Multilayered massive tissue body, production method thereof, and use thereof

Abstract

To provide means for quickly and easily producing a more massive and functional multilayered tissue body, to provide a tissue suitable for transplantation, especially to provide a multilayered myocardial tissue massive and functional to such an extent as to contribute to contractile force, and to provide a massive multilayered tissue body for use other than as a transplantation material including cultured meat.SOLUTION: A multilayered tissue scaffold comprising two or more fiber sheets each made of a biocompatible polymer and laminated at an interval of about 100 to about 200 μm, wherein the fiber sheet of the lowermost layer has a fiber density sufficient to hold cells, and the fiber sheet of the upper layer has such a fiber density that part of the seeded cells can reach the fiber sheet of the lowermost layer. The method for producing the multilayered tissue culture comprises seeding cells on the fiber sheet of the uppermost layer of the scaffold and culturing the cells in a liquid medium. The multilayered tissue culture is obtained by the method.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a multilayered, thick tissue, its manufacturing method, and its uses. More specifically, the present invention relates to a method for manufacturing a multilayered, thick tissue by seeding and culturing cells on a support having a plurality of fiber sheets stacked at appropriate intervals, a multilayered fiber sheet and a culture device for use in the method, a multilayered, thick tissue obtained by the method, and the use of the multilayered, thick tissue for disease treatment, efficacy and toxicity evaluation of drugs, and other uses. [Background technology]

[0002] There are approximately 800,000 patients with severe heart failure, including myocardial infarction, in Japan, and approximately 180,000 die from this disease annually. Adult cardiomyocytes have poor self-replicative ability, and when myocardial tissue is damaged, it is extremely difficult to repair it. At present, heart transplantation is the only effective treatment. However, there is a serious shortage of donors in Japan, and there are problems such as immune rejection.

[0003] In recent years, attempts have been made to transplant cardiac myocytes or tissues produced by cell or tissue culture into the affected area as an alternative treatment to heart transplantation. It is expected that the establishment of pluripotent stem cells (e.g., embryonic pluripotent stem cells (ES) and induced pluripotent stem cells (iPS)) will enable the recovery of myocardial function through cardiac cell replacement therapy.

[0004] Cardiomyocyte replacement therapy for severe heart failure can be broadly divided into two methods: injection method, injecting cardiomyocytes directly into the affected area, and patch method, attaching a sheet of cardiomyocytes to the affected area. However, a significant number of cardiomyocytes are lost in a failing heart, and both methods require a large amount of cardiomyocytes for treatment. In addition, the injection method has the disadvantage that the injected cells flow out, resulting in low transplant efficiency and low engraftment rate.

[0005] In contrast, the patch method involves attaching a cardiomyocyte sheet to the outside of the epicardium, and is definitely safer than the injection method. Since 2019, four clinical trials using cardiomyocyte sheets have been conducted. Current cardiomyocyte sheet technology has been shown to be effective against ischemic cardiomyopathy due to the paracrine effect mediated by cytokines secreted from the cells, but for severe cardiomyopathy in which cardiomyocyte function has been almost completely lost, the angiogenic effect of cytokines is insufficient, so there is an urgent need to develop highly effective myocardial tissue that can directly act mechanically on the recipient heart.

[0006] In the patch method, efforts are being made to develop three-dimensional tissue and thick myocardial tissue in order to transplant a larger number of cardiomyocytes. In fact, various methods have been attempted to make myocardial tissue three-dimensional, and it has been reported that transplanted tissue can survive for a longer period of time and that cardiac function has been improved (e.g., Non-Patent Documents 1 and 2, etc.). In these methods, cardiomyocyte sheet units are layered to prepare thick myocardial tissue pieces, but in conventional planar culture, as the thickness of the tissue increases, the center and the bottom layer in contact with the culture vessel are susceptible to cell damage due to low nutrition and low oxygen, and a simple layering method has a limit of three layers (approximately 80 μm) (Non-Patent Document 3).

[0007] In order to solve this problem, for example, attempts have been made to supply nutrients and oxygen to the inside of the tissue by co-culturing a cardiomyocyte sheet with vascular endothelial cells to form a blood vessel-like network within the sheet. However, because this endothelial cell network has an immature structure, it is necessary to introduce a vascular network into the tissue to supply nutrients and oxygen even in an in vitro culture system. In order to efficiently introduce a vascular network into a thick myocardial tissue, it has been reported that a tissue piece containing an artery and vein or a collagen gel having a microchannel is used as a vascular bed and perfusion culture is performed (Non-Patent Document 4). Alternatively, a method has been reported in which gelatin hydrogel microspheres are inserted between cardiovascular cell sheets consisting of cardiomyocytes, vascular endothelial cells, and mural cells, making it possible to supply an appropriate space and culture solution within the tissue (Non-Patent Document 5). However, all of these methods require complicated operations or special culture devices, and how to prepare a thick multi-layered myocardial tissue in vitro simply and in a good viable state is still an important problem to be solved in order to provide a myocardial tissue piece suitable for transplantation at a low cost.

[0008] The present inventors have succeeded in producing a 160 μm-thick multilayered myocardial tissue fragment in a single step using a fiber sheet scaffold in which biodegradable nanofibers are unidirectionally aligned, which was produced by electrospinning, and have demonstrated that transplantation of the tissue fragment into an ischemic cardiomyopathy model significantly restores cardiac function (Patent Document 1, Non-Patent Documents 6 and 7). However, the therapeutic effect is limited, and there is a demand for techniques to produce thicker, multi-layered myocardial tissue that can contribute to contractile force, in addition to the paracrine effect due to cytokine secretion.

[0009] In addition, regenerative medicine research using cell / tissue sheets is being conducted in many areas, not limited to myocardial tissue. For example, mesenchymal stem cells (MSCs) are widely used in transplantation therapy due to their pluripotency and ease of availability. The present inventors have produced tissue sheets (approximately 300 μm thick) of umbilical cord-derived MSCs (UC-MSCs) using the oriented fiber sheet and transplanted them into diabetic wound model mice, and found that they showed superior wound healing effects compared to injection methods (Non-Patent Document 8). However, in order to further improve the effects of transplantation therapy, a thick multi-layered tissue structure is still required.

[0010] Meanwhile, cultured meat has been attracting attention as an alternative to conventional meat due to its advantages of not requiring the slaughter of animals, allowing for strict hygiene control, placing a lower burden on the global environment compared to fattening animals for food, and reducing the risk of antibiotic-resistant bacteria, and currently over 70 startups have entered the cultured meat industry, with research and development ongoing for cultured meat of beef, pork, chicken, lamb, duck, fish, crustacean, eel, foie gras, scallops, and more. Many of these use cell sheet engineering, but here too, there is a need to develop manufacturing technology for thicker cultured meat to increase its commercial value. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] WO 2016 / 060260 [Non-patent literature]

[0012] [Non-Patent Document 1] Haraguchi, Y. et al., Nat. Protoc., 7(5): 850-858 (2012) [Non-Patent Document 2] Fleischer, S. et al., Proc. Natl. Acad. Sci. USA, 114(8): 1898-1903 (2017) [Non-Patent Document 3] Shimizu, T. et al., FASEB J, 20(6): 708-710 (2006) [Non-Patent Document 4] Matsuo, T. et al., Sci. Rep., 3: 1316 (2015) [Non-Patent Document 5] Sakaguchi, K. et al., Sci. Rep., 5: 16842 (2013) [Non-Patent Document 6] Li, J. et al., Stem Cell Rep., 9: 1546-1559 (2017) [Non-Patent Document 7] Suzuki, K. et al., J. Heart Lung Transplant., 40(8): 767-777 (2021) [Non-Patent Document 8] Zhang, J. et al., Int. J. Mol. Sci., 23: 12697 (2022) Summary of the Invention [Problem to be solved by the invention]

[0013] Therefore, an object of the present invention is to provide a means for quickly and easily producing a thicker and more functional multi-layered tissue, thereby providing a tissue suitable for transplantation, in particular to provide a multi-layered myocardial tissue that is thick and functional enough to contribute to contractile force. Another object of the present invention is to provide a thick multi-layered tissue, such as cultured meat, for uses other than as a transplant material. [Means for solving the problem]

[0014] As a result of extensive research to achieve the above-mentioned object, the inventors have succeeded in obtaining a multilayered myocardial tissue fragment having a maximum thickness of more than 1 mm, high viability, and improved contractile function and cytokine secretion ability by stacking one or more lower-density oriented fiber sheets at appropriate intervals on an oriented fiber sheet made of a biocompatible (especially biodegradable) polymer, and seeding the laminated sheets from above, where some of the cardiomyocytes pass through the upper fiber sheet and are retained in the lower fiber sheet. When these are cultured, the cardiomyocytes retained on each fiber sheet become multilayered and thickened, and the multilayered myocardial tissues adhere to each other and self-organize, thereby obtaining a multilayered myocardial tissue fragment having a maximum thickness of more than 1 mm, high viability, and improved contractile function and cytokine secretion ability. The inventors also succeeded in producing a multi-layered MSC tissue structure with a thickness of approximately 2 mm by seeding and culturing adipose tissue-derived mesenchymal stem cells (AD-MSCs) on the above-mentioned multi-layered fiber sheet, demonstrating that this technology can also be used to produce tissue sheets other than myocardium. Based on this finding, the present inventors conducted further research and have completed the present invention.

[0015] That is, the present invention provides the following. [Item 1] A multilayered tissue scaffold comprising two or more fiber sheets made of a biocompatible polymer laminated at intervals of about 100 to about 200 μm, the bottommost fiber sheet having a fiber density sufficient to retain cells, and the upper fiber sheets having a fiber density sufficient to allow some of the seeded cells to reach the bottommost fiber sheet. [Item 2] The scaffold according to item 1, wherein each fiber sheet has orientation. [Item 3] The scaffold according to item 1 or 2, wherein each fiber sheet is laminated at equal intervals. [Item 4] The scaffold according to any one of Items 1 to 3, which is formed by laminating 4 to 7 fiber sheets. [Item 5] The scaffold according to any one of Items 1 to 4, wherein the distance between the uppermost fiber sheet and the lowermost fiber sheet is about 500 to about 1000 μm. [Item 6] The scaffold according to any one of items 1 to 5, wherein the biocompatible polymer is biodegradable. [Item 7] The scaffold according to Item 6, wherein the biodegradable polymer is polylactic acid-polyglycolic acid copolymer (PLGA) or a biopolymer. [Item 8] A method for producing a multi-layered tissue culture, comprising seeding cells on the uppermost fiber sheet of the scaffold according to any one of items 1 to 7 and culturing the cells in a liquid medium. [Item 9] Seeding density is 0.7 to 3 × 10 7 cells / cm 2 The method according to item 8, wherein [Item 10] The method according to item 8 or 9, wherein the culture is dynamic culture. [Item 11] The method according to any one of Items 8 to 10, wherein the cell seeding is carried out in the presence of an extracellular matrix. [Item 12] The method according to any one of Items 8 to 11, wherein the cell is a somatic cell induced to differentiate from a pluripotent stem cell. [Item 13] The method according to item 12, wherein the pluripotent stem cells are induced pluripotent stem (iPS) cells. [Item 14] The method according to any one of Items 8 to 13, wherein the cells are cardiomyocytes or mesenchymal stem cells. [Item 15] The method according to any one of Items 8 to 14, wherein the cells are of human origin. [Item 16] The method according to any one of Items 8 to 14, wherein the cells are derived from an animal that produces food. [Item 17] A multi-layered tissue culture obtained by the method according to any one of items 8 to 16. [Item 18] A multi-layered tissue culture comprising the scaffold according to any one of items 1 to 7 and a multi-layered tissue formed across at least two or more fiber sheets in the scaffold, wherein the thickness of the tissue is greater than the thickness of the tissue formed when the same amount of cells is seeded on a single layer of fiber sheet. [Item 19] Seeding density is 0.7 to 3 × 10 7cells / cm 2 17. The tissue culture of claim 16, wherein the tissue culture is [Item 20] The tissue culture according to item 18 or 19, wherein the tissue is myocardial tissue or mesenchymal stem cell tissue. [Item 21] The tissue is myocardial tissue, and compared with a multi-layered myocardial tissue culture formed on a single layer of fiber sheet, the following (a) to (d): (a) High tissue contractility; (b) having a high secretion ability of one or more cytokines selected from vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and transforming growth factor β (TGFβ); (c) When transplanted into a subject with myocardial infarction, it has a high effect on restoring cardiac function. (d) When transplanted into subjects with myocardial infarction, the engraftment rate was high and the degree of fibrosis was low. 20. The tissue culture of item 18 or 19, having one or more characteristics selected from the following: [Item 22] The tissue culture according to Item 18 or 19, wherein the tissue is an edible tissue of an edible animal. [Item 23] A pharmaceutical composition comprising the tissue culture according to any one of items 17 to 21. [Item 24] The pharmaceutical composition according to Item 23, which is for use in transplantation therapy. [Item 25] A method for treating a disease in a mammal, comprising transplanting an effective amount of the tissue culture according to any one of items 17 to 21 into the mammal in need of treatment. [Item 26] A food comprising the tissue culture according to Item 22. [Item 27] ​​A culture device for producing a multilayer tissue culture, comprising a holder capable of fixing the scaffold described in any one of items 1 to 7 at an angle of 15 to 45° to a bottom surface of the device without contacting the bottom surface of the device, and a means for generating a flow of culture medium so that the culture medium flows from the lower fiber sheet side to the upper fiber sheet side. Effect of the Invention

[0016] According to the method for producing a multilayered myocardial tissue using the multilayered scaffold of the present invention, a myocardial tissue culture that is thicker and more functionally excellent than those obtained by conventional methods can be obtained in a single step, so that a myocardial tissue culture that is more suitable for transplantation can be provided quickly and easily. [Brief description of the drawings]

[0017] [Figure 1] The figures show the characteristics of the multi-layer scaffold (MLA) of the present invention, and the apparatus and culture conditions for its dynamic culture. (a) The fiber density (void ratio) of the lowermost high-density fiber sheet and the upper low-density fiber sheet is shown. (b) An overall image of a culture dish in which an MLA is installed and an enlarged photograph of the same. (c) The relationship between the rotation speed of the stirrer and the flow rate of the medium is shown. (d) The flow rate distribution in the culture vessel when the rotation speed is 250 rpm is shown. (e) The shear stress distribution in the culture vessel when the rotation speed is 250 rpm is shown. [Diagram 2] These figures show cross-sectional side views (top) of three types of MLA and a single-layer oriented fiber sheet, their corresponding scanning electron microscope (SEM) images (middle), and HE stained images of sections of multilayered myocardial tissue prepared using them (bottom). [Diagram 3] Side cross-sectional views (left) and SEM images (center) of an MLA in which fiber sheets are oriented perpendicular to each other in alternating layers (top row) and an MLA in which all fiber sheets are oriented in the same direction (bottom row), as well as TnT-stained images (right) of slices of multilayered myocardial tissue prepared using these MLAs. [Figure 4] This figure compares the efficiency of tissue construction into a fiber sheet (top row) and the electrophysiological properties of the resulting myocardial tissue (bottom row) when laminin was added and when it was not added at the time of seeding of cardiomyocytes. [Diagram 5] FIG. 1 shows the results of observing and comparing the myocardial tissues obtained by seeding cardiomyocytes at various seeding densities and culturing them by stationary culture or rotary culture, using HE staining. (a) HE staining image. (b) The thickness of the tissue at each seeding density. [Figure 6]This figure shows the immunostained images and TUNEL stained images of myocardial tissues obtained by seeding cardiomyocytes at various seeding densities and culturing them by dynamic culture. From the left, the seeding densities are 0.5×107 cells / cm2, 1×107 cells / cm2, and 2×107 cells / cm2. From the top, the images show double stained images of TnT2 and CX43, double stained images of α-actinin and MYL2, double stained images of type I collagen and type III collagen, double stained images of TnT2 and fibronectin, and TUNEL stained images. Nuclei were counterstained with DAPI in all cases. The lower row shows the apoptosis rate in myocardial tissues prepared at each seeding density. [Figure 7] FIG. 1 shows the results of in vitro pulsation analysis of myocardial tissues in the MLA group and the control group (monolayer). [Figure 8] 1 shows the concentrations of various cytokines secreted into the culture supernatant of myocardial tissue from the MLA group and the control group (monolayer), and are shown as relative concentrations, with the secretion level in the control group taken as 1. [Figure 9] FIG. 1 shows the results of transplantation of myocardial tissues from the MLA group and the control group into a rat model of myocardial infarction and echocardiographic analysis after transplantation. [Figure 10] Fig. 1 shows engraftment of myocardial tissues from the MLA group and the control group after transplantation into a rat model of myocardial infarction, (a) HE staining and TnT2 immunofluorescence staining images of cardiac sections, (b) Graft / left ventricular area ratio. [Figure 11] FIG. 1 shows the results of comparing tissue fibrosis (a, b), cell size in the border region (c, d), capillary density in the border region (e, f), and inflammatory response (g, h) after transplantation of myocardial tissue from the MLA group and the control group (monolayer) into a rat model of myocardial infarction. [Figure 12] Figure 1 shows an SEM image of the MLA (left) and a HE stained image of a section of a multi-layered MSC tissue produced using the MLA (right). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] [I] Multilayered tissue scaffold The present invention provides a scaffold for multilayered tissue (hereinafter, sometimes referred to as "scaffold of the present invention") in which two or more fiber sheets made of a biocompatible polymer are laminated at a necessary and sufficient interval for cells held in each fiber sheet to contact tissue formed in the adjacent fiber sheet through culture and to proliferate to an extent that they can self-organize. The interval varies depending on the proliferation rate and seeding density of the cells, and may be, for example, 40 to 1000 μm, preferably 80 to 500 μm. When the intended multilayered tissue is a transplant tissue such as myocardial tissue, the interval between the fiber sheets may be, for example, about 100 to about 200 μm. In the scaffold, the fiber sheet at the bottom layer has a fiber density sufficient to hold cells, and the fiber sheets above it have a fiber density such that some of the seeded cells can reach the fiber sheet at the bottom layer.

[0019] As used herein, the term "scaffold" refers to a base material that serves as a foothold for supporting cell adhesion and proliferation. The scaffold of the present invention has a structure in which two or more fiber sheets made of a biocompatible polymer are laminated at the above-mentioned intervals.

[0020] As used herein, the term "fiber sheet" refers to a sheet-like structure in which fibers are accumulated. The fiber sheet used in the present invention may be one in which fibers are randomly accumulated (random fiber sheet) or one in which fibers are accumulated so as to be oriented in one direction, but an oriented fiber sheet is preferred because it can closely mimic the myocardial structure in vivo.

[0021] In this specification, the term "oriented fiber sheet" refers to a fiber sheet in which fibers are accumulated so as to be oriented in one direction, and in particular refers to a sheet in which 60% or more of the fibers constituting the sheet are within ±20° of the orientation direction (0°). Preferably, an oriented fiber sheet is used in which 70% or more, more preferably 80% or more, and even more preferably 90% or more of the fibers constituting the sheet are within ±20° of the orientation direction.

[0022] The material constituting the fibers of the fiber sheet is a biocompatible polymer. In this specification, the term "biocompatible polymer" refers to a polymer that, when attached to or transplanted into a living body, does not cause adverse events such as side effects, and has the property of adapting to the body without being recognized as a foreign body and being eliminated. Depending on the purpose of use, the biocompatible polymer may be one that decomposes in the body (hereinafter referred to as "biodegradable") or one that does not decompose easily in the body. For example, when used to produce tissues to be used for transplantation, biodegradable polymers are preferably used. On the other hand, when used to produce tissues to be used as an evaluation system for the efficacy and / or toxicity of drugs, non-biodegradable polymers may also be preferably used.

[0023] Examples of biodegradable polymers include, but are not limited to, synthetic polymers such as polyvinyl alcohol (PVA), polyglycolic acid (PGA), polylactic acid (PLA), polyethylene glycol (PEG), polyethylene vinyl acetate (PEVA), polyethylene oxide (PE0), polylactic acid-polyglycolic acid copolymer (PLGA), and biopolymers such as gelatin, collagen, and cellulose. A preferred synthetic biodegradable polymer is PLGA. The decomposition rate of PLGA can be adjusted by the polymerization ratio of PLA and PGA. In one embodiment, a biopolymer may be preferably used as the biodegradable polymer.

[0024] Examples of non-biodegradable polymers include, but are not limited to, polystyrene (PS), polycarbonate (PC), polymethyl methacrylate (PMMA), polyvinyl chloride, polyethylene terephthalate (PET), polyamide (PA), and polymethyl glutarimide (PMGI).Preferably, PMGI and PS are included.

[0025] The molecular weight of the biocompatible polymer used in the present invention is not particularly limited as long as it is capable of forming fibers having the diameter described below. For example, in the case of PLGA or PMGI, it can be appropriately selected from the range of 20 to 200 kDa, preferably 50 to 150 kDa, and in the case of PS, it can be appropriately selected from the range of 50 to 500 kDa, preferably 100 to 400 kDa.

[0026] The diameter of the fiber may vary depending on the polymer material used, the concentration of the polymer solution, the manufacturing method, etc. Those skilled in the art can select an optimal diameter depending on the polymer material used and the intended use. For example, when PLGA or PS is used as the material, the fiber diameter is, for example, 2 to 4 μm. The thickness of the fiber sheet is, for example, 1 to 40 μm, preferably 2 to 30 μm, and more preferably 3 to 20 μm.

[0027] The fiber density of the fiber sheet may vary depending on the diameter of the fibers used, but for example, in the case of an oriented PLGA fiber sheet with a fiber diameter of 2 to 4 μm, one having a density of, for example, 10 to 3000 fibers per mm width, preferably 70 to 500 fibers per mm width can be used. Alternatively, the fiber density of the fiber sheet can be expressed by the porosity. For example, in the case of an oriented PLGA fiber sheet with a fiber diameter of 1 to 1.5 μm, one having a porosity of, for example, 20 to 80%, preferably 25 to 75% can be used.

[0028] The fiber sheet at the bottom layer has a fiber density sufficient to retain cells. If the fiber density of the fiber sheet is too low, the seeded cells will pass through without being retained, resulting in cell loss. The fiber density (porosity) of the fiber sheet at the bottom layer is, for example, 20 to 40%, preferably 25 to 35%, and more preferably about 30%. In this specification, "about X" means X±0.1X (X is an arbitrary numerical value).

[0029] The scaffold of the present invention is laminated with one or more fiber sheets in addition to the fiber sheet of the bottom layer. The number of fiber sheets of the upper layer is not particularly limited, and can be appropriately set so as to have an appropriate thickness depending on the intended use of the multilayered tissue. For example, when the multilayered tissue is a transplant tissue such as myocardial tissue, the number of fiber sheets of the upper layer is, for example, 1 to 9 sheets, preferably 2 to 7 sheets, and more preferably 3 to 6 sheets. Therefore, the number of fiber sheets of the scaffold of the present invention including the fiber sheet of the bottom layer is, for example, 2 to 10 sheets, preferably 3 to 8 sheets, and more preferably 4 to 7 sheets. On the other hand, when the multilayered tissue is cultured meat for consumption, a larger number of fiber sheets (for example, 10 to 20 sheets) can be laminated so as to have a thickness of the order of centimeters.

[0030] Each upper fiber sheet has a fiber density such that a part of the cardiomyocytes seeded on the uppermost fiber sheet can reach the lowermost fiber sheet, and therefore has a fiber density lower than that of the lowermost fiber sheet. On the other hand, each upper fiber sheet has a fiber density such that, when an appropriate amount of cardiomyocytes is seeded, the upper fiber sheet can retain a part of the cardiomyocytes and contribute to the formation of a multi-layered myocardial tissue. The fiber density (porosity) of the upper fiber sheet is, for example, 60 to 80%, preferably 65 to 75%, and more preferably about 70%. The fiber densities of the upper fiber sheets may be the same or different from each other as long as they are within the above range.

[0031] The intervals between the fiber sheets constituting the scaffold of the present invention can be appropriately set, for example, between 40 and 1000 μm, preferably between 80 and 500 μm. When the intended multilayer tissue is a transplant tissue such as myocardial tissue, the intervals between the fiber sheets can be appropriately set between about 100 and about 200 μm, preferably about 120 to about 180 μm, more preferably about 150 μm. The fiber sheets may be disposed at equal intervals or at different intervals, but are preferably disposed at equal intervals. The distance between the uppermost fiber sheet and the lowermost fiber sheet can vary depending on the number of fiber sheets used and the spacing between each fiber sheet, but can be appropriately selected, for example, from the range of 40 to 20,000 μm, preferably 200 to 10,000 μm. When the desired multilayer tissue is a transplant tissue such as myocardial tissue, the distance between the uppermost fiber sheet and the lowermost fiber sheet can be appropriately selected from the range of about 500 to about 1,000 μm.

[0032] The fiber sheets used in the scaffold of the present invention are preferably oriented fiber sheets, in which case the orientation of each oriented fiber sheet may be the same or different. For example, all fiber sheets may have the same orientation, or each layer may be oriented at 90°, but is not limited thereto.

[0033] The fiber sheet used in the present invention can be produced by, for example, electrospinning, dry spinning, conjugate melt spinning, melt blowing, etc., with electrospinning being preferred because it is simple and has a wide range of applications. In the case of the electrospinning method, first, the biocompatible polymer is dissolved in a suitable solvent. Any solvent, whether inorganic or organic, can be used as the solvent as long as it can dissolve the biocompatible polymer used. For example, hexafluoro-2-propanol, acetone, triacetone, dimethylformamide, dimethylacetamide, tetrahydrofuran, etc. can be used. A mixture of multiple solvents may be used. The concentration of the polymer solution varies depending on the type and molecular weight of the polymer and the solvent used, but in order to obtain a preferred fiber diameter and uniformity, it can be appropriately selected, for example, within the range of 0.1 to 40 wt%, preferably 10 to 40 wt%.

[0034] The electrospinning method can be carried out according to a method known per se. The principle of the electrospinning method is to spray a material by electrical force and make it into nano-sized fibers. A syringe is filled with a polymer solution, and a syringe pump is connected to the syringe with a needle-like nozzle at the tip to give a flow rate. A collector (which may be a flat plate or a winding type. A support to be described later may be placed on a flat collector, and nanofibers may be formed directly on the support to form a fiber sheet) is placed at a suitable distance from the nozzle, and the positive pole of a power source is connected to the nozzle side and the negative pole to the collector side. The syringe pump is turned on and a voltage is applied, so that the polymer is sprayed on the collector and fibers are formed. Here, the form and diameter of the fiber vary depending on the voltage, the distance from the nozzle to the collector, the inner diameter of the nozzle, etc., but a person skilled in the art can appropriately select these to produce a fiber having a desired fiber diameter and a uniform fiber. When producing an oriented fiber sheet, for example, a rotating drum can be used around which a sheet that acts as a fiber collector, such as a metal sheet such as aluminum tape, is wound, and the fibers sprayed from the nozzles are wound onto the rotating drum while the drum is rotating to obtain a fiber sheet, but this is not limited to the above.

[0035] The voltage to be applied may be appropriately set depending on the type and physical properties of the polymer used, and may be, for example, 0.1 to 50 kV, preferably 1 to 40 kV. The distance between the nozzle and the collector may be appropriately set depending on the type and physical properties of the polymer used and the applied voltage, and may be, for example, 10 to 1000 mm, preferably 30 to 300 mm.

[0036] The fiber density of the fiber sheet can be adjusted by appropriately changing the spinning time in electrospinning. A fiber sheet with higher density can be produced by extending the spinning time.

[0037] The size of the fiber sheet is not particularly limited, but may be, for example, 3 to 30 mm square.

[0038] In a preferred embodiment, each fiber sheet constituting the scaffold of the present invention is provided with a frame around it, and the frames are laminated so that they are in contact with each other, so that a predetermined interval can be provided between the fiber sheets according to the thickness of each frame. For example, by making the thickness of each frame the same, each fiber sheet can be arranged at equal intervals. There is no particular limit to the material of such a frame as long as it does not affect cell culture and can be easily peeled off from the fiber sheet, and examples of the material include polydimethylsiloxane (PDMS), cellophane tape, and glass. Examples of the method of attaching the frame include, but are not limited to, pressure bonding and adhesion using an adhesive. There is no particular limit to the adhesive as long as it does not affect cell culture and can be peeled off later, and examples of the adhesive that can be used include the polymer solution used in fiber production, PDMS, and commercially available biocompatible adhesives (e.g., silicone liquid condensation type RVT rubber (Shin-Etsu KE-45-T)).

[0039] [II] Method for culturing multi-layered tissues The present invention provides a method for producing a multi-layered tissue culture (hereinafter, sometimes referred to as the "production method of the present invention"), which comprises seeding cells on the topmost fiber sheet of the scaffold of the present invention and culturing them in a liquid medium. Here, the term "multilayered tissue" refers to a sheet-like tissue having a multilayered structure of two or more layers, and the multilayered tissue obtained by the method of the present invention, for example, in the case of myocardial tissue, has a multilayered structure of 20 or more layers, preferably 30 to 200 layers, and more preferably 40 to 150 layers, and in the case of mesenchymal stem cell (MSC) tissue, has a multilayered structure of 20 or more layers, preferably 30 to 600 layers, and more preferably 40 to 400 layers.

[0040] (A) Cell In the manufacturing method of the present invention, cells are first seeded on the uppermost fiber sheet of the scaffold of the present invention.

[0041] The cells to be seeded may be any cells, and the cells constituting the desired multi-layered tissue may be appropriately selected. For example, when the desired multi-layered tissue is a transplant tissue, the cells may include, but are not limited to, cells constituting tissues such as myocardium, mesenchymal stem cells (MSCs), corneal epithelium, oral mucosa, cartilage, nasal mucosa, periodontal ligament, fibroblasts, liver, brain, uterus, kidney, thyroid, and pancreatic islets. Alternatively, when the desired multi-layered tissue is edible cultured meat tissue, the cells may include, but are not limited to, cells of edible tissues such as myoblasts of edible animals such as cows, pigs, chickens, lambs, ducks, fish, crustaceans, eels, foie gras, and scallops. Hereinafter, myocardial tissue and MSC tissue will be described in detail as representative transplant tissues, but the present invention is not limited thereto.

[0042] As used herein, the term "cardiomyocyte" refers to a cell that beats autonomously and expresses the cardiomyocyte markers cTnT and / or CD172a. Cardiomyocytes may also express one or more other cardiomyocyte markers, such as FLK1, PDGFα receptor, EMILIN2, VCAM, etc. The cardiomyocytes used in the method of the present invention may be of any origin as long as they are a cell population containing cardiomyocytes, and examples thereof include, but are not limited to, cardiomyocytes induced to differentiate from pluripotent stem cells, myocardial tissue stem cells, etc., derived from mammals (e.g., humans, mice, rats, dogs, monkeys, pigs, etc.), preferably humans, or cardiomyocytes obtained by direct reprogramming from cardiac fibroblasts. Preferably, the cardiomyocytes are induced to differentiate from pluripotent stem cells. In this specification, unless technically inappropriate, the term "cardiomyocytes" is used to include not only purified cardiomyocytes but also cell populations containing cardiomyocytes.

[0043] (A-1) Preparation of cardiomyocytes from pluripotent stem cells The pluripotent stem cells used in the present invention are not particularly limited as long as they are undifferentiated cells that have the "self-renewal ability" of being able to proliferate while maintaining an undifferentiated state, and the "differentiation pluripotency" of being able to differentiate into all three germ layer lineages, and examples thereof include embryonic stem (ES) cells, iPS cells, as well as embryonic germ (EG) cells derived from primordial germ cells, mutipotent germline stem (mGS) cells isolated during the establishment and culture process of GS cells from testicular tissue, multipotent adult progenitor cells (MAPC) isolated from bone marrow, MUSE cells, etc. The ES cells may be ES cells generated by nuclear reprogramming from somatic cells. ES cells or iPS cells are preferred.

[0044] ES cells can be established by extracting the inner cell mass from the blastocyst of a mammalian fertilized egg and culturing the inner cell mass on a fibroblast feeder. Cells can be maintained by subculture in a culture medium supplemented with substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF). Methods for establishing and maintaining human and monkey ES cells are described in, for example, US Pat. No. 5,843,780; Thomson JA, et al. (1995), Proc Natl. Acad. Sci. USA 92:7844-7848; Thomson JA, et al. (1998), Science. 282:1145-1147; H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345:926-932; M. Ueno et al. (2006), Proc. Natl. Acad. Sci. USA, 103:9554-9559; H. Suemori et al. (2001), Dev. Dyn., 222:273-279; H. Kawasaki et al. (2002), Proc. Natl. Acad. Sci. USA, 99:1580-1585; Klimanskaya I, et al. (2006), Nature. 444:481-485. Human ES cell lines, for example WA01 (H1) and WA09 (H9), are available from WiCell Research Institute, and KhES-1, KhES-2 and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University (Kyoto, Japan).

[0045] iPS cells are artificial stem cells derived from somatic cells that can be produced by introducing specific reprogramming factors in the form of DNA or protein into somatic cells and have almost the same properties as ES cells, such as pluripotency and the ability to proliferate through self-renewal (K. Takahashi and S. Yamanaka (2006) Cell, 126:663-676; K. Takahashi et al. (2007), Cell, 131:861-872; J. Yu et al. (2007), Science, 318:1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26:101-106 (2008); International Publication WO 2007 / 069666). Here, the term "somatic cells" refers to any animal cell (preferably a mammalian cell, including a human cell) other than germline cells such as eggs, oocytes, and ES cells, or totipotent cells, and includes fetal (offspring) somatic cells, neonatal (offspring) somatic cells, and mature healthy or diseased somatic cells, as well as primary culture cells, passaged cells, and established cell lines. Specifically, somatic cells include, for example, (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue progenitor cells, and (3) differentiated cells such as lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, liver cells, gastric mucosa cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes.

[0046] When the obtained iPS cells are used for human regenerative medicine, it is particularly preferable to collect somatic cells from the patient himself or from another person with the same or substantially the same HLA type, from the viewpoint of preventing rejection. Here, "substantially the same" HLA type means that the HLA type matches to such an extent that the transplanted cells can be engrafted when cells obtained by inducing differentiation of the iPS cells derived from the somatic cells using immunosuppressants or the like are transplanted into a patient. For example, the main HLA (for example, the three loci of HLA-A, HLA-B, and HLA-DR, or the four loci including HLA-C) are the same.

[0047] The reprogramming factor may be composed of a gene specifically expressed in ES cells, its gene product or non-coding RNA, or a gene that plays an important role in maintaining the undifferentiated state of ES cells, its gene product or non-coding RNA, or a low molecular weight compound. Examples of genes contained in the reprogramming factor include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, or Glis1, and these reprogramming factors may be used alone or in combination.

[0048] Various methods are known for inducing cardiomyocytes from pluripotent stem cells (e.g., Burridge et al., Cell Stem Cell. 2012 Jan 6;10(1):16-28; Kattman et al., Cell Stem Cell 2011; 8: 228-240; Zhang et al., Circ Res 2012; 111: 1125-1136; Lian et al., Nat Protoc 2013; 8: 162-175; WO 2016 / 076368; WO 2013 / 111875; Minami et al., Cell Rep. 2012, 2(5): 1448-1460, etc.), including, for example, a method based on embryoid body formation, a method based on monolayer differentiation culture, and a method based on forced aggregation. In either method, the induction efficiency can be increased by sequentially acting on mesoderm induction factors (e.g., activin A, BMP4, bFGF, VEGF, SCF, etc.), cardiac determination factors (e.g., VEG F, DKK1, Wnt signal inhibitors (e.g., IWR-1, IWP-2, IWP-4, etc.), BMP signal inhibitors (e.g., NOGGIN, etc.), TGFβ / activin / NODAL signal inhibitors (e.g., SB431542, etc.), retinoic acid signal inhibitors, etc.), and cardiac differentiation factors (e.g., VEGF, bFGF, DKK1, etc.). In one aspect, differentiation of cardiomyocytes from pluripotent stem cells can be induced by sequentially acting on embryoid bodies formed under suspension culture with (1) BMP4, (2) BMP4, bFGF and activin A, (3) IWR-1, and (4) VEGF and bFGF.

[0049] (A-2) Preparation of cardiomyocytes from cells other than pluripotent stem cells Cardiac muscle stem cells can be obtained, for example, by selecting Lin-negative c-kit-positive cell fractions or Isl-1-positive cell fractions from cardiac muscle tissue. The obtained cardiac muscle stem cells can be induced to differentiate into cardiomyocytes by, for example, co-culturing with END2 cells. Direct reprogramming of cardiac fibroblasts to cardiomyocytes can be achieved by introducing GATA-4, MEF2c, and Tbx5 into cardiac fibroblasts using a technique similar to that used to generate iPS cells (Ieda et al., Cell, 142: 375-386, 2010).

[0050] (A-3) Purification of cardiomyocytes From the cell population containing cardiomyocytes prepared as described above, cardiomyocytes can be selected and their purity can be increased. For example, a cardiomyocyte-specific marker (e.g., cell surface marker, etc.) can be used to select cardiomyocytes by magnetic cell sorting (MACS), flow cytometry, affinity separation, etc. Examples of cardiomyocyte-specific cell surface markers include CD172a, KDR (FLK1), PDGFα receptor, EMILIN2, VCAM, etc. Alternatively, cardiomyocytes can be induced to differentiate from pluripotent stem cells into which a marker gene (e.g., a reporter gene such as a fluorescent protein, a drug resistance gene such as an antibiotic, etc.) under the control of a cardiomyocyte-specific promoter has been introduced, and the cardiomyocytes can be selected using the expression of the marker gene as an index. Examples of cardiomyocyte-specific promoters include promoters of the NKX2-5, MYH6, MLC2V, and ISL1 genes, etc. The cell population containing cardiomyocytes to be subjected to the production method of the present invention may be of any purity as long as the cardiomyocyte purity is 50% or more, but preferably has a purity of 70% or more, and more preferably 80% or more.

[0051] The cardiomyocytes used in the production method of the present invention may be the cell population itself obtained by performing a process to induce cardiomyocytes from pluripotent stem cells or the like, or may be cardiomyocytes whose purity has been increased by purifying the cardiomyocytes from the cell population after cardiomyocyte induction, or cardiomyocytes whose purity has been reduced by removing a portion of the cardiomyocytes from the cell population after cardiomyocyte induction or by mixing the purified cardiomyocytes with another cell population (e.g., a non-cardiomyocyte population remaining after cardiomyocyte purification).

[0052] The cardiomyocyte population used in the present invention may contain, in addition to cardiomyocytes, for example, vascular endothelial cells, smooth muscle cells, myofibroblasts, fibroblasts, etc. Vascular endothelial cells can be detected by CD-31 positivity, smooth muscle cells by α-SMA positivity, myofibroblasts by α-SMA and TE-7 positivity, and fibroblasts by TE-7 positivity. In constructing a thick myocardial tissue, in order to reduce cell damage caused by malnutrition and hypoxia in the central part, etc., attempts have been made to co-culture with vascular endothelial cells and form a network of vascular endothelial cells within the tissue. It is also known that the inclusion of the above-mentioned cells other than myocardial cells in the cardiomyocyte population increases cytokine production ability compared to the case where cardiomyocytes are contained at a high purity of 90% or more. However, in the present invention, by performing the scaffold culture of the present invention, preferably by performing dynamic culture, the survival state of the central part can be maintained well, so that it is also possible to contain cardiomyocytes with higher survival rate and purity.

[0053] As used herein, "mesenchymal stem cells (MSCs)" refer to cells that are present in bone marrow, adipose tissue, umbilical cord, dental pulp, etc., and have the multipotency to differentiate not only into mesodermal osteoblasts, adipocytes, muscle cells, and chondrocytes, but also into endodermal visceral tissues and ectodermal nerve cells, etc. As described above, MSCs can be relatively easily obtained from bone marrow, adipose tissue, umbilical cord, dental pulp, etc., using a method known per se, and can be purified using markers such as CD106, CD166, CD29, CD105, CD73, CD44, CD90, CD71, and Stro-1 as positive indicators, and markers such as CD31, CD18, CD56, CD45, CD34, CD14, CD11, CD80, CD86, and CD40 as negative indicators. Alternatively, MSCs can be produced from pluripotent stem cells such as iPS cells, for example, by a method described in npj Regenerative Medicine, 7: 47 (2022).

[0054] As for transplant cells other than cardiomyocytes and MSCs, for example, corneal epithelial cells, oral mucosa cells, chondrocytes, nasal mucosa cells, periodontal ligament cells, fibroblasts, etc., clinical studies using cell sheet engineering are underway, and they can be obtained by the methods used in those studies. Alternatively, for example, cells constituting tissues such as the liver, brain, uterus, kidney, thyroid, and pancreatic islets can be induced to differentiate from pluripotent stem cells such as iPS cells by methods known per se.

[0055] Furthermore, if the desired multilayered tissue is edible cultured meat tissue, the raw cells can be prepared according to information published by companies currently developing the tissue.

[0056] (B) Cell seeding onto the scaffold. The seeding of cells onto the scaffold can be carried out, for example, by placing the scaffold of the present invention in a culture vessel commonly used for cell culture (e.g., a dish, a Petri dish, a tissue culture dish, a low-adhesion dish, a multi-dish, a microplate, a microwell plate, a multi-plate, a multi-well plate, a chamber slide, a petri dish, a tube, a tray, a culture bag, etc.), dissociating the cells prepared as described above using an enzyme and / or a cell strainer, etc. as necessary, suspending the cells in a medium, and dropping the cell suspension onto the uppermost fiber sheet of the scaffold. The scaffold may or may not be attached to the bottom surface of the culture vessel. The scaffold can be attached by various methods, such as by pressure bonding or by using an adhesive. The adhesive is not particularly limited as long as it does not affect cell culture, and examples include the same polymer solution used in the production of the fiber, polydimethylsiloxane (PDMS), and commercially available biocompatible adhesives (e.g., silicone liquid condensation type RVT rubber (Shin-Etsu KE-45-T)).

[0057] The medium for cells can be prepared using a medium used for culturing animal cells as the basal medium. Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, StemPro34 (Invitrogen), and mixtures thereof. The medium may contain serum or may be serum-free. If necessary, the medium may contain one or more serum substitutes, such as, for example, albumin, transferrin, Knockout Serum Replacement (KSR) (serum substitute for FBS), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol (2ME), and thiolglycerol, and may also contain one or more substances, such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts. In a preferred embodiment, the medium may be DMEM or DMEM / F12 medium containing FBS. The concentration of FBS is not particularly limited, but is, for example, within the range of 1 to 30%, preferably 5 to 20%. However, in another preferred embodiment, the medium may be a serum-free medium. The medium may further contain a ROCK inhibitor (e.g., Y-27632, etc.) to suppress cell death, and an extracellular matrix such as laminin, collagen, or matrigel to promote cell adhesion to the fiber. Preferably, laminin can be used as the extracellular matrix.

[0058] Some of the cells seeded on the top fiber sheet of the scaffold of the present invention are retained on the fiber sheet, and the rest migrate to the lower fiber sheet. Some of the cells are also retained on the lower fiber sheet, and the rest migrate to the lower fiber sheet. This is repeated until some of the cells finally reach the bottom fiber sheet and are retained on the fiber sheet. The density of the cells seeded is not particularly limited as long as a multilayer tissue is formed across at least two or more fiber sheets in the scaffold, i.e., the bottom fiber sheet and at least one upper fiber sheet, and can be appropriately changed depending on the type of cells seeded, the number of fiber sheets used, the fiber density, the interval between the fiber sheets, etc., but for example, in the case of cardiomyocytes, it is 0.5 to 4 × 10 7 cells / cm 2 , preferably 0.7 to 3 × 10 7 cells / cm 2 , more preferably about 1 to about 2 × 10 7 cells / cm 2 , and more preferably about 2×10 7 cells / cm 2 Or, for example, in the case of MSC, it is 0.2 to 2 × 10 7 Cells, preferably about 0.5-1.5 x 10 7 cells, more preferably about 1×10 7 It is a cell.

[0059] (C) Cell culture If the seeded cells adhere to each fiber sheet, the cells can be cultured in a liquid medium to produce a culture of a multilayered tissue. The cells can adhere to each fiber sheet about 4 hours after seeding, but in a preferred embodiment, the cells seeded on the fiber sheet can be pre-cultured directly in a culture vessel. The pre-culture can be performed by static culture, shaking culture, etc., but static culture is preferred. The culture can be performed, for example, in a CO2 incubator in an atmosphere with a CO2 concentration of 1 to 10%, preferably 2 to 5%, at 30 to 40°C, preferably about 37°C, for 0.5 to 7 days, preferably 2 to 5 days.

[0060] The culture of cells held in the scaffold of the present invention is not particularly limited, and can be carried out, for example, by the method described in WO 2016 / 060260 or Stem Cell Rep., 9: 1546-1559 (2017), the method described in WO 2020 / 067479, the method described in Int. J. Mol. Sci., 23: 12697 (2022), etc. Examples of the culture medium include the same medium as exemplified when seeding cells in the scaffold and pre-culturing the cells in the scaffold. The culture may be performed by static culture, but in a preferred embodiment, dynamic culture can be performed in which the medium is given fluidity so that oxygen and nutrients in the medium are efficiently supplied to the inside of the multi-layered tissue and waste products are removed from the cells. In addition, it is expected that the function of the tissue (for example, contractility in the case of myocardial tissue) will be further improved by applying an appropriate mechanical stimulus (shear stress) to the cells. Examples of dynamic culture include, but are not limited to, stirring culture using a rotor (stirrer) or spinner flask, shaking culture using a shaker, etc. In dynamic culture, in order to avoid adverse effects of excessive shear stress on cells, it is preferable to set the flow of the medium from the lower fiber sheet side to the upper fiber sheet side, and for example, a holder is provided in the culture vessel, and the scaffold is fixed at an appropriate angle (for example, 15 to 45°, preferably about 30°) with respect to the bottom surface, and dynamic culture can be performed. The flow rate of the medium in dynamic culture can be appropriately set within the range of, for example, 2.5 to 13 cm / sec. The culture can be carried out in an atmosphere with a CO2 concentration of 1 to 10%, preferably 2 to 5%, at 30 to 40°C, preferably about 37°C, for 2 to 10 days.

[0061] As described above, by culturing cells seeded on the scaffold of the present invention, the cells held in each fiber sheet proliferate to form a multilayered tissue, and the multilayered tissues formed in each layer come into contact with each other and self-organize, allowing a thicker and more viable multilayered tissue culture to be produced quickly and easily in a single step.

[0062] (D) Culture device for dynamic culture As described above, in order to avoid adverse effects of excessive shear stress on cells in dynamic culture, a holder is provided in the culture vessel, and the scaffold is fixed at an appropriate angle to the bottom surface, so that dynamic culture can be performed. Therefore, the present invention also provides a culture device for producing a multi-layer tissue culture, which is provided with a holder that can fix the scaffold of the present invention at an angle of 15 to 45°, preferably about 30°, to the bottom surface of the device, and has a means for generating a flow of the culture medium so that the culture medium flows from the lower fiber sheet side to the upper fiber sheet side. Here, the "means for generating a flow of the culture medium" may be a dedicated accessory to the device, or a general-purpose product may be applied to the device by the user when performing dynamic culture. Examples of the means include stirring using a rotor (stirrer) or a spinner flask, shaking using a shaker, etc., and preferably a rotor. In addition, it is preferable to install the holder so that the scaffold of the present invention can be fixed without contacting the bottom surface of the device so as to prevent the flow of the culture medium from changing irregularly as much as possible.

[0063] [III] Multilayered tissue culture The tissue culture thus obtained (hereinafter also referred to as the "tissue culture of the present invention") contains a thick, multi-layered tissue that maintains a good viability throughout the tissue. The tissue culture of the present invention comprises the scaffold of the present invention and a multilayer tissue formed across at least two or more fiber sheets in the scaffold, and is characterized in that the thickness of the tissue is greater than the thickness of the tissue formed when the same amount of cells is seeded on a single layer of fiber sheet. Here, the term "tissue culture of the present invention" is used to include a multilayer tissue that has disappeared over time due to decomposition of the scaffold, as long as it is formed using the scaffold of the present invention as a scaffold.

[0064] The thickness of the tissue culture of the present invention is, for example, 200 to 20000 μm. The thickness of the tissue can be appropriately set depending on the application of the tissue culture, and a desired thickness can be achieved by appropriately adjusting the number of fiber sheets, the interval between fiber sheets, the seeding density of cells, and the proliferation rate of cells. When the tissue culture of the present invention is myocardial tissue, the thickness of the tissue is, for example, 200 to 2000 μm, preferably 300 to 1500 μm. When the tissue culture of the present invention is MSC tissue, the thickness of the tissue is, for example, 300 to 3000 μm, preferably 500 to 2500 μm. When the tissue culture of the present invention is edible cultured meat tissue, the thickness of the tissue is, for example, 5000 to 20000 μm, preferably 10000 to 20000 μm.

[0065] The thickness of the tissue in the tissue culture of the present invention depends on how many layers of fiber sheets the tissue is formed across in the scaffold. And how many layers of fiber sheets can be used depends on the initial cell density seeded on the scaffold. The seeding density in the tissue culture of the present invention is, for example, 0.5 to 4 × 10 in the case of myocardial tissue. 7 cells / cm 2 , preferably 0.7 to 3 × 10 7 cells / cm 2 , more preferably about 1 to about 2 × 10 7 cells / cm 2 , and more preferably about 2×10 7 cells / cm 2 Alternatively, for example, in the case of MSC tissue, it is 0.2 to 2 × 10 7 Cells, preferably about 0.5-1.5 x 10 7 cells, more preferably about 1×10 7Cells. If the number and spacing of fiber sheets are the same, it is thought that the higher the cell proliferation rate, the lower the seeding density tends to be. Therefore, when using the same scaffold to seed other cells, an appropriate seeding density can be selected based on the suitable seeding density of cardiomyocytes or MSCs and their specific proliferation rates. On the other hand, when the number of fiber sheets increases, a higher seeding density is required to utilize all of them. Therefore, for example, when it is necessary to form a thicker tissue using more fiber sheets, such as cultured meat, it is preferable to seed the raw material cells (myoblasts, etc.) at a seeding density higher than the suitable seeding density for cardiomyocytes or MSCs. If the seeding density is too low, only the bottom layer of the fiber sheet can be used, and only the thickness (approximately 200 μm or less) of the myocardial tissue culture formed on a conventional single-layer fiber sheet can be obtained, and there is no significant difference from the conventional product in terms of tissue function (for example, in the case of myocardial tissue, contractility, cytokine secretion ability, etc.). On the other hand, if the seeding density is too high, the cell viability rate decreases, and a thick multi-layered tissue culture cannot be obtained.

[0066] When the tissue culture of the present invention is a myocardial tissue, compared with a multi-layered myocardial tissue culture formed on a single layer of fiber sheet, (a) High tissue contractility; (b) having a high secretion ability of one or more cytokines selected from vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and transforming growth factor β (TGFβ); (c) When transplanted into a subject with myocardial infarction, it has a high effect on restoring cardiac function. (d) When transplanted into subjects with myocardial infarction, the engraftment rate was high and the degree of fibrosis was low. It has the following characteristics: The myocardial tissue culture of the present invention is also characterized in that, compared to a multilayered myocardial tissue culture formed on a single layer fiber sheet, expression of one or more of proteins that are specifically or highly expressed in myocardium, such as cardiac troponin T (TnT), connexin 43 (CX43), and α-actinin, cell adhesion factors such as fibronectin, and type III collagen is increased.

[0067] [IV] Uses of multi-layered tissue cultures The tissue culture of the present invention can be used for various purposes. For example, the tissue culture of the present invention, which is produced by seeding somatic cells collected from a patient or from another person having the same or substantially the same HLA type as the patient, or somatic cells differentiated from iPS cells induced using the somatic cells, on the scaffold of the present invention, which is particularly composed of a fiber sheet made of a biodegradable polymer, can be transplanted directly to the affected area of ​​the patient without removing the fiber sheet, making it possible to clinically apply it as a cell therapy agent for diseases by autologous or allogeneic transplantation. The tissue culture of the present invention is composed of a sheet-like tissue consisting of multiple layers, and is in a state where the extracellular matrix (ECM) is maintained, which plays the role of glue, so that it can be sufficiently engrafted to the surrounding tissue without treatment such as suturing, and a high engraftment rate can be achieved. Moreover, the tissue culture of the present invention has sufficient thickness and good viability throughout the tissue, as compared with tissues prepared by conventional methods. Furthermore, when the tissue culture of the present invention is myocardial tissue, it is possible to recover cardiac function not only by the paracrine effect of cytokine secretion but also by directly participating in contractility, thereby providing a sufficient amount of high-quality cardiomyocytes.

[0068] Cardiac diseases that can be treated using the myocardial tissue culture of the present invention include, for example, myocardial infarction (including chronic heart failure associated with myocardial infarction), dilated cardiomyopathy, ischemic cardiomyopathy, and cardiac diseases (e.g., heart failure, particularly chronic heart failure) associated with systolic dysfunction (e.g., left ventricular systolic dysfunction).

[0069] Even when the tissue culture of the present invention is a tissue that can be used for transplantation therapy other than myocardium, it can be used for the treatment of various diseases depending on the type of tissue. For example, the corneal limbus and oral mucosal epithelial tissue can be used for the treatment of corneal epithelial exhaustion. Oral mucosal epithelial tissue can also be used for the prevention of esophageal stenosis after endoscopic resection of esophageal cancer, and for the suppression of recurrence of esophageal stenosis after esophageal stenosis dilation. In addition, cartilage tissue can be used for cartilage regeneration therapy for osteoarthritis of the knee, for example. In addition, fibroblast tissue can be used for the prevention of pulmonary air leaks, such as postoperative air leaks.

[0070] In another preferred embodiment, the MSC tissue culture of the present invention can be used as a transplantation therapy for a wide range of diseases. MSCs can be collected relatively easily from bone marrow, adipose tissue, umbilical cord, dental pulp, etc., and are free of the ethical issues of ES cells and the risk of tumorigenesis of iPS cells. Furthermore, MSCs can be transplanted into various organs and tissues without terminal differentiation, making them highly versatile cells for regenerative therapy. Furthermore, since MSCs have an immunosuppressive effect, they can also be used to prevent rejection after transplantation.

[0071] The therapeutic effect of MSCs against diseases is believed to be due to a paracrine action caused by the secretion of cytokines and growth factors rather than differentiation into specific cells, which allows them to exert various physiological activities such as control of the immune system, angiogenesis, anti-inflammatory action, antioxidant action, anti-apoptotic action, tissue repair action, etc. Therefore, the MSC tissue culture of the present invention can be used to treat, for example, immune diseases such as graft-versus-host disease, spinal cord injury, wounds such as diabetic foot ulcers, heart diseases such as heart failure, inflammatory diseases, cerebrovascular diseases such as cerebral infarction, eye diseases such as optic nerve degeneration, and the like.

[0072] In another aspect of the present invention, the myocardial tissue culture of the present invention has a higher action potential, a larger pulsating force, and a higher expression of various marker genes that are considered to be an index of myocardial maturity than myocardial tissue prepared by conventional methods, and is therefore considered to closely reflect the state of myocardial tissue in vivo. Therefore, the myocardial tissue culture of the present invention can be suitably used as an in vitro evaluation system for the efficacy and cardiotoxicity of therapeutic drugs for heart disease. Furthermore, it can be suitably used as a tool for pathological research of heart diseases whose causes are unknown. Similarly, tissue cultures of the present invention other than myocardium are also considered to closely reflect the state of the corresponding tissue in the body, and therefore can be suitably used as in vitro evaluation systems for the efficacy of therapeutic agents for diseases in the tissue and the toxicity to the tissue.

[0073] When the myocardial tissue culture of the present invention is used as the in vitro evaluation system as described above, it can be performed by assaying myocardial pulsation and contractile force, action potential, electrokinetic velocity, Ca transient, mechanical stress response, cell cycle, cell death, etc., by a method known per se. Specific examples include various electrophysiological evaluations described in WO 2016 / 060260. For tissue cultures other than myocardial tissue, evaluation methods used in known in vitro evaluation systems for each tissue can be used.

[0074] When the tissue culture of the present invention is edible cultured meat tissue, the biodegradable polymer, preferably a biopolymer such as gelatin, collagen, cellulose, etc., can be degraded naturally or by enzymatic treatment or the like, and provided as edible meat, either as is, or processed into various foods.

[0075] The present invention will be described in more detail below with reference to examples, but it goes without saying that the present invention is not limited to these. EXAMPLES

[0076] Example 1 Preparation and evaluation of multi-layered myocardial tissue cultures (1) Preparation of human iPS cells and induction of their differentiation into cardiomyocytes HLA-homozygous human iPS cells (QHJI14s04, obtained from CiRA, Master Cell bank (MCB)) were maintained by standard methods and induced to differentiate into cardiomyocytes (hereinafter referred to as "iPSC-CMs") by the method described in https: / / doi.org / 10.3389 / fcvm.2022.950829 (2022). Experiments involving the use of human iPS cells were performed in accordance with the guidelines of Osaka University.

[0077] (2) Fabrication of multi-layered fiber scaffolds Oriented fiber sheets of polylactic acid-polyglycolic acid copolymer (PLGA) were produced by electrospinning using a commercially available electrospinning apparatus (NF-103, MECC, Japan). PLGA (PLA75 / PGA25, Sigma, USA) was mixed with hexafluoro-2-propanol (Fujifilm Wako Pure Chemical Industries, Japan) to a concentration of 20% (w / w). The PLGA solution was placed in a 5 mL syringe with a needle of 0.7 mm inner diameter. Aluminum foil was attached to the surface of a high-speed rotating drum and placed 150 mm away from the tip of the needle. A positive electrode was placed on the needle and a negative electrode was placed on the high-speed rotating drum, and the high-speed rotating drum was rotated at 700 rpm. A voltage of 20 kV was applied from the apparatus, and the fibers were oriented in one direction and sprayed onto the aluminum foil on the rotating drum. High density (H-AF; spraying time: 60 min) and low density (L-AF; spraying time: 10 min) oriented fiber sheets were fabricated by changing the spraying time. The fiber densities (porosity) of H-AF and L-AF were 69.52 ± 8.26% and 30.61% ± 1.85%, respectively (Fig. 1a; n = 7, p < 0.001). After fiber spraying, the aluminum foil on which the oriented fiber sheet was collected was collected and wrapped with Scotch tape (Scotch TMThe fibers were peeled off using a frame (external dimensions: 1.5 cm × 1.5 cm; internal dimensions: 1 cm × 1 cm) of a scotch tape (3M, USA). The frame of Scotch tape with H-AF, the frame without the fiber sheet, and the frame with L-AF were appropriately combined to prepare three types of multilayer fiber sheets (MLAs) with the same orientation (the distance between the topmost fiber sheet and the bottommost fiber sheet was all 600 μm). For comparison, a single-layer fiber sheet (Control) with only a frame with H-AF was prepared. MLA1: Upper layer fiber sheet (L-AF) x 3 + bottom layer fiber sheet (H-AF) (200 μm intervals) MLA2: Upper layer fiber sheet (L-AF) x 4 + bottom layer fiber sheet (H-AF) (150 μm intervals) MLA3: Upper layer fiber sheet (L-AF) x 6 + bottom layer fiber sheet (H-AF) (100 μm intervals) Figure 2 shows a schematic diagram of each MLA and single-layer fiber sheet viewed from the side (top) and an electron microscope photograph of their cross sections (middle).

[0078] (3) Preparation of Multilayered Myocardial Tissue Cultures The cardiomyocytes obtained in (1) above were passed through a 40 μm strainer (BD Falcon, USA) and resuspended in serum medium (40% high glucose Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich), 40% Iscove's modified Dulbecco's medium (IMDM; Sigma-Aldrich), 20% fetal bovine serum (FBS; Gibco, USA), 1% minimum essential medium non-essential amino acids (Sigma-Aldrich), 0.1% penicillin / streptomycin (Gibco), and 0.5% L-glutamine (Sigma-Aldrich)). 10 μM ROCK inhibitor (Wako) was added to enhance cell survival. The cell suspension was diluted to 2 × 10 7 cells / cm 2 (For single-layer fiber sheets, 5×10 6 cells / cm 2) onto the top layer of the fiber sheet of the MLA prepared in (2) above. After 1 hour, 1 mL of medium was added to the culture dish with the MLA placed in place. After another 3 hours, the MLA was inserted into a PDMS holder (Figure 1b) and 30 mL of medium was added to the culture dish. The culture dish was placed in an incubator and cultured stationarily for 1 day under conditions of 37°C and 5% CO2. From the next day, the cells were cultured for 5 days while stirring at 150 rpm using a magnetic stirrer (Figure 1b). Furthermore, the MLA was placed in a culture vessel without seeding cardiomyocytes, and the flow rate of the medium was changed by changing the rotation speed of the stirrer (Figure 1c). The distribution of the flow rate and shear stress in the culture vessel was measured (the results at a rotation speed of 250 rpm are shown in Figures 1d and 1e). The rotation speed of the stirrer was set so that the shear stress was kept at a safe level.

[0079] (4) Tissue staining The tissue obtained in (3) above was washed three times with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde in PBS, and embedded in paraffin. Ultrathin sections were prepared and stained with hematoxylin-eosin (HE). Each section was observed under a CKX41 microscope (Olympus). The results are shown in Figure 2 (bottom). When any of MLA1 to MLA3 was used, a thicker multi-layered myocardial tissue was obtained than when a single-layered fiber sheet was used. In particular, when MLA2 was used, a thicker multi-layered myocardial tissue with a thickness of about 1200 μm was obtained. This showed that a distance of 150 μm between each fiber sheet in MLA is particularly preferable.

[0080] Example 2 Effect of the orientation of each fiber sheet In Example 1(4), the thickest multilayered myocardial tissue was obtained when MLA2 [upper layer fiber sheet (L-AF) × 4 + bottom layer fiber sheet (H-AF) (150 μm intervals); Figure 2] was used, so the subsequent experiments were performed using MLA2. An MLA was created in which each fiber sheet was alternately stacked in a vertical orientation, and this was compared with MLA2, which had all the same orientation. After the culture was completed, the tissue was fixed and immunostained with an anti-TnT antibody to compare the thickness of the tissue. The results are shown in Figure 3. Regardless of the orientation of each fiber sheet, thick multi-layered myocardial tissue was obtained with all MLAs. These results indicate that it is not important to align the orientation of each fiber sheet.

[0081] Example 3 Effect of extracellular matrix The same experiment as in Example 1 was carried out with and without laminin added to the medium when the cardiomyocytes were seeded. The results are shown in Figure 4. When laminin was added to the medium when the cardiomyocytes were seeded, more cells adhered to the fiber sheet than when laminin was not added, and non-adherent floating cells were hardly observed (Figure 4, top). The obtained multi-layered myocardial tissue was also attached to a 64-channel multi-electrode array (USB-ME64-System, Multi-channel Systems, Germany) to record electrophysiological data. As a result, the addition of laminin showed a more uniform and higher amplification of the electrical signal than the absence of laminin. Therefore, the following experiments were carried out with laminin added to the medium when the cardiomyocytes were seeded.

[0082] Example 4 Effect of seeding density and dynamic culture The seeding density of cardiomyocytes was changed and cultured in the same manner as in Example 1. In addition, the dynamic culture was changed to a static culture and a similar experiment was performed. The results are shown in Figure 5. 7 cells / cm 2 When 1 × 10 cardiomyocytes were seeded, a multi-layered myocardial tissue of similar thickness to that obtained using a conventional single-layer fiber sheet was obtained, but when 1 × 10 cardiomyocytes were seeded, 7 cells / cm 2 Or 2×10 7 cells / cm 2 When 4 × 10 cardiomyocytes were seeded, thicker, multi-layered myocardial tissue was obtained (Figure 5a). However, when 4 × 10 7 cells / cm 2When cardiomyocytes were seeded at 100 μm, cell viability was significantly reduced (Figure 5a). Dynamic culture resulted in more dense and well-organized tissue formation than static culture. The thickness of the multi-layered myocardial tissue obtained by dynamic culture ranged from 226.51 ± 48.60 μm to 878.64 ± 193.45 μm (n = 4) depending on the seeding density (Figure 5b).

[0083] The tissues were then fixed with 4% paraformaldehyde for 0.5 h, permeabilized with 0.5% (v / v) Triton X-100 in Dulbecco's PBS (D-PBS) for 1 h, and then immersed in blocking solution overnight at 4°C. The tissues were then incubated with primary antibodies overnight at 4°C. (Primary antibody) Anti-α-actinin antibody (1:1000; A7811; Sigma-Aldrich) Anti-troponin T2 antibody (TnT2; 1:200; SC-20025; Santa Cruz Biotechnology, Dallas, TX, USA) Anti-connexin 43 antibody (Cx43; 1:200; C6219; Sigma-Aldrich) Anti-type I collagen antibody (1:200; C2456; Sigma-Aldrich) Anti-type III collagen antibody (1:200; ab7778; Abcam) The tissues were then washed with PBS and incubated with secondary antibodies diluted 1:300 in blocking solution at room temperature for 1 hour. (Secondary antibody) Alexa Fluor 594-conjugated anti-mouse IgG (715-586-150; Jackson Immuno Research, West Grove, PA, USA) DyLight-594-conjugated anti-mouse IgM (715-516-020; Jackson Immuno Research) Alexa Fluor 647-conjugated anti-rabbit IgG (A21245; ThermoFisher) Alexa Fluor 488-conjugated anti-rabbit IgG (A21206; ThermoFisher) Nuclei were counterstained with 300 nM DAPI (Fujifilm Wako Pure Chemicals) for 30 min. Images were captured using a confocal microscope (NIKON A1; Nikon). The immunostained images obtained are shown in Figure 6 (top). The multi-layered myocardial tissue showed high expression of proteins such as myocardial markers, intercellular adhesion factors, and extracellular matrix. Furthermore, compared with thin tissue, the expression of type III collagen was higher in the thicker multi-layered myocardial tissue.

[0084] The viability of cells in the tissue was measured by TUNEL staining using a conventional method. The results are shown in the middle and lower panels of Figure 6. 7 cells / cm 2 and 2 x 10 7 cells / cm 2 When 0.5×10 cardiomyocytes were seeded, the apoptosis rates were 10.59±2.47% and 14.68±6.75%, respectively, with no significant difference. 7 cells / cm 2 Although the apoptosis rate was higher than that of 6.64±1.8% when cardiomyocytes were seeded, it was shown that the survival rate was maintained at approximately 85% or higher.

[0085] Example 5. Characterization of multi-layered myocardial tissue obtained using MLA (1)-in vitro evaluation In the preceding examples, MLA2 [upper layer fiber sheet (L-AF) × 4 + bottom layer fiber sheet (H-AF) (150 μm interval)] was used as the MLA, and 2 × 10 7 cells / cm 2 The thickest multi-layered myocardial tissue with good viability was obtained when cardiomyocytes were seeded at 0.5 × 10 and cultured by dynamic culture. Therefore, in the subsequent experiments, the multi-layered myocardial tissues (MLA group) prepared under the above conditions were compared with the conventional single-layered fiber sheet, which has a seeding density of 0.5 × 10, which is equivalent to that used for the single-layered fiber sheet and has a similar thickness of the obtained myocardial tissue (approximately 200 μm). 7 cells / cm 2Various characteristics were evaluated and compared between the myocardial tissues produced by seeding the cardiomyocytes and culturing them in the same manner (control group).

[0086] (1) Pulse analysis The contractility of myocardial tissue was evaluated using a Cell Motion Imaging System (SI8000; Sony, Tokyo). Videos were recorded at a frame rate of 150 frames / s, a resolution of 1024 × 1024 pixels, and a depth of 8 bits. The results showed that the MLA group had significantly higher contractile velocity (75.91 ± 28.51 μm / s vs. 19.70 ± 5.81 μm / s), relaxation velocity (54.39 ± 15.12 μm / s vs. 14.74 ± 1.39 μm / s), and contractile deformation distance (8.14 ± 2.11 μm vs. 1.86 ± 0.73 μm) than the control group, indicating improved contractile activity (Figure 7).

[0087] (2) Cytokine secretion Next, the concentrations of various cytokines secreted into the culture supernatant were measured by ELISA and compared between the MLA group and the control group.The results showed that the MLA group secreted higher levels of VEGF, HGF, and TGF-β1 than the control group (Figure 8).

[0088] Example 6. Characterization of multi-layered myocardial tissue obtained using MLA (2)-in vivo evaluation (1) Creation of a rat model of myocardial infarction and transplantation of multi-layered myocardial tissue The animal experiment was approved by the ethical committee of Osaka University and was carried out in accordance with the guidelines of the university (approval number: 01-062-000). Rats (strain F344 / NJcl-rnu / rnu, sex: male, 8 weeks old, source: CLEA) were anesthetized and the chest was opened over the 4th and 5th intercostal spaces, and the left anterior descending coronary artery was ligated to induce myocardial infarction. Prior to transplantation, the myocardial tissue was treated with DMEM medium containing 1% iMatrix 511 (Nippi, Japan) and 20% FBS for 5 min at room temperature (Fig. 9a). Then, the frame was cut out, and the tissue sheet was placed on the epicardium and coated with fibrinogen-added factor XIII (Beriplast P; CSL Behring, USA) (Fig. 9c). Echocardiography was performed using an ultrasound system (Philips SONOS 7500, Amsterdam, Netherlands) equipped with a circular array probe at a frequency of 12 MHz (Fig. 9b). Ejection fraction (EF) was calculated using the following formula: LVEF(%)=100 X (LVEDV-LVESV) / LVEDV Four weeks after transplantation, the rats were sacrificed, and the hearts were harvested and 5 μm-thick frozen sections were prepared for histological analysis and immunostaining.

[0089] (2) Recovery of cardiac function in myocardial infarction models Echocardiography showed that the MLA group had a significant improvement in left ventricular ejection fraction (EF) after transplantation (0 weeks vs. 4 weeks: 38.8 ± 4.72 % vs. 53.1 ± 7.91 %, n=8, p = 0.0034), whereas the control group did not show a significant improvement (0 weeks vs. 4 weeks: 40.6 ± 5.4 % vs. 44.9 ± 4.1 %, n=8, p = 0.37) (Fig. 9d). The ejection fractions between the two groups were significantly different at 3 weeks (p = 0.0076) and 4 weeks (p = 0.02). The MLA group also showed a significant improvement in left ventricular fractional shortening (FS) (week 0 vs. week 4: 16.2±2.3% vs. 24.2±4.7%, n=8, p=0.0065), whereas the control group did not show a significant improvement (week 0 vs. week 4: 417.2±2.7% vs. 19.5±2.2%, n=8, p=0.027) (Fig. 9e). Left ventricular end-systolic dimensions (LVIDs) increased in the control group (0 weeks vs. 4 weeks: 0.607±0.045 cm vs. 0.704±0.048 cm, n=8, p=0.0048), whereas no significant increase was observed in the MLA group (0 weeks vs. 4 weeks: 0.598±0.08 cm vs. 0.66±0.098 cm, n=8, p=0.51).

[0090] (3) Improved engraftment rate and anti-fibrosis effect The survival of the grafted tissue was evaluated by HE staining and immunofluorescence staining of the frozen sections prepared in (1) above. The survival of the grafted myocardial tissue was confirmed by both staining in the MLA and control groups (Fig. 10a). The survival rate (ratio of graft / left ventricular area) of the MLA group was significantly higher than that of the control group (MLA vs. Control: 8.0±2.9% vs. 4.6±2.2%, n=5, p=0.028) (Fig. 10b). Furthermore, tissue fibrosis was evaluated by Sirius red staining. Capillary density and inflammatory response were examined by immunofluorescence staining using anti-isolectin antibody or anti-CD68 antibody. As a result, the rate of fibrosis was significantly lower in the MLA group than in the control group (MLA vs. Control: 10.8±3.1% vs. 16.4±5.9%, n=7, p=0.024) (Fig. 11a, b). There was no significant difference in the cell size (Fig. 11c, d) or capillary density (Fig. 11e, f) at the interface between the graft and the recipient heart between the two groups. CD68 staining showed no inflammatory response in either group 4 weeks after transplantation.

[0091] These results demonstrate that transplantation of high-dose multi-layered myocardial tissue using MLA shows superior cardiac function recovery, high engraftment rate, and reduced fibrosis in infarcted hearts compared to conventional methods.

[0092] Example 7 Preparation of multi-layered MSC tissue cultures A multilayered fiber scaffold similar to MLA2 prepared in Example 1(2) (SEM photograph of the cross section is shown in the left of Figure 12) was used to prepare a multilayered tissue culture of adipose tissue-derived mesenchymal stem cells (AD-MSCs). As a result, a thicker multi-layered MSC tissue (about 2 mm) was obtained compared to the case where a tissue culture of umbilical cord-derived mesenchymal stem cells (UC-MSC) was prepared using a single-layered fiber sheet (about 300 μm; Non-Patent Document 8). In other words, it was revealed that the multi-layered fiber scaffold of the present invention can also prepare a thick multi-layered tissue culture of tissues other than myocardium. [Industrial Applicability]

[0093] The multi-layered tissue obtained by the present invention is heavy and maintains a good viability, and is also functionally superior to those obtained by conventional methods, making it extremely useful as a tissue material more suitable for transplantation. Furthermore, the method for producing a multi-layered tissue culture of the present invention allows a heavy multi-layered tissue to be obtained quickly and easily in one step, which can greatly contribute to the practical application of transplant therapy for diseases using somatic cells induced to differentiate from iPS cells, MSCs, etc. Furthermore, by using the production method of the present invention to produce edible cultured meat tissue, it is possible to provide artificial meat that is heavier and has a better texture than those obtained by conventional methods.

Claims

1. A multilayered tissue scaffold comprising two or more fiber sheets made of a biocompatible polymer laminated at intervals of about 100 to about 200 μm, wherein the bottommost fiber sheet has a fiber density sufficient to retain cells, and the upper fiber sheets have fiber densities sufficient to allow some of the seeded cells to reach the bottommost fiber sheet.

2. 10. The scaffold of claim 1, wherein each fiber sheet has an orientation.

3. The scaffold according to claim 1 , wherein each fiber sheet is stacked at equal intervals.

4. The scaffold according to claim 1, wherein 4 to 7 fiber sheets are laminated.

5. 2. The scaffold according to claim 1, wherein the distance between the top fiber sheet and the bottom fiber sheet is about 500 to about 1000 μm.

6. The scaffold of claim 1 , wherein the biocompatible polymer is biodegradable.

7. The scaffold of claim 6, wherein the biodegradable polymer is polylactic acid-polyglycolic acid copolymer (PLGA) or a biopolymer.

8. A method for producing a multi-layered tissue culture, comprising seeding cells on the top fiber sheet of the scaffold according to any one of claims 1 to 7 and culturing the cells in a liquid medium.

9. Seeding density is 0.7 to 3 × 10 7 cells / cm 2 The method of claim 8, wherein

10. The method according to claim 8, wherein the culture is a dynamic culture.

11. The method of claim 8 , wherein the cell seeding is carried out in the presence of an extracellular matrix.

12. The method according to claim 8, wherein the cells are somatic cells induced to differentiate from pluripotent stem cells.

13. The method of claim 12, wherein the pluripotent stem cells are induced pluripotent stem (iPS) cells.

14. The method of claim 8 , wherein the cells are cardiomyocytes or mesenchymal stem cells.

15. The method of claim 8, wherein the cells are of human origin.

16. 9. The method of claim 8, wherein the cells are from a food animal.

17. A multi-layered tissue culture obtainable by the method of claim 8.

18. A multi-layered tissue culture comprising the scaffold according to any one of claims 1 to 7 and a multi-layered tissue formed across at least two or more fiber sheets in the scaffold, wherein the thickness of the tissue is greater than the thickness of the tissue formed when the same amount of cells is seeded on a single layer of fiber sheet.

19. Seeding density is 0.7 to 3 × 10 7 cells / cm 2 20. The tissue culture of claim 18, wherein

20. 19. The tissue culture of claim 18, wherein the tissue is cardiac muscle tissue or mesenchymal stem cell tissue.

21. When the tissue is myocardial tissue, the following (a) to (d) are obtained compared to a multi-layered myocardial tissue culture formed on a single layer of fiber sheet: (a) high tissue contractility; (b) having a high secretion ability of one or more cytokines selected from vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and transforming growth factor β (TGFβ); (c) When transplanted into a subject with myocardial infarction, it has a high effect on restoring cardiac function; (d) When transplanted into a subject with myocardial infarction, the engraftment rate is high and the degree of fibrosis is low.

20. The tissue culture of claim 18, having one or more characteristics selected from:

22. 20. The tissue culture of claim 18, wherein the tissue is an edible tissue of a food animal.

23. A pharmaceutical composition comprising the tissue culture of claim 18.

24. 24. The pharmaceutical composition of claim 23 for use in transplantation therapy.

25. 20. A method for treating a disease in a mammal in need thereof, comprising implanting an effective amount of the tissue culture of claim 18 into said mammal.

26. 23. A food product comprising the tissue culture of claim 22.

27. A culture device for producing a multilayered tissue culture, the culture device being provided with a holder capable of fixing the scaffold according to any one of claims 1 to 7 at an angle of 15 to 45 degrees to the bottom surface of the device without contacting the scaffold with the bottom surface of the device, and having a means for generating a flow of culture medium so that the culture medium flows from the lower fiber sheet side to the upper fiber sheet side.

Images