Abnormal flutter myocardial model, method for producing the same, forming agent for abnormal flutter myocardial model, and method for evaluating drug efficacy of therapeutic agent for heart disease

Abstract

The present invention relates to an abnormal beating myocardium model, which comprises a three-dimensional tissue containing cells, including cardiomyocytes, and collagen, with at least a portion of the cells adhering to the collagen.

Description

Technical Field

[0001] The present invention relates to an abnormal beating myocardial model, a method for producing the same, a forming agent for forming the abnormal beating myocardial model, and a method for evaluating the efficacy of a therapeutic agent for heart disease.

Background Art

[0002] In recent years, techniques for constructing three-dimensional tissue bodies of cells in vitro have been developed. Such three-dimensional tissue bodies can be applied to biological tissue models and the like that can be used as alternatives to experimental animals. Patent Documents 1 and 2 disclose three-dimensional tissue bodies of cardiomyocytes produced using collagen as a scaffold.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] An object of the present invention is to provide a novel abnormal beating myocardial model, a method for producing the same, and a forming agent that can be used for producing the abnormal beating myocardial model.

Means for Solving the Problems

[0005] As a result of intensive studies by the present inventors, it has been found that the above problems can be solved by the invention shown below. [1] An abnormal beating myocardial model comprising a three-dimensional tissue body containing cells including cardiomyocytes and collagen, wherein at least a part of the cells is adhered to the collagen. [2] The abnormal beating myocardial model according to [1], wherein the cells further contain collagen-producing cells. [3] The abnormal beating myocardial model according to [1] or [2], wherein the collagen content is 10% to 30% by weight based on the three-dimensional tissue. [4] The abnormal beating myocardial model according to any one of [1] to [3], wherein the collagen contains exogenous collagen. [5] The abnormal beating myocardial model according to any one of [1] to [4], wherein the collagen contains fragmented collagen derived from exogenous collagen. [6] A contacting step of contacting cells containing myocardial cells with exogenous collagen in an aqueous medium, and a culturing step of culturing the cells contacted with the exogenous collagen, comprising: wherein the amount of the exogenous collagen used in the contacting step is 1.0×10 5 ~ 10.0×10 5 mg or more per 0.1 mg of cells of cells, a method for producing an abnormal beating myocardial model. [7] The method for producing an abnormal beating myocardial model according to [6], wherein the cells further contain collagen-producing cells. [8] The production method according to [7], wherein the exogenous collagen contains fragmented collagen. [9] The production method according to [8], wherein the average length of the fragmented collagen is 100 nm to 200 μm.

[10] The production method according to [8] or [9], wherein the average diameter of the fragmented collagen is 50 nm to 30 μm.

[11] The production method according to any one of [6] to

[10] , further comprising a step of co-precipitating the exogenous collagen and the cells in the aqueous medium during the contacting step or the culturing step.

[12] The production method according to any one of [6] to

[11] , wherein the mass ratio of the exogenous collagen to the cells is 900:1 to 9:1.

[13] A forming agent for an abnormal beating myocardial model, comprising fragmented collagen, The forming agent for an abnormal beating myocardial model, wherein the average length of the fragmented collagen is 100 nm to 200 μm and the average diameter of the fragmented collagen is 50 nm to 30 μm. A method for evaluating the efficacy of a therapeutic drug for heart disease using the abnormal beating myocardial model according to any one of

[14] [1] to [5], comprising an administration step of administering the therapeutic drug for heart disease to the abnormal beating myocardial model, and an evaluation step of evaluating the efficacy based on the change in the beating behavior of the abnormal beating myocardial model administered with the therapeutic drug for heart disease.

[15] In the evaluation step, when the number of beats per unit time of the abnormal beating myocardial model administered with the therapeutic drug for heart disease is higher compared to the number of beats per unit time of the abnormal beating myocardial model not administered with the therapeutic drug for heart disease, it is evaluated as effective as a therapeutic drug for heart failure. When the number of beats per unit time of the abnormal beating myocardial model administered with the therapeutic drug for heart disease is lower compared to the number of beats per unit time of the abnormal beating myocardial model not administered with the therapeutic drug for heart disease, it is evaluated as ineffective as a therapeutic drug for heart failure. The method for evaluating the efficacy of the therapeutic drug for heart disease according to

[14] .

[16] The method for evaluating the efficacy of the therapeutic drug for heart disease according to

[14] or

[15] , wherein the evaluation step is performed multiple times.

Advantages of the Invention

[0006] According to the present invention, it becomes possible to provide a novel abnormal beating myocardial model and a method for manufacturing the same, and a forming agent that can be used for manufacturing the abnormal beating myocardial model.

Brief Description of the Drawings

[0007]

Figure 1

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Mode for Carrying Out the Invention

[0008] Hereinafter, the modes for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

[0009] <Abnormal Beating Myocardial Model> The abnormal beating myocardial model according to the present embodiment is composed of a three-dimensional tissue body containing cells (hereinafter, sometimes simply referred to as "cells") including myocardial cells and collagen, and at least a part of the cells is adhered to the collagen.

[0010] The "abnormal beating myocardial model" refers to a myocardial model composed of a three-dimensional tissue containing cardiomyocytes, and having abnormal beating behavior compared to a normal myocardial model. Examples of the beating behavior include beating interval, heart rate, beating force, contraction and / or relaxation rate, etc. The abnormal beating myocardial model may have irregular changes in the beating behavior, or may have enhanced or suppressed beating compared to the normal myocardial model. The abnormal beating myocardial model can be used as a heart disease model (for example, heart failure model, arrhythmia model, myocardial infarction model, etc.) caused by abnormal beating of the myocardium.

[0011] Here, the "three-dimensional tissue" means an aggregate of cells in which cells are three-dimensionally arranged via collagen such as fibrous collagen, and is an aggregate artificially created by cell culture. There is no particular limitation on the shape of the three-dimensional tissue, and examples include sheet-like, spherical, ellipsoidal, rectangular parallelepiped-like shapes, etc. Here, biological tissue includes blood vessels, etc., and its structure is more complex than that of the three-dimensional tissue. Therefore, the three-dimensional tissue and the biological tissue can be easily distinguished.

[0012] The three-dimensional tissue contains cells including cardiomyocytes. In the cardiomyocytes, examples of the animal species from which they are derived include humans, pigs, cows, mice, etc. For example, the cardiomyocytes may be human iPS cell-derived cardiomyocytes (iPS-CM), mouse iPS-derived cardiomyocytes, or ES cell-derived cardiomyocytes. Human iPS cell-derived cardiomyocytes can be obtained, for example, from the RIKEN BioResource Center, Takara Bio, etc. Also, since the reagents for reprogramming can be purchased from ReproCell, etc., iPS cells can be made by oneself.

[0013] The content rate of the cardiomyocytes may be 5 to 95% by mass, or may be 25% to 75% by mass based on the three-dimensional tissue.

[0014] The cells containing cardiomyocytes may further contain collagen-producing cells. That is, the three-dimensional tissue may contain endogenous collagen.

[0015] Here, the "collagen-producing cell" means a cell that secretes collagen such as fibrillar collagen. In collagen-producing cells, examples of the animal species from which they are derived include humans, pigs, cows, mice, etc. Examples of collagen-producing cells include mesenchymal cells such as fibroblasts (e.g., human dermal fibroblasts (NHDF), human cardiac fibroblasts (NHCF), human myofibroblasts), chondrocytes, and osteoblasts, and preferably fibroblasts. Preferred fibroblasts include, for example, human cardiac fibroblasts (NHCF) or human myofibroblasts.

[0016] In addition, the "endogenous collagen" means the collagen produced by collagen-producing cells. The endogenous collagen may be fibrillar collagen or non-fibrillar collagen.

[0017] The three-dimensional tissue construct contains collagen. Examples of collagen include fibrillar collagen or non-fibrillar collagen. Fibrillar collagen means collagen that is the main component of collagen fibers, and specifically includes type I collagen, type II collagen, type III collagen, etc. Examples of non-fibrillar collagen include type IV collagen.

[0018] In the three-dimensional tissue construct, at least a part of the cells including cardiomyocytes is adhered to collagen.

[0019] The collagen contained in the three-dimensional tissue construct may contain exogenous collagen. The collagen preferably contains fragmented collagen derived from exogenous collagen.

[0020] "Exogenous collagen" means collagen supplied from the outside, and specifically includes fibrous collagen, non-fibrous collagen, etc. In exogenous collagen, the animal species from which it is derived may be the same as or different from that of endogenous collagen. In exogenous collagen, examples of the animal species from which it is derived include humans, pigs, cows, etc. Further, exogenous collagen may be artificial collagen. Exogenous collagen is preferably fibrous collagen. Examples of the fibrous collagen include type I collagen, type II collagen, and type III collagen, and preferably type I collagen. The fibrous collagen may be commercially available collagen, and specific examples thereof include freeze-dried type I collagen derived from pig skin manufactured by Nippon Ham Foods Co., Ltd. Examples of exogenous non-fibrous collagen include type IV collagen.

[0021] In exogenous collagen, the animal species from which it is derived may be different from cardiomyocytes and cells containing the same. Further, when cells containing cardiomyocytes contain collagen-producing cells, in exogenous collagen, the animal species from which it is derived may be different from the collagen-producing cells. That is, exogenous collagen may be heterologous collagen.

[0022] "Fragmented collagen" means collagen such as fibrous collagen that has been fragmented and maintains a triple helix structure. The collagen from which the fragmented collagen is derived may be of one type or a combination of multiple types of collagen. Conventionally, collagen such as fibrous collagen has been dissolved in an acidic aqueous solution or the like, but the concentration is about 0.1 to 0.3% by weight and it could not be dissolved in large amounts. Therefore, it has been difficult to increase the amount of collagen such as fibrous collagen in a three-dimensional tissue by conventional methods. Fragmented collagen hardly dissolves in water, but it is presumed that by dispersing it in an aqueous medium, it becomes easier to contact cells in the aqueous medium and promotes the formation of a three-dimensional tissue.

[0023] The average length of fragmented collagen is preferably from 100 nm to 200 μm, more preferably from 22 μm to 200 μm, and even more preferably from 100 μm to 200 μm. The average diameter of fragmented collagen is preferably from 50 nm to 30 μm, more preferably from 4 μm to 30 μm, and even more preferably from 20 μm to 30 μm.

[0024] The method for fragmenting collagen such as fibrous collagen is not particularly limited. For example, a homogenizer such as an ultrasonic homogenizer, a stirring homogenizer, or a high-pressure homogenizer may be used to fragment collagen such as fibrous collagen. When using a stirring homogenizer, collagen such as fibrous collagen may be directly homogenized, or may be homogenized in an aqueous medium such as physiological saline. Also, it is possible to obtain fragmented collagen of millimeter size and nanometer size by adjusting the homogenization time, number of times, etc.

[0025] The diameter and length of fragmented collagen can be determined by analyzing individual fragmented collagen with an electron microscope.

[0026] The collagen content in the three-dimensional tissue may be from 0.01 to 90% by weight based on the three-dimensional tissue, preferably from 10 to 90% by weight, more preferably from 1 to 50% by weight, even more preferably from 10 to 40% by weight, and may be from 10 to 30% by weight. Here, "collagen in the three-dimensional tissue" means the collagen constituting the three-dimensional tissue, which may be endogenous collagen or exogenous collagen. That is, the concentration of collagen constituting the three-dimensional tissue means the combined concentration of endogenous collagen and exogenous collagen. The concentration of collagen in the three-dimensional tissue can be calculated from the volume of the obtained three-dimensional tissue and the mass of the decellularized three-dimensional tissue. Also, the collagen content in the three-dimensional tissue can also be measured by a method using an antigen-antibody reaction such as ELISA or a chemical detection method such as QuickZyme.

[0027] The three-dimensional tissue preferably has a survival rate of 70% or more, more preferably 80% or more, and even more preferably 90% or more after trypsin treatment at a trypsin concentration of 0.25%, a temperature of 37°C, a pH of 7.4, and a reaction time of 15 minutes. Such a three-dimensional tissue is less likely to be decomposed by an enzyme during or after culture and is stable. The above survival rate can be calculated from the mass of the three-dimensional tissue before and after trypsin treatment, for example.

[0028] The three-dimensional tissue preferably has a survival rate of 70% or more, more preferably 80% or more, and even more preferably 90% or more after collagenase treatment at a collagenase concentration of 0.25%, a temperature of 37°C, a pH of 7.4, and a reaction time of 15 minutes. Such a three-dimensional tissue is less likely to be decomposed by an enzyme during or after culture and is stable.

[0029] The three-dimensional tissue preferably has a thickness of 10 μm or more, more preferably 100 μm or more, and even more preferably 1000 μm or more. Such a three-dimensional tissue has a structure closer to that of a living tissue and is suitable as a substitute for experimental animals, etc. The upper limit of the thickness is not particularly limited, and for example, it may be 10 mm or less, 3 mm or less, 2 mm or less, 1.5 mm or less, or 1 mm or less.

[0030] Here, the "thickness of the three-dimensional tissue" means the distance between both ends in the direction perpendicular to the main surface when the three-dimensional tissue is in a sheet shape or a rectangular parallelepiped shape. When there are irregularities on the main surface, the thickness means the distance at the thinnest part of the main surface. When the three-dimensional tissue is spherical, it means its diameter. Furthermore, when the three-dimensional tissue is ellipsoidal, it means its minor axis. When the three-dimensional tissue is substantially spherical or substantially ellipsoidal and has irregularities on the surface, the thickness means the shortest distance between two points where a straight line passing through the center of gravity of the three-dimensional tissue intersects the above surface.

[0031] The cells constituting the three-dimensional tissue may further contain one or more other types of cells other than cardiomyocytes and collagen-producing cells.

[0032] The three-dimensional tissue may contain cells including cardiomyocytes and components other than collagen (other components). Examples of other components include elastin, collagen, proteoglycan, fibronectin, laminin, and the like.

[0033] The abnormal beating myocardial model composed of the three-dimensional tissue can be applied as a substitute for experimental animals (for example, a heart disease model caused by abnormal beating of the myocardium), a myocardial infarction model, a myocardial fibrosis model, and the like.

[0034] <Method for manufacturing an abnormal beating myocardial model> The method for manufacturing an abnormal beating myocardial model according to the present embodiment includes a contact step of bringing cells containing cardiomyocytes (hereinafter, sometimes simply referred to as "cells") and exogenous collagen into contact with each other in an aqueous medium, and a culture step of culturing the cells contacted with the exogenous collagen. The amount of exogenous collagen used in the contact step is 1.0×10 5 ~10.0×10 5 mg or more with respect to 1 cells.

[0035] The "aqueous medium" means a liquid having water as an essential constituent. The aqueous medium is not particularly limited as long as exogenous collagen and cells can stably exist therein. Examples thereof include physiological saline such as phosphate buffered saline (PBS), Dulbecco's Modified Eagle medium (DMEM), and liquid media such as endothelial cell-specific medium (EGM2). The liquid medium may be a mixed medium in which two types of media are mixed. From the viewpoint of reducing the burden on cells, the aqueous medium is preferably a liquid medium.

[0036] (Contact step) In an aqueous medium, there is no particular limitation on the method of bringing cells including cardiomyocytes into contact with exogenous collagen. For example, there may be mentioned a method of adding a dispersion of exogenous collagen to a culture solution containing cardiomyocytes, a method of adding cells to a medium dispersion of exogenous collagen, or a method of adding exogenous collagen and cardiomyocytes respectively to a previously prepared aqueous medium.

[0037] In the contacting step, the cells may further contain collagen-producing cells. In this case, the resulting three-dimensional tissue will be more stable and the cells will be more uniformly distributed. Although the details of the mechanism by which such a three-dimensional tissue is obtained are unclear, it is presumed as follows. First, the cells come into contact with and adhere to the exogenous collagen. Thereafter, the cells themselves produce proteins (such as collagen such as fibrillar collagen) that constitute the extracellular matrix (ECM). The produced proteins come into contact with and adhere to the exogenous collagen, thereby acting as a cross-linking agent between the exogenous collagens, and the structuring of fibrillar collagen and the like proceeds in an environment where the cells are uniformly present. As a result, a stable three-dimensional tissue in which the cells are uniformly distributed is obtained. However, the above presumption does not limit the present invention.

[0038] In the contacting step, the exogenous collagen may contain fragmented collagen derived from the exogenous collagen. As the exogenous collagen and the fragmented collagen, those described above can be used.

[0039] The concentration of the exogenous collagen in the aqueous medium in the contacting step can be appropriately determined according to the shape, thickness, size of the incubator, etc. of the target three-dimensional tissue (abnormal beating myocardium model). For example, the concentration of the exogenous collagen in the aqueous medium in the contacting step may be 0.1 to 90% by weight, or may be 1 to 30% by weight.

[0040] The amount of the exogenous collagen used in the contacting step is 1.0×10 5 ~10.0×10 5For the cells of [[ID=]], it may be 0.1 mg or more, and may be 0.5 mg or more, 1.0 mg or more, 2.0 mg or more, or 3.0 mg or more, or may be 100 mg or less, or 50 mg or less. The exogenous collagen is 2.0×10 5 ~8.0×10 5 cells, 3.0×10 5 ~6.0×10 5 cells, or 5×10 5 cells may be added to the cells so as to be within the above range.

[0041] It is preferable that the mass ratio (exogenous collagen: cells) of the exogenous collagen and the cells in the contact step is 1000:1 to 1:1, more preferably 900:1 to 9:1, and even more preferably 500:1 to 10:1.

[0042] When using cardiomyocytes and collagen-producing cells together, the ratio (number of cells) of cardiomyocytes: collagen-producing cells in the contact step may be 99:1 to 9:1, or may be 80:20 to 50:50.

[0043] During the contact step or the culture step, a step of co-precipitating fragmented collagen and cells in an aqueous medium (precipitation step) may be further included. By performing such a step, the distribution of exogenous collagen and cells in the three-dimensional tissue becomes more uniform. As a specific method, there is no particular limitation, but for example, a method of centrifuging a culture solution containing fragmented collagen and cells including cardiomyocytes can be mentioned.

[0044] (Culture step) The method of culturing the cells contacted with the fragmented collagen is not particularly limited, and can be performed by a suitable culturing method according to the type of cells to be cultured. For example, the culture temperature may be 20°C to 40°C, or may be 30°C to 37°C. The pH of the medium may be 6 to 8, or may be 7.2 to 7.4. The culture time may be 1 day to 2 weeks, or may be 1 week to 2 weeks.

[0045] The medium is not particularly limited, and a suitable medium can be selected according to the type of cells to be cultured. Examples of the medium include Eagle's MEM medium, DMEM, Modified Eagle medium (MEM), Minimum Essential medium, RPMI, and GlutaMax medium. The medium may be a medium supplemented with serum or a serum-free medium. The medium may also be a mixed medium obtained by mixing two types of media.

[0046] The cell density in the medium in the culturing step can be appropriately determined according to the shape, thickness of the target abnormal pulsatile myocardial model, the size of the incubator, etc. For example, the cell density in the medium in the culturing step may be 1 to 10 8 cells / ml, or may be 10 3 ~10 7 cells / ml. Also, the cell density in the medium in the culturing step may be the same as the cell density in the aqueous medium in the contacting step.

[0047] The three-dimensional tissue is preferably such that the shrinkage rate during culturing is 20% or less, more preferably 15% or less, and even more preferably 10% or less. The above shrinkage rate can be calculated, for example, by the following formula. In the formula, L1 represents the length of the longest part of the abnormal pulsatile myocardial model on the first day after culturing, and L3 represents the length of the corresponding part in the three-dimensional tissue on the third day after culturing. Shrinkage rate (%) = {(L1 - L3) / L1} × 100

[0048] <Agent for forming abnormal pulsatile myocardial model> The agent for forming an abnormal pulsatile myocardial model according to the present embodiment is an agent for forming an abnormal pulsatile model containing fragmented collagen, wherein the average length of the fragmented collagen is 100 nm to 200 μm, and the average diameter of the fragmented collagen is 50 nm to 30 μm. Further, regarding the length of the fragmented collagen, 95% of the entire fragmented collagen may be in the range of 100 nm to 200 μm. Furthermore, regarding the diameter of the fragmented collagen, 95% of the entire fragmented collagen may be in the range of 50 nm to 30 μm.

[0049] The "agent for forming an abnormal beating myocardial model" means a reagent for producing an abnormal beating myocardial model. The agent for forming an abnormal beating myocardial model may be in a powder state or in a dispersion state in which fragmented collagen is dispersed in an aqueous medium. Examples of the method for producing fragmented collagen and the method for using the above-mentioned forming agent include the same methods as those shown in the above (method for producing an abnormal beating myocardial model).

[0050] <Method for evaluating the efficacy of a therapeutic agent for heart disease> As one embodiment of the present invention, there is provided a method for evaluating the efficacy of a therapeutic agent for heart disease using an abnormal beating myocardial model, including an administration step of administering a therapeutic agent for heart disease to the abnormal beating myocardial model, and an evaluation step of evaluating the efficacy based on changes in the beating behavior of the abnormal beating myocardial model to which the therapeutic agent for heart disease has been administered. According to this embodiment, the efficacy of a therapeutic agent for heart disease that affects the beating of cardiomyocytes can be effectively evaluated.

[0051] In the administration step, a therapeutic agent for heart disease is administered to the abnormal beating myocardial model. Examples of the therapeutic agent for heart disease include therapeutic agents for heart failure such as isoproterenol, therapeutic agents for myocardial infarction such as β-blockers and nitrates, and antiarrhythmics such as amiodarone.

[0052] The administration of the therapeutic agent for heart disease may be carried out by using a medium containing the therapeutic agent for heart disease as the medium for culturing the three-dimensional tissue, or by adding the therapeutic agent for heart disease to the medium for culturing the three-dimensional tissue.

[0053] The abnormal beating myocardial model to which the therapeutic agent for heart disease is administered may be composed of a three-dimensional tissue cultured for 1 day or more, may be composed of a three-dimensional tissue cultured for 5 days or more, may be composed of a three-dimensional tissue cultured for 6 days or more, or may be composed of a three-dimensional tissue cultured for a longer number of days.

[0054] In the evaluation step, the efficacy of a cardiovascular disease therapeutic agent is evaluated based on changes in the behavior of the arrhythmic myocardium model administered with the agent. The efficacy can be evaluated using the behavior of the arrhythmia as an index. Examples of the behavior of the arrhythmia include the arrhythmia interval, the arrhythmia rate, the arrhythmia force, the contraction and / or relaxation rate, etc. The change in the behavior of the arrhythmia may be, for example, a change in the arrhythmia rate per unit time and / or a change in the arrhythmia interval (the time between arrhythmias). The efficacy may be evaluated based on a change in only one of the above indices, or may be evaluated based on two or more of the above indices.

[0055] The evaluation of the efficacy can be performed, for example, by comparing the behavior of the arrhythmic myocardium model composed of a three-dimensional tissue body administered with a cardiovascular disease therapeutic agent with the behavior of the arrhythmic myocardium model composed of a three-dimensional tissue body not administered with the cardiovascular disease therapeutic agent.

[0056] The evaluation step may be performed multiple times. That is, the evaluation of the efficacy may be performed multiple times at predetermined intervals after administration of the therapeutic agent.

[0057] In the evaluation step, when the arrhythmia rate per unit time of the arrhythmic myocardium model administered with a cardiovascular disease therapeutic agent is higher compared to the arrhythmia rate per unit time of the arrhythmic myocardium model not administered with the cardiovascular disease therapeutic agent, it may be evaluated that there is an effect as a heart failure therapeutic agent. When the arrhythmia rate per unit time of the arrhythmic myocardium model administered with a cardiovascular disease therapeutic agent is lower compared to the arrhythmia rate per unit time of the arrhythmic myocardium model not administered with the cardiovascular disease therapeutic agent, it may be evaluated that there is no effect as a heart failure therapeutic agent.

Example

[0058] <Production of Fragmented Collagen (CMF) Using Type I Collagen> The freeze-dried product of porcine skin-derived type I collagen manufactured by Nippon Ham Co., Ltd. was dispersed in 10-fold concentrated phosphate-buffered saline (X10 PBS), and homogenized for 2 minutes using a homogenizer to obtain fragmented collagen with a diameter of about 20 - 30 μm and a length of about 100 - 200 μm (Figure 1(A)). The diameter and length of the fragmented collagen were determined by analyzing individual fragmented collagen using an electron microscope. The obtained fragmented collagen was washed with serum-free medium (DMEM) to obtain a medium dispersion of the fragmented collagen. The obtained medium dispersion of the fragmented collagen could be stored at room temperature for 1 week. In the production of each of the abnormal beating myocardial models (three-dimensional tissue constructs) described later, fragmented collagen obtained by the same method was used. Also, in the above method, when the homogenization time was changed to 5 minutes, fragmented collagen with a diameter of about 950 nm - 16.8 μm and a length of about 9.9 μm - 78.6 μm was obtained (Table 1, Figure 1(B)). From this result, it was found that the size of the fragmented collagen could be controlled by adjusting the homogenization time.

[0059]

Table 1

[0060] <Production of three-dimensional tissue construct> The fragmented collagen was dispersed in a medium containing serum (DMEM) to a concentration of 10 mg / ml to prepare a dispersion containing the fragmented collagen.

[0061] (Example 1) The three-dimensional tissue was manufactured as shown in the schematic diagram of Fig. 2(A). That is, a dispersion containing the above-mentioned fragmented collagen, human cardiac fibroblasts (NHCF), and human iPS cell-derived cardiomyocytes (iPS-CM) (hereinafter, NHCF and iPS-CM are collectively also referred to as "cells") were added to a non-adhesive 96-well round-bottom plate, and the fragmented collagen and the cells were brought into contact (contact step). The dispersion containing the fragmented collagen was added such that the added amount of the fragmented collagen was 0.5 mg. NHCF and iPS-CM were mixed at a ratio of 25:75, and the total number of cells was added so as to be 5×10 5 cells. Then, it was cultured for a predetermined period (culture step) to produce three-dimensional tissue 1. Three-dimensional tissue 1 was subjected to Masson trichrome stain after culturing for 21 days. A photograph of three-dimensional tissue 1 taken by a phase-contrast microscope is shown in Fig. 2(C). Three-dimensional tissue 1 was spherical, and its diameter after culturing for 21 days was about 1.0 mm. In the left and center figures of Fig. 2(C), the darkly stained areas indicate collagen fibers, and the lightly stained areas indicate cytoplasm (the same applies hereinafter in Figs. 2(B), (D), and (E)).

[0062] (Example 2) A three-dimensional tissue 2 was produced in the same manner as in Example 1, except that the dispersion containing the fragmented collagen was added such that the added amount of the fragmented collagen was 1.0 mg. The observation result of the Masson trichrome-stained three-dimensional tissue 2 by a phase-contrast microscope is shown in Fig. 2(D). Three-dimensional tissue 2 was spherical, and its diameter after culturing for 21 days was about 1.2 mm.

[0063] (Example 3) A three-dimensional tissue 3 was obtained in the same manner as in Example 1, except that the dispersion containing the fragmented collagen was added such that the added amount of the fragmented collagen was 1.5 mg. The observation result of the Masson trichrome-stained three-dimensional tissue 3 by a phase-contrast microscope is shown in Fig. 2(E). Three-dimensional tissue 3 was spherical, and its diameter after culturing for 21 days was about 1.6 mm.

[0064] (Example 4) A three-dimensional tissue 4 was obtained in the same manner as in Example 1, except that a dispersion containing fragmented collagen was added such that the added amount of fragmented collagen was 3.0 mg. The diameter of the three-dimensional tissue 4 after culturing for 7 days was about 4 mm.

[0065] (Example 5) A three-dimensional tissue 5 was obtained in the same manner as in Example 1, except that a dispersion containing fragmented collagen was added such that the added amount of fragmented collagen was 5.0 mg. The diameter of the three-dimensional tissue 5 after culturing for 7 days was about 5 mm.

[0066] (Comparative Example 1) A comparative three-dimensional tissue 1 was obtained in the same manner as in Example 1, except that a dispersion containing fragmented collagen was not added. The observation result of the Masson's trichrome-stained comparative three-dimensional tissue 1 by a phase-contrast microscope is shown in Fig. 2(B). The comparative three-dimensional tissue 1 was spherical, and the diameter after culturing for 21 days was about 0.9 mm.

[0067] (Evaluation) (Pulse interval and pulse force of the myocardial model) The three-dimensional tissues 1 to 5 and the comparative three-dimensional tissue 1 obtained by the above method were used as the myocardial models of Examples 1 to 5 and Comparative Example 1, respectively. The pulse interval and pulse force of the myocardial model were evaluated by taking a video of the pulsation of the myocardial model after culturing for 7 days with an inverted microscope and observing it using image analysis (Image Pro). Specifically, the evaluation was performed based on the moving distance of the center of gravity that moves when the myocardial model pulsates and the time interval of the pulsation. Fig. 3 is a diagram for explaining the evaluation method of the pulse interval and pulse force of the myocardial model. The center of gravity of the myocardial model moves in response to the pulsation. The evaluation was performed by measuring the moving distance of the center of gravity when the myocardial model contracts (the distance from the center of gravity shown in Fig. 3(A) to the center of gravity shown in Fig. 3(B)) and the moving distance of the center of gravity when the myocardial model relaxes after contraction (the distance from the center of gravity shown in Fig. 3(B) to the center of gravity shown in Fig. 3(C)). The results are shown in Figs. 4 to 10.

[0068] Figure 4(A) shows the beating behavior of the myocardial model of Example 2 obtained by adding 1.0 mg of fragmented collagen. Figure 4(B) shows the time points at the start of observation (0 seconds), the time point when contraction occurred (0.67 seconds elapsed), and the time point when relaxation occurred (1.27 seconds elapsed). Figure 5(B) shows the beating behavior of the myocardial model obtained by adding 0 mg, 1.0 mg, and 2.0 mg of fragmented collagen.

[0069] As shown in Figures 4 and 5, when fragmented collagen was added, the beating (time interval and beating force of the beating) of the obtained myocardial model changed irregularly compared to the case where it was not added. Also, the beating force of the obtained myocardial model decreased when collagen was added compared to the case where no collagen was added. As shown in Figure 5(A), the beating behavior of the myocardial model without added collagen was close to normal.

[0070] (A) to (D) of Figures 6 and 8 show the beating behavior of myocardial models with 0, 1, 3, and 5 mg of fragmented collagen used, respectively. Figure 7(A) shows the result of the average beating interval calculated from the interval between the broken lines shown in Figures 6(A) to (D) (that is, the difference from the time point when one contraction-relaxation was completed to the time point when the next contraction-relaxation was completed) (unit: seconds). Figure 7(B) shows the average of the standard deviations (S.D.) from day 4 to day 7 of the culture period (an index of the irregularity of the beating). Figure 9 shows the time (unit: seconds) between contraction and relaxation of the myocardial model calculated from the interval indicated by the arrows in Figures 8(A) to (D) (that is, the time required for one contraction-relaxation) (unit: seconds). Figure 10 shows the measurement results of the beating interval according to the number of culture days of myocardial models with 0, 1, 3, and 5 mg of fragmented collagen used.

[0071] As shown in Fig. 7(A), the myocardial model obtained by adding fragmented collagen had a short average beat interval (i.e., a higher number of beats per unit time). As shown in Fig. 7(B), when 1 mg or 5 mg of fragmented collagen was added, the time interval between beats became more irregular. As shown in Fig. 9, in the myocardial model obtained by adding fragmented collagen, the time required for one contraction and relaxation became longer (i.e., the beating speed per beat decreased). As described above, it was suggested that the myocardial model produced by adding fragmented collagen approximated the model with the disease. Fig. 8 shows the beating behavior for each amount of collagen. In Fig. 9, the time taken for one contraction-relaxation was calculated from the results of Fig. 8 to examine the effect of collagen. As a result, the time required for beating tended to be significantly delayed depending on the amount of collagen (added amount) of 1 mg and 3 mg, but when the amount of collagen (added amount) was 5 mg, the time required for beating was almost the same as that of 1 mg. It was suggested that the time of the beating itself might be controllable by the amount of collagen. As shown in Fig. 10, the myocardial model obtained by adding fragmented collagen had a shorter beat interval and a higher number of beats over the culture period.

[0072] <Measurement of cell viability> (Construction of three-dimensional tissue) 5 mL of 10x phosphate-buffered saline (PBS) was added to 50 mg of type I collagen derived from porcine skin (provided by Nippon Ham Co., Ltd.) and homogenized with a homogenizer for 6 minutes. Then, it was centrifuged at 10,000 rpm for 3 minutes, and the supernatant was removed. 5 mL of serum-free DMEM was added here and washed by pipetting for 1 minute. After washing, it was centrifuged at 10,000 rpm for 3 minutes, and the supernatant was removed. 5 mL of DMEM containing serum was added here and pipetted to obtain a fragmented collagen (CMF) dispersion medium (CMF concentration 9.8 mg / mL). Dispersion liquids were weighed out from the CMF dispersion medium to obtain CMF of 0, 1, 2, 3 mg, and 5x10 5The cells were mixed with 75% iPS-CM / NHCF 25% and seeded into a 96-well round-bottom non-adherent plate (the medium volume was 300 μL). Then, centrifugation was performed at 1100 g for 5 minutes to precipitate the CMF and the cells. After centrifugation, the cells were cultured in an incubator at 37°C. The medium was changed once every two days by removing the old medium and adding 300 μL of fresh medium.

[0073] (Measurement of DNA amount) Kit used: DNeasy Blood & Tissue Kit (50) (69504, QIAGEN) 5x10 5 The DNA amount of 75% iPS-CM / NHCF 25% of 5x10 cells was measured using the above kit. The DNA amount at this time was taken as the reference (100%). The DNA amount in the three-dimensional tissue constructs of each CMF amount after 7-day culture was measured using the above kit. The cell viability (change rate of DNA amount) in the three-dimensional tissue constructs was calculated using the following formula. 5 Change rate of DNA amount (%) = (DNA amount of the three-dimensional tissue construct after 7-day culture) / (DNA amount of 75% iPS-CM / NHCF 25% of 5x10

[0074] The above kit is a commercially available kit containing DNeasy Mini Spin Column, collection tube, Buffer ATL, Buffer AL, Buffer AW1, Buffer AW2, Buffer AE, Proteinase K, etc. The DNA measurement was performed according to the following procedure.

[0075] The sample to be measured was placed in a 1.5 mL Eppendorf tube, and 180 μL of Buffer ATL was added. 20 μL of Proteinase K was added thereto, vortexed, and incubated at 56 °C until the tissue was completely lysed (overnight). Then, it was vortexed for 15 seconds, Buffer AL and ethanol were mixed in equal amounts, and 400 μL was added to 1 sample and vortexed. The solution was added into a DNeasy Mini Spin Column (hereinafter, may be simply referred to as column), and centrifuged at 8000 rpm for 1 min. The filtrate and the collection tube were discarded, the column was transferred to a new collection tube, and 500 μL of Buffer AW1 was added. Then, it was centrifuged at 8000 rpm for 1 min. The filtrate and the collection tube were discarded, the column was transferred to a new collection tube, and 500 μL of Buffer AW2 was added. After centrifugation at 14000 rpm for 3 min, the DNeasy membrane was completely dried. The filtrate and the collection tube were discarded, the column was transferred to an Eppendorf tube (Operation 1), 200 μL of Buffer AE was directly added onto the DNeasy membrane (Operation 2), and incubated at room temperature for 1 min (Operation 3). Then, it was centrifuged at 8000 rpm for 1 min (Operation 4). After repeating Operations 1 to 4, the collected filtrates were combined to make 400 μL. This was measured with a Nanodrop.

[0076] The results of the change rate of DNA amount are shown in Fig. 11. As shown in Fig. 11, as a result of culturing for 7 days, the number of cells in the three-dimensional tissue construct using CMF increased more compared to the tissue construct without using CMF. It is considered that more cells survived or cells survived for a longer period by culturing with CMF added. From the results of Figs. 10 and 11, the three-dimensional tissue construct using CMF showed a pulsatile behavior similar to that at the initial stage of culture even when cultured for a long period (at least 7 days), and the cell viability was also higher compared to the tissue construct (spheroid) without using CMF. Thus, the myocardial model composed of the three-dimensional tissue construct using CMF can perform drug efficacy evaluation over a long period because many cells survive or survive for a long time.

[0077] <Evaluation of drug responsiveness to isoproterenol> (Construction of three-dimensional tissue construct) Kit used: Total Collagen Assay Kit (QZBTOTCOL1, QuickZyme Biosciences) The three-dimensional tissue construct was constructed in the same manner as above. In the three-dimensional tissue construct constructed using 1 mg of CMF, the collagen content rate after 1-day culture was 34% by weight based on the freeze-dried three-dimensional tissue construct. The collagen content in the three-dimensional tissue construct was measured using the above kit according to the standard protocol of the above kit.

[0078] (Evaluation of drug responsiveness) After culturing the tissue mass for 5 to 6 days, the beating of the tissue mass was photographed using a SONY Motion analyzer, and the beating rate was measured. During the photographing, the inside of the microscope was maintained at 37°C and photographed for 15 to 20 seconds. After photographing, the plate was once taken out of the microscope to remove the culture medium, and 300 μL of DMEM (tissue without drug addition) or 300 μL of DMEM mixed with 100 nM isoproterenol was added respectively, and then it was put back into the microscope and incubated at 37°C. After incubating for 30 minutes, 60 minutes, and 80 minutes, the respective beatings were photographed in the same way, and the beating rate was measured. From the measurement results, the change rate of the beating rate due to the addition of isoproterenol (change rate of beating) was calculated by the following formula. The result was that the responsiveness to isoproterenol was good because the beating rate per unit time increased. Change rate of beating rate (%) = (Beating rate after each time of drug-added tissue / Beating rate before drug addition of drug-added tissue) / (Beating rate after each time of drug-free tissue / Beating rate before medium exchange of drug-free tissue) × 100 Note that the change rate of the beating rate due to the addition of isoproterenol is obtained by the upper formula, and this value is corrected by dividing it by the change rate of the beating rate without addition in the lower formula (= change in the beating rate due to medium exchange).

[0079] Figure 12 shows the results of the change rate of the beating rate. In the myocardial model, when the CMF usage amount was 0 mg, the drug responsiveness was poor, and when the CMF usage amounts were 1 mg and 3 mg, the drug responsiveness was shown. The myocardial model with a CMF usage amount of 1 mg showed better drug responsiveness.

[0080] The result of the drug efficacy evaluation using the myocardial model composed of a three-dimensional tissue mass using CMF is consistent with the finding of the effect of isoproterenol acting on the adrenergic receptor of the myocardium to enhance the contractility, indicating that the effect of the heart failure therapeutic drug can be correctly evaluated by the myocardial model composed of a three-dimensional tissue mass using CMF.

Industrial Applicability

[0081] According to the present invention, it becomes possible to provide a novel abnormal flutter myocardial model, and for example, it is expected to be applied to the development of drugs effective for diseases caused by abnormal heart flutter and the like.

Claims

1. It consists of a three-dimensional tissue containing cells including cardiomyocytes and collagen, with at least a part of the cells adhering to the collagen. The content rate of the collagen is 10 to 30% by weight based on the three-dimensional tissue. The collagen contains fragmented collagen derived from exogenous collagen. An abnormal beating myocardial model with an irregular beating interval, wherein the fragmented collagen is obtained by homogenizing collagen in an aqueous medium.

2. The abnormal beating myocardial model according to Claim 1, wherein the cells further contain collagen-producing cells.

3. A method for evaluating the efficacy of a therapeutic agent for heart disease using the abnormal beating myocardial model according to Claim 1 or 2, comprising: An administration step of administering the therapeutic agent for heart disease to the abnormal beating myocardial model; An evaluation step of evaluating the efficacy based on the change in the beating behavior of the abnormal beating myocardial model administered with the therapeutic agent for heart disease.

4. In the evaluation step, When the number of beats per unit time of the abnormal beating myocardial model administered with the therapeutic agent for heart disease is higher compared to the number of beats per unit time of the abnormal beating myocardial model not administered with the therapeutic agent for heart disease, it is evaluated that it is effective as a therapeutic agent for heart failure. The method for evaluating the efficacy of a therapeutic agent for heart disease according to Claim 3, wherein when the number of beats per unit time of the abnormal beating myocardial model administered with the therapeutic agent for heart disease is lower compared to the number of beats per unit time of the abnormal beating myocardial model not administered with the therapeutic agent for heart disease, it is evaluated that it is ineffective as a therapeutic agent for heart failure.

5. The method for evaluating the efficacy of a therapeutic agent for heart disease according to Claim 3 or 4, wherein the evaluation step is performed multiple times.

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