HEART-ON-CHIP DEVICE AND METHOD FOR EVALUATING CARDIAC FUNCTION, DRUG SCREENING METHOD, AND METHOD FOR EVALUATING CARDIAC TOXICITY USING SAME

Abstract

The present invention provides a heart-on-chip device provided with a microfluidic chip and a cardiac tissue sheet, wherein: the microfluidic chip comprises an oscillating section which is oscillatable in a definite direction, a flow channel in which a liquid flows due to the oscillation of the oscillating section, a diaphragm provided between the oscillating section and the flow channel, and a detection target dispersed in the liquid; and the cardiac tissue sheet, which contains cardiomyocytes, is in contact with the oscillating section in such a manner as to be able to oscillate the oscillating section.

Description

[Technical field]

[0001] The present invention relates to a heart-on-chip device comprising a microfluidic chip and a cardiac tissue sheet, as well as a method for evaluating cardiac function, a drug screening method, and a cardiotoxicity evaluation test method using the same. [Background technology]

[0002] Since adult cardiomyocytes hardly proliferate, replacement therapy has been proposed in which separately manufactured cardiomyocytes are administered when cardiomyocytes are lost due to disease, etc. In such replacement therapy, for example, a method of administering cardiomyocytes in the form of sheets has been investigated (H Masumoto, et al. "Human iPS cell-engineered cardiac tissue sheets with cardiomyocytes and vascular cells for cardiac regeneration" Scientific Reports volume 4, Article number:6716 (2014), Published:22 October 2014: Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] H Masumoto, et al. “Human iPS cell-engineered cardiac tissue sheets with cardiomyocytes and vascular cells for cardiac regeneration” Scientific Reports volume 4, Article number:6716 (2014), Published:22 October 2014 Summary of the Invention [Problem to be solved by the invention]

[0004] In drug discovery research for cardiac diseases or cardiotoxicity testing of new drugs, the effects of drugs on the heart are verified through animal testing. However, different species often differ in their responsiveness to drugs, making it difficult to determine the effects of drugs through animal testing. On the other hand, in toxicity evaluation tests performed on cultured cardiomyocytes, it is difficult to accurately evaluate toxicity on cardiac function.

[0005] An object of the present invention is to provide a heart-on-a-chip device capable of evaluating the contraction and relaxation of a cardiac tissue sheet as cardiac function. [Means for solving the problem]

[0006] The present invention provides the following [1] to

[12] . [1] A method for producing a cardiac tissue sheet comprising: The microfluidic chip includes a swinging part that can swing in a certain direction, a flow path through which a liquid flows by the swinging of the swinging part, a diaphragm provided between the swinging part and the flow path, and a detectable substance that is dispersed in the liquid; The cardiac tissue sheet comprises cardiomyocytes; A heart-on-a-chip device, wherein the cardiac tissue sheet is in contact with the rocking portion so as to be capable of rocking the rocking portion. [2] The heart-on-a-chip device according to [1], wherein the analyte is a fluorescent bead. [3] A heart-on-a-chip device according to [1] or [2], wherein the portion of the microfluidic chip that comes into contact with the cardiac tissue sheet has cell adhesive properties. [4] The cardiac tissue sheet further comprises vascular endothelial cells and vascular wall cells; The heart-on-chip device described in any of [1] to [3], wherein the cardiac tissue sheet comprises cardiomyocytes at 5% or more and 70% or less, vascular endothelial cells at more than 0% and 60% or less, and vascular wall cells at 1% or more and 60% or less. [5] The heart-on-a-chip device according to any one of [1] to [4], wherein the cardiac tissue sheet contains cells derived from induced pluripotent stem cells. [6] The heart-on-a-chip device according to any one of [1] to [5], wherein the cardiac tissue sheet contains cells in which a disease-related gene is deleted or mutated. [7] The cells contained in the cardiac tissue sheet include cells produced by at least one of production methods (A), (B), and (C), The production method (A) includes a culture step (A1) of culturing induced pluripotent stem cells in the presence of activin A and a GSK-3 inhibitor; A culture step (A2) of culturing the cells obtained in the culture step (A1) in the presence of BMP and bFGF; and A production method comprising a culture step (A3) of culturing the cells obtained in the culture step (A2) in the presence of VEGF, The production method (B) includes a culture step (B1) of culturing induced pluripotent stem cells in the presence of activin A and a GSK-3 inhibitor; A culture step (B2) of culturing the cells obtained in the culture step (B1) in the presence of BMP and bFGF; and A production method comprising a culture step (B3) of culturing the cells obtained in the culture step (B2) in the absence of VEGF and in the presence of insulin, The production method (C) includes a culture step (C1) of culturing induced pluripotent stem cells in the presence of activin A; A culture step (C2) of culturing the cells obtained in the culture step (C1) in the presence of BMP and bFGF; and A production method comprising a culture step (C3) of culturing the cells obtained in the culture step (C2) in the absence of VEGF and in the presence of serum. The heart-on-a-chip device according to any one of [1] to [6]. [8] The heart-on-a-chip device according to any one of [1] to [7], wherein the cardiac tissue sheet is a cardiac tissue sheet cultured with shaking. [9] The heart-on-chip device described in any of [1] to [8], further comprising a scaffold sheet for the cardiac tissue sheet.

[10] A method for evaluating cardiac function using the heart-on-chip device described in any one of [1] to [9].

[11] A drug screening method using the heart-on-chip device described in any one of [1] to [9].

[12] A method for evaluating cardiotoxicity using the heart-on-chip device described in any one of [1] to [9]. Effect of the Invention

[0007] According to the present invention, a heart-on-a-chip device capable of evaluating the contraction and relaxation of a cardiac tissue sheet as cardiac function can be provided. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 is a cross-sectional view showing an example of a heart-on-chip device of the present invention. [Diagram 2] FIG. 11 is a cross-sectional view showing an example of a heart-on-a-chip device when the oscillating part oscillates. [Diagram 3] FIG. 2 is a cross-sectional view showing another example of the heart-on-chip device of the present invention. [Figure 4A] FIG. 1 is a perspective view showing an example of a microfluidic chip. [Figure 4B] FIG. 4B is a cross-sectional view of the microfluidic chip shown in FIG. 4A along cross section AB. [Figure 4C] FIG. 1 is a cross-sectional view showing an example of a heart-on-a-chip device comprising a scaffold sheet. [Figure 4D] 1 is a perspective view (top and bottom) showing an example of a scaffolding sheet. [Figure 4E] FIG. 13 is a cross-sectional view showing another example of a heart-on-a-chip device having a scaffold sheet. [Figure 4F] 13 is a perspective view (top and bottom) showing another example of a scaffolding sheet. [Figure 4G] FIG. 11 is a perspective view showing another example of a scaffolding sheet. [Diagram 5] FIG. 1 is a diagram illustrating an example of a procedure for producing a cardiac tissue sheet. [Figure 6A] 1 is a photograph of the microfluidic chip produced in Example 1 and a schematic diagram of a channel. [Figure 6B]4 is a diagram showing a manufacturing method and stacking order of each component of the microfluidic chip in Example 1. FIG. [Figure 7] The upper part is a diagram that illustrates a schematic diagram of the procedure for producing cardiomyocytes, vascular endothelial cells, and vascular wall cells in Example 1. The lower part shows the proportion of each type of cell in the obtained cell population. [Figure 8] 1 is a micrograph of the cardiac tissue sheet produced in Example 1. The left image shows the entire cardiac tissue sheet, and the right image shows the peripheral portion of the cardiac tissue sheet. [Figure 9] FIG. 1 is a histological staining image of a cross section of a mature cardiac tissue sheet produced in Example 1. The dark areas indicate staining for cardiac troponin T (a marker for cardiomyocytes). The scale bar is 100 μm. [Figure 10] 2 is a graph showing the electrical stimulation responsiveness of the cardiac tissue sheet produced in Example 1. [Figure 11] 1 is a photograph showing an analyte in a flow channel in the heart-on-a-chip device of Example 1. [Figure 12] 1 is a graph showing electrical stimulation responsiveness of a cardiac tissue sheet detected by a heart-on-a-chip device in Experiment 1 of Example 1. [Figure 13A] 1 is a graph showing the isoproterenol responsiveness of a cardiac tissue sheet detected by a heart-on-a-chip device in Experiment 2 of Example 1. [Figure 13B] 1 is a graph showing the propranolol responsiveness of a cardiac tissue sheet detected by a heart-on-a-chip device in Experiment 2 of Example 1. [Figure 14A] 1 is a graph showing the responsiveness to isoproterenol in terms of the relationship between the movement frequency and the amount of displacement of the detection subject 14 in Experiment 2 of Example 1. [Figure 14B] 1 is a graph showing the responsiveness to isoproterenol in terms of the relationship between the movement frequency and the movement speed of the detection subject 14 in Experiment 2 of Example 1. [Figure 15] 1 is a graph showing the responsiveness of a cardiac tissue sheet to nifedipine detected by a heart-on-a-chip device in Experiment 3 of Example 1. [Figure 16] 1 is a graph showing the electrical stimulation responsiveness of a heart-on-chip device provided with cardiac tissue sheets having different proportions of vascular endothelial cells in Experiment 4 of Example 1. [Figure 17] 1 is a graph showing the electrical stimulation responsiveness of a heart-on-chip device provided with cardiac tissue sheets having different proportions of cardiomyocytes in Experiment 5 of Example 1. [Figure 18] 1 is a graph showing the electrical stimulation responsiveness of a heart-on-chip device provided with cardiac tissue sheets having different ratios of vascular wall cells in Experiment 6 of Example 1. [Figure 19] 1 shows the cellular composition of the cardiac tissue sheet produced in Example 2. [Figure 20] 1 shows histological staining of a cross section of the cardiac tissue sheet produced in Example 2. HE staining, SR staining, and immunostaining of cardiac troponin T are shown. The scale bar is 50 μm. [Figure 21] Fluorescent immunostaining of the cardiac tissue sheet prepared in Example 2. Double staining of cardiac troponin T and calponin, and nuclear staining with DAPI (4'6-diamidino-2-phenylindole) are shown. The scale bar is 100 μm. [Figure 22] Fluorescent immunostaining of the cardiac tissue sheet prepared in Example 2. Showing CD31 staining and DAPI nuclear staining. The scale bar is 100 μm. [Figure 23] FIG. 1 shows the structure of the heart-on-a-chip device fabricated in Example 2. The left image is an enlarged view of the microfluidic chip. The right image shows the microfluidic chip covered with a cardiac tissue sheet. The arrow indicates the cardiac tissue sheet. The dotted circle indicates the position of the oscillation part. The scale bar is 2 mm. [Figure 24] FIG. 1 is a top view schematic diagram of a heart-on-a-chip device comprising a microfluidic chip and a cardiac tissue sheet prepared in Example 2. [Diagram 25]The left image shows the microchannel of the heart-on-a-chip device of Example 2. The object to be detected (fluorescent particle) in the microchannel is detected by a fluorescent microscope. X indicates the axis of the position of the fluorescent particle. The right image is an enlarged view of the area enclosed by the square in the left image. The dotted circle in the right image indicates the same particle at end-diastole (top) and end-systole (bottom) of the pulsation of the cardiac tissue sheet. [Figure 26] 13 is a graph showing the displacement of a detection object over time in the heart-on-chip device of Example 2. [Figure 27] 13 is a graph showing electrical stimulation responsiveness of a cardiac tissue sheet detected by a heart-on-a-chip device in Experiment 7 of Example 2. [Figure 28] 1 is a graph showing the electrical stimulation responsiveness of the cardiac tissue sheet in the relationship between the frequency of electrical stimulation and the amount of displacement of the detection object in the entire pulsation cycle (the sum of the amount of displacement during systole and the amount of displacement during diastole) in experiment 7 of Example 2. The results of the regression analysis are shown in the upper right of the graph. [Figure 29] 1 is a graph showing the electrical stimulation responsiveness of a cardiac tissue sheet produced from human iPSC line GCaMP3-253G1 in Experiment 7 of Example 2, as a relationship between the frequency of electrical stimulation and the amount of displacement of the detectable object over the entire beating cycle (n=6). [Diagram 30] 1 is a graph showing the electrical stimulation responsiveness of a cardiac tissue sheet produced from human iPSC line FFI01s04 in Experiment 7 of Example 2, as a relationship between the frequency of electrical stimulation and the amount of displacement of the detectable object over the entire beating cycle (n=6). [Diagram 31] 1 is a graph showing the relationship between the frequency of electrical stimulation and the displacement or movement speed of the analyte in the cardiac tissue sheet produced from the human iPSC line GCaMP3-253G1 in Experiment 7 of Example 2 (n=7). *P<0.05, **P<0.01, ***<0.001. [Diagram 32] 1 is a graph showing the relationship between the frequency of electrical stimulation and the amount of displacement of the analyte or the moving speed of the analyte in the cardiac tissue sheet produced from the human iPSC line FFI01s04 in Experiment 7 of Example 2 (n=6). *P<0.05, **P<0.01, ***<0.001. [Diagram 33] 1 is a graph showing the electrical stimulation responsiveness of a cardiac tissue sheet produced from human iPSC line GCaMP3-253G1 during both the systole and diastole of the cardiac tissue sheet in Experiment 7 of Example 2 (n=7). *P<0.05, **P<0.01, ***<0.001. [Diagram 34] 1 is a graph showing the electrical stimulation responsiveness of a cardiac tissue sheet produced from human iPSC line FFI01s04 during both the systole and diastole of the cardiac tissue sheet in Experiment 7 of Example 2 (n=6). *P<0.05, **P<0.01, ***<0.001. [Diagram 35] 1 is a graph showing the isoproterenol responsiveness of a cardiac tissue sheet detected by a heart-on-a-chip device in Experiment 8 of Example 2. The graph shows the relationship between the isoproterenol concentration and the movement frequency of the detected object (bpm: beats per minute). [Diagram 36] 1 is a graph showing the isoproterenol responsiveness of a cardiac tissue sheet detected by a heart-on-a-chip device in Experiment 8 of Example 2. The graph shows the displacement of a detectable object when isoproterenol is added. [Figure 37] 1 is a graph showing the isoproterenol responsiveness of a cardiac tissue sheet in terms of the relationship between the movement frequency of the analyte and the amount of displacement of the analyte in the entire pulsation cycle in Experiment 8 of Example 2. The results of the regression analysis are shown in the upper right. [Figure 38] 13 is a graph showing the isoproterenol responsiveness of another cardiac tissue sheet in Experiment 8 of Example 2, as a relationship between the movement frequency of the analyte and the displacement of the analyte in the entire pulsation cycle. The results of the regression analysis are shown in the upper right. [Figure 39] 1 is a photograph of a cardiac tissue sheet adhered onto a scaffold sheet in Example 3. [Diagram 40] 1 is a photograph of a heart-on-a-chip device comprising a scaffold sheet and a cardiac tissue sheet in Example 3. [Diagram 41] 13 is a graph showing the spontaneous beating of a cardiac tissue sheet detected by a heart-on-a-chip device equipped with a scaffold sheet in Experiment 10 of Example 3. [Diagram 42] 13 is a graph showing the electrical stimulation responsiveness of a cardiac tissue sheet detected by a heart-on-a-chip device equipped with a scaffold sheet in Experiment 10 of Example 3. [Diagram 43] 13 is a graph showing the electrical stimulation responsiveness of a cardiac tissue sheet in terms of the relationship between the movement frequency of a detectable object and the amount of displacement of the detectable object in Experiment 10 of Example 3. The results of the regression analysis are shown in the upper right. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Hereinafter, an embodiment of the present invention will be described with reference to the drawings, but the present invention is not limited to the following embodiment. In the following embodiments, the same or common parts are denoted by the same reference numerals in the drawings, and the description thereof will not be repeated.

[0010] 1 is a cross-sectional view of a heart-on-a-chip device according to one embodiment of the present invention. The heart-on-a-chip device 100 shown in FIG. 1 includes a microfluidic chip 10 and a cardiac tissue sheet 20.

[0011] [Microfluidic chip] The microfluidic chip 10 comprises a oscillating part 11 capable of oscillating in a certain direction, a flow path 12 through which liquid flows as a result of the oscillation of the oscillating part 11, a diaphragm 13 provided between the oscillating part 11 and the flow path 12, and a detectable substance 14 dispersed in the liquid.

[0012] When the cardiac tissue sheet 20 contracts and relaxes, the oscillating unit 11 oscillates. In the heart-on-chip device 100 shown in FIG. 1, the cardiac tissue sheet 20 is relaxed and the oscillating unit 11 oscillates in the direction opposite to the diaphragm 13. FIG. 2 shows the heart-on-chip device when the oscillating unit 11 oscillates toward the diaphragm 13. At this time, the cardiac tissue sheet 20 contracts. As the oscillating unit 11 oscillates toward the diaphragm 13, the diaphragm 13 is pressed down by a distance z, and the liquid in the flow channel 12 facing the diaphragm 13 moves by a volume ΔV in the direction away from the oscillating unit 11. As shown in the enlarged view of the flow channel 12 in FIG. 2, the detectable substance 14 dispersed in the liquid also moves with the movement of the liquid in the flow channel 12. The movement of the detectable substance 14 is detected by the detector 15. The detector 15 can measure, for example, the movement frequency, displacement x, and movement speed of the detection object 14, which can be evaluated as values ​​reflecting cardiac function, in particular the heart rate, pulsation magnitude, and pulsation speed.

[0013] The movement frequency represents the number of times that the detectable object 14 moves within a certain period of time. When the detectable object 14 repeatedly moves back and forth, one round trip is counted as one movement. The displacement amount represents the distance that the detectable object 14 moves in one movement. When the detectable object 14 moves back and forth, the displacement amount is the distance moved in one direction. The movement speed is the value obtained by dividing the distance moved in one movement of the detectable object 14 by the time required for one movement. The displacement amount and movement speed are average values ​​obtained by measuring the movement of the detectable object 14 four times.

[0014] The microfluidic chip 10 may be made from glass or synthetic polymeric materials.

[0015] Examples of glass include soft glass (soda-lime glass, potash glass, lead glass, etc.) and hard glass (borosilicate glass, quartz glass, alkali-free glass, aluminosilicate glass, etc.), and borosilicate glass is preferably used. The above glass may be a commercially available product. Examples of commercially available glass include Tempax (borosilicate glass) manufactured by Matsunami Glass Industry Co., Ltd., Preclean Water Edge Polish (soda-lime glass) manufactured by Matsunami Glass Industry Co., Ltd., and VIOSIL (quartz glass) manufactured by Shin-Etsu Chemical Co., Ltd.

[0016] Examples of synthetic polymer materials include silicon-based elastomers, polystyrene, polyethylene, and polyethylene terephthalate. Examples of silicon-based elastomers include polyalkylsiloxanes such as polydimethylsiloxane (PDMS), polyalkylvinylsiloxanes such as polymethylvinylsiloxane, polyalkylphenylsiloxanes, and polyalkylhydrogensiloxanes, and PDMS and polymethylvinylsiloxane are preferably used. PDMS has advantages such as good oxygen permeability, excellent transparency, low autofluorescence, and the ability to faithfully reproduce a mold with a fine structure. From the viewpoint of being able to more clearly observe the non-detection object 14 with a microscope, it is preferable that the diaphragm 13 is formed from PDMS. The silicon-based elastomer may be a commercially available product. An example of a commercially available silicon-based elastomer is SILPOT 184 (PDMS) manufactured by Dow Corning Toray Co., Ltd.

[0017] From the viewpoint of culturing the cardiac tissue sheet 20 and filling the channels 12 of the microfluidic chip 10 with liquid, the entire heart-on-a-chip device 100 may be immersed in liquid. The liquid may be a balanced salt solution or a liquid culture medium.

[0018] [Swinging part] The oscillating part 11 can oscillate in a certain direction. The certain direction is not particularly limited as long as it is a direction that can deform the diaphragm 13, but from the viewpoint of efficiently reflecting the oscillation of the oscillating part 11 in the movement of the fluid, it is preferably a direction perpendicular to the main surface of the diaphragm 13. The oscillating part 11 can oscillate by directly or indirectly receiving the contraction and relaxation movements of the cardiac tissue sheet 20.

[0019] The shape of the oscillating part 11 is not particularly limited and may be a disk, cylinder, sphere, ellipsoid, rectangular parallelepiped, etc., and from the viewpoint of the adhesive area with the cardiac tissue sheet 20 and from the viewpoint of preventing damage to the cardiac tissue sheet 20, it is preferable that the planar shape is circular. From the viewpoint of oscillating the oscillating part 11 by contraction and relaxation of the cardiac tissue sheet 20, it is preferable that the oscillating part 11 has a shape that generates a portion that does not come into contact with the cardiac tissue sheet 20, and for example, the oscillating part 11 may have a convex part on the diaphragm 13 side or the opposite side, and preferably has a convex part on the diaphragm 13 side.

[0020] The size of the oscillating portion 11 (the diameter, major axis, or length of the major side when viewed in a direction perpendicular to the main surface of the diaphragm 13) is not particularly limited, but can be, for example, 0.5 mm or more and 8 mm or less, and preferably 3 mm or more and 5 mm or less.

[0021] The thickness of the oscillating portion 11 (the length in the direction perpendicular to the main surface of the diaphragm 13) is not particularly limited, but may be, for example, 50 μm or more and 600 μm or less, and preferably 100 μm or more and 400 μm or less.

[0022] [Flow path] The flow channel 12 is filled with a liquid, and the liquid moves due to the oscillation of the oscillation unit 11. The liquid is not particularly limited as long as it has a viscosity that allows it to flow through the flow channel, but is preferably a liquid that has little effect on the cells that constitute the cardiac tissue sheet 20, and may be a phosphate buffer solution, a balanced salt solution such as physiological saline, water, or a liquid culture medium.

[0023] The number of flow channels 12 may be one or two or more. One or both ends of the flow channels 12 communicate with the outside of the microfluidic chip 10 via a communication port 121. The flow channels 12 may be formed from a support 17 that supports the diaphragm 13 and a substrate 18 that has a groove for forming the flow channel. The cardiac tissue sheet 20 is in contact with the microfluidic chip 10 so as not to block the communication port 121, which is the inlet and outlet for the fluid flowing through the flow channel 12.

[0024] The length of the flow path 12 is not particularly limited, but from the viewpoint of allowing the liquid to flow easily within the flow path 12 and making it easy to detect the detectable substance 14, it can be, for example, 5 mm or more and 100 mm or less, 10 mm or more and 50 mm or less, and preferably 15 mm or more and 25 mm or less.

[0025] The flow channel 12 may have a chamber 122 which is an area in contact with the diaphragm 13, and a fine flow channel 123 which is an area not in contact with the diaphragm 13. When the diaphragm 13 is pressed by the swinging part 11, the volume of the chamber 122 decreases by ΔV. When the volume of the chamber 122 decreases by ΔV, the same volume of liquid flows from the chamber 122 to the fine flow channel 123. The flow of liquid from the chamber 122 to the fine flow channel 123 is detected as the movement of the detection target 14 in the fine flow channel 123. From the viewpoint of improving the detection sensitivity of the movement of the detection target 14, it is preferable that the chamber 122 has a structure capable of holding a larger amount of liquid than the fine flow channel 123, and it is preferable that the cross-sectional area of ​​the fine flow channel 123 is smaller than that of the chamber 122. In order to smoothly move the liquid, the inflow portion from the chamber 122 to the fine flow channel 123 may be fan-shaped or funnel-shaped.

[0026] The cross-sectional shape of the microchannel 123 may be circular, elliptical or rectangular, and may vary depending on the location. The diameter or length of the long side and cross-sectional area of ​​the cross section of the microchannel 123 are not particularly limited as long as they are large enough to allow fluid to flow easily and to detect the movement of the detection target 14 in the channel 12, but the diameter or length of the long side may be, for example, 0.1 mm or more or 0.2 mm or more, and may be 5 mm or less or 3 mm or less. The cross-sectional area of ​​the microchannel 123 is, for example, 0.02 mm 2 It may be more than 0.6 mm 2 It may be the following.

[0027] As another embodiment, the microfluidic chip 10 may include check valves 16a and 16b in the flow channel 12. FIGS. 3, 4A, and 4B are cross-sectional views of a heart-on-a-chip device according to an embodiment of the present invention, including check valves 16a and 16b. One check valve 16a is installed so as to be openable and closable in the direction toward the oscillating part 11, and the other check valve 16b is installed so as to be openable and closable in the direction opposite to the oscillating part 11, thereby allowing the liquid in the flow channel 12 to flow in one direction. When the check valves 16a and 16b are provided in the flow channel 12, the contraction and relaxation of the cardiac tissue sheet 20 can be detected as the movement of the detection object 14 with higher sensitivity. The flow channel 12 may be divided into a chamber 122 and a fine flow channel 123 via the check valves 16a and 16b.

[0028] [diaphragm] The diaphragm 13 is provided between the oscillating part 11 and the flow path 12. The diaphragm 13 separates the oscillating part 11 and the flow path 12. The diaphragm 13 transmits the movement of the oscillating part 11 as the movement of the liquid in the flow path 12. The diaphragm 13 preferably has a structure or property that allows it to be recessed toward the flow path 12 side by the oscillation of the oscillating part 11, and is preferably made of a material that is deformable by the oscillation of the oscillating part 11. The diaphragm 13 may cover a part of or the entirety of the surface of the support part 17 on the oscillating part 11 side to which the diaphragm 13 is attached or which is provided integrally with the diaphragm 13.

[0029] The size of diaphragm 13 is not particularly limited, but it is preferably larger than the surface of oscillating part 11 adhering to diaphragm 13 and large enough to allow cardiac tissue sheet 20 to be adequately adhered thereto. When diaphragm 13 is in the form of a square sheet, the size (length of a side) of diaphragm 13 can be, for example, 5 mm or more and 6 cm or less, and preferably 2 cm or more and 4 cm or less. When diaphragm 13 is circular, the size (diameter) of diaphragm 13 can be, for example, 5 mm or more and 8 cm or less, and preferably 1.5 cm or more and 4 cm or less.

[0030] The thickness of the diaphragm 13 is not particularly limited, but it is preferable that the diaphragm 13 has a thickness that allows it to be easily deformed by the oscillation of the oscillation part, and can be, for example, 10 μm to 2 mm, and preferably 50 μm to 1 mm.

[0031] [Detection target] The detectable substance 14 is dispersed in the liquid filling the flow channel 12. The shape, size, density, etc. of the detectable substance 14 are not particularly limited as long as the movement of the detectable substance 14 can be detected as a liquid flow, but it may be, for example, spherical, have a diameter of 0.5 μm or more and 5 μm or less, and have a density of 25 particles / cubic mm or more and 5000 particles / cubic mm or less. The detectable substance 14 may be introduced into the flow channel 12 using a syringe or the like.

[0032] The movement of the object to be detected 14 can be observed and measured, for example, using a microscope as the detector 15. The object to be detected 14 may be fluorescent beads. For example, fluorescent microspheres (Fruoresbrite Fluorescent Microspheres, Polysciences) can be used as the fluorescent beads. When the object to be detected 14 is a fluorescent bead, it is preferable to use a device capable of measuring fluorescence as the detector 15, for example, a culture microscope CKX53 (Olympus Corporation) can be used.

[0033] From the movement of the object 14 measured by the detector 15, the frequency of movement, the amount of displacement, and the movement speed of the object 14 can be calculated using, for example, MATLAB (registered trademark) (MathWorks, Inc.).

[0034] The microfluidic chip 10 preferably has cell adhesiveness in the portion that comes into contact with the cardiac tissue sheet 20. The entire surface of the microfluidic chip 10 may have cell adhesiveness. The cardiac tissue sheet 20 having cell adhesiveness means that the cardiac tissue sheet 20 adheres to the microfluidic chip 10. In the microfluidic chip 10, at least the diaphragm 13 preferably has cell adhesiveness, and the portion of the diaphragm 13 that comes into contact with the cardiac tissue sheet 20 preferably has cell adhesiveness. If the diaphragm 13 has poor cell adhesiveness, the cardiac tissue sheet 20 easily slides on the diaphragm 13, and the force generated by the contraction and relaxation of the cardiac tissue sheet 20 is difficult to transmit as a force for rocking the rocking part 11.

[0035] In order to make the microfluidic chip 10 have cell adhesiveness, a method generally used for culturing cells in an adhesive manner in a culture vessel can be used. Examples of such a method include a method of forming the microfluidic chip 10 from a synthetic polymer material (such as polylactic acid) that has high cell adhesiveness, and a method of subjecting the microfluidic chip 10 to a surface treatment. Feeder cells may be cultured as a scaffold. These methods may be combined.

[0036] The surface treatment that can be performed on the microfluidic chip 10 is preferably to introduce a polar group to enhance hydrophilicity, and examples of such treatment include chemical treatments such as plasma treatment, corona treatment, amination, and cationization. The microfluidic chip 10 may be coated with an extracellular matrix. Examples of the extracellular matrix include fibronectin, laminin, collagen, proteoglycan, and poly-L-lysine, and it is preferable to use fibronectin in view of its high affinity with the surface of cardiomyocytes. Examples of commercially available extracellular matrices include bovine serum fibronectin (Sigma) and Matrigel (Corning).

[0037] [Scaffolding sheet] The microfluidic chip may further include a scaffold sheet 19 for the cardiac tissue sheet. A microfluidic chip according to another embodiment of the present invention includes a rocking part capable of rocking in a fixed direction, a flow path through which liquid flows by rocking of the rocking part, a diaphragm provided between the rocking part and the flow path, and a scaffold sheet for the cardiac tissue sheet. A microfluidic chip according to another embodiment of the present invention includes a rocking part capable of rocking in a fixed direction, a flow path through which liquid flows by rocking of the rocking part, a diaphragm provided between the rocking part and the flow path, a detectable substance dispersed in the liquid, and a scaffold sheet for the cardiac tissue sheet.

[0038] An example of a heart-on-a-chip device 100 having a scaffold sheet 19 is shown in FIG. 4C. The scaffold sheet 19 is in contact with the oscillating part 11 so that the oscillating part 11 can be oscillated, for example. The cardiac tissue sheet 20 is in contact with the oscillating part 11 via the scaffold sheet 19 or integrally with the scaffold sheet 19 (scaffold sheet 192 on which the cardiac tissue sheet is formed). By having the scaffold sheet 19, the cardiac tissue sheet 20 can be easily moved. In addition, by having the scaffold sheet 19, the strength of the cardiac tissue sheet 20 is increased, making it less likely to break. The scaffold sheet 19 oscillates the oscillating part 11 without inhibiting the contraction and relaxation of the cardiac tissue sheet 20, and moves the liquid in the channel 12 through the diaphragm 13.

[0039] The cardiac tissue sheet 20 may be prepared by culturing cells constituting the cardiac tissue sheet 20 on the scaffold sheet 19, or a cardiac tissue sheet 20 prepared according to the method described below may be layered on the scaffold sheet 19. The scaffold sheet 19 may be fixed to the microfluidic chip 10 in advance before combining with the cardiac tissue sheet 20, but preferably the scaffold sheet 19 and the cardiac tissue sheet 20 are combined and fixed to the microfluidic chip 10 so as to cover the swinging part 11. If the cells constituting the cardiac tissue sheet are cultured on the scaffold sheet 19, the cardiac tissue sheet 20 can be produced more stably. The scaffold sheet 19 may be directly fixed to the diaphragm 13 or the support part 17, but is preferably fixed to the microfluidic chip 10 using a frame described below.

[0040] As shown in Fig. 4D, the scaffold sheet 19 preferably has a flat shape before being placed on the oscillating part 11. By placing the scaffold sheet 19 on the oscillating part 11 protruding beyond the diaphragm 13, the scaffold sheet 19 is deformed into a shape that conforms to the shape of the upper surface of the oscillating part 11 as shown in Fig. 14C. The scaffold sheet 19 may be, for example, a fiber sheet, a cell scaffold, or a cell culture holding member described in International Publication No. WO 2019 / 221172, WO 2019 / 167960, or WO 2020 / 085423.

[0041] The scaffold sheet 19 preferably has orientation. An oriented sheet is a sheet in which the fibers constituting the sheet are arranged along one direction. When the cardiac tissue sheet 20 is produced on the oriented scaffold sheet 19, an oriented cardiac tissue sheet 20 can be obtained.

[0042] In the scaffold sheet 19, when the angle of one direction (orientation axis) is set to 0°, 80% or more of the fibers, preferably 95% or more of the fibers, are arranged along an angle within the range of ±5°, preferably within the range of ±1°. The orientation angle is measured, for example, by the following method. Using a digital microscope (e.g., VHX-5000, manufactured by Keyence Corporation) as a photographing device, five locations in the sheet are selected and photographed at a magnification of 2000 times. From each photograph, 20 fibers are randomly selected, the angle with respect to the fiber production direction is measured, and the average is calculated.

[0043] The oriented scaffold sheet 19 can be manufactured, for example, by electrospinning a solution containing a polymeric material. The method for manufacturing the oriented scaffold sheet 19 is not particularly limited, but it can be manufactured, for example, by using a rotating drum, spraying a solution containing a polymeric material onto the rotating surface of the drum from a nozzle while rotating the drum, and winding up the fibers formed on the rotating drum.

[0044] The average diameter of the orthogonal cross section of the fibers constituting the scaffold sheet 19 is, for example, 1 μm or more and 7 μm or less, preferably 2 μm or more and 6 μm or less, and more preferably 3 μm or more and 5 μm or less. When the average diameter of the orthogonal cross section of the fibers is within this range, the cardiac tissue sheet 20 can be more stably produced. The diameter of the orthogonal cross section of the fibers is measured, for example, by the following method. Using a digital microscope (e.g., VHX-5000, manufactured by Keyence Corporation) as the photographing device, five locations in the sheet are randomly selected and photographed at a magnification of 2000 times. From each photograph taken, 20 fibers are randomly selected, the diameter of the orthogonal cross section of each selected fiber is measured, and the average is calculated.

[0045] The elastic modulus of the fibers constituting the scaffold sheet 19 is preferably 2000 MPa or less, more preferably 1000 MPa or less, and even more preferably 500 MPa or less. If fibers with a low elastic modulus are used, the pulsation of the cardiac tissue sheet 20 can be easily detected. The elastic modulus of the fibers is measured, for example, by preparing a test piece with a width of 20 mm and a length of 40 mm and using a tensile tester (load cell: 50 N). The chuck distance is set to 20 mm, and the test speed is set to 10 mm / min.

[0046] The pitch of the scaffold sheet 19 is, for example, 6 μm or more and 60 μm or less, preferably 6 μm or more and 50 μm or less, and more preferably 6 μm or more and 30 μm or less. The pitch of the scaffold sheet 19 is the distance between the core wires of adjacent fibers among the fibers constituting the scaffold sheet 19. The pitch of the scaffold sheet 19 is measured, for example, by the following method. Using a digital microscope (for example, VHX-5000, manufactured by Keyence Corporation) as a photographing device, five points in the sheet are randomly selected and photographed at a magnification of 2000 times. From each photograph taken, the distance from the core wire of a certain fiber to the core wire of an adjacent fiber is randomly selected and measured at 20 points, and the average is calculated.

[0047] The scaffold sheet 19 may be composed of one sheet layer (single layer) or two or more sheet layers (laminated or multi-layered, for example, 2 layers, 3 layers, 4 layers, 5 layers, 6 layers, etc.) in the direction perpendicular to the sheet plane. When the sheets are laminated, the upper and lower sheets are in contact with each other. The orientation axes of the upper and lower sheets intersect at, for example, 5° to 25°, preferably 10° to 20°, more preferably 13° to 17°.

[0048] The thickness of the scaffold sheet 19 is, for example, 4 μm to 70 μm, preferably 5 μm to 60 μm, more preferably 5 μm to 40 μm, and even more preferably 10 μm to 30 μm. The thickness of the sheet is measured, for example, by the following method. Using a digital indicator (ABS digital indicator ID-CX, ID-C112XBS), the thickness of the spinning base material alone and the thickness of the part where the scaffold sheet 19 is adhered are measured at five random points. The difference between the two is averaged and calculated.

[0049] The scaffold sheet 19 has a size capable of covering the swinging portion 11. The scaffold sheet 19 is preferably larger than the cardiac tissue sheet 20, and may be, for example, 1 mm or more larger than the cardiac tissue sheet 20, or 2 mm or more larger.

[0050] The porosity of the scaffold sheet 19 is, for example, 10% to 60%, preferably 15% to 50%, more preferably 20% to 40%, and even more preferably 30% to 40%. The porosity of the scaffold sheet 19 is the ratio of the area where no fibers are present to a certain area of ​​the fiber sheet plane in a sheet that is one layer in the direction perpendicular to the plane of the scaffold sheet 19. The porosity is measured, for example, by the following method. Using a digital microscope (for example, VHX-5000, manufactured by Keyence Corporation) as a photographing device, five locations in the sheet are selected and photographed at a magnification of 2000 times. From each photograph taken, the brightness measurement of automatic area measurement is performed using image processing software installed in the VHX-5000, and the porosity is calculated by measuring the ratio of brightness to the whole, and the average is calculated.

[0051] The scaffold sheet 19 is, for example, a sheet made of a polymer material. The fibers forming the sheet are prepared from a polymer material. The polymer material may be any material that does not exhibit cytotoxicity, and may be a biodegradable or non-biodegradable polymer material.

[0052] Biodegradable polymeric materials include, but are not limited to, copolymers of polylactic acid and polyglycolic acid (PLGA), polyglycolic acid (PGA), polybutyric acid (PLA), polyvinyl alcohol (PVA), polyethylene glycol (PEG), polyethylene vinyl acetate (PEVA), polyethylene oxide (PEO), etc. PLGA is a highly safe material that is known to be hydrolyzed in vivo to lactic acid and glycolic acid that are originally present in the body, and then decomposed into water and carbon dioxide and excreted from the body, and is particularly preferably used. PLGA's in vivo decomposition rate can be adjusted by changing the combination ratio of PLA (polylactic acid) and PGA (polyglycolic acid).

[0053] The non-biodegradable polymeric material is not particularly limited, but examples thereof include polystyrene (PS), polycarbonate (PC), polymethyl methacrylate (PMMA), polyvinyl chloride, polyethylene terephthalate (PET), polyamide (PA), polymethyl glutarimide (PMGI), thermoplastic polyester elastomers (e.g., Hytrel (registered trademark)), and the like. Polystyrene (PS), which is a material with low cytotoxicity, is particularly preferably used.

[0054] 4D, the scaffold sheet 19 may have a frame 191 for fixing the periphery of the sheet. By having the frame 191, it becomes easier to move the scaffold sheet 19 and the cardiac tissue sheet 20. The entire periphery or a part of the scaffold sheet 19 may be fixed to the frame 191. When a part of the periphery of the scaffold sheet 19 is fixed to the frame 191, a gap exists between the scaffold sheet 19 and the frame 191. The scaffold sheet 19 is preferably fixed to the frame 191 in the orientation direction of the sheet, and is not fixed in a direction intersecting the orientation direction.

[0055] The material of the frame 191 is not particularly limited as long as it does not have cytotoxicity, and examples thereof include polydimethylsiloxane (PDMS), PS, polycarbonate, stainless steel, etc. The specific gravity of the frame 191 is preferably greater than that of the culture solution, and more preferably is large enough that the frame 191 and the scaffold sheet 19 can sink in the culture solution and be fixed to the microfluidic chip 10 by their own weight. The specific gravity of the frame 191 is, for example, 1.05 or more, and preferably 1.1 or more.

[0056] As shown in FIG. 4E and FIG. 4F, the frame 191, the diaphragm 13, and / or the support 17 may be provided with a fixing portion 193 to fix the frame 191 and the scaffold sheet 19 to the microfluidic chip 10. When the frame 191 has the fixing portion 193, the position of the scaffold sheet 19 can be determined and fixed more precisely. When the frame 191 or the like has the fixing portion 193, the specific gravity of the frame 191 is not particularly limited. The fixing portion 193 may be made of the same material as the frame 191 and may be formed continuously. The shape of the fixing portion 193 is not particularly limited as long as it has a shape that allows the frame 191 to be fixed in the microfluidic chip 10. The fixing portion 193 may be a convex portion provided on the frame 191, and in this case, the diaphragm 13 and the support 17 are provided with a concave portion corresponding to the convex portion. The frame 191 may have a concave portion, and in this case, the diaphragm 13 and / or the support 17 are provided with a convex portion corresponding to the concave portion.

[0057] The shape of the frame 191 is not particularly limited as long as the scaffolding sheet 19 is exposed, and may be, for example, an annular or circular hollow shape. The frame 191 is, for example, a thin flat plate. The thickness of the frame 191 (height in a direction perpendicular to the surface of the scaffolding sheet 19) may be 1 cm or less or 5 mm or less, for example, 0.5 mm or more, and may be 1 mm or more. The inner diameter of the frame 191 is larger than the swinging part 11, and may be, for example, 1 mm or more larger than the swinging part 11, or 2 mm or more larger. The thickness of the frame 191 in the planar direction (when the frame 191 is circular and hollow, the difference between the outer diameter and the inner diameter is divided by 2) is, for example, 1 mm or more, may be 2 mm or more, or may be 3 mm or more. The frame 191 may have a protrusion 194, for example, upward from a flat base (opposite the scaffolding sheet 19) in order to improve operability, as shown in FIG. 4G.

[0058] The scaffold sheet 19 is fixed to the bottom surface of the frame 191 by, for example, a silicone adhesive (for example, silicone one-liquid condensation type RVT rubber Shin-Etsu Chemical, catalog number KE-45). It is preferable that the adhesive is absorbed into the gaps in the scaffold sheet 19 when the scaffold sheet 19 and the frame 191 are pressed together, and becomes one with the scaffold sheet 19. The thickness of the adhesive layer is preferably the same as the thickness of the scaffold sheet 19.

[0059] [Method of manufacturing microfluidic chips] The oscillating part 11, the diaphragm 13, the check valves 16a and 16b, the support part 17 and the substrate 18 used in the manufacture of the microfluidic chip can be manufactured from a polymer material such as PDMS or glass by a replica molding method based on a mold. The mold can be manufactured by photolithography using, for example, a photoresist. SU-8 3000 (Nippon Kayaku Co., Ltd.) can be used as the photoresist.

[0060] The components of the fabricated microfluidic chip are stacked under a microscope to achieve the desired overlap. Each component is treated with O2 plasma and heated to complete the stacking process.

[0061] [Cardiac tissue sheet] The cardiac tissue sheet 20 includes cardiomyocytes and may include other cells that form the heart or blood vessels. The cardiac tissue sheet 20 is a sheet-shaped cell aggregate in which cells are connected to each other by intercellular bonds. The cardiac tissue sheet 20 is preferably self-pulsating, has electrical bonds and orientation between cells, and has a uniform change in calcium ion concentration gradient in response to pulsation. The cardiac tissue sheet 20 is in contact with the oscillating unit 11 so that the oscillating unit 11 can be oscillated. Contraction and relaxation of the cardiac tissue sheet are derived from contraction and relaxation of the cardiomyocytes that constitute the cardiac tissue sheet 20.

[0062] The size of the cardiac tissue sheet 20 can be adjusted according to the size of the microfluidic chip 10, and is preferably large enough to cover the oscillating portion 11 and to be sufficiently attached to the diaphragm 13. The size of the cardiac tissue sheet 20 may be, for example, 3 mm or more and 30 mm or less in the longest part of the cardiac tissue sheet 20, and is preferably 5 mm or more and 20 mm or less. The area of ​​the cardiac tissue sheet 20 is, for example, 5 mm 2 More than 800mm 2 It may be the following.

[0063] The thickness of the cardiac tissue sheet 20 may be, for example, 30 μm or more and 1000 μm or less, and preferably 40 μm or more and 600 μm or less. The cardiac tissue sheet 20 may be composed of one cell layer, or two or more cell layers. When the thickness of the cardiac tissue sheet 20 is 30 μm or more, the cardiac tissue sheet 20 has sufficient strength and is less likely to break when placed on the microfluidic chip 10, and the structure of the cardiac tissue sheet 20 becomes more mature, the contractile force of the cardiac tissue sheet 20 becomes larger, and contraction and relaxation can be easily detected.

[0064] The cardiac tissue sheet 20 is preferably adhered to the microfluidic chip 10 so as to cover the oscillating portion 11. Methods for adhering the cardiac tissue sheet 20 include a method in which the cardiac tissue sheet 20 is placed over the microfluidic chip 10 in the absence of a culture medium, and allowed to stand at a humidity of 50% to 99%, a temperature of 30°C to 40°C, and for 30 minutes to 24 hours.

[0065] The cardiac tissue sheet 20 may contain differentiated cells such as cardiomyocytes isolated from a living body, but preferably contains cells differentiated from pluripotent stem cells, and more preferably contains cells derived from induced pluripotent stem cells.

[0066] The cardiac tissue sheet 20 may contain cells in which a disease-related gene is deleted or mutated. The disease-related gene may be, for example, a gene associated with a heart disease such as heart failure, ischemic heart disease, myocardial infarction, cardiomyopathy, myocarditis, hypertrophic cardiomyopathy, dilated phase hypertrophic cardiomyopathy, dilated cardiomyopathy, long QT syndrome, Timothy syndrome, or a gene associated with a disease other than a heart disease such as Marfan syndrome. Examples of the heart disease-related gene include δ-sarcoglycan, KCNQ1, SCN5A, and the like, and examples of the gene associated with a disease other than a heart disease include FBN1, and the like.

[0067] Cells in which a disease-related gene is deleted or mutated include cells in which the disease-related gene is not expressed, cells in which the expression level of the gene has changed, and cells in which the localization or function of the molecule (protein, RNA, etc.) expressed from the disease-related gene has changed. The deletion or mutation of the disease-related gene may or may not be accompanied by a genome mutation.

[0068] Cells in which a disease-associated gene is deleted or mutated can be obtained, for example, by a method of isolating cells from a patient with a disease, a method of preparing induced pluripotent stem cells from cells of a patient with a disease and differentiating them into cells of interest, a method of deleting or mutating a disease-associated gene in a pluripotent stem cell by a known method and differentiating it into cells of interest, etc. Whether or not a disease-associated gene is deleted or mutated can be determined by a method known to those skilled in the art, such as a method of analyzing a genome sequence, a method of quantifying the expression level of RNA or protein, or an immunostaining method.

[0069] Each of the cells capable of constituting the cardiac tissue sheet will be described in detail below. [Pluripotent stem cells] Pluripotent stem cells are not particularly limited, but examples thereof include the following cells:

[0070] <1.Embryonic stem cells> Embryonic stem cells (ES cells) are stem cells that are established from the inner cell mass of early mammalian embryos (eg, blastocysts) such as humans and mice, and have the pluripotency and ability to proliferate through self-renewal.

[0071] ES cells are embryo-derived stem cells that originate from the inner cell mass of a blastocyst, which is the embryo at the eight-cell stage of a fertilized egg, or after the morula stage. They have the ability to differentiate into any cell that makes up an adult, known as pluripotency, and the ability to proliferate by self-renewal. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292:154-156), and ES cell lines were subsequently established in humans, monkeys, and other primates (JA Thomson et al. (1998), Science 282:1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; JA Thomson et al. (1996), Biol. Reprod., 55:254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38:133-165).

[0072] ES cells can be established by extracting the inner cell mass from the blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. Cells can be maintained by subculture in a culture medium supplemented with substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF). Methods for establishing and maintaining human and monkey ES cells are described, for example, in US Pat. No. 5,843,780; Thomson JA, et al. (1995), Proc Natl. Acad. Sci. USA. 92: 7844-7848; Thomson JA, et al. (1998), Science. 282: 1145-1147; H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345: 926-932; M. Ueno et al. (2006), Proc. Natl. Acad. Sci. USA, 103: 9554-9559; H. Suemori et al. (2001), Dev. Dyn., 222: 273-279; H. Kawasaki et al. (2002), Proc. Natl. Acad. Sci. USA, 99:1580-1585; Klimanskaya I, et al. (2006), Nature. 444:481-485, etc.

[0073] As a culture medium for preparing ES cells, for example, DMEM / F-12 culture medium supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acids, 2 mM L-glutamic acid, 20% KSR and 4 ng / ml bFGF can be used, and human ES cells can be maintained at 37°C, 5% CO2, and in a humid atmosphere (H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345:926-932). In addition, ES cells need to be passaged every 3 to 4 days, and passage can be performed using, for example, 0.25% trypsin and 0.1 mg / ml collagenase IV in PBS containing 1 mM CaCl2 and 20% KSR.

[0074] ES cells can generally be selected by real-time PCR using the expression of gene markers such as alkaline phosphatase, Oct-3 / 4, Nanog, etc. In particular, human ES cells can be selected using the expression of gene markers such as OCT-3 / 4, NANOG, ECAD, etc. (E. Kroon et al. (2008), Nat. Biotechnol., 26: 443-452).

[0075] Human ES cell lines, for example WA01 (H1) and WA09 (H9), are available from WiCell Research Institute, and KhES-1, KhES-2 and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University (Kyoto, Japan).

[0076] <2.Sperm stem cells> Spermatogonial stem cells are pluripotent stem cells derived from the testis, and are the source of spermatogenesis. These cells can be induced to differentiate into various lineages of cells, just like ES cells, and have properties such as the ability to produce chimeric mice when transplanted into mouse blastocysts (M. Kanatsu-Shinohara et al. (2003) Biol. Reprod., 69:612-616; K. Shinohara et al. (2004), Cell, 119:1001-1012). They can self-replicate in a culture medium containing glial cell line-derived neurotrophic factor (GDNF), and spermatogonial stem cells can be obtained by repeated passage under the same culture conditions as ES cells (Takebayashi, Masanori et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Extra Edition), pp. 41-46, Yodosha (Tokyo, Japan)).

[0077] <3.Embryonic germ cells> Embryonic germ cells are cells that are established from primordial germ cells during the fetal period and have pluripotency similar to that of ES cells. They can be established by culturing primordial germ cells in the presence of substances such as LIF, bFGF, and stem cell factor (Y. Matsui et al. (1992), Cell, 70:841-847; JL Lesnick et al. (1992), Nature, 359:550-551).

[0078] <4. Induced pluripotent stem cells> Induced pluripotent stem (iPS) cells can be produced by introducing specific reprogramming factors in the form of DNA, RNA, or protein into somatic cells. Induced pluripotent stem cells have almost the same characteristics as ES cells, and have pluripotency and the ability to proliferate through self-renewal (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al. (2007), Cell, 131: 861-872; J. Yu et al. (2007), Science, 318: 1917-1920; Nakagawa, M. et al., Nat. Biotechnol.26:101-106(2008); International Publication WO2007 / 069666). The reprogramming factor may be composed of a gene specifically expressed in ES cells, its gene product or non-coding RNA, or a gene that plays an important role in maintaining the undifferentiated state of ES cells, its gene product or non-coding RNA, or a low molecular weight compound. Examples of genes contained in the reprogramming factor include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, or Glis1, and these reprogramming factors may be used alone or in combination. Combinations of reprogramming factors include WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO 2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO2010 / 056831, WO2010 / 0689 55, WO2010 / 098419, WO2010 / 102267, WO2010 / 111409, WO2010 / 111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D,et al.(2008),Nat.Biotechnol.,26:795-797,Shi Y,et al.(2008),Cell Stem Cell,2:525-528,Eminli S,et al.(2008),Stem Cells.26:2467-2474、Huangfu D,et al.(2008),Nat Biotechnol.26:1269-1275、Shi Y,et al.(2008),Cell Stem Cell,3,568-574、Zhao Y,et al.(2008),Cell Stem Cell,3:475-479、Marson A,(2008),Cell Stem Cell,3,132-135、Feng B,et al.(2009),Nat Cell Biol.11:197-203、RLJudson et al.,(2009),Nat.Biotech.,27:459-461、Lyssiotis CA,et al.(2009),Proc Natl Acad Sci USA.106:8912-8917、Kim JB, et al. al.(2009),Nature.461:649-643、Ichida JK,et al.(2009),Cell Stem Cell.5:491-503、Heng JC,et al.(2010),Cell Stem Cell.6:167-74、Han J,et al.(2010),Nature.463:1096-100、Mali P,et al. al.(2010),Stem Cells.28:713-720、Maekawa M,et al.(2011),Nature.474:225-9.

[0079] The above-mentioned reprogramming factors include histone deacetylase (HDAC) inhibitors (e.g., small molecule inhibitors such as valproic acid (VPA), trichostatin A, sodium butyrate, MC1293, M344, etc., and nucleic acid expression inhibitors such as siRNA and shRNA against HDAC (e.g., HDAC1 siRNA Smartpool (registered trademark) (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene)), etc.), MEK inhibitors (e.g., PD184352, PD98059, U0126, SL327, and PD0325901), glycogen synthase inhibitors (e.g., PD184352, PD98059, U0126, SL327, and PD0325901), and the like. kinase-3 inhibitors (e.g., Bio and CHIR99021), DNA methyltransferase inhibitors (e.g., 5-azacytidine), histone methyltransferase inhibitors (e.g., small molecule inhibitors such as BIX-01294, nucleic acid expression inhibitors such as siRNA and shRNA against Suv39hl, Suv39h2, SetDBl, and G9a), L-channel calcium agonists (e.g., Bayk8644), butyric acid, TGFβ inhibitors or ALK5 inhibitors (e.g., LY364947, SB431542, 616453, and A-83-01), p53 inhibitors (e.g., siRNA and shRNA against p53), ARID3A inhibitors (e.g., siRNA and shRNA against ARID3A), miRNAs such as miR-291-3p, miR-294, miR-295, and mir-302, Wnt Signaling (e.g., soluble Also included may be factors used to enhance establishment efficiency, such as Wnt3a), neuropeptide Y, prostaglandins (e.g., prostaglandin E2 and prostaglandin J2), hTERT, SV40LT, UTF1, IRX6, GLISl, PITX2, and DMRTBl.

[0080] When the reprogramming factor is in the form of a protein, it may be introduced into a somatic cell by techniques such as lipofection, fusion with a cell membrane-permeable peptide (eg, HIV-derived TAT and polyarginine), and microinjection.

[0081] When the reprogramming factor is in the form of DNA, it can be introduced into somatic cells by vectors such as viruses, plasmids, and artificial chromosomes, lipofection, liposomes, and microinjection. Examples of viral vectors include retroviral vectors, lentiviral vectors (Cell, 126, pp. 663-676, 2006; Cell, 131, pp. 861-872, 2007; Science, 318, pp. 1917-1920, 2007), adenoviral vectors (Science, 322, 945-949, 2008), adeno-associated virus vectors, and Sendai virus vectors (WO2010 / 008054). Examples of artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC). As the plasmid, a plasmid for mammalian cells may be used (Science, 322: 949-953, 2008). The vector may contain control sequences such as a promoter, an enhancer, a ribosome binding sequence, a terminator, and a polyadenylation site so that the nuclear reprogramming substance can be expressed. The vector may further contain a selection marker sequence such as a drug resistance gene (e.g., a kanamycin resistance gene, an ampicillin resistance gene, a puromycin resistance gene, etc.), a thymidine kinase gene, a diphtheria toxin gene, and a reporter gene sequence such as green fluorescent protein (GFP), β-glucuronidase (GUS), and FLAG, as necessary. In addition, the above vector may have LoxP sequences before and after a gene encoding a reprogramming factor or a promoter and a gene encoding a reprogramming factor bound thereto, in order to excise both of them after introduction into a somatic cell.

[0082] When the reprogramming factor is in the form of RNA, it may be introduced into somatic cells by techniques such as lipofection or microinjection, and RNA incorporating 5-methylcytidine and pseudouridine (TriLink Biotechnologies) may be used to suppress degradation (Warren L, (2010) Cell Stem Cell. 7:618-630).

[0083] As a medium for inducing iPS cells, for example, DMEM, DMEM / F12 or DME culture medium containing 10-15% FBS (fetal bovine serum) can be used. The medium can further contain LIF, penicillin / streptomycin, puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc., as appropriate. As a medium, a commercially available culture medium, for example, a culture medium for mouse ES cell culture (TX-WES culture medium, ThromboX), a culture medium for primate ES cell culture (culture medium for primate ES / iPS cells, ReproCell), a serum-free medium (mTeSR, Stemcell Technology), etc. may be used.

[0084] In a method for inducing iPS cells, for example, somatic cells are contacted with reprogramming factors in DMEM or DMEM / F12 culture medium containing 10% FBS at 37°C in the presence of 5% CO2 and cultured for about 4 to 7 days, and then the cells are seeded onto feeder cells (mitomycin C-treated STO cells, SNL cells, etc.) and cultured in a bFGF-containing culture medium for primate ES cell culture from about 10 days after contacting the somatic cells with the reprogramming factors, and iPS-like colonies can be generated about 30 to 45 days or more after the contact.

[0085] Another method for inducing iPS cells is, for example, culturing on feeder cells (mitomycin C-treated STO cells, SNL cells, etc.) in 10% FBS-containing DMEM culture medium (which may further contain LIF, penicillin / streptomycin, puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc., as appropriate) at 37°C and in the presence of 5% CO2, and ES-like colonies can be generated after about 25 to about 30 days or more. Instead of feeder cells, the somatic cells to be reprogrammed themselves may be used (Takahashi K, et al. (2009), PLoS One.4:e8067 or WO2010 / 137746), or extracellular matrix (e.g., Laminin-5 (WO2009 / 123349) and Matrigel (BD)).

[0086] Another example of an iPS cell induction method is a method in which iPS cells are cultured using a serum-free medium (Sun N, et al. (2009), Proc Natl Acad Sci USA. 106: 15720-15725). To increase the efficiency of establishment, iPS cells may be established under low oxygen conditions (oxygen concentration of 0.1% or more and 15% or less) (Yoshida Y, et al. (2009), Cell Stem Cell. 5: 237-241 or WO2010 / 013845).

[0087] During the above culture, the culture medium is replaced with fresh medium once a day from the second day of culture onward. The number of somatic cells used for nuclear reprogramming is not particularly limited. For example, the number of somatic cells used for nuclear reprogramming in a 100 cm culture dish is 2 Approximately 5 x 10 3 ~Approx. 5×10 6 It is the range of cells.

[0088] iPS cells can be selected based on the shape of the colony formed. On the other hand, when a drug resistance gene that is expressed in conjunction with a gene (e.g., Oct3 / 4, Nanog) that is expressed when somatic cells are reprogrammed is introduced as a marker gene, the established iPS cells can be selected by culturing in a culture medium (selection culture medium) containing the corresponding drug. In addition, when the marker gene is a fluorescent protein gene, iPS cells can be selected by observing with a fluorescent microscope, when it is a luminescent enzyme gene, by adding a luminescent substrate, and when it is a chromogenic enzyme gene, by adding a chromogenic substrate.

[0089] The term "somatic cells" as used herein refers to any animal cell (preferably a mammalian cell, including a human cell) excluding germline cells such as eggs, oocytes, and ES cells, or totipotent cells. Somatic cells include, but are not limited to, fetal (baby) somatic cells, neonatal (baby) somatic cells, and mature healthy or diseased somatic cells, as well as primary culture cells, passaged cells, and established cell lines. Specifically, somatic cells include differentiated cells such as tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, tissue progenitor cells, lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, liver cells, gastric mucosa cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes.

[0090] <5. ES cells derived from cloned embryos obtained by nuclear transfer> Cloned embryo-derived ES cells obtained by nuclear transfer (nt ES cells) are cloned embryo-derived ES cells produced by nuclear transfer technology, and have almost the same characteristics as fertilized egg-derived ES cells (T. Wakayama et al. (2001), Science, 292: 740-743; S. Wakayama et al. (2005), Biol. Reprod., 72: 932-936; J. Byrne et al. (2007), Nature, 450: 497-502). In other words, nt ES (nuclear transfer ES) cells are ES cells established from the inner cell mass of a cloned embryo-derived blastocyst obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell. To produce nt ES cells, a combination of nuclear transfer technology (JB Cibelli et al. (1998), Nature Biotechnol., 16:642-646) and ES cell production technology is used (Sayoka Wakayama et al. (2008), Experimental Medicine, Vol. 26, No. 5 (special issue), pp. 47-52). In nuclear transfer, the nucleus of a somatic cell is injected into an enucleated unfertilized egg of a mammal, and the egg can be initialized by culturing for several hours.

[0091] <6.Multilineage-differentiating Stress Enduring cells (Muse cells)> Muse cells are pluripotent stem cells produced by the method described in WO2011 / 007900. More specifically, Muse cells are pluripotent cells obtained by trypsinizing fibroblasts or bone marrow stromal cells for a long period of time, preferably 8 or 16 hours, followed by suspension culture, and are positive for SSEA-3 and CD105.

[0092] [Cardiomyocytes] In the present invention, cardiomyocytes refer to cells expressing at least cardiac troponin (cTnT) or αMHC. Examples of cTnT in humans include NCBI accession number NM_000364, and in mice include NM_001130174. Examples of αMHC in humans include NCBI accession number NM_002471, and in mice include NM_001164171.

[0093] The method for inducing cardiomyocytes from pluripotent stem cells is not particularly limited as long as it is a known method, and examples thereof include a method performed in the absence of feeder cells and a method performed in the presence of feeder cells.

[0094] <Method for inducing cardiomyocytes from pluripotent stem cells in the absence of feeder cells> In the present invention, an example of a method for inducing cardiomyocytes from pluripotent stem cells in the absence of feeder cells includes (i) culturing induced pluripotent stem cells in a medium containing Activin A, and (ii) after step (i), further culturing the induced pluripotent stem cells in a medium containing BMP4 and bFGF.

[0095] (i) culturing in a medium containing Activin A In this step, the pluripotent stem cells are separated by any method and may be cultured by suspension culture or may be cultured by adhesion using a coated culture dish. Preferably, the pluripotent stem cells are cultured by adhesion. Methods for separating the pluripotent stem cells into single cells include mechanical methods, or methods using a separation solution having protease activity and collagenase activity (Accutase(TM) and Accumax(TM), etc.) or a separation solution having only collagenase activity. A preferred method is to dissociate the pluripotent stem cells using a separation solution having only collagenase activity, and mechanically separate them into small pieces, preferably single cells. It is preferable to use a colony that has been cultured until it becomes about 80% confluent as the pluripotent stem cells used for differentiation induction.

[0096] Suspension culture refers to culturing cells in a non-adherent state on a culture dish, and can be performed using, but is not limited to, culture dishes that have not been artificially treated to improve adhesion to the cells (e.g., coating with extracellular matrix, etc.), or culture dishes that have been artificially treated to suppress adhesion (e.g., coating with polyhydroxyethyl methacrylate (poly-HEMA)).

[0097] Adherence culture is a culture method performed in a coated culture dish in any medium. Examples of coating agents include Matrigel (BD), collagen, gelatin, laminin, heparan sulfate proteoglycan, or entactin, and combinations thereof. Matrigel is preferred. More preferably, the adhesion culture is performed by the Matrigel sandwich method, in which induced pluripotent stem cells are attached to a culture dish coated with Matrigel, and Matrigel is further added to the medium to coat the entire pluripotent stem cells with Matrigel.

[0098] The medium in step (i) can be prepared by adding Activin A to a medium used for culturing animal cells as a basal medium.

[0099] Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Doulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof. RPMI 1640 medium is preferable. The basal medium may contain serum or may be serum-free. If necessary, the medium may contain one or more serum substitutes such as albumin, transferrin, Knockout Serum Replacement (KSR) (a serum substitute for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc., and may also contain one or more substances such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc. An example of a preferred basal medium in step (i) is RPMI medium containing L-glutamine and B27 supplement.

[0100] The medium in step (i) may further contain one or more growth factors selected from the group consisting of Wnt1, Wnt3, Wnt3a, Wnt4, Wnt7a, TGF-β, Nodal, BMP2, BMP4, BMP6, BMP7, GDF, bFGF, and VEGF (vascular endothelial growth factor) in addition to Activin A. A preferred growth factor is Wnt3a.

[0101] The concentration of Activin A added to the medium may be 10 ng / mL or more and 300 ng / mL or less, for example, 10 ng / mL, 25 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 175 ng / mL, or 200 ng / mL, but is not limited thereto. The concentration of Activin A added to the medium is preferably 100 ng / mL.

[0102] The concentration of Wnt3a added to the medium may be 10 mg / mL or more and 300 mg / mL or less, for example, 10 mg / mL, 25 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 175 mg / mL, or 200 mg / mL, but is not limited thereto. Preferably, the concentration of Wnt3a added to the medium is 100 mg / mL.

[0103] The culture temperature is, but not limited to, about 30 to 40° C., preferably about 37° C., and the culture is performed in an atmosphere of CO2-containing air, with the CO2 concentration being preferably about 2 to 5%. The culture time is, for example, 1 to 5 days, preferably 1 day.

[0104] (ii) culturing in a medium containing BMP and bFGF In step (ii), when step (i) is performed in suspension culture, the obtained cell population may be transferred directly to a coated culture dish and cultured in any medium. Examples of coating agents include Matrigel (BD), collagen, gelatin, laminin, heparan sulfate proteoglycan, entactin, and combinations thereof. The coating material is preferably Matrigel. When step (i) is performed in adhesion culture, step (ii) may be followed by changing the medium.

[0105] The medium used in step (ii) can be prepared by adding BMP and bFGF to a medium used for culturing animal cells as a basal medium. The basal medium can be the same as that used in step (i) above.

[0106] The BMP used in step (ii) is preferably a BMP belonging to the TGFβ superfamily, and examples thereof include BMP2, BMP4, and BMP7. A preferred BMP is BMP4.

[0107] The concentration of BMP4 added to the medium may be 0.1ng / mL or more and 50ng / mL or less, for example, 0.1ng / mL, 0.5ng / mL, 1ng / mL, 2.5ng / mL, 5ng / mL, 6ng / mL, 7ng / mL, 8ng / mL, 9ng / mL, 10ng / mL, 11ng / mL, 12ng / mL, 13ng / mL, 14ng / mL, 15ng / mL, 17.5ng / mL, 20ng / mL, 30ng / mL, 40ng / mL or 50ng / mL, but is not limited thereto. The concentration of BMP4 added to the medium is preferably 10ng / mL.

[0108] The concentration of bFGF added to the medium may be 0.1 ng / mL or more and 50 ng / mL or less, for example, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 17.5 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL or 50 ng / mL, but is not limited thereto. The concentration of bFGF added to the medium is preferably 10 ng / mL.

[0109] The culture temperature is, but not limited to, about 30 to 40° C., preferably about 37° C., and culture is performed in an atmosphere of CO2-containing air, with the CO2 concentration being preferably about 2 to 5%. The culture time is, for example, 1 to 10 days, preferably 4 days.

[0110] <Method for inducing cardiomyocytes from pluripotent stem cells in the presence of feeder cells> An example of a method for inducing cardiomyocytes from pluripotent stem cells in the presence of feeder cells is a method of co-culturing OP9 cells (Nishikawa, SI et al., Development 125, 1747-1757 (1998)) or END-2 cells (Mummery C, et al., Circulation. 107: 2733-40 (2003)) with pluripotent stem cells or Flk1-positive cells derived from pluripotent stem cells.

[0111] The medium for co-culture with feeder cells can be prepared by adding appropriate additives to a medium used for culturing animal cells as a basal medium.

[0112] The basal medium may be any of the basal media exemplified in the above step (i). Preferred basal media include α-MEM medium containing 10% FBS and DMEM medium containing 10% FBS.

[0113] Examples of additives to the basal medium in co-culture with feeder cells include 1 to 3 μg / mL cyclosporine A, activin A, and BMP4.

[0114] The culture temperature is, but not limited to, about 30 to 40° C., preferably about 37° C., and the culture is performed in an atmosphere of CO2-containing air, with the CO2 concentration being preferably about 2 to 5%. The culture time is the number of days required for the expression of cardiac troponin and / or αMHC, for example, 10 to 20 days.

[0115] Preferred conditions include culturing Flk1-positive cells in αMEM medium containing 10% FBS for 4 days, isolating the cells, and co-culturing them with OP9 cells for 6 days using αMEM medium containing 3 μg / mL cyclosporine A and 10% FBS, or co-culturing them with END-2 cells for 16 days using DMEM medium containing 10% FBS.

[0116] The obtained cardiomyocytes may be further cultured in a basal medium supplemented with VEGF (hereinafter also referred to as "additional culture"). The concentration of VEGF added to the medium may be within the range of, for example, 10 ng / mL to 500 ng / mL, 25 ng / mL to 300 ng / mL, 40 ng / mL to 200 ng / mL, 50 ng / mL to 100 ng / mL, 60 ng / mL to 90 ng / mL, or 65 ng / mL to 85 ng / mL. Preferably, the concentration of VEGF added to the medium is 50 ng / mL to 100 ng / mL. The concentration of VEGF added to the medium may be, but is not limited to, 10ng / mL, 25ng / mL, 50ng / mL, 55ng / mL, 60ng / mL, 65ng / mL, 70ng / mL, 75ng / mL, 80ng / mL, 85ng / mL, 90ng / mL, 95ng / mL, 100ng / mL, 110ng / mL, 120ng / mL, 130ng / mL, 140ng / mL, 150ng / mL, or 200ng / mL. Preferably, the concentration of VEGF added to the medium is 75ng / mL.

[0117] During the additional culture, a Wnt inhibitor may be added. Examples of Wnt inhibitors include IWP2, IWP3, IWP4, and XAV939. When IWP4 is used as a Wnt inhibitor, the concentration in the medium can be, for example, 0.25 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 5 μM, 7.5 μM, or 10 μM, and is preferably 2.5 μM. When XAV939 is used as a Wnt inhibitor, the concentration in the medium can be, for example, 0.5 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 15 μM, or 20 μM, and is preferably 5 μM.

[0118] The culture temperature is, but not limited to, about 30 to 40° C., preferably about 37° C., and the culture is performed in an atmosphere of CO2-containing air, with the CO2 concentration being preferably about 2 to 5%. The culture time is, for example, 4 to 20 days, preferably 10 days.

[0119] An example of a preferred method for producing cardiomyocytes and vascular endothelial cells is a production method (A) including a culture step (A1) of culturing induced pluripotent stem cells in the presence of activin A and a GSK-3 inhibitor, a culture step (A2) of culturing the cells obtained in the culture step (A1) in the presence of BMP and bFGF, and a culture step (A3) of culturing the cells obtained in the culture step (A2) in the presence of VEGF.

[0120] The culture step (A1) can be carried out under the same conditions as the above step (i) except for adding a GSK-3 inhibitor. The concentration of the GSK-3 inhibitor added to the medium may be 0.1 μM or more and 50 μM or less, for example, 0.25 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 4.5 μM, 6 μM, 8 μM, 10 μM, 15 μM, 20 μM, 25 μM, or 30 μM, but is not limited thereto. The concentration of the GSK-3 inhibitor added to the medium is preferably 3 μM. Examples of GSK-3 inhibitors include CHIR-99021.

[0121] The culture step (A2) can be performed under the same conditions as the above-mentioned step (ii), and the culture step (A3) can be performed under the same conditions as the additional culture of the above-mentioned step (ii). The medium used in the culture steps (A1) to (A3) is preferably a basal medium containing RPMI1640, B27 supplement, and no insulin. At the start of the culture step (A1), CHIR-99021 is preferably added. At the start of the culture step (A3), IWP4 and XAV939 are preferably added. According to the production method (A), cardiomyocytes and vascular endothelial cells can be obtained more efficiently, and for example, a cell population in which 20% to 80% of the cells are cardiomyocytes and 3% to 50% are vascular endothelial cells can be obtained.

[0122] An example of a preferred method for producing cardiomyocytes is a production method (B) comprising a culture step (B1) of culturing induced pluripotent stem cells in the presence of activin A and a GSK-3 inhibitor, a culture step (B2) of culturing the cells obtained in the culture step (B1) in the presence of BMP and bFGF, and a culture step (B3) of culturing the cells obtained in the culture step (B2) in the absence of VEGF and in the presence of insulin.

[0123] The culture steps (B1) and (B2) can be carried out under the same conditions as the above-mentioned culture steps (A1) and (A2). The culture step (B3) preferably does not contain VEGF but contains insulin.

[0124] The medium used in the culture steps (B1) to (B3) preferably contains RPMI1640 as a basal medium and a B27 supplement. In the culture step (B3), cells can be cultured in the presence of insulin by adding an insulin-containing B27 supplement to the basal medium. CHIR-99021 is preferably added at the start of the culture step (B1). IWP4 and XAV939 are preferably added at the start of the culture step (B3). According to the production method (B), cardiomyocytes can be obtained more efficiently, and for example, a cell population in which 20% to 80% of the cells are cardiomyocytes can be obtained. The obtained cell population may contain 1% to 70% of vascular wall cells.

[0125] [Vascular endothelial cells] The cardiac tissue sheet 20 preferably further comprises vascular endothelial cells. In the present invention, vascular endothelial cells refer to cells expressing at least one of PE-CAM, VE-cadherin, and von Willebrand factor (vWF). PE-CAM is exemplified by NCBI accession number NM_000442 in the case of humans, and NM_001032378 in the case of mice. VE-cadherin is exemplified by NCBI accession number NM_001795 in the case of humans, and NM_009868 in the case of mice. vWF is exemplified by NCBI accession number NM_000552 in the case of humans, and NM_011708 in the case of mice.

[0126] The method for inducing vascular endothelial cells from pluripotent stem cells is not particularly limited as long as it is a known method, and an example thereof includes the steps of: (a) culturing pluripotent stem cells in a medium containing Activin A and Wnt3a; (b) culturing the cells obtained in step (a) in a medium containing BMP and bFGF; and (c) culturing the cells obtained in step (b) in a medium containing VEGF.

[0127] Steps (a), (b) and (c) may be performed in the same manner as steps (i) and (ii) of the above-mentioned method for inducing cardiomyocytes, and the additional culture in the presence of VEGF. Thus, steps (a), (b) and (c) can induce cardiomyocytes and vascular endothelial cells simultaneously.

[0128] In step (c), cAMP may be further added to the medium. The concentration of cAMP is, for example, in the range of more than 0.5 mM and less than 2 mM, for example, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, but is not limited thereto, and is preferably 1 mM. The period for adding cAMP is not particularly limited, but is preferably 1 to 5 days, and particularly preferably 3 days. By adding cAMP, vascular endothelial cells can be obtained more efficiently.

[0129] An example of a preferred method for producing vascular endothelial cells is a production method (D) including a culture step (D1) of culturing induced pluripotent stem cells in the presence of activin A and a GSK-3 inhibitor, a culture step (D2) of culturing the cells obtained in the culture step (D1) in the presence of BMP and bFGF, and a culture step (D3) of culturing the cells obtained in the culture step (D2) in the presence of VEGF and cAMP.

[0130] The culture steps (D1) and (D2) can be carried out under the same conditions as the above-mentioned culture steps (A1) and (A2). The culture step (D3) can be carried out under the same conditions as the culture step (A3) except that cAMP is added. In addition, in the culture step (D3), the concentration of VEGF is preferably high. According to the production method (D), vascular endothelial cells can be obtained more efficiently, and for example, a cell population in which 1% to 40% of the cells are cardiomyocytes and 10% to 99% of the cells are vascular endothelial cells can be obtained.

[0131] [Vessel wall cells] The cardiac tissue sheet 20 preferably further comprises vascular wall cells. In the present invention, vascular wall cells refer to cells expressing smooth muscle actin (SMA) and / or PDGFRB. Examples of SMA in humans include NCBI accession number NM_001141945, and in mice include NM_007392. Examples of PDGFRB in humans include NCBI accession number NM_002609, and in mice include NM_001146268.

[0132] The method for inducing vascular wall cells from pluripotent stem cells is not particularly limited as long as it is a known method, and examples thereof include a method comprising the steps of: (I) culturing pluripotent stem cells in a medium containing Activin A; (II) culturing the cells obtained in step (I) in a medium containing BMP and bFGF; and (III) culturing the cells obtained in step (II) in a medium not containing VEGF.

[0133] Steps (I) and (II) may be performed using the same methods as steps (i) and (ii) of the above-mentioned method for inducing cardiomyocytes.

[0134] (III) culturing in a medium not containing VEGF The cells obtained in step (II) are cultured in a basal medium that is a medium used for culturing animal cells that does not contain VEGF. The VEGF-free medium may be a medium that does not substantially contain VEGF, for example, a medium with a VEGF concentration of less than 1 ng / mL, preferably less than 0.1 ng / mL, more preferably 0 ng / mL. The above-mentioned medium can be used as the basal medium, and an example of a preferred basal medium is RPMI medium containing 10% FBS.

[0135] An example of a preferred method for producing vascular wall cells is a production method (C) comprising a culture step (C1) of culturing induced pluripotent stem cells in the presence of activin A, a culture step (C2) of culturing the cells obtained in the culture step (C1) in the presence of BMP and bFGF, and a culture step (C3) of culturing the cells obtained in the culture step (C2) in the absence of VEGF and in the presence of serum.

[0136] The culture steps (C1) and (C2) can be carried out under the same conditions as the above steps (I) and (II). The culture step (C3) is carried out by adding serum to the above step (III). An example of the serum is FBS. The concentration of serum added to the medium may be 1% or more and 20% or less, for example, but not limited to, 1%, 2%, 5%, 8%, 10%, 13%, 15%, and 20%. The concentration of FBS added to the medium is preferably 10%.

[0137] The culture steps (C1) and (C2) are preferably performed using RPMI1640 as the basal medium, containing a B27 supplement but not containing insulin. The culture step (C3) may be started 3 days after the start of the culture step (C1). According to the production method (C), vascular wall cells can be obtained more efficiently, and for example, a cell population in which 30% to 99% of the cells are vascular wall cells can be obtained. The cell population may contain 1% to 20% cardiomyocytes.

[0138] [Method for simultaneously inducing cardiomyocytes, vascular endothelial cells, and vascular wall cells] Cardiomyocytes, vascular endothelial cells, and vascular wall cells can also be induced simultaneously. Examples of such a culture method include a method comprising the steps of (1) producing cardiomyocytes from pluripotent stem cells, and (2) culturing the cardiomyocytes obtained in step (1) in the presence of VEGF.

[0139] (1) The step of producing cardiomyocytes from pluripotent stem cells can be carried out in the same manner as the steps (i) and (ii) of inducing cardiomyocytes described above.

[0140] (2) culturing cardiomyocytes in the presence of VEGF; In step (2), the cardiomyocytes obtained by the above-mentioned method are further cultured in the presence of VEGF to produce mixed cells containing cardiomyocytes, vascular endothelial cells, and vascular wall cells in a desired ratio.

[0141] In the case where the previous step is a cell population after suspension culture, the obtained cardiomyocytes may be cultured in any medium on a coated culture dish. Examples of the coating agent include Matrigel (BD), collagen, gelatin, laminin, heparan sulfate proteoglycan, entactin, and combinations thereof. Matrigel is preferable. Alternatively, the cells obtained by adhesion culture in the previous step may be continued to be cultured by replacing the medium.

[0142] The medium used in this step can be prepared using the above-mentioned medium as the basal medium. A preferred medium in step (2) is, for example, RPMI 1640 medium containing L-glutamine, B27 supplement and VEGF.

[0143] The concentration of VEGF added to the medium may be within the range of, for example, 10 ng / mL to 500 ng / mL, 25 ng / mL to 300 ng / mL, 40 ng / mL to 200 ng / mL, 50 ng / mL to 100 ng / mL, 60 ng / mL to 90 ng / mL, or 65 ng / mL to 85 ng / mL. Preferably, the concentration of VEGF added to the medium is 50 ng / mL to 100 ng / mL. The concentration of VEGF added to the medium may be, but is not limited to, 10ng / mL, 25ng / mL, 50ng / mL, 55ng / mL, 60ng / mL, 65ng / mL, 70ng / mL, 75ng / mL, 80ng / mL, 85ng / mL, 90ng / mL, 95ng / mL, 100ng / mL, 110ng / mL, 120ng / mL, 130ng / mL, 140ng / mL, 150ng / mL, or 200ng / mL. The concentration of VEGF added to the medium is preferably 75ng / mL.

[0144] The culture temperature is not particularly limited, but is about 30 to 40° C., preferably about 37° C., and culture is performed in an atmosphere of CO2-containing air, with the CO2 concentration being preferably about 2 to 5%. The culture time is, for example, 4 to 20 days (e.g., 5 to 15 days), preferably 10 days.

[0145] The cell composition ratio of cardiomyocytes, vascular endothelial cells and vascular wall cells produced by this method is not particularly limited, but may be, for example, 40% to 80% cardiomyocytes, 1% to 20% vascular endothelial cells, 1% to 40% vascular wall cells and 0.1% to 10% undifferentiated cells. The content of vascular endothelial cells is preferably 3% or more. The composition ratio of cardiomyocytes, vascular endothelial cells and vascular wall cells is, for example, a mixed cell composition ratio of 62.7% cardiomyocytes, 7.9% vascular endothelial cells, 18.3% vascular wall cells and 2.7% undifferentiated cells. The cell composition ratio of cardiomyocytes, vascular endothelial cells and vascular wall cells according to the present invention can be arbitrarily changed by the concentration of VEGF and various other culture conditions, and can be arbitrarily changed within a range in which an appropriate strength can be maintained when the cells are made into a sheet.

[0146] [Process for removing undifferentiated cells] The method for producing cardiomyocytes, vascular endothelial cells, and vascular wall cells can include a step of removing undifferentiated cells from the mixed cells of cardiomyocytes, vascular endothelial cells, and vascular wall cells produced by the above-mentioned method.

[0147] In this step, any method capable of separating cardiomyocytes, vascular endothelial cells, and vascular wall cells from undifferentiated cells in the mixed cells can be adopted. Separation of cardiomyocytes, vascular endothelial cells, and vascular wall cells from undifferentiated cells may be a method of extracting only undifferentiated cells from the mixed cells based on an indicator of undifferentiated cells, or a method of extracting cardiomyocytes, vascular endothelial cells, and vascular wall cells from the mixed cells based on an indicator of cardiomyocytes, vascular endothelial cells, and vascular wall cells. Preferably, the former method is used in this step.

[0148] The indicator of undifferentiated cells may be, for example, a gene or protein that is specifically expressed in undifferentiated cells. These genes or proteins are well known in the art (Cell., 2005 Sep 23; 122(6): 947-56, Stem Cells., 2004; 22(1): 51-64, Mol Biol Cell., 2002 Apr; 13(4): 1274-81), and include, but are not limited to, Oct3 / 4, Nanog (all transcription factors), SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 (all cell surface antigens). The indicator used in this step is preferably a cell surface antigen, and TRA-1-60 can be used as an indicator of undifferentiated cells.

[0149] Examples of indicators for cardiomyocytes, vascular endothelial cells, and vascular wall cells include, but are not limited to, cardiactroponin-T (cTnT) (cardiomyocytes), VE-cadherin (vascular endothelial cells), and PDGFRb (vascular wall cells).

[0150] In this step, the removal of undifferentiated cells is carried out based on the above-mentioned indicators using a method such as flow cytometry (FACS) or magnetic cell sorting (MACS), preferably MACS.

[0151] In a preferred embodiment of the present invention, the step of removing undifferentiated cells from mixed cells is carried out by capturing undifferentiated cells with TRA-1-60 antibody and removing the captured undifferentiated cells (TRA-1-60 positive cells) by an immunomagnetic method (MACS).

[0152] The mixed cells after the step of removing undifferentiated cells may consist of only cardiomyocytes, vascular endothelial cells and vascular wall cells, or may contain any cells in addition to cardiomyocytes, vascular endothelial cells and vascular wall cells. The any cells may contain undifferentiated cells.

[0153] [Method of manufacturing cardiac tissue sheet] An example of a method for producing a cardiac tissue sheet 20 is shown in Figure 5. In the production method shown in Figure 5, first, undifferentiated human iPS cells are induced to differentiate to obtain cardiomyocytes, vascular endothelial cells, and vascular wall cells. The obtained mixed cells of cardiomyocytes, vascular endothelial cells, and vascular wall cells are further cultured to obtain a cardiac tissue sheet 20. In producing the cardiac tissue sheet 20, shaking culture may be performed as described in the maturation method for a cardiac tissue sheet described below.

[0154] The cardiomyocytes, vascular endothelial cells, and vascular wall cells for constituting the cardiac tissue sheet 20 may be prepared separately or simultaneously. The cardiomyocytes, vascular endothelial cells, and vascular wall cells are preferably used in the manufacture of the cardiac tissue sheet 20 after adjusting the cell numbers to a desired ratio. The cell number or cell ratio can be adjusted by counting the cell number or cell concentration by FACS or MACS using cardiactroponin-T (cTnT) (cardiomyocytes), VE-cadherin (vascular endothelial cells), PDGFRb (vascular wall cells), etc. as markers for each cell. When the cardiomyocytes, vascular endothelial cells, and vascular wall cells are differentiated simultaneously by the above-mentioned method, the differentiated cells may be used as they are as mixed cells in the manufacture of the cardiac tissue sheet 20.

[0155] The cardiac tissue sheet 20 preferably contains cardiomyocytes at 5% or more and 70% or less, vascular endothelial cells at more than 0% and 60% or less, and vascular wall cells at 1% or more and 60% or less. The proportion of cardiomyocytes contained in the cardiac tissue sheet 20 is more preferably 20% or more, even more preferably 25% or more, and even more preferably 30% or more. The proportion of vascular endothelial cells contained in the cardiac tissue sheet 20 is more preferably 10% or more, and even more preferably 20% or more. The proportion of vascular wall cells contained in the cardiac tissue sheet 20 is more preferably 2% or more, even more preferably 5% or more, and even more preferably 10% or more. The proportion of vascular wall cells contained in the cardiac tissue sheet 20 is more preferably 30% or less, and even more preferably 20% or less. When the proportion of cells constituting the cardiac tissue sheet 20 is within this range, the sheet has excellent contractile ability, excellent structural strength as a tissue, and excellent durability against tests such as drug administration.

[0156] For the culture of mixed cells, a culture vessel coated with a temperature-responsive polymer obtained by polymerizing a (meth)acrylamide compound, an N-(or N,N-di)alkyl-substituted (meth)acrylamide derivative (JP Patent Publication 2010-255001 A), or a vinyl ether derivative may be used, and preferably a culture vessel with poly-N-isopropylacrylamide immobilized thereon is used. The temperature-sensitive culture dish is hydrophobic at, for example, 37°C and hydrophilic at room temperature (25°C). The culture vessel can also be purchased as UpCell from WAKO Corporation.

[0157] The mixed cell culture equipment may be further coated with any coating agent, such as Matrigel (BD), collagen, gelatin, laminin, heparan sulfate proteoglycan, entactin, or combinations thereof, preferably gelatin.

[0158] The mixed cells may be cultured directly on the scaffold sheet 19. The scaffold sheet 19 may be placed on a culture vessel, and a suspension of the mixed cells may be added thereon. The frame 191 of the scaffold sheet 19 may be used as a wall for storing the culture solution. In this case, the suspension of the mixed cells is dropped into the space surrounded by the frame 191 and the scaffold sheet 19, for example, using a pipette, and the cells are cultured.

[0159] The medium used for culturing mixed cells can be prepared using a medium used for culturing animal cells as the basal medium. The basal medium includes the above-mentioned basal medium, and is preferably αMEM medium or RPMI 1640 medium. The medium desirably contains serum, but may be replaced as necessary with, for example, albumin, transferrin, sodium selenite, ITS-X (Invitrogen) (containing insulin, transferrin, and sodium selenite), Knockout Serum Replacement (KSR) (a serum substitute for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acid, insulin, collagen precursor, trace elements, etc. The medium may further contain one or more substances such as 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, Glutamax, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts. Growth factors in this step include Wnt1, Wnt3, Wnt3a, Wnt4, Wnt7a, TGF-β, Activin A, Nodal, BMP2, BMP4, BMP6, BMP7, GDF, bFGF and VEGF, and preferably VEGF.

[0160] The medium may further contain a Rho kinase (ROCK) inhibitor as a low molecular weight compound. The ROCK inhibitor is not particularly limited as long as it can suppress the function of Rho kinase (ROCK), and examples of the ROCK inhibitor include Y-27632 (see, for example, Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000); Narumiya et al., Methods Enzymol. 325, 273-284 (2000)), Fasudil / HA1077 (see, for example, Uenata et al., Nature 389:990-994 (1997)), H-1152 (see, for example, Sasaki et al., Pharmacol. Ther. 93:225-232 (2002)), Wf-536 (see, for example, Nakajima et al., Cancer Chemother 1999, 2002), and the like. Pharmacol. 52(4):319-324(2003)) and derivatives thereof, as well as antisense nucleic acids against ROCK, RNA interference-inducing nucleic acids (eg, siRNAs), dominant negative mutants, and expression vectors thereof. Other low molecular weight compounds are also known as ROCK inhibitors, and such compounds or their derivatives can also be used in the present invention (see, for example, U.S. Patent Application Publication Nos. 20050209261, 20050192304, 20040014755, 20040002508, 20040002507, 20030125344, 20030087919, and International Publication Nos. 2003 / 062227, 2003 / 059913, 2003 / 062225, 2002 / 076976, and 2004 / 039796). In the present method, one or more ROCK inhibitors can be used.

[0161] In the present invention, preferred media include αMEM medium containing serum, Y-27632 and VEGF, or RPMI 1640 medium containing serum and VEGF.

[0162] In this step, the number of cells to be cultured can be appropriately changed depending on the desired size of the sheet. For example, 4~1×10 8 However, the size of the cardiac tissue sheet 20 can be changed depending on the culture equipment. The culture period may be from 1 to 10 days, and is preferably 4 days.

[0163] In culturing mixed cells for producing the cardiac tissue sheet 20, it is preferable to perform the culturing while excluding undifferentiated cells that retain pluripotency, from the viewpoint of preventing tumor formation after production of the cardiac tissue sheet 20. Undifferentiated cells that retain pluripotency can be recognized by, for example, Nanog or Oct3 / 4.

[0164] The cardiac tissue sheet 20 may be used in a laminated state, for example, three layers may be laminated to be used as the cardiac tissue sheet 20. The cardiac tissue sheets 20 may be laminated in a culture solution (preferably, the cardiac tissue sheets 20 are laminated with each sheet shifted), and then the culture solution is removed and the sheets are allowed to stand for a certain period of time to bond the sheets. When multiple sheets are laminated, the same procedure may be performed all at once, but preferably the same procedure is performed for each layer.

[0165] [Method for maturation of cardiac tissue sheets] The cardiac tissue sheet 20 may be a shaking-cultured cardiac tissue sheet 20. By further shaking-culture the cardiac tissue sheet 20 produced as above, a mature cardiac tissue sheet 20 can be obtained.

[0166] Shaking can be performed, for example, at 10 rpm or more and 200 rpm or less, and may be at 30 rpm or more and 100 rpm or less. The shaking direction may be rotational, reciprocating (horizontal shaking), figure eight, etc. The shaking frequency and medium amount are preferably such that the cardiac tissue sheet 20 is kept immersed in the medium by shaking.

[0167] The shaking culture period is, for example, from 5 to 20 days, and may be from 9 to 13 days. The medium, culture temperature, and the like during the shaking culture may be the same as those used in the production of the cardiac tissue sheet 20 described above.

[0168] The thickness of the mature cardiac tissue sheet 20 is usually thicker than that of the cardiac tissue sheet 20 before shaking culture, and may be, for example, 30 μm or more and 1000 μm or less, and preferably 50 μm or more and 300 μm or less. When the thickness of the mature cardiac tissue sheet 20 is within this range, it has excellent adhesiveness to the microfluidic chip 10 and is excellent in transmitting the movement of the oscillating part 11 to the diaphragm 13 with high sensitivity.

[0169] In the mature cardiac tissue sheet 20, the cardiomyocytes are enlarged compared to the cardiac tissue sheet 20 before the shaking culture, and the adhesion between the cells in the sheet is also increased. The enlargement of the cardiomyocytes can be observed under a microscope. The adhesion between the cells can be confirmed, for example, by observing under a microscope and detecting the increase in the expression of cardiac troponin T by a known method. In the mature cardiac tissue sheet 20, the cardiomyocytes are also maturing, for example, the expression amount of β-receptors is also increased. The structure of the cardiac tissue sheet 20 is also maturing. The maturation of the structure of the cardiac tissue sheet 20 refers to a structural change in which, for example, the cardiomyocytes are packed closer together and integrated as myocardial tissue.

[0170] The mature cardiac tissue sheet 20 can contract in synchronization with electrical stimulation, and has increased electrical stimulation responsiveness. For example, when the mature cardiac tissue sheet 20 is subjected to electrical stimulation (voltage 30 V) with a period of 1500 msec to 100 msec in a liquid in which the cardiac tissue sheet 20 is immersed, the contraction and relaxation of the cardiac tissue sheet 20 according to the frequency of the stimulation is measured. Generally, as the frequency of electrical stimulation increases, the magnitude of contraction (width of contraction) of the cardiac tissue sheet 20 decreases, but the magnitude of contraction of the mature cardiac tissue sheet 20 is less likely to decrease.

[0171] The mature cardiac tissue sheet 20 also has improved circulatory agonist responsiveness. When a drug (circulatory agonist) is administered to cardiac tissue, the heart's pulsation rate, pulsation magnitude, pulsation speed, etc. can change. A cardiac tissue sheet 20 with excellent circulatory agonist responsiveness can change the contraction rate, contraction magnitude, and contraction speed of the cardiac tissue sheet 20 in response to changes in the heart's pulsation rate, pulsation magnitude, and pulsation speed. Examples of circulatory agonists include β agonists, β blockers, calcium antagonists, catecholamines, etc. Examples of β agonists include isoproterenol, adrenaline, metaproterenol, trimetoquinol, fenoterol, procaterol, salbutamol, formoterol, salmeterol, indacaterol, etc. Examples of β blockers include propranolol, carteolol, betaxolol, bisoprolol, metoprolol, nadolol, atenolol, acebutolol, etc. Examples of calcium antagonists include nifedipine, amlodipine, efonidipine, nicardipine, diltiazem, verapamil, felodipine, etc. Circulatory agonists may act on any of the heart's pulsation rate, pulsation magnitude, and pulsation speed among the functions of the heart, and administration of a circulatory agonist to a mature cardiac tissue sheet 20 may change any of the values ​​of the contraction rate, contraction magnitude, and contraction speed of the cardiac tissue sheet 20 corresponding to the heart's pulsation rate, pulsation magnitude, and pulsation speed.

[0172] [Method of evaluating cardiac function] The above-mentioned heart-on-a-chip device 100 can be used to evaluate cardiac function. The method for evaluating cardiac function includes a detection step of detecting the movement of the detectable object 14 of the heart-on-a-chip device 100, and an evaluation step of evaluating the detected movement as cardiac function. In the detection step, contraction and relaxation of the cardiac tissue sheet 20 can be detected as the movement of the detectable object 14. In the evaluation step, cardiac function can be evaluated by using the movement of the detectable object 14, particularly the movement frequency, displacement amount, and movement speed of the detectable object 14, as values ​​reflecting the heart's pulsation rate, pulsation magnitude, and pulsation speed.

[0173] Usually, the contraction and relaxation of the cardiac tissue sheet 20 can be observed under a microscope, but it is difficult to predict cardiac function. The heart-on-a-chip device 100, when combined with the microfluidic chip 10, which is inexpensive and has a simple structure, can convert the contraction and relaxation of the cardiac tissue sheet 20 into the pumping function of the heart and measure it.

[0174] Furthermore, when cardiac function is evaluated using the cardiac tissue sheet 20 containing cardiomyocytes in which a specific gene is deleted or mutated in the heart-on-chip device 100, it is possible to examine whether and how the specific gene is affecting cardiac function by detecting the movement of the detection object 14. The specific gene may be a cardiac disease-related gene or a gene associated with another disease.

[0175] The heart-on-a-chip device 100 can reproduce diseases caused by problems in cardiac function, such as heart failure, ischemic heart disease, myocardial infarction, cardiomyopathy, myocarditis, hypertrophic cardiomyopathy, dilated phase hypertrophic cardiomyopathy, dilated cardiomyopathy, long QT syndrome, Timothy's syndrome, and the like, and can evaluate the cardiac function.

[0176] The heart-on-a-chip device 100 is preferably used immersed in a culture medium at a temperature of 30° C. or higher and 40° C. or lower.

[0177] [Drug screening methods] The heart-on-chip device 100 can be used for drug screening. The drug screening method includes a contact step of contacting the cardiac tissue sheet 20 with a drug to be evaluated, a detection step of detecting the movement of the detectable object 14 of the heart-on-chip device 100, and an evaluation step of evaluating how the drug affects the cardiac tissue sheet 20 based on the movement of the detectable object 14.

[0178] In the contact step, the contact method is not particularly limited as long as it can bring the cardiac tissue sheet 20 into contact with the drug, and examples of the contact method include a method of adding a drug to a medium in which the cardiac tissue sheet 20 is immersed, a method of culturing the cardiac tissue sheet 20 in a medium to which the drug has been added, a method of immersing the heart-on-chip device 100 provided with the cardiac tissue sheet 20 in a medium to which the drug has been added, etc. The concentration of the drug is not particularly limited as long as it is within a range of concentrations expected to be used in actual treatment of the subject to be evaluated.

[0179] In the detection step, contraction and relaxation of the cardiac tissue sheet 20 can be detected as the movement of the detectable object 14. The detected movement of the detectable object 14 can be evaluated in the evaluation step as the effect of the drug on the cardiac tissue sheet 20 and the cells that make up the cardiac tissue sheet 20. Furthermore, the effect of the drug on the cardiac tissue sheet 20 can be inferred as the effect of the drug on the heart. By using the heart-on-chip device 100 equipped with the cardiac tissue sheet 20, it is possible to screen for therapeutic drugs that are effective for diseases related to cardiac function.

[0180] The cardiac tissue sheet 20 provided in the heart-on-chip device 100 may be a cardiac tissue sheet 20 in which a cardiac disease such as that exemplified above is reproduced, or may be a cardiac tissue sheet 20 containing cardiomyocytes in which a cardiac disease-related gene is deleted or mutated.

[0181] [Cardiotoxicity evaluation test method] The above-mentioned heart-on-a-chip device 100 can be used in a cardiotoxicity evaluation test. It is known that some drugs used as therapeutic agents for cardiac disease or diseases other than cardiac disease affect cardiac function. In a drug evaluation test performed on cultured cardiomyocytes, although toxicity to individual cardiomyocytes can be evaluated, it is difficult to accurately measure the effect of the drug on cardiac function. In addition, the properties of individually cultured cardiomyocytes may be different from those of cardiomyocytes in tissue.

[0182] The cardiotoxicity evaluation test method using the heart-on-a-chip device 100 includes a contact step of bringing the cardiac tissue sheet 20 into contact with the drug to be evaluated, a detection step of detecting the movement of the detectable object 14 of the heart-on-a-chip device 100, and an evaluation step of evaluating the effect of the drug on the cardiac tissue sheet 20 based on the movement of the detectable object 14.

[0183] Each step can be performed in the same manner as in the drug screening method described above. By using the heart-on-a-chip device 100 equipped with the cardiac tissue sheet 20, it is possible to predict the effect of a drug on cardiac function. EXAMPLES

[0184] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these.

[0185] <Example 1> [Microfluidic chip manufacturing] First, a mold was made from photoresist. First, a silicon wafer was spin-coated with SU-8 to a thickness of 200 to 500 μm. Next, it was placed on a hot plate and the solvent was evaporated. The photoresist was cooled at room temperature to prevent it from adhering to the mask. Parallel ultraviolet light was passed through a transparent high-resolution OHP. SU-8 was crosslinked at 65°C using a hot plate, and then light was applied to reach 95°C to promote crosslinking. The unpolymerized parts of the photoresist were removed using SU-8 Developer (developing solution, Nippon Kayaku Co., Ltd.) and dried under a nitrogen atmosphere. Finally, the mold was exposed to the vapor of a surface modifier (Novec EGC-1700, 3M Co.) for 1 minute to prevent the mold from sticking to the PDMS during the molding of the elastomer. The surface was washed with distilled water and dried under a nitrogen atmosphere. Parts made of PDMS curable elastomer were molded from the mold. Uncured PDMS prepolymer was poured onto the microfabricated silicon wafer and slowly placed under vacuum conditions to cure at 120 °C for 1 h before the replica was removed from the mold.

[0186] The replicas were stacked in order as shown in FIG. 4B to obtain the microfluidic chip 10 shown in the photograph of FIG. 6A. The size of the substrate 18 was a square of 2 cm×2 cm. The oscillating part 11 was cylindrical, with the diameter of the surface not in contact with the diaphragm 13 being 4 mm, and the diameter of the surface in contact with the diaphragm 13 being 2 mm. The chamber 122 had a diameter of 4 mm and a depth of 500 μm. The height and width of the microchannel 123 were each 200 μm. The check valves 16a and 16b were placed between the chamber 122 and the microchannel 123 so that they would not peel off even when water pressure was generated.

[0187] The manufacturing method and stacking order of each part will be described in more detail with reference to FIG. 6B. First, a mold for the flow path of the microfluidic chip was made of SU-8. From this mold, (a) a PDMS substrate 18 was made. Next, a PDMS sheet with a thickness of 100 μm was prepared, and (b) a support part 17 was made. The substrate 18 and the support part 17 were bonded by vacuum O2 plasma (product name: PLASMA ETCHING SYSTEM Model FA-1 (Samco Corporation), 15 W, 30 seconds, O2: 5 mL / min), and further baked at 85°C for 4 hours using product name: DIGITAL HOT PLATE HP-1SA (As One Corporation). As a result, (c) a bonded body of the substrate and the support part having a flow path was obtained.

[0188] A PDMS sheet with a thickness of 200 μm was produced, and circular PDMS cylinders with diameters of 2 mm and 4 mm were obtained using a biopsy punch. These were then baked in the same manner as above to obtain (d) the oscillation part 11. A PDMS sheet with a thickness of 100 μm was produced to obtain (e) the diaphragm 13. The oscillation part 11 and the diaphragm 13 were baked to obtain (f) the diaphragm with an oscillation part. The bonded body of (c) the substrate and the support part and the diaphragm with an oscillation part (f) were baked to obtain (g) a microfluidic chip made of PDMS.

[0189] [Maintenance culture of pluripotent stem cells] Human iPS cells were used as pluripotent stem cells. Human iPS cells (201B6) were obtained from the Center for iPS Cell Research and Application, Kyoto University. Maintenance culture was performed as described in Uosaki H. et al. PLoS One 2011;6:e23657. Details are as follows. Human iPS cells were seeded on FALCON culture dishes (10 cm) coated with Matrigel (growth factor reduced, 1:60 dilution, Invitrogen). The medium used was a conditioned medium (hereinafter referred to as "MEF-CM") obtained by culturing mouse embryonic fibroblasts (MEF) in a basal medium, to which 4 ng / mL of recombinant human bFGF (hbFGF, WAKO) was added. The basal medium used was a mixture of 471 mL of Knockout DMEM (Life technologies), 120 mL of Knockout serum replacement (KSR) (Life technologies), 6 mL of NEAA (Sigma-Aldrich), 3 mL of 200 mM L-Glutamine (Life technologies), and 55 mM 2-ME (mercaptoethanol) (Life technologies). MEFs were treated with Mitomycin-C (MMC) (WAKO) for 2.5 hours.

[0190] Human iPS cells were passaged every 4-6 days. The passage was performed by detaching cell colonies from the culture dish using a 1:1 mixture of TrypLE Select (1x) (Thermo Fisher Scientific) and 0.5 mM ETDA solution (adjusted with phosphate buffered saline (PBS)) (hereinafter referred to as "dissociation solution A"), and then dissociating the cells into single cells using a cell strainer. When seeding, iMatrix-511 silk (Nippi Corporation) was added at a concentration of 0.125 mg / mL.

[0191] [Production of cells that compose cardiac tissue sheets (1)] Cardiomyocytes, vascular endothelial cells, and vascular wall cells were produced according to the procedure shown in the upper part of Figure 7. Specifically, confluent human iPS cells were detached from the culture dish by incubating them with dissociation solution A at 37°C for 3 to 5 minutes. After aspirating dissociation solution A, StemFit AK02N (Ajinomoto Co., Inc.) was added and pipetted to recover single cells, which were then centrifuged and the number of cells was counted. Human iPS cells were obtained at a density of approximately 3 x 10 per 10 cm dish. 6 Approximately 1 × 10 cells were collected on a 6-well plate coated with Matrigel (Growth Factor Reduced Matrigel, BD Biosciences). 5 cells / cm 2 Human iPS cells were seeded at approximately 1:10 ...

[0192] After 24 hours, the medium was replaced with RPMI+B27 medium (RPMI1640 (GIBCO), 2 mM L-glutamine, 1×B27 supplement without insulin (GIBCO)) supplemented with 100 ng / mL Activin A (ActA, R&D Systems), and differentiation induction into cardiomyocytes was initiated. This day was designated as day 0 of differentiation induction. After 24 hours of culture, the medium was replaced with RPMI+B27 medium supplemented with 10 ng / mL human bone morphogenetic protein 4 (BMP4, R&D) and 10 ng / mL hbFGF (day 1 of differentiation induction). After 4 days of culture, the medium was replaced with RPMI+B27 medium supplemented with three concentrations (0 ng / mL, 50 ng / mL, or 100 ng / mL) of VEGF (rhVEGF, WAKO) to further induce endothelial cells and vascular wall cells (day 5 of differentiation induction). The medium was further supplemented with IWP4 at a concentration of 2.5 μM and XAV93 at a concentration of 5 μM. From the 7th day of differentiation induction onwards, the medium was replaced with the same medium (but without IWP4 and XAV939) every 2 days. After culturing for 10 days, the cells were harvested using AccuMax (Innovative Cell Technologies) (15th day of differentiation induction).

[0193] A portion of the collected cells was used for FACS analysis. The following antibodies were used for FACS analysis. For cardiomyocytes, anti-cardiac isoform of Troponin-T (cTnT) antibody (clone 13211, Thermo Fisher Scientific) was used, which was labeled with Alexa Fluor 488 (1:50) using Zenon technology (invirogen). For vascular endothelial cells, anti-VE-cadherin-conjugated-FITC (clone 55-7h1, 1:100 dilution, BD) was used. For vascular wall cells, anti-human PDGFRb-conjugated-PE (clone 28d4, 1:100 dilution, BD) was used. The composition ratio of collected cells is shown in the lower part of Figure 7. Under 0 ng / mL VEGF conditions, cardiomyocytes (CM) accounted for 55.7%, vascular endothelial cells (EC) for 0.4%, and vascular wall cells (MC) for 34.4%. Under 50ng / mL VEGF conditions, the CMs were 54.1%, ECs were 13.4%, and MCs were 16.9%. Under 100ng / mL VEGF conditions, the CMs were 35.7%, ECs were 27.4%, and MCs were 18.0%.

[0194] [Production of cells that compose cardiac tissue sheets (2)] To efficiently produce cardiomyocytes and vascular endothelial cells, human iPS cells were induced to differentiate by another method. The production of cells constituting cardiac tissue sheets (2) was the same as the production of cells constituting cardiac tissue sheets (1) except that on day 0 of differentiation induction in the production of cells constituting cardiac tissue sheets (1), CHIR-99021 (TOCRIS, model number 4423, 3 μM) was further added to the RPMI+B27 medium supplemented with Activin A and cultured. The concentration of VEGF added from day 5 of differentiation induction was 50 ng / mL.

[0195] Cells were harvested 15 days after differentiation induction and analyzed by FACS. Cardiomyocytes (CM) accounted for 60.9%, vascular endothelial cells (EC) for 22.6%, and vascular wall cells (MC) for 2.3%.

[0196] [Production of cells that compose cardiac tissue sheets (3)] In order to efficiently produce cardiomyocytes, human iPS cells were induced to differentiate by another method. Until the fourth day of differentiation induction, the cells were cultured in the same manner as in the above-mentioned Production of Cells Constituting Cardiac Tissue Sheets (2). On the fifth day of differentiation induction, the medium was replaced with RPMI medium containing no VEGF and supplemented with insulin-containing B27 supplement, IWP4 at a concentration of 2.5 μM, and XAV93 at a concentration of 5 μM, and the cells were cultured for 10 days. From the seventh day of differentiation induction onwards, the medium was replaced once every two days with RPMI + insulin-containing B27 supplement medium without IWP4 and XAV939.

[0197] Cells were harvested on the 15th day after differentiation induction and analyzed by FACS. Cardiomyocytes (CM) accounted for 68.6%, endothelial cells (EC) for 0.1%, and vascular wall cells (MC) for 18.2%.

[0198] [Production of cells that compose cardiac tissue sheets (4)] To efficiently produce vascular wall cells, human iPS cells were induced to differentiate using another method. Until the second day of differentiation induction, the cells were cultured in the same manner as in the above-mentioned Production of cells that constitute cardiac tissue sheets (1). On the third day of differentiation induction, the medium was changed to one that did not contain VEGF but contained 10% FBS (BIOWEST, model number BWT-S1760-500), and the cells were cultured for 12 days. The medium was changed every other day using the same medium.

[0199] Cells were harvested 15 days after differentiation induction and analyzed by FACS. Cardiomyocytes (CM) accounted for 1.2%, vascular endothelial cells (EC) for 0.3%, and vascular wall cells (MC) for 96.5%.

[0200] [Production of cells that compose cardiac tissue sheets (5)] To efficiently produce vascular endothelial cells, human iPS cells were induced to differentiate by another method. The cells were cultured in the same manner as in the above-mentioned Production of cells constituting cardiac tissue sheets (2), except that 75 ng / mL VEGF and 1 mM cAMP were added to the medium from day 5 onwards after differentiation induction.

[0201] The cells were harvested on the 15th day after differentiation induction, and the proportion of the constituent cells was analyzed, revealing that vascular endothelial cells (EC) accounted for 98.4%.

[0202] [Manufacturing of cardiac tissue sheets] The mixed cells of cardiomyocytes, vascular endothelial cells and vascular wall cells obtained by the above-mentioned method (cardiomyocytes 55.4%, vascular endothelial cells 20.3%, vascular wall cells 11.4%) were cultured on a temperature-sensitive culture dish (UpCell, WAKO) 12-multiwell plate coated with FBS at 2.6 × 10 5 The cells were seeded at 100 cells / well and cultured at 37°C. The medium consisted of αMEM (invirogen) with 10% FBS and 5 × 10 -5 A medium containing 50 nM VEGF and 10 μM Y-27632 (Rock inhibitor, WAKO) added to the medium containing M2-ME (hereinafter also referred to as "adhesion medium") was added at 2 mL per well. After 2 days of culture, VEGF was added to a concentration of 50 ng / mL. A photograph of the obtained cardiac tissue sheet 20 is shown in FIG. 8. The left image in FIG. 8 is an overall image of the cardiac tissue sheet 20, which had a diameter of about 10 mm and a thickness of about 50 μm. The right image in FIG. 8 is an enlarged photograph of the peripheral portion of the cardiac tissue sheet 20.

[0203] The obtained cardiac tissue sheet 20 was further cultured with shaking. The culture was performed with changing the medium every two days. The shaking frequency was 60 rpm. After 10 days of shaking culture, the UpCell was moved from 37°C to room temperature, and the cells were peeled off in a sheet form to obtain a mature cardiac tissue sheet 20. Figure 9 shows a histological staining of the cross section of the obtained cardiac tissue sheet 20. The diameter of the mature cardiac tissue sheet 20 was about 12 mm. The thickness of the mature cardiac tissue sheet 20 was about 200 μm, which was thicker than before the shaking culture. The dark colored parts in Figure 9 indicate staining of cardiomyocytes. The mature cardiac tissue sheet 20 had a thicker layer of cardiomyocytes and was in close contact with each other, and the structure was mature.

[0204] The electrical stimulation responsiveness of the mature cardiac tissue sheet 20 was measured. A voltage of 30 V with a pulse width of 4 ms was applied to the medium in which the cardiac tissue sheet 20 was immersed using a cell sheet electrical stimulation unit (Strex), and the contraction behavior of the cardiac tissue sheet 20 was observed under a microscope. The results of analyzing the contraction behavior of the cardiac tissue sheet 20 using MATLAB (registered trademark) (Mathworks) are shown in FIG. 10. It was found that the mature cardiac tissue sheet 20 can contract in synchronization with electrical stimulation with a period from 1000 msec to 100 msec. It was also found that the contraction amplitude does not easily decrease even if the electrical stimulation frequency increases, and a cardiac tissue sheet 20 suitable for evaluating cardiac function was obtained.

[0205] [Fabrication of Heart-on-a-Chip Device] The obtained cardiac tissue sheet 20 was attached to the microfluidic chip 10 to obtain a heart-on-chip device 100. Before attachment, the microfluidic chip 10 was coated with bovine serum fibronectin (Sigma, diluted 100 times). The cardiac tissue sheet 20 was placed over the oscillating part 11 of the microfluidic chip 10, and left to stand for 60 minutes at a humidity of 95% and a temperature of 37°C without being immersed in a culture medium. Thereafter, the sheet was immersed in a culture medium for adhesion, and fluorescent microspheres (Fruoresbrite Fluorescent Microspheres, Polyscience, diluted 100 times) as the object to be detected 14 were added to the culture medium to obtain a heart-on-chip device 100.

[0206] A 488 nm laser beam was irradiated onto the micro flow channel 123, and the detectable objects 14 in the flow channel were detected using a microscope (CKX53, Olympus). The detectable objects 14 in the micro flow channel 123 were detected as shown in FIG. 11. The movements of the individual detectable objects 14 enclosed in squares were recognized using MATLAB (registered trademark), and the movement frequency, displacement amount, and movement speed of the detectable objects 14 were calculated. In the following experiments, the movements of the detectable objects 14 were detected using a similar method.

[0207] [Experiment 1: Responsiveness of cardiac tissue sheets to electrical stimulation] It was verified whether the electrical stimulation responsiveness of the cardiac tissue sheet 20 can be detected by the heart-on-chip device 100. A cell sheet electrical stimulation unit (Strex) was used to apply a voltage of 30 V with a pulse width of 4 ms to the culture medium in which the heart-on-chip device 100 was immersed, and the displacement of the detection object was detected. As shown in FIG. 12, the displacement of the detection object 14 was detected in synchronization with the electrical stimulation with a period of 1000 msec to 600 msec. It was found that the use of the heart-on-chip device 100 allows the self-pulsation of the cardiac tissue sheet 20 and the contraction and relaxation due to electrical stimulation to be detected as the movement of the detection object 14.

[0208] [Experiment 2: Circulatory agonist response of cardiac tissue sheets (1)] We verified whether the circulatory agonist responsiveness of the cardiac tissue sheet 20 can be detected by the heart-on-a-chip device 100. Isoproterenol (Iso) and propranolol (Prop) were used as circulatory agonists. It is known that isoproterenol increases the heart rate, and propranolol decreases the heart rate. The heart-on-a-chip device 100 equipped with the cardiac tissue sheet 20 was exposed to a concentration of 10 -9 M to 10 -6 M isoproterenol or concentration 10 -7 M to 10 -4 The heart-on-chip device 100 was then immersed for 10 minutes in an adhesion medium containing M propranolol. Thereafter, the heart-on-chip device 100 was transferred to a medium containing the analyte 14, and the movement of the analyte 14 was detected.

[0209] As shown in FIG. 13A, the addition of isoproterenol increased the frequency of movement of the detectable object 14 in a concentration-dependent manner. It is believed that isoproterenol increased the frequency of contraction and relaxation of the cardiac tissue sheet 20, and therefore increased the frequency of movement of the detectable object 14. Furthermore, as shown in FIG. 13B, the addition of propranolol decreased the frequency of movement of the detectable object 14 in a concentration-dependent manner. It is believed that propranolol decreased the frequency of contraction and relaxation of the cardiac tissue sheet 20, and therefore decreased the frequency of movement of the detectable object 14. It was found that the mature cardiac tissue sheet 20 responded to circulatory agonists in the same way as a living heart, and that the change could be detected as the movement of the detectable object 14 in the heart-on-a-chip device 100.

[0210] The relationship between the movement frequency and the displacement of the detectable substance 14 when isoproterenol is added is shown in Fig. 14A, and the relationship between the movement frequency and the movement speed is shown in Fig. 14B. As the amount of isoproterenol added increased and the movement frequency of the detectable substance 14 increased, the displacement of the detectable substance 14 decreased. Furthermore, as the movement frequency decreased, the movement speed also decreased gradually. The displacement and movement speed changed in correlation with the increase in the movement frequency of the detectable substance 14, and it was shown that the heart-on-chip device 100 visualizes and evaluates the contractile force of the cardiac tissue sheet 20 with good correlation.

[0211] [Experiment 3: Circulatory agonist response of cardiac tissue sheets (2)] Nifedipine, an L-type calcium channel inhibitor, was used as a circulatory agonist to detect the responsiveness of the cardiac tissue sheet 20. Nifedipine is known to reduce the magnitude and speed of cardiac pulsation without significantly changing the cardiac rate. The heart-on-chip device 100 equipped with the cardiac tissue sheet 20 was placed in a 10 -7 The heart-on-a-chip device 100 was then immersed for 10 minutes in an adhesion medium containing nifedipine M. Thereafter, the heart-on-a-chip device 100 was transferred to a medium containing the analyte 14, and the movement of the analyte 14 was detected.

[0212] As shown in Fig. 15, the addition of nifedipine slightly decreased the frequency of movement of the detection object 14 in the heart-on-a-chip device 100, and significantly decreased the amount of displacement and the speed of movement. This change was similar to the responsiveness of a living heart when nifedipine was administered. It was found that the mature cardiac tissue sheet 20 showed a responsiveness to circulatory agonists similar to that of a living heart, and that this responsiveness could be detected by the heart-on-a-chip device 100.

[0213] [Experiment 4: Electrical stimulation response of cardiac tissue sheets with different proportions of vascular endothelial cells] The electrical stimulation responsiveness of the cardiac tissue sheet 20 was examined by changing the ratio of vascular endothelial cells among the cells constituting the cardiac tissue sheet 20. The cardiac tissue sheet 20 was produced by the same method as in the above [Method for producing cardiac tissue sheet] with a mixture ratio of cardiomyocytes, vascular endothelial cells and vascular wall cells of 43.7% cardiomyocytes, 18.2% vascular endothelial cells and 4.0% vascular wall cells, and was designated as cardiac tissue sheet A. The cardiac tissue sheet A was treated with AccuMax (Innovative Cell Technologies) and separated into single cells by pipetting. FACS was performed using the same method and antibodies as above to examine the ratio of cells constituting the cardiac tissue sheet A. Cardiomyocytes (CM) were 29.1%, vascular endothelial cells (EC) were 25.0%, and vascular wall cells (MC) were 5.5%.

[0214] A cardiac tissue sheet 20 was prepared in the same manner as in the above [Method of manufacturing cardiac tissue sheet] with a mixture ratio of cardiomyocytes, vascular endothelial cells and vascular wall cells of 37.8% cardiomyocytes, 8.4% vascular endothelial cells and 3.6% vascular wall cells, designated as cardiac tissue sheet B. The ratios of cells constituting the prepared cardiac tissue sheet B were 25.6% cardiomyocytes (CM), 2.9% vascular endothelial cells (EC) and 1.9% vascular wall cells (MC).

[0215] A heart-on-chip device 100 including a cardiac tissue sheet A or a cardiac tissue sheet B was produced. A cell sheet electrical stimulation unit (Strex) was used to apply a voltage of 30 V, a pulse width of 4 ms, and a period of 1000 msec, 900 msec, or 800 msec to the medium in which the heart-on-chip device 100 was immersed, and the displacement of the detection target 14 was detected. As shown in FIG. 16, when an electrical stimulation with a period of 900 msec was applied, the heart-on-chip device 100 including the cardiac tissue sheet A did not show any change in the amount of displacement of the detection target 14 compared to the heart-on-chip device 100 including the cardiac tissue sheet B. When an electrical stimulation with a period of 1000 msec and 800 msec was applied, the heart-on-chip device 100 including the cardiac tissue sheet A showed an increased amount of displacement of the detection target 14 compared to the heart-on-chip device 100 including the cardiac tissue sheet B. The cardiac tissue sheet A had a significantly higher proportion of vascular endothelial cells than the cardiac tissue sheet B, and the proportion was 20% or more. It was found that the cardiac tissue sheet 20 containing 20% ​​or more of vascular endothelial cells exhibited improved responsiveness to electrical stimulation.

[0216] [Experiment 5: Electrical stimulation response of cardiac tissue sheets with different proportions of cardiomyocytes] A cardiac tissue sheet 20 was prepared in the same manner as in the above [Method of manufacturing cardiac tissue sheet] with a mixture ratio of cardiomyocytes, vascular endothelial cells and vascular wall cells of 58.1% cardiomyocytes, 26.2% vascular endothelial cells and 2.6% vascular wall cells, which is designated as cardiac tissue sheet C. The ratios of cells constituting the prepared cardiac tissue sheet C were 9.2% cardiomyocytes (CM), 37.9% vascular endothelial cells (EC) and 1.2% vascular wall cells (MC).

[0217] A heart-on-chip device 100 including a cardiac tissue sheet A or a cardiac tissue sheet C was produced. A voltage of 30 V, a pulse width of 4 ms, and a period of 1000 msec, 900 msec, or 800 msec was applied to the medium in which the heart-on-chip device 100 was immersed, using a cell sheet electrical stimulation unit (Strex), to detect the displacement of the detection target 14. As shown in FIG. 17, when electrical stimulation with periods of 1000 msec, 900 msec, and 800 msec was applied, the heart-on-chip device 100 including the cardiac tissue sheet A had a larger displacement of the detection target 14 than the heart-on-chip device 100 including the cardiac tissue sheet C. The cardiac tissue sheet A had a significantly higher proportion of cardiomyocytes than the cardiac tissue sheet C, the proportion being 20% ​​or more. It was found that the cardiac tissue sheet 20 including 20% ​​or more of cardiomyocytes had improved electrical stimulation responsiveness.

[0218] [Experiment 6: Electrical stimulation response of cardiac tissue sheets with different proportions of cardiomyocytes] A cardiac tissue sheet 20 was prepared in the same manner as in the above [Method of manufacturing cardiac tissue sheet] with a mixture ratio of cardiomyocytes, vascular endothelial cells and vascular wall cells of 46.8% cardiomyocytes, 14.2% vascular endothelial cells and 37.0% vascular wall cells, designated cardiac tissue sheet D. The ratios of cells constituting the prepared cardiac tissue sheet D were 28.3% cardiomyocytes (CM), 10.5% vascular endothelial cells (EC) and 57.9% vascular wall cells (MC).

[0219] A heart-on-chip device 100 including a cardiac tissue sheet D was produced. A cell sheet electrical stimulation unit (Strex) was used to apply a voltage of 30 V, a pulse width of 4 ms, and a period of 1000 msec, 900 msec, 800 msec, 700 msec, or 600 msec to the culture medium in which the heart-on-chip device 100 was immersed, and the displacement of the detection object 14 was detected. As shown in FIG. 18, as the frequency of electrical stimulation increased from the period of 1000 msec, the amount of displacement decreased in correlation. However, the amount of displacement was smaller than that of cardiac tissue sheets A, B, or C. It was found that cardiac tissue sheet D, in which the proportion of vascular wall cells exceeds 40%, had the ability to reflect the contractile force, but the amount of displacement itself decreased.

[0220] <Example 2> [Microfluidic chip manufacturing] The microfluidic chip was manufactured in the same manner as in Example 1.

[0221] [Production of cells that compose cardiac tissue sheets (6)] The production of cells constituting a cardiac tissue sheet (6) is similar to the production of cells constituting a cardiac tissue sheet (2). The production of cells constituting a cardiac tissue sheet (6) was the same as the production of cells constituting a cardiac tissue sheet (1), except that on day 0 of differentiation induction in the production of cells constituting a cardiac tissue sheet (1), CHIR-99021 (TOCRIS, model number 4423, 3 to 5 μM) was further added to the RPMI + B27 medium supplemented with Activin A and cultured. The concentration of VEGF added from day 5 of differentiation induction was 50 ng / mL. The cells were collected 8 to 12 days after the addition of VEGF (13 to 17 days after differentiation induction). GCaMP3-253G1 or FFI01s04 was used as the human iPSC line.

[0222] [Production of cells that compose cardiac tissue sheets (7)] The production of cells constituting cardiac tissue sheets (7) is similar to the production of cells constituting cardiac tissue sheets (4). Until the second day of differentiation induction, cells were cultured in the same manner as in the production of cells constituting cardiac tissue sheets (1). On the third day of differentiation induction, the medium was changed to one containing 10% FBS (BIOWEST, model number BWT-S1760-500) without VEGF. The same medium was used for medium change every other day. Cells were collected 13 to 17 days after differentiation induction. GCaMP3-253G1 or FFI01s04 was used as the human iPSC line.

[0223] [Manufacturing of cardiac tissue sheets] The cardiomyocytes, vascular endothelial cells, and vascular wall cells produced in the above-mentioned production of cells constituting the cardiac tissue sheet (6) and (7) were mixed in an optimal ratio, and a cardiac tissue sheet 20 was produced in the same manner as in Example 1. FACS analysis of the cellular composition of the cardiac tissue sheet revealed that cardiomyocytes (CM) accounted for 37±15%, vascular endothelial cells (EC) for 15±12%, and vascular wall cells (MC) for 20±19% (FIG. 19).

[0224] The cross section of the obtained cardiac tissue sheet 20 was stained with hematoxylin-eosin (HE), Sirius red (SR) and immunostained with cardiac troponin T (cTnT) (FIG. 20). HE staining was performed using Musashi Chemical Co., Ltd., product numbers: 30151 and 32042, SR staining was performed using Polysciences, product number: 09400, and cardiac troponin T staining was performed using anti-cardiac isoform of troponin T antibody (clone 13-11), Thermo Fisher Scientific, product number: MS-295-P0. The results of the tissue staining showed that the cardiac tissue sheet 20 was composed of about 15 cell layers. Furthermore, it was found that the myocardial cell layer accounted for more than half of the total cell layers, and that the sheet had a structure in which extracellular matrix proteins were held in layers inside.

[0225] The results of fluorescent immunostaining of the peripheral portion of the cardiac tissue sheet 20 with cardiac troponin T, calponin, and CD31 are shown in Figures 21 and 22. Calponin is expressed in vascular wall cells, and CD31 is expressed in vascular endothelial cells. The anti-cardiac troponin T antibody was manufactured by Thermo Fisher Scientific, product number MS-295-P0, diluted at a concentration of 1:500, the anti-calponin antibody was manufactured by Abcam, product number ab46794, diluted at a concentration of 1:500, and the anti-CD31 antibody was manufactured by R&D, product number BBA7, diluted at a concentration of 1:50. The secondary antibodies used were manufactured by Thermo Fisher Scientific, product numbers A-11008 and A-11003, diluted at a concentration of 1:500. It was confirmed that a cardiac tissue sheet 20 containing cardiac myocytes, vascular endothelial cells, and vascular wall cells was produced.

[0226] [Fabrication of Heart-on-a-Chip Device] The cardiac tissue sheet 20 was attached to the microfluidic chip 10 in the same manner as in Example 1 to obtain a heart-on-chip device 100. As shown in FIG. 23, the cardiac tissue sheet 20 was placed so as to cover the oscillating portion 11 of the microfluidic chip 10. A schematic diagram of the heart-on-chip device 100 viewed from the top is shown in FIG. 24. The communication port 121 is an inlet and an outlet of the fine channel 123. After the cardiac tissue sheet 20 was attached to the microfluidic chip 10, the heart-on-chip device 100 was immersed in a culture medium, and fluorescent microspheres as the analyte 14 were added to the channel 12 using a syringe.

[0227] A 488 nm laser beam was irradiated, and the microchannel 123 was observed using a microscope (CKX53, Olympus) (FIG. 25). The right side of FIG. 25 shows an enlarged view of the area enclosed by a square in the left side of FIG. 25. The detectable object 14 enclosed by a circle in the right side of FIG. 25 moved in the X-axis direction at end-diastole (top) and end-systole (bottom) during the pulsation of the cardiac tissue sheet 20. The displacement of the detectable object 14 is shown in FIG. 26. It was observed that the detectable object 14 repeatedly moved between end-diastole and end-systole. It was found that the heart-on-a-chip device 100 reflected the pulsation of the heart, and cardiac function could be detected and evaluated as the movement of the detectable object 14.

[0228] [Experiment 7: Electrical stimulation response of cardiac tissue sheets] The electrical stimulation responsiveness of the cardiac tissue sheet 20 was detected by the heart-on-chip device 100. A cell sheet electrical stimulation unit (Strex) was used to apply a voltage of 30 V with a pulse width of 4 ms to the culture medium in which the heart-on-chip device 100 was immersed, and the displacement of the detection target was detected. As shown in FIG. 27, the displacement of the detection target 14 was detected in synchronization with the electrical stimulation with a period of 1000 msec to 600 msec. With the electrical stimulation with a period of 500 msec, the cardiac tissue sheet pulsated once with two electrical stimulations, and a pulsation equivalent to an interval stimulation of 1000 msec was detected. The relationship between the frequency of electrical stimulation and the amount of displacement of the detection target 14 is shown in FIG. 28. As the frequency of electrical stimulation increased, the amount of displacement of the detection target 14 tended to decrease.

[0229] The electrical stimulation responsiveness of cardiac tissue sheets 20 produced from human iPSC lines GCaMP3-253G1 and FFI01s04 was detected by the heart-on-chip device 100. As shown in Figures 29 and 30, cardiac tissue sheets produced from both cell lines exhibited electrical stimulation responsiveness, which could be detected as a change in the position of the detection target 14. As the frequency of electrical stimulation increased, the amount of displacement of the detection target 14 tended to decrease.

[0230] For each human iPSC line, the relationship between the frequency of electrical stimulation and the amount of displacement of the detectable object 14, as well as the relationship between the frequency of electrical stimulation and the migration speed of the detectable object 14, are shown in Figures 31 and 32. In cardiac tissue sheets produced from both cell lines, the migration speed of the detectable object 14 tended to decrease as the frequency of electrical stimulation increased. In both human iPSC lines, the displacement and migration speed of the detectable object 14 decreased during both the systole and diastole of the cardiac tissue sheet 20 (Figures 33 and 34).

[0231] [Experiment 8: Cardiac agonist response of cardiac tissue sheets] The responsiveness of the cardiac tissue sheet 20 when isoproterenol was added was detected by the heart-on-chip device 100. In the same manner as in Experiment 2 of Example 1, the heart-on-chip device of Example 2 was immersed in a medium containing isoproterenol, and the movement of the detectable object 14 was detected. As shown in FIG. 35, when isoproterenol was added, the movement frequency of the detectable object 14 increased in a concentration-dependent manner. As shown in FIG. 36, the addition of isoproterenol also reduced the amount of displacement of the detectable object 14. The relationship between the movement frequency of the detectable object 14 and the amount of displacement of the detectable object 14 due to the addition of isoproterenol is shown in FIG. 37. As the movement frequency of the detectable object 14 increased, the amount of displacement of the detectable object 14 decreased. When Experiment 3 was performed using multiple heart-on-chip devices, in all cases, as the movement frequency of the detectable object 14 increased, the amount of displacement of the detectable object 14 tended to decrease (FIG. 38).

[0232] [Experiment 9: Method for calculating physiological parameters] In the heart-on-a-chip device of Example 2, when the radius of the chamber is r, the width of the microchannel 123 is w, the depth of the microchannel 123 is d, and the displacement of the object to be detected is x, the physiological parameters are expressed as follows: Stroke amount ΔV=0.94wdx Displacement of the diaphragm ΔV=(1 / 3)πr 2 z Vertical pressure on the membrane P=(C1σt / r 2 )z+(C2Et / r 4 )z 3 Force on the membrane F=πr 2 P For example, when x = 10 μm, P is calculated to be 6.8 Pa, F is calculated to be 0.048 mN, and the systolic volume ΔV is calculated to be 0.38 nL (= 0.94 wdx).

[0233] <Example 3> [Microfluidic chip manufacturing] The microfluidic chip was manufactured in the same manner as in Example 1.

[0234] [Production of cells that compose cardiac tissue sheets (8)] Preparation of cells constituting cardiac tissue sheets Cells were prepared in the same manner as in (6) and (7).

[0235] [Production of cardiac tissue sheets (1)] A cardiac tissue sheet was prepared using cardiomyocytes, vascular endothelial cells, and vascular wall cells in the same manner as in Example 1. The prepared cardiac tissue sheet 20 was attached to a scaffold sheet 19, and the cardiac tissue sheet 20 was further cultured on the scaffold sheet 19. The medium was αMEM (invirogen) with 10% FBS, 5×10 -5 A medium containing M2-ME and 10 μM Y-27632 was used. After one day of culture, the medium was replaced with the above-mentioned medium that did not contain Y-27632. Thereafter, the medium was replaced with the same medium every other day. At the same time, shaking culture was performed for 5 days in the same manner as in Example 1, starting from two days after culture.

[0236] The scaffold sheet 19 had orientation, was made of thermoplastic polyester elastomer, was multi-layered (4 layers), and had the orientation axes of the upper and lower sheets crossed at 15°, had a porosity of about 27%, a material modulus of elasticity of 20 MPa, a thickness of about 50 μm, and an average diameter of the fibers constituting the sheet of 2.6 μm. The scaffold sheet 19 had a circular hollow frame 191 made of SUS304 stainless steel. The frame 191 and the scaffold sheet 19 were bonded with a silicone adhesive (Shin-Etsu Chemical, catalog number KE-45). The circular hollow frame 191 had an inner diameter of 5 mm x outer diameter of 11.5 mm x height of 1 mm, an inner diameter of 6 mm x outer diameter of 13 mm x height of 1 mm, or an inner diameter of 8 mm x outer diameter of 13 mm x height of 1 mm.

[0237] [Production of cardiac tissue sheets (2)] Cardiac tissue sheets 20 were prepared by seeding cardiomyocytes, vascular endothelial cells, and vascular wall cells onto a scaffold sheet 19. 3.6 million cells / 200 μL of culture medium were seeded on each scaffold sheet 19 and cultured at 37° C. The culture medium was αMEM (invirogen) with 10% FBS and 5×10 -5A medium containing M2-ME and 10 μM Y-27632 was used. After one day of culture, the medium was replaced with the same medium as above except that it did not contain Y-27632. Thereafter, the medium was replaced with the same medium every day. After one day of culture, shaking culture was performed for 10 days in the same manner as in Example 1, and cardiac tissue sheet 20 formed in the shape of scaffold sheet 19 was obtained. The scaffold sheet 19 used was the one described above.

[0238] [Fabrication of Heart-on-a-Chip Device] A cardiac tissue sheet 20 formed on a scaffold sheet 19 produced by the method of producing a cardiac tissue sheet (1) was placed on a microfluidic chip 10 to obtain a heart-on-a-chip device 100. The object to be detected 14 used was the same as in Example 1.

[0239] [Experiment 10: Self-pulsation and electrical stimulation response of cardiac tissue sheets] The self-pulsation and electrical stimulation responsiveness of the cardiac tissue sheet 20 were measured under the same measurement conditions as in Experiment 1. The results are shown in Figures 41 and 42, respectively. As shown in Figure 41, even when the heart-on-chip 100 with the scaffold sheet 19 was used, the self-pulsation pattern of the cardiac tissue sheet was detected as the displacement of the detection target 14, just as when the scaffold sheet 19 was not used. As shown in Figure 42, even when the heart-on-chip 100 with the scaffold sheet 19 was used, the electrical stimulation responsiveness of the cardiac tissue sheet 20 was detected as the displacement of the detection target 14, and the change in the amount of displacement due to electrical stimulation was detected as a pattern similar to when the scaffold sheet 19 was not used. As in the case when the scaffold sheet 19 was not used, the amount of displacement of the detection target 14 tended to decrease as the frequency of movement of the detection target 14 increased (Figure 43). [Explanation of symbols]

[0240] 100 heart-on-a-chip device, 10 microfluidic chip, 20 cardiac tissue sheet, 11 oscillating portion, 12 flow path, 121 communication port, 122 chamber, 123 micro flow path, 13 diaphragm, 14 object to be detected, 15 detector, 16a, 16b check valve, 17 support portion, 18 substrate, 19 scaffold sheet, 191 frame, 192 scaffold sheet on which cardiac tissue sheet is formed, 193 fixing portion, 194 protrusion.

Claims

1. A microfluidic chip and a cardiac tissue sheet are provided, the microfluidic chip includes a swinging part capable of swinging in a certain direction, a flow path through which a liquid flows by the swinging of the swinging part, a diaphragm provided between the swinging part and the flow path, and a detectable substance dispersed in the liquid; The cardiac tissue sheet comprises cardiomyocytes; A heart-on-a-chip device, wherein the cardiac tissue sheet is in contact with the oscillating portion so as to be able to oscillate the oscillating portion.

2. The heart-on-a-chip device according to claim 1 , wherein the analyte is a fluorescent bead.

3. The heart-on-a-chip device according to claim 1 or 2, wherein the portion of the microfluidic chip that comes into contact with the cardiac tissue sheet has cell adhesive properties.

4. The cardiac tissue sheet further comprises vascular endothelial cells and vascular wall cells, The heart-on-a-chip device according to any one of claims 1 to 3, wherein the cardiac tissue sheet comprises cardiomyocytes at 5% or more and 70% or less, vascular endothelial cells at more than 0% and 60% or less, and vascular wall cells at 1% or more and 60% or less.

5. The heart-on-a-chip device according to any one of claims 1 to 4, wherein the cardiac tissue sheet comprises cells derived from induced pluripotent stem cells.

6. The heart-on-a-chip device according to any one of claims 1 to 5, wherein the cardiac tissue sheet comprises cells in which a disease-related gene is deleted or mutated.

7. The heart-on-a-chip device according to any one of claims 1 to 6, wherein the cardiac tissue sheet is a cardiac tissue sheet cultured with shaking.

8. The heart-on-a-chip device of any one of claims 1 to 7, further comprising a scaffold sheet of said cardiac tissue sheet.

9. A method for evaluating cardiac function using a heart-on-chip device described in any one of claims 1 to 8.

10. A drug screening method using a heart-on-chip device described in any one of claims 1 to 8.

11. A method for evaluating cardiotoxicity using a heart-on-a-chip device described in any one of claims 1 to 8.

Images