Culture cell sheet, method for producing same, method for evaluating compound or drug, and method for evaluating quality of culture cell sheet

Abstract

The present invention addresses the problem of providing: a mature cultured cell sheet comprising cardiac muscle cells without using a specific drug; and a method for producing the mature cultured cell sheet. Also provided are a method for evaluating a compound or a drug using the cultured cell sheet, and a method for evaluating the quality of a cultured cell sheet comprising myocardial cells. The cultured cell sheet according to the present invention is a cultured cell sheet comprising myocardial cells, the myocardial cells being arranged so as to have orientation, and the cultured cell sheet being obtained by immunostaining the cultured cell sheet with an anti-alpha-actin antibody and observing the same with a microscope, in an image obtained by subjecting the cultured cell sheet to immunostaining with the anti-alpha-actin antibody. When the angle in the longitudinal direction of the rod-like structure, which is detected by an alpha-actin antibody, is measured within the measurement range (71.6 [mu] m * 71.6 [mu] m) when the image horizontal direction is set as 0 degree, and the degree of orientation, which is obtained by formula (1), is 23% or more, said degree of orientation being the frequency of the rod-like structure contained within the mode + / -15 degrees. Degree of orientation (%) = (number of rod-shaped structures included within mode + / -15 degrees) / (total number of rod-shaped structures) * 100 (1)

Description

Technical Field

[0001] The present invention relates to a cultured cell sheet composed of cardiomyocytes and a method for producing the same, a method for evaluating a compound or a drug using the cultured cell sheet, and a method for evaluating the quality of the cultured cell sheet. Background Art

[0002] From the perspective of reducing the cost and time of drug development and replacing animal experiments, it is expected that cells differentiated from stem cells such as human iPS cells will be used in the development of innovative drugs. The advantage is that experiments using these human cells can be conducted from the initial stage of development, and the differences in drug responsiveness caused by species differences can be eliminated by replacing the experiments previously conducted in animal experiments with experiments based on stem cell-derived cells such as human iPS cells. In particular, cardiomyocytes derived from stem cells such as human iPS cells are required to be used in the development of innovative drugs, which are difficult to obtain from cells derived from living organisms.

[0003] However, currently available cardiomyocytes derived from stem cells such as human iPS cells are immature, and their physiological activity and motor function are close to those of fetal myocardium. Therefore, there are reports that the responsiveness to drugs is different from that of the organism, and a method for maturing cardiomyocytes derived from stem cells such as human iPS cells is needed.

[0004] For example, Patent Document 1 provides a culture medium composition of known composition that can promote the maturation of fetal-like (immature) cardiomyocytes derived from pluripotent embryonic stem cells in in vitro culture, and discloses a serum-free aqueous culture medium composition containing a thyroid hormone-like compound, a lipid mixture, and a carnitine compound in order to provide a method for producing adult-like cardiomyocytes by in vitro culture that is more efficient and helps to produce adult-like cardiomyocytes that meet general requirements for cardiac research and clinical use on a large scale, thereby eliminating the main limitations in the art.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent Application Publication No. 2020-022460 Summary of the invention

[0008] Problem that the invention aims to solve

[0009] Patent Document 1 uses a specific culture medium to mature cardiomyocytes into adult-like cardiomyocytes, but the possibility that components contained in the culture medium may have effects on cardiomyocytes other than maturation cannot be ruled out.

[0010] The object of the present invention is to provide a mature cultured cell sheet composed of cardiomyocytes without using a specific drug and a method for producing the same. In addition, the object of the present invention is to provide a method for evaluating a compound or drug (protein, peptide, antibody, DNA, RNA, etc.) using the cultured cell sheet. Furthermore, the object of the present invention is to provide a method for evaluating the quality of a cultured cell sheet composed of cardiomyocytes.

[0011] Solutions for solving problems

[0012] The above-mentioned problems of the present invention can be solved by the following configurations <1> to <19>.

[0013] <1> A cultured cell sheet comprising cardiomyocytes, wherein the cardiomyocytes are arranged in an orientation, wherein in an image obtained by immunostaining the cultured cell sheet with an anti-α-actinin antibody and observing the image with a microscope, the angle of the length direction of rod-like structures detected by the α-actinin antibody within a measurement range (71.6 μm×71.6 μm) when the horizontal direction of the image is set to 0° is measured, and the frequency of the rod-like structures within ±15° of the mode value, that is, the orientation degree obtained according to the following formula (1), is 23% or more.

[0014] Orientation degree (%) = (number of rod-like structures within ±15° of the mode value) / (total number of rod-like structures) × 100 (1)

[0015] <2> The cultured cell sheet according to <1>, wherein the number of vectors showing an angle of ±5° from a mode value of a motion vector (degree of orientation) in live cell imaging is 12% or more.

[0016] <3> The cultured cell sheet according to <1> or <2>, wherein the cardiomyocytes are stem cell-derived cardiomyocytes.

[0017] <4> The cultured cell sheet according to any one of <1> to <3>, which satisfies at least one of the following requirements A1 and A2.

[0018] Requirement A1: The ratio of the expression level of the MYL3 (myosin light chain 3) gene to the expression level of the ACTB (β-actin) gene (MYL3 / ACTB) is 12.0 or more.

[0019] Requirement A2: The ratio of the expression level of the MYH7 (myosin heavy chain 7) gene to the expression level of the MYH6 (myosin heavy chain 6) gene (MYH7 / MYH6) is 6.0 or more.

[0020] <5> The cultured cell sheet according to any one of <1> to <4>, which satisfies at least one of the following requirements B1 and B2.

[0021] Requirement B1: The ratio of the gene expression level of CKM (creatine kinase, M type) to the gene expression level of ACTB (β-actin) (CKM / ACTB) is 1.80 or more.

[0022] Requirement B2: The ratio of the gene expression level of LDHA (lactate dehydrogenase A subunit) to the gene expression level of ACTB (LDHA / ACTB) is 0.80 or more.

[0023] <6> The cultured cell sheet according to any one of <1> to <5>, which satisfies at least any one of the following requirements C1 to C4.

[0024] Requirement C1: The ratio of the gene expression level of CACNA2D1 (voltage-gated calcium channel auxiliary subunit α2δ1) to the gene expression level of ATP1A1 (ATPase Na+ / K+ transport subunit α1) (CACNA2D1 / ATP1A1) is 0.085 or more on any day from the 15th day to the 30th day of culture.

[0025] Requirement C2: The ratio of the gene expression level of KCNJ2 (inwardly rectifier potassium channel subfamily J member 2) to the gene expression level of ATP1A1 (ATPase Na+ / K+ transport subunit α1) (KCNJ2 / ATP1A1) is 0.038 or more on any day from the 15th day to the 45th day of culture.

[0026] Requirement C3: The ratio of the gene expression level of KCNE1 (voltage-gated potassium channel subfamily E regulatory subunit 1) to the gene expression level of ATP1A1 (ATPase Na+ / K+ transport subunit α1) (KCNE1 / ATP1A1) is 0.003 or more on any day from the 15th day to the 45th day of culture.

[0027] Requirement C4: The ratio of the gene expression level of SCN5A (voltage-gated sodium channel α subunit 5) to the gene expression level of ATP1A1 (ATPase Na+ / K+ transport subunit α1) (SCN5A / ATP1A1) is 0.87 or more on any day from the 15th day to the 45th day of culture.

[0028] <7> The cultured cell sheet according to any one of <1> to <6>, which satisfies at least any one of the following requirements D1 to D4.

[0029] Requirement D1: The ratio of the gene expression level of LPL (lipoprotein lipase) to the gene expression level of ACTB (β-actin) (LPL / ACTB) is 0.45 or more on any day from the 15th day to the 45th day of culture.

[0030] Requirement D2: The ratio of the gene expression level of ACAT1 (acetyl-CoA acetyltransferase 1) to the gene expression level of ACTB (β-actin) (ACAT1 / ACTB) is 0.43 or more on any day from the 15th day to the 30th day of culture.

[0031] Requirement D3: The ratio of the gene expression level of HADHA (hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit α) to the gene expression level of ACTB (β-actin) (HADHA / ACTB) is 0.75 or more on any day from the 15th day to the 30th day of culture.

[0032] Requirement D4: The ratio of the gene expression level of HADHB (hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit β) to the gene expression level of ACTB (β-actin) (HADHB / ACTB) is 0.95 or more on any day from the 15th day to the 30th day of culture.

[0033] <8> The cultured cell sheet according to any one of <1> to <7>, which satisfies at least any one of the following requirements E1 to E8.

[0034] Requirement E1: The ratio of the gene expression level of PPARGC1A (PPARG (peroxisome proliferator-activated receptor γ) coactivator 1α) to the gene expression level of HIF1A (hypoxia-inducible factor 1α) (PPARGC1A / HIF1A) is 0.80 or more on any day of culture.

[0035] Requirement E2: The ratio of the gene expression level of ESRRA (estrogen related receptor α) to the gene expression level of HIF1A (hypoxia inducible factor 1α) (ESRRA / HIF1A) is 0.55 or more on any day of culture.

[0036] Requirement E3: The ratio of the gene expression level of VEGFA (vascular endothelial growth factor A) to the gene expression level of HIF1A (hypoxia inducible factor 1α) (VEGFA / HIF1A) is 0.65 or more on any day of culture.

[0037] Requirement E4: The ratio of the gene expression level of APLN (Apelin) to the gene expression level of HIF1A (hypoxia-inducible factor 1α) (APLN / HIF1A) is 0.005 or more on any day of culture.

[0038] Requirement E5: The ratio of the gene expression level of FABP3 (fatty acid binding protein 3) to the gene expression level of HIF1A (hypoxia inducible factor 1α) (FABP3 / HIF1A) is 5.0 or more on any day of culture.

[0039] Requirement E6: The ratio of the gene expression level of ESM1 (endothelial cell-specific molecule 1) to the gene expression level of HIF1A (hypoxia-inducible factor 1α) (ESM1 / HIF1A) is 0.002 or more on any day of culture.

[0040] Requirement E7: The ratio of the gene expression level of EMCN (endothelial mucin) to the gene expression level of HIF1A (hypoxia-inducible factor 1α) (EMCN / HIF1A) is 0.002 or more on any day after the 7th day of culture.

[0041] Requirement E8: The ratio of the gene expression level of BCL2 (BCL2 apoptosis regulator) to the gene expression level of HIF1A (hypoxia-inducible factor 1α) (BCL2 / HIF1A) is 0.013 or more on any day after the 7th day of culture.

[0042] <9> The cultured cell sheet according to any one of <1> to <8>, wherein the area of ​​mitochondria per cell is 200 μm 2 above.

[0043] <10> The cultured cell sheet according to any one of <1> to <9>, wherein the following formula (2) is satisfied when the contraction velocity detected in live cell imaging is CV (m / sec) and the relaxation velocity is RV (m / sec).

[0044] 1.0≤CV / RV≤2.8 (2)

[0045] <11> The cultured cell sheet according to any one of <1> to <10>, wherein in calcium imaging analysis, when the peak height of the waveform of the calcium transient is set to 100%, the waveform width of 20% height is set to Duration (seconds), and the interval between peaks is set to Interval (seconds), the following formula (3) is satisfied.

[0046] Duration / (Interval) 1 / 2 ≥0.68 (3)

[0047] <12> The cultured cell sheet according to any one of <1> to <11>, wherein the cells constituting the cultured cell sheet have an action potential 80% repolarization duration of 600 msec or longer and a maximum diastolic potential of -60 mV or less in a patch clamp test in a 25°C solution.

[0048] <13> A cultured cell sheet according to any one of <1> to <12>, wherein, in an oxygen consumption rate measurement for measuring the rate at which cells use oxygen contained in a culture medium, the oxygen consumption rate is reduced to less than 20% compared to before the addition of the β-oxidation inhibitor by adding a β-oxidation inhibitor that inhibits the β-oxidation activity of fatty acids.

[0049] <14> The cultured cell sheet according to any one of <1> to <13>, which is used for regenerative medicine.

[0050] <15> A method for manufacturing a cultured cell sheet, which is the method for manufacturing a cultured cell sheet described in any one of <1> to <14>, the method comprising the step of culturing cardiomyocytes using a cell sheet forming member, the cell sheet forming member having a surface for forming a cultured cell sheet, the surface having a plurality of flat portions and a plurality of concave-convex portions, each flat portion having a shape extending along a first direction, and the plurality of flat portions being arranged along a second direction intersecting the first direction on the entire surface, each concave-convex portion comprising a plurality of height difference structures filling between the adjacent flat portions, the pitch of the height difference structures being greater than or equal to 100 nm and less than or equal to 10 μm, the height difference structures being convex portions, the concave-convex portions having a plurality of convex portions on the bottom surface of a concave portion clamped by the adjacent flat portions, and in the thickness direction of the cell sheet forming member, the difference between the height of the front end surface in the concave-convex portion and the height of the flat portion is less than or equal to 0.5 μm.

[0051] <16> A method for evaluating a compound or a drug, comprising allowing the compound or drug to act on the cultured cell sheet according to any one of <1> to <14>.

[0052] <17> The method for evaluating a compound or drug according to <16>, wherein at least one change selected from the group consisting of a change in a physiological property and a change in a motor function of a cultured cell sheet is evaluated.

[0053] <18> A method for evaluating a compound or drug according to <16> or <17>, wherein the compound or drug to be evaluated is at least one existing compound or a candidate compound thereof selected from the group consisting of INa blockers, Ikr blockers, Iks blockers, ICa blockers, 5-HT4 receptor agonists, α receptor blockers, β receptor blockers, α receptor agonists and β receptor agonists, toxic substances, anticancer agents, lipid metabolism inhibitors and other physiologically active substances.

[0054] <19> A quality evaluation method for a cultured cell sheet composed of cardiomyocytes, the quality evaluation method evaluating any one of the following (i) to (xii).

[0055] (i) In an image obtained by immunostaining the aforementioned cultured cell sheet with an anti-α-actinin antibody and observing it under a microscope, the angle of the length direction of the rod-like structures detected by the α-actinin antibody within the measurement range (71.6 μm×71.6 μm) when the horizontal direction of the image is set to 0° is measured, and the frequency of the rod-like structures contained within ±15° of the mode value, that is, the orientation degree calculated according to the following formula (1), is 23% or more.

[0056] Orientation degree (%) = (number of rod-like structures within ±15° of the mode value) / (total number of rod-like structures) × 100 (1)

[0057] (ii) In live cell imaging, the number of vectors showing an angle of ±5° from the angle of the mode value of the motion vector (degree of orientation) is 12% or more.

[0058] (iii) Satisfy at least one of the following requirements A1 and A2.

[0059] Requirement A1: The ratio of the expression level of the MYL3 (myosin light chain 3) gene to the expression level of the ACTB (β-actin) gene (MYL3 / ACTB) is 12.0 or more.

[0060] Requirement A2: The ratio of the expression level of the MYH7 (myosin heavy chain 7) gene to the expression level of the MYH6 (myosin heavy chain 6) gene (MYH7 / MYH6) is 6.0 or more.

[0061] (iv) Satisfy at least one of the following requirements B1 and B2.

[0062] Requirement B1: The ratio of the gene expression level of CKM (creatine kinase, M type) to the gene expression level of ACTB (β-actin) (CKM / ACTB) is 1.80 or more.

[0063] Requirement B2: The ratio of the gene expression level of LDHA (lactate dehydrogenase A subunit) to the gene expression level of ACTB (LDHA / ACTB) is 0.80 or more.

[0064] (v) Satisfy at least any one of the following requirements C1 to C4.

[0065] Requirement C1: The ratio of the gene expression level of CACNA2D1 (voltage-gated calcium channel auxiliary subunit α2δ1) to the gene expression level of ATP1A1 (ATPase Na+ / K+ transport subunit α1) (CACNA2D1 / ATP1A1) is 0.085 or more on any day from the 15th day to the 30th day of culture.

[0066] Requirement C2: The ratio of the gene expression level of KCNJ2 (inwardly rectifier potassium channel subfamily J member 2) to the gene expression level of ATP1A1 (ATPase Na+ / K+ transport subunit α1) (KCNJ2 / ATP1A1) is 0.038 or more on any day from the 15th day to the 45th day of culture.

[0067] Requirement C3: The ratio of the gene expression level of KCNE1 (voltage-gated potassium channel subfamily E regulatory subunit 1) to the gene expression level of ATP1A1 (ATPase Na+ / K+ transport subunit α1) (KCNE1 / ATP1A1) is 0.003 or more on any day from the 15th day to the 45th day of culture.

[0068] Requirement C4: The ratio of the gene expression level of SCN5A (voltage-gated sodium channel α subunit 5) to the gene expression level of ATP1A1 (ATPase Na+ / K+ transport subunit α1) (SCN5A / ATP1A1) is 0.87 or more on any day from the 15th day to the 45th day of culture.

[0069] (vi) Satisfy at least any one of the following requirements D1 to D4.

[0070] Requirement D1: The ratio of the gene expression level of LPL (lipoprotein lipase) to the gene expression level of ACTB (β-actin) (LPL / ACTB) is 0.45 or more on any day from the 15th day to the 45th day of culture.

[0071] Requirement D2: The ratio of the gene expression level of ACAT1 (acetyl-CoA acetyltransferase 1) to the gene expression level of ACTB (β-actin) (ACAT1 / ACTB) is 0.43 or more on any day from the 15th day to the 30th day of culture.

[0072] Requirement D3: The ratio of the gene expression level of HADHA (hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit α) to the gene expression level of ACTB (β-actin) (HADHA / ACTB) is 0.75 or more on any day from the 15th day to the 30th day of culture.

[0073] Requirement D4: The ratio of the gene expression level of HADHB (hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit β) to the gene expression level of ACTB (β-actin) (HADHB / ACTB) is 0.95 or more on any day from the 15th day to the 30th day of culture.

[0074] (vii) Satisfy at least any one of the following requirements E1 to E8.

[0075] Requirement E1: The ratio of the gene expression level of PPARGC1A (PPARG (peroxisome proliferator-activated receptor γ) coactivator 1α) to the gene expression level of HIF1A (hypoxia-inducible factor 1α) (PPARGC1A / HIF1A) is 0.80 or more on any day of culture.

[0076] Requirement E2: The ratio of the gene expression level of ESRRA (estrogen related receptor α) to the gene expression level of HIF1A (hypoxia inducible factor 1α) (ESRRA / HIF1A) is 0.55 or more on any day of culture.

[0077] Requirement E3: The ratio of the gene expression level of VEGFA (vascular endothelial growth factor A) to the gene expression level of HIF1A (hypoxia-inducible factor 1α) (VEGFA / HIF1A) is 0.65 or more on any day of culture.

[0078] Requirement E4: The ratio of the gene expression level of APLN (Apelin) to the gene expression level of HIF1A (hypoxia-inducible factor 1α) (APLN / HIF1A) is 0.005 or more on any day of culture.

[0079] Requirement E5: The ratio of the gene expression level of FABP3 (fatty acid binding protein 3) to the gene expression level of HIF1A (hypoxia inducible factor 1α) (FABP3 / HIF1A) is 5.0 or more on any day of culture.

[0080] Requirement E6: The ratio of the gene expression level of ESM1 (endothelial cell-specific molecule 1) to the gene expression level of HIF1A (hypoxia-inducible factor 1α) (ESM1 / HIF1A) is 0.002 or more on any day of culture.

[0081] Requirement E7: The ratio of the gene expression level of EMCN (endothelial mucin) to the gene expression level of HIF1A (hypoxia-inducible factor 1α) (EMCN / HIF1A) is 0.002 or more on any day after the seventh day of culture.

[0082] Requirement E8: The ratio of the gene expression level of BCL2 (BCL2 apoptosis regulator) to the gene expression level of HIF1A (hypoxia-inducible factor 1α) (BCL2 / HIF1A) is 0.013 or more on any day after the 7th day of culture.

[0083] (viii) The area of ​​mitochondria in each cell is 200 μm 2 above.

[0084] (ix) When the contraction velocity detected in live cell imaging is denoted as CV (m / sec) and the relaxation velocity is denoted as RV (m / sec), the following formula (2) is satisfied.

[0085] 1.0≤CV / RV≤2.8 (2)

[0086] (x) In calcium imaging analysis, when the peak height of the calcium transient waveform is 100%, the waveform width of 20% height is Duration (seconds), and the interval between peaks is Interval (seconds), the following formula (3) is satisfied.

[0087] Duration / (Interval) 1 / 2 ≥0.68 (3)

[0088] (xi) The duration of 80% repolarization of the action potential of the cells constituting the cultured cell sheet in a patch clamp test in a 25°C solution is 600 msec or longer, and the maximum diastolic potential is -60 mV or less.

[0089] (xii) In an oxygen consumption rate measurement that measures the rate at which cells use oxygen contained in a culture medium, the oxygen consumption rate is reduced to 20% or less compared to before the addition of the β-oxidation inhibitor by adding a β-oxidation inhibitor that inhibits the β-oxidation activity of fatty acids.

[0090] Effects of the Invention

[0091] According to the present invention, a mature cultured cell sheet composed of cardiomyocytes without using a specific drug and a method for producing the same can be provided. In addition, according to the present invention, a method for evaluating a compound or drug using the cultured cell sheet can be provided. Furthermore, according to the present invention, a method for evaluating the quality of a cultured cell sheet composed of cardiomyocytes can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 The figures show the structure of a cell sheet forming member in one embodiment of the member, (a) is a stereoscopic view showing the structure of the cell sheet forming member together with a shallow base plate, (b) is a stereoscopic view showing a part of the surface of the cell sheet forming member in an enlarged manner, (c) is a top view showing a part of the surface of the cell sheet forming member in an enlarged manner, and (d) is a partial cross-sectional view showing a part of the cell sheet forming member in an enlarged manner.

[0093] Figure 2 This is a process diagram for explaining an example of a method for producing a cell sheet forming member.

[0094] Figure 3 (a) to (c) are schematic diagrams for explaining the process of manufacturing a cell sheet.

[0095] Figure 4 It is a graph showing the orientation degree on the 15th day, the 30th day, the 45th day, and the 60th day of culture.

[0096] Figure 5 It is a graph showing the time-dependent changes in the ratio (MYL3 / ACTB) of the expression level of the MYL3 (myosin light chain 3) gene to the expression level of the ACTB (β-actin) gene.

[0097] Figure 6 This is a graph showing the temporal changes in the ratio of the expression level of the MYH7 (myosin heavy chain 7) gene to the expression level of the MYH6 (myosin heavy chain 6) gene (MYH7 / MYH6).

[0098] Figure 7-1 The graph shows the temporal changes in the expression levels of the CKM (creatine kinase, M type) gene, the COX6A2 (cytochrome C oxidase subunit 6A2) gene, the CKMT2 (mitochondrial creatine kinase 2) gene, and the LDHA (lactate dehydrogenase A subunit) gene.

[0099] Figure 7-2 It is a graph showing the temporal changes in the ratio of the expression level of the CKM gene to the expression level of the ACTB (β-actin) gene (CKM / ACTB), the ratio of the expression level of the COX6A2 gene to the expression level of the ACTB gene (COX6A2 / ACTB), the ratio of the expression level of the CKMT2 gene to the expression level of the ACTB gene (CKMT2 / ACTB), and the ratio of the expression level of the LDHA gene to the expression level of the ACTB gene (LDHA / ACTB).

[0100] Figure 7-3 The graph shows the temporal changes in the expression levels of CACNA2D1 (voltage-gated calcium channel auxiliary subunit α2δ1) gene, KCNJ2 (inwardly rectifier potassium channel subfamily J member 2) gene, KCNE1 (voltage-gated potassium channel subfamily E regulatory subunit 1) gene, and SCN5A (voltage-gated sodium channel α subunit 5) gene.

[0101] Figure 7-4 It is a graph showing the temporal changes in the ratio of the expression level of the CACNA2D1 gene to the expression level of the ATP1A1 (ATPase Na+ / K+ transport subunit α1) gene (CACNA2D1 / ATP1A1), the ratio of the expression level of the KCNJ2 gene to the expression level of the ATP1A1 gene (KCNJ2 / ATP1A1), the ratio of the expression level of the KCNE1 gene to the expression level of the ATP1A1 gene (KCNE1 / ATP1A1), and the ratio of the expression level of the SCN5A gene to the expression level of the ATP1A1 gene (SCN5A / ATP1A1).

[0102] Figure 7-5The graph shows the temporal changes in the expression levels of the LPL (lipoprotein lipase) gene, the ACAT1 (acetyl-CoA acetyltransferase 1) gene, the HADHA (hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit α) gene, and the HADHB (hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit β) gene.

[0103] Figure 7-6 It is a graph showing the temporal changes in the ratio of the expression level of the LPL gene to the expression level of the ACTB (β-actin) gene (LPL / ACTB), the ratio of the expression level of the ACAT1 gene to the expression level of the ACTB gene (ACAT1 / ACTB), the ratio of the expression level of the HADHA gene to the expression level of the ACTB gene (HADHA / ACTB), and the ratio of the expression level of the HADHB gene to the expression level of the ACTB gene (HADHB / ACTB).

[0104] Figure 7-7 The graph shows the time-dependent changes in the expression levels of PPARGC1A (PPARG (peroxisome proliferator-activated receptor γ) coactivator 1α) gene, ESRRA (estrogen-related receptor α) gene, VEGFA (vascular endothelial growth factor A) gene, and APLN (Apelin) gene.

[0105] Figure 7-8 It is a graph showing the temporal changes in the ratio of the expression level of the PPARGC1A gene to the expression level of the HIF1A (hypoxia-inducible factor 1α) gene (PPARGC1A / HIF1A), the ratio of the expression level of the ESRRA gene to the expression level of the HIF1A gene (ESRRA / HIF1A), the ratio of the expression level of the VEGFA gene to the expression level of the HIF1A gene (VEGFA / HIF1A), and the ratio of the expression level of the APLN gene to the expression level of the HIF1A gene (APLN / HIF1A).

[0106] Figure 7-9 The graph shows the time-dependent changes in the expression levels of FABP3 (fatty acid binding protein 3) gene, ESM1 (endothelial cell-specific molecule 1) gene, EMCN (endothelial mucin) gene, and BCL2 (BCL2 apoptosis regulator) gene.

[0107] Figure 7-10It is a graph showing the temporal changes in the ratio of the expression level of the FABP3 gene to the expression level of the HIF1A gene (FABP3 / HIF1A), the ratio of the expression level of the ESM1 gene to the expression level of the HIF1A gene (ESM1 / HIF1A), the ratio of the expression level of the EMCN gene to the expression level of the HIF1A gene (EMCN / HIF1A), and the ratio of the expression level of the BCL2 gene to the expression level of the HIF1A gene (BCL2 / HIF1A).

[0108] Figure 8 It is a graph showing the mitochondrial area and mitochondrial activity of each cell.

[0109] Fig. 9 This is a fluorescent image of the nucleus and mitochondria.

[0110] Fig.10 is a graph showing the angle-based frequency of motion vectors in a live cell imaging setup.

[0111] Figure 11-1 The graph shows the temporal changes in heart rate (BR), contractile velocity (CV), relaxation velocity (RV), contraction-relaxation duration (CRD), and orientation measured using a live cell imaging device.

[0112] Figure 11-2 This is a graph showing the temporal changes in the ratio of contraction velocity to relaxation velocity (CV / RV) measured using a live cell imaging device.

[0113] Fig.12 The Duration, Interval, and Duration / (Interval) values ​​on the 5th day of culture were measured using calcium imaging analysis. 1 / 2 of charts.

[0114] Fig.13 is a graph showing the results of the patch clamp experiment.

[0115] Fig.14 This is a graph showing the effects of each compound on a cultured cell sheet measured by live cell imaging analysis.

[0116] Figure 15-1 This is a graph showing the effect of a compound (E-4031) on a cultured cell sheet measured by calcium imaging analysis.

[0117] Figure 15-2This is a graph showing the effect of a compound (quinidine) on a cultured cell sheet measured by calcium imaging analysis.

[0118] Figure 15-3 This is a graph showing the effect of a compound (cisapride) on a cultured cell sheet measured by calcium imaging analysis.

[0119] Figure 15-4 This is a graph showing the effect of a compound (isoproterenol) on a cultured cell sheet measured by calcium imaging analysis.

[0120] Figure 15-5 This is a graph showing the effect of a compound (DMSO) on a cultured cell sheet measured by calcium imaging analysis.

[0121] Figure 16-1 This is a graph showing the effect of a compound (isoproterenol) on a cultured cell sheet on day 15 of culture, measured by live cell imaging analysis.

[0122] Figure 16-2 This is a graph showing the effect of a compound (milrinone) on a cultured cell sheet on the 15th day of culture, measured by live cell imaging analysis.

[0123] Fig.17 This is a graph showing the effect of a compound (doxorubicin) on a cultured cell sheet on day 15 of culture, measured by live cell imaging analysis.

[0124] Fig.18 This is a graph showing changes in oxygen consumption rate caused by the addition of etomoxir as a β-oxidation inhibitor. DETAILED DESCRIPTION

[0125] Hereinafter, preferred embodiments of the present invention will be described. It should be noted that, in this specification, "X to Y" indicating a range means "more than X and less than Y". When a numerical range is described in sections, the upper and lower limits of each numerical range can be arbitrarily combined.

[0126] [Cultured cell sheets]

[0127] The cultured cell sheet of the present invention is a cultured cell sheet composed of cardiomyocytes, wherein the cardiomyocytes are arranged in an orientation. In an image obtained by immunostaining the aforementioned cultured cell sheet with an anti-α-actinin antibody and observing it under a microscope, the angle of the length direction of the rod-like structure (Z band of the myofibril) detected by the α-actinin antibody within the measurement range of 71.6μm×71.6μm area when the horizontal direction of the image is set to 0° is measured, and the frequency of the rod-like structure contained within ±15° of the mode value, that is, the orientation degree obtained according to the following formula (1) is 23% or more.

[0128] Orientation degree (%) = (number of rod-like structures within ±15° of the mode value) / (total number of rod-like structures) × 100 (1)

[0129] The rod-like structure refers to the Z band of the myofibril detected by α-actinin. The Z band exists straight relative to the direction of the myofibril. When the cardiomyocytes are aligned in the long axis direction and oriented in one direction, the Z band shows an angle of 90° with respect to the long axis direction of the cell.

[0130] Regarding the image analysis of rod-like structures, the obtained image was divided into 9 sections (one section was an area of ​​71.6 μm×71.6 μm) divided into three equal parts vertically and horizontally, and three sections (upper left, center, and lower right sections) were used. When more than three images were obtained, one section in the upper left of each image was measured (n=3 or more), and when less than two images were obtained, three sections (upper left, center, and lower right sections) were measured from one representative image. On average, 10 cells were included in one section. For the rod-like structures on the image, the angle distribution analysis of the image analysis software (A-ZO-KUN, manufactured by Asahi Kasei Engineering Corporation) or the like was used to detect the rod-like structures detected by α-actinin, and the angle distribution (0 to 180°) of the rod-like structures detected by α-actinin was calculated when the horizontal direction of the image was set to 0°. Alternatively, the rod-like structure may be marked with straight lines using image analysis software (e.g., Photoshop, manufactured by Adobe), and then only the marked straight lines may be saved as an image, followed by analyzing the angle distribution of the straight lines using analysis software (A-ZO-KUN, manufactured by Asahi Kasei Engineering Corporation).

[0131] The angle distribution is expressed as a frequency of every 10 degrees between 0 and 180 degrees.

[0132] It should be noted that the cultured cell sheet refers to a cell sheet that is not a cell sheet existing in a living body but a cell sheet produced in vitro.

[0133] As a means of maturing cardiomyocytes, it is believed that culturing cells in a state close to that of a living body is effective. The myocardium in a living body exists in a state of being oriented in one direction, and performs functions such as contraction and relaxation by conducting electrical conduction between cells mediated by intercellular junctions.

[0134] In this embodiment, it is found that by using an oriented culture substrate, the myocardial cells are cultured in an oriented state, so that the maturation of myocardial cells (physiological activity and motor function improvement) can be promoted. And it is found that by using cells cultured by this method, the myocardial cells derived from human iPS cells and the like can be used in toxicity tests and effectiveness tests in the field of innovative drugs. In addition, the maturation of the cultured cell sheet is promoted to be closer to the state in the body, so it is also expected to be used in regenerative medicine.

[0135] From the viewpoint of promoting the maturation of cardiomyocytes, the degree of orientation represented by the above formula (1) is 23% or more, preferably 24% or more, more preferably 25% or more, and further preferably 26% or more. The upper limit is not particularly limited, but from the viewpoint of ease of manufacturing, it is 50% or less.

[0136] It should be noted that the orientation degree represented by the above formula (1) only needs to satisfy the above range at any number of culture days.

[0137] <Myocardial cells>

[0138] The cultured cell sheet of the present embodiment is composed of cardiomyocytes.

[0139] As the aforementioned cardiomyocytes, cardiomyocytes of invertebrates, vertebrates including humans and non-humans can be exemplified, and vertebrates include fish, amphibians, reptiles, birds, and mammals. Specifically, for example, as mammals, rodents such as mice, rats, ferrets, hamsters, guinea pigs, or rabbits, dogs, cats, sheep, pigs, cattle, horses, or primates including rhesus monkeys, chimpanzees, orangutans, humans, etc. In addition, in addition to mammals, fish, birds including poultry, reptiles, etc. are also included.

[0140] Among them, preferred are cardiomyocytes of mammals including humans and mice, and more preferred are human cardiomyocytes.

[0141] In this embodiment, the cardiomyocytes can be cardiomyocytes differentiated and induced by pluripotent stem cells (stem cells) by using a method for differentiation induction factors for various mesoderm systems. In addition, in this embodiment, the cardiomyocytes can also be induced cardiomyocytes (iCM, cardiomyocytes obtained by direct reprogramming) induced by introducing transcription factors into fibroblasts and blood cells of patients, etc. Furthermore, the cardiomyocytes of this embodiment can also be primary culture (Primary Cell Culture) cardiomyocytes obtained from the heart of patients, etc.

[0142] Among them, the cardiomyocytes are preferably cardiomyocytes derived from stem cells.

[0143] Here, stem cells include, for example, stem cells (Stem Cell) with multi-differentiation ability that can be differentiated into various cells in organisms such as primates including humans and mammals other than primates. Stem cells are suitable for having the following properties: they can be passed down, and even after passage, they remain in a state of not being differentiated, and the karyotype, etc. are not easy to change or the epigenetic phenotype is not easy to change. In addition, in connection with this, stem cells are also suitable for having sufficient proliferation ability in vitro. As a specific example of such stem cells, embryonic stem cells (Embryonic Stem Cell, hereinafter referred to as "ES cells"), induced pluripotent stem cells (Induced Pluripotent Stem Cell, hereinafter referred to as "iPS cells"), other artificially generated or selected stem cells with pluripotency, etc. can be listed. These stem cells can be stem cells made by reprogramming somatic cells using various vectors, RNA, low molecular weight compounds, etc., such as retroviruses, adenoviruses, plasmids, etc., containing specific genes.

[0144] It should be noted that, as stem cells, although it is not necessarily necessary to have cells with multipotency close to omnipotence (pluripotent), it is preferred to use cells with multipotency (multipotency) with multipotency higher than usual. In addition, stem cells can be cells made from cells obtained from disease patients, cells as other disease models, cells integrated with reporter genes (reporter cells), cells that can be conditionally knocked out (conditionally knocked out), other genetically recombined cells, etc. The genetic recombination includes: the addition, modification or deletion of genes in chromosomes; the addition of genes, etc. using various vectors, artificial chromosomes; changes in epigenetic regulation; the addition of artificial genetic materials such as PNA; other genetic recombination.

[0145] Among them, in the present embodiment, the cardiomyocytes are preferably cardiomyocytes derived from ES cells or iPS cells from the viewpoint of easy availability and inducibility to differentiate into cardiomyocytes.

[0146] <Gene expression>

[0147] The cultured cell sheet of this embodiment preferably undergoes characteristic changes in gene expression levels associated with the maturation of cardiomyocytes. Such characteristic changes in gene expression levels are described, for example, in reference 1 (Circ. Res. 2020 Apr 10: 126 (8), 1086-1106) and reference 2 (Nat Rev Cardiol. 2020 Jun; 17 (6): 341-359).

[0148] In the method of the present invention, the gene expression level is measured by RNA expression information. The method for obtaining RNA expression information is not particularly limited, and examples thereof include: converting RNA contained in the sample into cDNA by reverse transcription, and measuring the cDNA or its amplification product to obtain RNA expression information. As means for measuring expression levels, microarray, quantitative RT-PCR, RNA-Seq, etc. are listed, and RNA-Seq is preferred.

[0149] Regarding the expression level of RNA, when using microarray analysis, it is quantified by the signal intensity ratio. When RNA-seq is analyzed, it is quantified by the number of sequence reads mapped to the genome (read count value). For the number of reads mapped to each transcript, correction is performed by the length of the transcript, the total number of reads obtained in the analysis, etc., and after the operation called standardization, it is used for analysis. As a specific method of standardization, for example: in order to correct the difference in the total number of reads between samples, the total number of reads is corrected to 1 million CPM (Counts Per Million); and the length of the transcript is set to 1kb, the total number of reads is set to 1 million, and the number of reads corrected by the gene length of each gene is RPKM (Reads Per Kilobase of exon per Million mapped sequence reads) value. Similarly, the total number of reads is set to 1 million and the number of fragments corrected by the gene length of each gene, that is, the FPKM (Fragments Per Kilobase ofexon per Million mapped sequence reads) value, and the TPM (Transcripts Per Million) value, which represents the number of transcripts when the number of reads of each transcript is corrected by the gene length and the total number of reads is set to 1 million, is also widely used. In this embodiment, CPM and TPM are used, but it is not limited to this.

[0150] The cultured cell sheet of the present embodiment preferably has a large amount of MYL3 (myosin light chain 3) gene expression. When assuming that the expression of the ACTB (β-actin) gene is a constant amount of gene expression regardless of the tissue or the culture period, the ratio of the gene expression of MYL3 to the expression of the ACTB gene (MYL3 / ACTB) is preferably 12.0 or more, more preferably 12.5 or more. In addition, although it depends on the number of days of culture, it is further preferably 15.0 or more. The upper limit is not particularly limited, and is generally 30.0 or less, preferably 25.0 or less. It should be noted that in the cultured cell sheet of the present embodiment, although the change in gene expression will be confirmed according to the difference in the number of days of culture, it is sufficient to achieve the above-mentioned ratio of gene expression at any number of days of culture.

[0151] In addition, it has been reported that isoform changes occur in cardiomyocytes as they mature (see the above-mentioned reference 1), and as such isoform changes, transition from MYH6 (myosin heavy chain 6) to MYH7 (myosin heavy chain 7) is known.

[0152] In the cultured cell sheet of the present embodiment, the ratio of the expression level of the MYH7 gene to the expression level of the MYH6 gene (MYH7 / MYH6) is preferably 6.0 or more, more preferably 7.0 or more. In addition, the upper limit is not particularly limited, generally 15.0 or less, preferably 10.0 or less. It should be noted that in the cultured cell sheet of the present embodiment, although the change in gene expression level will be confirmed according to the different culture days, it is sufficient to achieve the above-mentioned gene expression level ratio at any culture day.

[0153] And then, in the cultured cell sheet, the gene expression of CKM (creatine kinase, M type) related to myocardial contraction is preferably increased. In the cultured cell sheet of the present embodiment, the expression of CKM gene is preferably more than 900TPM (Transcript Per Kilobase Million) during any culture days, more preferably more than 1000TPM, and further preferably more than 1100TPM. In addition, its upper limit is not particularly limited, but is generally less than 1500TPM.

[0154] The expression ratio of the CKM gene to the ACTB gene (CKM / ACTB) is preferably 1.80 or more, more preferably 2.00 or more, and further preferably 2.10 or more at any culture day. The upper limit is not particularly limited, but is generally 5.0 or less, and preferably 4.5 or less.

[0155] In the cultured cell sheet, the gene expression level of COX6A2 (cytochrome C oxidase subunit 6A2), a protein localized in mitochondria, is preferably increased. In the cultured cell sheet of the present embodiment, the expression level of the COX6A2 gene is preferably 850 TPM or more, more preferably 875 TPM or more, and further preferably 900 TPM or more at any culture day, and the upper limit is not particularly limited, for example, 2000 TPM or less, preferably 1500 TPM or less.

[0156] The expression ratio of the COX6A2 gene to the ACTB gene (COX6A2 / ACTB) is preferably 1.95 or more, more preferably 2.20 or more, and further preferably 2.40 or more at any culture day. The upper limit is not particularly limited, but is generally 5.00 or less, and preferably 3.50 or less.

[0157] In the cultured cell sheet, the gene expression of CKMT2 (mitochondrial creatine kinase 2), a protein preferably located in mitochondria, is increased. In the cultured cell sheet of the present embodiment, the expression of the CKMT2 gene is preferably more than 330 TPM at any culture day, more preferably more than 350 TPM, and the upper limit is not particularly limited, for example, less than 1000 TPM, preferably less than 800 TPM.

[0158] The expression ratio of the CKMT2 gene to the ACTB gene (CKMT2 / ACTB) is preferably 0.60 or more, more preferably 0.65 or more, on the 15th to 30th day of culture, and the upper limit is not particularly limited, but is generally 3.0 or less, preferably 1.5 or less. In addition, CKMT2 / ACTB is preferably 0.80 or more at any culture day.

[0159] In the cultured cell sheet, the gene expression level of LDHA (lactate dehydrogenase A subunit), which is a glycolytic enzyme, is preferably increased. This is considered to be because the energy production increases with cell activation, thereby increasing the gene expression level of LDHA.

[0160] In the cultured cell sheet of this embodiment, the expression level of the LDHA gene is preferably 550 TPM or more, more preferably 600 TPM or more, and further preferably 650 TPM or more at any culture day. The upper limit is not particularly limited, for example, 2000 TPM or less, preferably 1500 TPM or less.

[0161] The expression ratio of the LDHA gene to the ACTB gene (LDHA / ACTB) is preferably 0.80 or more, more preferably 1.00 or more, and further preferably 1.20 or more. The upper limit is not particularly limited, but is generally 5.0 or less, and preferably 4.0 or less.

[0162] In the cultured cell sheet, the expression levels of CACNA2D1 (voltage-gated calcium channel auxiliary subunit α2δ1) related to the formation of myocardial calcium ion channels, KCNJ2 (inward rectifier potassium channel subfamily J member 2) related to the formation of myocardial potassium ion channels, KCNE1 (voltage-gated potassium channel subfamily E regulatory subunit 1), and SCN5A (voltage-gated sodium channel α subunit 5) related to the formation of myocardial sodium ion channels are preferably increased over time. This is believed to be because: as myocardial cells mature, the contraction and relaxation movements of myocardial cells are also activated, and the expression levels of genes related to the formation of ion channels increase accordingly.

[0163] It should be noted that in the case of CACNA2D1 gene, KCNJ2 gene, KCNE1 gene, and SCN5A gene, ATP1A1 (ATPase Na+ / K+ transport subunit α1) which is involved in the generation of Na+ and K+ gradients inside and outside the cell membrane is used instead of the aforementioned ACTB. The reason for selecting it is that ATP1A1 is reported to be a stably expressed myocardial cell membrane protein, and the expression level in myocardial cells derived from iPS cells, fetal myocardium, and adult myocardium varies little (see Okai, et al., Video-based assessment of drug-induced effects on contractile motion properties using human induced pluripotent stem cell-derived cardiomyocytes, Journal of Pharmacological and Toxicological Methods, Volume 105, September 2020, 106893).

[0164] In the cultured cell sheet of the present embodiment, the expression level of the CACNA2D1 gene associated with the formation of myocardial calcium ion channels is preferably 2100 CPM or more, more preferably 2200 CPM or more, further preferably 2300 CPM or more, and further preferably 2400 CPM or more at any culture day. The upper limit is not particularly limited, for example, it is 3500 CPM or less, and preferably 3000 CPM or less.

[0165] The expression ratio of the CACNA2D1 gene to the ATP1A1 gene (CACNA2D1 / ATP1A1) is preferably 0.08 or more, more preferably 0.085 or more, further preferably 0.09 or more, and further preferably 0.095 or more on any day from the 15th day to the 30th day of culture. The upper limit is not particularly limited, but is generally 0.50 or less, and preferably 0.20 or less.

[0166] In the cultured cell sheet of this embodiment, the expression level of the KCNJ2 gene is preferably above 950 CPM, more preferably above 1000 CPM, and further preferably above 1200 CPM at any culture day. Moreover, the upper limit is not particularly limited, for example, it is below 3000 CPM, and preferably below 2000 CPM.

[0167] The expression ratio of the KCNJ2 gene to the ATP1A1 gene (KCNJ2 / ATP1A1) is preferably 0.038 or more, more preferably 0.040 or more, and further preferably 0.045 or more on any day from the 15th to the 45th day of culture. The upper limit is not particularly limited, but is generally 0.15 or less, and preferably 0.10 or less.

[0168] In the cultured cell sheet of the present embodiment, the expression level of the KCNE1 gene is preferably 50 CPM or more on any day from the 15th day to the 45th day of culture, more preferably 60 CPM or more, further preferably 70 CPM or more, and further preferably 75 CPM or more. The upper limit is not particularly limited, for example, it is 200 CPM or less, preferably 150 CPM or less.

[0169] The expression ratio of the KCNE1 gene to the ATP1A1 gene (KCNE1 / ATP1A1) is preferably 0.0025 or more, more preferably 0.003 or more, and further preferably 0.0035 or more on any day from the 15th to the 45th day of culture. The upper limit is not particularly limited, but is generally 0.01 or less, and preferably 0.007 or less.

[0170] In the cultured cell sheet of this embodiment, the expression level of the SCN5A gene is preferably above 22000 CPM, more preferably above 23000 CPM, and further preferably above 23500 CPM on any day from the 15th day to the 45th day of culture. Moreover, the upper limit is not particularly limited, for example, it is below 50000 CPM, and preferably below 40000 CPM.

[0171] The expression ratio of the SCN5A gene to the ATP1A1 gene (SCN5A / ATP1A1) is preferably 0.85 or more, more preferably 0.90 or more, and further preferably 0.95 or more on any day from the 15th to the 45th day of culture. The upper limit is not particularly limited, but is generally 5.0 or less, and preferably 2.0 or less.

[0172] In the cultured cell sheet, the expression of LPL (lipoprotein lipase) present on the cell surface to promote lipid uptake into the cell, ACAT1 (acetyl-CoA acetyltransferase 1) located in the mitochondria in the cell and related to the β-oxidation of fatty acids, HADHA (hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit α), and HADHB (hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit β) is preferably increased over time. With the maturation of myocardial cells, motor function improves and ATP consumption increases. It is believed that in order to produce ATP more efficiently, the expression of these genes will increase.

[0173] In the cultured cell sheet of the present embodiment, the expression level of the LPL gene is preferably above 3100 CPM on any day from the 15th day to the 45th day of culture, more preferably above 3500 CPM, further preferably above 4000 CPM, and further preferably above 4500 CPM. Moreover, the upper limit is not particularly limited, for example, it is below 9000 CPM, and preferably below 7500 CPM.

[0174] The expression ratio of the LPL gene to the ACTB gene (LPL / ACTB) is preferably 0.45 or more, more preferably 0.50 or more, and further preferably 0.60 or more on any day from the 15th day to the 45th day of culture. The upper limit is not particularly limited, but is generally 2.0 or less, preferably 1.5 or less, and more preferably 1.0 or less.

[0175] In the cultured cell sheet of this embodiment, the expression level of the ACAT1 gene is preferably 3500 CPM or more, more preferably 3800 CPM or more on any day from the 30th to the 45th day of culture, and the upper limit is not particularly limited, for example, 8000 CPM or less, preferably 6000 CPM or less.

[0176] The expression ratio of the ACAT1 gene to the ACTB gene (ACAT1 / ACTB) is preferably 0.43 or more, more preferably 0.45 or more, and further preferably 0.50 or more on any day from the 15th to the 30th day of culture. The upper limit is not particularly limited, but is generally 2.0 or less, preferably 1.5 or less, and more preferably 1.0 or less.

[0177] In the cultured cell sheet of the present embodiment, the expression level of the HADHA gene is preferably 6000 CPM or more, more preferably 6200 CPM or more, and further preferably 6500 CPM or more on any day from the 30th day to the 45th day of culture. The upper limit is not particularly limited, for example, it is 12000 CPM or less, and preferably 10000 CPM or less.

[0178] The expression ratio of the HADHA gene to the ACTB gene (HADHA / ACTB) is preferably 0.75 or more, more preferably 0.80 or more, and further preferably 0.90 or more on any day from the 15th day to the 30th day of culture. The upper limit is not particularly limited, but is generally 2.5 or less, preferably 2.0 or less, and more preferably 1.5 or less.

[0179] In the cultured cell sheet of this embodiment, the expression level of the HADHB gene is preferably above 8200 CPM, more preferably above 8300 CPM, and further preferably above 8500 CPM on any day from the 15th day to the 45th day of culture. Moreover, the upper limit is not particularly limited, for example, it is below 20000 CPM, and preferably below 15000 CPM.

[0180] The expression ratio of the HADHB gene to the ACTB gene (HADHB / ACTB) is preferably 0.95 or more, more preferably 1.00 or more, further preferably 1.10 or more, and further preferably 1.20 or more on any day from the 15th day to the 30th day of culture. The upper limit is not particularly limited, but is generally 2.5 or less, preferably 2.0 or less, and more preferably 1.8 or less.

[0181] In the cultured cell sheet, the expression levels of the PPARGC1A gene (PPARG (peroxisome proliferator-activated receptor γ) coactivator 1α) encoding the transcription factor PGC-1α that functions as a master regulator of mitochondrial biogenesis, the ESRRA gene (estrogen-related receptor α) encoding the estrogen-related receptor ERR-α associated with the maturation of cardiomyocytes, the VEGFA gene (vascular endothelial growth factor A) encoding the vascular growth factor, the APLN gene (Apelin) encoding the myokine that stimulates cardiac contraction in the myocardium, the FABP3 gene (fatty acid binding protein 3) encoding the myokine that regulates the uptake of long-chain fatty acids in the myocardium, the ESM1 gene (endothelial cell-specific molecule 1) that is specifically expressed in endothelial cells, and the EMCN gene (endothelial mucin) encoding the mucin-like glycoprotein secreted by epithelial cells and the like are preferably increased over time, and the expression level of the BCL2 gene (BCL2 apoptosis regulator) associated with the differentiation of cardiomyocytes is preferably maintained unchanged.

[0182] The reason is believed to be that as cardiomyocytes mature, motor function becomes hyperactive, thereby increasing the expression levels of the PPARGC1A gene involved in mitochondrial functionalization and the VEGFA gene involved in angiogenesis. In addition, the expression levels of genes encoding myokines such as APLN and FABP3 increase, and further, the expression levels of endothelial cell-specific genes such as ESM1 and EMCN increase. In addition, it is believed that the expression of the BCL2 gene related to cardiomyocyte differentiation remains unchanged.

[0183] In the cultured cell sheet of the present embodiment, the expression level of the PPARGC1A gene is preferably 4500 CPM or more, more preferably 4700 CPM or more, and further preferably 5000 CPM or more on any day from the 15th day to the 45th day of culture. The upper limit is not particularly limited, for example, it is 20000 CPM or less, preferably 15000 CPM or less, and more preferably 8000 CPM or less.

[0184] The expression ratio of the PPARGC1A gene to the HIF1A gene (PPARGC1A / HIF1A) is preferably 0.80 or more, more preferably 0.85 or more, and further preferably 0.90 or more on any day of culture. The upper limit is not particularly limited, but is generally 2.5 or less, preferably 2.0 or less, and more preferably 1.5 or less.

[0185] It should be noted that in the case of PPARGC1A gene, ESRRA gene, VEGF gene, APLN gene, FABP3 gene, ESM1 gene, EMCN gene, and BCL2 gene, HIF1A (hypoxia-inducible factor 1α), a transcription factor induced under the hypoxic state of cells, is used instead of the aforementioned ACTB. The reason is that it is reported that the hyperangiogenesis described in this project is induced by hypoxia or exercise stimulation. In addition, it is believed that although any environment can be adopted under cell culture conditions, in cardiomyocytes in good condition (functionally mature), the expression of each factor caused by exercise stimulation will increase (refer to Arany, et al., HIF-independent regulation of VEGF and angiogenesis by the transcriptional coactivator PGC-1a, Vol 451 | 21 February 2008).

[0186] In the cultured cell sheet of the present embodiment, the expression level of the ESRRA gene is preferably 2750 CPM or more, more preferably 2900 CPM or more, and further preferably 3000 CPM or more on any day from the 15th day to the 45th day of culture. The upper limit is not particularly limited, and is, for example, 10000 CPM or less, preferably 6000 CPM or less, and more preferably 4000 CPM or less.

[0187] The expression ratio of the ESRRA gene to the HIF1A gene (ESRRA / HIF1A) is preferably 0.55 or more, more preferably 0.57 or more, and further preferably 0.60 or more on any day of culture. The upper limit is not particularly limited, but is generally 2.0 or less, preferably 1.5 or less, and more preferably 1.0 or less.

[0188] In the cultured cell sheet of the present embodiment, the expression level of the VEGFA gene on any day of culture is preferably above 3300 CPM, more preferably above 3700 CPM, further preferably above 4500 CPM, and further preferably above 6000 CPM. Moreover, the upper limit is not particularly limited, for example, it is below 25000 CPM, preferably below 15000 CPM, and more preferably below 10000 CPM.

[0189] The expression ratio of the VEGFA gene to the HIF1A gene (VEGFA / HIF1A) on any day of culture is preferably 0.65 or more, more preferably 0.70 or more, further preferably 0.90 or more, further preferably 1.2 or more, particularly preferably 1.5 or more, and the upper limit is not particularly limited, but is generally 5.0 or less, preferably 3.0 or less, and more preferably 2.0 or less.

[0190] In the cultured cell sheet of this embodiment, the expression level of the APLN gene on any day of culture is preferably above 100 CPM, more preferably above 150 CPM, and further preferably above 200 CPM. The upper limit is not particularly limited, for example, it is below 50,000 CPM, preferably below 3,000 CPM, and more preferably below 2,000 CPM.

[0191] The expression ratio of the APLN gene to the HIF1A gene (APLN / HIF1A) is preferably 0.005 or more, more preferably 0.020 or more, further preferably 0.030 or more, and further preferably 0.040 or more on any day of culture. The upper limit is not particularly limited, but is generally 1.0 or less, preferably 0.50 or less, and more preferably 0.30 or less.

[0192] In the cultured cell sheet of this embodiment, the expression level of the FABP3 gene on any day of culture is preferably above 30,000 CPM, more preferably above 31,000 CPM, and further preferably above 32,000 CPM. The upper limit is not particularly limited, for example, below 100,000 CPM, preferably below 60,000 CPM, and more preferably below 40,000 CPM.

[0193] The expression ratio of the FABP3 gene to the HIF1A gene (FABP3 / HIF1A) is preferably 5.0 or more, more preferably 5.5 or more, and further preferably 6.0 or more on any day of culture. The upper limit is not particularly limited, but is generally 15.0 or less, preferably 12.0 or less, and more preferably 8.0 or less.

[0194] In the cultured cell sheet of the present embodiment, the expression level of the ESM1 gene on any day of culture is preferably 5 CPM or more, more preferably 25 CPM or more, further preferably 40 CPM or more, further preferably 100 CPM or more, particularly preferably 200 CPM or more, and the upper limit is not particularly limited, for example, 3000 CPM or less, preferably 1000 CPM or less, more preferably 500 CPM or less.

[0195] The expression ratio of the ESM1 gene to the HIF1A gene (ESM1 / HIF1A) is preferably 0.002 or more, more preferably 0.005 or more, and further preferably 0.010 or more on any day of culture. The upper limit is not particularly limited, but is generally 0.200 or less, preferably 0.150 or less, and more preferably 0.100 or less.

[0196] In the cultured cell sheet of the present embodiment, the expression level of the EMCN gene on any day after the 7th day of culture is preferably above 10 CPM, more preferably above 20 CPM, further preferably above 40 CPM, and further preferably above 60 CPM. Moreover, the upper limit is not particularly limited, for example, it is below 1000 CPM, preferably below 500 CPM, and more preferably below 300 CPM.

[0197] The expression ratio of the EMCN gene to the HIF1A gene (EMCN / HIF1A) is preferably 0.002 or more, more preferably 0.004 or more, further preferably 0.005 or more, and further preferably 0.010 or more on any day after the 7th day of culture. The upper limit is not particularly limited, but is generally 0.100 or less, preferably 0.070 or less, and more preferably 0.050 or less.

[0198] In the cultured cell sheet of the present embodiment, the expression level of the BCL2 gene on any day after the 7th day of culture is preferably 60 CPM or more, more preferably 70 CPM or more, further preferably 80 CPM or more, and further preferably 90 CPM or more. The upper limit is not particularly limited, for example, it is 1000 CPM or less, preferably 500 CPM or less, and more preferably 300 CPM or less.

[0199] The ratio of the expression levels of the BCL2 gene to the HIF1A gene (BCL2 / HIF1A) is preferably 0.013 or more, more preferably 0.015 or more, and further preferably 0.017 or more on any day after the 7th day of culture. The upper limit is not particularly limited, but is generally 0.100 or less, preferably 0.070 or less, and more preferably 0.050 or less.

[0200] <Mitochondrial volume>

[0201] There is a tendency for the amount and activity of mitochondria to increase as cardiomyocytes mature.

[0202] The cultured cell sheet of this embodiment is stained with a fluorescent dye that selectively labels mitochondria based on membrane potential and a fluorescent dye that selectively labels nuclei, and a fluorescent image is obtained to measure the area and brightness of mitochondria and nuclei.

[0203] Examples of fluorescent dyes that selectively label mitochondria based on membrane potential include Mito-tracker series manufactured by Thermo Fisher Scientific Inc. In addition, examples of fluorescent dyes that selectively label nuclei include Hoechst 33342.

[0204] At this time, the area (Area) and brightness (Intensity) of mitochondria per cell are calculated by dividing by the number of nuclei, that is, the number of cells. It should be noted that the area per cell reflects the number of mitochondria, and the brightness per cell reflects the mitochondrial activity.

[0205] The area of ​​mitochondria per cell is preferably 200 μm 2 More preferably 300 μm 2 More preferably, 400 μm 2 More preferably, 450 μm 2 The upper limit is not particularly limited, but is generally 1000 μm. 2 Below, preferably 800 μm 2 the following.

[0206] The brightness of each cell's mitochondria is preferably 2×10 6 More preferably 3×10 6 More preferably, 4×10 6 The upper limit is not particularly limited, but is generally 10×10 6 Below, preferably 8×10 6 It should be noted that the brightness of each cell is the brightness measured by the method described in the Examples.

[0207] In addition, the cell seeding condition for measuring the mitochondrial area and mitochondrial brightness per cell was 6×10 4 cells / well, culture days 9 (after day 5), area of ​​96 wells (0.33 cm 2 ).

[0208] <Live Cell Imaging>

[0209] For a cultured cell sheet composed of cardiomyocytes, the movement changes associated with the contraction / relaxation of the cardiomyocytes can be detected. In vivo, cardiomyocytes are observed to be oriented in one direction and to contract / relax along the oriented direction.

[0210] Using a live cell imaging device (e.g., SI8000, manufactured by Sony Corporation), cultured cell sheets (living cells) are photographed under culture conditions to obtain animation data. Based on the animation data, the motion vectors (speed, direction, and number) associated with the contraction / relaxation of cardiomyocytes are detected, thereby enabling the heart rate (BR: beating rate), contractile velocity (CV: contractile velocity), and relaxation velocity (RV:

[0211] For the measurement of BR, CV, RV, CRD, etc. based on live cell imaging, see, for example, Methods in Molecular Biology, vol. 2320, Chapter 15.

[0212] In addition, regarding these measuring methods, the methods described in Examples can be referred to.

[0213] Here, the degree of orientation is calculated by finding the angle of the motion vector when the horizontal direction of the image is set to 0°, and then finding the ratio of the number of vectors showing ±5° from the angle of the mode value of the vector angle to the total number of detected vectors. The degree of orientation obtained by the living cell imaging device is calculated using the following formula.

[0214] Orientation degree (%)

[0215] = (the number of vectors within the range of ±5° of the mode value) / (the total number of vectors) × 100

[0216] The degree of orientation of the cultured cell sheet of this embodiment measured using live cell imaging is preferably 10% or more, more preferably 12% or more, further preferably 16% or more, further preferably 18% or more, and particularly preferably 20% or more. The upper limit of the degree of orientation is not particularly limited.

[0217] In live cell imaging, there is the following tendency: the heart rate of an adult is 60 beats / minute on average. The closer the heart rate (BR) is to 60 beats / minute, the closer it is to normal mature cardiomyocytes. The faster the contraction velocity (CV), the closer it is to normal mature cardiomyocytes. The faster the relaxation velocity (RV), the closer it is to mature cardiomyocytes. The shorter the beat duration (CRD), the closer it is to normal mature cardiomyocytes.

[0218] In addition, the smaller the ratio (CV / RV) of the contraction velocity (CV) to the relaxation velocity (RV), the more preferable it is. The CV / RV of the cultured cell sheet of this embodiment is preferably 2.8 or less, more preferably 2.6 or less, and preferably 1.0 or more. That is, CV / RV preferably satisfies the following formula (2).

[0219] 1.0≤CV / RV≤2.8 (2)

[0220] <Ca-imaging analysis>

[0221] In myocardial cells, it is observed that the Ca concentration of the whole cytoplasm caused by the action potential rises roughly at the same time as the myocardial contraction, which is called a phenomenon of calcium transient. The cultured cell sheet of the present embodiment is treated with a fluorescent Ca indicator and then observed with a confocal quantitative imaging cytometer (Ca-imaging analysis), thereby being able to observe calcium transients over time. As a method for making a waveform diagram based on the signal information of calcium transients and analyzing the parameters recorded below, a dedicated analysis software is used.

[0222] In immature cardiomyocytes and cardiomyocytes with some abnormality due to diseases, the line width (Duration) at 20% height of the waveform peak of calcium transient tends to be longer than that of normal mature cardiomyocytes. The line width (Duration) at 20% height varies according to the heart rate, so the relationship between Duration (seconds) and Interval (seconds) to the power of 1 / 2 preferably satisfies the following formula (3).

[0223] Duration / (Interval) 1 / 2 ≥0.68 (3)

[0224] It should be noted that it is reported that the line width (Duration) at 20% of the height of the duration of calcium transient is correlated with the duration of action potential. In cardiomyocytes, there is a tendency that the action potential duration becomes longer as maturation progresses. Interval is affected by the heartbeat. Usually, in the analysis of electrocardiograms, the action potential duration uses a value corrected by the heart rate, so Duration / (Interval) is used as shown in formula (3). 1 / 2 .

[0225] In the present embodiment, it is preferred that the cells constituting the cultured cell sheet have an action potential 80% repolarization duration of 600 msec or longer and a maximum diastolic potential of -60 mV or less in a patch clamp test in a 25°C solution.

[0226] In the patch clamp test, a current of 20 pA to 100 pA is injected into the cardiomyocytes at 0.2 Hz in a current clamp at 25°C Tyrode's solution to generate an action potential, and the change in potential is measured. It should be noted that the current can be appropriately selected within the range that can generate an appropriate action potential.

[0227] It should be noted that the action potential 80% repolarization duration is used as an indicator of the action potential duration. This is the time required to reach a potential equivalent to 80% repolarization when the difference between the peak height of the action potential displayed as the first phase after the application of current and the maximum diastolic potential is set to 100%. In other words, this is the time required from the rise time of the action potential to the action potential reaching a potential equivalent to 80% repolarization.

[0228] As cardiomyocytes mature, there is a tendency for the action potential to last longer and the maximum diastolic potential to be deeper than in immature cardiomyocytes.

[0229] The duration of 80% repolarization of the action potential of the cells constituting the cultured cell sheet of the present embodiment is preferably 600 msec or longer, more preferably 650 msec or longer, further preferably 700 msec or longer, and further preferably 750 msec or longer. Furthermore, the upper limit is not particularly limited, but is preferably the same as that of cardiomyocytes in a living body. From this point of view, it is preferably 1200 msec or shorter, more preferably 1050 msec or shorter, and further preferably 900 msec or shorter.

[0230] In addition, the maximum diastolic potential of the cells constituting the cultured cell sheet of the present embodiment is preferably closer to the value of new cells in the body, and from this point of view, it is preferably below -60 mV, more preferably below -62.5 mV, further preferably below -65 mV, and further preferably below -67.5 mV. In addition, the lower limit is not particularly limited, but since the ventricular cardiomyocytes of adults are about -80 mV, a value close to -80 mV is preferably used.

[0231] Note that, regarding the action potential duration and maximum diastolic potential (resting membrane potential) of normal isolated ventricular myocytes from humans, reference can be made to Circulation, 2013; 127: 575-584.

[0232] <Reduction of oxygen consumption rate by β-oxidation inhibitor>

[0233] In this embodiment, it is preferred that: in an oxygen consumption rate measurement that measures the rate at which cells constituting the cultured cell sheet use oxygen contained in the culture medium, by adding a β-oxidation inhibitor that inhibits the β-oxidation activity of fatty acids, the oxygen consumption rate is reduced to less than 20% compared to before the addition of the β-oxidation inhibitor.

[0234] Oxygen contained in the culture medium is taken up by cells and consumed in the pathway of producing ATP through oxidative phosphorylation in mitochondria. Therefore, the oxygen consumption rate (OCR) of the culture medium can be used as an indicator for analyzing mitochondrial function. Metabolism in mitochondria mainly includes pathways using glucose, fatty acids, and glutamine, and the metabolic pathways can be inferred by adding inhibitors of enzymes involved in each metabolism.

[0235] The oxygen consumption amount can be measured using a commercially available oxygen consumption rate plate test kit (manufactured by Dojin Chemical Co., Ltd.) or the like.

[0236] Mature cardiomyocytes are more dependent on fatty acid metabolism than immature cardiomyocytes.

[0237] In an oxygen consumption rate measurement for measuring the rate at which cells constituting the cultured cell sheet of the present embodiment use oxygen contained in the culture medium, the oxygen consumption rate obtained based on the addition of a β-oxidation inhibitor that inhibits the β-oxidation activity of fatty acids is preferably 30% or less, more preferably 20% or less, and even more preferably 15% or less, when the rate before the addition of the β-oxidation inhibitor is set to 100%.

[0238] <Applications of cultured cell sheets>

[0239] The cultured cell sheet of this embodiment can be used for various purposes, for example, for regenerative medicine. The cultured cell sheet may be a single-layer sheet formed of one layer of cells, or may be a multi-layer sheet formed of two or more layers of cells.

[0240] Specifically, the cultured cell sheet can be directly attached to the damaged part of the myocardial tissue of the heart (hereinafter also referred to as "damaged heart") damaged by ischemic diseases such as myocardial infarction and angina pectoris in the same direction as the contraction of the cardiac tissue, and sutured or inserted after attachment. When used for regenerative medicine, a single-layer cell sheet or a multi-layer cell sheet can be stacked and used for regenerative medicine. It should be noted that when stacking, it is preferred to stack in an aligned orientation direction. Specifically, it is sufficient to stack in an aligned contraction direction or orientation direction of the cultured cell sheet.

[0241] Furthermore, the cultured cell sheet of the present embodiment can be used for evaluating the effect of a compound or drug on cardiomyocytes by allowing the compound or drug to act as an evaluation target, as described later.

[0242] [Method for producing cultured cell sheet]

[0243] The manufacturing method of a cultured cell sheet composed of cardiomyocytes according to the present embodiment includes the step of culturing cardiomyocytes using a specific cell sheet forming component, the cell sheet forming component having a surface for forming a cultured cell sheet, the surface having a plurality of flat portions and a plurality of concave-convex portions, each flat portion having a shape extending along a first direction, and the plurality of flat portions being arranged along a second direction intersecting the first direction on the entire surface, each concave-convex portion including a plurality of height difference structures filling between the adjacent flat portions, the pitch of the height difference structures being greater than 100 nm and less than 10 μm, the height difference structures being convex portions, the concave-convex portions having a plurality of convex portions on the bottom surface of a concave portion clamped by the adjacent flat portions, and in the thickness direction of the cell sheet forming component, the difference between the height of the front end surface in the concave-convex portion and the height of the flat portion is less than 0.5 μm.

[0244] like Figure 1 As shown in (a), the cell sheet forming member 100 is, for example, a sheet of a culture dish 110 placed on a shallow bottom plate. The shallow bottom plate holds a cell suspension in a space surrounded by the culture dish 110 and the cover 120. It should be noted that the bottom of the shallow bottom plate can be processed into the above-mentioned surface shape having a specific flat portion and concave-convex portion. In this case, the shallow bottom plate itself is the cell sheet forming member.

[0245] like Figure 1 As shown in (b), the surface 111 of the cell sheet forming member 100 has a plurality of flat portions 130 and a plurality of concave-convex portions 140. The concave-convex portion 140 is composed of a plurality of height difference structures, and the plurality of height difference structures fill between adjacent flat portions 130. The height difference structure is a convex portion. It should be noted that the concave-convex portion 140 has a concave portion sandwiched by adjacent flat portions 130 and a plurality of convex portions 141 located on the bottom surface of the concave portion.

[0246] like Figure 1 As shown in (c), each flat portion 130 is along one direction, namely, the first direction ( Figure 1 Each flat portion 130 is formed on the entire surface 111 along a second direction ( Figure 1 Each concave-convex portion 140 also extends in the first direction and is arranged in the second direction on the entire surface 111.

[0247] Each convex portion 141 constituting the concavoconvex portion 140 is located at each vertex of a triangular lattice, for example, when viewed from a direction opposite to the surface 111. Each concavoconvex portion 140 repeats such an arrangement of the convex portions 141 along the first direction and the second direction. If the concavoconvex portion 140 has the convex portions 141 located at each vertex of the triangular lattice, the original plate for forming the convex portions 141 can be formed by using a mask suitable for forming a minute repetitive structure, for example, by an etching method using a single particle film as a mask.

[0248] Each convex portion 141 has, for example, a circular shape when viewed from the direction opposite to the surface 111. The mode value of the distance between the centers of adjacent convex portions 141 is the pitch of the convex portions 141. The maximum width of the convex portion 141 in the plan view is the diameter of the convex portion 141.

[0249] From the viewpoint of aligning the elongation direction of the cardiomyocytes with the first direction, it is appropriate that the pitch of the convex portions 141 satisfies the following (A) and (B). That is, from the viewpoint of clearly distinguishing the quality of adhesion to the cardiomyocytes between the flat portion 130 and the concave-convex portion 140, it is appropriate that the pitch of the convex portions 141 satisfies the following (A) and (B).

[0250] (A) Pitch of protrusions 141: 100 nm or more and 10 μm or less

[0251] (B) Diameter of the convex portion 141: 50% or more and 100% or less of the pitch of the convex portion 141

[0252] The length of each flat portion 130 in the second direction (short-side direction) is the width of the flat portion 130 . In addition, the length between adjacent flat portions 130 in the second direction (short-side direction) is the width of the concavo-convex portion 140 .

[0253] The width of the flat portion 130 and the width of the concavoconvex portion 140 are, for example, 1 / 10 times or more and 10 times or less the size of the cells to be cultured (5 μm or more and 100 μm or less). From the viewpoint of aligning the elongation direction of the cardiomyocytes with the first direction, it is suitable that the width of the flat portion 130 and the width of the concavoconvex portion 140 satisfy the following (C) and (D).

[0254] (C) Width of flat portion 130: 10 μm or more and 50 μm or less

[0255] (D) Width of the concavo-convex portion 140: 10 μm or more and 50 μm or less

[0256] like Figure 1As shown in (d), the concave-convex portion 140 may include concave portions 142 between adjacent convex portions 141 and between the flat portion 130 and the convex portion 141 adjacent thereto. Since a plurality of convex portions 141 are dispersed in the concave-convex portion 140, the spaces between the convex portions 141, i.e., the concave portions 142, are connected in the concave-convex portion 140 along the first direction and the second direction.

[0257] In the thickness direction of the cell sheet forming member 100, the length between the bottom surface of the concave portion 142 and the flat portion 130 is the height of the flat portion 130. In addition, in the thickness direction of the cell sheet forming member 100, the height difference between the front end surface of each convex portion 141 and the flat portion 130 is the boundary height difference. The height difference between the bottom surface of the concave portion 142 and the front end surface of each convex portion 141 is the height of the convex portion 141. In a configuration in which the front end surface of each convex portion 141 and the flat portion 130 are in the same plane, the height of the flat portion 130 and the height of the convex portion 141 are equal to each other. The ratio of the pitch of the convex portion 141 to the height of the convex portion 141 is the aspect ratio of the convex portion 141.

[0258] From the perspective of improving the flatness of the cell sheet, it is appropriate that the boundary height difference satisfies the following (E). From the perspective of improving the structural stability of the concavoconvex portion 140 and facilitating the formation of the concavoconvex portion 140, it is appropriate that the height of the convex portion 141 satisfies the following (F), and the height ratio of the convex portion 141 satisfies the following (G).

[0259] (E) Boundary height difference: 0.5 μm or less, preferably 0.3 μm or less

[0260] (F) Height of the protrusion 141: 50 nm or more and 5 μm or less

[0261] (G) Ratio of height of convex portion 141: 0.1 or more and 10 or less

[0262] Furthermore, if the configurations satisfying the above (A) and (B) are satisfied, the cells will preferentially adhere to one structure and less adhere to the other structure, regardless of whether they are cells that are more adherent to the flat portion 130 or cells that are more adherent to the concave-convex portion 140. Accordingly, the elongation direction of the cells will be aligned with the extension direction of the two structures, i.e., the first direction. As a result, in the cell sheet developed in the two-dimensional direction along the surface 111, the elongation direction of the cells can be aligned with the one-dimensional direction, i.e., the orientation of the cells can be improved.

[0263] In addition, the configuration satisfying the above (E), especially the configuration in which the front end surface of each convex portion 141 is flush with the flat portion 130, can improve the flatness of the cell sheet formed so as to cover the concave-convex portion 140 and the flat portion 130. Furthermore, the configuration satisfying the above (F) can further improve the flatness of the cell sheet.

[0264] It should be noted that since the surface 111 of the cell sheet forming member 100 includes the flat portion 130 and the concavo-convex portion 140, the same cell sheet forming member 100 can be applied to both cells that adhere predominantly to the flat portion 130 and cells that adhere predominantly to the concavo-convex portion 140. That is, the versatility of the cell sheet forming member 100 can also be improved.

[0265] In addition, the surface 111 of the cell sheet forming member 100 may be coated with an organic substance for the purpose of improving cell adhesion, or may be a surface composed of metal, wherein the organic substance contains, for example, an extracellular matrix such as laminin, collagen, gelatin, fibronectin, polylysine (PDL or PLL), hyaluronic acid, a polymer, an adhesion factor such as gel, etc. In addition, the surface 111 of the cell sheet forming member 100 may have hydrophilicity or hydrophobicity for the purpose of improving cell adhesion and flatness of the cell sheet.

[0266] In addition, in order to easily peel off / recover the cell sheet after the cell sheet is formed, a stimulus-responsive material can be applied. As a stimulus-responsive material, it is preferably a temperature-responsive polymer whose water affinity changes according to temperature changes. Specifically, poly-N-isopropylacrylamide (PIPAAm) is preferably used. The stimulus-responsive material can be applied to the substrate using a conventional coating method, or the substrate treated with the stimulus-responsive material can be structurally processed using the method described below.

[0267] <Method for producing a cell sheet forming member>

[0268] An example of a method for producing a cell sheet forming member will be described. In the following description, an example in which the surface 111 of the cell sheet forming member is formed by transfer of the intaglio plate 150 using the nanoimprint method will be described.

[0269] like Figure 2 As shown, the method for manufacturing the member for forming a cell sheet includes a step of forming the intaglio plate 150 and a step of forming the surface 111 of the member for forming a cell sheet 100 by transferring the intaglio plate 150 .

[0270] The lower surface of the intaglio plate 150 includes: a plurality of flat portions having a shape extending in a first direction (a direction perpendicular to the paper surface) and arranged in a second direction (a left-right direction of the paper surface) intersecting the first direction; and a concave-convex portion consisting of a plurality of height difference structures filling in between adjacent flat portions. The flat portion of the intaglio plate 150 is a portion for forming the flat portion 130 of the cell sheet forming member 100 by transfer. The concave-convex portion of the intaglio plate 150 is a portion for forming the concave-convex portion 140 of the cell sheet forming member 100 by transfer.

[0271] The height difference structure of the gravure 150 is a convex portion or a concave portion. It should be noted that the height difference structure of the gravure 150 in the present embodiment is a concave portion 151 for forming the convex portion 141, and the spacing of the concave portion 151 is greater than 100nm and less than 10μm. In the process of forming the gravure 150, for example, for the silicon substrate used to form the gravure 150, at least one of photolithography, colloidal photolithography, anodization and interference exposure is used to form the concave and convex portions. In addition, the gravure 150 itself can be obtained by transferring from the original plate once or multiple times. For example, for the silicon substrate, a shape corresponding to the surface shape of the gravure 150 is embedded in the original plate using at least one of photolithography, colloidal photolithography, anodization and interference exposure.

[0272] Next, the lower surface of the gravure 150 is made to face the surface 111 of the substrate 160 for forming the cell sheet forming member 100. The forming material of the substrate 160 is, for example, a thermoplastic resin or a photocurable resin. In addition, the lower surface of the gravure 150 is pressed against the surface 111 of the substrate 160 while the substrate 160 has fluidity. Next, the gravure 150 is demolded from the surface 111 of the substrate 160 while suppressing the fluidity of the substrate 160. Thus, the concave portion 151 of the gravure 150 is transferred to the surface 111 of the substrate 160, forming the flat portion 130 and the concave-convex portion 140.

[0273] In order to improve the adhesion of cells, an organic substance containing, for example, an extracellular matrix such as laminin, collagen, gelatin, fibronectin, polylysine (PDL or PLL), hyaluronic acid, a polymer, or an adhesion factor such as gel can be coated on the surface of the thermoplastic resin or photocurable resin forming the substrate 160. In addition, as the forming material of the substrate 160, a biomaterial such as polysaccharide or protein can be used.

[0274] <Method for producing cultured cell sheet>

[0275] A cell sheet produced using the cell sheet forming member 100 will be described.

[0276] In the cell sheet forming substrate satisfying the above (A), if Figure 3As shown in (a), the cells in the cell suspension held in the cell sheet forming member 100 are cells S1 that preferentially adhere to the flat portion 130, and cells S2 that are inferior to the flat portion 130 but allowed to adhere to the concave-convex portion 140. Alternatively, the cells in the cell suspension held in the cell sheet forming member 100 are cells S2 that preferentially adhere to the concave-convex portion 140, and cells S1 that are inferior to the concave-convex portion 140 but allowed to adhere to the flat portion 130.

[0277] In this case, if Figure 3 As shown in (b), the flat portion 130 and the concave-convex portion 140 extend in the first direction and are alternately arranged in the second direction. Therefore, on the surface 111 of the cell sheet forming member, for example, the orientation of the cell S1 that preferentially adheres to the flat portion 130 is regulated by the structure of the flat portion 130 and the structure of the concave-convex portion 140 that divides the flat portion 130.

[0278] Furthermore, in the concavoconvex portion 140 sandwiched between the adjacent flat portions 130, the cell S2 that is inferior to the flat portion 130 but adheres to the concavoconvex portion 140 reflects the orientation control by the flat portion 130. Figure 3 As shown in (c), a cultured cell sheet SA is formed in which the cells S1 and S2 whose orientation is regulated to the first direction are spread over the entire surface 111.

[0279] Alternatively, the orientation of the cell S2 that preferentially adheres to the concave-convex portion 140 is regulated by the structure of the concave-convex portion 140 and the structure of the flat portion 130 that divides it. Furthermore, in the flat portion 130 sandwiched between the adjacent concave-convex portions 140, the orientation regulation by the concave-convex portion 140 is reflected by the cell S1 that adheres to the flat portion 130 despite being at a disadvantage compared to the concave-convex portion 140. The result is as follows Figure 3 As shown in (c), a cultured cell sheet SA is formed in which the cells S1 and S2 whose orientation is regulated to the first direction are spread over the entire surface 111.

[0280] [Evaluation Methods for Compounds or Drugs]

[0281] In the method for evaluating a compound or a drug according to the present embodiment, the compound or drug to be evaluated is allowed to act on the above-mentioned cultured cell sheet.

[0282] Preferably, the compound or drug is evaluated by causing the compound or drug to act on the cultured cell sheet and evaluating at least one change selected from the changes in physiological characteristics and motor function of the cultured cell sheet. As changes in physiological characteristics, changes in BR, Duration, Interval, peak height (Intensity) of calcium transients, etc. in the above-mentioned calcium transient observation can be exemplified, and as changes in motor function, changes in BR, CV, RV, CRD, contraction duration, relaxation duration, etc. obtained based on the above-mentioned live cell imaging observation can be exemplified.

[0283] The number of days the cultured cell sheet is cultured during evaluation is not particularly limited, but from the viewpoint of achieving a higher degree of maturity of cardiomyocytes, it is suitable to use a cultured cell sheet cultured for about 15 days for evaluation.

[0284] The compound or drug to be evaluated is not particularly limited, but is preferably an existing compound or a candidate compound thereof that is known to have an effect on cardiomyocytes, preferably, for example, at least one existing compound or a candidate compound thereof selected from the group consisting of INa blockers, Ikr blockers, Iks blockers, ICa blockers, 5-HT4 receptor agonists, α receptor blockers, β receptor blockers, α receptor agonists, β receptor agonists, toxic substances, anticancer agents, lipid metabolism inhibitors, phosphodiesterase 3 inhibitors and other physiologically active substances (such as proteins, peptides, antibodies, DNA, RNA, etc.).

[0285] Examples of existing INa blockers include quinidine and propranolol.

[0286] Examples of existing Ikr inhibitors include E-4031, quinidine, sotalol, cisapride, and bepridil.

[0287] Examples of existing Iks inhibitors include bepridil and chromanol.

[0288] Examples of existing beta blockers include sotalol, propranolol, and carvedilol.

[0289] As a conventional α blocker, carvedilol can be exemplified.

[0290] Examples of existing β receptor agonists include isoproterenol.

[0291] Examples of existing phosphodiesterase 3 inhibitors include milrinone.

[0292] [Quality evaluation method of cultured cell sheets]

[0293] According to the quality evaluation method of the cultured cell sheet of the present embodiment, a quality evaluation method of a cultured cell sheet can be provided for evaluating whether a cultured cell sheet composed of cardiomyocytes is a cultured cell sheet for maturation advancement and regenerative medicine, or a cultured cell sheet that can be suitable for a compound or a drug. In addition, by having the above-mentioned quality evaluation method as a process in the manufacturing method of the cultured cell sheet, a cultured cell sheet with better quality can be provided. That is, in the present embodiment, it is also preferred to integrate the above-mentioned quality evaluation method as a process in the manufacturing method of the cultured cell sheet.

[0294] It is preferred that at least one of the following (i) to (xii) is satisfied, more preferred that at least (i) is satisfied and at least one of (ii) to (xii) is satisfied, and even more preferred that all of (i) to (xii) are satisfied.

[0295] (i) In an image obtained by immunostaining the aforementioned cultured cell sheet with an anti-α-actinin antibody and observing it under a microscope, the angle of the length direction of the rod-like structures detected by the α-actinin antibody within the measurement range (71.6 μm×71.6 μm) when the horizontal direction of the image is set to 0° is measured, and the frequency of the rod-like structures contained within ±15° of the mode value, that is, the orientation degree calculated according to the following formula (1), is 23% or more.

[0296] Orientation degree (%) = (number of rod-like structures within ±15° of the mode value) / (total number of rod-like structures) × 100 (1)

[0297] (ii) In live cell imaging, the number of vectors showing an angle of ±5° from the angle of the mode value of the motion vector (degree of orientation) is 12% or more.

[0298] (iii) Satisfy at least one of the following requirements A1 and A2.

[0299] Requirement A1: The ratio of the expression level of the MYL3 (myosin light chain 3) gene to the expression level of the ACTB (β-actin) gene (MYL3 / ACTB) is 12.0 or more. After the seventh day of culture, MYL3 / ACTB is preferably 12.0 or more.

[0300] Requirement A2: The ratio of the expression level of the MYH7 (myosin heavy chain 7) gene to the expression level of the MYH6 (myosin heavy chain 6) gene (MYH7 / MYH6) is 6.0 or more. After the seventh day of culture, MYH7 / MYH6 is preferably 6.0 or more.

[0301] (iv) Satisfy at least one of the following requirements B1 and B2.

[0302] Requirement B1: The ratio of the gene expression level of CKM (creatine kinase, M type) to the gene expression level of ACTB (β-actin) (CKM / ACTB) is 1.80 or more. On the 15th to 45th day of culture, CKM / ACTB is preferably 1.80 or more.

[0303] Requirement B2: The ratio of the gene expression level of LDHA (lactate dehydrogenase A subunit) to the gene expression level of ACTB (LDHA / ACTB) is 0.80 or more. After the seventh day of culture, LDHA / ACTB is preferably 0.80 or more, and more preferably 1.2 or more.

[0304] (v) Satisfy at least any one of the following requirements C1 to C4.

[0305] Requirement C1: The ratio of the gene expression level of CACNA2D1 (voltage-gated calcium channel auxiliary subunit α2δ1) to the gene expression level of ATP1A1 (ATPase Na+ / K+ transport subunit α1) (CACNA2D1 / ATP1A1) is 0.085 or more on any day from the 15th day to the 30th day of culture.

[0306] Requirement C2: The ratio of the gene expression level of KCNJ2 (inwardly rectifier potassium channel subfamily J member 2) to the gene expression level of ATP1A1 (ATPase Na+ / K+ transport subunit α1) (KCNJ2 / ATP1A1) is 0.038 or more on any day from the 15th day to the 30th day of culture.

[0307] Requirement C3: The ratio of the gene expression level of KCNE1 (voltage-gated potassium channel subfamily E regulatory subunit 1) to the gene expression level of ATP1A1 (ATPase Na+ / K+ transport subunit α1) (KCNE1 / ATP1A1) is 0.003 or more on any day from the 15th day to the 30th day of culture.

[0308] Requirement C4: The ratio of the gene expression level of SCN5A (voltage-gated sodium channel α subunit 5) to the gene expression level of ATP1A1 (ATPase Na+ / K+ transport subunit α1) (SCN5A / ATP1A1) is 0.87 or more on any day from the 15th day to the 30th day of culture.

[0309] (vi) Satisfy at least any one of the following requirements D1 to D4.

[0310] Requirement D1: The ratio of the gene expression level of LPL (lipoprotein lipase) to the gene expression level of ACTB (β-actin) (LPL / ACTB) is 0.45 or more on any day from the 15th day to the 45th day of culture.

[0311] Requirement D2: The ratio of the gene expression level of ACAT1 (acetyl-CoA acetyltransferase 1) to the gene expression level of ACTB (β-actin) (ACAT1 / ACTB) is 0.43 or more on any day from the 15th day to the 30th day of culture.

[0312] Requirement D3: The ratio of the gene expression level of HADHA (hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit α) to the gene expression level of ACTB (β-actin) (HADHA / ACTB) is 0.75 or more on any day from the 15th day to the 30th day of culture.

[0313] Requirement D4: The ratio of the gene expression level of HADHB (hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit β) to the gene expression level of ACTB (β-actin) (HADHB / ACTB) is 0.95 or more on any day from the 15th day to the 30th day of culture.

[0314] (vii) Satisfy at least any one of the following requirements E1 to E8.

[0315] Requirement E1: The ratio of the gene expression level of PPARGC1A (PPARG (peroxisome proliferator-activated receptor γ) coactivator 1α) to the gene expression level of HIF1A (hypoxia-inducible factor 1α) (PPARGC1A / HIF1A) is 0.80 or more on any day of culture.

[0316] Requirement E2: The ratio of the gene expression level of ESRRA (estrogen related receptor α) to the gene expression level of HIF1A (hypoxia inducible factor 1α) (ESRRA / HIF1A) is 0.55 or more on any day of culture.

[0317] Requirement E3: The ratio of the gene expression level of VEGFA (vascular endothelial growth factor A) to the gene expression level of HIF1A (hypoxia-inducible factor 1α) (VEGFA / HIF1A) is 0.65 or more on any day of culture.

[0318] Requirement E4: The ratio of the gene expression level of APLN (Apelin) to the gene expression level of HIF1A (hypoxia-inducible factor 1α) (APLN / HIF1A) is 0.005 or more on any day of culture.

[0319] Requirement E5: The ratio of the gene expression level of FABP3 (fatty acid binding protein 3) to the gene expression level of HIF1A (hypoxia inducible factor 1α) (FABP3 / HIF1A) is 5.0 or more on any day of culture.

[0320] Requirement E6: The ratio of the gene expression level of ESM1 (endothelial cell-specific molecule 1) to the gene expression level of HIF1A (hypoxia-inducible factor 1α) (ESM1 / HIF1A) is 0.002 or more on any day of culture.

[0321] Requirement E7: The ratio of the gene expression level of EMCN (endothelial mucin) to the gene expression level of HIF1A (hypoxia-inducible factor 1α) (EMCN / HIF1A) is 0.002 or more on any day after the 7th day of culture.

[0322] Requirement E8: The ratio of the gene expression level of BCL2 (BCL2 apoptosis regulator) to the gene expression level of HIF1A (hypoxia-inducible factor 1α) (BCL2 / HIF1A) is 0.013 or more on any day after the 7th day of culture.

[0323] (viii) The area of ​​each mitochondria is 200 μm 2 above.

[0324] (ix) When the contraction velocity detected in live cell imaging is denoted as CV (m / sec) and the relaxation velocity is denoted as RV (m / sec), the following formula (2) is satisfied.

[0325] 1.0≤CV / RV≤2.8 (2)

[0326] (x) In calcium imaging analysis, when the peak height of the calcium transient waveform is 100%, the waveform width of 20% height is Duration (seconds), and the interval between peaks is Interval (seconds), the following formula (3) is satisfied.

[0327] Duration / (Interval) 1 / 2 ≥0.68 (3)

[0328] (xi) The duration of 80% repolarization of the action potential of the cells constituting the cultured cell sheet in a patch clamp test in a 25°C solution is 600 msec or longer, and the maximum diastolic potential is -60 mV or less.

[0329] (xii) In an oxygen consumption rate measurement that measures the rate at which cells use oxygen contained in a culture medium, the oxygen consumption rate is reduced to 20% or less compared to before the addition of the β-oxidation inhibitor by adding a β-oxidation inhibitor that inhibits the β-oxidation activity of fatty acids.

[0330] Example

[0331] Hereinafter, although an Example is given in order to demonstrate this invention concretely, this invention is not limited to these Examples.

[0332] (1) Culture of cardiomyocytes derived from iPS cells-1 (CM1)

[0333] As human iPS cell-derived cardiomyocytes, iCell cardiomyocytes (iCell Cardiomyocyte v1.0, manufactured by FUJIFILM Cellular Dynamics, Inc.) (hereinafter referred to as CM1) were used. Cultivation was performed according to the following procedure.

[0334] The frozen cell sample was thawed by heating in a 37°C water bath for 3 minutes. The cell solution was transferred to a 50 mL centrifuge tube, and 9 mL of a plate culture medium (iCell cardiomyocyte thawing medium, manufactured by FUJIFILM Cellular Dynamics) pre-warmed to 37°C was added and mixed to dilute the cell solution.

[0335] A portion of the diluted cell suspension was collected and mixed with an equal amount of Trypan Blue, and the number of viable cells was determined using a hemocytometer. The cell concentration was adjusted to 1×10 6 cells / 2mL(=5×10 5 / mL), and 2mL of each was inoculated into a 6-well plate pre-coated with gelatin.

[0336] For gelatin coating of culture vessels, sterilized 0.1% gelatin solution was added to the vessel (2 mL / well) and allowed to stand for one hour at 37° C. The gelatin solution was removed before use and cells were seeded.

[0337] After inoculation, the cells were incubated at 37°C in a CO 2 The cells were left to stand in an incubator, with the day of inoculation designated as day 0, and the medium was replaced on day 3 of culture.

[0338] In order to re-seed on the fifth day of culture, the cells were detached and recovered with trypsin. A portion of the recovered cell suspension was collected and mixed with an equal amount of trypan blue to determine the number of viable cells. The cells were adjusted to a concentration of 8 × 10 4 40 μL of 0.05 mg / mL fibronectin was added to a 96-well plate coated with 0.1 mL of fibronectin for more than 2 hours. The 96-well plate was prepared using ND Cell Aligner (alignment culture plate, 96-well plate type). As a control, a commercially available 96-well plate with a flat culture surface (flat culture plate) was used.

[0339] After inoculation, the cells were incubated at 37°C in a CO 2 The cells were left to stand in an incubator for 4 hours, and the medium was replaced with a maintenance medium (iCell cardiomyocyte maintenance medium, manufactured by FUJIFILM Cellular Dynamics) every two days, and maintenance culture was performed for a predetermined period of time.

[0340] (2) Culture of cardiomyocytes derived from iPS cells-2 (CM2)

[0341] As human iPS cell-derived cardiomyocytes, iCell cardiomyocytes (iCell Cardiomyocyte v2.0, manufactured by FUJIFILM Cellular Dynamics, Inc.) (hereinafter referred to as CM2) were used. Cultivation was performed according to the following procedure.

[0342] The frozen cell sample was thawed by heating in a 37°C water bath for 3 minutes. The cell suspension was transferred to a 50 mL centrifuge tube, and 9 mL of plate culture medium pre-warmed to 37°C was added and mixed to dilute the cell suspension. A portion of the diluted cell suspension was collected and mixed with an equal amount of trypan blue, and the number of live cells was determined using a hemocytometer.

[0343] The cell concentration was adjusted to 6 × 10 cells / mL using plating medium. 4 The cells were seeded at a concentration of 10 cells / well in a 96-well plate pre-coated with fibronectin. The 96-well plate was ND Cell Aligner (alignment culture plate, 96-well plate type), and as a control, the cells were seeded in a commercially available 96-well plate with a flat culture surface (flat culture plate).

[0344] at 37°C in CO 2 After standing in the incubator for 4 hours, the medium was replaced with a maintenance medium. The medium of the cells after the medium replacement was replaced every two days, and the cells were maintained for a predetermined period of time.

[0345] The ND Cell Aligner used in (1) and (2) above has a flat portion and a concave-convex portion, each flat portion has a shape extending in a first direction, and is arranged in a second direction intersecting the first direction on the entire surface, and the width of each flat portion (the length in the second direction) is 10μm. Each concave-convex portion is composed of a plurality of height difference structures filling adjacent flat portions, the length in the second direction between each flat portion is 10μm, and the spacing between the convex portions in the concave-convex portion is 300nm. The result of measuring the height of each convex portion in the concave-convex portion using AFM is as follows: the average height from the bottom surface of the concave portion to the front end of the convex portion is 446nm. In addition, the average height from the bottom surface of the concave portion to the flat portion is 455nm.

[0346] (3) Analysis of Orientation

[0347] Using iCell cardiomyocytes CM2 maintained in culture, the sarcomere structure was immunostained with an anti-α-actinin antibody and the nucleus was stained with Hoechst 33342 on days 15, 30, 45, and 60. Fluorescence images were obtained using a fluorescence microscope.

[0348] Immunostaining was performed according to the following steps. The culture medium was removed from the well plate after the specified number of days of culture, and the cells were washed with PBS. Next, 4% paraformaldehyde was added for fixation. After washing the cells with PBST (0.1% tween 20), an anti-α-actinin antibody (manufactured by abcam, Anti-Sarcomeric α Actinin antibody [EA-53]) was added as the primary antibody and allowed to react overnight at 4°C. After washing with PBST (0.1% tween 20) and removing the primary antibody, a secondary antibody (Alexa Fluor 647 labeled, manufactured by life technologies, Alexa Fluor(R) 647 F{ab} 2 Goat anti-mouse IgG (H+L) antibody fragment) was reacted, and then diluted Hoechst 33342 was added to stain the nucleus.

[0349] Fluorescence images were acquired at 60 times (objective lens), sarcomeres were detected at an excitation wavelength of 640 nm / fluorescence wavelength of 685 nm, and nuclei were detected at an excitation wavelength of 405 nm / fluorescence wavelength of 461 nm.

[0350] From the image observed with a 60x objective lens (size: area of ​​215 μm×215 μm), angle distribution analysis using image analysis software (A-ZO-KUN, manufactured by Asahi Kasei Engineering Corporation) was used to detect the rod-like structures detected using α-actinin, and the angle distribution (0 to 180°) of the rod-like structures was obtained when the horizontal direction of the image was set to 0°.

[0351] The degree of orientation was determined by the following formula from the number of rod-like structures contained in the range of ±15° of the angle of the mode value of the angle distribution and the total number of rod-like structures contained in the measurement range.

[0352] Orientation degree (%) = (number of rod-like structures within ±15° of the mode value) / (total number of rod-like structures) × 100

[0353] Image analysis was performed at n = 3 to 6, and the average value was obtained. On the 15th and 30th days of culture, images obtained from different multiple wells (the upper left partition of the 9 equally divided partitions) were used for analysis. On the 45th and 60th days of culture, images obtained from one well were divided into 9 equally divided images, and analysis was performed using 3 partitions (the upper left, center, and lower right partitions).

[0354] The results of the angular distribution analysis were expressed as the orientation of the rod-like structures contained in every 10°, and the orientation of the oriented culture plate and the flat culture plate within the range of ±15° including the maximum value were compared. Regarding the statistical test, a T test was performed on the orientation of the oriented culture plate and the flat culture plate, and it was confirmed that: after 30 days of culture, the orientation of the oriented culture plate and the flat culture plate had a significant difference at a risk rate of 5%.

[0355] The results of the orientation measurement showed that after the 15th day of culture, the orientation of CM2 cultured on the oriented culture plate was higher than that of CM2 cultured on the flat culture plate. On the 30th to 60th day of culture, the orientation of the oriented culture plate was more than 23%. On the other hand, the orientation of the flat culture plate was less than 21%. Figure 4 shown.

[0356] (4) Maturation confirmation test (gene expression)

[0357] The cells of iCell cardiomyocytes (CM1) cultured on orientation culture plates and flat culture plates were collected on the 1st, 7th, 15th, 30th, and 45th days of culture and analyzed for gene expression.

[0358] The collected cells were broken to extract RNA (using NucleoSpinRNAPlus XS manufactured by Takara Bio Inc.), and the SMART-Seq v4 Low Input Kit was used for reverse transcription and library construction. Next, RNA sequencing (comprehensive gene expression analysis) was performed to obtain gene expression data. As an analysis program, RaNA-seq (Reference 3: Bioinformatics, 2020 Mar 10: 36 (6), 1955-1956) was used. Using the characteristic gene expression changes in the mature stage of myocardium derived from human iPS cells (Reference 4: Circ. Res. 2020 Apr 10: 126 (8), 1086-1106) as an evaluation index, the time-dependent changes in gene expression levels of cardiomyocytes cultured on oriented culture plates and flat culture plates were compared.

[0359] Regarding (4-1) to (4-3) below, samples used for RNA sequencing were prepared by mixing equal amounts of RNA extracted from five wells (based on the mass of RNA) and using them in the experiment. The results of gene expression analysis based on RNA sequencing are considered to be the average value of the five wells.

[0360] In addition, regarding the following (4-4) to (4-6), the samples used for RNA sequencing were RNA extracted from three wells and RNA sequencing was performed to obtain the average value of the expression level (CPM). For the expression level (CPM) of each gene, a T test was performed to detect the difference in gene expression levels in the flat culture plate and the oriented culture plate at each culture day. The case where the P value is 0.01≤P<0.05 is indicated as * in the figure, and the case where the P value is P<0.01 is indicated as ** in the figure.

[0361] As a result of gene expression analysis based on RNA sequencing, the following results were obtained.

[0362] (4-1)MYL3 / ACTB

[0363] The CM1 cultured on the oriented culture plate showed an increase in the gene expression of MYL3 (myosin light chain 3) expressed in mature myocardium compared to the CM1 cultured on the flat culture plate. On the other hand, the expression level of the ACTB (β-actin) gene, which is assumed to be expressed at a constant level regardless of the tissue and culture period, was at the same level in the CM1 cultured on the oriented culture plate and the CM1 cultured on the flat culture plate.

[0364] In each culture plate, the ratio of the expression level of the MYL3 gene to the expression level of the ACTB gene after the 7th day of culture was 12.5 or more in the oriented culture plate and 11.4 or less in the flat culture plate ( Figure 5 ).

[0365] (4-2)MYH7 / MYH6

[0366] As an isomeric change accompanying the maturation of the fetal myocardium to the adult myocardium, the isomeric change from MYH6 (myosin heavy chain 6) to MYH7 (myosin heavy chain 7) was confirmed to be more significant. In each culture plate, the ratio of the expression level of the MYH7 gene to the expression level of the MYH6 gene after the 7th day of culture was 7.1 or more in CM1 cultured on the oriented culture plate, and 5.4 or less in CM1 cultured on the flat culture plate ( Figure 6 ).

[0367] (4-3)CKM, CKM / ACTB, COX6A2, COX6A2 / ACTB, CKMT2, CKMT2 / ACTB, LDHA, LDHA / ACTB

[0368] Compared with CM1 cultured on a flat plate, the expression levels of CKM (creatine kinase, M-type) related to myocardial contraction, COX6A2 (cytochrome C oxidase subunit 6A2) localized in mitochondria, CKMT2 (mitochondrial creatine kinase 2), and glycolytic enzyme LDHA (lactate dehydrogenase A subunit) increased over time in CM1 cultured on an oriented plate. Figure 7-1 , Figure 7-2 ).

[0369] After the 15th day of culture, the expression level of the CKM gene was 1161 TPM (Transcripts Per Kilobase Million) or more in CM1 cultured on the oriented culture plate, while it was 824 TPM or less in CM1 cultured on the flat culture plate. Figure 7-1 (A)).

[0370] After the 15th day of culture, the expression level of COX6A2 gene was 875 TPM or more in CM1 cultured on the oriented culture plate, while it was 842 TPM or less in CM1 cultured on the flat culture plate ( Figure 7-1 (B)).

[0371] After the 30th day of culture, the expression level of CMKT2 gene was 377 TPM or more in CM1 cultured on the oriented culture plate, while it was 325 TPM or less in CM1 cultured on the flat culture plate ( Figure 7-1 (C)).

[0372] In addition, after the 7th day of culture, the expression level of LDHA gene was 682 TPM or more in CM1 cultured on the oriented culture plate, while it was 372 TPM or less in CM1 cultured on the flat culture plate ( Figure 7-1 (D)).

[0373] In each culture plate, the ratio of the expression level of the CKM gene to the expression level of the ACTB gene after the 15th day of culture was 2.1 or more in CM1 cultured on the oriented culture plate, and was 1.7 or less in CM1 cultured on the flat culture plate ( Figure 7-2 (A)).

[0374] The ratio of the expression level of COX6A2 gene to the expression level of ACTB gene after the 15th day of culture was 1.95 or more in CM1 cultured on the oriented culture plate, and was 1.92 or less in CM1 cultured on the flat culture plate ( Figure 7-2 (B)).

[0375] In addition, the ratio of the expression level of the CKMT2 gene to the expression level of the ACTB gene on the 15th to 30th day of culture was 0.68 or more in CM1 cultured on the oriented culture plate, and was 0.57 or less in CM1 cultured on the flat culture plate ( Figure 7-2 (C)).

[0376] Furthermore, the ratio of the expression level of the LDHA gene to the expression level of the ACTB gene after the 7th day of culture was 1.3 or more in CM1 cultured on the oriented culture plate, and was 0.6 or less in CM1 cultured on the flat culture plate ( Figure 7-2 (D)).

[0377] (4-4)CACNA2D1, CACNA2D1 / ATP1A1, KCNJ2, KCNJ2 / ATP1A1, KCNE1, KCNE1 / ATP1A1, SCN5A, SCN5A / ATP1A1

[0378] Compared with CM1 cultured on flat culture plates, the expression levels of CACNA2D1 (voltage-gated calcium channel auxiliary subunit α2δ1) related to the formation of cardiac calcium channels, KCNJ2 (inward rectifier potassium channel subfamily J member 2) related to the formation of cardiac potassium channels, KCNE1 (voltage-gated potassium channel subfamily E regulatory subunit 1) related to the formation of cardiac sodium channels, and SCN5A (voltage-gated sodium channel α subunit 5) related to the formation of cardiac sodium channels increased over time in CM1 cultured on oriented culture plates ( Figure 7-3 , Figure 7-4 ).

[0379] On the 15th to 30th day of culture, the expression level of the CACNA2D1 gene was 2469 CPM (counts per million) or more in CM1 cultured on the oriented culture plate, while it was 2004 CPM or less in CM1 cultured on the flat culture plate ( Figure 7-3 (A)).

[0380] After the 15th day of culture, the expression level of KCNJ2 gene was 1026 CPM or more in CM1 cultured on the oriented culture plate, while it was 926 CPM or less in CM1 cultured on the flat culture plate ( Figure 7-3 (B)).

[0381] On the 15th to 45th day of culture, the expression level of the KCNE1 gene was 79 CPM or more in CM1 cultured on the oriented culture plate, while it was 42 CPM or less in CM1 cultured on the flat culture plate ( Figure 7-3 (C)).

[0382] In addition, on the 15th to 45th day of culture, the expression level of the SCN5A gene was 23580 CPM or more in CM1 cultured on the oriented culture plate, while it was 21571 CPM or less in CM1 cultured on the flat culture plate ( Figure 7-3 (D)).

[0383] In each culture plate, the ratio of the expression level of the CACNA2D1 gene to the expression level of the ATP1A1 gene on the 15th to 30th day of culture was 0.085 or more in CM1 cultured on the oriented culture plate, and was 0.23 or less in CM1 cultured on the flat culture plate ( Figure 7-4 (A)).

[0384] The ratio of the expression level of the KCNJ2 gene to the expression level of the ATP1A1 gene on the 15th to 30th day of culture was 0.038 or more in CM1 cultured on the oriented culture plate, and was 0.115 or less in CM1 cultured on the flat culture plate ( Figure 7-4 (B)).

[0385] In addition, the ratio of the expression level of the KCNE1 gene to the expression level of the ATP1A1 gene after the 15th day of culture was 0.003 or more in CM1 cultured on the oriented culture plate, and was 0.007 or less in CM1 cultured on the flat culture plate ( Figure 7-4 (C)).

[0386] Furthermore, the ratio of the expression level of the SCN5A gene to the expression level of the ATP1A1 gene on the 15th to 30th day of culture was 0.87 or more in CM1 cultured on the oriented culture plate, and was 2.6 or less in CM1 cultured on the flat culture plate ( Figure 7-4 (D)).

[0387] (4-5)LPL, LPL / ACTB, ACAT1, ACAT1 / ACTB, HADHA, HADHA / ACTB, HADHB, HADHB / ACTB

[0388] Compared with CM1 cultured on a flat plate, the expression levels of LPL (lipoprotein lipase), ACAT1 (acetyl-CoA acetyltransferase 1), HADHA (hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit α), and HADHB (hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit β) localized in the mitochondria of the myocardium and related to β-oxidation of fatty acids increased over time in CM1 cultured on an oriented plate. Figure 7-5 , Figure 7-6 ).

[0389] After the 15th day of culture, the expression level of LPL gene was 3832 CPM (count per million) or more in CM1 cultured on the oriented culture plate, while it was 3024 CPM or less in CM1 cultured on the flat culture plate ( Figure 7-5 (A)).

[0390] On the 30th to 45th day of culture, the expression level of the ACAT1 gene was 3663 CPM or more in CM1 cultured on the oriented culture plate, while it was 3357 CPM or less in CM1 cultured on the flat culture plate ( Figure 7-5 (B)).

[0391] On the 30th to 45th day of culture, the expression level of the HADHA gene was 6240 CPM or more in CM1 cultured on the oriented culture plate, while it was 5920 CPM or less in CM1 cultured on the flat culture plate ( Figure 7-5 (C)).

[0392] In addition, after the 15th day of culture, the expression level of the HADHB gene was 8505 CPM or more in CM1 cultured on the oriented culture plate, while it was 8072 CPM or less in CM1 cultured on the flat culture plate ( Figure 7-5 (D)).

[0393] In each culture plate, the ratio of the expression level of the LPL gene to the expression level of the ACTB gene after the 15th day of culture was 0.46 or more in CM1 cultured on the oriented culture plate, and was 0.43 or less in CM1 cultured on the flat culture plate ( Figure 7-6 (A)).

[0394] The ratio of the expression level of the ACAT1 gene to the expression level of the ACTB gene on the 15th to 30th day of culture was 0.47 or more in CM1 cultured on the oriented culture plate, and was 0.42 or less in CM1 cultured on the flat culture plate ( Figure 7-6 (B)).

[0395] In addition, the ratio of the expression level of the HADHA gene to the expression level of the ACTB gene on the 15th to 30th day of culture was 0.79 or more in CM1 cultured on the oriented culture plate, and was 0.74 or less in CM1 cultured on the flat culture plate ( Figure 7-6 (C)).

[0396] Furthermore, the ratio of the expression level of the HADHB gene to the expression level of the ACTB gene on the 15th to 30th day of culture was 1.00 or more in CM1 cultured on the oriented culture plate, and was 0.91 or less in CM1 cultured on the flat culture plate ( Figure 7-6 (D)).

[0397] (4-6)PPARGC1A, PPARGC1A / HIF1, ESRRA, ESRRA / HIF1, VEGFA, VEGFA / HIF1, APLN, APLN / HIF1, FABP3, FABP3 / HIF1, ESM1, ESM1 / HIF1, EMCN, EMCN / HIF1, BCL2, BCL2 / ACTB

[0398] Compared with CM1 cultured on flat culture plates, the expression levels of PPARGC1A gene (PPARG coactivator 1α) encoding PGC-1α related to mitochondrial biogenesis and function, ESRRA gene (estrogen related receptor α) encoding estrogen related receptor ERR related to cardiomyocyte maturation, VEGFA gene (vascular endothelial growth factor A) encoding vascular growth factor, APLN gene (Apelin) encoding myokine related to cardiac contraction in myocardial tissue, FABP3 gene (fatty acid binding protein 3) encoding myokine regulating the uptake of long-chain fatty acids in the myocardium, ESM1 gene (endothelial cell specific molecule 1) expressed specifically in endothelial cells, EMCN gene (endothelial mucin) encoding mucin-like glycoprotein secreted by epithelial cells, and BCL2 gene (BCL2 apoptosis regulator) related to mitochondrial function regulation increased over time in CM1 cultured on oriented culture plates ( Figure 7-7 to Figure 7-10 ).

[0399] After the 15th day of culture, the expression level of the PPARGC1A gene was 4577 CPM (count per million) or more in CM1 cultured on the oriented culture plate, while it was 4282 CPM or less in CM1 cultured on the flat culture plate ( Figure 7-7 (A)).

[0400] After the 15th day of culture, the expression level of the ESRRA gene was 3081 CPM or more in CM1 cultured on the oriented culture plate, while it was 3061 CPM or less in CM1 cultured on the flat culture plate ( Figure 7-7 (B)).

[0401] After the 7th day of culture, the expression level of the VEGFA gene was 3737 CPM or more in CM1 cultured on the oriented culture plate, while it was 3237 CPM or less in CM1 cultured on the flat culture plate ( Figure 7-7 (C)).

[0402] In addition, after the 7th day of culture, the expression level of the APLN gene was 28.7 CPM or more in CM1 cultured on the oriented culture plate, while it was 20.7 CPM or less in CM1 cultured on the flat culture plate ( Figure 7-7 (D)).

[0403] After the 15th day of culture, the expression level of FABP3 gene was 28788 CPM or more in CM1 cultured on the oriented culture plate, while it was 28529 CPM or less in CM1 cultured on the flat culture plate ( Figure 7-9 (A)).

[0404] After the 30th day of culture, the expression level of the ESM1 gene was 40 CPM or more in CM1 cultured on the oriented culture plate, while it was 1.3 CPM or less in CM1 cultured on the flat culture plate ( Figure 7-9 (B)).

[0405] After the 15th day of culture, the expression level of the EMCN gene was 24.6 CPM or more in CM1 cultured on the oriented culture plate, while it was 1 CPM or less in CM1 cultured on the flat culture plate ( Figure 7-9 (C)).

[0406] After the 7th day of culture, the expression level of the BCL2 gene was 83.3 CPM or more in CM1 cultured on the oriented culture plate, while it was 58.3 CPM or less in CM1 cultured on the flat culture plate ( Figure 7-9 (D)).

[0407] In each culture plate, the ratio of the expression level of the PPARGC1A gene to the expression level of the HIF1 gene after the 7th day of culture was 0.86 or more in CM1 cultured on the oriented culture plate, and was 0.80 or less in CM1 cultured on the flat culture plate ( Figure 7-8 (A)).

[0408] The ratio of the expression level of the ESRRA gene to the expression level of the HIF1 gene after the 7th day of culture was 0.57 or more in CM1 cultured on the oriented culture plate, and was 0.54 or less in CM1 cultured on the flat culture plate ( Figure 7-8 (B)).

[0409] In addition, the ratio of the expression level of the VEGFA gene to the expression level of the HIF1 gene after the 7th day of culture was 0.71 or more in CM1 cultured on the oriented culture plate, and was 0.64 or less in CM1 cultured on the flat culture plate ( Figure 7-8 (C)).

[0410] Furthermore, the ratio of the expression level of the APLN gene to the expression level of the HIF1 gene after the 7th day of culture was 0.013 or more in CM1 cultured on the oriented culture plate, and was 0.004 or less in CM1 cultured on the flat culture plate ( Figure 7-8 (D)).

[0411] Furthermore, the ratio of the expression level of the FABP3 gene to the expression level of the HIF1 gene after the 7th day of culture was 5.3 or more in CM1 cultured on the oriented culture plate, and was 5.2 or less in CM1 cultured on the flat culture plate ( Figure 7-10 (A)).

[0412] Furthermore, the ratio of the expression level of the ESM1 gene to the expression level of the HIF1 gene after the 7th day of culture was 0.001 or more in CM1 cultured on the oriented culture plate, and was 0.0003 or less in CM1 cultured on the flat culture plate ( Figure 7-10 (B)).

[0413] Furthermore, the ratio of the expression level of the EMCN gene to the expression level of the HIF1 gene after the 7th day of culture was 0.003 or more in CM1 cultured on the oriented culture plate, and was 0.001 or less in CM1 cultured on the flat culture plate ( Figure 7-10 (C)).

[0414] Furthermore, the ratio of the expression level of the BCL2 gene to the expression level of the HIF1 gene on the 7th to 30th day of culture was 0.018 or more in CM1 cultured on the oriented culture plate, and was 0.012 or less in CM1 cultured on the flat culture plate ( Figure 7-10 (D)).

[0415] (5) Maturation confirmation test Confirmation of mitochondrial activity

[0416] Using iCell cardiomyocyte CM2 cells that had been maintained in culture, cells on day 9 were stained with membrane potential-dependent fluorescent dye Mito-tracker Red (M7510, manufactured by Thermo Fisher Scientific) to stain mitochondria, and Hoechst 33342 to stain nuclei. For staining, the medium was removed from the well plate, and Mito-tracker Red diluted to a predetermined concentration with the maintenance medium was added, and the cells were incubated at 37°C under CO 2 After standing in the incubator for 30 minutes, Hoechst 33342 diluted to a specified concentration using maintenance medium was added and the cells were incubated at 37°C with CO 2 The cells were left to stand in an incubator for 30 minutes. The cells were washed with a culture medium before observation, and fluorescent images were obtained using a confocal fluorescence microscope (Confocal Quantitative Imaging Cytometer CQ1, manufactured by Yokogawa Electric Corporation).

[0417] Images were acquired at a magnification of 60 times (objective lens), mitochondria were detected at an excitation wavelength of 561 nm / fluorescence wavelength of 617 nm, and nuclei were detected at an excitation wavelength of 405 nm / fluorescence wavelength of 461 nm.

[0418] The image was analyzed using analysis software (CellPathfinder, manufactured by Yokogawa Electric Corporation). The area and intensity detected at a fluorescence wavelength of 617 nm were measured from the measurement range of the visual field observed at a magnification of 60 times the objective lens (size: area of ​​215 μm×215 μm), and the number of nuclei detected at a fluorescence wavelength of 461 nm was measured.

[0419] It should be noted that the detection value of the fluorescence wavelength 617nm is the value detected by the detector of the camera of CQ1, and the area (Area) is the total area detected by the fluorescence wavelength 617nm (unit: μm 2 ), Intensity is the sum of the brightness values ​​detected by the detector. Intensity is measured at full scale under the same conditions.

[0420] The analysis was performed at n = 6 to 10. The area (Area) and brightness (Intensity) were corrected by the number of nuclei, and the area (Area) and brightness (Intensity) of each cell were compared. The statistical test was performed by T-test, and a significant difference was confirmed at a risk rate of 1%.

[0421] It should be noted that area refers to the number of mitochondria, and intensity refers to the activity of mitochondria. Figure 8 Error bars show standard error.

[0422] like Figure 8 As shown, on the 9th day of culture, the number and activity of mitochondria in CM2 cultured on oriented culture plates were significantly higher than those in CM2 cultured on flat culture plates.

[0423] In addition, Fig. 9 Figure 2 shows fluorescent images of CM2 cultured on the oriented culture plate on day 9 and CM2 cultured on the flat culture plate on day 9. Nuclei are shown in blue and mitochondria are shown in red. As can be seen from the photographs, the number of mitochondria and mitochondrial activity increased in CM2 cultured on the oriented culture plate.

[0424] (6) Maturation confirmation test Analysis based on motility test method using live cell imaging device

[0425] In order to detect the changes in movement associated with contraction / relaxation of cardiomyocytes inoculated on each culture plate, animated data were obtained using SI8000 (manufactured by Sony Corporation). Regarding the measurement conditions, phase contrast images, magnification 10 times (objective lens), 150 frames / second, resolution 2048×2048 pixels, and 8-bit depth were used. The image acquisition time was 10 seconds. The cells used CM2, which was cultured on the 15th, 31st, 45th, and 61st days with the day of inoculation as day 0.

[0426] The motion vectors (speed, direction, and number) associated with the contraction / relaxation of myocardial cells were detected from the animation data, and each parameter (heart rate (beating rate; BR), contractile velocity (contractile velocity; CV), relaxation velocity (relaxation velocity; RV), contraction-relaxation duration (contraction-relaxation duration; CRD), and orientation) were analyzed.

[0427] Regarding the orientation degree, the angle of the motion vector when the horizontal direction of the image is set to 0° is obtained, and the ratio of the number of vectors showing an angle of ±5° from the angle of the mode value of the vector angle to the total number of detected vectors is calculated according to the following formula.

[0428] Orientation degree (%)

[0429] = (the number of vectors within the ±5° range of the mode value) / (the total number of vectors) × 100

[0430] exist Fig.10 An example of the measurement results of the angle of the motion vector and its frequency is shown in FIG.

[0431] exist Figure 11-1 The results of CM2 in which cells were seeded on each culture plate and maintained in culture are shown in FIG.

[0432] Compared with CM2 cultured on a flat culture plate, heart rate (BR) and relaxation velocity (RV) of CM2 cultured on the oriented culture plate increased significantly from day 15 to day 61, and a tendency of shortening of the beat duration (CRD) was observed after day 31. The vector direction distribution (mode value ± 5°) indicating the orientation of cells was maintained at a significantly high level from day 15 to day 61.

[0433] It should be noted that the higher the heart rate (BR), the closer it is to mature cardiomyocytes; the faster the contraction velocity (CV) and relaxation velocity (RV), the closer it is to mature cardiomyocytes; and the shorter the beat duration (CRD), the closer it is to mature cardiomyocytes.

[0434] The ratio of the contraction velocity (CV) to the relaxation velocity (RV) is Figure 11-2 In CM2 cultured on the oriented culture plate, the ratio of contraction velocity (CV) to relaxation velocity (RV) (CV / RV) was 1.8 to 2.5 from the 15th to the 45th day of culture. On the other hand, in CM2 cultured on the flat culture plate, it was 3.1 to 3.5.

[0435] Based on the above results, the ratio (CV / RV) of the contraction velocity (CV) to the relaxation velocity (RV) in CM2 cultured on the orientation culture plate is within the following range.

[0436] (Formula) 1.0≤CV / RV≤2.8

[0437] It should be noted that, regarding the vector direction distribution, the orientation degrees at the mode values ​​of ±1.25°, ±5°, and ±10° on the 15th day are shown in Table 1. At the mode value of ±1.25°, the ratio of the oriented culture plate / plane culture plate is the highest, and the ratio of the plane culture plate to the theoretical value is the highest. The small orientation angle of the analyzed object is believed to be due to the increased influence of noise. In the comparison of the mode values ​​of ±5° and ±10°, the ratio of the plane culture plate to the theoretical value is ±5°>±10°, but the ratio of the oriented culture plate / plane culture plate is ±5°>±10°, so the orientation degree of the mode value of ±5° was evaluated as the range in which the orientation degree tendency is likely to occur.

[0438] [Table 1]

[0439] Table 1

[0440]

[0441] (7) Ca-imaging analysis using confocal imaging device for maturation confirmation test

[0442] The change in Ca fluorescence intensity accompanying the contraction of the cardiomyocytes seeded on each culture plate was measured using CQ1 (confocal fluorescence microscope, manufactured by Yokogawa Electric Corporation).

[0443] In order to visualize Ca ion flux, a fluorescent Ca indicator (EarlyToxcarditotoxicity kit, manufactured by Molecular Devices) was added to CM2 and allowed to stand at 37°C for 15 minutes. The measurement conditions for CQ1 were an excitation wavelength of 488 nm / fluorescence wavelength of 525 nm and a data acquisition time of 60 seconds. In addition, CellPathfinder was used for analysis.

[0444] CM2 cells were used, with the day of seeding being designated as day 0 and maintained in culture until day 5. From the measurement data, the line width (Duration) at 20% of the height of the calcium transient waveform peak and the interval (Interval) of the waveform peak were selected as parameters for analysis.

[0445] Compared with CM2 cultured on a flat culture plate, the line width (Duration) of the 20% height of the calcium transient waveform peak and the interval (Interval) of the calcium transient waveform of CM2 cultured on an oriented culture plate were significantly prolonged ( Fig.12 (A)).

[0446] In addition, regarding the relationship between Duration and the square root of Interval, such as Fig.12 (B) shown.

[0447] Based on the above results, in CM2 cultured on an orientation culture plate, the ratio of the line width (Duration) at 20% height of the peak to the square root of the interval (Interval) of the waveform is within the following range.

[0448] Duration / (Interval) 1 / 2 ≥0.68

[0449] It is reported that the duration of calcium transient (line width at 20% height (Duration)) is correlated with the duration of action potential. Generally speaking, the higher the differentiation degree of cultured cardiomyocytes, the longer the duration of action potential. Therefore, it is believed that the oriented culture plate promotes maturation more than the flat culture plate.

[0450] The interval time is affected by the heartbeat, so the value corrected by the heart rate is usually used. 1 / 2 The numerical value is used to calculate.

[0451] (8) Maturation confirmation test: Measurement of intracellular potential by patch clamp method

[0452] Follow the above steps to convert CM2 into 6×10 4 The concentration of cells / well was inoculated into oriented culture plates and flat culture plates. At this time, each culture plate was not a 96-well plate, but a small piece of the bottom membrane was cut into a size of 4mm×4mm and placed on the bottom of a commercially available 96-well plate for use. Regarding the determination of intracellular potential based on the patch clamp test, for CM2 in Tyrode solution at 25°C, a current of 20pA to 100pA was injected into the cell at 0.2Hz under current clamp to generate an action potential. It should be noted that the current provided selects the current at which normal potential changes can be observed.

[0453] As a result, two representative examples are shown in Fig.13 The results for CM2 cultured on the oriented plate are from cells cultured on day 8, and the results for CM2 cultured on the flat plate are from cells cultured on day 22. The intracellular potential of CM2 cultured on the oriented plate was longer in duration of action potential and deeper in maximum diastolic potential than that of CM2 cultured on the flat plate.

[0454] The action potential 80% repolarization duration of CM2 cultured on the oriented culture plate was 791 ms, and the maximum diastolic potential was -68.6 mV. On the other hand, the action potential 80% repolarization duration of CM2 cultured on the flat culture plate was 478 ms, and the maximum diastolic potential was -57.9 mV.

[0455] The action potential in normal isolated ventricular myocytes from humans lasted as long as that in CM2 cultured on oriented culture plates, and the maximum diastolic potential (resting membrane potential) was deeper than that in CM2 cultured on oriented culture plates (Circulation, 2013; 127: 575-584). Therefore, it can be said that the action potential of CM2 cultured on oriented culture plates is closer to that of normal myocardial cells than that of CM2 cultured on flat culture plates.

[0456] (9) Drug safety test-1

[0457] Using iCell cardiomyocytes CM2, the volume-dependent effects of four pharmacological agents commonly used to induce myocardial side effects were investigated.

[0458] Follow the above steps to convert CM2 into 5×10 4 The concentration of cells / well was inoculated into oriented culture plates (96-well plates, NDCell Aligner) and flat culture plates (96-well plates). 2 The cells were maintained in an incubator, with the day of inoculation being designated as day 0, and the drugs being administered on day 5 or 6.

[0459] The drug used was E-4031, Quinidine, Cisapride, and Isoproterenol. The drug was dissolved in DMSO and administered cumulatively. First, as dose 0, the drug was measured without adding the drug. Immediately after the measurement, the drug dissolved in DMSO was added to each well at the concentration of dose 1, and the measurement was performed after 15 minutes. Then, immediately after the measurement, the drug corresponding to each well was added to the same well in a manner that became the concentration of dose 2, and the measurement was performed after 15 minutes. About dose 3, the same operation was performed.

[0460] The types of drugs, their mechanisms of action and their concentrations are shown in the table below.

[0461] [Table 2]

[0462] Table 2

[0463]

[0464] Regarding the responsiveness of cells after drug administration, an arrhythmia measurement method based on Ca-imaging using a confocal imaging device (CQ1, manufactured by Yokogawa Electric Corporation) and an exercise test method using a living cell imaging device (SI8000, manufactured by Sony Corporation) were implemented to investigate the effects on the beat rate and the line width of the beat peak.

[0465] (9-1) Measurement by motility test method using a live cell imaging device

[0466] Follow the steps below to do so.

[0467] Using CM2 on the fifth day of culture, first, as dose 0, SI8000 (manufactured by Sony Corporation) was used to obtain animation data without adding drugs. Immediately after the measurement, various drugs at the concentration of dose 1 were added to the same well plate, and animation data was obtained 15 minutes later. For doses 2 and 3, drugs were cumulatively administered in the same manner to obtain animation data. The addition test of each drug was performed at n=6.

[0468] The beat rate (BR), contraction velocity (CV), relaxation velocity (RV), and contraction-relaxation interval (CRD) were analyzed from the animation data of SI8000, and the effects of the drugs on cell motility were compared. In order to correct the fluctuation caused by the beat rate (BR), the contraction-relaxation interval (CRD) was corrected according to the following formula with reference to the Frederica formula.

[0469] CRD / (60 / BR) 1 / 3

[0470] Regarding the test of significance of difference, T-test was performed between the DMSO-added subject group and the drug-added group in the flat culture plate and the oriented culture plate.

[0471] The side effects observed from the parameter changes of SI8000 include early after depolarization (EAD) and delayed after depolarization (DAD), small regular waveform (VT-like), and disappearance of response (waveform). Waveform changes not classified as EAD or DAD are recorded as arrhythmia. In addition, the "-" in the table indicates that arrhythmia and the like do not occur.

[0472] Regarding the degree of arrhythmia, EAD is mild, DAD is moderate, and vt-like is severe. The case recorded as "arrhythmia" is mild.

[0473] E-4031 (Ikr blocker)

[0474] The addition of E-4031 reduced the beat rate (BR), contraction velocity (CV), and relaxation velocity (RV) of CM2 cultured on flat plates and CM2 cultured on oriented plates. In both oriented plates and flat plates, CRD (corrected by heart rate) showed a significant shortening at medium concentrations (dose 2, 0.3 μM) and high concentrations (dose 3, 1 μM). Fig.14 (A) ). The shortening is more pronounced in flat culture plates.

[0475] In CM2 cultured on a flat plate, arrhythmia occurred in 6 out of 6 wells, but in CM2 cultured on an oriented plate, arrhythmia occurred in 1 out of 6 wells (Table 3-1, Table 3-2).

[0476] Regarding the incidence of arrhythmia, in CM2 cultured on oriented plates, the incidence was 1 / 6 at low concentration (dose 1, 0.1 μM), 1 / 6 at medium concentration (dose 2, 0.3 μM), and 1 / 6 at high concentration (dose 3, 1 μM). On the other hand, in CM2 cultured on flat plates, the incidence was 3 / 6 at low concentration (dose 1, 0.1 μM), 5 / 6 at medium concentration (dose 2, 0.3 μM), and 6 / 6 at high concentration (dose 3, 1 μM), with a tendency to be low in CM2 cultured on oriented plates and high in CM2 cultured on flat plates.

[0477] [Table 3-1]

[0478] Table 3-1

[0479]

[0480] [Table 3-2]

[0481] Table 3-2

[0482]

[0483] Isoproterenol (beta-agonist)

[0484] 1, 3, and 10 μM isoproterenol was applied. BR and RV tended to increase in CM2 cultured on oriented plates and CM2 cultured on flat plates, and CV did not change significantly in CM2 cultured on flat plates and CM2 cultured on oriented plates. CRD (corrected by heart rate) was prolonged at low concentrations (dose 1, 1 μM), medium concentrations (dose 2, 3 μM), and high concentrations (dose 3, 10 μM) in oriented plates and flat plates. In oriented plates, changes similar to those of the solvent (DMSO) were shown, but the difference between the flat plate and the solvent (DMSO) was large, which is believed to be due to the influence of the drug ( Fig.14 (B)).

[0485] In addition, no arrhythmia was observed at any concentration.

[0486] (9-2) Arrhythmia determination based on Ca-imaging

[0487] Follow the steps below to do so.

[0488] A fluorescent Ca indicator (EarlyTox carditotoxicity kit, manufactured by Molecular Devices) was added to CM2 on the 6th day of maintenance culture, and the living cells were stained. First, as dose 0, the change in Ca fluorescence intensity associated with cardiomyocyte contraction was measured using CQ1 without adding the drug. Immediately after the measurement, various drugs at the concentration of dose 1 were added to the same well plate, and the change in Ca fluorescence intensity was measured after 15 minutes. For doses 2 and 3, the drugs were cumulatively administered in the same manner, and the change in Ca fluorescence intensity was measured. The addition test of each drug was performed at n=6.

[0489] Using the data of 20% waveform width (Duration), interval between peaks (Interval), and peak height (calcium transient peak) when the peak of the waveform of calcium transient measured by CQ1 was set as 100%, the effects of the drugs were compared in CM2 inoculated on a flat plate and an oriented plate and cultured for 6 days. The significance test was performed between the CM2 cultured on a flat plate and the CM2 cultured on an oriented plate, and between the DMSO-added subject group and the drug-added group.

[0490] Regarding the side effects observed from the changes in the waveform of calcium transients, early after depolarization (EAD) and delayed after depolarization (DAD), small regular waveform (VT-like), and disappearance of the response (waveform) were recorded. In addition, the "-" in the table shows that there is no arrhythmia. Regarding the degree of arrhythmia, EAD is mild, DAD is moderate, and VT-like is severe.

[0491] E-4031 (Ikr blocker)

[0492] Regarding the Duration of calcium transients, at 0 μM, CM2 cultured on oriented culture plates was slightly longer (significantly) than CM2 cultured on flat culture plates, at 0.1 μM, CM2 cultured on flat culture plates was slightly longer, at 0.3 μM, CM2 cultured on flat culture plates was significantly longer than CM2 cultured on oriented culture plates, and at 1 μM, although not significantly, CM2 cultured on flat culture plates was significantly prolonged.

[0493] From these results, it can be said that the effect of E-4031 addition on IKr blockade is more likely to occur in CM2 cultured on flat culture plates, but less in CM2 cultured on oriented culture plates ( Figure 15-1 ).

[0494] Regarding the incidence of moderate to severe arrhythmias (DAD, VT-like), in CM2 cultured on oriented plates, it was 0 / 6 at low concentration (dose 1, 0.1 μM), 1 / 6 at medium concentration (dose 2, 0.3 μM), and 4 / 6 at high concentration (dose 3, 1 μM). In CM2 cultured on flat plates, it was 3 / 6 at low concentration (dose 1, 0.1 μM), 6 / 6 at medium concentration (dose 2, 0.3 μM), and 6 / 6 at high concentration (dose 3, 1 μM), with a tendency to be low in CM2 cultured on oriented plates and high in CM2 cultured on flat plates (Table 4-1, Table 4-2).

[0495] [Table 4-1]

[0496] Table 4-1

[0497]

[0498] [Table 4-2]

[0499] Table 4-2

[0500]

[0501] Quinidine (INa blocker, Ikr blocker)

[0502] The results of the same study on the effect of quinidine at 3, 10, and 30 μM are as follows: Figure 15-2 As shown. At medium concentrations (dose 2, 10 μM), a stronger extension of Duration and Interval and a significantly weaker decrease in height were observed in CM2 cultured on a flat plate than in CM2 cultured on an oriented plate. At high concentrations (dose 3, 30 μM), the contractile force was extremely reduced and changed to a high-frequency waveform, so it is not shown in the figure.

[0503] Regarding the incidence of moderate to severe arrhythmias (DAD, VT-like), in CM2 cultured on oriented culture plates, it was 0 / 6 at low concentrations (dose 1, 3 μM), 0 / 6 at medium concentrations (dose 2, 10 μM), and 6 / 6 at high concentrations (dose 3, 30 μM). In CM2 cultured on flat culture plates, it was 0 / 6 at low concentrations (dose 1, 3 μM), 0 / 6 at medium concentrations (dose 2, 10 μM), and 4 / 6 at high concentrations (dose 3, 30 μM), with a tendency to be slightly higher in oriented culture plates (Table 5-1, Table 5-2). Although the incidence of arrhythmias tends to be slightly higher in oriented culture plates, the changes in Duration and Interval are smaller in CM2 cultured on oriented culture plates, so it is believed that the influence of drugs is less likely to occur.

[0504] [Table 5-1]

[0505] Table 5-1

[0506]

[0507] [Table 5-2]

[0508] Table 5-2

[0509]

[0510] Cisapride (Ikr blocker, 5-HT4 receptor agonist)

[0511] The effects of 0.01, 0.03, and 0.1 μM cisapride were studied. Duration and Interval were prolonged in a dose-dependent manner ( Figure 15-3 ). In CM2 cultured on the oriented culture plate, the difference between the value shown at dose 0 and the value shown at dose 3 was smaller than that of CM2 cultured on the flat culture plate. This is believed to be because CM2 cultured on the oriented culture plate has tolerance to cisapride compared to CM2 cultured on the flat culture plate.

[0512] The occurrence of arrhythmia in the oriented culture plate was 1 / 6 at low concentrations (dose 1, 0.01 μM) to high concentrations (dose 3, 0.1 μM), while no arrhythmia occurred in the flat culture plate (Table 6-1, Table 6-2).

[0513] The incidence of arrhythmia tended to be slightly higher in the oriented culture plate, but the changes in Duration and Interval were smaller in CM2 cultured on the oriented culture plate, which is considered to be less likely to be affected by the drug.

[0514] [Table 6-1]

[0515] Table 6-1

[0516]

[0517] [Table 6-2]

[0518] Table 6-2

[0519]

[0520] Isoproterenol (beta-agonist)

[0521] The effects of 1, 3, and 10 μM isoproterenol were studied. A decrease in Duration and Interval was observed ( Figure 15-4 ). However, the decrease in Duration in the flat culture plate was smaller. This decrease in Duration is a typical response that can be observed in β-receptor stimulation.

[0522] Isoproterenol did not cause arrhythmia even at high concentrations (3, 10 μM). CM2 cultured on oriented plates had the same tendency as CM2 cultured on flat plates.

[0523] DMSO (Solvent)

[0524] In the case of solvent, Duration was slightly prolonged. This effect did not have a significant impact on the drug effect ( Figure 15-5 ).

[0525] When focusing on E-4031, the most typical IKr blocker that prolongs the duration of action potential, and similar effects, it is less likely to confirm the duration prolongation effect of high-dose E-4031 in CM2 cultured on oriented culture plates than in CM2 cultured on flat culture plates. Therefore, it is believed that there is a tendency for side effects to occur more easily in CM2 cultured on flat culture plates than in CM2 cultured on oriented culture plates.

[0526] In most experimental systems, the duration of calcium transients before drug adaptation was longer in CM2 cultured on oriented plates and shorter in CM2 cultured on flat plates, suggesting that the duration of action potentials was also the same. The extension of this duration is generally associated with the maturation of cardiomyocytes, and even on day 6, functional changes were clearly observed in CM2 cultured on oriented plates and CM2 cultured on flat plates (differentiation promotion by oriented plates).

[0527] (10) Drug safety test-2

[0528] iCell cardiomyocytes CM2 were cultured for 15 days, in a more mature state, to study the volume-dependent effects of two agents commonly used to induce myocardial side effects.

[0529] Follow the above steps to convert CM2 into 6×10 4 The concentration of cells / well was inoculated into oriented culture plates (96-well plates, NDCell Aligner) and flat culture plates (96-well plates). 2 The cells were maintained in an incubator, with the day of inoculation being designated as day 0 and the drug being administered on day 15.

[0530] Isoproterenol and milrinone were used as the drug. The drug was dissolved in DMSO and administered. First, as dose 0, the measurement was performed without adding the drug. Immediately after the measurement, the drug dissolved in DMSO was added to each well according to the concentration of each dose, and the measurement was performed after a certain period of time. The type, mechanism of action and concentration of the drug are shown in the following table.

[0531] [Table 7]

[0532] Table 7

[0533]

[0534] Regarding the responsiveness of cells after drug administration, an exercise test method using a live cell imaging device (SI8000, manufactured by Sony Corporation) was performed to examine the effects on the pulsation rate, contraction velocity, and relaxation velocity.

[0535] (10-1) Measurement by motility test method using a live cell imaging device

[0536] Follow the steps below to do so.

[0537] Using CM2 on the 15th day of culture, first, as dose 0, in the state where no drug was added, animation data was obtained using SI8000 (manufactured by Sony Corporation). Immediately after the measurement, various drugs of different concentrations were added to the same well plate, and animation data was obtained after 120 minutes for isoproterenol and 15 minutes for milrinone. The addition test of each drug was performed at n=5.

[0538] The beat rate (BR), contraction velocity (CV), and relaxation velocity (RV) were analyzed from the animated data of SI8000, and the effects of drugs on cell motility were compared.

[0539] The measurement data were corrected using the average value of the dose 0 without adding the drug (each measurement value / the measurement value of the dose 0), and the relative value when the measurement value of the dose 0 was set to 1 was obtained.

[0540] Regarding the test of significance of difference, the results of the flat culture plates and oriented culture plates at each concentration of drug addition were compared with the results of dose 0, and a T test was performed. In addition, a T test was performed between the flat culture plates and oriented culture plates in the drug addition group. The comparison groups with a P value of less than 0.01 were marked in the chart.

[0541] Isoproterenol (beta-agonist)

[0542] The addition of isoproterenol increased the beating rate (BR) and relaxation velocity (RV) of CM2 cultured on flat plates and CM2 cultured on oriented plates. Regarding the relaxation velocity, the oriented plates at doses 2 and 3 increased it compared to the flat plates.

[0543] The contraction velocity (CV) increased in CM2 cultured on the oriented culture plate, but tended to decrease in the flat culture plate. The contraction velocity (CV) of the oriented culture plate at dose 2 increased significantly compared to before the addition of the drug, and, in comparison with the flat culture plate, the contraction velocity (CV) of the oriented culture plate at doses 2 and 3 increased significantly ( Figure 16-1 ).

[0544] Milrinone (phosphodiesterase 3 inhibitor)

[0545] The addition of milrinone increased the pulsation rate (BR), contraction velocity (CV), and relaxation velocity (RV) of CM2 cultured on the oriented culture plate. In CM2 cultured on the flat culture plate, the pulsation rate did not change due to drug administration, but the contraction velocity and relaxation velocity tended to decrease.

[0546] The beating rate (BR) of CM2 cultured on the oriented culture plate was significantly increased at all doses compared with before the addition of the drug. The relaxation velocity (RV) was significantly increased in the oriented culture plate at dose 3 compared with before the addition of the drug. In addition, it was significantly increased compared with the flat culture plate. The contraction velocity (CV) was significantly increased in the oriented culture plate at dose 3 compared with the flat culture plate ( Figure 16-2 ).

[0547] DMSO (Solvent)

[0548] In the case of the solvent, no significant effect on the drug effect was observed.

[0549] As shown in (10) above, iCell cardiomyocytes CM2 were cultured for 15 days in a more mature state, and the volume-dependent effects of two drugs commonly used to cause myocardial side effects were studied. The results obtained showed that by using an oriented culture plate, a significant increase in the contraction velocity was observed, and the use of an oriented culture plate made it possible to detect the positive inotropic effect, which was difficult to verify in iCell cardiomyocytes.

[0550] (11) Drug safety test-3 (anticancer drugs)

[0551] iCell cardiomyocytes CM2 were cultured for 15 days, and the volume-dependent effects of anticancer drugs that cause side effects on the myocardium (toxic myocarditis, allergic myocarditis) were studied.

[0552] Follow the same steps as (10) above to convert CM2 into 6×10 4 The concentration of cells / well was inoculated into oriented culture plates (96-well plates, ND Cell Aligner) and flat culture plates (96-well plates). 2 The cells were maintained in an incubator, with the day of inoculation being designated as day 0 and the drug being administered on day 15.

[0553] The drug used is doxorubicin (Doxorubicin, anthracycline anticancer agent). The drug is dissolved in DMSO and administered. First, as a prior measurement, the measurement is performed without adding the drug. Immediately after the measurement, the drug dissolved in DMSO is added to each well at a concentration of 3 μM, and the measurement is performed after 24 hours and 48 hours. The measurement is performed in the same manner as the measurement performed by the motility test method using a live cell imaging device described above (10-1).

[0554] The measurement data were corrected for the measured value at a concentration of 3 μM using the average value of a previous measurement without adding a drug (measured value at 3 μM / measured value in previous measurement), and the change rate was calculated when the measured value in previous measurement was set to 1.

[0555] Regarding the difference significance test, the results of the planar culture plates and oriented culture plates in the drug addition group at each time were compared with the DMSO addition group at each time, and a T test was performed. In addition, a T test was performed between the planar culture plates and oriented culture plates in the drug addition group. For the comparison groups with a P value of less than 0.01, a mark was made in the chart.

[0556] DMSO was added as a solvent at a final concentration of 0.01%. The effect of DMSO was confirmed by measuring the group with 0.01% DMSO addition 24 hours and 48 hours later. The measurement data were processed in the same manner as the drug administration group.

[0557] Doxorubicin (anticancer agent)

[0558] The addition of doxorubicin increased the beating rate (BR) of CM2 cultured on the flat culture plate and CM2 cultured on the oriented culture plate. The measurement results after 48 hours increased compared with the measurement results after 24 hours, and the beating rate increased over time. In particular, in CM2 cultured on the oriented culture plate, the beating rate increased significantly by adding 3μM compared with the flat culture plate. The addition of DMSO showed a tendency to reduce the beating rate of both the flat culture plate and the oriented culture plate ( Fig.17 ).

[0559] Regarding CV (contraction velocity), an increase was confirmed in CM2 cultured on the oriented culture plate. In the measurement after 24 hours, the CV increased significantly by adding 3 μM compared with the DMSO administration group. In the measurement after 24 hours and 48 hours, the CV increased significantly in CM2 cultured on the oriented culture plate compared with the flat culture plate. The addition of DMSO showed a tendency to reduce the contraction velocity on the flat culture plate, but there was no change in the oriented culture plate compared with before the drug administration ( Fig.17 ).

[0560] The addition of doxorubicin increased the RV (relaxation velocity) of CM2 cultured on the flat plate and CM2 cultured on the oriented plate. In the measurement after 48 hours, the RV of CM2 cultured on the oriented plate increased significantly compared with the flat plate. The addition of DMSO showed a tendency to increase the RV in the oriented plate, but there was no change in the flat plate compared with before the drug administration ( Fig.17 ).

[0561] As shown in (11) above, the volume-dependent effect of the anticancer agent doxorubicin was studied, and the results showed that the use of oriented culture plates resulted in significant increases in the beat rate, contraction velocity, and relaxation velocity, and that the use of oriented culture plates could be used to detect myocardial toxicity.

[0562] (12) Determination of oxygen consumption rate

[0563] Follow the above steps to convert CM2 into 6×10 4 The concentration of cells / well was inoculated into oriented culture plates (96-well plates, NDCell Aligner) and flat culture plates (96-well plates). 2 The cells were maintained in an incubator, with the day of inoculation designated as day 0, and the oxygen consumption rate was measured on day 27.

[0564] The oxygen consumption rate plate assay kit (manufactured by Dojin Chemical Co., Ltd., E297) was used for the measurement.

[0565] An oxygen probe diluted with a culture medium was added to the well plate containing CM2, and the plate was left to stand for 30 minutes in a plate reader pre-warmed to 37°C. Next, a sample solution containing etomoxir, a β-oxidation inhibitor that inhibits fatty acid metabolic enzymes, was added at a final concentration of 50 μM, and mineral oil was immediately added dropwise to block the oxygen supply.

[0566] After standing for 5 minutes in a plate reader kept at 37° C., the fluorescence intensity was measured over time. The measurement conditions of the fluorescence plate reader were excitation wavelength 495 nm / fluorescence wavelength 650 nm, and data were acquired every 10 minutes for at least 168 minutes.

[0567] The measurement was performed by adding 50 μM etomoxir to 3 wells while no etomoxir was added to 6 wells as a control.

[0568] The oxygen concentration (pmol) was calculated using the calculation sheet attached to the oxygen consumption rate plate test kit, and the oxygen consumption rate (pmol / min) was calculated from the decrease in oxygen concentration within 60 minutes after 10 to 70 minutes from the start of the measurement.

[0569] The measurement results showed that in the flat culture plate, the OCR was 79.1 pmol / min when no etomoxir was added and 77.8 pmol / min when etomoxir was added. The addition of etomoxir increased the OCR to 98.3% compared to the OCR before addition.

[0570] On the other hand, in the oriented culture plate, the OCR was 63.6 pmol / min when no etomoxir was added and 7.0 pmol / min when etomoxir was added. The addition of etomoxir reduced the OCR to 11.0% compared to the OCR before addition.

[0571] The results are shown in Fig. 16. In Fig. 16, the vertical axis takes the OCR without the addition of etomoxir as 1.0.

[0572] Based on the above results, it is considered that the cultured cell sheet of the present embodiment is more mature than conventional cultured cell sheets, and as a result, the evaluation method of the compound or drug to be evaluated can be closer to in vivo evaluation by using the cultured cell sheet.

Claims

1. A cultured cell sheet comprising cardiomyocytes. Cardiac myocytes are arranged in a directional manner. In an image obtained by immunostaining the cultured cell sheet with an anti-α-actinin antibody and observing the image under a microscope, the angles in the longitudinal directions of rod-like structures detected with the α-actinin antibody within a measurement range (71.6 μm × 71.6 μm) are measured with the horizontal direction of the image set to 0°, and the frequency of rod-like structures within ±15° of the mode value, i.e., the degree of orientation determined according to the following formula (1), is 23% or more. Orientation degree (%) = (number of rod-like structures included within ±15° of the mode value) / (total number of rod-like structures)×100 (1).

2. The cultured cell sheet according to claim 1, wherein In live cell imaging, the number of vectors showing an angle of ±5° from the angle of the mode value of the motion vector (degree of orientation) is 12% or more.

3. The cultured cell sheet according to claim 1, wherein The cardiomyocytes are stem cell-derived cardiomyocytes.

4. The cultured cell sheet according to claim 1, which satisfies at least one of the following requirements A1 and A2: Requirement A1: The ratio of the expression level of the MYL3 (myosin light chain 3) gene to the expression level of the ACTB (β-actin) gene (MYL3 / ACTB) is 12.0 or more, Requirement A2: The ratio of the expression level of the MYH7 (myosin heavy chain 7) gene to the expression level of the MYH6 (myosin heavy chain 6) gene (MYH7 / MYH6) is 6.0 or more.

5. The cultured cell sheet according to claim 1, which satisfies at least one of the following requirements B1 and B2: Requirement B1: The ratio of the gene expression level of CKM (creatine kinase, M type) to the gene expression level of ACTB (β-actin) (CKM / ACTB) is 1.80 or higher, Requirement B2: The ratio of the gene expression level of LDHA (lactate dehydrogenase subunit A) to the gene expression level of ACTB (LDHA / ACTB) is 0.80 or higher. The cultured cell sheet according to claim 1 , wherein The area of ​​each mitochondria in a cell is 200 μm 2 above.

7. The cultured cell sheet according to claim 1, wherein When the contraction velocity detected in live cell imaging is defined as CV (m / sec) and the relaxation velocity is defined as RV (m / sec), the following formula (2) is satisfied: 1.0≤CV / RV≤2.8 (2).

8. The cultured cell sheet according to claim 1, wherein In calcium imaging analysis, when the peak height of the calcium transient waveform is set to 100%, the waveform width at 20% of the height is set to Duration (seconds), and the interval between peaks is set to Interval (seconds), the following formula (3) is satisfied: Duration / (Interval) 1 / 2 ≥0.68 (3)。 9. The cultured cell sheet according to claim 1, wherein The cells constituting the cultured cell sheet had an action potential 80% repolarization duration of 600 msec or longer and a maximum diastolic potential of -60 mV or less in a patch clamp test in a 25°C solution.

10. The cultured cell sheet according to claim 1, which is used for regenerative medicine.

11. A method for producing a cultured cell sheet, which is the method for producing a cultured cell sheet according to any one of claims 1 to 10, The manufacturing method includes the step of culturing cardiomyocytes using a cell sheet-forming member, The cell sheet forming member comprises a surface for forming a cultured cell sheet, The surface has a plurality of flat portions and a plurality of concave and convex portions, Each flat portion has a shape extending in a first direction, and the plurality of flat portions are arranged along a second direction intersecting the first direction over the entire surface. Each concave-convex portion includes a plurality of level difference structures filling between the adjacent flat portions, and a pitch between the level difference structures is 100 nm or more and 10 μm or less. The height difference structure is a convex portion, The concave-convex portion includes a plurality of convex portions on the bottom surface of a concave portion sandwiched between the adjacent flat portions. In the thickness direction of the cell sheet forming member, a difference between the height of the front end surface of the concavo-convex portion and the height of the flat portion is 0.5 μm or less. 12 . A method for evaluating a compound or a drug, comprising allowing the compound or drug to act on the cultured cell sheet according to claim 1 . 13 . The method for evaluating a compound or a drug according to claim 12 , wherein at least one change selected from the group consisting of changes in physiological properties and changes in motor function of a cultured cell sheet is evaluated.

14. The method for evaluating a compound or a drug according to claim 12, wherein: The compound or drug to be evaluated is at least one existing compound or candidate compound thereof selected from the group consisting of INa blockers, Ikr blockers, Iks blockers, ICa blockers, 5-HT4 receptor agonists, α receptor blockers, β receptor blockers, α receptor agonists, β receptor agonists, toxic substances, anticancer agents and other physiologically active substances.

15. A quality evaluation method for a cultured cell sheet composed of cardiomyocytes, wherein the quality evaluation method evaluates any one of the following (i) to (viii): (i) in an image obtained by immunostaining the cultured cell sheet with an anti-α-actinin antibody and observing the image under a microscope, the angles in the longitudinal direction of rod-like structures detected with the α-actinin antibody within a measurement range (71.6 μm × 71.6 μm) are measured with the horizontal direction of the image set to 0°, and the frequency of rod-like structures within ±15° of the mode value, i.e., the degree of orientation determined according to the following formula (1), is 23% or more; Orientation degree (%) = (number of rod-like structures within ±15° of the mode value) / (total number of rod-like structures) × 100 (1); (ii) in live cell imaging, the number of vectors showing an angle of ±5° from the angle of the mode value of the motion vector (degree of orientation) is 12% or more; (iii) Satisfy at least one of the following requirements A1 and A2, Requirement A1: The ratio of the expression level of the MYL3 (myosin light chain 3) gene to the expression level of the ACTB (β-actin) gene (MYL3 / ACTB) is 12.0 or more, Requirement A2: The ratio of the expression level of the MYH7 (myosin heavy chain 7) gene to the expression level of the MYH6 (myosin heavy chain 6) gene (MYH7 / MYH6) is 6.0 or higher; (iv) Satisfy at least one of the following requirements B1 and B2, Requirement B1: The ratio of the gene expression level of CKM (creatine kinase, M type) to the gene expression level of ACTB (β-actin) (CKM / ACTB) is 1.80 or higher, Requirement B2: The ratio of the gene expression level of LDHA (lactate dehydrogenase subunit A) to the gene expression level of ACTB (LDHA / ACTB) is 0.80 or higher; (v) The area of ​​mitochondria in each cell is 200 μm 2 above; (vi) When the contraction velocity detected in live cell imaging is defined as CV (m / sec) and the relaxation velocity is defined as RV (m / sec), the following formula (2) is satisfied: 1.0≤CV / RV≤2.8 (2) (vii) In calcium imaging analysis, when the peak height of the calcium transient waveform is set to 100%, the waveform width at 20% of the height is set to Duration (seconds), and the interval between peaks is set to Interval (seconds), the following formula (3) is satisfied: Duration / (Interval) 1 / 2 ≥0.68 (3) (viii) The duration of 80% repolarization of the action potential of cells constituting the cultured cell sheet in a patch clamp test in a 25°C solution is 600 msec or longer, and the maximum diastolic potential is -60 mV or less.

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