Cell culture substrate with layered cardiomyocyte sheet, method for producing the same, and method for evaluating normal cardiomyocyte sheet for transplantation.

Abstract

To provide a cell culture substrate with a laminated cardiomyocyte sheet for evaluating the healing effect of living body transplantation using a normal cardiomyocyte sheet, and to provide a method for producing the same, as well as, furthermore, to provide an evaluation method of a normal cardiomyocyte sheet for living body transplantation.SOLUTION: Provided is a cell culture substrate with a laminated cardiomyocyte sheet, the cell culture substrate with a laminated cardiomyocyte sheet having two or more layers of cardiomyocyte sheets on the cell culture substrate, the cell culture substrate having an alternating arrangement portion in which band-shaped cell adhesion regions and band-shaped cell adhesion suppression regions are arranged alternately; having two or more layers of cardiomyocyte sheets on at least the alternating arrangement portion of the cell culture substrate; and having, as the cardiomyocyte sheet, each at least one layer of a diseased cardiomyocyte sheet consisting of a differentiation-induced cardiomyocyte derived from a diseased patient, and a normal cardiomyocyte sheet consisting of a differentiation-induced cardiomyocyte derived from a healthy individual.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a cell culture substrate with a layered cardiomyocyte sheet, a method for producing the same, and a method for evaluating a normal cardiomyocyte sheet for transplantation. [Background technology]

[0002] A treatment method has been developed in which a sheet of cardiomyocytes is created outside the body and transplanted into the affected area of ​​the heart of a patient with heart disease to restore cardiac function (see, for example, Patent Document 1). Cardiomyocyte sheets for transplantation may be made using autologous cells derived from the patient, which are created by generating iPS cells from the patient's own somatic cells and then differentiating them into cardiomyocytes, or they may be made using allogeneic cells derived from stocked iPS cells from healthy individuals and then differentiating them into cardiomyocytes, which are of a different origin than the patient's. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2003-306434 [Overview of the project] [Problems that the invention aims to solve]

[0004] Whether using autologous or allogeneic cells as the cardiomyocyte sheet for transplantation, the preparation of the cardiomyocyte sheet requires time-consuming and costly steps, including collecting cell samples from patients or healthy individuals, inducing differentiation of iPS cells into cardiomyocytes, and fabricating the cell sheet. Despite these costs, a challenge remains: the therapeutic effect of cardiomyocyte sheet transplantation cannot be evaluated until the transplant treatment is actually performed. Therefore, there is a need for a method to evaluate the transplantation effect in advance.

[0005] The present invention aims to provide a cell culture substrate with a laminated cardiomyocyte sheet and a method for producing the same, for evaluating the healing effect of transplanting normal cardiomyocyte sheets into living organisms. Furthermore, the present invention aims to provide a method for evaluating the healing effect of normal cardiomyocyte sheets for transplantation into living organisms. [Means for solving the problem]

[0006] The above problems of the present invention are as follows: <1> ~ <7> This can be resolved by the configuration. <1> A cell culture substrate with a layered cardiomyocyte sheet, having two or more layers of cardiomyocyte sheets on a cell culture substrate, wherein the cell culture substrate has alternating arrangement portions in which strip-shaped cell adhesion regions and strip-shaped cell adhesion inhibition regions are arranged alternately, and at least two or more layers of cardiomyocyte sheets are provided on at least the alternating arrangement portions of the cell culture substrate, and the cardiomyocyte sheets consist of at least one disease cardiomyocyte sheet made of differentiated cardiomyocytes derived from diseased patients and at least one normal cardiomyocyte sheet made of differentiated cardiomyocytes derived from healthy individuals. <2> The normal cardiomyocyte sheet is attached to the cell culture substrate, and the diseased cardiomyocyte sheet is laminated on the normal cardiomyocyte sheet. <1> Cell culture substrate with layered cardiomyocyte sheets as described above. <3> The diseased cardiomyocyte sheet is attached to the cell culture substrate, and the normal cardiomyocyte sheet is laminated on the diseased cardiomyocyte sheet. <1> Cell culture substrate with layered cardiomyocyte sheets as described above. <4> A method for producing a cell culture substrate with a laminated cardiomyocyte sheet, comprising the steps of: preparing a cell culture substrate having alternating arrangement portions in which band-shaped cell adhesion regions and band-shaped cell adhesion inhibition regions are arranged alternately; forming a normal cardiomyocyte sheet consisting of differentiated cardiomyocytes derived from a healthy person on at least the alternating arrangement portion of the cell culture substrate; and forming a diseased cardiomyocyte sheet consisting of differentiated cardiomyocytes derived from a diseased patient on the normal cardiomyocyte sheet. <5> A method for producing a cell culture substrate with a laminated cardiomyocyte sheet, comprising the steps of: preparing a cell culture substrate having alternating arrangement portions in which band-shaped cell adhesion regions and band-shaped cell adhesion inhibition regions are arranged alternately; forming a diseased cardiomyocyte sheet consisting of differentiated cardiomyocytes derived from a diseased patient on at least the alternating arrangement portion of the cell culture substrate; and forming a normal cardiomyocyte sheet consisting of differentiated cardiomyocytes derived from a healthy person on the diseased cardiomyocyte sheet. <6> A method for producing a cell culture substrate with a laminated cardiomyocyte sheet, comprising the steps of: forming a diseased cardiomyocyte sheet made of differentiated cardiomyocytes derived from a diseased patient on at least the alternating arrangement portion of a cell culture substrate having alternating arrangement portions of band-shaped cell adhesion regions and band-shaped cell adhesion inhibition regions; forming a normal cardiomyocyte sheet made of differentiated cardiomyocytes derived from a healthy person on at least the alternating arrangement portion of a cell culture substrate having alternating arrangement portions of band-shaped cell adhesion regions and band-shaped cell adhesion inhibition regions; and separating either the diseased cardiomyocyte sheet or the normal cardiomyocyte sheet from the cell culture substrate and laminating it onto the other cardiomyocyte sheet. <7> <1> ~ <3> A method for evaluating a normal cardiomyocyte sheet for transplantation in a living organism, comprising the step of evaluating at least one change selected from changes in motor function and changes in physiological properties of a diseased cardiomyocyte sheet in a cell culture substrate with a cardiomyocyte sheet as described in any one of the above. [Effects of the Invention]

[0007] According to the present invention, it is possible to provide a cell culture substrate with a laminated cardiomyocyte sheet and a method for producing the same for evaluating the healing effect of transplanting normal cardiomyocyte sheets into living organisms. Furthermore, according to the present invention, a method for evaluating the healing effect of normal cardiomyocyte sheets for transplantation into living organisms can be provided. [Brief explanation of the drawing]

[0008] [Figure 1]This figure shows the configuration of the member in one embodiment of the alternating arrangement portion, where (a) is a perspective view showing the structure of the alternating arrangement portion together with a petri dish, (b) is a perspective view showing a magnified portion of the surface of the alternating arrangement portion, (c) is a plan view showing a magnified portion of the surface of the alternating arrangement portion, and (d) is a partial cross-sectional view showing a magnified portion of the alternating arrangement portion. [Figure 2] This is a process diagram illustrating an example of a manufacturing method for the alternating arrangement portion. [Figure 3] (a) to (c) are schematic diagrams illustrating the manufacturing process of cell sheets. [Figure 4] This graph shows the heart rate (BR) results measured using a live cell imaging device. [Figure 5] This graph shows the results of contraction velocity (CV) measured using a live-cell imaging device. [Figure 6] This graph shows the results of relaxation velocity (RV) measured using a live-cell imaging device. [Figure 7] This graph shows the results of the contraction / relaxation duration (corrected value) (CRD(corrected value)) measured using a live cell imaging device. [Figure 8] This graph shows the results of the degree of orientation measured using a live cell imaging device. [Figure 9] This graph shows the line width (Duration) at 50% height on day 21 of culture, as measured by calcium imaging analysis. [Figure 10] This graph shows the Duration / (Interval) 1 / 2 calculated from the line width at 50% height (Duration) and the interval between waveform peaks (Interval) measured by calcium imaging analysis on day 21 of culture. [Modes for carrying out the invention]

[0009] Hereinafter, preferred embodiments of the present invention will be described. In this specification, "X~Y" indicating a range means "X or more and Y or less". When numerical ranges are described stepwise, the upper and lower limits of each numerical range can be arbitrarily combined.

[0010] [Cell culture substrate with laminated cardiomyocyte sheets] The cell culture substrate with laminated cardiomyocyte sheets of the present invention is a cell culture substrate with laminated cardiomyocyte sheets having two or more layers of cardiomyocyte sheets on a cell culture substrate. The cell culture substrate has an alternating arrangement portion in which strip-shaped cell adhesion regions and strip-shaped cell adhesion suppression regions are alternately arranged, and has two or more layers of cardiomyocyte sheets on at least the alternating arrangement portion of the cell culture substrate. As the cardiomyocyte sheets, it has at least one layer each of a diseased cardiomyocyte sheet composed of differentiated cardiomyocytes derived from a diseased patient and a normal cardiomyocyte sheet composed of differentiated cardiomyocytes derived from a healthy person. That is, the two or more layers of cardiomyocyte sheets have at least one layer of a diseased cardiomyocyte sheet composed of differentiated cardiomyocytes derived from a diseased patient and at least one layer of a normal cardiomyocyte sheet composed of differentiated cardiomyocytes derived from a healthy person. The cell culture substrate with laminated cardiomyocyte sheets of the present invention can be suitably used for evaluating normal cardiomyocyte sheets for living body transplantation.

[0011] By using a cell culture substrate with laminated cardiomyocyte sheets formed by laminating a normal cardiomyocyte sheet composed of differentiated cardiomyocytes derived from a healthy person (hereinafter also referred to as "normal cardiomyocytes") and a diseased cardiomyocyte sheet composed of differentiated cardiomyocytes derived from a diseased patient (hereinafter also referred to as "diseased cardiomyocytes") on a specific cell culture substrate as a model after living body transplantation, the healing effect of a normal cardiomyocyte sheet for living body transplantation can be evaluated more accurately. The detailed mechanism by which the above effects are obtained is unclear, but part of it is considered as follows. The cell culture substrate with a layered cardiomyocyte sheet of this embodiment has a cell culture substrate (hereinafter also referred to as the "orientation culture substrate") on which alternating arrangement portions of band-shaped cell adhesion regions and band-shaped cell adhesion inhibition regions are arranged alternately. As a result, the cardiomyocytes are cultured with their long axes aligned in one direction. In other words, by forming a cell sheet in which the cardiomyocytes are oriented in one direction, the cardiomyocytes are cultured in a state closer to that in vivo. Consequently, it is considered possible to evaluate the effects of transplanting a normal cardiomyocyte sheet in an environment closer to that in vivo. Furthermore, when diseased cardiomyocytes are cultured in a monolayer on an oriented culture substrate, their function on the oriented substrate is significantly closer to that of normal cardiomyocytes compared to when they are cultured in a monolayer on a planar culture substrate. By forming a cardiomyocyte sheet with diseased cardiomyocytes oriented in one direction, the cardiomyocytes are cultured in an environment much closer to that in vivo, leading to a shift toward normalization of myocardial function. Therefore, by analyzing cell sheets obtained by monoculture of diseased cardiomyocytes on an oriented substrate, it is possible to evaluate the therapeutic effect of transplanting normal cardiomyocytes monocultured on an oriented substrate. Furthermore, these results suggest that this oriented substrate with alternating arrangement can be used as a device that directly attaches to diseased cardiomyocyte tissue to the affected area, demonstrating a therapeutic effect in normalizing diseased cardiac function.

[0012] Furthermore, culturing cells in conditions similar to those of a living organism is considered an effective method for maturing cardiomyocytes. In living organisms, cardiomyocytes exist in a unidirectional orientation, and electrical conduction between cells occurs via intercellular junctions, allowing them to perform functions such as contraction and / or relaxation. In this embodiment, by using an oriented culture substrate and culturing cardiomyocytes in an oriented state, the maturation of cardiomyocytes (improvement of physiological activity and motor function) is promoted, and the healing effect of the normal cardiomyocyte sheet for transplantation can be evaluated in an environment closer to that in vivo.

[0013] <cardiomyocytes> In this embodiment, the cardiomyocyte sheet is composed of cardiomyocytes. Examples of cardiomyocytes include cardiomyocytes of vertebrates, including invertebrates, humans, and non-humans, and vertebrates include fish, amphibians, reptiles, birds, and mammals. Specifically, for example, mammals may include rodents such as mice, rats, ferrets, hamsters, guinea pigs, or rabbits, or primates such as dogs, cats, sheep, pigs, cows, horses, or rhesus monkeys, chimpanzees, orangutans, and humans. In addition to mammals, it also includes fish, birds including poultry, reptiles, and the like. Among these, the cardiomyocytes are preferably those of mammals, including humans and mice, and more preferably human cardiomyocytes.

[0014] In this embodiment, the cardiomyocytes are differentiation-induced cells derived from healthy individuals or patients with diseases. The cardiomyocytes may also be cardiomyocytes differentiated from pluripotent stem cells (stem cells) using methods involving differentiation-inducing factors for various mesodermal lines. In addition, in this embodiment, the cardiomyocytes may be induced cardiomyocytes (iCM, cardiomyocytes obtained by direct reprogramming) induced by introducing transcription factors into fibroblasts or blood cells of a patient or the like. Among these, cardiomyocytes are preferably those derived from stem cells. Here, stem cells are cells that possess pluripotency and self-renewal capabilities, and include, for example, pluripotent stem cells (Stem Cells) that can differentiate into various cell types in organisms such as primates including humans and mammals other than primates. Preferably, stem cells are passageable, maintain a state where differentiation does not progress even after passage, and have properties that make it difficult for the karyotype to change or the epigenetic phenotype to change. In addition, it is preferable that stem cells have sufficient proliferative capacity in vitro. Specific examples of such stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and other artificially generated or selected pluripotent stem cells. These stem cells may also be stem cells produced by reprogramming somatic cells with various vectors such as retroviruses, adenoviruses, plasmids, RNA, or small molecule compounds containing specific genes. Furthermore, while stem cells do not necessarily need to be cells with multipotency close to totipotency (pluripotency), it is preferable to use multipotency cells that have a higher-than-usual multipotency. Stem cells may also be cells produced from cells obtained from patients with a disease, cells that serve as models for other diseases, cells into which a reporter gene has been incorporated (reporter cells), cells that can be conditionally knocked out, or other genetically modified cells. This genetic modification includes the addition, modification, or deletion of genes within chromosomes, the addition of genes using various vectors or artificial chromosomes, alteration of epigenetic regulation, the addition of artificial genetic materials such as PNA, and other forms of genetic modification. Among these, in this embodiment, cardiomyocytes are preferably cardiomyocytes derived from ES cells or iPS cells, from the viewpoint of ease of availability and ability to induce differentiation into cardiomyocytes.

[0015] (Normal cardiomyocytes) Normal cardiomyocytes are differentiated cells derived from healthy individuals; that is, cardiomyocytes differentiated from cells collected from healthy individuals. Here, differentiated normal cardiomyocytes refer to cardiomyocytes that do not appear to have a specific disease, and are the cardiomyocytes that make up cardiomyocyte sheets for transplantation aimed at treating diseases. If the target heart disease is a hereditary heart disease, the normal cardiomyocytes may be normal cardiomyocytes derived from allogeneic cells, or they may be normal cardiomyocytes that have been normalized by correcting gene mutations through genome editing or the like. Furthermore, if the target heart disease is not a hereditary heart disease, the normal cardiomyocytes may be allogeneic normal cardiomyocytes or autologous normal cardiomyocytes.

[0016] (Disease cardiomyocytes) Disease cardiomyocytes are cardiomyocytes derived from patients with a disease. Here, differentiated disease cardiomyocytes are cardiomyocytes obtained from patients with a disease (heart disease), and disease-derived cardiomyocytes and cardiomyocyte sheets possess the pathological characteristics of the disease and can be used as a model. Examples of heart diseases include cardiomyopathy (hypertrophic cardiomyopathy, dilated cardiomyopathy, restrictive cardiomyopathy), heart failure (chronic heart failure, severe heart failure, congestive heart failure), ischemic heart disease (myocardial infarction, angina pectoris), arrhythmias, and cardiac hypertrophy due to sports. Among these, from the viewpoint that it is preferable for the disease to be a target for transplantation in regenerative medicine, disease cardiomyocytes are preferably cardiomyocytes established from cells of a patient having one of the following diseases selected from the group consisting of hereditary heart diseases such as hypertrophic cardiomyopathy, dilated cardiomyopathy, and restrictive cardiomyopathy, as well as acquired heart diseases such as cardiac hypertrophy due to sports.

[0017] Furthermore, diseased cardiomyocytes or diseased cardiomyocyte sheets, depending on the disease, exhibit changes in motor function and physiological characteristics compared to normal cardiomyocyte sheets or normal cardiomyocyte sheets, such as a decrease in heart rate (BR), contractile velocity (CV), relaxation velocity (RV), and contraction-relaxation duration (CRD).

[0018] <Lamination of cardiomyocyte sheets> In this embodiment, the oriented culture substrate has two or more layers of cardiomyocyte sheets, each consisting of at least one layer of diseased cardiomyocyte sheets made from differentiated cardiomyocytes derived from diseased patients, and at least one layer of normal cardiomyocyte sheets made from differentiated cardiomyocytes derived from healthy individuals. Furthermore, the diseased cardiomyocyte sheets and the normal cardiomyocyte sheets are in contact on at least one surface. The layered cardiomyocyte sheet is not particularly limited as long as it is composed of two or more layers of cardiomyocyte sheets, and may be composed of three or more layers of cardiomyocyte sheets. From the viewpoint of ease of manufacturing of cell culture substrates with layered cardiomyocyte sheets, the number of layers of cardiomyocyte sheets constituting the layered cardiomyocyte sheet is preferably two to ten layers, more preferably two to five layers, even more preferably two to three layers, and even more preferably two layers. In this case, if the structure consists of three or more layers of cardiomyocyte sheets, it may have a configuration in which identical cardiomyocyte sheets are stacked, such as diseased cardiomyocyte sheet / diseased cardiomyocyte sheet / normal cardiomyocyte sheet or normal cardiomyocyte sheet / normal cardiomyocyte sheet / disease cardiomyocyte sheet, that is, a configuration in which identical cardiomyocyte sheets are superimposed within the stacked structure.

[0019] Here, the cardiomyocyte sheet consists of one or more layers each of normal cardiomyocyte sheets and diseased cardiomyocyte sheets. The cell culture substrate with a layered cardiomyocyte sheet of the first embodiment has a configuration in which a normal cardiomyocyte sheet is attached to an oriented substrate, and a diseased cardiomyocyte sheet is layered on top of the normal cardiomyocyte sheet. With this configuration, diseased cardiomyocytes can be seeded onto the oriented normal cardiomyocyte sheet, making it easier for the diseased cardiomyocytes to orient themselves, and enabling evaluation in an environment closer to that of a living organism. In addition, since the diseased cardiomyocyte sheet is on top, it is easier to observe the diseased cardiomyocyte sheet.

[0020] The cell culture substrate with a layered cardiomyocyte sheet of the second embodiment may have a configuration in which a diseased cardiomyocyte sheet is attached to an oriented substrate, and a normal cardiomyocyte sheet is layered on top of the diseased cardiomyocyte sheet. With the above configuration, when actually transplanting a cardiomyocyte sheet in regenerative medicine, it is thought that a normal cardiomyocyte sheet will be transplanted onto a diseased heart, and it is thought that evaluation can be performed in an environment closer to the actual state of regenerative medicine.

[0021] <Oriented Substrate> In this embodiment, the cell culture substrate of the cell culture substrate with a laminated cardiomyocyte sheet has alternating arrangement portions in which strip-shaped cell adhesion regions and strip-shaped cell adhesion inhibition regions are arranged alternately. The cell attachment region is a flat portion, and the cell attachment region is also an uneven portion having multiple irregularities. Each flat portion has a shape extending in a first direction, and the multiple flat portions are arranged in a second direction intersecting the first direction across the entire surface, and each uneven portion includes multiple stepped structures that fill the spaces between adjacent flat portions. In other words, it has an alternating arrangement portion in which the band-shaped flat portion, which is the cell attachment region, and the uneven portion, which is the cell attachment inhibition region, are arranged alternately. The pitch of the stepped structure in the uneven portion is preferably 100 nm or more and 10 μm or less. The stepped structure is a convex portion, and the uneven portion has a plurality of convex portions on the bottom surface of the recess sandwiched between the adjacent flat portions, and in the thickness direction of the alternating arrangement portion, the difference between the height of the tip surface of the uneven portion and the height of the flat portion is preferably 0.5 μm or less. As shown in Figure 1(a), the alternating arrangement portion 100 is, for example, a sheet material placed on the culture dish 110 of a petri dish. The petri dish holds the cell suspension in the space enclosed by the culture dish 110 and the lid 120. The bottom of the petri dish may be processed to have the surface shape having the specific flat and uneven portions described above, in which case the petri dish itself is a cell culture substrate having the alternating arrangement portion. As shown in Figure 1(b), the surface 111 of the alternating arrangement portion 100 comprises a plurality of flat portions 130 and a plurality of uneven portions 140. The uneven portions 140 are composed of a plurality of stepped structures, which fill the spaces between adjacent flat portions 130. The stepped structures are convex portions. The uneven portions 140 comprise a recess sandwiched between adjacent flat portions 130 and a plurality of convex portions 141 located on the bottom surface of the recess.

[0022] As shown in Figure 1(c), each flat portion 130 is a flat surface extending in a single direction, the first direction (the vertical direction in Figure 1(c)). Each flat portion 130 is aligned in a second direction (the horizontal direction in Figure 1(c)) perpendicular to the first direction across the entire surface 111. Each uneven portion 140 also extends in the first direction and is aligned in the second direction across the entire surface 111.

[0023] Each protrusion 141 constituting the uneven surface 140 is located, for example, at each vertex of a triangular lattice when viewed from a direction opposite to the surface 111. Each uneven surface 140 repeats this arrangement of protrusions 141 in a first direction and a second direction. If the uneven surface 140 has protrusions 141 located at each vertex of a triangular lattice, the master plate for forming the protrusions 141 can be formed by an etching method using a mask suitable for forming minute repeating structures, such as a single-particle film as the mask.

[0024] When viewed from a direction opposite to the surface 111, each protrusion 141 has, for example, a circular shape. The mode of the distance between the centers of adjacent protrusions 141 is the pitch of the protrusions 141. Also, the maximum width of the protrusion in the plan view shape of the protrusion 141 is the diameter of the protrusion 141.

[0025] A configuration in which the pitch of the protrusions 141 satisfies the following (A) and (B) is preferable from the viewpoint of aligning the extension direction of cardiomyocytes with the first direction. That is, a configuration in which the pitch of the protrusions 141 satisfies the following (A) and (B) is preferable from the viewpoint of clearly defining the superiority or inferiority of cardiomyocyte adhesion between the flat portion 130 and the uneven portion 140. (A) Pitch of the protrusion 141: 100 nm or more and 10 μm or less (B) Diameter of the protrusion 141: 50% or more and 100% or less of the pitch of the protrusion 141

[0026] The length of each flat section 130 in the second direction (short side direction) is the width of the flat section 130. Also, the length between adjacent flat sections 130 in the second direction (short side direction) is the width of the uneven section 140. The width of the flat portion 130 and the width of the uneven portion 140 are, for example, 1 / 10 to 10 times the size of the cells to be cultured (5 μm to 100 μm). A configuration in which the width of the flat portion 130 and the width of the uneven portion 140 satisfy the following (C) and (D) is preferable in terms of facilitating the alignment of the cardiomyocytes in the direction of extension to the first direction. (C) Width of flat section 130: 10 μm or more and 50 μm or less (D) Width of uneven portion 140: 10 μm or more and 50 μm or less

[0027] As shown in Figure 1(d), the uneven portion 140 may include recesses 142 between adjacent protrusions 141 and between the flat portion 130 and the adjacent protrusions 141. Since multiple protrusions 141 are scattered on the uneven portion 140, the recesses 142, which are spaces between the protrusions 141, are connected in the first and second directions on the uneven portion 140.

[0028] In the thickness direction of the alternating arrangement portion 100, the length between the bottom surface of the recess 142 and the flat portion 130 is the height of the flat portion 130. Also, in the thickness direction of the alternating arrangement portion 100, the height difference between the tip surface of each convex portion 141 and the flat portion 130 is the boundary step. The height difference between the bottom surface of the recess 142 and the tip surface of each convex portion 141 is the height of the convex portion 141. In a configuration where the tip surface of each convex portion 141 and the flat portion 130 are flush, the height of the flat portion 130 and the height of the convex portion 141 are equal to each other. The ratio of the pitch of the convex portion 141 to the height of the convex portion 141 is the aspect ratio of the convex portion 141.

[0029] A configuration in which the boundary step satisfies (E) below is preferable from the viewpoint of improving the flatness of the cardiomyocyte sheet. A configuration in which the height of the protrusion 141 satisfies (F) below, and a configuration in which the aspect ratio of the protrusion 141 satisfies (G) below, is preferable from the viewpoint of improving the structural stability of the uneven portion 140 and facilitating the formation of the uneven portion 140. (E) Boundary step height: 0.5 μm or less, preferably 0.3 μm or less (F) Height of protrusion 141: 50 nm or more and 5 μm or less (G) Aspect ratio of the convex portion 141: 0.1 or more and 10 or less

[0030] Furthermore, if the configuration satisfies conditions (A) and (B) above, cells that are predominantly adhering to the flat portion 130 will preferentially adhere to the flat portion, and this, combined with the inferiority of adhesion to the uneven portion 140, aligns the cell elongation direction with the first direction, which is the extension direction of both structures. As a result, in a cardiomyocyte sheet that extends in a two-dimensional direction along the surface 111, it becomes possible to align the cell elongation direction in a one-dimensional direction, that is, to improve the orientation of the cells.

[0031] Furthermore, a configuration that satisfies (E) above, in particular, a configuration in which the tip surface of each protrusion 141 and the flat portion 130 are flush, makes it possible to improve the flatness of a cardiomyocyte sheet formed to cover the uneven portion 140 and the flat portion 130. Moreover, a configuration that satisfies (F) above makes it possible to further improve the flatness of the cardiomyocyte sheet.

[0032] Furthermore, the surface 111 of the alternating arrangement portion 100 may be coated with organic materials containing adhesion factors such as extracellular matrix, polymers, gels, etc., including laminin, collagen, gelatin, fibronectin, polylysine (PDL or PLL), hyaluronic acid, or it may be a surface made of metal, for the purpose of enhancing cell adhesion. In addition, the surface 111 of the alternating arrangement portion 100 may be hydrophilic or hydrophobic for the purpose of enhancing cell adhesion and the flatness of the cell sheet.

[0033] Furthermore, a stimulus-responsive material may be applied to facilitate the peeling and retrieval of the cell sheet after its formation. A temperature-responsive polymer, whose water affinity changes with temperature, is preferred as the stimulus-responsive material. Specifically, poly-N-isopropylacrylamide (PIPAAm) is preferred. The stimulus-responsive material may be applied to the substrate using conventional application methods, or the substrate treated with the stimulus-responsive material may be processed using the methods described below.

[0034] (Method for producing cell culture substrates having alternatingly arranged portions) An example of a method for producing a cell culture substrate having alternating arrangement portions is described below. In the following description, an example is described in which the surface 111 of the alternating arrangement portion is formed by transferring an intaglio plate 150 using a nanoimprint method. As shown in Figure 2, the method for producing a cell culture substrate having alternating arrangement portions includes the steps of forming a grooved plate 150 and forming the surface 111 of the alternating arrangement portions 100 by transferring the grooved plate 150.

[0035] The lower surface of the intaglio plate 150 has a shape that extends in a first direction (a direction perpendicular to the paper plane) and comprises a plurality of flat portions arranged in a second direction (the left-right direction of the paper plane) intersecting the first direction, and a plurality of stepped structures that fill the spaces between adjacent flat portions. The flat portions of the intaglio plate 150 are for forming the flat portions 130 of the alternating arrangement portion 100 by transfer. The uneven portions of the intaglio plate 150 are for forming the uneven portions 140 of the alternating arrangement portion 100 by transfer.

[0036] The stepped structure of the intaglio plate 150 is either a convex portion or a concave portion. In this embodiment, the stepped structure of the intaglio plate 150 is a concave portion 151 for forming a convex portion 141, and the pitch of the concave portion 151 is 100 nm or more and 10 μm or less. In the process of forming the intaglio plate 150, the uneven portion is formed by using, for example, at least one of the following methods on a silicon substrate for forming the intaglio plate 150: photolithography, colloidal lithography, anodizing, and interference exposure. Alternatively, the intaglio plate 150 itself may be obtained by one or more transfers from a master plate. The master plate has a shape corresponding to the surface shape of the intaglio plate 150 created on it using, for example, at least one of the following methods on a silicon substrate: photolithography, colloidal lithography, anodizing, and interference exposure.

[0037] Next, the lower surface of the intaglio plate 150 is placed against the surface 111 of the base material 160 for forming the alternating arrangement portion 100. The forming material of the base material 160 is, for example, a thermoplastic resin or a photocurable resin. Then, while the base material 160 is fluid, the lower surface of the intaglio plate 150 is pressed against the surface 111 of the base material 160. Subsequently, while suppressing the fluidity of the base material 160, the intaglio plate 150 is released from the surface 111 of the base material 160. As a result, the recesses 151 of the intaglio plate 150 are transferred to the surface 111 of the base material 160, forming a flat portion 130 and an uneven portion 140.

[0038] The surface of the thermoplastic resin or photocurable resin forming material of the base material 160 may be coated with organic materials containing adhesion factors such as extracellular matrix, polymers, or gels, for example, laminin, collagen, gelatin, fibronectin, polylysine (PDL or PLL), hyaluronic acid, etc., for the purpose of enhancing cell adhesion. Alternatively, biomaterials such as polysaccharides or proteins may be used as the forming material of the base material 160.

[0039] <Method for manufacturing cell culture substrate with layered cardiomyocyte sheet> A method for producing a cell culture substrate with a layered cardiomyocyte sheet, which is manufactured using a cell culture substrate having alternating arrangement portions 100, is described below. In a cell culture substrate having alternating arrangement portions that satisfy the above (A), as shown in Figure 3(a), the cells of the cell suspension held in the alternating arrangement portion 100 are cells S1 that preferentially adhere to the flat portion 130, and are also cells S2 that are less likely to adhere to the flat portion 130 but are allowed to adhere to the uneven portion 140. In this case, as shown in Figure 3(b), the flat portion 130 and the uneven portion 140 extend in a first direction and are arranged alternately in a second direction. Therefore, on the surface 111 of the alternating arrangement portion, for example, the orientation of cells S1 that are preferentially attached to the flat portion 130 is controlled by the structure of the flat portion 130 and the structure of the uneven portion 140 that divides it. Furthermore, in the uneven portion 140 sandwiched between adjacent flat portions 130, although less pronounced than in the flat portion 130, the orientation control by the flat portion 130 is reflected in the cells S2 adhering to the uneven portion 140. As a result, as shown in Figure 3(c), cells S1 and S2 whose orientation is controlled in the first direction form a cultured cell sheet SA that spreads across the entire surface 111. Therefore, a cardiomyocyte sheet on an oriented substrate imparts orientation to the cardiomyocytes.

[0040] The cell culture substrate with the layered cardiomyocyte sheet of this embodiment is preferably manufactured by any of the following methods 1 to 3. (Method 1) A step of preparing a cell culture substrate having alternating arrangement portions in which band-shaped cell adhesion regions and band-shaped cell adhesion inhibition regions are arranged alternately, A step of forming a normal cardiomyocyte sheet consisting of differentiated cardiomyocytes derived from a healthy person on at least the alternating arrangement portion of the cell culture substrate, The process includes, in this order, forming a diseased cardiomyocyte sheet consisting of differentiated cardiomyocytes derived from a diseased patient on the normal cardiomyocyte sheet, A method for producing a cell culture substrate with a layered cardiomyocyte sheet. (Method 2) A step of preparing a cell culture substrate having alternating arrangement portions in which band-shaped cell adhesion regions and band-shaped cell adhesion inhibition regions are arranged alternately, A step of forming a diseased cardiomyocyte sheet consisting of differentiated cardiomyocytes derived from diseased patients on at least the alternating arrangement portion of the cell culture substrate, The process includes, in this order, forming a sheet of normal cardiomyocytes consisting of differentiated cardiomyocytes derived from a healthy person on the diseased cardiomyocyte sheet, A method for producing a cell culture substrate with a layered cardiomyocyte sheet. (Method 3) A step of forming a diseased cardiomyocyte sheet consisting of differentiated cardiomyocytes derived from a diseased patient on at least the alternating arrangement portion of a cell culture substrate having alternating arrangement portions of band-shaped cell adhesion regions and band-shaped cell adhesion inhibition regions, A step of forming a normal cardiomyocyte sheet consisting of differentiated cardiomyocytes derived from a healthy person on at least the alternating arrangement portion of a cell culture substrate having alternating arrangement portions of band-shaped cell adhesion regions and band-shaped cell adhesion inhibition regions, The process includes the step of separating either the diseased cardiomyocyte sheet or the normal cardiomyocyte sheet from the cell culture substrate and laminating it onto the other cardiomyocyte sheet. A method for producing a cell culture substrate with a layered cardiomyocyte sheet.

[0041] In Method 1, a normal cardiomyocyte sheet is formed on at least the alternating arrangement portion of a cell culture substrate having the alternating arrangement portion described above. That is, normal cardiomyocytes are seeded and cultured so as to be in contact with the alternating arrangement portion to form a normal cardiomyocyte sheet. Note that it is sufficient to form the normal cardiomyocyte sheet on at least the alternating arrangement portion, and the normal cardiomyocyte sheet may be formed over the entire surface of the cell culture substrate. When a normal cardiomyocyte sheet is formed on the alternating arrangement portion, a normal cardiomyocyte sheet is formed in which the cells are oriented in the first direction described above. After a sheet of normal cardiomyocytes is formed, diseased cardiomyocytes are seeded and cultured to form a sheet of diseased cardiomyocytes on top of the normal cardiomyocyte sheet. The diseased cardiomyocyte sheet only needs to be formed on the normal cardiomyocyte sheet formed on at least the alternating arrangement portion; it may also be formed across the entire surface of the cell culture substrate. By forming the diseased cardiomyocyte sheet on an oriented normal cardiomyocyte sheet, the diseased cardiomyocyte sheet will also be oriented in the same way as the normal cardiomyocyte sheet. In Method 1, a layered cardiomyocyte sheet is manufactured by stacking a cell culture substrate, a normal cardiomyocyte sheet, and a diseased cardiomyocyte sheet in that order.

[0042] In Method 2, a diseased cardiomyocyte sheet is formed on at least the alternating arrangement portion of the cell culture substrate having the alternating arrangement portion described above. That is, diseased cardiomyocytes are seeded and cultured so as to be in contact with the alternating arrangement portion to form a diseased cardiomyocyte sheet. Note that it is sufficient to form the diseased cardiomyocyte sheet on at least the alternating arrangement portion, and the diseased cardiomyocyte sheet may be formed over the entire surface of the cell culture substrate. When a diseased cardiomyocyte sheet is formed on the alternating arrangement portion, a diseased cardiomyocyte sheet is formed in which the cells are oriented in the first direction described above. After the diseased cardiomyocyte sheet is formed, normal cardiomyocytes are seeded and cultured to form a normal cardiomyocyte sheet on top of the diseased cardiomyocyte sheet. The normal cardiomyocyte sheet only needs to be formed on the diseased cardiomyocyte sheet formed on at least the alternating arrangement portion; it may also be formed across the entire surface of the cell culture substrate. By forming the normal cardiomyocyte sheet on the oriented diseased cardiomyocyte sheet, the normal cardiomyocyte sheet will also be oriented in the same way as the diseased cardiomyocyte sheet. In Method 2, a layered cardiomyocyte sheet is manufactured by stacking a cell culture substrate, a diseased cardiomyocyte sheet, and a normal cardiomyocyte sheet in that order.

[0043] In Method 3, diseased cardiomyocytes are seeded and cultured on at least the alternating arrangement portion of a cell culture substrate having an alternating arrangement portion to form a diseased cardiomyocyte sheet. This results in the formation of an oriented diseased cardiomyocyte sheet. Furthermore, normal cardiomyocytes are seeded and cultured on at least the alternating arrangement portion of a cell culture substrate having an alternating arrangement portion to form a normal cardiomyocyte sheet. This results in the formation of an oriented normal cardiomyocyte sheet. Furthermore, diseased cardiomyocyte sheets and normal cardiomyocyte sheets are formed on separate cell culture substrates. Subsequently, either the diseased cardiomyocyte sheet or the normal cardiomyocyte sheet is separated from the cell culture substrate and laminated onto the other cardiomyocyte sheet. Separation from the cell culture substrate is performed while the cardiomyocytes maintain their sheet-like structure. One example of such a method is to pre-coat the cell culture substrate with a stimulus-responsive material to facilitate the peeling and recovery of the cell sheet. As the stimulus-responsive material, a temperature-responsive polymer whose water affinity changes with temperature is preferred, and poly-N-isopropylacrylamide (PIPAAm) is an example. The stimulus-responsive material may be applied to the substrate using a conventional coating method, or a textured structure may be formed on a planar substrate treated with the stimulus-responsive material using the method described above. It is preferable to stack the cardiomyocyte sheets detached from the cell culture substrate so that their orientation direction is the same as that of the cardiomyocyte sheets on the cell culture substrate. Furthermore, when diseased cardiomyocyte sheets are detached from the cell culture substrate and laminated onto normal cardiomyocyte sheets, a cell culture substrate with laminated cardiomyocyte sheets is obtained, in which the cell culture substrate, normal cardiomyocyte sheets, and diseased cardiomyocyte sheets are laminated in this order. Furthermore, when a normal cardiomyocyte sheet is detached from the cell culture substrate and laminated onto a diseased cardiomyocyte sheet, a cell culture substrate with a laminated cardiomyocyte sheet is obtained, in which the cell culture substrate, the diseased cardiomyocyte sheet, and the normal cardiomyocyte sheet are laminated in this order.

[0044] [Evaluation Method for Normal Cardiomyocyte Sheets for Organ Transplantation] The cell culture substrate with cardiomyocyte sheets of this embodiment consists of a laminated sheet of diseased cardiomyocytes and a sheet of normal cardiomyocytes, and can be used as an evaluation model for the disease-healing effect when a sheet of normal cardiomyocytes is transplanted into the heart of a patient with heart disease. It is preferable to have a step of evaluating at least one change selected from changes in motor function and physiological characteristics of diseased cardiomyocyte sheets laminated with normal cardiomyocyte sheets. Examples of the aforementioned changes in motor function include those observable by live-cell imaging. Examples of physiological characteristics include electrophysiological changes measured by Ca-imaging analysis, patch-clamp methods, and changes in state such as vitality and shape. Furthermore, if the motor function and physiological characteristics of diseased cardiomyocyte sheets alone approach those of normal cardiomyocytes, a healing effect is considered to have occurred. In this case, the motor function and physiological characteristics of normal cardiomyocyte sheets alone may be used as a positive control.

[0045] <Live Cell Imaging> Cardiac cell sheets, composed of cardiomyocytes, allow for the detection of changes in movement associated with the contraction and relaxation of cardiomyocytes. In vivo, cardiomyocytes are observed to be oriented in one direction, and contraction and relaxation occur in the direction of their orientation. By using a live cell imaging system equipped with a microscope and a camera capable of video recording (e.g., SI8000, manufactured by Sony Corporation), it is possible to analyze heart rate (BR), contractile velocity (CV), relaxation velocity (RV), contraction-relaxation duration (CRD), and degree of orientation from video data obtained by imaging cultured cell sheets (living cells) under culture conditions. For more information on measuring BR, CV, RV, CRD, etc. using live cell imaging, see, for example, Methods in Molecular Biology, vol. 2320, Chapter 15. Furthermore, the methods described in the examples are referenced for these measurement methods. Here, the degree of orientation is calculated by determining the angle of the motion vector when the horizontal direction of the screen of the image obtained by measurement is set to 0°, and finding the ratio of the number of vectors indicating ±5° from the angle of the most frequent value of the vector angle to the total number of detected vectors. The degree of orientation obtained from the live cell imaging device is determined by the following formula. Degree of orientation (%) =(Number of vectors included in the range of ±5° of the most frequent value) / (Total number of vectors)×100 In the present embodiment, in any layer of the laminated cardiomyocyte sheet, the degree of orientation measured using live cell imaging is preferably 10% or more, more preferably 12% or more, still more preferably 16% or more, even more preferably 18% or more, and particularly preferably 20% or more on the orientation substrate. The upper limit of the degree of orientation is not particularly limited. When the degree of orientation is within the above range, the cardiomyocyte sheet is cultured in a state closer to that in vivo, and evaluation in an environment closer to that in vivo becomes possible.

[0046] In live cell imaging, since the average heart rate of an adult is 60 beats / min, generally, a heart rate (BR) closer to 60 beats / min is closer to normal mature cardiomyocytes. Also, it is considered that a contraction velocity (CV), relaxation velocity (RV), and contraction-relaxation duration (CRD) according to the normal heart rate are closer to normal cardiomyocytes. Normal cardiomyocytes may be cultured in the same method, and the measured BR, CV, RV, CRD, etc. may be used as a positive control (target value for normalization). When BR, CV, RV, CRD, etc. measured for the diseased cardiomyocyte sheet alone change to a state closer to normal by the lamination of the normal cardiomyocyte sheet, it is considered that a therapeutic effect for the disease can be obtained when the normal cardiomyocyte sheet is transplanted in vivo. Also, it is possible to measure the therapeutic effect according to the degree of improvement.

[0047] <Ca-imaging analysis> In cardiomyocytes, a phenomenon called calcium transient is observed, in which the Ca concentration throughout the cytoplasm rises almost simultaneously, triggered by the action potential during myocardial contraction. By treating the cardiomyocyte sheet of this embodiment with a fluorescent Ca indicator and observing it with a confocal quantitative image cytometer (Ca-imaging analysis), it is possible to observe the calcium transient over time. A waveform graph is created from the signal information of the calcium transient, and dedicated analysis software is used to analyze the parameters described below. In immature cardiomyocytes or cardiomyocytes exhibiting abnormalities due to disease, the linewidth (duration) at 50% height of the calcium transient waveform peak tends to be prolonged compared to normal, mature cardiomyocytes. The line width at 50% height (Duration) varies with heart rate, so divide Duration (seconds) by the square root of Interval (seconds): Duration / (Interval) 1 / 2 It is also preferable to use [this]. Generally, the shorter the 50% height line width (Duration), which reflects the calcium transient duration, the closer the result is to a mature cardiomyocyte. A shorter 50% height line width indicates that diseased cardiomyocytes are becoming closer to normal cardiomyocytes. Interval is affected by heart rate. Typically, in electrocardiogram analysis, the action potential duration is corrected for heart rate, and therefore, as mentioned above, Duration / (Interval) 1 / 2 You may adopt this approach. Therefore, if the duration of calcium transients obtained from diseased cardiomyocyte sheets becomes equivalent to that of normal cardiomyocytes through the layering of normal cardiomyocyte sheets, it is considered that a healing effect can be obtained through transplantation of normal cardiomyocyte sheets.

[0048] <Patch Clamp Method> The patch-clamp test involves applying a current of 20 pA to 100 pA at 0.2 Hz to cardiomyocytes in a Tyrode solution at 25°C under current clamp, generating action potentials, and measuring the change in potential. The current should be appropriately selected within the range that generates appropriate action potentials. Examples of physiological characteristics include the maximum diastolic potential and the duration of the action potential. Furthermore, the duration of 80% repolarization of the action potential is often cited as an indicator of action potential duration. This is the time required from the time a current is applied until the potential reaches 80% repolarization, assuming that the difference between the peak height of the action potential (shown as the first phase) and the maximum diastolic potential is 100%. In other words, it is the time required from the rise time of the action potential until the action potential reaches a potential equivalent to 80% repolarization. As cardiomyocytes mature, they tend to have a longer action potential duration and a deeper diastolic potential compared to immature cardiomyocytes. In this embodiment, the duration of the 80% repolarization of the action potential of diseased cardiomyocytes in the stacked cardiomyocyte sheet is preferably 600 msec or more, more preferably 650 msec or more, even more preferably 700 msec or more, and even more preferably 750 msec or more. While there is no particular upper limit, from the viewpoint of preferring an action potential duration similar to that of cardiomyocytes in vivo, it is preferably 1200 msec or less, more preferably 1050 msec or less, and even more preferably 900 msec or less. Furthermore, from the viewpoint of preferring that the maximum diastolic potential of diseased cardiomyocytes constituting the cell culture substrate with the layered cell sheet of this embodiment be closer to the value of novel cells in vivo, it is preferably -60mV or less, more preferably -62.5mV or less, even more preferably -65mV or less, and even more preferably -67.5mV or less. Also, there is no particular lower limit, but since adult ventricular cardiomyocytes are approximately -80mV, it is preferable that the value be close to -80mV. For information on action potential duration and maximum diastolic potential (resting membrane potential) in normal isolated human ventricular myocytes, please refer to Circulation, 2013;127:575-584.

[0049] <Other> One of the changes selected from the changes in motor function and physiological characteristics of diseased cardiomyocyte sheets may be evaluated to assess the cytotoxic effect on diseased cardiomyocyte sheets. Alternatively, the absence of a cytotoxic effect on diseased cardiomyocyte sheets may be evaluated by stacking normal cardiomyocyte sheets using trypan blue staining or the like. Alternatively, changes in the shape of diseased cardiomyocytes in diseased cardiomyocyte sheets may be evaluated.

[0050] <Applications of cell culture substrates with layered cardiomyocyte sheets> The cell culture substrate with laminated cardiomyocyte sheets of this embodiment can be used for various purposes, for example, in evaluating the healing effect in regenerative medicine. By laminating a normal cardiomyocyte sheet onto a diseased cardiomyocyte sheet, it is possible to evaluate how closely the motor function and physiological characteristics change to those of the normal cardiomyocyte sheet. Furthermore, it is possible to evaluate whether the lamination of the normal cardiomyocyte sheet causes changes in the shape of the cardiomyocytes constituting the diseased cardiomyocyte sheet or a decrease in the number of viable cells. Specifically, it can be used to evaluate the healing effect when a sheet of normal myocardial cells is directly attached to the damaged area of ​​myocardial tissue in a heart damaged by ischemic diseases such as myocardial infarction and angina pectoris (hereinafter also referred to as a "damaged heart") in the same direction as the contraction of the cardiac tissue, and then transplanted by methods such as suturing or insertion after attachment. When actually using it in regenerative medicine, a single layer of normal cardiomyocyte sheets may be used, or several layers of normal cardiomyocyte sheets may be stacked and laminated before use. When laminating, it is preferable to align the orientation direction of the sheets; specifically, the direction of contraction or orientation of the normal cardiomyocyte sheets should be aligned when laminating. [Examples]

[0051] Examples are given below to illustrate the present invention in detail, but the present invention is not limited to these examples.

[0052] In this example, iPS cell-derived hypertrophic cardiomyopathy (HCM) cardiomyocytes were used as a model for heart disease, and iPS cell-derived cardiomyocytes (CM2) were used as normal cardiomyocytes. First, to reproduce the environment of the heart in vivo, normal cardiomyocytes were seeded and cultured on an oriented substrate to create a normal cardiomyocyte sheet with the long axis aligned in one direction. After forming the normal cardiomyocyte sheet with oriented cells, hypertrophic cardiomyopathy (HCM) cardiomyocytes were seeded onto the normal cardiomyocyte sheet to form a hypertrophic cardiomyopathy cardiomyocyte sheet on the normal cardiomyocyte sheet. Maintenance culture was performed for a predetermined number of days, and the function of the hypertrophic cardiomyopathy cardiomyocyte sheet was measured to evaluate the therapeutic effect of transplantation. As a control, cell culture substrates with diseased cardiomyocyte sheets were prepared by seeding and culturing HCM in a single layer on a flat or oriented substrate, and cell culture substrates with layered normal cardiomyocyte sheets were prepared by seeding and culturing CM2 in layers on a flat or oriented substrate, and were evaluated in the same manner. Furthermore, the results obtained using a cell culture substrate with a single-layer normal cardiomyocyte sheet, prepared by seeding and culturing CM2 in a single layer, were equivalent to the results obtained using a cell culture substrate with a layered normal cardiomyocyte sheet, prepared by layering CM2.

[0053] (1) Culture of human iPS cell-derived cardiomyocytes (normal cardiomyocytes) (CM2) iCell Cardiomyocyte v2.0 (FUJIFILM Cellular Dynamics) (hereinafter referred to as CM2) was used as the cardiomyocyte derived from human iPS cells. Culture was performed according to the following procedure. The frozen cell samples were thawed by warming them in a 37°C water bath for 3 minutes. The cell solution was transferred to a 50 mL centrifuge tube, and 9 mL of pre-warmed 37°C plating medium (iCell cardiomyocyte thawing medium, FUJIFILM Cellular Dynamics) was added and mixed to dilute the cell solution. A portion of the diluted cell suspension was collected, mixed with an equal volume of trypan blue, and the number of viable cells was measured using a hemocytometer. The cell concentration was adjusted using plating medium, and 6 × 104 Cells were seeded at a concentration of cells / well in 96-well plates pre-coated with fibronectin. The 96-well plates used were those with an orientation substrate (orientation plate, ND Cell Aligner, 96-well plate type), and as a control, commercially available 96-well plates with a flat culture surface (flat plate) were used. After seeding, the cells were incubated in a 37°C CO2 incubator for 4 hours, and then the culture medium was changed using Maintenance Medium (iCell cardiomyocyte maintenance medium, FUJIFILM Cellular Dynamics). After the medium change, the cells were maintained in culture for a predetermined period, with the medium being changed every two days.

[0054] The ND Cell Aligner used in (1) above and the examples described below had a band-shaped flat portion (cell adhesion region) and a band-shaped uneven portion (cell adhesion inhibition region). Each flat portion had a shape extending in a first direction and was aligned in a second direction intersecting the first direction on the entire surface, with a width (length in the second direction) of 10 μm for each flat portion. Each uneven portion consisted of multiple stepped structures filling the gaps between adjacent flat portions, with a length in the second direction of 10 μm between each flat portion, and a pitch of convex portions in the uneven portion being 300 nm. The height of each convex portion in the uneven portion was measured using AFM, and the average height from the bottom of the recess to the tip of the convex portion was 446 nm. The average height from the bottom of the recess to the flat portion was 455 nm.

[0055] (2) Culture of diseased human iPS cells (disease-derived cardiomyocytes) (HCM) As cardiomyocytes exhibiting the pathogenesis of hypertrophic cardiomyopathy, we used cardiomyocytes derived from diseased human iPS cells (Mycell cardiomyocytes (MYH7 R403Q) 01178 (FUJIFILM Cellular Dynamics)) (hereinafter referred to as HCM). These cells are diseased cardiomyocytes of hypertrophic cardiomyopathy, differentiated from iPS cells of diseased patients with a missense mutation (R403Q) in the 403rd residue of the β-myosin heavy chain 7 gene, where arginine is replaced with glutamine. Culture was performed using the same procedure as for CM2. The cell saturation, in which the number of viable cells was measured, was adjusted for cell concentration using plating medium, resulting in 6 × 10⁶ cells. 4 The cells were seeded into 96-well culture vessels at a concentration of cells / well.

[0056] (3) Performing monolayer culture HCM was cultured on flat plates and oriented plates, and the motility of cell sheets cultured on each culture substrate was measured and evaluated. Using the procedure described in (2) above, apply 6 × 10 HCM to the flat plate and the oriented plate. 4 Seeds were seeded at a concentration of cells / well and cultured. Day 0 was defined as the day of sowing, and various parameters were measured on day 5 of culture using a live cell imaging system (SI8000, manufactured by Sony Corporation). In addition, various parameters were measured on day 21 using a confocal fluorescence microscope (confocal quantitative image cytometer CQ1, manufactured by Yokogawa Electric Corporation).

[0057] (4) Implementation of layered culture In the layered culture method, the lower layer cells (CM2) were initially seeded and cultured, with the seeding day designated as day 0. On day 10, the upper layer cells (CM2 or HCM) were seeded and cultured on top of the lower layer cells. Following the procedure described in (1) above, 6 × 10 CM2 layers were placed on the flat plate and the oriented plate to form the lower layer. 4 Seeds were seeded at a concentration of cells / well. On day 10, CM2 or HCM was added as an upper layer on top of the cultured CM2, in a layer of 6 × 10⁶. 4 Cells were seeded and cultured at a concentration of cells / well. Day 0 was set as the start day of layered culture, and image data was acquired on day 5 using a live cell imaging system (SI8000, Sony Corporation). Additionally, image data was acquired on day 21 using a confocal fluorescence microscope (confocal quantitative image cytometer CQ1, Yokogawa Electric Corporation).

[0058] (5) Maturation confirmation test: Analysis using motion testing method with live cell imaging device To detect changes in the movement of cardiomyocytes seeded in each culture plate during contraction and relaxation, video data was acquired using a live cell imaging system (SI8000, manufactured by Sony Corporation). The measurement conditions were phase contrast imaging, 10x magnification (objective lens), 150 frames / second, resolution of 2048 x 2048 pixels, and 8-bit depth. Image acquisition time was 10 seconds. From video data, motion vectors (velocity, direction, and quantity) associated with the contraction and relaxation of cardiomyocytes were detected, and each parameter (heart rate (beating rate; BR), contractile velocity (CV), relaxation velocity (RV), contraction-relaxation duration (CRD), and orientation) were analyzed. To correct for variations due to heart rate (BR), the contraction-relaxation duration (CRD) was corrected using the following formula, referencing Frederica's formula. CRD (corrected value) = CRD / (60 / BR) × (1 / 3)

[0059] The degree of orientation was calculated based on the following formula by determining the angle of the motion vector when the horizontal direction of the screen is set to 0°, and then finding the ratio of the number of vectors that show angles within ±5° of the mode of the vector angle to the total number of detected vectors. Orientation degree (%) = (Number of vectors within ±5° of the mode) / (Total number of vectors) × 100 Generally speaking, the higher the heart rate (BR), the closer the cells are to mature cardiomyocytes; the faster the contraction velocity (CV) and relaxation velocity (RV), the closer the cells are to mature cardiomyocytes; and the shorter the contraction-relaxation duration (CRD), the closer the cells are to mature cardiomyocytes. The measurement results using the live cell imaging system are shown in Figures 4 to 8.

[0060] (6) Maturation confirmation test: Ca-imaging analysis using a confocal imaging device The change in Ca fluorescence intensity associated with the contraction of cardiomyocytes seeded on each culture plate was measured using a CQ1 (confocal fluorescence microscope, Yokogawa Electric Corporation). To visualize the Ca ion flux, a fluorescent Ca indicator (EarlyTox carditotoxicity kit, manufactured by Molecular Devices) was added to the cardiomyocyte sheet and left standing at 37°C for 15 minutes. The measurement conditions for CQ1 were an excitation wavelength of 488 nm / emission wavelength of 525 nm, and a data acquisition time of 60 seconds. Also, CellPath Finder was used for analysis. From the measurement data, as parameters, the line width (Duration) at 50% height of the Ca transient waveform peak and the interval (Interval) between waveform peaks were selected for analysis. The line width (Duration) at 50% height of the Ca transient waveform peak is usually corrected for the variation in the interval (Interval) between waveform peaks. In the analysis of electrocardiograms, the corrected value of the action potential duration is used according to the heart rate. Therefore, Duration / (Interval) 1 / 2 was adopted. Duration (corrected value) = Duration / (Interval) 1 / 2 The measurement results using the confocal imaging device are shown in Figures 9 and 10.

[0061] <Results of the layered culture of iPS-derived cardiomyocytes (CM2) on the orientation plate> For the layered culture, first, the cells (CM2) that would become the lower layer were seeded and cultured. The seeding day was designated as day 0. On day 10, the cells (CM2) that would become the upper layer were seeded on top of the lower layer CM2 and cultured. The seeding day of the upper layer cells was designated as day 0, and on day 5, image data was acquired using a live cell imaging device (SI8000, manufactured by Sony Corporation). Also, on day 21, image data was acquired using CQ1 (confocal fluorescence microscope, manufactured by Yokogawa Electric Corporation). Measurement results from motion vector analysis using SI8000 showed that CM2 cultured in stacked plates on orientation plates had significantly increased heart rate (BR), relaxation velocity (RV), and systolic-relaxation duration (CRD, corrected for heart rate) on day 5 of culture compared with CM2 cultured in stacked plates (all p<0.01), and the degree of cell orientation (mode ±5°) was significantly higher (p<0.01). On the other hand, CM2 cultured in stacked plates on orientation plates had significantly decreased contraction velocity (CV) compared with CM2 cultured in stacked plates (p<0.01) (comparison of flat CM2 / CM2 and orientation CM2 / CM2 in each graph (Figures 4-8)). Measurements using the SI8000 focused on the top layer of cultured cells to acquire data. Therefore, the measured values ​​reflect the results of observing the upper layer of cells in a layered culture.

[0062] From the CQ1 measurement results, CM2 cultured in stacked oriented plates showed a significantly shorter line width at 50% height of the Ca ion transient peak compared to CM2 cultured in stacked flat plates. Furthermore, the line width (Duration) at 50% height of the waveform peaks (corrected value), corrected for the interval between waveform peaks, was significantly shorter for CM2 cultured in stacked layers on oriented plates compared to CM2 cultured in stacked layers on flat plates (p<0.05). Measurements using CQ1 focused on the top layer of cultured cells, and data was acquired from these cells. Therefore, the measured values ​​reflect the results of observing the upper layer of cells in the layered culture.

[0063] Layered culture of CM2 / CM2 cells cultured on orientation plates cultures normal cardiomyocytes in a state similar to that of cardiomyocytes in vivo, with the cells oriented in one direction. If diseased cells are treated in some way and the symptoms improve, the measurement results of the diseased cells are thought to approach the values ​​of CM2 / CM2 layer culture cultured on an oriented plate. In this example, the approach of each measurement value of HCM to the value of CM2 indicates an improvement in the disease state of HCM.

[0064] <Results of monolayer culture of disease-related human iPS cell-derived cardiomyocytes (HCM) on oriented plates> Measurement results from motion vector analysis using SI8000 showed that HCM cultured in a single layer on an orientation plate had significantly increased heart rate (BR), relaxation velocity (RV), and systolic-relaxation duration (CRD, corrected for heart rate) on day 5 of culture, and the degree of cell orientation (mode ±5°) was significantly higher compared to HCM cultured on a flat plate (all p<0.01). On the other hand, HCM cultured in a single layer on an orientation plate had decreased contraction velocity (CV) compared to HCM cultured on a flat plate (p<0.01) (comparison of flat HCM and orientation HCM in each graph (Figures 4-8)).

[0065] Measurement results for CQ1 on day 21 of culture showed that the line width at 50% height of the Ca ion transient peak was similar for HCM cultured in a monolayer on an oriented plate compared to HCM cultured in a monolayer on a flat plate. Furthermore, the line width (Duration) at 50% height of the waveform peaks (corrected value), after correction for the interval between waveform peaks, was similar for HCM cultured in monolayers on oriented plates and HCM cultured in monolayers on flat plates.

[0066] Based on these results, culturing HCM cells from cardiac disease on oriented plates resulted in improvements in heart rate and relaxation speed, which affects arrhythmias. This indicates that culturing cells in an oriented state using oriented plates is a culture method that improves arrhythmias in hypertrophic cardiomyopathy cardiomyocytes. However, HCM cultured in monolayers on orientation plates did not reach the measured values ​​of CM2 cultured in stacked layers on orientation plates in all parameters except for the degree of orientation. It is thought that a combination of orientation culture and other treatments is necessary for further improvement of HCM symptoms.

[0067] <Results of layered culture of HCM on normal cardiomyocytes CM2 on flat plates and oriented plates> In the layered culture, the lower layer cells (CM2) were initially seeded and cultured. Day 0 was designated as the seeding day, and on day 10, the upper layer cells (HCM) were seeded on top of the lower layer CM2 and cultured. Day 0 was designated as the seeding day of the upper layer cells, and image data was acquired on day 5 using SI8000. Image data was also acquired on day 21 using CQ1. Measurement results from motion vector analysis using SI8000 showed that HCM cultured in an orientation plate stacked on top of CM2 cells had significantly increased heart rate (BR), relaxation velocity (RV), and systolic-relaxation duration (CRD, corrected for heart rate) on day 5 of culture, compared to HCM cultured in a flat plate stacked on top of CM2 cells. The degree of cell orientation (mode ± 5°) was also significantly higher. On the other hand, HCM cultured in a layered manner by stacking it on an oriented plate showed a reduced shrinkage rate (CV) compared to HCM cultured in a layered manner by stacking it on a flat plate.

[0068] From the measurement results of CQ1, the line width at 50% height of the Ca ion transient peak was significantly shorter in HCM cultured in an oriented plate stacked on top of CM2 compared to HCM cultured in a flat plate stacked on top of CM2. Furthermore, the line width (Duration) at 50% height of the waveform peaks (corrected value), corrected for the interval between waveform peaks, was significantly shorter for HCM cultured in an oriented plate stacked on top of CM2 compared to HCM cultured in a flat plate stacked on top of CM2. Based on these results, layered culture of HCM cells from cardiac disease cells on top of CM2 cells in an oriented plate resulted in improvements in heart rate, relaxation speed (which affects arrhythmias), and systolic-relaxation duration. This indicates that layered culture of HCM cells from cardiac disease cells on top of CM2 cells using an oriented plate is a culture method that improves arrhythmias in hypertrophic cardiomyopathy cardiomyocytes.

[0069] Based on these results, it has become clear that a culture method in which normal cardiomyocytes and cardiomyocytes from cardiac disease are layered on an oriented plate can be used as a model system for cardiomyocyte sheet transplantation. By layering cardiomyocytes derived from cardiac disease patients on top of a layer of normal cardiomyocytes cultured using an oriented plate, and then measuring their motor function and physiological activity and analyzing the parameters, it is possible to evaluate whether cardiomyocytes derived from cardiac disease patients can be improved by transplanting oriented normal cardiomyocytes. Furthermore, the oriented plate was shown to have potential as a tool for treating arrhythmias by creating cardiomyocyte sheets that perform oriented movements for the treatment of heart failure, and then transplanting these cardiomyocyte sheets. [Explanation of symbols]

[0070] 100 Alternate array part 111 Surface 110 Culture dish 120 Lid 130 Flat area 140 Uneven part 141 Convex part 142 recess

Claims

1. A cell culture substrate with a layered cardiomyocyte sheet having two or more layers of cardiomyocyte sheets on a cell culture substrate, The cell culture substrate has alternating arrangement portions in which strip-shaped cell adhesion regions and strip-shaped cell adhesion inhibition regions are arranged alternately. The cell culture substrate has at least two layers of cardiomyocyte sheets on top of the alternating arrangement portion, The cardiomyocyte sheet comprises at least one layer each of diseased cardiomyocytes derived from diseased patients and normal cardiomyocytes derived from healthy individuals. The normal cardiomyocyte sheet is attached to the cell culture substrate and the diseased cardiomyocyte sheet is laminated on the normal cardiomyocyte sheet, or the diseased cardiomyocyte sheet is attached to the cell culture substrate and the normal cardiomyocyte sheet is laminated on the diseased cardiomyocyte sheet. Cell culture substrate with layered cardiomyocyte sheets.

2. A step of preparing a cell culture substrate having alternating arrangement portions in which band-shaped cell adhesion regions and band-shaped cell adhesion inhibition regions are arranged alternately, A step of forming a normal cardiomyocyte sheet consisting of differentiated cardiomyocytes derived from a healthy person on at least the alternating arrangement portion of the cell culture substrate, The process includes, in this order, forming a diseased cardiomyocyte sheet consisting of differentiated cardiomyocytes derived from a diseased patient on the normal cardiomyocyte sheet, A method for producing a cell culture substrate with a layered cardiomyocyte sheet.

3. A step of preparing a cell culture substrate having alternating arrangement portions in which band-shaped cell adhesion regions and band-shaped cell adhesion inhibition regions are arranged alternately, A step of forming a diseased cardiomyocyte sheet consisting of differentiated cardiomyocytes derived from diseased patients on at least the alternating arrangement portion of the cell culture substrate, The process includes, in this order, forming a sheet of normal cardiomyocytes consisting of differentiated cardiomyocytes derived from a healthy person on the diseased cardiomyocyte sheet, A method for producing a cell culture substrate with a layered cardiomyocyte sheet.

4. A step of forming a diseased cardiomyocyte sheet consisting of differentiated cardiomyocytes derived from a diseased patient on at least the alternating arrangement portion of a cell culture substrate having alternating arrangement portions of band-shaped cell adhesion regions and band-shaped cell adhesion inhibition regions, A step of forming a normal cardiomyocyte sheet consisting of differentiated cardiomyocytes derived from a healthy person on at least the alternating arrangement portion of a cell culture substrate having alternating arrangement portions of band-shaped cell adhesion regions and band-shaped cell adhesion inhibition regions, The process includes the step of separating either the diseased cardiomyocyte sheet or the normal cardiomyocyte sheet from the cell culture substrate and laminating it onto the other cardiomyocyte sheet. A method for producing a cell culture substrate with a layered cardiomyocyte sheet.

5. A method for evaluating a normal cardiomyocyte sheet for transplantation, comprising the step of evaluating at least one change selected from changes in motor function and changes in physiological characteristics of a diseased cardiomyocyte sheet in a cell culture substrate with a cardiomyocyte sheet as described in claim 1.

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