Lipid-toxic culture medium

Abstract

The present invention provides a culture medium for culturing cardiac tissue, a method for inducing the disease phenotype of diabetic cardiomyopathy in cardiac tissue, affected cardiac tissue, and a kit. An exemplary culture medium for culturing cardiac tissue comprises 100-300 pM palmitate, 100-300 pM oleate, and 0.5-15 nM endothelin-1.

Description

Technical Field

[0001] The present invention relates to a culture medium for culturing heart tissue, a method for inducing a disease phenotype of diabetic cardiomyopathy in heart tissue, diseased heart tissue produced by said method, diseased heart tissue, and a kit.

Background Art

[0002] Heart failure currently affects more than 64 million people worldwide and its prevalence appears to be on the rise. Experts predict that in the United States alone, the prevalence of heart failure will increase by 46% by 2030. Among all cases of heart failure, heart failure with preserved ejection fraction (HFpEF) accounts for approximately 50% of cases, and the proportion of heart failure patients with HFpEF has recently been increasing. In addition to the fact that HFpEF is a global epidemic, due to the large burden of symptoms caused by HFpEF, it has become an urgent task to identify pharmaceutical candidates that can treat and prevent HFpEF.

[0003] HFpEF typically is associated with comorbidities such as diabetes, obesity, metabolic syndrome, and approximately 65% of HFpEF patients have at least one of these diseases. The main characteristic of the metabolic abnormalities presumed to be the cause of HFpEF is dyslipidemia, which is a state in which an abnormally large amount of lipids and fatty acids circulate in the blood. Dyslipidemia has an adverse effect on cell metabolism, increases fatty acid oxidation, and decreases glucose oxidation. The increase in lipids is involved in insulin resistance, which is associated with an increase in serum glucose and insulin concentrations. Furthermore, lipids such as diacylglycerol and ceramide accumulate in heart cells over time, affecting cell morphology and function, and may cause a pathological condition called diabetic cardiomyopathy.

[0004] Diabetic cardiomyopathy can be formally defined as cardiac dysfunction in the absence of other risk factors such as hypertension, ischemic heart disease, and valvular insufficiency. In clinical practice, patients with diabetic cardiomyopathy may initially present asymptomatically, but may later exhibit fibrosis, increased ventricular stiffness, and diastolic dysfunction. This disease may later progress to left ventricular hypertrophy, worsening diastolic dysfunction, and heart failure with reduced ejection fraction (HFpEF). In extreme cases, heart failure with reduced ejection fraction (HFrEF) may also be observed.

[0005] Several 2D in vitro models of diabetic cardiomyopathy exist (Geraets et al. "Human embryonic stem cell-derived cardiomyocytes as an in vitro model to study cardiac insulin resistance," Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, vol.1864, no.5, Part B, pp.1960-1967, 2018; Graneli et al. "Diabetic Cardiomyopathy Modelling Using Induced Pluripotent Stem," Stem Cell Reviews and Reports, vol.15, pp.13-22, 2019). However, these models do not adequately represent diabetic cardiomyopathy.

[0006] There is a need to deepen our understanding of lipid toxicity mechanisms and provide improved models of diabetic cardiomyopathy that can be used for drug discovery and development. [Overview of the project]

[0007] According to a first aspect of the present disclosure, a culture medium for culturing cardiac tissue is provided, the culture medium comprising 100-300 μM of palmitate, 100-300 μM of oleate, and 0.5-15 nM of endothelin-1.

[0008] The inventors have developed a lipid-toxic culture medium that can be used to culture cardiac tissue to induce the phenotype of diabetic cardiomyopathy. Advantageously, this cultured tissue can be used as an in vitro or ex vivo three-dimensional (3D) model to reproduce the main features of diabetic cardiomyopathy, and therefore can be used to better understand the lipid toxicity mechanism and for drug discovery and development. It was not possible from the prior art to predict that the combination of palmitate, oleate, and endothelin-1, particularly at the concentrations described, could be used to induce the phenotype of diabetic cardiomyopathy in cardiac tissue. Furthermore, it was not possible from the prior art to predict that endothelin-1, when combined with palmitate and oleate, would induce the phenotype of diabetic cardiomyopathy in cardiac tissue.

[0009] As used herein, the term “culture medium” refers to a solution containing factors and nutrients, such as growth factors, energy sources, amino acids, organic salts, and inorganic salts, which are used for the maintenance and proliferation of cells in ex vivo or in vitro cultures. Culture media are often buffered to a near-neutral pH (e.g., pH 6.6–pH 7.8) and may contain one or more antibiotics to prevent the growth of bacterial and / or fungal contaminants. Any suitable basal culture medium may be used, such as StemPro®-34 serum-free medium, MEM, DMEM, RPMI1640, Advanced MEM, BME, Neurobasal medium, cardiomyocyte selective medium, sodium bicarbonate buffered medium 199, myocyte growth medium, cardiomyocyte growth medium, and cardiomyocyte maintenance medium.

[0010] As used herein, the term “lipid toxicity” is intended to mean that a culture medium causes the accumulation of lipids and lipid intermediates within the cultured cardiac tissue, resulting in adverse effects such as cellular dysfunction (e.g., manifesting as increased tissue relaxation time) and / or cell death.

[0011] The terms “growth” or “culture” refer to the cultivation, maintenance, growth, differentiation, or maturation of cells or tissues in vitro or ex vivo on or in culture media to obtain a desired phenotype, structure, and / or functionality.

[0012] As used herein, the term “palmitate” is intended to encompass salts and esters of palmitic acid, including, for example, sodium palmitate and calcium palmitate. In some embodiments, palmitates form complexes with other proteins, such as bovine serum albumin (BSA). In some embodiments, the palmitate is palmitic acid.

[0013] As used herein, the term “oleate” is intended to encompass salts and esters of oleic acid, including, for example, sodium oleate and calcium oleate. In some embodiments, oleates form complexes with other proteins, such as bovine serum albumin (BSA). In some embodiments, the oleate is oleic acid. Oleic acid may also be called cis-9-octadecanoic acid.

[0014] Endothelin-1 is a vasoconstrictive peptide. In some embodiments, endothelin-1 is human endothelin-1. In some embodiments, endothelin-1 is preproendothelin-1 or proendothelin-1.

[0015] As used herein, the term “cardiac tissue” is intended to encompass ex vivo cardiac tissue and in vitro cultured tissue, including both 2D and 3D cardiac cell cultures. Cardiac tissue may be healthy or diseased, and / or may be modified in any way (e.g., by diagnostic agents, therapeutic agents, genetic modification, etc.).

[0016] In some embodiments, the culture medium is for culturing cardiac tissue produced using the systems defined in WO 2015 / 061907 Al, WO 2021 / 158233 Al and / or WO 2016 / 183143 Al, which are incorporated herein by reference in their entirety. In some embodiments, the culture medium is for culturing cardiac tissue (e.g., cardiac organoids) as defined in WO 2016 / 183143 Al.

[0017] In some embodiments, cardiac tissue is generated from a cell line (e.g., a human cell line). In some embodiments, cardiac tissue is generated from ex vivo primary cardiomyocytes (e.g., human cardiomyocytes). In some embodiments, cardiac tissue is generated from ex vivo pluripotent stem cell-derived cardiomyocytes (e.g., human iPSCs). In some embodiments, cardiac tissue is human cardiac tissue. In some embodiments, cardiac tissue comprises cardiomyocytes genetically modified to give a desired phenotype.

[0018] In some embodiments, the cardiac tissue includes a population of cardiac fibroblasts in addition to a population of cardiomyocytes. The ratio of cardiomyocytes to cardiac fibroblasts may be between approximately 1:3 and 15:1. The ratio of cardiomyocytes to cardiac fibroblasts may be between approximately 1:1 and 10:1. In some embodiments, the ratio of cardiomyocytes to cardiac fibroblasts is approximately 4:1. In some embodiments, the ratio of cardiomyocytes to cardiac fibroblasts is approximately 10:1.

[0019] In some embodiments, the cardiac tissue includes a population of endothelial cells, in addition to a population of cardiomyocytes (and optionally cardiac fibroblasts). The ratio of cardiomyocytes to endothelial cells may be between approximately 1:10 and 4:1. The ratio of cardiomyocytes to endothelial cells may be between approximately 1:6 and 2:1. In some embodiments, the ratio of cardiomyocytes to cardiac fibroblasts to endothelial cells is 1:3:6, 2:1:1, 4:1:2, or 2:2:1.

[0020] In some embodiments, the culture medium is used to induce a disease phenotype of diabetic cardiomyopathy in cultured cardiac tissue. As used herein, the term “disease phenotype of diabetic cardiomyopathy” is intended to mean an increase in the relaxation time of cardiac tissue, which is analogous to delayed relaxation of the left ventricle of the heart.

[0021] In some embodiments, the culture medium contains 120-280 μM of palmitate. In some embodiments, the culture medium contains 140-260 μM of palmitate. In some embodiments, the culture medium contains 160-240 μM of palmitate. In some embodiments, the culture medium contains 180-220 μM of palmitate. In some embodiments, the culture medium contains 190-210 μM of palmitate. In some embodiments, the culture medium contains 195-205 μM of palmitate. In some embodiments, the culture medium contains approximately 200 μM of palmitate. In some embodiments, the culture medium contains 200 μM of palmitate.

[0022] In some embodiments, the culture medium contains 120-280 μM of oleate. In some embodiments, the culture medium contains 140-260 μM of oleate. In some embodiments, the culture medium contains 160-240 μM of oleate. In some embodiments, the culture medium contains 180-220 μM of oleate. In some embodiments, the culture medium contains 190-210 μM of oleate. In some embodiments, the culture medium contains 195-205 μM of oleate. In some embodiments, the culture medium contains approximately 200 μM of oleate. In some embodiments, the culture medium contains 200 μM of oleate.

[0023] In some embodiments, the culture medium contains 0.5 to 15 nM of endothelin-1. In some embodiments, the culture medium contains 0.6 to 12 nM of endothelin-1. In some embodiments, the culture medium contains 0.7 to 10 nM of endothelin-1. In some embodiments, the culture medium contains 0.8 to 8 nM of endothelin-1. In some embodiments, the culture medium contains 0.9 to 6 nM of endothelin-1. In some embodiments, the culture medium contains 1 to 5 nM of endothelin-1. In some embodiments, the culture medium contains 1 nM or 5 nM of endothelin-1.

[0024] In some formulations, the culture medium contains 100-250 μM palmitate, 100-250 μM oleate, and 0.5-15 nM endothelin-1. In some embodiments, the culture medium contains 100-225 μM palmitate, 100-225 μM oleate, and 0.5-15 nM endothelin-1.

[0025] In some embodiments, the culture medium contains 120-280 μM palmitate, 120-280 μM oleate, and 0.5-15 nM endothelin-1. In some embodiments, the culture medium contains 180-220 μM palmitate, 180-220 μM oleate, and 0.5-10 nM endothelin-1. In some embodiments, the culture medium contains 200 μM palmitate, 200 μM oleate, and 1-5 nM endothelin-1.

[0026] In some embodiments, the culture medium does not contain glucocorticoids such as cortisol, cortisone, or hydrocortisone. In some embodiments, the culture medium contains less than 0.2 μM of glucocorticoids. In some embodiments, the culture medium contains less than 0.1 μM of glucocorticoids. In some embodiments, the culture medium contains less than 0.2 μM of cortisol or cortisone. In some embodiments, the culture medium contains less than 0.1 μM of cortisol or cortisone. In some embodiments, the culture medium does not contain cortisol.

[0027] In some embodiments, the culture medium further comprises StemPro™-34 Serum-Free Basal Medium (e.g., supplied by Gibco™). One of ordinary skill in the art will understand that other suitable basal media can also be used as an alternative.

[0028] In some embodiments, the culture medium further comprises one or more of a buffer, a sugar (e.g., glucose), an amino acid (e.g., L-glutamine), an antibiotic, a vitamin, a serum, a growth factor, a cytokine, sodium pyruvate, a recombinant protein, an iron transport protein, or a combination thereof.

[0029] In some embodiments, the culture medium is (i) GlutaMAX™ Supplement (e.g., supplied by Gibco™), optionally 0.1 - 2% (v / v) or 1% (v / v) GlutaMAX, (ii) HEPES (e.g., supplied by Gibco™), optionally 10 - 30 mM or 20 mM HEPES, (iii) Penicillin - Streptomycin (e.g., supplied by Gibco™), optionally 0.1 - 2% or 1% penicillin - streptomycin, (iv) Transferrin solution (e.g., supplied by Sigma - Aldrich), optionally 0.05 - 0.25 mg / mL or 0.15 mg / mL transferrin solution, (v) Ascorbic acid solution (e.g., supplied by Sigma-Aldrich), optionally 0.1-0.4 mg / mL or 0.256 mg / mL of ascorbic acid solution, and (vi) Further comprising one or more StemPro®-34 dietary supplements (e.g., supplied by Gibco®), optionally in concentrations of 1-4% (v / v) or 2.6% (v / v). In some embodiments, the culture medium is (i) 0.1-2% (v / v) of GlutaMAX (trademark) and (ii) 10-30 mM HEPES and (iii) 0.1-2% penicillin-streptomycin and (iv) A transferrin solution containing 0.05 to 0.25 mg / mL, (v) Ascorbic acid solution at 0.1-0.4 mg / mL, (vi) StemPro™-34 nutritional supplement in 1-4% (v / v) concentrations, Includes.

[0030] In some embodiments, the culture medium includes cardiac tissue.

[0031] A second aspect of this disclosure provides a method for inducing a disease phenotype of diabetic cardiomyopathy in cardiac tissue, the method comprising culturing cardiac tissue in the culture medium described in relation to the first aspect of this disclosure.

[0032] In some embodiments, the method includes generating cardiac tissue using systems defined in WO 2015 / 061907 Al, WO 2021 / 158233 Al, and / or WO 2016 / 183143 Al. In some embodiments, the method includes providing cardiac tissue (e.g., cardiac organoids) as defined in WO 2016 / 183143 Al.

[0033] In some embodiments, this cardiac tissue includes a mature ultrastructure. In some embodiments, this ultrastructure is selected from the group consisting of sarcomeres, mitochondria, T-tubules, sarcoplasmic reticulum, and combinations thereof. In some embodiments, this cardiac tissue includes T-tubules.

[0034] In some embodiments, this cardiac tissue exhibits a positive force-frequency relationship. In some embodiments, this force is approximately 0.25 to approximately 2 mN / mm at frequencies of approximately 0 to 6 Hz. 2 That is the case.

[0035] This heart tissue may be cultured in a culture medium for any period of time, provided it is appropriate. In some embodiments, this heart tissue is cultured in a culture medium for 3 to 100 days. In some embodiments, this heart tissue is cultured in a culture medium for 4 to 90 days. In some embodiments, this heart tissue is cultured in a culture medium for 5 to 80 days. In some embodiments, this heart tissue is cultured in a culture medium for 6 to 70 days. In some embodiments, this heart tissue is cultured in a culture medium for 7 to 60 days. In some embodiments, this heart tissue is cultured in a culture medium for 9 to 50 days. In some embodiments, this heart tissue is cultured in a culture medium for 14 to 45 days. In some embodiments, this heart tissue is cultured in a culture medium for 18 to 40 days. In some embodiments, this heart tissue is cultured in a culture medium for 21 to 35 days. In some embodiments, this heart tissue is cultured in a culture medium for about 7 days. In some embodiments, this heart tissue is cultured in a culture medium for about 14 days. In some embodiments, this heart tissue is cultured in a culture medium for about 21 days. In some embodiments, the cardiac tissue is cultured in culture medium for about 28 days. In some embodiments, the cardiac tissue is cultured in culture medium for about 35 days.

[0036] Those skilled in the art will understand that, in accordance with standard cell culture techniques, the culture medium may be replenished / replaced at intervals.

[0037] In some embodiments, the method includes the step of culturing cells in a growth medium to increase their size. These cells may be ex vivo primary cardiomyocytes, cell lines, or stem cells (e.g., iPSCs such as human iPSCs). The growth medium may be any medium that promotes cell growth. Exemplary growth media include mTeSRl (Stemcell Technologies), mTeSR Plus (Stemcell Technologies), TeSR-E8 (Stemcell Technologies), Essential 8® Medium (Gibco), and StemFlex® Medium (Gibco). In these embodiments, this growth step is performed prior to culturing cardiac tissue in a culture medium as defined with respect to the first aspect of this disclosure.

[0038] Cells may be cultured in growth medium for any appropriate period. In some embodiments, cells are cultured in growth medium for 1 day to 75 weeks. In some embodiments, cells are cultured in growth medium for 1 to 24 days. In some embodiments, cells are cultured in growth medium for 2 to 21 days. In some embodiments, cells are cultured in growth medium for 3 to 18 days. In some embodiments, cells are cultured in growth medium for 4 to 15 days. In some embodiments, cells are cultured in growth medium for 5 to 12 days. In some embodiments, cells are cultured in growth medium for 6 to 11 days. In some embodiments, cells are cultured in growth medium for 7 to 10 days. In some embodiments, cells are cultured in growth medium for 8 to 9 days.

[0039] In some embodiments, the method includes the step of culturing cells in a differentiation medium and differentiating these cells into cardiomyocytes for the formation of cardiac tissue. These cells may be cell lines or stem cells (e.g., iPSCs such as human iPSCs). In some embodiments, the method includes the step of culturing pluripotent stem cells in a differentiation medium to provide cardiomyocytes. In these embodiments, this differentiation step is performed before culturing cardiac tissue in a medium as defined with respect to the first aspect of this disclosure. Typically, this differentiation step is performed after culturing cells in a growth medium.

[0040] For example, stem cells may be embryonic stem cells (ESCs), fetal stem cells (FSCs), and / or adult (or somatic) stem cells (SSCs). Stem cells can be classified in terms of their differentiation potential as totipotent (also called pluripotent) (stem cells that can differentiate into embryonic and extraembryonic cell types), pluripotent stem cells (stem cells that can differentiate into almost all cell types), pluripotent stem cells (stem cells that can differentiate into many cell types), oligopluripotent stem cells (stem cells that can differentiate into only a few cell types), or unipotent stem cells (stem cells that produce only one cell type). Stem cells can be obtained commercially, directly from / isolated from patients, or from any other suitable source.

[0041] In some embodiments, these stem cells are induced pluripotent stem cells (iPSCs). In some embodiments, these pluripotent stem cells are human iPSCs. In some embodiments, these pluripotent stem cells are autologous.

[0042] The differentiation medium may be any medium suitable for resulting differentiation of stem cells into the cardiac lineage. In some embodiments, this differentiation medium includes activin A and bone morphogenetic protein 4 (BMP4). An exemplary differentiation medium is RPMI / B27 insulin-free medium supplemented with activin A (e.g., 50 ng / ml) and BMP4 (e.g., 25 ng / ml). In some embodiments, this differentiation medium includes a Gsk3β inhibitor and a Wnt inhibitor. An exemplary differentiation medium is described in WO 2016 / 183143 A1.

[0043] Cells may be cultured in differentiation medium for any appropriate period. In some embodiments, cells are cultured in differentiation medium for 1 to 30 days. In some embodiments, cells are cultured in differentiation medium for 2 to 27 days. In some embodiments, cells are cultured in differentiation medium for 3 to 24 days. In some embodiments, cells are cultured in differentiation medium for 4 to 21 days. In some embodiments, cells are cultured in differentiation medium for 5 to 18 days. In some embodiments, cells are cultured in differentiation medium for 6 to 15 days. In some embodiments, cells are cultured in differentiation medium for 7 to 12 days. In some embodiments, cells are cultured in differentiation medium for 8 to 10 days. Typically, cells are cultured in differentiation medium for 21 to 24 days.

[0044] Typically, the growth step and / or differentiation step are performed before the culture step in culture medium according to the first aspect of the present disclosure. Typically, the growth step is performed before any differentiation step.

[0045] In some embodiments, the method includes a step of encapsulating cells within a hydrogel. This encapsulation step may be performed 10 to 30 days after the cells have been grown in growth medium and / or differentiation medium. This encapsulation step may also be performed 15 to 25 days after the cells have been grown in growth medium and / or differentiation medium. Typically, this encapsulation step is performed about 20 days after the cells have been grown in growth medium and / or differentiation medium. As an example, cells may be encapsulated in a hydrogel as described in WO 2016 / 183143 Al.

[0046] As used herein, the term “hydrogel” refers to a physically or chemically crosslinked polymer network capable of absorbing large amounts of water, and is a common material for forming scaffolds in tissue engineering. These can be classified into different categories depending on various parameters, including preparation methods, charge, and mechanical and structural properties. See S. Van Vlierberghe et al., “Biopolymer-Based Hydrogels As Scaffolds for Tissue Engineering Applications: A Review,” Biomacromolecules, 2011, 12(5), pp. 1387–1408. This entire document is incorporated herein by reference. Hydrogels may include polyvinyl alcohol, sodium polyacrylate, hydrophilic acrylate polymers and copolymers, and other materials. Natural hydrogel materials include agarose, methylcellulose, hyaluronic acid, and other naturally derived polymers.

[0047] In some embodiments, the method further includes a step of electromechanically conditioning cells. A suitable conditioning regimen can be selected by those skilled in the art. As an example, cells may be electromechanically conditioned as described in WO 2016 / 183143 Al. In some embodiments, the method further includes a step of electromechanically conditioning cardiomyocytes by exposing them to electromechanical stimulation of increasing intensity over a period of time, thereby forming cardiac tissue having molecular, structural, and functional properties that mimic innate adult cardiac tissue. This has the advantage of inducing cell maturation. Typically, the electromechanical conditioning step is performed before the culture step in culture medium according to the first embodiment of the present disclosure. In alternative embodiments, the electromechanical conditioning step is performed concurrently with the culture step in culture medium according to the first embodiment of the present disclosure.

[0048] In some embodiments, the period is 1 to 6 weeks. In some embodiments, the period is 2 to 5 weeks. In some embodiments, the period is 3 to 4 weeks.

[0049] In some embodiments, the method further includes measuring one or more parameters of cardiac tissue contractility.

[0050] In some embodiments, one or more parameters of this contractility are selected from the group consisting of relaxation time, spasm amplitude, contraction time, and combinations thereof. Relaxation time is the time it takes for the tissue to relax from a fully contracted state (peak amplitude) to a relaxed state (10% of peak amplitude). Spasm amplitude is the sum of the forces exerted by the tissue during contraction (peak amplitude). Contraction time is the time it takes for the tissue to contract from a relaxed state (10% of peak amplitude) to a fully contracted state (peak amplitude). Methods for measuring contractility are known and routinely used in the art (see, for example, WO 2016 / 183143 Al or Zhao et al. “A Platform for Generation of Chamber-Specific Cardiac Tissues and Disease Modeling.” Cell. 2019 Feb 7;176(4):913-927.e18. These documents are incorporated herein by reference in their entirety).

[0051] In some embodiments, one or more parameters are measured before, during, and / or after the culture step.

[0052] In some embodiments, the method further includes characterizing the tissue morphology of cardiac tissue.

[0053] In some embodiments, this tissue morphology is characterized by microscopic examination with or without tissue staining. In some embodiments, this tissue morphology is characterized by microscopic examination of tissue stained with lipid staining (e.g., HCS LipidTOX® Deep Red Neutral Lipid Stain (Thermo Fisher Scientific, catalog no. H34477)). In some embodiments, this tissue dye is an anti-α-actinin (sarcomere) antibody (i.e., for staining sarcomeres) or an anti-TOM20 antibody (i.e., for staining mitochondria).

[0054] In some embodiments, this tissue morphology is characterized before, during, and / or after the culture step.

[0055] In some embodiments, the disease phenotype of diabetic cardiomyopathy is induced within cardiac tissue, and the method further includes characterizing one or more endpoints of cardiac tissue selected from the group consisting of lipidomics, phosphorylated Akt Western blotting, RNA sequencing, proteomics, intracellular calcium transient analysis, and combinations thereof.

[0056] In some embodiments, one or more evaluation items are characterized before the culture step, during the culture step, and / or after the culture step.

[0057] In some embodiments, the method further includes the step of incubating disease-phenotypic cardiac tissue with a diagnostic agent. A “diagnostic agent” is any substance whose ability to diagnose, cure, alleviate, treat, prevent, modify, or promote a disease of interest is evaluated. In one embodiment, a diagnostic agent may be a “medicine” as defined under Section 321(g)(1) of the Food, Drug, and Cosmetic Act. Diagnostic agents include, but are not limited to, chemical compounds, biological agents, proteins, peptides, antibodies, nucleic acids, lipids, polysaccharides, nutritional supplements, diagnostic agents, and immunomodulators, and these diagnostic agents may also be called “pharmacological substances.” Any suitable diagnostic agent may be tested, including anti-inflammatory agents such as opioid analgesics, antihistamines and non-steroidal anti-inflammatory drugs (NSAIDs); diuretics such as carbonic anhydrase inhibitors, loop diuretics, hyperbasic diuretics, thiazides and thiazide analogs, and potassium-sparing diuretics; agents affecting the cardiovascular system such as angiotensin-converting enzyme inhibitors; cardiovascular agents such as organonitrates, calcium channel blockers, sympathomimetic agents, vasodilators, beta-adrenergic receptor agonists and antagonists, alpha-adrenergic receptor agonists and antagonists, cardiac glycosides, and antiarrhythmic agents; agents affecting hyperlipoproteinemia such as 3-hydroxymethylglutaryl coenzyme A (HMG-CoA) inhibitors; antitumor agents such as alkylating agents, antimetabolites, natural products, antibiotics and other agents; immunomodulators, antidiabetic agents; and antimicrobial agents such as antibacterial agents, antiviral agents, antifungal agents, antiprotozoal agents and anthelmintic agents.

[0058] Generally, the diagnostic agent can be incubated with cardiac tissue exhibiting the disease phenotype at a dose range estimated to produce an effect, and for a sufficient period to produce an effect (e.g., a metabolic effect or an effect demonstrating toxicity or efficacy). The incubation time is typically between 1 hour and 24 hours, or can be extended to several days or weeks if necessary. Incubation conditions typically include standard conditions known in the art, including an incubation temperature of 37 degrees Celsius.

[0059] Using cardiac tissue exhibiting the disease phenotype, the dosage range for the diagnostic agent can be determined. The effects on cardiac tissue as the concentration (i.e., dosage) of the diagnostic agent is increased can be monitored to detect its effectiveness.

[0060] Various doses of individual test agents and combinations of test agents can be screened in a panel of cardiac tissues with diverse genetic backgrounds (e.g., those derived from different donors and / or those containing genetically modified cells) to determine the pharmacogenetic efficacy profile of the test agents. For example, multiple doses of a test agent, or combinations with test agents, can be screened for specific efficacy or lack thereof in one or more genetic backgrounds.

[0061] A third aspect of this disclosure provides diseased cardiac tissue produced by a method according to a second aspect of this disclosure, which exhibits a disease phenotype of diabetic cardiomyopathy.

[0062] In some embodiments, the affected cardiac tissue is produced directly by the methods described herein.

[0063] A fourth aspect of the present disclosure provides diseased cardiac tissue comprising a population of cardiomyocytes in a culture medium according to the first aspect of the present disclosure.

[0064] In some embodiments, this cardiac tissue is produced using the systems defined in WO 2015 / 061907 Al, WO 2021 / 158233 Al, and / or WO 2016 / 183143 Al.

[0065] In some embodiments, the cardiac tissue further includes two or more scaffold elements positioned within the cardiac tissue. In some embodiments, these scaffold elements are opposite each other (they may be formed from a single element or from separate elements) and function to form anchor points of the cardiac tissue formed between them. The cardiac tissue is not limited to having two scaffold elements and may include more than two, for example, three, four, five, six, seven, eight, nine, or ten or more such scaffold elements. Deformable scaffold elements may be provided in any number, as long as the cardiac tissue has the ability to form a 3D tissue positioned between the scaffold elements by being formed around each of the scaffold elements and joined together. In some embodiments, the cardiac tissue includes two scaffold elements positioned at or near opposite ends of the longitudinal axis of the cardiac tissue.

[0066] In some embodiments, these scaffold elements are deflectable, deformable, or bendable, and are further configured to allow measurement of the contractile force exerted by cardiac tissue on the scaffold elements. The shape, thickness, length, orientation, and surface morphological properties of the scaffold elements allow deformation, bending, or other shape changes in response to the contractile action or activity of the scaffold elements and the cardiac tissue connected thereto, and any number of suitable methods can be varied as long as such deformation, bending, or other shape changes can be reliably measured. In some embodiments, the scaffold elements have elasticity of about 20 kPa to 0.5 MPa.

[0067] Scaffold elements may be made from any suitable material, including, for example, poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PMMA), and polystyrene. Scaffold elements may also be made from biodegradable materials. Other suitable materials include poly(glycerol sebacate), POMaC without citrate, poly(lactic acid), poly(glycolic acid), poly(e-caprolactone), various polyurethanes and their copolymers, silk, microstructured materials, nanofabricated materials, and / or nanostructure-doped materials such as nanorods or quantum dots, among others. Optionally, in certain embodiments, the scaffold element material may be permeable, thereby allowing the exchange and / or passage of water and molecules, including proteins, pharmaceuticals, nutrients, and metabolic waste materials. In other certain embodiments, permeability may be achieved through the formation of pores in the scaffold element material. In further embodiments, the scaffold elements may be fabricated by any suitable means, including but not limited to microfabrication, soft lithography processes (including step-and-flash imprint lithography (STIL)), 3D printing (i.e., additive manufacturing), hot embossing, extrusion, injection molding, phase-shift edge lithography, and nanoskiving.

[0068] In some embodiments, this cardiac tissue contains mature ultrastructure. In some embodiments, this ultrastructure is selected from the group consisting of sarcomeres, mitochondria, T-tubules, sarcoplasmic reticulum, and combinations thereof.

[0069] In some embodiments, this cardiac tissue exhibits a positive force-frequency relationship. In some embodiments, this force is approximately 0.25 to approximately 2 mN / mm at frequencies of approximately 0 to 6 Hz. 2 That is the case.

[0070] A fifth aspect of the present disclosure provides a kit comprising a culture medium and a bioreactor for culturing cardiac tissue according to a first aspect of the present disclosure, wherein the bioreactor comprises a plurality of wells, each well comprising a chamber configured for growing cardiac tissue therein and one or more deformable scaffold elements attached to each chamber.

[0071] In some embodiments, the bioreactor is as described in WO 2015 / 061907 Al.

[0072] In some embodiments, the bioreactor is a multiwell plate. In some embodiments, the bioreactor is a multiwell plate using 12 wells, 96 wells, 384 wells, or 1536 wells.

[0073] In some embodiments, the bioreactor is composed of a polymer. In some embodiments, the polymer is a biodegradable polymer. In some embodiments, the biodegradable polymer is polylactic acid, poly(lactic acid-co-glycol) acid, or poly(caprolactone), polyglycolide, polylactide, polyhydroxobutyric acid, polyhydroxyalkanoic acid, chitosan, hyaluronic acid, hydrogel, poly(2-hydroxyethyl methacrylate), poly(ethylene glycol), poly(L-lactide) (PLA), or any combination thereof. In some embodiments, the polymer is poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PMMA), polystyrene, poly(glycerol sebacate), citric acid-free POMaC, poly(e-caprolactone), polyurethane, silk, or nanoprocessed materials, or copolymers or mixtures thereof. In some embodiments, the polymer is doped with nanostructures.

[0074] In some embodiments, the deformable scaffold elements are made of metal, silk, or polymer. In some embodiments, the deformable scaffold elements are made of intestinal material, monoacrylic, polyglycol, prolene, polyglutinin, polydioxanone, polypropylene, nylon, or polyester.

[0075] In some embodiments, the chamber is configured to be seeded with cardiomyocytes.

[0076] In some embodiments, the deformable scaffold elements attached to each chamber are oriented substantially perpendicular, substantially parallel, or substantially oblique to the orientation of the longitudinal axis of the chamber.

[0077] In some embodiments, the deformable scaffold elements are configured to be embedded or partially embedded by cardiac tissue during cardiac tissue growth.

[0078] In some embodiments, the deformable scaffold element is configured to be encapsulated or partially encapsulated by cardiac tissue and attached to this cardiac tissue, so that the cardiac tissue moves in conjunction with the movement of the deformable scaffold element.

[0079] In some embodiments, the bioreactor further includes a pair of electrodes configured to generate an electric current through the growth chamber of the bioreactor.

[0080] A sixth aspect of this disclosure provides a method for evaluating the safety and / or efficacy of a diagnostic agent to cardiac tissue, comprising: (a) culturing cardiac tissue in a culture medium described herein; (b) contacting the cardiac tissue with the diagnostic agent; (c) measuring the effect on one or more physiological parameters indicating safety and / or efficacy; and (d) comparing (c) with the same physiological parameters measured from control cardiac tissue not exposed to the diagnostic agent, wherein a statistically significant change in the physiological parameters in (c) compared to (d) indicates that the diagnostic agent lacks safety and / or efficacy.

[0081] In some embodiments, this cardiac tissue is produced using the systems defined in WO 2015 / 061907 Al, WO 2021 / 158233 Al, and / or WO 2016 / 183143 Al.

[0082] Where applicable, or unless specifically denied, any of the embodiments described herein are intended to be combined with any one or more other embodiments, even if those embodiments are described under different aspects of this disclosure. The other purposes, features, and advantages of this disclosure will become more apparent from the following detailed description, which will proceed with reference to the accompanying drawings.

[0083] Next, this disclosure will be described in detail by illustration only, with reference to the following figures. [Brief explanation of the drawing]

[0084] [Figure 1] A schematic diagram of the contractile force over time is shown, specifying the single contraction amplitude (peak height), contraction time (time from 10% of peak height to amplitude), relaxation time (time from amplitude to 10% of peak height), and passive tension (force between peaks). [Figure 2]This diagram shows the time course of contractility in cardiac tissue treated with different media. The upper panel shows relative changes in Δsingle contraction amplitude, Δcontraction time, and Δrelaxation time for different treatment groups. Data are presented as median (circle) changes relative to the untreated response (day 0). The lower panel shows raw data for single contraction amplitude, contraction time, and relaxation time for different treatment groups. Data are presented as median (circle). Tissues were switched from I3M medium to treatment groups on day 3. The I3M+BSA control group was supplemented with 250 μM BSA control. [Figure 3] This diagram shows the time course of contractility in cardiac tissue treated with different media. The upper panel shows relative changes in Δsingle contraction amplitude, Δcontraction time, and Δrelaxation time for different treatment groups. Data are presented as median (circle) changes relative to the untreated response (day 0). The lower panel shows raw data for single contraction amplitude, contraction time, and relaxation time for different treatment groups. Data are presented as median (circle). Tissues were switched from I3M medium to treatment groups on day 2. N=3 tissues per group, and shading represents 95% confidence intervals. 250 μM BSA control was added to the I3M (control) group. [Figure 4] The Δ-single contraction amplitude for various treatment groups is shown. Tissues were switched from I3M medium to the treatment group on day 2. Data are presented as the median change ±95% confidence interval compared to the untreated group (day 0). N=3 tissues per group. [Figure 5] This shows the Δ-contraction time for various treatment groups. Tissues were switched from I3M medium to the treatment group on day 2. Data are presented as the median change ±95% confidence interval compared to the untreated group (day 0). N=3 tissues per group. [Figure 6] This shows the Δ-relaxation time for various treatment groups. Tissues were switched from I3M medium to the treatment group on day 2. Data are presented as the median change in response compared to the untreated group (day 0) ± 95% confidence interval. N=3 tissues per group. [Figure 7] The spontaneous heart rate and stimulation threshold for various treatment groups are shown over time. [Figure 8] This shows the Δ-relaxation time for various treatment groups. Tissues were switched from I3M medium to the treatment group on day 2. Data are presented as the median change in response compared to the untreated group (day 0) ± 95% confidence interval. N=6 tissues per group. [Figure 9] This graph shows the time course of contractility in cardiac tissue treated with different media after endothelin-1 was added to the culture medium on day 0. The upper panel shows data for Δsingle contraction amplitude, Δrelaxation time, and relative changes in Δrelaxation time for the different treatment groups. Data are presented as the change in median (circle) relative to the untreated response (day 0). The lower panel shows raw data for single contraction amplitude, contraction time, and relaxation time for the different treatment groups. Data are presented as median (circle). Tissues were switched from I3M medium to the treatment group on day 2, and washout was initiated on day 30. N=3 tissues per group, and shading represents the 95% confidence interval. [Figure 10] This graph shows the time course of contractility of cardiac tissue treated with different media when endothelin-1 was added to the culture medium during each medium change. The upper panel shows data for Δsingle contraction amplitude, Δrelaxation time, and relative changes in Δrelaxation time for different treatment groups. Data are presented as the change in median (circle) relative to the untreated response (day 0). The lower panel shows raw data for single contraction amplitude, contraction time, and relaxation time for different treatment groups. Data are presented as median (circle). Tissues were switched from I3M medium to the treatment group on day 2, and washout was initiated on day 30. N=3 tissues per group, and shading represents the 95% confidence interval. [Figure 11] The images show tissue samples from day 0 of treatment (upper panel) and day 30 of treatment (lower panel) for different treatment groups. [Figure 12] The images show tissue samples from day 0 (upper panel) and day 30 (lower panel) of the I3M + 200 μM palmitate + 200 μM oleate + 5 nM endothelin-1 treatment group. [Figure 13]The image shows 40x confocal stacked images of different treatment groups using lipid droplet staining (red) and nuclear staining (blue). Features circled in the image are lipid droplets larger than 10 μm. [Figure 14] This shows the time course of contractility between a group treated with a control definition medium (RPMI1640 + 5 mM glucose + 1.4 mM CaCl2 + 50 μM palmitate + 50 μM oleate + B27 without insulin + 1 nM insulin) and a group treated with a lipid-toxic medium (definition medium + 150 μM palmitate + 150 μM oleate + 1 nM endothelin-1). The upper panel shows the relative changes in Δsingle contraction amplitude, Δcontraction time, and Δrelaxation time for the different treatment groups. The data are presented as the change in median (circle) relative to the response at no treatment (day 0). The lower panel shows the raw data for single contraction amplitude, contraction time, and relaxation time for the different treatment groups. The data are presented as median (circle). N = 3 tissues in the definition medium group, N = 2 tissues in the definition medium + 150 μM palmitate + 150 μM oleate + 1 nM endothelin - 1 group (1 tissue was damaged during the experiment). Shading indicates 95% confidence intervals. Tissues were switched from I3M medium to definition medium on day 2. After treating the tissues in definition medium for 10 days, they were switched to their respective treatment groups on day 12. [Figure 15] A shows the measured change in passive tension from day 0 to day 14 or 15. The bar represents the median. N=27 represents tissue from three independent experiments for each group. B shows the measured change in tissue area from day 0 to day 14 or 15. The bar represents the median. N=27 represents tissue from three independent experiments for each group. [Figure 16] This shows a tissue ruler that detects the contours of tissues in order to calculate tissue area. [Figure 17] This chart shows the measured change in stimulus threshold (ET) from day 0 to day 14 or 15. The bar represents the median. N=27 is tissue from three independent experiments for each group. [Figure 18]A shows APD90 (action potential duration from peak to 90% repolarization) measured at the end of processing. The bar represents the median. N=6 represents tissue from two independent experiments for each group. B shows APD90 measured from cardiac myocardial action potential traces via MATLAB®. [Figure 19] A shows the time to 50% calcium transient decay measured at the end of the treatment. The bars represent the median ± 95% confidence interval. N=9 represents tissue from three independent experiments for each group. B shows the time to 50% calcium transient decay measured from cardiac calcium transient traces via MATLAB®. [Modes for carrying out the invention]

[0085] With the unique ability to visualize and quantify cardiac tissue contractile parameters using the system described in WO 2015 / 061907 Al, WO 2021 / 158233 Al and / or WO 2016 / 183143 Al, also known as the Biowire® system, we were able to measure more robust contractility indicators, such as increased relaxation time, which function as a similar sign of diastolic dysfunction, a characteristic feature of lipid-toxic diabetic cardiomyopathy, that other groups have not demonstrated in their models. The three-dimensional in vitro lipid-toxic cardiomyopathy model is the first metabolism-driven in vitro model of diastolic dysfunction.

[0086] The concentrations of each lipid-toxic component added to the basic culture medium, namely palmitate, oleate, and endothelin-1, were strictly and specifically optimized to produce the desired phenotype of lipid-toxic diabetic cardiomyopathy in cardiac tissue. [Examples]

[0087] The following examples are provided to illustrate specific features and / or embodiments. These examples should not be construed as limiting this disclosure to the specific features or embodiments described.

[0088] Example 1 In general terms, the development of our lipid-toxic cardiomyopathy model involved culturing healthy cardiac tissue in the Biowire® II platform and exposing these tissues to lipid-toxic media for 28 days to induce the phenotype of lipid-toxic cardiomyopathy within them. During treatment with the lipid-toxic media, we recorded changes in tissue contractility over time. One of the main parameters used to measure tissue contractility was relaxation time (i.e., the time it takes for the tissue to relax from a fully contracted state). This is because an increase in relaxation time in cardiac tissue is analogous to delayed relaxation of the left ventricle (Figure 1). In addition to relaxation time, we also measured single contraction amplitude, which is the sum of the forces exerted by the tissue during contraction, and contraction time, which is the time it takes for the tissue to reach the peak of contraction from a relaxed state (Figure 1). After the completion of the series of treatments, we collected these tissues and performed a more detailed characterization of the model to confirm that these tissues exhibited other important features of lipid-toxic cardiomyopathy.

[0089] The inventors have found that cardiac tissue treated with their own lipid-toxic culture medium can achieve a consistent contractile phenotype consistent with lipid-toxic diabetic cardiomyopathy.

[0090] Lipid-toxic culture medium treatment group The inventors first added the following components to StemPro-34® basic culture medium (Life Technologies, catalog number 10639011): 1% (v / v) GlutaMAX (Life Technologies, catalog number 35050-061) and 2% (v / v) 1M HEPES (Life Technologies). (Technologies, catalog number 15630-080, final concentration 20 mM), 1% (v / v) penicillin-streptomycin (Gibco, catalog number 15140-122), 0.5% (v / v) 30 mg / mL transferrin solution (prepared by dissolving n-transferrin powder (Sigma-Aldrich, catalog number T8158) in IMDM (Gibco, catalog number 12440-053), final concentration 0.15 mg / mL), 0.4% (v / v) 64 mg / mL ascorbic acid solution (prepared by dissolving 2-phosphate-L-ascorbic acid trisodium salt (Sigma-Aldrich, catalog number 49752) in PBS (Sigma-Aldrich, catalog number D8537), final concentration 0.256 mg / mL), and 2.6% (v / v) StemPro™-34 nutritional supplement (Life A basic culture medium was prepared by adding (Technologies, catalog number 10639011). This basic culture medium is called "I3M". It will be obvious to those skilled in the art that other media may be suitable as basic culture media.

[0091] Next, the inventors prepared various test media by adding various components to these basic media at various concentrations, as summarized in the table below. [Table 1]

[0092] Generation of heart tissue Cardiac tissue was generated by culturing hiPSC-derived cardiomyocytes in I3M Media using the Biowire® system, and according to a 7-week maturation protocol described by Feric et al., incorporated herein by reference ("Engineered Cardiac Tissues Generated in the Biowire II: A Platform for Human-Based Drug Discovery." Toxicol Sci. 2019 Nov 1;172(1):89-97. doi:10.1093 / toxsci / kfz168). The inventors then transferred the matured tissue to a chronic test chamber, which divided the tissue into six wells (three tissues assigned to each well). This tissue was exposed to a test lipid-toxic medium for 28 days.

[0093] result In preliminary experiments in which cortisol was added to a lipid-toxic medium, the inventors demonstrated that 1 μM cortisol significantly and sustainably increased the single contraction amplitude of Biowire® tissue, which was inconsistent with the expected phenotype. Therefore, supplementation with 1 μM cortisol was excluded from all future replicates of the lipid-toxic medium.

[0094] The inventors demonstrated that the I3M + 250 μM palmitate + 10 nM endothelin-1 group induced an increase in contraction and relaxation times, along with a decrease in single contraction amplitude, after 14 days of treatment. The inventors also found that a similar effect on tissue contractility was achieved even when the concentration of endothelin-1 was reduced to 5 nM (I3M + 250 μM palmitate + 5 nM endothelin-1) (Figure 2). Therefore, subsequent experiments used a lower concentration of 5 nM endothelin-1. However, those skilled in the art will recognize that higher concentrations of endothelin-1 are also suitable.

[0095] All tissues treated with I3M + 250 μM palmitate + 5 nM or 10 nM endothelin-1 ceased pulsation within 24 days (Figure 2). This is thought to be due to the toxic effects of the high concentration of palmitate added to the culture medium. To address this observed toxicity, the inventors employed two approaches: (1) an approach in which the inventors treated tissues with low concentrations of palmitate (100, 150, or 200 μM) for 28 days, and (2) an approach in which the inventors started treatment with 250 μM palmitate for the first 10 days, then switched to low concentrations of palmitate (100 or 150 μM). The inventors found that the I3M + 200 μM palmitate + 5 nM endothelin-1 treatment group sustainably increased relaxation time (Figure 3). However, although the tissue treated with this culture medium survived longer than the tissue in previous experiments, it stopped pulsating by the 30th day after treatment.

[0096] Therefore, the inventors sought additional approaches to counteract the toxic effects observed with treatment at high concentrations of palmitate. In the following experiment, the inventors began by adding oleate (Ol) in a 1:1 ratio with palmitate. The inventors believed that a 1:1 ratio of oleate to palmitate would reduce the toxicity of palmitate without significantly increasing the total fatty acid content in the culture medium, because a significant increase in total fatty acid content could have other adverse effects on the tissue and prevent it from exhibiting the desired phenotype.

[0097] The inventors found that in tissues treated with oleate and palmitate in a 1:1 ratio over time, pulsation did not cease or single contraction amplitude did not decrease significantly even after 28 days of treatment (Figure 4). These same groups, i.e. I3M + 150 μM palmitate + 150 μM oleate + 5 nM endothelin-1, I3M + 200 μM palmitate + 200 μM oleate + 5 nM endothelin-1, and The treatment regimen was changed from I3M + 250 μM palmitate + 250 μM oleate + 5 nM endothelin-1 (10 days) to I3M + 100 μM palmitate + 100 μM oleate + 5 nM endothelin-1. These treatments increased relaxation time in the tissues by an average of 5%, 13%, and 15%, respectively, but did not adversely affect other metrics of tissue health and maturity, such as stimulation threshold (the minimum voltage required for tissue to respond to external stimuli) and spontaneous beating (a beating initiated without electrical stimulation) (Figures 6 and 7). A significant increase in either stimulation threshold or spontaneous beating would indicate an extreme decline in tissue health, and in this particular experiment, tissues not treated with oleate over time exhibited this characteristic (Figure 7).

[0098] At the end of the time course, the inventors washed out the lipid-toxic medium with standard basal medium for 7 days and observed that the contractile effect was partially or completely reversed in tissues treated with both palmitate and oleate during the time course. This indicates that lipid-toxic mediums containing both palmitate and oleate do not cause irreversible damage to these tissues. This supports the idea that the lipid-toxic phenotype in cardiac tissue may be reversed with the addition of compounds that mechanistically inhibit lipid production.

[0099] The inventors also have a group of treatments that do not contain endothelin-1, specifically, I3M + 200 μM palmitate + 200 μM oleate, and The regimen will transition from I3M + 250 μM palmitate + 250 μM oleate (10 days) to I3M + 100 μM palmitate + 100 μM oleate. However, we also found that these treatments resulted in an average increase of 15% and 13% in relaxation time within the tissue (Figure 6). These amounts are comparable to the increases achieved by treatment groups containing both fatty acids and endothelin-1. Therefore, we hypothesized that the increase in relaxation time observed within the tissue during the treatment period was primarily caused by fatty acids (and not endothelin-1).

[0100] The inventors repeated treatment groups from previous experiments that (1) had the greatest effect on relaxation time and (2) did not adversely affect the stimulation threshold, spontaneous pulse, or single contraction amplitude. Of these groups, the groups that received I3M + 200 μM palmitate + 200 μM oleate + 5 nM endothelin-1 and the groups that received I3M + 250 μM palmitate + 250 μM oleate + 5 nM endothelin-1 (for 10 days) followed by I3M + 100 μM palmitate + 100 μM oleate + 5 nM endothelin-1 induced an average increase of 18% and 12%, respectively, in maximum relaxation time by the end of the treatment (Figure 8). This effect decayed toward baseline values ​​in both groups by performing a washout with standard basal medium at the end of the time course.

[0101] For future iterations of the lipid-toxic medium, the inventors used a concentration of 200 μM palmitate + 200 μM oleate. This is because these concentrations consistently increased the relaxation time by more than 10% in two independent experiments and also offer the simplicity of remaining at the same concentration over time (i.e., the concentrations of palmitate and oleate do not need to be changed to lower concentrations after 10 days of treatment). However, those skilled in the art will recognize that there may be variability in these concentrations.

[0102] This experiment reaffirmed that treatment with fatty acids alone may increase the relaxation time of cardiac tissue. However, the increase tends to be more pronounced when both fatty acids and endothelin-1 are administered to the tissue. This was revealed when the inventors compared a treatment group containing both fatty acids and endothelin-1 with a treatment group without endothelin-1. For example, in this experiment, the I3M + 200 μM palmitate + 200 μM oleate + 5 nM endothelin-1 treatment group showed an average increase of 18% in relaxation time by the end of the time course, while the I3M + 200 μM palmitate + 200 μM oleate treatment group achieved an average increase of only 10%. Similarly, in the treatment group that switched from I3M + 250 μM palmitate + 250 μM oleate + 5 nM endothelin-1 (10 days) to I3M + 100 μM palmitate + 100 μM oleate + 5 nM endothelin-1, relaxation time increased by an average of 12%, whereas in the treatment group that switched from I3M + 250 μM palmitate + 250 μM oleate (10 days) to I3M + 100 μM palmitate + 100 μM oleate, the increase was only an average of 8%.

[0103] The inventors investigated the individual contributions of fatty acids and endothelin-1 to the contractile effect observed in cardiac tissue by including a treatment group containing only endothelin-1 (I3M + 5nM endothelin-1) and a treatment group containing only fatty acids (I3M + 200 μM palmitate + 200 μM oleate). The inventors also aimed to determine whether endothelin-1 receptors in the tissue desensitize over time, as repeated exposure to high concentrations of endothelin-1 may cause its effect on contractile parameters to diminish over time. Therefore, the inventors included two additional treatment groups. Specifically, one group contains a low concentration of 1 nM endothelin-1 (I3M + 200 μM palmitate + 200 μM oleate + 1 nM endothelin-1), and the other group adds endothelin-1 to the tissue every 96 hours instead of every 48 hours (I3M + 200 μM palmitate + 200 μM oleate + 5 nM endothelin-1 (alternating)). In this experiment, the inventors found that while the treatment groups without endothelin-1 (I3M + BSA control, and I3M + 200 μM palmitate + 200 μM oleate) did not appear to have a significant effect on contractility, the treatment groups containing endothelin-1 (I3M + 5 nM endothelin-1, I3M + 200 μM palmitate + 200 μM oleate + 1 nM endothelin-1, I3M + 200 μM palmitate + 200 μM oleate + 5 nM endothelin-1, and I3M + 200 μM palmitate + 200 μM oleate + 5 nM endothelin-1 (alternating)) caused a sustained increase in contraction time, and the initial increase in single contraction amplitude disappeared by the end of the treatment (Figure 9). Importantly, the relaxation time did not increase in the 5 nM endothelin-1 group, leading the inventors to conclude that endothelin-1 treatment alone cannot produce a phenotype consistent with lipid-toxic cardiomyopathy.The inventors also found that adding fatty acids and endothelin-1 with each medium change (I3M + 200 μM palmitate + 200 μM oleate + 1 nM endothelin-1, and I3M + 200 μM palmitate + 200 μM oleate + 5 nM endothelin-1) resulted in maximum increases in relaxation time of 15% and 8%, respectively (Figure 9).

[0104] The inventors reconfirmed that after a 7-day washout at the end of the time course, the effect of all treatment groups on contraction and relaxation times was attenuated, while single contraction amplitude decreased over time regardless of the presence or absence of washout. The I3M + 200 μM palmitate + 200 μM oleate + 5 nM endothelin-1 group consistently produced an increase in relaxation time across three independent experiments, leading the inventors to conclude that this group and the 1 nM endothelin-1 group are particularly effective lipid-toxic culture media. In this experiment, the medium containing I3M + 200 μM palmitate + 200 μM oleate + 1 nM endothelin-1 produced the greatest increase in relaxation time, and therefore this medium was used for subsequent model characterization.

[0105] I3M + 200 μM palmitate + 200 μM oleate + 1 nM endothelin-1 preparation The inventors added 4% (v / v) 5 mM BSA-palmitate saturated fatty acid complex (Cayman Chemical, catalog number 29558, final concentration 200 μM), 4% (v / v) 5 mM BSA-oleate monounsaturated fatty acid complex (Cayman Chemical, catalog number 29557, final concentration 200 μM), and 0.1% (v / v) 1 μM endothelin-1 (prepared by dissolving endothelin-1 powder (Sigma-Aldrich, catalog number E7764-10UG) in water for cell culture (Corning, catalog number 25-055-CI, final concentration 1 nM) to I3M medium.

[0106] Model Characteristics Evaluation Bright-field imaging In addition to measuring changes in shrinkage induced by lipid-toxic media, the inventors also characterized changes in tissue morphology by taking bright-field images of the tissue at the beginning and end of the time course. These images showed that tissues treated with both palmitate and oleate exhibited rough, jagged edges along their periphery that were not present before treatment with the lipid-toxic media (Figure 11). These jagged edges consistently appeared across three independent experiments in tissues treated with a medium containing 200 μM palmitate + 200 μM oleate + 5 nM endothelin-1 (Figure 12).

[0107] Lipid staining The inventors also performed lipid staining on the same set of tissues shown in Figure 11 using HCS LipidTOX® Deep Red Neutral Lipid Stain (Thermo Fisher Scientific, catalog number H34477). The resulting confocal images showed a greater proportion of lipid droplets larger than 10 μm in size within the tissues treated with both palmitate and oleate (Figure 13), and these tissues were the same as those that showed rough, uneven edges along the periphery in the bright-field images (Figure 11).

[0108] Example 2 The inventors further demonstrated that the basal culture medium is not limited to I3M medium by conducting experiments using alternative basal culture media. As described above, cardiac tissue was generated in I3M medium on the Biowire® platform and switched on day 2 to definition medium (RPMI 1640 + 5 mM glucose + 1.4 mM CaCl2 + 50 μM palmitate + 50 μM oleate + insulin-free B27 + 1 nM insulin). After treating the cardiac tissue with definition medium for 10 days, they were switched to their respective treatment groups, namely, definition medium (control group) or lipid-toxic definition medium (definition medium + 150 μM palmitate + 150 μM oleate + 1 nM endothelin-1). As shown in Figure 14 and Tables 1 and 2 below, treatment with lipid-toxic definition medium increased relaxation time compared to the control group, demonstrating that the lipid-toxic component has the ability to induce disease phenotypes in other basal media. [Table 2] [Table 3] The data is presented as mean ± standard deviation.

[0109] Example 3 Control medium and lipid-toxic culture medium I3M was used as the basic medium in the preparation of the control medium and the lipid toxicity medium. To prepare the I3M medium, the inventors added the following components to StemPro-34 basic medium (Life Technologies catalog number 10639011): 1% (v / v) GlutaMAX (Life Technologies, catalog number 35050-061) and 2% (v / v) 1M HEPES (Life Technologies). (Technologies, catalog number 15630-080, final concentration 20 mM), 1% (v / v) penicillin-streptomycin (Gibco, catalog number 15140-122), 0.5% (v / v) 30 mg / mL transferrin solution (prepared by dissolving transferrin powder (Sigma-Aldrich, catalog number T8158) in IMDM (Gibco, catalog number 12440-053), final concentration 0.15 mg / mL), 0.4% (v / v) 64 mg / mL ascorbic acid solution (prepared by dissolving 2-phosphate-L-ascorbic acid trisodium salt (Sigma-Aldrich, catalog number 49752) in PBS (Sigma-Aldrich, catalog number D8537), final concentration 0.256 mg / mL), and 2.6% (v / v) StemPro nutritional supplement (Life Technologies (catalog number 10639011).

[0110] In all experiments, the control medium consisted of I3M + 64uM BSA control. These media were prepared by adding 8% (v / v) of 0.8mM BSA Control for Fatty Acid Complexes (Cayman Chemical Company, catalog no. 29556) to I3M medium.

[0111] In all experiments, the lipid toxicity medium consisted of I3M + 200 μM palmitate + 200 μM oleate + 1 nM endothelin-1. These media were prepared by adding the following components to I3M: 4% (v / v) 5 mM BSA-Palmitate Saturated Fatty Acid Complex (Cayman Chemical, catalog no. 29558, final concentration 200 μM), 4% (v / v) 5 mM BSA-Oleate Monounsaturated Fatty Acid Complex (Cayman Chemical, catalog no. 29557, final concentration 200 μM), and 0.1% (v / v) 1 μM endothelin-1 (prepared by dissolving endothelin-1 powder (Sigma-Aldrich, catalog no. E7764-10UG) in Cell Culture Grade Water (Corning, catalog no. 25-055-CI), final concentration 1 nM).

[0112] method Chronic force experiment During the chronic force experiment, tissues were treated with control medium or lipotoxic medium for 14–15 days. 100% of the medium was changed every 2–3 days during the treatment period. Tissues were evaluated every 2–5 days to measure changes in contractility parameters. During evaluation, tissues were moved from an incubator (5% CO2, 37°C) to an environmental chamber (5% CO2, 37°C) housing the microscope objective lens. Electrical stimulation was initiated at 1 Hz (pulse duration 2 milliseconds, single phase, 3 V). The tissues were equilibrated in the chamber for 30 minutes before video recording. During video recording, polymer wires in a Biowire® II platform were exposed to an excitation wavelength of 405 nm, resulting in fluorescence of the polymer wires. While these tissues were stimulated at 1 Hz, 10-second videos of the wires were recorded. After obtaining videos of all wires, the tissues were returned to the incubator and stimulation was continued until the next evaluation. This process was repeated until the end of the treatment.

[0113] The video was analyzed using custom MATLAB® code to track the position of the polymer wire, and the displacement of the wire in each video frame was measured. This displacement was converted into force values ​​using an experimentally derived force-displacement equation. The single contraction amplitude was calculated as the height of the force peak. The contraction time was calculated by measuring the time from 10% of the peak height to the maximum peak amplitude, and the relaxation time was calculated by measuring the time from the maximum peak amplitude to 90% (or 10% of the peak height) relaxation. Passive tension was calculated by measuring the magnitude of the force in the wire between contractions (i.e., the flat line between peaks).

[0114] Measurement of tissue area To measure tissue area, bright-field images of the tissue were acquired at 2x magnification at each evaluation. These images were processed using custom tissue measurement software, Tissue Ruler. Tissue Ruler determined the contour of each tissue by contrasting the dark areas that make up the tissue against the bright background of the platform (see Figure 16). The area of ​​the tissue was then calculated using this outline.

[0115] Measurement of stimulus threshold The stimulation threshold for each tissue was determined by observing the tissue under 2x bright-field magnification, setting the stimulation frequency to 2 Hz, and then gradually decreasing the stimulation voltage from the starting voltage of 3 V until the tissue stopped contracting at a frequency of 2 Hz. The minimum voltage required to maintain tissue pulsation at 2 Hz was recorded as the stimulation threshold.

[0116] Action potential measurement Action potentials were measured by incubating tissue with membrane potential-sensitive dyes, resulting in cell fluorescence based on membrane potential. During myocardial action potentials, the cell membrane potential fluctuates, and consequently, the cell fluorescence also fluctuates. The membrane potential-sensitive dyes used in the experiment caused cells to fluoresce more brightly when the membrane potential was hyperpolarized and to dimmer when the membrane potential was depolarized.

[0117] After incubating the tissue with dye, 10-second videos of these tissues were acquired at a stimulation frequency of 1 Hz. Next, using a custom MATLAB® program, action potential traces were generated based on the measured fluorescence. Using the same MATLAB® program, APD 90 We also calculated the time required from the peak of the action potential to 90% repolarization (see Figure 18B).

[0118] Calcium transient measurement Calcium transients were measured by incubating tissue with a calcium-sensitive dye. This dye enters cells, binds to calcium, and emits fluorescence. During calcium transients in myocardium, the fluorescence of the calcium dye increased with increasing intracellular calcium (during contraction) and decreased with decreasing intracellular calcium (during relaxation).

[0119] After incubating the tissue with a dye, a 7-second video of the tissue was acquired at a stimulation frequency of 1 Hz. A custom MATLAB® program was then used to generate traces of the calcium transient based on the measured fluorescence. The same MATLAB® program was also used to calculate the time to 50% calcium transient decay (the time required from the transient peak to 50% transient decay) (see Figure 19B).

[0120] result Figure 15A shows the changes in passive tension (i.e., tension in the polymer wire when the tissue is not actively contracting) measured from day 0 to the end of treatment for Biowire® tissues treated with control medium or lipotoxic medium. Tissues treated with lipotoxic medium demonstrated a significant increase in passive tension by day 14 / 15 compared to tissues treated with control medium. The increase in passive tension in lipotoxic tissues may be evidence of increased ECM crosslinking, fibrosis, and / or increased fibroblast contractile activity, which is consistent with the increased myocardial rigidity observed in patients with lipotoxic cardiomyopathy.

[0121] Figure 15B shows measured changes in tissue area from day 0 to the end of treatment in Biowire tissue treated with either control medium or lipotoxic medium. Tissue treated with lipotoxic medium showed a significant increase in tissue area by day 14 / 15 compared to tissue treated with control medium. This may be evidence of hypertrophy, a symptom commonly associated with lipid-toxic cardiomyopathy.

[0122] Figure 17 shows measured changes in the stimulation threshold (the minimum voltage required to induce tissue contraction at a set stimulation frequency) from day 0 to the end of treatment in Biowire® tissue treated with control medium or lipotoxic medium. Tissue treated with lipotoxic medium showed a significant increase in stimulation threshold by day 14 / 15 compared to tissue treated with control medium. Since cardiac cells rely on intercellular interactions via gap junctions and adhesion complexes for effective electrical conduction, the increase in stimulation threshold observed in lipotoxic tissue may indicate a breakdown in intercellular interactions and / or a decline in cellular health, and while we do not wish to be bound by theory, we hypothesize that this may be related to the increase in relaxation time similarly observed in lipotoxic tissue.

[0123] Figure 18A shows the APD measured at the end of treatment in Biowire® tissue treated with either control medium or lipotoxic medium. 90 (Action potential duration from peak to 90% repolarization) is shown. Tissue treated with lipotoxic medium shows APD compared to tissue treated with control medium. 90The values ​​were significantly higher, which is consistent with the increased relaxation time similarly observed in lipid-toxic tissues. While we do not wish to be bound by theory, the increased action potential duration observed in lipid-toxic tissues may be a result of impaired ion channel function downstream of pathways activated by metabolic disorders (see N. Ozturk, S. Uslu and S. Ozdemir, “Diabetes-induced changes in cardiac voltage-gated ion channels,” World Journal of Diabetes, vol.12, no.1, pp.1-18, 2021, and Z. Lu, Y.-P. Jiang, X.-H. Xu, LMBallou, ISCohen and RZLin, “Decreased l-Type Ca2+Current in Cardiac Myocytes of Type 1 Diabetic Akita Mice Due to Reduced Phosphatidylinositol 3-Kinase Signaling,” Diabetes, vol.56, no.11, pp.2780-2789, 2007).

[0124] Figure 19A shows the time to 50% calcium transient decay measured at the end of treatment in Biowire® tissue treated with control medium or lipotoxic medium. Tissue treated with lipotoxic medium showed a significantly longer time to 50% transient decay compared to tissue treated with control medium. This observation is consistent with our hypothesis that the increased relaxation time in lipotoxic tissue is caused by impaired calcium handling, specifically by known mechanisms leading to longer decay in calcium transients, i.e., impaired diastolic function (see RHRitchie and EDAbel, “Basic Mechanisms of Diabetic Heart Disease,” Circulation Research, vol.126, no.11, pp.1501-1525, 2020).

Claims

1. A culture medium for culturing cardiac tissue, wherein the culture medium is 100-300 μM palmitate, 100-300 μM oleate, Endothelin-1 at 0.5-15 nM, A culture medium containing the following:

2. The culture medium according to claim 1, wherein the culture medium is for inducing a disease phenotype of diabetic cardiomyopathy in the cultured cardiac tissue.

3. The culture medium according to any of the preceding claims, wherein the culture medium contains 180 to 220 μM of palmitate.

4. The culture medium according to claim 3, wherein the culture medium contains 200 μM palmitate.

5. The culture medium according to any of the preceding claims, wherein the culture medium contains 180 to 220 μM of oleate.

6. The culture medium according to claim 5, wherein the culture medium contains 200 μM of oleate.

7. The culture medium according to any of the preceding claims, wherein the culture medium contains 1 to 5 nM endothelin-1.

8. The culture medium according to any one of the preceding claims, wherein the culture medium comprises 1 nM or 5 nM endothelin-1.

9. The culture medium is 180-220 μM palmitate, 180-220 μM oleate, Endothelin-1 at 0.5-10 nM, A culture medium according to claim 1, comprising:

10. The culture medium is 200 μM palmitate and 200 μM oleate and Endothelin-1 at 1-5 nM, A culture medium according to claim 1, comprising:

11. The culture medium according to any of the preceding claims, wherein the culture medium does not contain cortisol.

12. A culture medium according to any of the preceding claims, further comprising StemPro-34 serum-free basic medium.

13. (i) GlutaMAX, GlutaMAX of 0.1-2% (v / v) as optional, and GlutaMAX of 1% (v / v) as optional, (ii) HEPES, optionally selected from 10 to 30 mM HEPES, and optionally selected from 20 mM HEPES, (iii) Penicillin-streptomycin, optionally 0.1-2% penicillin-streptomycin, and optionally 1% penicillin-streptomycin, (iv) Transferrin solution, optionally a transferrin solution of 0.05 to 0.25 mg / mL, and optionally a transferrin solution of 0.15 mg / mL. (v) Ascorbic acid solution, optionally 0.1 to 0.4 mg / mL ascorbic acid solution, and optionally 0.256 mg / mL ascorbic acid solution, (vi) StemPro-34 nutritional supplement, optionally 1-4% (v / v) StemPro-34 nutritional supplement, and optionally 2.6% (v / v) StemPro-34 nutritional supplement, A culture medium according to any of the prior claims, further comprising one or more of the above.

14. (i) GlutaMAX at 0.1-2% (v / v), (ii) HEPES of 10-30 mM, (iii) 0.1-2% penicillin-streptomycin and (iv) A transferrin solution of 0.05–0.25 mg / mL, (v) Ascorbic acid solution at 0.1–0.4 mg / mL, (vi) 1-4% (v / v) StemPro-34 nutritional supplement, The culture medium according to claim 13, comprising:

15. A method for inducing the disease phenotype of diabetic cardiomyopathy in cardiac tissue, wherein the method is A method comprising culturing cardiac tissue in the culture medium described in any one of claims 1 to 14.

16. The method according to claim 15, wherein the cardiac tissue is cultured in the culture medium for 3 to 80 days.

17. The method according to claim 16, wherein the cardiac tissue is cultured in the culture medium for 21 to 35 days.

18. The method according to any one of claims 15 to 17, further comprising the step of culturing pluripotent stem cells in a differentiation medium to provide cardiomyocytes.

19. The method according to claim 18, wherein the pluripotent stem cells are human induced pluripotent stem cells.

20. The method according to claim 18 or claim 19, wherein the pluripotent stem cells are of autologous origin.

21. The method according to any one of claims 18 to 20, further comprising the step of electromechanically conditioning the cardiomyocytes by exposing them to electromechanical stimulation that increases in intensity over time, thereby forming cardiac tissue having molecular, structural, and functional properties that mimic innate adult cardiac tissue.

22. The method according to claim 21, wherein the cardiac tissue includes T-tubules.

23. The method according to claim 21, wherein the cardiac tissue exhibits a positive force-frequency relationship.

24. The method described above is Further comprising measuring one or more parameters of the contractility of the cardiac tissue, The method according to any one of claims 15 to 23.

25. The method according to claim 24, wherein one or more of the contractility parameters are selected from the group consisting of relaxation time, single contraction amplitude, contraction time, and combinations thereof.

26. The method according to claim 24 or 25, wherein one or more of the parameters are measured before the culture step, during the culture step, and / or after the culture step.

27. The method described above is Further comprising characterizing the tissue morphology of the aforementioned cardiac tissue, The method according to any one of claims 15 to 26.

28. The method according to claim 27, wherein the tissue morphology is characterized by microscopic examination with or without tissue staining.

29. The method according to claim 27 or 28, wherein the tissue morphology is characterized before the culture step, during the culture step, and / or after the culture step.

30. The method described above is Lipidomics, phosphorylated Akt Western blotting, RNA sequencing, proteomics, intracellular calcium transient analysis, and combinations thereof. The method according to any one of claims 15 to 29, further comprising characterizing one or more evaluation items of the cardiac tissue selected from the group consisting of the following.

31. The method according to claim 30, wherein one or more of the evaluation items are characterized before the culture step, during the culture step, and / or after the culture step.

32. Affected cardiac tissue produced by the method according to any one of claims 15 to 31, wherein the affected cardiac tissue exhibits a disease phenotype of diabetic cardiomyopathy.

33. A diseased cardiac tissue comprising the population of cardiomyocytes in the culture medium according to any one of claims 1 to 14.

34. The diseased cardiac tissue according to claim 33, further comprising two or more scaffold elements disposed within the cardiac tissue.

35. The diseased cardiac tissue according to claim 33 or claim 34, wherein the cardiac tissue comprises a mature ultrastructure.

36. The diseased cardiac tissue according to any one of claims 33 to 35, wherein the ultrastructure is selected from the group consisting of sarcomeres, mitochondria, T-tubules, sarcoplasmic reticulum, and combinations thereof.

37. The diseased cardiac tissue according to any one of claims 33 to 36, wherein the cardiac tissue exhibits a positive force-frequency relationship.

38. The aforementioned force is approximately 0.25 to approximately 2 mN / mm at a frequency of approximately 0 to 6 Hz. ２ The diseased cardiac tissue according to claim 37.

39. A kit comprising a culture medium according to any one of claims 1 to 14 and a bioreactor for culturing cardiac tissue, wherein the bioreactor comprises a plurality of wells, each well comprising a chamber configured therein for culturing cardiac tissue, and two or more deformable scaffold elements attached to each chamber.

40. The kit according to claim 39, wherein the bioreactor is a multiwell plate.

41. The kit according to claim 40, wherein the bioreactor is a multiwell plate having 12 wells, 96 wells, 384 wells, or 1536 wells.

42. The kit according to any one of claims 39 to 41, wherein the bioreactor is composed of a polymer.

43. The kit according to claim 42, wherein the polymer is a biodegradable polymer.

44. The kit according to claim 43, wherein the biodegradable polymer is polylactic acid, poly(lactic acid-co-glycol) acid, or poly(caprolactone), polyglycolide, polylactide, polyhydroxobutyrate, polyhydroxyalkanoic acid, chitosan, hyaluronic acid, hydrogel, poly(2-hydroxyethyl methacrylate), poly(ethylene glycol), poly(L-lactide) (PLA), or a combination thereof.

45. The kit according to claim 44, wherein the polymer is poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PMMA), polystyrene, poly(glycerol sebacate), POMaC without citric acid, poly(e-caprolactone), polyurethane, silk, or nanoprocessed material, or copolymers or blends thereof.

46. The kit according to claim 45, wherein the polymer is doped with a nanostructure.

47. The kit according to any one of claims 39 to 46, wherein the deformable scaffold element is made of metal, silk, or polymer.

48. The kit according to any one of claims 39 to 46, wherein the deformable scaffold element is composed of intestinal material, monoacrylic, polyglycol, prolene, polyglucin, polydioxanone, polypropylene, nylon, or polyester.

49. The kit according to any one of claims 39 to 48, wherein the chamber is configured to be seeded by cardiomyocytes.

50. The kit according to any one of claims 39 to 49, wherein the deformable scaffold elements attached to each chamber are oriented substantially perpendicular, substantially parallel, or substantially oblique to the orientation of the longitudinal axis of the chamber.

51. The kit according to any one of claims 39 to 50, wherein the deformable scaffold element is configured to be embedded or partially embedded by the cardiac tissue during the growth of the cardiac tissue.

52. The kit according to any one of claims 39 to 51, wherein the deformable scaffold element is configured to be encapsulated or partially encapsulated by the cardiac tissue and attached to the cardiac tissue, so that the cardiac tissue moves in conjunction with the movement of the deformable scaffold element.

53. The kit according to any one of claims 39 to 52, further comprising a pair of electrodes configured to generate an electric current through the growth chamber of the bioreactor.

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