catalog. Generally, as used herein, the medium incorporates appropriate amounts of glucose, lactate, and / or fatty acids as essential nutrients. Glucose, lactate, and / or fatty acids ensure that cells embedded in the reconstitution mixture receive an adequate supply of nutrients.

[0051] If deemed appropriate, amino acids can be supplemented into the basal medium. For example, if αMEM is used as the basal medium, there is no need to supplement non-essential amino acids into the basal medium. Non-essential amino acids can be purchased commercially as combination supplements. For example, such supplements contain 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.

[0052] In a particularly preferred embodiment, the reconstitution medium further comprises (c) 35-790 μM ascorbic acid, (d) 5-500 ng / ml IGF-1, (e) 0.3-26 ng / ml VEGF, (f) 0.5-53 ng / ml FGF-2, and (g) 0.5-10 ng / ml TGFβ1. In an even more preferred embodiment, the reconstitution medium further comprises (c) 75-300 μM ascorbic acid, (d) 26-105 ng / ml IGF-1, (e) 1.3-5.2 ng / ml VEGF, (f) 2.6-10.5 ng / ml FGF-2, and (g) 1-6 ng / ml TGFβ1. In a more preferred embodiment, the culture 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.

[0053] Of particular preference is VEGF. 165 Those skilled in the art are familiar with published schemes for appropriate reconfiguration media, such as those by Tiburcy et al. (2017) or Tiburcy et al. (2020), and are able to identify equally applicable schemes.

[0054] As used herein, the “mould” is freely selectable and can be geometrically variable. The key is that the mold must contain the reconstructed mixture, and that the reconstructed mixture must gel within the mold. The mold can be customized for its intended use. For example, if the purpose is an implant for human implantation, a customized mold can be matched to a myocardial defect. If the purpose of the MEHM is testing, such as drug efficacy, different shapes can be used. Technicians can customize the shape of the mold according to their needs or purposes. For example, a technician can use a 3D printer to individually customize the shape of the mold. In a preferred embodiment, the mold is formed to facilitate the formation of a planar MEHM, a bag-shaped MEHM, or a cylindrical MEHM. In a preferred embodiment, the mold has the form of a patch, a bag, or a cylinder. More preferably, the MEHM has the form of a patch, a bag, or a cylinder. In a more preferred embodiment, the patch has the form of a disc.

[0055] If the mold is formed to facilitate the formation of a planar MEHM, the mold may be in the form of a disc, preferably a circular or polygonal disc, and more preferably a hexagonal disc. Figure 2 and Figure 7 The exemplary hexagonal disk disclosed herein is also shown and used to form the MEHM as described in Example 2.

[0056] If the mold is formed to facilitate the formation of a bag-shaped MEHM, the mold can be in the form of a bag, preferably a spherical or elliptical bag. In a particularly preferred embodiment, the mold is formed by inner and outer spherical or ellipsoidal walls, and more preferably, the inner and outer spherical or ellipsoidal walls are inflatable. Examples of bag-shaped molds can be found in the descriptions disclosed herein. Figure 8 As seen in A. Furthermore, WO2008 / 058917 A1 and EP2842581 A1 disclose methods for generating sac-like engineered heart tissue, as described therein. Figure 1 As shown. Therefore, those skilled in the art are generally aware of the generation of pouch-shaped engineered heart tissue. However, those skilled in the art are unaware of multilayer pouch-shaped EHMs, in which the reconstructed mixture (forming the MEHM) is perforated by at least two rod-like structures. As mentioned above, the mold can be 3D printed to adjust, for example, the radius of the pouch. After the pouch-shaped generation of the MEHM is completed, the pouch is separated from the mold and can be cut (if necessary).

[0057] If the mold is formed to facilitate the formation of a cylindrical MEHM, the mold can have a cylindrical form; preferably, the mold is a cylindrical mold. In a particularly preferred embodiment, the cylindrical mold is formed by a cylindrical inner wall and a cylindrical outer wall. In another embodiment, the cylindrical mold is formed by a single cylindrical wall. The single cylindrical wall can be covered with a reconstituted mixture by centrifugation. Exemplary cylindrical molds are shown as disclosed herein. Figure 8 In section B, as mentioned above, the mold can be 3D printed to adjust, for example, the length or diameter of a cylinder. A major advantage of cylindrical MEHMs is that larger MEHMs can be produced with a relatively small footprint. Therefore, multiple MEHMs can be generated in parallel. Cylindrical MEHMs can be cut along their long axis before potential use, such as in a patient.

[0058] As exemplary Figure 2 , Figure 7 and Figure 8As shown, the reconstructed mixture is perforated by at least two rods. In one embodiment, the rods are flexible, thereby enabling the introduction of mechanical loads onto the MEHM. Generally, rods are crucial in the fabrication of MEHMs because they serve at least two purposes: first, the rods penetrating the MEHM create channels to provide adequate oxygen and nutrient supply to the cells within the reconstructed mixture / MEHM. Second, the rods provide physical stimulation to the developing muscle. Those skilled in the art know that chemical and physical stimulation is essential for the development of engineered muscle. In other words, the rods support the force development of the MEHM, as muscle training is crucial for muscle development. In this embodiment, the channels created by the perforated rods allow the MEHM to be perfused with culture medium in steps (ii) and (iii), and optionally with maturation medium in step (iv), and with oxygen in steps (ii), (iii), and optionally (iv). In a particularly preferred embodiment, during steps (ii), (iii) and / or optionally (iv), the perfusion of culture medium and / or maturation medium results in adequate nutritional support for cardiomyocytes and non-myocellular cells.

[0059] In a preferred embodiment, the reconstituted mixture is perforated by at least two rods, wherein the perforated rods introduce channels in the reconstituted mixture / MEHM, thereby increasing the surface area of ​​the reconstituted mixture / MEHM. In a further preferred embodiment, the reconstituted mixture / MEHM is perforated by at least two rods, wherein the increase in MEHM surface area controls the diffusion distance of oxygen and nutrients within the MEHM. This increase in surface area provides a reduced average distance from the cells to the surface of the reconstituted mixture / MEHM, allowing the cells to receive adequate oxygen and / or nutrient supply, which can be determined, for example, by a hypoxia sensor (e.g., Hesse et al., 2014) or by cell death (e.g., using well-known viability assay dyes), as further described below. In another preferred embodiment, the interpole 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 The strongly preferred embodiment is shown. Optimal rod spacing is particularly important for MEHMs with a thickness greater than rod spacing, because the distance from a cell to the next perforated rod may be shorter than the distance to the top or bottom surface.

[0060] In one embodiment, the reconstituted mixture is perforated from the bottom and / or from the top by at least two rods. Specifically, the reconstituted mixture can be perforated over its entire thickness. This means the rods extend through the reconstituted mixture. In a particularly preferred embodiment, at least two rods perforate the reconstituted mixture from the bottom, and the mold is in the form of a disc. Furthermore, at least two rods extend from the bottom of the mold through the reconstituted mixture. Even more preferably, the rods are permanently attached to the mold or a base plate. For example, the rods can be securely attached to the casting area of ​​the mold or to a removable base plate. The base plate can also be transferred to a different mold. The advantage of a base plate is that it can be removed if needed, thereby transferring to a different mold (e.g., see...). Figure 8 For example, rods can be an essential part of the mold as permanently attached or removable elements. More preferably, the mold and at least two rod elements are 3D printed, and even more preferably, the mold with at least two rods is 3D printed as a single solid. Even more preferably, the mold including at least two rods is 3D printed as a single solid. The advantage of 3D printing is that the mold (e.g., a disc) can be custom-made and / or tailored. The advantage of the substrate is that the MEHM is removable and can be easily transferred from the mold to the place of use, such as an operating room, in a suitable transport container.

[0061] In another embodiment, at least two rods perforate the reconstitution mixture from the top, and the mold has the form of a disc. In a particularly preferred embodiment, at least two rods are inserted into the liquid reconstitution mixture from the top. For example, at least two rods can be inserted from the top as a grid-shaped comb.

[0062] In another embodiment, at least two rods perforate the reconstituted mixture from the bottom, and the mold is designed to support the formation of the bag. In a particularly preferred embodiment, the bag is formed by inner and outer spherical or ellipsoidal walls. More preferably, at least two rods extend from the inner spherical or ellipsoidal wall, thereby perforating the reconstituted mixture. In a further preferred embodiment, at least two rods are permanently attached to the inner spherical or ellipsoidal wall.

[0063] In another embodiment, at least two rods perforate the reconstituted mixture from the top, and the mold is designed to support the formation of the bag. In a particularly preferred embodiment, the bag is formed by inner and outer spherical or ellipsoidal walls. In a further preferred embodiment, at least two rods extend from the outer spherical or ellipsoidal wall, thereby perforating the reconstituted mixture. Even more preferably, at least two rods are permanently attached to the outer spherical or ellipsoidal wall.

[0064] In another embodiment, at least two rods perforate the reconstructed mixture from the bottom, and the mold is a cylindrical mold. In a preferred embodiment, the cylindrical mold is formed by an inner cylindrical wall and an outer cylindrical wall. More preferably, at least two rods extend from the outer cylindrical wall through the reconstructed mixture, thereby perforating the mixture. In a more preferred embodiment, the rods are permanently attached to the outer cylindrical wall. In particular, the outer cylindrical wall comprising at least two rods can be 3D printed as a solid. The advantage of 3D printing is that it can create any shape, such as a cylinder with at least two rods, and the dimensions can be individually adjusted.

[0065] In another embodiment, at least two rods perforate the reconstructed mixture from the top, and the mold is a cylindrical mold. In a preferred embodiment, the cylindrical mold is formed by an inner cylindrical wall and an outer cylindrical wall. In a more preferred embodiment, at least two rods extend from the inner cylindrical wall through the reconstructed mixture. In an even more preferred embodiment, the rods are permanently attached to the inner cylindrical wall. In particular, the inner cylindrical wall comprising at least two rods can be 3D printed as a solid. The advantage of 3D printing is that any shape can be created, such as a cylinder with at least two rods, and the dimensions can be individually adjusted. The mold and / or rods can be produced by any suitable engineering method, including 3D printing, casting, milling, or glass blowing. Specifically, the mold can be manufactured by injection molding or milling in, for example, a PTFE block. In a strongly preferred embodiment, the mold and / or rods are 3D printed.

[0066] The reconstitution mixture is poured into a mold, completely or partially filling the mold, i.e., the casting area. A technician can determine the optimal volume based on the mold dimensions. Typically, the thickness of a liquid reconstitution mixture is from 1 mm to about 10 mm. The optimal volume can be determined by simple multiplication based on the casting area and the desired thickness. In a preferred embodiment, the volume of each liquid reconstitution mixture is 0.5-200 ml, preferably 1-150 ml, more preferably 1.5-100 ml, more preferably 2-50 ml, more preferably 2.5-40 ml, more preferably 3-30 ml, more preferably 3.5-25 ml, more preferably 4-20 ml, more preferably 4.5-15 ml, more preferably 5-11.5 ml, more preferably 6-9.5 ml, even more preferably 6.5-8.5 ml, and most preferably about 8 ml. As disclosed herein, in Figure 2 An exemplary EHM using an 8 ml volume is shown in the figure.

[0067] The mold can hold volumes ranging from 1 to 400 ml. The volume a mold can hold can be determined by multiplying the pouring area by the possible height of the MEHM. For example, if the mold can form a disc, the mold volume can be calculated by multiplying the pouring area by the mold height. Figure 7 A illustrates an exemplary casting mold for producing a hexagonal disk. If the mold is in the form of a bag, the volume of the mold is the difference between the volumes of the outer and inner walls of a spherical or ellipsoidal shape. Similarly, if a cylindrical mold has an outer wall and an inner wall, the volume of the mold can be determined by the volume difference between the outer and inner walls of the cylinder. In the case where the cylindrical mold includes only one outer wall, the cylindrical mold can theoretically accommodate the entire volume of the cylinder. In a preferred embodiment, the volume of the mold is 2-300 ml, more preferably 3-200 ml, more preferably 4-100 ml, more preferably 5-80 ml, more preferably 6-60 ml, more preferably 6.5-50 ml, more preferably 7-40 ml, more preferably 7.5-30 ml, more preferably 8-23 ml, more preferably 13-19 ml, even more preferably 13-17 ml, and even more preferably about 15 ml. As described above, the mold can be patch-shaped, bag-shaped, or cylindrical.

[0068] As described above, the mold has a gating area. The liquid reconstituted mixture is poured into the gating area. In other words, the size of the mold describes the size of the gating area. Furthermore, a technician can determine the ideal gating area of ​​the mold according to their needs. For example, if MEHM is desired to be used as an implant, the mold can be customized using, for example, a 3D printer. In a preferred embodiment, the mold size is 1-400 cm. 2 Preferred size: 2-300 cm 2More preferably 3-200 cm 2 More preferably 4-100cm 2 More preferably 5-80 cm 2 More preferably 6-60 cm 2 More preferably 7-50 cm 2 More preferably 8-40 cm 2 More preferably 9-30 cm 2 More preferably 10-23 cm 2 More preferably 12-19 cm 2 Even better is 13-17 cm. 2 More preferably, about 13-17 cm 2 In a strongly preferred embodiment, a mold is used to form a patch-like MEHM.

[0069] As described above, the reconstructed mixture is perforated by at least two rods. In a preferred embodiment, the reconstructed mixture is perforated by at least three rods. In a more preferred embodiment, the rods are arranged in a grid, more preferably, wherein the rods are arranged in a triangular or rectangular grid, and even more preferably, in a triangular grid. Exemplary triangular grid arrangements are, for example, those disclosed herein. Figure 7 As shown in the diagram. However, technicians are able to determine other suitable grid arrangements.

[0070] The appropriate number and diameter of the perforated rods in the reconstructed mixture depend on the size of the mold. The perforated rods serve two purposes: first, they ensure the formation of channels to provide sufficient oxygen and nutrients to the cells within the reconstructed mixture; second, they support the training of developing muscles, i.e., they support muscle exertion. Technicians can determine the optimal number of rods based on the mold size. Through simple experiments, those skilled in the art can determine whether cells are not receiving adequate oxygen and / or nutrient supply. For example, Hesse et al. (2014) described the use of hypoxia sensors in engineered tissues such as engineered cardiomyocytes to determine if cells are suffering from hypoxia. Furthermore, if cells do not receive sufficient oxygen and / or nutrient supply, they may die, which can also be determined experimentally using staining methods. Additionally, technicians can determine the optimal number and diameter of rods by assessing the contractile force of the MEHM. As mentioned above, contraction can be measured by FAC or FOC, as described, for example, in Tiburcy et al. (2017) or Zimmermann et al. (2000). In a preferred embodiment, the reconstituted mixture is perforated by at least 5 rods, more preferably at least 7 rods, more preferably at least 14 rods, even more preferably at least 20 rods, even more preferably at least 30 rods, even more preferably at least 37 rods, and most preferably at least 52 rods. As described herein... Figure 7 A and Figure 7 As shown in C, the exemplary casting molds illustrate 52 rods and 14 rods, respectively. To the inventor's knowledge, there is no fixed upper limit to the number of rods. For example, the reconstructed mixture can have up to 2000 rods, preferably up to 1500, and even more preferably up to 1000 perforations. According to the invention, those skilled in the art can freely combine the limits of the number of rods. In another particularly preferred embodiment, the diameter of the rods 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. Figure 7 As further shown in C, different diameters from 0.8 to 1.1 mm have also been experimentally tested. In a particularly preferred embodiment, the reconstructed mixture can be perforated by at least seven rods, wherein the rods are arranged in a grid, and wherein the diameters of the rods form a gradient along a planar axis. An example of a diameter gradient is disclosed herein. Figure 7 As shown in C. Furthermore, the gradient of the diameter produces a technical effect where the FAC measured along this gradient is greater than the FAC measured along a rod of the same rod thickness. Experimental data also support this finding, such as... Figure 7 As shown in C.

[0071] For example, such as Figure 7 The casting mold shown in A comprises 52 rods, and the volume of each liquid reconstitution mixture is 8 ml. Those skilled in the art can also deduce the volume of the reconstitution mixture and the number of rods from this embodiment.

[0072] In a preferred embodiment, each rod has a base area, and the base can be circular, rectangular, elliptical, triangular, or polygonal; preferably, the base area is circular. Circular base areas of the rods are also shown, for example, herein. Figure 7 and Figure 8 middle.

[0073] Advantageously, the rod generates a restoring force to simulate heart wall stress in normal and / or infarcted hearts during the contraction cycle. A restoring force is a force that acts to bring the object to its equilibrium position. As mentioned above, the rod is typically flexible to introduce a mechanical load onto the MEHM. Therefore, the rod ideally possesses elastic properties (flexibility). Generally, the elastic properties of the rod are quantified by the elastic modulus, or Young's modulus, which defines the magnitude of stress required to achieve a unit strain. For example, a higher modulus indicates that the material is less prone to deformation. Typically, the SI unit for this modulus is Pascal (Pa). For example, Rump et al. (2007) and Pislaru et al. (2014) described the viscoelastic properties of normal and infarcted myocardium. Ideally, engineered tissue simulates the contractile properties of normal and / or infarcted myocardium (approximately 0.3–60 mN / mm²). 2 (Wiegerinck et al., 2009; Muleri et al., 1992) and viscoelastic (5 to 27 kPa; Rump et al., 2007) properties.

[0074] In a particularly preferred embodiment, the restoring force of the rod ideally matches the wall tension of normal and / or diseased myocardium (10 to 200 kDain / cm). 2Or 1 to 20 kPa; Fujita et al., 1993). The elastic properties can be defined by the working spring rate. In this case, the working spring rate can be evaluated at the point where the MEHM contacts the rod. Generally, spring rate is defined as the magnitude of the force required to deform the spring by one unit distance. The unit of measurement for spring rate is typically N / mm (Newtons per millimeter) or mN / mm (millinewtons per millimeter). In a strongly preferred embodiment, the elastic properties of the rod, defined by the 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 rod matches the wall tension of a normal and / or diseased human heart, even more preferably, where the wall tension is 1 to 20 kPa. Fujita et al. (1993) also disclosed the aforementioned range.

[0075] The elastic properties of rods can exhibit different elastic moduli to establish biomechanical anisotropy. In other words, the elastic modulus varies among rods. In another embodiment, the elastic properties of rods exhibit similar elastic moduli to establish biomechanical isotropy. In other words, the elastic modulus is uniform among rods.

[0076] In another embodiment, the lower part of the rod is surrounded by a circumferential wall, which forms a ring-shaped ramp around the rod, and the ring-shaped ramp tapers gradually from the bottom upwards. Figure 4 B describes exemplary dimensions of the circumferential wall for a ring-shaped EHM. Furthermore, WO 2017 / 207431 A1 describes the circumferential wall in detail. The advantage of the circumferential wall at the base of the rod is that the densified reconstructed mixture moves upward along the slope due to the development of densification and / or muscular forces. This upward sliding due to the development of densification and / or muscular forces facilitates the addition of additional reconstructed mixture from the bottom, as described in step (iii) of the method.

[0077] In another embodiment, the rod tapers in a conical shape, with its diameter reaching its maximum at the bottom. This gradual taper from bottom to top has similar advantages to circumferential walls: the EHM can be separated from the mold and slid upwards to facilitate the addition of additional remodeling mixtures, particularly from the bottom.

[0078] The technician knows the appropriate conditions for performing tissue culture experiments. In a preferred embodiment, steps (i), (ii), and / or optional (iv) are performed in a temperature range of 36.4-37.6°C, preferably in a temperature range of 36.6-37.4°C, more preferably in a temperature range of 36.8-37.2°C, and more preferably at about 37°C. Furthermore, in another preferred embodiment, steps (i), (ii), and / or optional (iv) are performed in a humidified cell culture 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. Generally, culturing can be carried out under ambient oxygen or an oxygen carrier, such as hemoglobin-based oxygen carrier (HBOC) and perfluorocarbon-based oxygen carrier (PFOC) conditions (Iyer et al., 2007). In another preferred embodiment, steps (i), (ii), and / or optionally (iv) are carried out in a humidified cell culture 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 understand from common sense that the stated temperature and CO2 ranges are standard tissue culture conditions in the art. Additionally, 21% O2 corresponds to the typical concentration of oxygen in dry air.

[0079] In step (i), the liquid reconstituted mixture gels. When the reconstituted mixture gels, it is no longer a liquid—in other words, it is non-liquid. In a preferred embodiment, the gelation is characterized by the reconstituted mixture obtained by step (i) being gel-like. The gelation of the reconstituted mixture is due to the collagen in it. For example, if highly purified bovine corium (skin) type I acid-soluble collagen is used in the reconstituted mixture, the pH of the reconstituted mixture is adjusted to a physiological pH, for example, between 7.0 and 8.0. At this pH shift, the collagen forms a gel. In fact, the ideal conditions for collagen to form a gel (3D scaffold) are a pH of around 7. Biochemically, natural collagen molecules are covalently cross-linked hydroxylysine and lysine residues. Therefore, in vitro, by cross-linking at 37°C and a near-neutral pH, natural collagen molecules are able to self-assemble into collagen fibers and form hydrogels. This self-assembly gelation process can be initiated by a pH shift and occurs optimally at 37°C. The physicochemical properties of collagen hydrogels are influenced by collagen concentration, polymerization pH, and ionic strength during crosslinking. This process is known to those skilled in the art and has been used in various tissue engineering applications to form 3D scaffolds (e.g., Tiburcy et al., 2017). In a particularly preferred embodiment, 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 approximately 7.4.

[0080] In another preferred embodiment, the gelation in step (i) is characterized in that, if added to a mold, the culture medium surrounds the reconstituted mixture and does not dissolve it. A technician can assess the gelation of the reconstituted mixture, for example, by testing whether the culture medium dissolves the reconstituted mixture after different time points. This test can also be performed in experimental tubes during the manufacture of the MEHM. Furthermore, the gelation in step (i) can be characterized by the reconstituted mixture being opaque, preferably by methods such as those described by Tiburcy M et al. (2014). Figure 3 The visual inspection shown in B is used to determine this.

[0081] In the strongly preferred embodiment, step (i) is performed for at least 15 minutes, preferably up to 24 hours, more preferably 20 minutes to 15 hours, more preferably 30 minutes to 8 hours, even more preferably 45 minutes to 1.5 hours, and most preferably about 1 hour.

[0082] In another embodiment, 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. However, those skilled in the art can determine suitable culture media. For example, Tiburcy et al. (2017), (2020), and Schlick et al. (2019) provide exemplary media suitable for culturing EHM. Each factor, such as IGF-1, VEGF, and / or TGFβ1, can be replaced by factors with the same or similar effects. For example, TGFβ1 can be replaced with any other factor that stimulates non-myocellular cells (particularly stromal cells) to initiate densification of the remodeling mixture.

[0083] In a preferred embodiment, the basal medium for the culture medium is selected from Iscove medium, αMEM medium, DMEM medium, and RPMI medium, preferably Iscove medium or αMEM medium, more preferably Iscove medium. However, any suitable basal medium may be used in this method. The basal medium is commercially available or can be produced according to publicly available formulations, such as those in the ATCC catalog. Amino acids may be supplemented to the basal medium if deemed appropriate. In another preferred embodiment, the medium may contain a serum-free supplement. The serum-free supplement may provide the final concentration as defined above in the culture medium. In another preferred embodiment, the culture medium in step (ii) may contain 0.4-10 mM L-glutamine, preferably 0.8-6 mM L-glutamine, more preferably 1.2-5 mM L-glutamine, more preferably 1.5-4 mM L-glutamine, more preferably 1.7-3 mM L-glutamine, and most preferably about 2 mM L-glutamine. In a very strongly preferred embodiment, the culture medium in step (ii) may contain 30-3000 µM ascorbic acid or a derivative thereof, preferably 100-1000 µM ascorbic acid or a derivative thereof, more preferably 180-500 µM ascorbic acid or a derivative thereof, more preferably 220-370 µM ascorbic acid or a derivative thereof, more preferably 270-330 µM ascorbic acid or a derivative thereof, and most preferably about 300 µM ascorbic acid or a derivative thereof. An example of an ascorbic acid derivative is ascorbic acid-2-phosphate.

[0084] In another preferred embodiment, the culture medium in step (ii) comprises 10-1000 ng / ml IGF1, preferably 50-500 ng / ml IGF1, more preferably 70-200 ng / ml IGF1, even more preferably 90-120 ng / ml IGF1, and most preferably about 100 ng / ml IGF1. In particular, the IGF1 may be human IGF1.

[0085] In another preferred embodiment, the culture medium in step (ii) contains 2.5-10 ng / ml VEGF, preferably 3-9 ng / ml VEGF, more preferably 3.5-8 ng / ml VEGF, more preferably 4-7 ng / ml VEGF, more preferably 4.5-6 ng / ml VEGF, and most preferably about 5 ng / ml VEGF. Specifically, the VEGF can be human VEGF, preferably, wherein the VEGF is VEGF. 165 .

[0086] In another preferred embodiment, the culture medium in step (ii) comprises 5-20 ng / ml FGF-2, preferably 6-18 ng / ml FGF-2, more preferably 7-16 ng / ml FGF-2, more preferably 8-14 ng / ml, more preferably 9-12 ng / ml FGF-2, and most preferably about 10 ng / ml FGF-2. In particular, the FGF-2 may be human FGF-2.

[0087] In another preferred embodiment, the culture medium in step (ii) contains 2-8 ng / ml of TGFβ1, preferably 3-7 ng / ml of TGFβ1, more preferably 4-6 ng / ml of TGFβ1, even more preferably 4.5-5.5 ng / ml of TGFβ1, even more preferably about 5 ng / ml of TGFβ1, and most preferably, the TGFβ1 is human TGFβ1.

[0088] In another preferred embodiment, 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.

[0089] In step (ii), the reconstructed mixture is densified. Step (ii) yields a densified reconstructed mixture. In a preferred embodiment, the densification of the reconstructed mixture in step (ii) reduces the volume of the original reconstructed mixture 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%, as preferably determined by visual inspection and / or video-optical analysis. For example, assuming the EHM volume densification is symmetrical in all dimensions, the EHM volume can be evaluated by a planimetric method. Furthermore, the thickness can be precisely determined, for example, by ultrasound, laser interferometry, or optical coherence tomography. The EHM volume can be determined by combining planar area and thickness measurements. In a preferred embodiment, completion of step (ii) is determined by the following steps: (a) generating an engineered heart muscle (EHM) in a ring-like form parallel to the MEHM by performing steps (i) and (ii), and (b) determining that the engineered heart muscle (EHM) in the ring-like form is detached from the mold, as in Tiburcy M et al., Collagen-based engineered heartmuscle, Methods Mol Biol. 2014;1181:167-176. Figure 3 As described in C. The detachment from the mold can be easily evaluated by generating the ring-shaped EHM in parallel, since the detachment is readily visible. For example, in this paper… Figure 5 C also shows a clear detachment of the annular EHM from the mold. In a further preferred embodiment, the completion of step (ii) can be further determined by generating an annular EHM parallel to the MEHM through steps (i) and (ii), wherein the densification of the annular reconstructed mixture reduces the cross-sectional area of ​​the original reconstructed mixture 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%. This densification can be evaluated by visual inspection and / or video-optical analysis of the EHM. Figure 5 C serves as an example to demonstrate the optimal densification of the toroidal form of EHM.

[0090] In a particularly preferred embodiment, step (ii) is performed for at least 12 hours, preferably up to 15 days, more preferably 12 hours to 7 days, more 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.

[0091] In step (iii), an additional reconstitution mixture is added from the top and / or bottom to the reconstitution mixture of step (ii). In a preferred embodiment, in step (iii) a), an additional reconstitution mixture is added to the top of the densified reconstitution mixture of step (ii). In another preferred embodiment, in step (iii) a), an additional reconstitution mixture is added from the bottom to the densified reconstitution mixture of step (ii). In another preferred embodiment, in step (iii) a), an additional reconstitution mixture is added from both the top and bottom to the densified reconstitution mixture of step (ii). In a more preferred embodiment, in step (iii) a), an additional reconstitution mixture is coated from the top and / or bottom onto the densified reconstitution mixture of step (ii). For example, the surface areas of the densified reconstitution mixture of step (ii) and the additional reconstitution mixture of step (iii) a) are the same. For ease of illustration, Figure 3 The five-layer EHM shown is generated by coating the densified reconstructed mixture of step (ii), wherein the additional reconstructed mixture of step (iii) a) has the same surface area as the densified reconstructed mixture of step (ii). In another example, in step (iii) a), the additional reconstructed mixture is coated from the top or bottom onto the restricted area of ​​the densified reconstructed mixture of step ii). The surface area of ​​the additional reconstructed mixture of step (iii) a) may be smaller than the surface area of ​​the reconstructed mixture of step (ii). For ease of illustration, Figure 8 C2 describes an inlet that can be placed on the densified remodeling mixture of step (ii) to provide a smaller-sized liquid remodeling mixture on top of the densified remodeling mixture of step (ii). For example, the smaller surface area of ​​the remodeling mixture of step (iii) a) can be adjusted according to the patient's specific defect or another desired shape. The advantage of doing so is that the MEHM for cardiac repair can be more enforced in a specific defined area compared to the rest of the MEHM.

[0092] In another embodiment, the surface area of ​​the additional reconstituted mixture is greater than that of the densified reconstituted mixture of step (ii). For example, in step (iii) b), the different molds have a larger casting area than the mold of step (i), preferably wherein the additional reconstituted mixture coats the densified reconstituted mixture of step (ii) at least from the top or from the bottom. More preferably, the additional reconstituted mixture coats the densified reconstituted mixture of step (ii) from the top or from the bottom and fills the larger casting area of ​​the different molds. For ease of illustration, Figure 8 C1 describes how a smaller, denser remodeling mixture has been transferred into a mold with a larger surface area. For example, in step (iii) b), the larger surface area of ​​the different molds can be adjustable according to the patient's specific defect. The advantage of doing so is that the MEHM for cardiac repair can be customized to specific needs.

[0093] Additional EHM layers are created by adding a further reconstruction mixture in step (iii). Thus, adding the additional reconstruction mixture thickens the EHM layer by layer. The desired thickness can be determined by a technician. For example, MEHM can be used as an implant in the human heart. Preferably, the desired thickness and size can be determined by imaging the human defect using existing imaging techniques such as echocardiography, magnetic resonance imaging, or computed tomography. In a preferred embodiment, step (iii) a) or (iii) b) is repeated at least twice, 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, still more preferably at least nine times, and most preferably at least ten times. In a more preferred embodiment, step (iii) a) or (iii) b) is repeated 2-200 times, preferably 2-100 times, more preferably 2-80 times, more preferably 2-70 times, more preferably 2-60 times, more preferably 2-50 times, more preferably 2-40 times, more preferably 3-30 times, more preferably 3-25 times, more preferably 4-20 times, more preferably 4-15 times, more preferably 4-10 times, even more preferably 5-9 times, and most preferably 5-8 times. However, those skilled in the art can determine how many times step (iii) should be repeated, and whether step (iii) a) or (iii) b) should be performed at all. Those skilled in the art will use the desired dimensions and geometry as a guide to assess whether these layers should have the same surface area, or whether individual layers should be smaller or larger. For example, a technician may require a cardiac implant. Therefore, the surface area of ​​a single layer will be customized according to the size of the myocardial defect. Furthermore, the thickness of the MEHM is ideally matched to the thickness of healthy tissue. For example, the thickness of a densified remodeling mixture can be, for example, about 500 µm. To achieve the desired heart wall thickness of up to 10 mm (Kawel et al. (2012)), 20 layers of compacted remodeling mixtures can be layered. In another instance, the compacted remodeling mixture can be thinner than 500 µm. Therefore, it might be necessary to layer 50 remodeling mixtures to obtain the typical heart wall thickness. Technicians can determine how many remodeling mixtures are needed to obtain a MEHM with typical human heart thickness and / or patient myocardial defect thickness.

[0094] After completing step (iii), the EHM can be further matured in a maturation medium. Exemplary compositions of maturation media can be found, for example, in Tiburcy et al. (2017) or Tiburcy et al. (2020). In a particularly preferred embodiment, the maturation medium in step (iv) is limited to the aforementioned culture medium, except that TGFβ1 is omitted from the maturation medium compared to the culture medium. In an even more preferred embodiment, step (iv) is carried out for 4-200 days, preferably 6-150 days, more preferably 8-120 days, more preferably 9-110 days, more preferably 10-90 days, more preferably 15-70 days, more preferably 20-50 days, and most preferably 28-42 days.

[0095] In a preferred embodiment, the MEHM of step (iii) is cultured in a suitable maturation medium in step (iv), thereby the MEHM of step (iv) exhibits enhanced maturation, as determined by an increase in the abundance of cardiomyocyte sarcomeric proteins, such as those determined by fluorescence microscopy after antibody labeling of one or more of the proteins, for example, α-sarcomylin, myosin heavy chain protein, myosin light chain protein and troponin, Western blotting, flow cytometry or RNA sequencing to determine the abundance of their respective protein-coding transcripts, as described in Tiburcy et al. Circulation 135(19)1832-1847 (2017). Furthermore, when compared with the MEHM of step (iii), the MEHM of step (iv) may exhibit enhanced force exertion, wherein the enhanced force exertion is determined by measuring the fractional area change (FAC) using the method described in Circulation 135(19)1832-1847 (2017) by Tiburcy et al., and wherein the fractional area change (FAC) of the MEHM of step (iv) upon electrical stimulation 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%.

[0096] A particular advantage of the method disclosed herein is that the cells within the reconstructed mixture / MEHM receive adequate nutrition and oxygen at all times. This adequate nutrient supply is ideally achieved by consistently creating channels through perforated rods during the manufacturing process to ensure a minimum acceptable surface-tissue diffusion distance, while still producing a thickened MEHM, which can be used, for example, as an implant. For instance, the distance between the rods can be 3.5 mm, resulting in a diffusion distance of 1.75 mm, from the rod (tissue surface) to the tissue core (the center between two rods). Figure 8 The distance (as described further above) is the diffusion distance from the tissue surface (rods or top / bottom surfaces) to the tissue core. In other words, the diffusion distance is kept optimal. This diffusion distance can be adjusted by the number and design of the perforated rods. The optimal surface-tissue diffusion distance can be determined by tissue hypoxia measurements (e.g., using the ODD-Luc hypoxia reporter gene described in Hesse AR et al. (2014)) or cell viability assays (e.g., using well-known viability dyes). Viability dyes, also known as live / dead stains, are known in the art and are available from various suppliers. For example, intracellular esterases can hydrolyze dyes in living cells to produce hydrophilic, strongly fluorescent compounds, allowing fluorescence signals to be measured in living cells at, for example, Ex / Em = 485 / 530 nm. In dead cells, the dye enters the damaged cell membrane and may undergo a 40-fold fluorescence enhancement upon binding to nucleic acids, resulting in bright red fluorescence in dead cells (Ex / Em = 495 / 635 nm). In MEHM formulations, the surface-tissue distance is defined by the distance between the perforated rods. Figure 8 The example shown is a suitable 3.5 mm inter-rod spacing, resulting in a maximum surface-core diffusion distance of 1.75 mm. For instance, if a 3.5 mm inter-rod spacing is chosen, the maximum surface-tissue core diffusion distance is 1.75 mm regardless of the MEHM thickness. Those skilled in the art can determine and adjust the optimal inter-rod spacing in the MEHM as needed based on the information provided herein to ensure oxygen and nutrients are provided throughout the MEHM of the desired size and thickness. For example, when a monolayer EHM is produced via steps (i), (ii), and (iv), an 8 ml liquid reconstitution mixture is densified to approximately 0.8 ml, as... Figure 3As shown on the right side of C. Such monolayer EHMs can be between 0.5 mm and 1 mm thick. Therefore, the remodeling mixture can be densified by approximately 90% throughout the process. However, ideally, the thickness of clinically used artificial heart tissue is at least 5 mm to maintain the stress of the beating heart and support its rhythm. As a thought experiment, and to obtain an EHM with a thickness of at least 5 mm using a single remodeling mixture, the technician would need to pour at least 80 ml of the remodeling mixture, which corresponds to a thickness of 50 mm (5 cm) of liquid remodeling mixture. However, cells at the center of a single remodeling mixture of this thickness will not survive in this method because they will suffer necrosis at the core of the remodeling mixture due to hypoxia and lack of nutrients, subsequently leading to tissue densification. The introduction of perforated rods circumvents this limitation because it establishes the required optimal surface-tissue-core diffusion distance to ensure adequate oxygen and nutrient supply throughout the MEHM.

[0097] Another key advantage of the method disclosed herein is that the layers are added sequentially. Thus, each additional reconstructed mixture is fused into the densified reconstructed mixture of step (ii). Due to this fusion, the resulting MEHM originates from several layers, yet these layers are inseparable. In other words, the formed MEHM behaves like a single layer of muscle because the individual layers fuse to form a single entity. This is a particular advantage compared to the fusion of individually stacked engineered cardiac muscle disclosed in WO2007054286, Zimmermann et al. 2006, and Naito et al. 2006.

[0098] In a preferred embodiment, hypoxia is measured using a hypoxia reporter gene. In a particularly preferred embodiment, cardiomyocytes in the MEHM receive adequate oxygen supply, preferably wherein oxygen supply is measured by the ODD-Luc hypoxia reporter gene, as described in Hesse AR et al. (2014). Figure 3 As shown in D, this implementation scheme is also supported by experimental evidence. In the experiment, the cardiomyocytes of the innermost layer of MEHM and the cardiomyocytes of the EHM monolayer contained the ODD-Lux hypoxia reporter gene. From Figure 3As can be seen from the comparison of D, monolayer EHM actually exhibits greater hypoxia than 5-layer EHM. In a particularly preferred embodiment, cardiomyocytes in MEHM receive adequate oxygen supply, as determined by the ODD-Luc hypoxia reporter gene as described in Hesse AR et al. (2014). Preferably, adequate oxygen supply is ensured if the mean 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, obtained by the steps (i), (ii), and optional (iv) of this method. In an even more preferred embodiment, cardiomyocytes in MEHM receive adequate oxygen supply, as determined by the ODD-Luc hypoxia reporter gene as described in Hesse AR et al. (2014), and wherein the mean relative luminescence of cardiomyocytes in MEHM is not greater than the mean relative luminescence measured in cardiomyocytes of monolayer EHM, for example in Figure 3 This was experimentally demonstrated in C.

[0099] On the other hand, multilayer engineered myocardium (MEHM) obtained by the methods disclosed herein is also described. Furthermore, multilayer engineered myocardium (MEHM) obtainable by the methods disclosed herein is also described.

[0100] In another aspect, a multilayer engineered myocardium (MEHM) is disclosed, wherein the MEHM comprises (a) collagen and (b) a mixture of cardiomyocytes and non-myocellular cells, and wherein the MEHM comprises at least two layers. The MEHM is preferably a patch, pouch, or cylinder. More preferably, the thickness of the MEHM is at least 0.2 mm. Further preferably, the MEHM is produced by a repetitive and sequential layering method, wherein the MEHM originates from 2-200 layers. Further preferably, these layers have been fused together, thereby increasing the thickness of the MEHM. Those skilled in the art will understand that the cell mixture requires a culture medium to provide nutrients to the cells. Even more preferably, each layer of the MEHM is derived from a reconstructed mixture, wherein the reconstructed mixture comprises (a) collagen, (b) a mixture of cardiomyocytes and non-myocellular cells, and (c) a suitable reconstructing medium.

[0101] In a preferred embodiment, a multilayer engineered myocardium (MEHM) is described, wherein the MEHM is characterized by having been generated by a repeated and sequential layering method, wherein the MEHM originates from 2-200 layers, wherein these layers have been fused together and thereby increased the thickness of the MEHM, wherein each layer originates from a remodeling mixture, and wherein each remodeling mixture comprises (a) collagen, (b) a mixture of cardiomyocytes and non-myocellular cells, and (c) a suitable remodeling medium.

[0102] More preferably, the MEHM is capable of contraction, as measured by determining the fractional area change (FAC) of the MEHM using the method described by Tiburcy et al. in Circulation 135(19)1832-1847 (2017). In an even more preferred embodiment, the FAC is at least 0.5% during 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%.

[0103] In a highly preferred embodiment, the MEHM is derived from 2-100 layers, preferably 2-80 layers, more preferably 2-70 layers, more preferably 2-60 layers, more preferably 2-50 layers, more preferably 2-40 layers, more preferably 3-30 layers, more preferably 3-25 layers, and even more preferably 4-20 layers. For example, for manufacturing MEHMs for use as human implants, 20 layers with an individual fully compacted thickness of 0.5 mm for each remodeling mixture may be an ideal setting.

[0104] Since there are at least two layers, the thickness of MEHM can be from 0.2 mm to about 30 mm, preferably from about 0.3 mm to about 30 mm, more preferably from about 0.5 mm to about 30 mm, more preferably from about 0.7 mm to about 25 mm, more preferably from about 0.9 mm to about 20 mm, more preferably from about 1 mm to about 17 mm, more preferably from about 2.3 mm to about 15 mm, more preferably from about 2.8 mm to about 14 mm, more preferably from about 3.3 mm to about 13 mm, more preferably from about 3.8 mm to about 12 mm, more preferably from about 4.2 mm to about 11 mm, more preferably from about 4.6 mm to about 10.5 mm, and most preferably from about 5 mm to about 10 mm.

[0105] More preferably, the MEHM is not vascularized and / or is not controlled by the central nervous system. In a preferred embodiment, the MEHM is perforated by at least two rod-shaped perforations of similar diffusion distance to ensure nutrient and oxygen supply to the cellular mixture. For example, cardiomyocytes in the MEHM receive adequate oxygen supply, preferably wherein oxygen supply is determined by the ODD-Luc hypoxia reporter gene, as described by Hesse AR et al. (2014). Specifically, cardiomyocytes in MEHM can receive adequate oxygen supply, as determined by the ODD-Luc hypoxia reporter gene as described in Hesse AR et al. (2014). Preferably, adequate 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 higher than that in monolayer EHM. Monolayer EHM can be obtained by the steps (i), (ii), and optionally (iv) of the method disclosed herein.

[0106] Particularly preferred is that the cardiomyocytes in MEHM receive adequate oxygen supply, as determined by the ODD-Luc hypoxia reporter gene described in Hesse AR et al. (2014), and that the mean relative luminescence of the cardiomyocytes in MEHM is no greater than the mean relative luminescence measured in cardiomyocytes in a monolayer EHM.

[0107] In a preferred embodiment, the MEHM is obtained through any embodiment or combination of embodiments of the methods disclosed herein. The MEHM disclosed herein may exhibit any characteristics relating to the methods disclosed herein.

[0108] On the other hand, MEHM is obtained by performing steps (i)-(iii) of the method disclosed herein.

[0109] In another aspect, an engineered heart muscle, preferably an engineered heart patch, an engineered heart bag, or an engineered heart cylinder, is disclosed, wherein the EHM comprises (a) collagen, (b) a mixture of cardiomyocytes and non-myocellular cells, and wherein the EHM has a thickness of at least about 0.6 mm. Preferably, the EHM is a MEHM and comprises at least two layers.

[0110] On the other hand, the use of MEHM obtained by the methods disclosed herein, or MEHM disclosed herein, in in vitro models for drug screening is also described. In particular, MEHM can be used for drug toxicity screening or drug efficacy screening in in vitro models.

[0111] Furthermore, the use of MEHMs obtained by the methods disclosed herein, or MEHMs disclosed herein, in the in vitro fabrication of engineered human myocardium is described. In a preferred embodiment, the MEHM has a three-dimensional shape to match a patient-specific myocardial defect, more preferably, wherein the patient-specific myocardial defect is assessed by MRI, ultrasound, computed tomography, positron emission tomography, and / or optical coherence tomography. All of these methods are known to those skilled in the art because they are standard practice in the field. After assessing the three-dimensional patient-specific myocardial defect, those skilled in the art can generate a MEHM with customized dimensions. For example, those skilled in the art can produce the MEHM based on the patient's myocardial wall thickness, preferably based on the patient's myocardial wall thickness before the defect occurred.

[0112] MEHMs obtained using the methods disclosed herein, or MEHMs disclosed herein, can also be used as research tools.

[0113] In another aspect, the use of MEHM obtained through the methods disclosed herein, or the MEHM disclosed herein, is also contemplated for pharmaceutical purposes. In particular, the use of MEHM obtained through the methods disclosed herein, or the MEHM disclosed herein, is contemplated for cardiac repair. For example, the MEHM can be an implant. This implant can be used to treat patients suffering from heart failure. In a preferred embodiment, the MEHM is permanently applied to the patient's heart. This permanent application of the MEHM to the heart can be achieved through standard surgical procedures, such as suturing, stapling, or gluing. For example, the MEHM can be sutured to the patient's heart.

[0114] Finally, a method for treating an individual suffering from heart failure is also disclosed, comprising administering MEHM obtained by the method disclosed herein to the patient's heart. In a preferred embodiment, the method comprises attaching MEHM to the individual's heart, and wherein attachment preferably comprises suturing the MEHM to the heart.

[0115] The present invention is further described through the following embodiments:

[0116] 1. A method for manufacturing multilayer engineered myocardial tissue (MEHM), the method comprising the following steps: (i) A liquid reconstitution mixture is provided in a mold, wherein the reconstitution mixture is perforated by at least two rods. The reconstitution mixture comprises (a) collagen, (b) a mixture of cardiomyocytes and non-cardiomyocytes, and (c) a suitable reconstitution medium, wherein the reconstitution mixture gels in a mold. (ii) The mixture obtained in step (i) in the mold is cultured in a suitable culture medium, thereby reconstructing the densification of the mixture in the mold; (iii) a) Adding a further liquid reconstitution mixture as defined in step (i) from the top and / or bottom to the densified reconstitution mixture obtained in step (ii), whereby the further liquid reconstitution mixture gels, and then incubating under the same conditions as in step (ii), whereby the further reconstitution mixture densifies in a mold; or b) Transfer the densified reconstituted mixture obtained from step (ii) into different molds, wherein the reconstituted mixture is perforated by at least two rods, and then step (iii) a) is performed in the different molds; This allows for the creation of multilayer engineered myocardium (MEHM). Preferably, the MEHM is thickened by repeating step (iii) a) and / or step (iii) b) at least once, and (iv) Optionally, the MEHM from step (iii) in the mold is cultured in a suitable maturation medium. The MEHM is capable of contraction.

[0117] 2. The method according to embodiment 1, wherein the contractile capacity of the MEHM is assessed by visual inspection and / or by determining the ability of the reconstructed mixture of step (i) to form a force-generating engineered myocardium by performing steps (i), (ii), and optionally (iv), and wherein the engineered myocardium is capable of generating a contractile force of at least 0.05 mN (FOC), as in the appendix to Circulation 135(19)1832-1847(2017) by Tiburcy et al. Figure 6 The measurements were taken under the standard equidistant conditions in C.

[0118] 3. The method according to embodiment 2, wherein the contraction of the MEHM is further determined by parallel evaluation of the ability of the reconstructed mixture of step (i) to form a ring-shaped engineered human muscle generating force by performing steps (i), (ii) and (iv), as described in Tiburcy et al., Circulation 135(19)1832-1847(2017), and wherein the ring-shaped engineered myocardium generates a contractile force (FOC) of at least 0.05 mN, as described in Tiburcy et al., Circulation 135(19)1832-1847(2017). Figure 6 The result measured in C.

[0119] 4. The method according to any one of the foregoing embodiments, wherein the contraction force (FOC) generated by the MEHM is 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.

[0120] 5. The method according to any one of the foregoing embodiments, wherein the thickness of each layer of MEHM is 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.

[0121] 6. The method according to any one of the foregoing embodiments, wherein the thickness of the MEHM is about 0.2 mm to about 30 mm, preferably about 0.3 mm to about 30 mm, 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 1.5 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.

[0122] 7. The method according to any one of the foregoing embodiments, wherein the shrinkage of MEHM is measured video-optically by a fractional area change (FAC) measurement.

[0123] 8. The method according to any one of the foregoing embodiments, wherein the contraction is determined by measuring the fractional area change (FAC) of the MEHM using the method described in Circulation 135(19)1832-1847(2017) by Tiburcy et al., and wherein the fractional area change (FAC) is at least 0.5% during 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%.

[0124] 9. The method according to any one of the foregoing embodiments, wherein the pH of the reconstituted mixture is 7.0 to 7.8, preferably 7.2 to 7.6, more preferably 7.3 to 7.5, and most preferably, wherein the pH of the reconstituted mixture is about 7.4.

[0125] 10. The method according to any one of the foregoing embodiments, wherein the reconstituted mixture provides a final concentration of collagen of 0.5 mg / ml to 3 mg / ml, preferably 0.55 mg / ml to 2.5 mg / ml, more preferably 0.6 mg / ml to 2.2 mg / ml, more preferably 0.65 mg / ml to 2 mg / ml, more preferably 0.7 mg / ml to 1.75 mg / ml, more preferably 0.75 mg / ml to 1.5 mg / ml, more preferably 0.8 mg / ml to 1.2 mg / ml, even more preferably 0.85 mg / ml to 1 mg / ml, and most preferably about 0.9 mg / ml.

[0126] 11. The method according to any one of the foregoing embodiments, wherein the collagen in step (i) is selected from 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.

[0127] 12. The method according to any one of the foregoing embodiments, wherein at least 90% of the collagen in the reconstituted mixture of step (i) is type I collagen.

[0128] 13. The method according to any one of the foregoing embodiments, wherein the collagen in the reconstituted mixture of step (i) is medical grade.

[0129] 14. The method according to any one of the foregoing embodiments, wherein the collagen in the reconstituted mixture of step (i) is of bovine, equine, human or marine origin, preferably of bovine origin.

[0130] 15. The method according to any one of the foregoing embodiments, 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 naturally occurring or synthetic extracellular matrix components, more preferably wherein the extracellular matrix components are selected from elastin, laminin, entactin, nidogen, proteoglycans, glycosaminoglycans and fibronectin or synthetic mimics thereof.

[0131] 16. The method according to any one of the foregoing embodiments, wherein the cell mixture in step (i) provides 1 × 10⁻⁶ cells in the reconstituted mixture. 6 -26.5×10 6 cells / ml, preferably 2×10⁻⁶ cells / ml 6-13.2×10 6 cells / ml, more preferably 3×10⁻⁶ cells / ml 6 -10×10 6 Cells / ml, more preferably 3.5 × 10⁻⁶ cells / ml. 6 -9×10 6 Cells / ml, more preferably 4×10⁻⁶ cells / ml 6 -8×10 6 Cells / ml, more preferably 4.3 × 10⁻⁶ cells / ml. 6 -7×10 6 Cells / ml, more preferably 4.6 × 10⁶ cells / ml. 6 -6×10 6 Cells / ml, more preferably about 5×10⁻⁶ cells / ml. 6 The final cell concentration is cells / ml.

[0132] 17. The method according to any one of the foregoing embodiments, wherein the cardiomyocytes are human cardiomyocytes.

[0133] 18. The method according to any one of the foregoing embodiments, wherein the cardiomyocytes are derived from embryonic stem cells, wherein the cells are not produced using methods involving modification of human germline genetic characteristics or methods involving the use of human embryos for industrial or commercial purposes.

[0134] 19. The method according to any one of the foregoing embodiments, 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.

[0135] 20. The method according to any one of the foregoing embodiments, wherein cardiomyocytes are obtained by serum-free differentiation, preferably wherein the cardiomyocytes are obtained by following the protocol of WO2015 / 040142 or alternatives such as those reviewed 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).

[0136] 21. The method according to any one of the foregoing embodiments, wherein the cardiomyocytes are non-human primate stem cell-derived, fetal, or neonatal cardiomyocytes.

[0137] 22. The method according to any one of the foregoing embodiments, wherein cardiomyocytes express ACTN2, TTN, RYR2 and troponin, as determined by flow cytometry or RNA sequencing.

[0138] 23. The method according to any one of the foregoing embodiments, wherein the cell mixture in step (i) comprises at least 10% cardiomyocytes, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, and most preferably at least 50%.

[0139] 24. The method according to any one of the foregoing embodiments, wherein the contraction of the MEHM is caused by myocardial cells, thereby generating the contractile force of the MEHM.

[0140] 25. The method according to any one of the foregoing embodiments, wherein the non-muscle cells are selected from one or more of stromal cells, endothelial cells, smooth muscle cells and mesenchymal stem cells, preferably wherein the non-muscle cells are stromal cells or endothelial cells, more preferably wherein the non-muscle cells are stromal cells, even more preferably wherein the stromal cells are cardiac stromal cells, even more preferably wherein the cardiac stromal cells have fibroblast characteristics, and even more preferably wherein the cardiac stromal cells are fibroblasts.

[0141] 26. The method according to any one of the foregoing embodiments, wherein non-muscle cells express CD90, as determined by flow cytometry, preferably wherein non-muscle cells express CD90, CD74 and CD44, and more preferably wherein non-muscle cells secrete extracellular matrix proteins, particularly collagen, and / or express integrins to promote cell-matrix interactions, wherein non-muscle cells compact the reconstitution mixture.

[0142] 27. The method according to any one of the foregoing embodiments, wherein the non-muscle cells are derived from embryonic stem cells, wherein the cells are not produced using methods involving modification of human germline genetic characteristics or methods involving the use of human embryos for industrial or commercial purposes.

[0143] 28. The method according to any one of the foregoing embodiments, 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 wherein the non-muscle cells are derived from induced pluripotent stem cells.

[0144] 29. The method according to any one of the foregoing embodiments, wherein the non-muscle cells are derived from living tissue obtained from a human individual, preferably from a patient, and even more preferably from a patient for autologous or allogeneic administration of MEHM.

[0145] 30. The method according to any one of the foregoing embodiments, wherein the non-muscle cells are obtained by serum-free differentiation, preferably, wherein the non-muscle cells are obtained by following 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. The scheme obtained under fully defined conditions, Nat Protoc. 2017Sep;12(9):1890-1900, was obtained.

[0146] 31. The method according to any one of the foregoing embodiments, wherein the non-muscular cells of the cell mixture in step (i) are stromal cells, preferably, wherein the stromal cells are human stromal cells.

[0147] 32. The method according to embodiment 31, wherein the cell mixture in 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% matrix cells.

[0148] 33. The method according to any one of the foregoing embodiments, wherein non-muscular cells, preferably stromal cells, are capable of densifying the reconstructed mixture in step (ii).

[0149] 34. The method according to any one of the foregoing embodiments, wherein the reconstitution medium comprises (a) a basal culture medium and (b) a serum-free supplement.

[0150] 35. The method according to embodiment 34, wherein the serum-free supplement provides albumin at a final concentration of 0.5-50 mg / ml, transferrin at 1-100 μg / ml, ethanolamine at 0.1-10 μg / ml, sodium selenite at 0.003-0.3 μg / ml, L-carnitine hydrochloride at 0.4-40 μg / ml, hydrocortisone at 0.1-10 μg / ml, fatty acid supplement at 0.05-5 μl / ml, and triiodo-L-thyroxine (T3) at 0.0001-0.1 μg / ml.

[0151] 36. The method according to embodiment 34 or 35, wherein the serum-free supplement further comprises one or more of the following components: vitamin A, D-galactose, linoleic acid, linolenic acid, progesterone, and putrescine.

[0152] 37. The method according to any one of embodiments 34-36, wherein the serum-free supplement is an insulin-free B27 supplement.

[0153] 38. The method according to any one of embodiments 34-37, wherein the serum-free supplement is 2-6% (v / v) of an insulin-free B27 supplement, preferably wherein the serum-free supplement is 4% (v / v) of an insulin-free B27 supplement.

[0154] 39. The method according to any one of embodiments 34-38, wherein the basal medium in step (i) is selected from Iscove medium, RPMI, αMEM and DMEM or a mixture thereof, preferably, wherein the basal medium is selected from Iscove medium, RPMI, αMEM or a mixture thereof, more preferably, wherein the basal medium is a mixture of Iscove medium and RPMI.

[0155] 40. The method according to any one of embodiments 34-39, wherein the reconstitution medium further comprises (c) 35-790 μM ascorbic acid, (d) 5-500 ng / ml IGF-1, (e) 0.3-26 ng / ml VEGF, (f) 0.5-53 ng / ml FGF-2 and (g) 0.5-10 ng / ml TGFβ1; Preferably, the reconstructing medium further comprises (c) 75-300 uM ascorbic acid, (d) 26-105 ng / ml IGF-1, (e) 1.3-5.2 ng / ml VEGF, (f) 2.6-10.5 ng / ml FGF-2 and (g) 1-6 ng / ml TGFβ1; More preferably, 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.

[0156] 41. The method according to embodiment 40, wherein VEGF is VEGF 165 .

[0157] 42. The method according to any one of the foregoing embodiments, wherein the mold is formed to facilitate the formation of a planar EHM, a bag-shaped EHM, or a cylindrical EHM.

[0158] 43. The method according to embodiment 42, wherein the mold is formed to facilitate the formation of a planar EHM, preferably wherein the mold is in the form of a disc, preferably wherein the disc is in the form of a circular or polygonal disc, more preferably wherein the mold is in the form of a hexagonal disc, most preferably wherein the disc is in the form of a patch-like shape; or wherein the mold forms a confined three-dimensional space, preferably wherein the three-dimensional space is in the form of a circular or polygonal three-dimensional space, more preferably wherein the mold is in the form of a hexagonal three-dimensional space, and even more preferably wherein the three-dimensional space is in the form of a patch-like shape.

[0159] 44. The method according to embodiment 42, wherein the mold is formed to facilitate the formation of a bag-shaped EHM, preferably wherein the mold has the form of a bag, more preferably wherein the mold has the form of a spherical or elliptical bag, even more preferably wherein the mold is formed by inner and outer spherical or ellipsoidal walls, and more preferably wherein the inner and outer spherical or ellipsoidal walls are inflatable.

[0160] 45. The method according to embodiment 42, wherein the mold is formed to facilitate the formation of a cylindrical EHM, preferably wherein the mold has a cylindrical form, more preferably wherein the mold is a cylindrical mold, even more preferably wherein the cylindrical mold is formed by inner and outer spherical or ellipsoidal walls; or wherein the cylindrical mold is formed by a single cylindrical wall, and wherein the cylindrical mold is covered by the reconstructed mixture by centrifugation.

[0161] 46. ​​The method according to any one of the foregoing embodiments, wherein the reconstructed mixture is perforated by at least two rods, and wherein the rods are flexible, thereby enabling the introduction of mechanical loads onto the MEHM.

[0162] 47. The method according to any one of the foregoing embodiments, wherein the rod allows MEHM to be perfused with culture medium in steps (ii) and (iii), and optionally, MEHM to be perfused with maturation medium in step (iv), and MEHM to be perfused with oxygen in steps (ii), (iii) and optionally (iv), preferably wherein the culture medium perfusion and maturation medium perfusion result in nutritional support for cardiomyocytes and non-myocellular cells during steps (ii), (iii) and / or optionally (iv).

[0163] 48. The method according to any one of the foregoing embodiments, wherein the reconstructed mixture is perforated from the bottom or from the top by the at least two rods, preferably wherein the reconstructed mixture is perforated over its entire thickness.

[0164] 49. The method according to embodiment 48, wherein at least two rods perforate the reconstructed mixture from the bottom, and wherein the mold is in the form of a disc, preferably wherein at least two rods extend from the bottom of the mold through the reconstructed mixture, more preferably wherein the rods are permanently attached to the mold or base plate, more preferably wherein the rods are permanently attached to a casting area of ​​the mold or to a removable base plate of the mold, and even more preferably wherein (a) the mold and at least two rods or (b) the removable base plate and at least two rods are 3D printed as a single entity.

[0165] 50. The method according to embodiment 48, wherein at least two rods perforate the reconstitution mixture from the top, and wherein the mold is in the form of a disc, preferably wherein at least two rods are inserted into the liquid reconstitution mixture from the top.

[0166] 51. The method according to embodiment 48, wherein at least two rods perforate the reconstituted mixture from the bottom, and wherein the mold has the form of a bag, preferably wherein the bag is formed by inner and outer spherical or ellipsoidal walls, more preferably wherein at least two rods extend from the inner and outer spherical or ellipsoidal walls, and more preferably wherein the rods are permanently attached to the inner and outer spherical or ellipsoidal walls.

[0167] 52. The method according to embodiment 48, wherein at least two rods perforate the reconstituted mixture from the top, and wherein the mold has the form of a bag, preferably wherein the bag is formed by inner and outer spherical or ellipsoidal walls, more preferably wherein at least two rods extend from the inner and outer spherical or ellipsoidal walls, and more preferably wherein the rods are permanently attached to the outer spherical or ellipsoidal wall.

[0168] 53. The method according to embodiment 48, wherein at least two rods perforate the reconstructed mixture from the bottom, wherein the mold is a cylindrical mold, and wherein the cylindrical mold is formed by inner and outer spherical or ellipsoidal walls, preferably wherein at least two rods extend through the reconstructed mixture from the outer cylindrical wall, more preferably wherein the rods are permanently attached to the outer cylindrical wall, and even more preferably wherein the outer cylindrical wall of the at least two rods is 3D printed as a single entity.

[0169] 54. The method according to embodiment 48, wherein at least two rods perforate the reconstructed mixture from the top, wherein the mold is a cylindrical mold, and wherein the cylindrical mold is formed by an inner cylindrical wall and an outer cylindrical wall, preferably wherein at least two rods extend through the reconstructed mixture from the inner cylindrical wall, more preferably wherein the rods are permanently attached to the inner cylindrical wall, and even more preferably wherein the inner cylindrical wall of the at least two rods is 3D printed as a solid.

[0170] 55. The method according to any one of the foregoing embodiments, wherein the volume of each reconstituted mixture is 0.5-200 ml, preferably 1-150 ml, more preferably 1.5-100 ml, more preferably 2-50 ml, more preferably 2.5-40 ml, more preferably 3-30 ml, more preferably 3.5-25 ml, more preferably 4-20 ml, more preferably 4.5-15 ml, more preferably 5-11.5 ml, more preferably 6-9.5 ml, even more preferably 6.5-8.5 ml, and most preferably about 8 ml.

[0171] 56. The method according to any one of the foregoing embodiments, wherein the volume of the mold is 1-400 ml, preferably 2-300 ml, more preferably 3-200 ml, more preferably 4-100 ml, more preferably 5-80 ml, more preferably 6-60 ml, more preferably 6.5-50 ml, more preferably 7-40 ml, more preferably 7.5-30 ml, more preferably 8-23 ml, more preferably 13-19 ml, even more preferably 13-17 ml, even more preferably 15 ml, and most preferably wherein the mold is used to form an EHM, which is patch-shaped, bag-shaped or cylindrical.

[0172] 57. The method according to any one of the foregoing embodiments, wherein the size of the mold is 1-400 cm. 2 Preferred size: 2-300 cm 2 More preferably 3-200 cm 2 More preferably 4-100 cm 2 More preferably 5-80 cm 2 More preferably 6-60 cm 2 More preferably 7-50 cm 2 More preferably 8-40 cm 2 More preferably 9-30 cm 2 More preferably 10-23 cm 2 More preferably 12-19 cm 2 Even better is 13-17 cm. 2 Even better is 16 cm. 2 The dimensions are preferably chosen such that the mold is used to form the patch-shaped EHM.

[0173] 58. The method according to any one of the foregoing embodiments, wherein the reconstituted mixture is perforated by at least three rods.

[0174] 59. The method according to any one of the foregoing embodiments, wherein the rods are arranged in a grid, preferably, wherein the rods are arranged in a triangular or rectangular grid, preferably, in a triangular grid.

[0175] 60. The method according to any one of the foregoing embodiments, wherein the reconstructed mixture is perforated by at least 5 rods, more preferably at least 7 rods, more preferably at least 14 rods, even more preferably at least 20 rods, even more preferably at least 30 rods, even more preferably at least 37 rods, most preferably at least 52 rods, and most preferably at least 61 rods.

[0176] 61. The method according to any one of the foregoing embodiments, wherein each rod has a base area, and wherein the base is circular, rectangular, elliptical, triangular or polygonal, preferably wherein the base area is circular.

[0177] 62. The method according to any one of the foregoing embodiments, wherein the rods have similar or different elastic properties.

[0178] 63. The method according to any one of the foregoing embodiments, wherein the elastic characteristics of the rod are defined by the working spring stiffness at the point of contact between the MEHM and the rod, preferably wherein the restoring force is 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.

[0179] 64. The method according to any one of the foregoing embodiments, wherein the elastic properties of the rod simulate the wall tension in the human heart, preferably wherein the wall tension is 1 to 20 kPa.

[0180] 65. The method according to any one of the foregoing embodiments, wherein the elastic properties of the rod exhibit different elastic moduli to establish biomechanical anisotropy.

[0181] 66. The method according to any one of embodiments 1-64, wherein the elastic properties of the rods exhibit similar elastic moduli to establish biomechanical isotropy.

[0182] 67. The method according to any one of the foregoing embodiments, wherein the lower part of the rod is surrounded by a circumferential wall, wherein the circumferential wall forms an annular ramp around the rod, and wherein the annular ramp gradually tapers from the bottom upwards.

[0183] 68. The method according to any one of the foregoing embodiments, wherein the rod tapers tapering in a conical shape, and wherein the rod has its maximum diameter at the bottom.

[0184] 69. The method according to any one of the foregoing embodiments, wherein the diameter of the rod 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.

[0185] 70. The method according to embodiment 69, wherein the reconstructed mixture is perforated by at least seven rods, wherein the rods are arranged in a grid, and wherein the diameters of the rods form a gradient along a planar axis, particularly as shown in... Figure 7 As described in C, preferably, the FAC measured along the gradient is greater than the FAC measured along a rod having the same rod thickness.

[0186] 71. The method according to any one of the foregoing embodiments, wherein steps (i), (ii) and / or optional (iv) are performed in a temperature range of 36.4-37.6°C, preferably in a temperature range of 36.6-37.4°C, more preferably in a temperature range of 36.8-37.2°C, and more preferably at about 37°C.

[0187] 72. The method according to any one of the foregoing embodiments, wherein steps (i), (ii) and / or optional (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.

[0188] 73. The method according to any one of the foregoing embodiments, wherein steps (i), (ii) and / or optional (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.

[0189] 74. The method according to any one of the foregoing embodiments, wherein the gelation in step (i) is characterized in that the reconstituted mixture obtained by step (i) is gel-like.

[0190] 75. The method according to any one of the foregoing embodiments, wherein the gelation in step (i) is characterized in that if a culture medium is added to the mold, the culture medium surrounds the reconstituted mixture and does not dissolve the reconstituted mixture.

[0191] 76. The method according to any one of the foregoing embodiments, wherein the gelation in step (i) is characterized by the reconstituted mixture being opaque, preferably by means of, for example, Tiburcy M, Meyer T, Soong PL, Zimmermann WH, Collagen-based engineered heart muscle. Methods Mol Biol. 2014;1181:167-76. Figure 3 The visual inspection measurement shown in B.

[0192] 77. The method according to any one of the foregoing embodiments, wherein step (i) is performed for at least 15 minutes, preferably up to 24 hours, more preferably 20 minutes to 15 hours, more preferably 30 minutes to 8 hours, even more preferably 45 minutes to 1.5 hours, and most preferably about 1 hour.

[0193] 78. The method according to any one of the foregoing embodiments, wherein 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.

[0194] 79. The method according to embodiment 78, wherein (a) the basal medium is selected from Iscove medium, αMEM, DMEM and RPMI, preferably, wherein the basal medium is Iscove medium or αMEM, more preferably, wherein the basal medium is Iscove medium.

[0195] 80. The method according to embodiment 78 or 79, wherein (b) the serum-free supplement is defined in any one of embodiments 35-38.

[0196] 81. The method according to any one of embodiments 78-80, wherein the culture medium in step (ii) comprises 0.4-10 mM L-glutamine, preferably 0.8-6 mM L-glutamine, more preferably 1.2-5 mM L-glutamine, more preferably 1.5-4 mM L-glutamine, more preferably 1.7-3 mM L-glutamine, and most preferably about 2 mM L-glutamine.

[0197] 82. The method according to any one of embodiments 78-81, wherein the culture medium in step (ii) comprises 30-3000 μM ascorbic acid or a derivative thereof, preferably 100-1000 μM ascorbic acid or a derivative thereof, more preferably 180-500 μM ascorbic acid or a derivative thereof, more preferably 220-370 μM ascorbic acid or a derivative thereof, more preferably 270-330 μM ascorbic acid or a derivative thereof, and most preferably about 300 μM ascorbic acid or a derivative thereof.

[0198] 83. The method according to embodiment 82, wherein the derivative of ascorbic acid is ascorbic acid-2-phosphate.

[0199] 84. The method according to any one of embodiments 78-83, wherein the culture medium in step (ii) comprises 10-1000 ng / ml IGF1, preferably 50-500 ng / ml IGF1, more preferably 70-200 ng / ml IGF1, even more preferably 90-120 ng / ml IGF1, and most preferably about 100 ng / ml IGF1.

[0200] 85. The method according to implementation scheme 84, wherein IGF1 is human IGF1.

[0201] 86. The method according to any one of embodiments 78-85, wherein the culture medium in step (ii) contains 2.5-10 ng / ml VEGF, preferably 3-9 ng / ml VEGF, more preferably 3.5-8 ng / ml VEGF, more preferably 4-7 ng / ml VEGF, more preferably 4.5-6 ng / ml VEGF, and most preferably about 5 ng / ml VEGF.

[0202] 87. The method according to embodiment 86, wherein the VEGF is a human VEGF, preferably wherein the VEGF is a VEGF. 165 .

[0203] 88. The method according to any one of embodiments 78-87, wherein the culture medium in step (ii) contains 5-20 ng / ml FGF, preferably 6-18 ng / ml FGF, more preferably 7-16 ng / ml FGF, more preferably 8-14 ng / ml, more preferably 9-12 ng / ml FGF, and most preferably about 10 ng / ml FGF.

[0204] 89. The method according to any one of embodiments 78-88, wherein FGF-2 is human FGF-2.

[0205] 90. The method according to any one of embodiments 78-89, wherein the culture medium in step (ii) comprises 2-8 ng / ml of TGFβ1, preferably 3-7 ng / ml of TGFβ1, more preferably 4-6 ng / ml of TGFβ1, even more preferably 4.5-5.5 ng / ml of TGFβ1, even more preferably about 5 ng / ml of TGFβ1, and most preferably wherein the TGFβ1 is human TGFβ1.

[0206] 91. The method according to any one of embodiments 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.

[0207] 92. The method according to any one of the foregoing embodiments, wherein, as preferably determined by visual inspection or video optical analysis of the EHM volume, the densification of the reconstructed mixture in step (ii) reduces the volume of the original reconstructed mixture 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%, 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%.

[0208] 93. The method according to any one of the foregoing embodiments, wherein the completion of step (ii) is determined by following steps (i) and (ii) to generate an engineered heart muscle (EHM) in a ring-like form parallel to the MEHM, and by determining the disengagement of the engineered heart muscle (EHM) in the ring-like form from the mold after step (ii) is completed, as in Tiburcy M, Meyer T, Soong PL, Zimmermann WH. Collagen-based engineered heart muscle. Methods Mol Biol. 2014;1181:167-176. Figure 3 As described in C.

[0209] 94. The method according to any one of the foregoing embodiments, wherein step (ii) is performed for at least 12 hours, preferably up to 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.

[0210] 95. The method according to any one of the foregoing embodiments, wherein in step (iii) a), an additional reconstitution mixture is added on top of the densified reconstitution mixture of step (ii).

[0211] 96. The method according to any one of embodiments 1-94, wherein in step (iii) a), an additional reconstitution mixture is added from the bottom to the densified reconstitution mixture of step (ii).

[0212] 97. The method according to any one of embodiments 1-94, wherein in step (iii) a), an additional reconstitution mixture is simultaneously added from the top and bottom to coat the densified reconstitution mixture of step (ii).

[0213] 98. The method according to any one of the foregoing embodiments, wherein in step (iii) a), an additional reconstitution mixture is applied from the top and / or bottom to the densified reconstitution mixture of step (ii), preferably wherein the surface area of ​​the densified reconstitution mixture of step (ii) and the additional reconstitution mixture of step (iii) a) is the same.

[0214] 99. The method according to any one of embodiments 1-97, wherein in step (iii) a), an additional reconstitution mixture is applied from the top or bottom to the defined area of ​​the densified reconstitution mixture of step (ii), preferably wherein the surface area of ​​the additional reconstitution mixture of step (iii) a) is smaller than the surface area of ​​the reconstitution mixture of step (ii).

[0215] 100. The method according to any one of embodiments 1-97, wherein in step (iii) b), the different molds have a larger casting area than the mold in step (i), preferably, wherein in step (iii) b), an additional reconstitution mixture is applied from at least the top or bottom to the densified reconstitution mixture of step (ii), more preferably, wherein in step (iii) b), an additional reconstitution mixture is applied from the top or bottom to the densified reconstitution mixture of step (ii) and fills the larger casting area of ​​the different molds, more preferably, wherein the surface area of ​​the additional reconstitution mixture in step (iii) b) is greater than the surface area of ​​the densified reconstitution mixture in step (ii).

[0216] 101. The method according to any one of the foregoing 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.

[0217] 102. The method according to any one of the foregoing embodiments, wherein step (iii)a) or step (iii)b) is repeated 2-200 times, preferably 2-100 times, more preferably 2-80 times, more preferably 2-70 times, more preferably 2-60 times, more preferably 2-50 times, more preferably 2-40 times, more preferably 3-30 times, more preferably 3-25 times, more preferably 4-20 times, more preferably 4-15 times, more preferably 4-10 times, even more preferably 5-9 times, and most preferably 5-8 times.

[0218] 103. The method according to any one of the foregoing embodiments, wherein the maturation medium in step (iv) is defined as the culture medium described in any one of embodiments 60-71 and 73, except that TGFβ1 is omitted from the maturation medium compared to the culture medium.

[0219] 104. The method according to any one of the foregoing embodiments, wherein step (iv) is performed for 4-200 days, preferably 6-150 days, more preferably 8-120 days, more preferably 9-110 days, more preferably 10-90 days, more preferably 15-70 days, more preferably 20-50 days, and most preferably 28-42 days.

[0220] 105. The method according to any one of the foregoing embodiments, wherein in step (iv), the MEHM of step (iii) is cultured in a suitable maturation medium, whereby the MEHM of step (iv) exhibits increased force development compared to the MEHM of step (iii), wherein the increased force development is determined by measuring area change (FAC) using the method described in Tiburcy et al., Circulation 135(19)1832-1847(2017), and wherein the area change fraction (FAC) of the MEHM of step (iv) is 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%.

[0221] 106. The method according to any one of the foregoing embodiments, wherein the cardiomyocytes in the MEHM are adequately supplied with oxygen, preferably wherein the oxygen supply is determined by the ODD-Luc hypoxia reporter gene 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.

[0222] 107. The method according to any one of the foregoing embodiments, wherein the cardiomyocytes in the MEHM are adequately supplied with oxygen as determined by the ODD-Luc hypoxia reporter gene as described in Hesse AR et al. (2014), preferably wherein adequate oxygen supply is ensured if the average relative luminescence of the cardiomyocytes in the 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, by means of the steps (i), (ii) and optional (iv) of the method.

[0223] 108. The method according to any one of the foregoing embodiments, wherein cardiomyocytes in MEHM are adequately supplied with oxygen as determined by the ODD-Luc hypoxia reporter gene as described in Hesse AR et al. (2014), and wherein the mean relative luminescence of cardiomyocytes in MEHM is not greater than the mean relative luminescence measured in cardiomyocytes in a monolayer EHM.

[0224] 109. A multilayer engineered myocardium (MEHM) obtained by the method according to any one of embodiments 1-108.

[0225] 110. A multilayer engineered myocardium (MEHM) that can be obtained by the method according to any one of embodiments 1-108.

[0226] 111. A multilayer engineered heart muscle (MEHM), preferably an engineered heart patch, an engineered heart bag, or an engineered heart cylinder, wherein the MEHM comprises (a) a mixture of collagen and (b) cardiomyocytes and non-myocellular cells, and wherein the MEHM comprises at least two layers.

[0227] 112. The MEHM according to embodiment 111, wherein the thickness of the MEHM is at least 0.2 mm.

[0228] 113. The MEHM according to embodiment 111 or 112, wherein the MEHM is generated by a repeated and sequential layering method. MEHM is derived from layers 2-200. These layers have fused together, thus increasing the thickness of the MEHM.

[0229] 114. The MEHM according to any one of embodiments 111-113, wherein each layer is derived from a reconstructed mixture. Each of these remodeling mixtures comprises (a) collagen, (b) a mixture of cardiomyocytes and non-myocellular cells, and (c) a suitable remodeling medium. MEHMs can shrink, as measured by the fractional change in area (FAC) of MEHMs as described in Tiburcy et al., Circulation 135(19)1832-1847(2017).

[0230] 115. The MEHM according to any one of embodiments 111-114, wherein the MEHM is derived from 2-100 layers, preferably 2-80 layers, more preferably 2-70 layers, more preferably 2-60 layers, more preferably 2-50 layers, more preferably 2-40 layers, more preferably 3-30 layers, more preferably 3-25 layers, and even more preferably 4-20 layers.

[0231] 116. The MEHM according to any one of embodiments 111-115, wherein the FAC is at least 0.5% during 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%.

[0232] 117. The MEHM according to any one of embodiments 111-116, wherein the thickness of the MEHM is from 0.2 mm to about 30 mm, preferably from about 0.3 mm to about 30 mm, more preferably from about 0.5 mm to about 30 mm, more preferably from about 0.7 mm to about 25 mm, more preferably from about 0.9 mm to about 20 mm, more preferably from about 1 mm to about 17 mm, more preferably from about 2.3 mm to about 15 mm, more preferably from about 2.8 mm to about 14 mm, more preferably from about 3.3 mm to about 13 mm, more preferably from about 3.8 mm to about 12 mm, more preferably from about 4.2 mm to about 11 mm, more preferably from about 4.6 mm to about 10.5 mm, and most preferably from about 5 mm to about 10 mm.

[0233] 118. The MEHM according to any one of embodiments 111-117, wherein the MEHM is not vascularized and / or the MEHM is not controlled by the central nervous system.

[0234] 119. The MEHM according to any one of embodiments 111-118, wherein the MEHM is perforated by at least two rod-shaped perforations having similar diffusion distances to ensure the supply of nutrients and oxygen to the cell mixture.

[0235] 120. The MEHM according to any one of embodiments 111-119, wherein the cardiomyocytes in the MEHM receive adequate oxygen supply, preferably wherein the oxygen supply is determined by the ODD-Luc hypoxia reporter gene 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.

[0236] 121. The MEHM according to any one of embodiments 111-120, wherein the cardiomyocytes in the MEHM are adequately supplied with oxygen as determined by the ODD-Luc hypoxia reporter gene as described in Hesse AR et al. (2014), preferably wherein adequate oxygen supply is ensured if the mean relative luminescence of the cardiomyocytes in the 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.

[0237] 122. The MEHM according to any one of embodiments 111-121, wherein the cardiomyocytes in the MEHM are adequately supplied with oxygen as determined by the ODD-Luc hypoxia reporter gene as described in Hesse AR et al. (2014), and wherein the mean relative luminescence of the cardiomyocytes in the MEHM is not greater than the mean relative luminescence measured in the cardiomyocytes of a monolayer EHM.

[0238] 123. The MEHM according to any one of embodiments 111-122, wherein the MEHM is obtained by the method according to any one of embodiments 1-95.

[0239] 124. A multilayer engineered myocardium (MEHM) obtained by performing steps (i)-(iii) of the method according to any one of embodiments 1-102.

[0240] 125. An engineered heart muscle (EHM), preferably an engineered heart patch, an engineered heart bag, or an engineered heart cylinder, wherein the EHM comprises (a) collagen, (b) a mixture of cardiomyocytes and non-myocellular cells, and wherein the thickness of the EHM is at least about 0.6 mm, preferably wherein the thickness of the EHM is at least about 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.

[0241] 126. The EHM according to embodiment 125, wherein the EHM is a multi-layer EHM and includes at least 2 layers.

[0242] 127. Use of MEHM obtained by the method according to any one of embodiments 1-108 or MEHM according to any one of embodiments 109-126 in an in vitro model for drug screening.

[0243] 128. Use of Implementation Scheme 127 in in vitro models for drug toxicity screening or drug efficacy screening.

[0244] 129. Use of a MEHM obtained by the method according to any one of embodiments 1-108 or a MEHM according to any one of embodiments 109-126 in the in vitro fabrication of engineered human myocardium, preferably wherein the MEHM has a three-dimensional shape to match patient-specific myocardial defects, more preferably wherein the patient-specific myocardial defects are evaluated by MRI, ultrasound, computed tomography, positron emission tomography and / or optical coherence tomography.

[0245] 130. The use of a MEHM obtained by the method according to any one of embodiments 1-108 or a MEHM according to any one of embodiments 109-126 as a research tool.

[0246] 131. Use of a multilayer EHM (MEHM) obtained by the method according to any one of embodiments 1-108 or the MEHM according to any one of embodiments 109-126 for pharmaceutical purposes.

[0247] 132. The use of a multilayer EHM (MEHM) obtained by the method according to any one of embodiments 1-108 or the MEHM according to any one of embodiments 109-126 for cardiac repair, preferably for treating patients with heart failure.

[0248] 133. The MEHM according to embodiment 132, wherein the MEHM is permanently applied to the patient's heart, preferably wherein the MEHM is sutured to the patient's heart.

[0249] 134. A method for treating an individual suffering from heart failure, the method comprising administering MEHM to the patient's heart by means of a method according to any one of embodiments 1-108 or as defined in any one of embodiments 109-126.

[0250] 135. The method according to embodiment 134, wherein the method includes attaching MEHM to the heart of an individual, and wherein attachment preferably includes suturing MEHM to the heart.

[0251] Of course, all embodiments disclosed herein can be used alone or in combination with other embodiments. Furthermore, all embodiments disclosed herein are applicable to methods for manufacturing MEHM and to in vitro and in vivo uses of MEHM. Attached Figure Description

[0252] Figure 1 This paper describes an overview of the multilayer engineered myocardium (MEHM) disclosed herein, involving repeated stratification. Typically, newly added layers (reconstruction mixtures) fuse with the previous layer (dense reconstruction mixture). Through repeated stratification, thick and robust engineered myocardium is obtained. “Top stratification” is achieved by adding new reconstruction mixtures on top of the dense reconstruction mixture. An “onion pattern” is obtained by immersing the first layer and the continuously formed fused layer stack in newly added reconstruction mixtures. “Bottom-down stratification” is achieved by adding new reconstruction mixtures from the bottom to the dense reconstruction mixture. This stratification can be repeated indefinitely, taking into account the requirements for oxygen and nutrient diffusion within the cells. Oxygen and nutrient supply is achieved through immersion in an oxygenated culturing medium containing nutrients (particularly glucose, lactate, and / or fatty acids) and by designing to increase tissue surface area, i.e., by creating channels via perforated rods (not shown in the schematic diagram; see [link to diagram]). Figure 2 and Figure 3 The initial first layer is represented by a dashed line; subsequent layers are represented by black.

[0253] Figure 2Scalability of engineered heart muscle. The photographs depict typical engineered heart muscle formats used by the inventors for disease modeling, drug screening, and cardiac repair. The labeled volumes represent the initial volumes of the respective engineered heart muscle remodeling mixtures. Each remodeling mixture can have these exemplary volumes in the layered method for producing MEHMs as disclosed herein. Mechanical loading is achieved by integrating flexible rods into the respective tissue formulations. Geometry and scale can be further adjusted as needed. After completing the self-organization process of one layer of EHM, the EHM thickness (z-dimensional) in the illustrated article is typically 0.5 ± 0.1 mm. By repeating layering, the MEHM is thickened and strengthened as disclosed herein. Scale bar: 1 cm.

[0254] Figure 3 A: The thickness of the EHM is increased through continuous / repeated layering and adequate oxygen and nutrient supply. A: A custom-designed casting mold with a stretch device (composed of 3D-printed flexible rods with a specified spacing fixed to a solid substrate) enables perforated EHM designs and helps developing EHMs or MEHMs overcome specified resistance to produce tensile contraction. B: Schematic diagram of a layered perforated patch design. C: Top and side views of a 5-layer (left) and a monolayer (right) EHM; the 5-layer EHM is approximately 5-6 mm thick, and the monolayer EHM is approximately 1 mm thick. D: Using the human ODD-Luc hypoxia reporter gene iPSC line to construct the intermediate layer in a 5-layer construct (both the 5-layer and monolayer EHMs contain the same amount of ODD-Luc cardiomyocytes, ODD-Lux hypoxia method by Hesse et al., 2014), there is no evidence of enhanced hypoxia in the significantly thickened 5-layer design. The gray shade code on the right represents low and high ODD-Luc activity, a hallmark of hypoxia sensing. The gray scale depicts the luminescence of the reporter cell line. Compared to the background ODD-Luc signal in a single patch (right), the ODD-Luc signal in a 5-layer EHM (left) is lower. In other words, the intensity of the luminescence signal in a single-layer EHM appears to be at least the same, if not brighter, than in a multi-layer EHM. Therefore, the 5-layer EHM is not affected by hypoxia.

[0255] Figure 4 : Multi-well plate format for automated casting in annular form. A: Custom stretcher (flexible rods fixed to the substrate) to support auxotonic contractions of engineered myocardium in annular form, showing typical dimensions of the rods and their arrangement. B: Multi-well plate format for automated casting, parallel culture, and video-optical analysis of 48 engineered myocardium in annular form, with a close-up view of a single well showing the cavity for casting and the flexible rods for maturation under defined mechanical loads (see Tiburcy et al., 2020 for more details).

[0256] Figure 5 EHM formation in a ring-like form. A: EHM is automatically poured into a well plate. B: One hour after pouring, EHM transitions to a non-liquid state (collagen gelation causes the EHM reconstituted mixture to appear opaque). C: Twenty-four hours after pouring, EHM becomes significantly densified and suspends on flexible rods used for applying mechanical loading. D: On day 18 of cultivation, densified EHM is completely suspended on flexible rods used for applying bidirectional mechanical loading and supporting tensile contraction under preload conditions. Scale bar: 5 mm.

[0257] Figure 6 A: Video-optical analysis of EHM contraction with or without stimulation. A: Screenshots from a custom video-optical analysis tool for functional phenotypic analysis of EHM via fractional area change (FAC) measurements. B: A device for electrically stimulating the EHM to induce contraction at a defined rate. Increase the electric field stimulation (typically up to 2 V / cm) until the EHM contracts at a defined beating rate. (C) Alternatively, localized point stimulation of the EHM with contact electrodes can be applied to induce synchronous contraction of the EHM for FAC analysis.

[0258] Figure 7A: A multi-layer EHM casting mold in patch form. A: A custom-designed casting mold with two recesses for securing a base plate (bottom) with rods and a culture medium (top). In the left recess with flexible rods, a densified reconstituted mixture is depicted for illustration. Multiple layers are stacked on top, inserted beneath or around the first layer. B: A cross-section of a recess in the casting mold with vertically perforated rods (grey and white), where the layered EHM is shown in black. C: Examples of rod designs with and without rod thickness gradients; note that the mechanical load applied to the EHM can be controlled by designing the rods (elastic properties, diameter, or rod geometry) to be uniform (no gradient, all rods in this example have a diameter of 0.9 mm) and non-uniform (an example with a gradient from top to bottom, where the rods have diameters ranging from 1.1 mm to 0.7 mm in 0.1 mm steps). D: An example of an EHM that develops isotropic (no gradient; where the rods have the same diameter) or anisotropic (vertical gradient; where the rods have a gradient in diameter) contraction modes. The contraction mode was analyzed by comparing the FAC of the upper and lower parts of the EHM. When all rods have the same diameter, the upper and lower parts of the EHM show similar contraction of approximately 1% FAC. In contrast, the vertical gradient results in an increase in upper contraction force of approximately 1.2% FAC (for rods with larger diameters) and a decrease in lower contraction force of approximately 0.7% FAC (for rods with smaller diameters).

[0259] Figure 8 Multilayer engineered heart muscle tissue (MEHM) in various geometries. Examples of MEHM design. MEHMs can be fabricated in pouch form (A), cylindrical form (B), and patch form (C) with different thicknesses and xy-dimensions (taking the patch form in C as an example). In the geometry of the MEHM pouch (A), the MEHM is cast into a circular or spherical mold with a central element (a rigid or flexible biomechanical loading for densifying the EHM; similar to that described in Zimmermann WH, Yildirim Y, Eschenhagen T: Pouch-like engineered heart muscletissue, WO2008058917 and Yildirim et al. (2007) Circulation). In the example shown, the distance between the rods is 3.5 mm, and the diameter of the rods is 1.5 mm, similar to the preferred embodiment described for the EHM patch ( Figure 7The size, geometry, and spacing between rods can be adjusted as needed to create optimal load and perforation for oxygen and nutrient supply. In the case of a cylindrical geometry (B), the rods can be manufactured pointing inwards or outwards, and preferably, the entire cylinder has outward-pointing rods. Lamination can be supported by centrifugal force (>2 g; "spin coating") caused by controlled rotation. (C) Heterogeneous ("patch-in-patch") multilayer strategies are demonstrated by positioning the perforated patch assembly (including perforated rod elements) in a new mold to accommodate additional lamination (C1) or by isolating a defined casting volume in a larger casting mold design using inserts (C2). This allows for the free design of MEHMs with different thicknesses and xy dimensions to facilitate the fabrication of patches that are matched / individualized to the patient's heart. Example

[0260] The following embodiments are intended to further illustrate the invention, but are not limited thereto. These embodiments describe many technical features, and the invention also relates to combinations of the technical features described in this section.

[0261] Example 1 - Large-scale production of engineered heart muscle (EHM) in ring and patch forms

[0262] As previously described by the inventors in Tiburcy et al. (2017) and Tiburcy et al. (2020), engineered human myocardium can be produced in a ring form as well as as a patch. Engineered cardiac patches are particularly important for use in in vivo for cardiac repair. Figure 2 An overview of the different sizes and formats that have been previously produced and published is provided (Tiburcy et al. (2017) and Tiburcy et al. (2020)).

[0263] Example 2- The EHM thickness is increased by sequential and repeated layering supported by perforated rods to ensure mechanical integrity. Load and adequate supply of oxygen and nutrients

[0264] To produce myocardium for treating patients with heart failure, engineered myocardium ideally needs to provide the strength to support the human heartbeat. The heart wall to be treated is typically 5-10 mm thick in the patient's body, ideally achieving this thickness through implantation, and geometrically suited to meet the specific mechanical support needs of patients with underperforming heart walls.

[0265] To produce thickened (and reinforced) multilayer EHMs, the inventors have developed a continuous and repetitive layering method. Specifically, Figure 1Three different repetitive and sequential layering methods are shown to obtain thickened engineered cardiac patches (note that perforated rods are not included for simplicity). As a general principle, after the first remodeling mixture is densified into a defined layer, it is coated with an additional remodeling mixture. Figure 1 As schematically shown, coating can be applied from the top, bottom, or sides. This process is repeated until the desired tissue thickness is achieved. Any shape and desired XY dimensions (personalized patch design) can be created by designing the casting mold. A prerequisite for the coating / layering process is that the first and subsequent layers form compact tissue. This compact tissue formation is achieved through repeated and sequential layering, as the individual reconstructed mixtures merge together. Tissue compaction depends on the type and content of stromal cells (Tiburcy et al. (2017), Schlick et al. (2019)). The choice of stromal cell type and content is crucial to achieve a compaction reduction of ≥20% of the original reconstructed mixture. Figure 5 BC exemplarily demonstrates the compaction of EHM in a ring-shaped form. This provides structural and physical space to coat the compacted EHM with an additional layer or two surface coating layers from the top and / or bottom. The inventors typically add a new layer 24 hours after the previous layer is cast. These times can be shortened or extended as needed and determined experimentally, as the desired tissue compaction process depends on the stromal cell type and density, the general cell composition, and the composition of the hydrogel and culture medium (e.g., adding an effective concentration of TGFb1 can accelerate the process, as developed for EHM formation in Tiburcy et al. (2017) and WO2017 / 207431). Additional remodeling mixtures can be added to the mold to coat the compacted multilayer engineered myocardium. By performing this method, the EHM thickens layer by layer, thus achieving clinically desired thicknesses of 5 mm to 10 mm or even more for multilayer EHMs. Since the thickness of human cell walls ranges from 5 mm to 10 mm, it is believed that this thickened cardiac patch will also maintain sufficient mechanical stability to support cardiac wall function. Furthermore, the contractility of cardiomyocytes embedded with the EHM is expected to support the contraction of the natural heart.

[0266] Figure 3A depicts a casting mold with a stretching device (including 3D-printed flexible rods with defined spacing fixed to a rigid, removable substrate) to enable perforated EHM designs and facilitate tensile shrinkage of the developing patch against defined resistance. Of course, the dimensions of the casting mold, as well as the number and size of the rods, can also be adjusted. Figure 3 B illustrates a schematic overview of a multi-tiered EHM as disclosed herein. Figure 3 In C, the generated 5-layer EHM and single-layer EHM are shown adjacent to each other. Figure 3 The lower part of C provides a detailed comparison of EHM thickness, showing that the thickness of 5-layer EHM is approximately 5-6 mm. By using the introduced perforated layered design, the overall tissue thickness is unlimited due to the channels created by the perforated rods, provided the diffusion distance is constant. Of course, the distance and size of the rods can be adjusted to increase the number of channels and change the channel size and inter-channel distance.

[0267] A key challenge in obtaining such thick, multilayer engineered cardiomyocytes (MEHMs) is ensuring adequate oxygen and nutrient supply to the cells embedded within the engineered cardiomyocytes. Through a perforated EHM design, the inventors have ensured sufficient oxygen and nutrient supply in any given multilayer design. This is illustrated by the inventors' use of a human ODD-Luc hypoxia reporter gene model (developed by the inventors based on previous experience in mouse models; Hesse et al. (2014)). Cardiomyocytes derived from the ODD-Luc human pluripotent stem cell line in both monolayer and 5-layer (5–6 mm thick) configurations did not exhibit differences in ODD-Luc signal intensity above the background (reported in monolayer EHMs). In the demonstrated EHM design, channel diameter and density are defined by the rod diameter (1.5 mm) and the circumference-to-circumference distance of the rods (3.5 mm). Under these conditions (effective maximum surface distance in the EHM ≤ 1.75 mm), no hypoxia was observed. The type of perforated patch corresponds to the vascular density in the human heart (2,000-5,000 capillaries / mm). 2 This contrasts sharply with the fact that the intercapillary distance is <25 µm. Therefore, this is surprising and contrary to the theoretical assumption that supplying oxygen via diffusion over distances >100 µm would lead to cardiomyocytes sensing hypoxic conditions (Radisic et al., 2005). This inconsistency can be explained by the high hypoxia resistance of pluripotent stem cell-derived cardiomyocytes in the presence of a culture medium containing nutrients (glucose, lactate, and / or fatty acids).

[0268] By performing repeated and sequential layering, and particularly by designing perforated patches, it is ensured that cells within the EHM do not suffer from hypoxia and receive adequate nutrition. Another theoretical option for producing, for example, a 10 mm thick EHM is to directly cast a large volume of the reconstructed mixture in a single casting step. To obtain a 10 mm thick EHM, the thickness of a single reconstructed mixture needs to reach approximately 100 mm. Depending on the stage and degree of tissue densification, oxygen and nutrients need to be supplied to the cells in the mixture at a distance of 1 to 10 cm. Because the EHM is not vascularized, cells embedded in centimeter-scale EHMs will suffer from hypoxia-induced apoptosis and necrosis. To overcome this obstacle, the inventors have developed a method for performing repeated and sequential layering in a casting mold equipped with perforated rods. Figure 3 and Figure 7 This method, as disclosed herein, is superior to other methods because other methods cannot ensure an adequate supply of oxygen and nutrients.

[0269] In addition, it is also crucial to install mechanical support elements through rods to support tension-increasing contractions.

[0270] Materials and methods in Example 2

[0271] Generation of 5-layer EHM Using the reconstructed mixtures described in Tiburcy et al. (2017) and WO2015025030, EHMs were constructed in a custom casting mold. Figure 7 ). Cast the structural mixture (8 mL; see Figure 2 Afterward, within 3 days, EHM compaction was observed to be less than 50% of the original reconstituted volume. After removing the culture medium, a second layer (consisting of 6 mL of the reconstituted mixture—the volume can be adjusted according to the mold design / surface area and intended use, and can also be adjusted according to the EHM compaction) was transferred into the casting mold using a pipette to cover the compacted EHM from top to bottom. For example, a volume of 8 ml or less (e.g., 2 ml) can also be added to cover a defined area (see Example 5 for further explanation). After approximately 1 hour, gelation of the newly transferred reconstituted mixture was complete, and culture medium was added to the mold. This process was repeated until 5 layers were obtained. The volume and timing of the reconstituted mixture used for repeated layering depend on the casting mold size and the compaction of the stromal cell-mediated EHM.

[0272] The generation of hypoxia reporter systems.The piggybac-ODD-Luc plasmid was cloned, containing a firefly luciferase fused to the oxygen-dependent degradation domain (ODD) of HIF1α under the control of the chicken actin promoter (CAG). Under normoxic conditions, the ODD domain is rapidly degraded by the ubiquitin-proteasome system. Under hypoxic conditions, the ODD domain stabilizes, resulting in measurable luminescence (Hesse et al. (2014)). TC1133 iPSCs were electroporated with the piggyBAC-ODDLuc construct and the transposase vector. Forty-eight hours after electroporation, cells were selected for seven days with increasing concentrations of neomycin (250–500–750 μg / mL), followed by manual selection and colony amplification.

[0273] Validation of the hypoxia report system. Pluripotent stem cells expressing firefly luciferase fused to the oxygen-dependent degradation domain (ODD) of HIF1α were lysed under hypoxic conditions (5% or 1% O2) in 1× passive lysis buffer (Dual-Luciferase® Reporter Assay Systems, Promega) containing protease and phosphatase inhibitors (both from Roche). Lysates containing cells or cells-free (lysate buffer only) were mixed with luciferase assay kit II (Dual-Luciferase® Reporter Assay Systems, Promega), and luminescence was measured using a FlexStation 3 multimode microplate reader (molecular device). Mean ± SEM; n=3:

[0274] The ODD-Luc reporter gene from stable genetic integration responded to an increase in luminescence signaling at lower oxygen concentrations, demonstrating the utility of the ODD-Luc model in hypoxia perception in a human pluripotent stem cell model.

[0275] Bioluminescent imaging of the hypoxia reporter gene EHM. ODD-Luc EHMs were immersed in phosphate-buffered saline containing 1 mg / ml XenoLight D-Luciferin (PerkinElmer). The luminescence of the EHMs was imaged at 37°C using an IVISLumina III system (PerkinElmer). The luminescence was observed in… Figure 3The values ​​in D are represented in grayscale. A 5-layer EHM shows less luminescence than a single-layer EHM.

[0276] Example 3- The formation of ring-shaped EHMs in a multi-well plate indicates coagulation in engineered human myocardium. (condensation) process

[0277] Figure 5 A proof-of-principle overview is provided for the coagulation (after 1 hour of gelation) and densification (after 24 hours and 18 days) of the reconstituted mixture to form a ring-shaped force-generating EHM. For example, a ring-shaped EHM can be generated on a custom-designed stretcher (a flexible rod fixed to a substrate) to support tonic contraction of engineered human myocardium. Figure 4 A). As previously described in Tiburcy et al. (2020), 48 EHMs in a ring-shaped configuration can be produced in parallel using a defined perforated plate. Figure 4 B). Porous plates are further described in WO 2017 / 207431. The porous plates are characterized in that each mold is in the form of an annular channel. Each EHM is cast into an annular channel. During the coagulation and densification of the reconstituted mixture, the engineered human myocardium detaches from the annular channel, moves upward, and tightly wraps around two rod-like structures.

[0278] In the first step, as previously described in Tiburcy et al. 2017 or Tiburcy et al. 2020, a reconstructed mixture is provided in a mold of a multi-well plate. When the reconstructed mixture is poured into the mold, it is a viscous, semi-transparent liquid solution. After 1 hour of incubation at 37°C in a humidified incubator containing 5% CO2, the engineered human cardiomyocyte becomes opaque as the reconstructed mixture solidifies primarily through the gelation of a cell-independent collagen hydrogel (see also Tiburcy et al. (2014), Schlick et al. (2019)). Solidification (lasting, for example, 1 hour) Figure 5 B) After that, culture medium is added to the mold, which provides nutrients and growth factors (see, for example, exemplary culture medium compositions in Tiburcy et al. (2017) and Tiburcy et al. (2020)). After incubation, for example, for 24 hours, the loop detaches from the mold, making the space between the reconstituted mixture and the mold visible. Furthermore, after 24 hours, the reconstituted mixture detaches from the annular channel and tightly wraps around the two rods (…). Figure 5 C). The EHM formation process around the flexible rods depends on the stromal cell composition and can be further promoted by adding an effective concentration of TGFb1. Engineered human cardiomyocytes were placed on the flexible rods and maintained in engineered human cardiomyocyte maintenance medium without TGFb1. Over the next 17 days, the mixture further coagulated around the two rods, as... Figure 5As shown in D, a compact and contracting engineered human myocardium is formed. The rods are developed to bend in response to EHM contraction and stretch the EHM to a defined resting length during the EHM relaxation phase to mimic the heart's natural tonic contraction cycle.

[0279] Example 4- Excitability of engineered myocardium (EHM)

[0280] Excitation of cardiomyocytes typically leads to their contraction. To measure contraction within the EHM, Tiburcy et al. (2017) described the fractional area change (FAC) of the patch during contraction. FAC is a video-optical measurement in which the surface area of ​​the patch is compared in both non-contracted and contracted states. Phenotypic analysis and comparison of EHMs can be performed using the aforementioned video analysis tool. Figure 6 A describes a custom-designed video optical analysis tool to facilitate measurement. Figure 6 B illustrates a device for electrically stimulating the EHM to induce its contraction at a defined frequency. The intensity of the electric field stimulation is increased (typically to 2 V / cm). 2 This continues until the EHM is induced to contract at a defined pulsation frequency (typically controlled at 0.1–3 Hz). Alternatively, local point stimulation can be performed using electrodes contacting the EHM to induce synchronous contraction of the EHM at the desired pulsation frequency, followed by FAC analysis. Figure 6 C).

[0281] The fractional area change (FAC) is measured by comparing the EHM surface area during video optical imaging at the maximum contraction (maximum contraction state) and maximum relaxation (maximum relaxation state). The change in surface area is recorded as a function of time.

[0282] Example 5— Various geometries of multilayer engineered myocardial design

[0283] Typically, MEHMs can be generated in various three-dimensional shapes. The general principle is to equip the mold with perforated rods of uniform or non-uniform shape and size to (i) apply mechanical resistance to the forming EHM, and (ii) create perforated channels for nutrient and oxygen supply. In the geometry of the EHM bag (such as...) Figure 8As shown in Figure A), the EHM is cast into a circular or spherical mold with a central element (rigid or flexible, used to apply a biomechanical load to densify the 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). Unlike WO2008058917, a perforated rod-like structure is designed at the top of the central element to reliably generate, for example, a pouch with controlled muscle formation and a thickness >1 mm. Figure 8 In the example shown in A, the distance between the rods is 3.5 mm, and the diameter of the rods is 1.5 mm, similar to the preferred embodiment described for the EHM patch. Figure 7 The size, geometry, and spacing of the rods can be adjusted as needed to produce optimal load and perforation for oxygen and nutrient supply.

[0284] In another instance, MEHM can also be in the form of a cylinder. In the case of a cylindrical geometry ( Figure 8B) The rods can be manufactured to point inward or outward to perforate the reconstitution mixture. A key advantage of cylindrical EHMs is that many EHMs can be produced in parallel with a relatively small footprint. After the cylindrical MEHM is produced, one side of the MEHM can be cut to obtain a patch, or it can be left intact to obtain a MEHM cylinder / tube of the desired size. The MEHM size can be adjusted by freely adjusting the circumference of the inner and / or outer mold cylinder with inward or outward extending / radiating rods. The mold in the form of a cylinder with perforated rods is produced, for example, by 3D printing of the prototype to fit a container. The container then provides the outer wall (e.g., a 50 ml polypropylene or glass tube) for the production of the cylindrical EHM. Sufficient reconstitution mixture is filled into the container to cover the inner or outer side of the cylinder. Layering can be supported by centrifugal force (>2 g; "spin coating") caused by controlled rotation. The production of tubular / cylindrical EHMs in a cylindrical, longitudinally rotating bioreactor should be carried out at >2 g. The insert-type tubular stretcher contains radially projecting rods for perforating the EHM. The centrifugal force experienced by the EHM can be controlled by adjusting the rotation speed to simulate the increase in diastolic pressure during maturation. Furthermore, centrifugation ensures uniform dispersion of the reconstituted mixture, guaranteeing equal EHM thickness. In an outwardly radiating cylindrical design, uniform coating can be further improved by using an integral inner mold cylinder to ensure even distribution of the reconstituted mixture during spin coating. After coating and gelation, culture medium can be added to promote densification. Multilayering can be repeated until the desired layer composition is achieved. Multiple layers can be added to the top and / or bottom of the previous reconstituted mixture.

[0285] Figure 8 C demonstrates another example of MEHM design. Figure 8C illustrates a non-uniform (“patch within a patch”) multilayer strategy to create MEHMs with variable thicknesses to meet the needs of personalized MEHM production (e.g., clinical use for cardiac repair): first, by positioning the perforated patch assembly into a new mold to accommodate additional layering (C1), and second, by isolating a defined casting volume within a larger casting mold design using inserts (C2). This allows the MEHM to be freely designed with (i) variable thickness and (ii) xy dimensions to facilitate the fabrication of a patch that matches / personalizes the patient’s heart. The components can be fabricated by prototyping 3D printing, casting, or other suitable engineering methods, as described by Tiburcy et al. (2017). Molds are typically created by milling or casting from polytetrafluoroethylene, PDMS, or agarose, while the perforated rod assembly is typically printed using 3D printing, for example, using a Connex 350 (Stratasys) 3D printer, or by casting. For 3D printing, biocompatible MED610 polymers can be used as rigid components (e.g., substrates in perforated patch designs or outer mold elements in bag designs) and combined with TangoBlack polymers as flexible components, such as rods and cylinders. sheet Table 1: Composition of serum-free supplement "Insulin-Removing B27" (50× concentration, liquid) 20 ml of 'insulin-removed B27' per 500 ml of culture medium is equivalent to 4% insulin-removed B27 (v / v); Reference List EP2099508 EP20188364.2 EP2842581 A1 EP2840132 B1 WO 2007 / 054286 WO 2008 / 058917 A1 WO 2015 / 025030 WO 2017 / 207431 Bao X, et al. Long-term self-renewing human epicardial cells generated from pluripotent stem cells under defined xeno-free conditions. 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Claims

1. A method for manufacturing multilayer engineered myocardial tissue (MEHM), the method comprising the following steps: (i) A liquid reconstitution mixture is provided in a mold, wherein the reconstitution mixture is perforated by at least two rods. The reconstitution mixture comprises (a) collagen, (b) a mixture of cardiomyocytes and non-cardiomyocytes, and (c) a suitable reconstitution medium, wherein the reconstitution mixture gels in the mold. (ii) The mixture obtained in the mold by step (i) is cultured in a suitable culture medium, thereby densifying the reconstituted mixture in the mold and reducing its volume by at least 50% of the original reconstituted mixture volume; (iii) a) Adding a further liquid reconstituted mixture as defined in step (i) from top and / or bottom to the densified reconstituted mixture obtained in step (ii), whereby the further liquid reconstituted mixture gels, and then incubating under the same conditions as in step (ii), whereby the further reconstituted mixture densifies in the mold; or b) Transfer the densified reconstructed mixture obtained in step (ii) to different molds, wherein the reconstructed mixture is perforated by at least two rods, and then step (iii) a) is performed in the different molds; This allows for the creation of multilayer engineered myocardial tissue (MEHM), and (iv) Optionally, the MEHM from step (iii) in the mold is cultured in a suitable maturation medium. The MEHM described therein is capable of contraction. The MEHM has a thickness of 2 mm to 13 mm, and the rods have a spacing of 2.5 to 6 mm between them. In the multilayer engineered myocardium, the layers have been fused together.

2. The method according to claim 1, wherein the MEHM is thickened by repeating step (iii) a) and / or step (iii) b) at least once.

3. The method of claim 1, wherein the ability of the MEHM to shrink is assessed by visual inspection.

4. The method according to claim 1 or 2, wherein the thickness of the MEHM is 2.8 mm to 13 mm.

5. The method according to claim 1 or 2, wherein the thickness of the MEHM is 3.3 mm to 13 mm.

6. The method according to claim 1 or 2, wherein the thickness of the MEHM is 3.8 mm to 12 mm.

7. The method according to claim 1 or 2, wherein the thickness of the MEHM is from 4.2 mm to 11 mm.

8. The method according to claim 1 or 2, wherein the thickness of the MEHM is from 4.6 mm to 10.5 mm.

9. The method according to claim 1 or 2, wherein the thickness of the MEHM is 5 mm to 10 mm.

10. The method according to claim 1 or 2, wherein the non-muscle cells are selected from one or more of the following: stromal cells, endothelial cells, smooth muscle cells, and mesenchymal stem cells.

11. The method according to claim 1 or 2, wherein the non-muscular cells are stromal cells or endothelial cells.

12. The method according to claim 1 or 2, wherein the non-muscle cells are stromal cells.

13. The method of claim 12, wherein the stromal cells are cardiac stromal cells.

14. The method of claim 13, wherein the cardiac stromal cells have fibroblast characteristics.

15. The method of claim 14, wherein the cardiac stromal cells are fibroblasts.

16. The method according to claim 1 or 2, wherein the MEHM is in the form of a patch, a bag, or a cylinder.

17. The method according to claim 1 or 2, wherein the gel in step (i) is characterized in that the reconstituted mixture is opaque.

18. The method according to claim 1 or 2, wherein the diameter of the rod is from 0.5 mm to 3 mm.

19. The method according to claim 1 or 2, wherein the diameter of the rod is from 0.6 mm to 2.5 mm.

20. The method according to claim 1 or 2, wherein the diameter of the rod is from 0.65 mm to 2 mm.

21. The method according to claim 1 or 2, wherein the diameter of the rod is from 0.7 mm to 1.7 mm.

22. The method according to claim 1 or 2, wherein the diameter of the rod is from 0.8 mm to 1.5 mm.

23. The method according to claim 1 or 2, wherein step (iii) a) or step (iii) b) is repeated 2-200 times.

24. The method according to claim 1 or 2, wherein step (iii) a) or step (iii) b) is repeated 2-100 times.

25. The method according to claim 1 or 2, wherein step (iii) a) or step (iii) b) is repeated 2-80 times.

26. The method according to claim 1 or 2, wherein step (iii) a) or step (iii) b) is repeated 2-70 times.

27. The method according to claim 1 or 2, wherein step (iii) a) or step (iii) b) is repeated 2-60 times.

28. The method according to claim 1 or 2, wherein step (iii) a) or step (iii) b) is repeated 2-50 times.

29. The method according to claim 1 or 2, wherein step (iii) a) or step (iii) b) is repeated 2-40 times.

30. The method according to claim 1 or 2, wherein step (iii) a) or step (iii) b) is repeated 3-30 times.

31. The method according to claim 1 or 2, wherein step (iii) a) or step (iii) b) is repeated 3-25 times.

32. The method according to claim 1 or 2, wherein step (iii) a) or step (iii) b) is repeated 4 to 20 times.

33. The method according to claim 1 or 2, wherein step (iii) a) or step (iii) b) is repeated 4-15 times.

34. The method according to claim 1 or 2, wherein step (iii) a) or step (iii) b) is repeated 4-10 times.

35. The method according to claim 1 or 2, wherein step (iii) a) or step (iii) b) is repeated 5-9 times.

36. The method according to claim 1 or 2, wherein step (iii) a) or step (iii) b) is repeated 5-8 times.

37. A multilayer engineered myocardium (MEHM) obtained by the method according to any one of claims 1-36, wherein the MEHM has a thickness of 2 mm to 13 mm and wherein the MEHM has a rod-to-rod spacing of 2.5 to 6 mm.

38. A multilayer engineered myocardium (MEHM), wherein the MEHM comprises (a) a mixture of collagen and (b) cardiomyocytes and non-myocellular cells, and wherein the MEHM comprises at least two layers and wherein the thickness of the MEHM is 2 mm to 13 mm, and wherein the MEHM has a rod-to-rod spacing of 2.5 to 6 mm. The MEHM mentioned above is generated through a repeated and sequential layering method. The MEHM described therein originates from 2-200 layers, and These layers have fused together, thus increasing the thickness of the MEHM.

39. The MEHM of claim 38, wherein the cardiomyocytes in the MEHM are adequately supplied with oxygen.

40. The MEHM according to claim 38, wherein the MEHM is an engineered heart patch, an engineered heart bag, or an engineered heart cylinder.

41. Use of MEHM obtained by the method of claim 1 or MEHM of claim 38 in the in vitro fabrication of engineered human myocardium.

42. The use according to claim 41, wherein the MEHM has a three-dimensional shape that matches a patient-specific myocardial defect.

43. The use according to claim 42, wherein the patient-specific defect is assessed by MRI, ultrasound, computed tomography, positron emission tomography and / or optical coherence tomography.

44. Use of the multilayer engineered myocardium (MEHM) obtained by the method of claim 1 or the MEHM of claim 38 in the preparation of materials for cardiac repair.

45. The use according to claim 44, wherein the material is used to treat a patient suffering from heart failure.

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