Lipotoxic medium

Abstract

The invention provides a culture medium for culturing heart tissue, a method for inducing diabetic cardiomyopathy disease phenotype in heart tissue, diseased heart tissue and a kit. An exemplary culture medium for culturing cardiac tissue comprises 100 to 300 M of palmitic acid / salt / ester, 100 to 300 M of oleic acid / salt / ester, and 0.5 to 15 nM of endothelin-1.

Description

Technical Field

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

[0003] Currently, heart failure affects more than 64 million people worldwide, and its prevalence appears to be rising. In the United States alone, health experts predict that the prevalence of heart failure will increase by 46% by 2030. Of all heart failure cases, heart failure with preserved ejection fraction (HFpEF) accounts for approximately 50%, and the percentage of heart failure patients with HFpEF has been increasing recently. Given the global prevalence of HFpEF and the heavy symptomatic burden it imposes, there is an urgent need to identify candidate drugs for the treatment and prevention of HFpEF.

[0004] Heart disease with peripheral angina pectoris (HFpEF) is often associated with other comorbidities such as diabetes, obesity, and metabolic syndrome, with approximately 65% ​​of patients with HFpEF having at least one of these conditions. A key feature of the metabolic disorder identified as contributing to HFpEF is hyperlipidemia, a condition in which abnormally high levels of lipids and fatty acids circulate in the blood. Hyperlipidemia negatively impacts cellular metabolism, leading to higher fatty acid oxidation and lower glucose oxidation. Elevated lipids contribute to insulin resistance, which is associated with elevated serum glucose and insulin levels. Furthermore, lipids such as diglycerides and ceramides accumulate in heart cells over time, affecting cell morphology and function, which may contribute to a condition known as diabetic cardiomyopathy.

[0005] Diabetic cardiomyopathy can be formally defined as cardiac dysfunction in the absence of any other risk factors, such as hypertension, ischemic heart disease, and / or valvular dysfunction. In a clinical setting, patients with diabetic cardiomyopathy may initially present as asymptomatic but with fibrosis, increased ventricular stiffness, and diastolic dysfunction. The disease may subsequently progress to include 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.

[0006] 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; Granéli 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 provide good representation of diabetic cardiomyopathy.

[0007] There is a need to provide improved models of diabetic cardiomyopathy that can be used to better understand the mechanisms of lipotoxicity and for drug discovery and development. Summary of the Invention

[0008] According to a first aspect of this disclosure, a culture medium for culturing cardiac tissue is provided, wherein the culture medium comprises: 100-300 µM palmitic acid / salt / ester; 100-300 µM oleic acid / salt / ester and 0.5-15 nM endothelin-1.

[0009] The inventors have developed a lipotoxic culture medium that can be used to culture cardiac tissue to induce a diabetic cardiomyopathy phenotype. Advantageously, the cultured tissue can be used as a three-dimensional (3D) in vitro or ex vivo model to reproduce the key features of diabetic cardiomyopathy, and thus can be used to better understand the mechanisms of lipotoxicity, and can be used for drug discovery and development. The prior art could not have anticipated or foreseen that the combination of palmitic acid / salt / ester, oleic acid / salt / ester, and endothelin-1, and especially at the described concentrations, could be used to induce a diabetic cardiomyopathy phenotype in cardiac tissue. Furthermore, the prior art could not have anticipated or foreseen that the combination of endothelin-1 with palmitic acid / salt / ester and oleic acid / salt / ester would induce a diabetic cardiomyopathy phenotype in cardiac tissue.

[0010] As used herein, the term "culture medium" refers to a solution containing factors and nutrients, including, for example, growth factors, energy sources, amino acids, and organic and inorganic salts, used to maintain and grow cells in vitro or in vitro. Culture media are typically buffered to approximately neutral pH (e.g., a pH from approximately 6.6 to approximately 7.8) and may be supplemented with one or more antibiotics to prevent the growth of bacterial and / or fungal contaminants. Any suitable basal medium may be used, such as StemPro™-34 serum-free medium, MEM, DMEM, RPMI 1640, Advanced MEM, BME, Neurobasal medium, cardiomyocyte selective medium, sodium bicarbonate buffered medium 199, myocyte growth medium, cardiomyocyte maintenance medium.

[0011] As used herein, the term “lipotoxicity” is intended to refer to the accumulation of lipids and lipid intermediates in cultured cardiac tissue caused by the culture medium, resulting in harmful effects such as cellular dysfunction (e.g., manifested as increased tissue relaxation time) and / or cell death.

[0012] The term "cultivating" or "culturing" refers to the incubation, maintenance, growth, differentiation, or maturation of cells or tissues in vitro or in a culture medium to obtain the desired phenotype, structure, and / or function.

[0013] As used herein, the term "palmitic acid / salt / ester" is intended to encompass salts and esters of palmitic acid, including, for example, sodium palmitate and calcium palmitate. In some embodiments, palmitic acid / salt / ester is complexed with other proteins, such as bovine serum albumin (BSA). In some embodiments, palmitic acid / salt / ester is palmitic acid itself.

[0014] As used herein, the term "oleic acid / salt / ester" is intended to encompass salts and esters of oleic acid, including, for example, sodium oleate and calcium oleate. In some embodiments, oleic acid / salt / ester is compounded with other proteins, such as bovine serum albumin (BSA). In some embodiments, oleic acid / salt / ester is oleic acid. Oleic acid may also be referred to as cis-9-octadecenoic acid.

[0015] Endothelin-1 is a vasoconstrictive peptide. In some embodiments, endothelin-1 is human endothelin-1. In some embodiments, endothelin-1 is proendothelin-1 or proendothelin-1.

[0016] As used herein, the term "cardiac tissue" is intended to encompass both ex vivo cardiac tissue and in vitro engineered tissue, including both 2D and 3D cardiac cell cultures. Cardiac tissue may be healthy or diseased and / or modified in some way (e.g., by assays, therapeutics, genetic modifications, etc.).

[0017] In some embodiments, the culture medium is used to culture cardiac tissue produced using systems defined in WO 2015 / 061907 A1, WO 2021 / 158233 A1, and / or WO 2016 / 183143 A1, which are incorporated herein by reference in their entirety. In some embodiments, the culture medium is used to culture cardiac tissue (e.g., cardiac organoids) as defined in WO 2016 / 183143 A1.

[0018] In some embodiments, the cardiac tissue is generated from a cell line (e.g., a human cell line). In some embodiments, the cardiac tissue is generated from ex vivo primary cardiomyocytes (e.g., human cardiomyocytes). In some embodiments, the cardiac tissue is generated from ex vivo pluripotent stem cell-derived cardiomyocytes (e.g., human iPSCs). In some embodiments, the cardiac tissue is human cardiac tissue. In some embodiments, the cardiac tissue comprises cardiomyocytes that have been genetically modified to achieve a desired phenotype.

[0019] In some embodiments, the cardiac tissue comprises a population of cardiac fibroblasts in addition to a population of cardiomyocytes. The ratio of cardiomyocytes to cardiac fibroblasts can be between about 1:3 and 15:1. The ratio of cardiomyocytes to cardiac fibroblasts can be between about 1:1 and 10:1. In some embodiments, the ratio of cardiomyocytes to cardiac fibroblasts is about 4:1. In some embodiments, the ratio of cardiomyocytes to cardiac fibroblasts is about 10:1.

[0020] In some embodiments, the cardiac tissue comprises a population of cardiomyocytes (and optionally cardiac fibroblasts) as well as a population of endothelial cells. The ratio of cardiomyocytes to endothelial cells may be between about 1:10 and 4:1. The ratio of cardiomyocytes to endothelial cells may be between about 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.

[0021] In some embodiments, the culture medium is used to induce a diabetic cardiomyopathy phenotype in cultured cardiac tissue. As used herein, the term "diabetic cardiomyopathy phenotype" is intended to refer to an increased cardiac tissue relaxation time, which is similar to delayed relaxation of the left ventricle of the heart.

[0022] In some embodiments, the culture medium contains 120-280 µM palmitic acid / salt / ester. In some embodiments, the culture medium contains 140-260 µM palmitic acid / salt / ester. In some embodiments, the culture medium contains 160-240 µM palmitic acid / salt / ester. In some embodiments, the culture medium contains 180-220 µM palmitic acid / salt / ester. In some embodiments, the culture medium contains 190-210 µM palmitic acid / salt / ester. In some embodiments, the culture medium contains 195-205 µM palmitic acid / salt / ester. In some embodiments, the culture medium contains about 200 µM palmitic acid / salt / ester. In some embodiments, the culture medium contains 200 µM palmitic acid / salt / ester.

[0023] In some embodiments, the culture medium contains 120-280 µM oleic acid / salt / ester. In some embodiments, the culture medium contains 140-260 µM oleic acid / salt / ester. In some embodiments, the culture medium contains 160-240 µM oleic acid / salt / ester. In some embodiments, the culture medium contains 180-220 µM oleic acid / salt / ester. In some embodiments, the culture medium contains 190-210 µM oleic acid / salt / ester. In some embodiments, the culture medium contains 195-205 µM oleic acid / salt / ester. In some embodiments, the culture medium contains about 200 µM oleic acid / salt / ester. In some embodiments, the culture medium contains 200 µM oleic acid / salt / ester.

[0024] In some embodiments, the culture medium contains 0.5-15 nM endothelin-1. In some embodiments, the culture medium contains 0.6-12 nM endothelin-1. In some embodiments, the culture medium contains 0.7-10 nM endothelin-1. In some embodiments, the culture medium contains 0.8-8 nM endothelin-1. In some embodiments, the culture medium contains 0.9-6 nM endothelin-1. In some embodiments, the culture medium contains 1-5 nM endothelin-1. In some embodiments, the culture medium contains 1 nM or 5 nM endothelin-1.

[0025] In some embodiments, the culture medium comprises 100-250 µM palmitic acid / salt / ester; 100-250 µM oleic acid / salt / ester; and 0.5-15 nM endothelin-1. In some embodiments, the culture medium comprises 100-225 µM palmitic acid / salt / ester; 100-225 µM oleic acid / salt / ester; and 0.5-15 nM endothelin-1.

[0026] In some embodiments, the culture medium comprises: 120-280 µM palmitic acid / salt / ester; 120-280 µM oleic acid / salt / ester; and 0.5-15 nM endothelin-1. In some embodiments, the culture medium comprises: 180-220 µM palmitic acid / salt / ester; 180-220 µM oleic acid / salt / ester; and 0.5-10 nM endothelin-1. In some embodiments, the culture medium comprises: 200 µM palmitic acid / salt / ester; 200 µM oleic acid / salt / ester; and 1-5 nM endothelin-1.

[0027] 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.

[0028] In some embodiments, the culture medium further comprises StemPro™-34 serum-free basal medium (e.g., provided by Gibco™). Those skilled in the art will understand that any other suitable basal medium may be used alternatively.

[0029] In some embodiments, the culture medium further comprises one or more of the following: buffer, sugar (e.g., glucose), amino acids (e.g., L-glutamine), antibiotics, vitamins, serum, growth factors, cytokines, sodium pyruvate, recombinant proteins, iron transporters, and combinations thereof.

[0030] In some embodiments, the culture medium further comprises one or more of the following:

[0031] (i) GlutaMAX™ supplements (provided by, for example, Gibco™), optionally 0.1%–2% (v / v) or 1% (v / v) GlutaMAX;

[0032] (ii) HEPES (provided by, for example, Gibco™), optionally 10-30 mM or 20 mM HEPES;

[0033] (iii) Penicillin-streptomycin (provided by, for example, Gibco™), optionally 0.1%-2% or 1% penicillin-streptomycin;

[0034] (iv) Transferrin solution (provided by, for example, Sigma-Aldrich), optionally 0.05–0.25 mg / mL or 0.15 mg / mL transferrin solution;

[0035] (v) Ascorbic acid solution (provided by, for example, Sigma-Aldrich), optionally 0.1–0.4 mg / mL or 0.256 mg / mL ascorbic acid solution; and

[0036] (vi) StemPro™-34 nutritional supplement (provided by, for example, Gibco™), optionally 1-4% (v / v) or 2.6% (v / v) StemPro™-34 nutritional supplement.

[0037] In some embodiments, the culture medium comprises:

[0038] (i) 0.1%-2% (v / v) GlutaMAX™;

[0039] (ii) 10-30 mM HEPES;

[0040] (iii) 0.1%-2% penicillin-streptomycin;

[0041] (iv) 0.05–0.25 mg / mL transferrin solution;

[0042] (v) 0.1–0.4 mg / mL ascorbic acid solution; and

[0043] (vi) 1%-4% (v / v) StemPro™-34 nutritional supplement.

[0044] In some embodiments, the culture medium contains heart tissue.

[0045] According to a second aspect of this disclosure, a method for inducing a diabetic cardiomyopathy phenotype in cardiac tissue is provided, wherein the method includes culturing cardiac tissue in a culture medium as described in the first aspect of this disclosure.

[0046] In some embodiments, the method includes generating cardiac tissue using a system defined in WO 2015 / 061907 A1, WO 2021 / 158233 A1, and / or WO 2016 / 183143 A1. In some embodiments, the method includes providing cardiac tissue (e.g., cardiac organoids) as defined in WO2016 / 183143 A1.

[0047] In some embodiments, cardiac tissue comprises mature ultrastructures. In some embodiments, the ultrastructures are selected from the group consisting of sarcomeres, mitochondria, T tubules, sarcoplasmic reticulum, and combinations thereof. In some embodiments, cardiac tissue comprises T tubules.

[0048] In some embodiments, cardiac tissue exhibits a positive force-frequency relationship. In some embodiments, the force is approximately 0.25 to approximately 2 mN / mm at frequencies of approximately 0 to 6 Hz. 2 .

[0049] Heart tissue can be cultured in a culture medium for any suitable length of time. In some embodiments, heart tissue is cultured in a culture medium for 3-100 days. In some embodiments, heart tissue is cultured in a culture medium for 4-90 days. In some embodiments, heart tissue is cultured in a culture medium for 5-80 days. In some embodiments, heart tissue is cultured in a culture medium for 6-70 days. In some embodiments, heart tissue is cultured in a culture medium for 7-60 days. In some embodiments, heart tissue is cultured in a culture medium for 9-50 days. In some embodiments, heart tissue is cultured in a culture medium for 14-45 days. In some embodiments, heart tissue is cultured in a culture medium for 18-40 days. In some embodiments, heart tissue is cultured in a culture medium for 21-35 days. In some embodiments, heart tissue is cultured in a culture medium for about 7 days. In some embodiments, heart tissue is cultured in a culture medium for about 14 days. In some embodiments, heart tissue is cultured in a culture medium for about 21 days. In some embodiments, heart tissue is cultured in a culture medium for about 28 days. In some embodiments, heart tissue is cultured in a culture medium for about 35 days.

[0050] Those skilled in the art will understand that the culture medium can be replenished / replaced periodically according to standard cell culture techniques.

[0051] In some embodiments, the method includes the step of culturing cells in a growth medium to expand the cells. The cells may be isolated primary cardiomyocytes, cell lines, or stem cells (e.g., iPSCs, such as human iPSCs). The growth medium may be any suitable medium that promotes cell expansion. Exemplary growth media are mTeSR1 (Stemcell Technologies), mTeSR Plus (Stemcell Technologies), TeSR-E8 (Stemcell Technologies), Essential 8™ medium (Gibco), and StemFlex™ medium (Gibco). In these embodiments, the growth step occurs prior to the culture of cardiac tissue in a medium as defined with respect to the first aspect of this disclosure.

[0052] Cells can be cultured in growth medium for any suitable length of time. 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.

[0053] In some embodiments, the method includes the step of culturing cells in a differentiation medium to differentiate the cells into cardiomyocytes to form cardiac tissue. The 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, the differentiation step occurs before the cardiac tissue is cultured in a medium as defined with respect to the first aspect of this disclosure. Typically, the differentiation step occurs after the cells are cultured in a growth medium.

[0054] As examples, stem cells can be embryonic stem cells (ESCs), fetal stem cells (FSCs), and / or adult (or somatic) stem cells (SSCs). In terms of potential, stem cells can be totipotent (also known as pluripotent stem cells) (stem cells that can differentiate into embryonic and extraembryonic cell types), pluripotent stem cells (stem cells that can differentiate into almost any cell type), multipotent stem cells (stem cells that can differentiate into multiple cell types), oligopotent stem cells (stem cells that can differentiate into only a few cell types), or unipotent stem cells (stem cells that can produce only one cell type). Stem cells can be obtained commercially or directly from patients or any other suitable source.

[0055] In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs). In some embodiments, the pluripotent stem cells are human iPSCs. In some embodiments, the pluripotent stem cells are autologous.

[0056] The differentiation medium can be any suitable medium that leads to the differentiation of stem cells into the cardiac lineage. In some embodiments, the differentiation medium contains activin A and bone morphogenetic protein 4 (BMP4). An exemplary differentiation medium is insulin-free RPMI / B27 medium supplemented with activin A (e.g., 50 ng / ml) and BMP4 (e.g., 25 ng / ml). In some embodiments, the differentiation medium contains a Gsk3β inhibitor and a Wnt inhibitor. Exemplary differentiation media are described in WO 2016 / 183143 A1.

[0057] Cells can be cultured in differentiation medium for any suitable length of time. In some embodiments, cells are cultured in differentiation medium for 1–30 days. In some embodiments, cells are cultured in differentiation medium for 2–27 days. In some embodiments, cells are cultured in differentiation medium for 3–24 days. In some embodiments, cells are cultured in differentiation medium for 4–21 days. In some embodiments, cells are cultured in differentiation medium for 5–18 days. In some embodiments, cells are cultured in differentiation medium for 6–15 days. In some embodiments, cells are cultured in differentiation medium for 7–12 days. In some embodiments, cells are cultured in differentiation medium for 8–10 days. Typically, cells are cultured in differentiation medium for 21–24 days.

[0058] Typically, the growth step and / or differentiation step occurs prior to the culture step in the culture medium according to the first aspect of this disclosure. Generally, any growth step occurs prior to any differentiation step.

[0059] In some embodiments, the method includes the step of encapsulating cells in a hydrogel. This encapsulation step may occur after the cells have been grown in a growth and / or differentiation medium for 10–30 days. Alternatively, it may occur after the cells have been grown in a growth and / or differentiation medium for 15–25 days. Typically, the encapsulation step occurs after the cells have been grown in a growth and / or differentiation medium for approximately 20 days. As an example, cells may be encapsulated in a hydrogel, as described in WO 2016 / 183143 A1.

[0060] As used herein, the term "hydrogel" refers to a physically or chemically cross-linked polymer network capable of absorbing large amounts of water and is a common material for forming tissue engineering scaffolds. They can be categorized into different classes based on various parameters, including preparation method, charge, and mechanical and structural characteristics. See S. Van Vlierberghe et al., "Biopolymer-Based Hydrogels As Scaffolds for Tissue Engineering Applications: A Review," Biomacromolecules, 2011, 12(5), pp. 1387-1408, which is incorporated herein by reference. Hydrogels can include materials such as polyvinyl alcohol, sodium polyacrylate, acrylate polymers, and copolymers with abundant hydrophilic groups. Natural hydrogel materials include agarose, methylcellulose, hyaluronic acid, and other naturally derived polymers.

[0061] In some embodiments, the method further includes a step of electromechanical modulation of the cells. Those skilled in the art can choose a suitable modulation scheme. As an example, cells can be electromechanically modulated as described in WO 2016 / 183143 A1. In some embodiments, the method further includes the step of electromechanically modulating cardiomyocytes by exposing them to electromechanical stimulation of increasing intensity over a period of time, thereby forming heart tissue with molecular, structural, and functional characteristics mimicking natural adult heart tissue. This provides the advantage of inducing cell maturation. Typically, the electromechanical modulation step is performed prior to the culture step in the culture medium according to the first aspect of this disclosure. In alternative embodiments, the electromechanical modulation step is performed simultaneously with the culture step in the culture medium according to the first aspect of this disclosure.

[0062] In some embodiments, the time period is 1-6 weeks. In some embodiments, the time period is 2-5 weeks. In some embodiments, the time period is 3-4 weeks.

[0063] In some embodiments, the method further includes measuring one or more contractility parameters of cardiac tissue.

[0064] In some embodiments, one or more contractility parameters are selected from the group consisting of: relaxation time, twitching amplitude, contraction time, and combinations thereof. Relaxation time is the time taken for a tissue to relax from a fully contracted state (peak amplitude) to a relaxed state (10% of peak amplitude). Twitching amplitude is the total force exerted by the tissue during contraction (peak amplitude). Contraction time is the time taken 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 in the art and are conventionally used (see, for example, WO 2016 / 183143 A1 or Zhao et al., “APlatform for Generation of Chamber-Specific Cardiac Tissues and Disease Modeling.” Cell. 2019 Feb 7;176(4):913-927.e18, which are incorporated herein by reference).

[0065] In some embodiments, one or more parameters are measured before, during, and / or after the culture step.

[0066] In some embodiments, the method further includes: characterizing the tissue morphology of cardiac tissue.

[0067] In some embodiments, tissue morphology is characterized by microscopic examination with or without tissue staining. In some embodiments, tissue morphology is characterized by microscopy of tissue stained with a lipid staining agent (e.g., HCS LipidTOX™ Deep Red Neutral Lipid Stain (Thermo Fisher Scientific, Cat.#H34477)). In some embodiments, the tissue staining agent is an anti-α-actin (sarcomere) antibody (i.e., staining sarcomeres) or an anti-TOM20 antibody (i.e., staining mitochondria).

[0068] In some embodiments, tissue morphology is characterized before, during, and / or after the culture step.

[0069] In some embodiments, a diabetic cardiomyopathy phenotype is induced in cardiac tissue, and the method further includes characterizing one or more endpoints of the cardiac tissue selected from the group consisting of: lipidomics, phosphorylated Akt Western blot, RNA sequencing, proteomics, intracellular calcium transient analysis, and combinations thereof.

[0070] In some embodiments, one or more endpoints are characterized before, during, and / or after the culture step.

[0071] In some embodiments, the method further includes the step of incubating cardiac tissue of a disease phenotype together with the test agent. A “test agent” is any substance that assesses the ability of a substance to diagnose, cure, alleviate, treat, prevent, alter, or promote a disease in a subject. The test agent in the embodiments may be a “drug” as defined in section 321(g)(1) of the Food, Drug and Cosmetic Act. Test agents include, but are not limited to, chemical compounds, biological agents, proteins, peptides, antibodies, nucleic acids, lipids, polysaccharides, supplements, diagnostic agents, and immunomodulators, and may also be referred to as “pharmacological agents.” It can test any suitable reagents, including opioid analgesics, anti-inflammatory drugs such as antihistamines and nonsteroidal anti-inflammatory drugs (NSAIDs), diuretics such as carbonic anhydrase inhibitors, loop diuretics, potent diuretics, thiazides and thiazide-like agents, and potassium-sparing diuretics, agents affecting the cardiovascular system such as angiotensin-converting enzyme inhibitors, cardiac drugs such as organic nitrates, calcium channel blockers, sympathetic nerve blockers, vasodilators, β-adrenergic receptor agonists and antagonists, α-adrenergic receptor agonists and antagonists, cardiac glycosides, antiarrhythmic drugs, agents affecting hyperlipoproteinemia such as 3-hydroxymethylglutaryl-CoA (HMG-CoA) inhibitors, antitumor drugs such as alkylating agents, antimetabolites, natural products, antibiotics and other drugs, immunomodulators, antidiabetic agents, and antimicrobial agents such as antibacterial agents, antiviral agents, antifungal agents, antiprotozoal agents, and anthelmintics.

[0072] Generally, the test reagent can be incubated with disease-phenotype cardiac tissue at a dose range estimated to produce an effect, for a duration sufficient to produce an effect (e.g., a metabolic effect or an effect indicative of toxicity or efficacy). Incubation time can range from about 1 hour to 24 hours, or can be extended to several days or even weeks as needed. Incubation conditions typically involve standard culture conditions known in the art, including a culture temperature of about 37 degrees Celsius.

[0073] Disease phenotypes in cardiac tissue can be used to determine the effective dose range of the test agent. The effect of increasing the concentration (e.g., dose) of the test agent on cardiac tissue can be monitored to determine efficacy.

[0074] Different doses of individual test agents and combinations of test agents can be screened in groups of cardiac tissues with different genetic backgrounds (e.g., derived from different donors and / or containing genetically modified cells) to determine the pharmacogenetic efficacy profile of the test agents. For example, multiple doses of test agents or combinations of test agents can be screened for efficacy or lack of efficacy against one or more genetic backgrounds.

[0075] According to a third aspect of this disclosure, diseased cardiac tissue produced by a method according to a second aspect of this disclosure is provided, wherein the diseased cardiac tissue exhibits a diabetic cardiomyopathy phenotype.

[0076] In some embodiments, diseased cardiac tissue is generated directly by the methods described herein.

[0077] According to a fourth aspect of this disclosure, a diseased cardiac tissue is provided, comprising a population of cardiomyocytes in a culture medium according to a first aspect of this disclosure.

[0078] In some embodiments, the cardiac tissue is generated using a system defined in WO 2015 / 061907 A1, WO 2021 / 158233 A1 and / or WO 2016 / 183143 A1.

[0079] In some embodiments, the cardiac tissue further comprises two or more scaffold elements disposed within the cardiac tissue. In some embodiments, the scaffold elements are opposite each other (they may be formed from a single element or multiple separate elements) and serve to form anchor points for the cardiac tissue formed therebetween. The cardiac tissue is not limited to having two scaffold elements, but may contain more than two, such as three, four, five, six, seven, eight, nine, or ten or more such scaffold elements. Any number of deformable scaffold elements may be provided, as long as it is possible to form 3D tissue surrounding and connected therebetween each scaffold element, such that the cardiac tissue is disposed between the scaffold elements. In some embodiments, the cardiac tissue comprises two scaffold elements disposed at or near opposite ends of the longitudinal axis of the cardiac tissue.

[0080] In some embodiments, the stent element may be deflectable, deformable, bendable, etc., and is further configured to allow measurement of the contractile forces exerted on the stent element by cardiac tissue. The shape, thickness, length, orientation, and surface morphology characteristics of the stent element may vary in any number of suitable ways, as long as the stent element is able to deform, bend, or otherwise change shape in response to the contractile action or activity of the cardiac tissue connected therein, and such deformation, bending, or other shape changes can be reliably measured. In some embodiments, the stent element has an elasticity of about 20 kPa to 0.5 MPa.

[0081] The scaffold element can be made of any suitable material, including, for example, poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PMMA), and polystyrene. The scaffold element can be made of biodegradable materials. Other suitable materials may include poly(glycerol sebacic acid), citric acid-free POMaC, poly(lactic acid), poly(glycolic acid), poly(e-caprolactone), various polyurethanes and their copolymers, silk, microstructured nanofabrication materials, and / or materials doped with nanostructures such as nanorods or quantum dots. Optionally, in some embodiments, the scaffold element material can be permeated to allow the exchange and / or passage of water and molecules, including proteins, drugs, nutrients, and metabolic waste materials. In some other embodiments, permeability can be achieved by forming pores in the scaffold element material. In still other embodiments, the scaffold element can be manufactured by any suitable means, including microfabrication, soft lithography processes (including but not limited to step-flash lithography (STIL), 3D printing (i.e., additive manufacturing), thermal embossing, extrusion, injection molding, phase-shifting edge lithography, and nanomachining).

[0082] In some embodiments, cardiac tissue comprises mature ultrastructures. In some embodiments, the ultrastructures are selected from the group consisting of sarcomeres, mitochondria, T tubules, sarcoplasmic reticulum, and combinations thereof.

[0083] In some embodiments, cardiac tissue exhibits a positive force-frequency relationship. In some embodiments, the force is approximately 0.25 to approximately 2 mN / mm at frequencies of approximately 0 to 6 Hz. 2 .

[0084] According to a fifth aspect of this disclosure, a kit is provided comprising a culture medium according to a first aspect of this disclosure and a bioreactor for culturing heart tissue, wherein the bioreactor comprises a plurality of pores, wherein each pore comprises a chamber configured for growing heart tissue therein and one or more deformable scaffold elements fixed to each chamber.

[0085] In some embodiments, the bioreactor is as described in WO 2015 / 061907 A1.

[0086] In some embodiments, the bioreactor is a porous plate. In some embodiments, the bioreactor is a porous plate having 12, 96, 384, or 1536 pores.

[0087] In some embodiments, the bioreactor is constructed of a polymer. In some embodiments, the polymer is a biodegradable polymer. In some embodiments, the biodegradable polymer is polylactic acid, poly(lactic-co-glycolic acid), or poly(caprolactone), polyglycolic acid, polylactide, polylactide, polyhydroxybutyrate, 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 sebate), citric acid-free POMaC, poly(e-caprolactone), polyurethane, silk, or nanomaterials, or copolymers or blends thereof. In some embodiments, the polymer is doped with nanostructures.

[0088] In some embodiments, the deformable scaffold element is made of metal, silk, or polymer. In some embodiments, the deformable scaffold element is made of intestinal material, monocryl, polyglycolic acid, prolene, polyglactin, polydioxanone, polypropylene, nylon, or polyester.

[0089] In some embodiments, the chamber is configured to be seeded with cardiomyocytes.

[0090] In some embodiments, the deformable support elements fixed to each chamber are oriented substantially perpendicularly, substantially parallelly, or substantially diagonally relative to the longitudinal axis of the chamber.

[0091] In some embodiments, the deformable scaffold element is configured to be embedded or partially embedded in the heart tissue as the heart tissue grows.

[0092] In some embodiments, the deformable stent element is configured to be encapsulated or partially encapsulated by cardiac tissue and attached to the cardiac tissue such that movement of the cardiac tissue coincides with movement of the deformable stent element.

[0093] In some embodiments, the bioreactor further includes a pair of electrodes configured to generate an electric current through the growth chamber of the bioreactor.

[0094] According to a sixth aspect of this disclosure, a method for evaluating the safety and / or efficacy of an assay agent on cardiac tissue is provided, the method comprising: (a) culturing cardiac tissue in a culture medium described herein; (b) contacting the cardiac tissue with the assay agent; (c) measuring the effect on one or more physiological parameters indicative of safety and / or efficacy; and (d) comparing (c) with the same physiological parameters measured in control cardiac tissue not exposed to the assay agent, wherein a statistically significant change in the physiological parameters in (c) compared to (d) indicates a lack of safety and / or efficacy of the assay agent.

[0095] In some embodiments, the cardiac tissue is generated using a system defined in WO 2015 / 061907 A1, WO 2021 / 158233 A1 and / or WO 2016 / 183143 A1.

[0096] Where applicable or not expressly denied, any of the embodiments described herein are contemplated to be combined with one or more other embodiments, even if those embodiments are described under different aspects of this disclosure.

[0097] The above and other objects, features and advantages of this disclosure will become more apparent from the following detailed description taken with reference to the accompanying drawings. Attached Figure Description

[0098] This disclosure will now be described in detail by way of example and with reference to the accompanying drawings, wherein:

[0099] Figure 1 A schematic diagram showing the change of contractile force over time is shown, indicating the twitch amplitude (peak height), contraction time (time from 10% of the peak height to the amplitude), relaxation time (time from the amplitude to 10% of the peak height), and passive tension (force between peaks).

[0100] Figure 2 The changes in contractility of cardiac tissue treated with different culture media over time are shown. The top inset plots show relative changes in data: Δtwitch amplitude, Δcontraction time, and Δrelaxation time for different treatment groups. Data are presented as median (circles) changes relative to the untreated response (day 0). The bottom inset plots show raw data: twitch amplitude, contraction time, and relaxation time for different treatment groups. Data are presented as median (circles). On day 3, tissues were transferred from I3M medium to the treatment groups. The I3M + BSA control group received 250 µM BSA as a control.

[0101] Figure 3 The changes in contractility of cardiac tissue treated with different culture media over time are shown. The top inset plots show relative changes in data: Δtwitch amplitude, Δcontraction time, and Δrelaxation time for each treatment group. Data are presented as median (circles) changes relative to the untreated response (day 0). The bottom inset plots show raw data: twitch amplitude, contraction time, and relaxation time for each treatment group. Data are presented as median (circles). On day 2, tissues were transferred from I3M medium to the treatment groups. N = 3 tissues per group; shading indicates 95% confidence intervals. The I3M (control) group received 250 µM BSA as a control.

[0102] Figure 4The Δtwitch amplitude for each treatment group is shown. On day 2, tissues were transferred from I3M medium to the treatment groups. Data are presented as median change relative to the untreated response (day 0) ± 95% CI. N = 3 tissues per group.

[0103] Figure 5 The Δ shrinkage time for each treatment group is shown. On day 2, tissues were transferred from I3M medium to the treatment groups. Data are presented as median change ± 95% CI relative to the untreated response (day 0). N = 3 tissues per group.

[0104] Figure 6 The Δ relaxation time for each treatment group is shown. On day 2, tissues were transferred from I3M medium to the treatment groups. Data are presented as median change ± 95% CI relative to the untreated response (day 0). N = 3 tissues per group.

[0105] Figure 7 The changes in spontaneous pulsation frequency and excitation threshold over time are shown for each treatment group.

[0106] Figure 8 The Δ relaxation time for each treatment group is shown. On day 2, tissues were transferred from I3M medium to the treatment groups. Data are presented as median change ± 95% CI relative to the untreated response (day 0). N = 6 tissues per group.

[0107] Figure 9 The changes in contractility of cardiac tissue treated with different media over time are shown after the addition of ET-1 to the medium on day 0. The top inset plots show relative changes in data: Δtwitch amplitude, Δcontraction time, and Δrelaxation time for different treatment groups. Data are presented as median (circles) changes relative to the untreated response (day 0). The bottom inset plots show raw data: twitch amplitude, contraction time, and relaxation time for different treatment groups. Data are presented as median (circles). On day 2, tissues were transferred from I3M medium to the treatment groups, and elution began on day 30. N = 3 tissues per group; shading indicates 95% CI.

[0108] Figure 10The changes in contractility of cardiac tissue treated with different media over time are shown after the addition of ET-1 to the medium at each medium change. The top inset plots show relative changes in data: Δtwitch amplitude, Δcontraction time, and Δrelaxation time for different treatment groups. Data are presented as median (circles) changes relative to the untreated response (day 0). The bottom inset plots show raw data: twitch amplitude, contraction time, and relaxation time for different treatment groups. Data are presented as median (circles). On day 2, tissues were transferred from I3M medium to the treatment groups, and elution began on day 30. N = 3 tissues per group; shading indicates 95% CI.

[0109] Figure 11 Tissue images are shown for different treatment groups on day 0 (top thumbnail) and day 30 (bottom thumbnail).

[0110] Figure 12 Tissue images of the I3M + 200 µM Palm + 200 µM Ol + 5 nM ET-1 treatment group on day 0 (top thumbnail) and day 30 (bottom thumbnail) are shown.

[0111] Figure 13 40x confocal stacked images of different treatment groups with lipid droplet staining (red) and cell nuclear staining (blue) are shown. Features circled are lipid droplets > 10 µm.

[0112] Figure 14 The changes in contractility over time are shown in the control composition-determined medium (RPMI 1640 + 5 mM glucose + 1.4 mM CaCl2 + 50 µM palmitate / salt / ester + 50 µM oleic acid / salt / ester + insulin-free B27 + 1 nM insulin) compared to the group treated with the lipotoxic medium (composition-determined medium + 150 µM palmitate / salt / ester + 150 µM oleic acid / salt / ester + 1 nMET-1). The top inset shows the relative changes: Δtwitch amplitude, Δcontraction time, and Δrelaxation time for each treatment group. Data are presented as median (circles) changes relative to the untreated response (day 0). The bottom inset shows the raw data: twitch amplitude, contraction time, and relaxation time for each treatment group. Data are presented as median (circles). In the composition determination medium group, N = 3 tissues; in the composition determination medium + 150 µM Palm + 150 µM Ol + 1 nM ET-1 group, N = 2 tissues (one tissue was damaged during the experiment). Shaded areas represent 95% confidence intervals. Tissues were transferred from I3M medium to composition determination medium on day 2. Tissues were treated with composition determination medium for 10 days and then transferred to their respective treatment groups on day 12.

[0113] Figure 15 A shows the measurements of passive tension changes from day 0 to day 14 or day 15. Bars represent the median. In 3 independent experiments, N = 27 tissues were represented in each group. Figure 15 B shows the measurements of tissue area change from day 0 to day 14 or day 15. The bars represent the median. In 3 independent experiments, N = 27 tissues were analyzed in each group.

[0114] Figure 16 The tissue scale is shown to detect the tissue outline in order to calculate the tissue area.

[0115] Figure 17 The measurements of the change in the activation threshold (ET) from day 0 to day 14 or day 15 are shown. The bars represent the median. There were N = 27 tissues in each of the three independent experiments.

[0116] Figure 18 A shows the APD measured at the end of the processing. 90 (Duration of action potential from peak to 90% repolarization). Bars represent the median. In two independent experiments, N = 6 tissues were represented in each group. Figure 18 B shows the APD measured from cardiac action potential trajectory via MATLAB. 90 .

[0117] Figure 19 A shows the time to 50% calcium transient decay measured at the end of treatment. Bars represent the median ± 95% confidence interval. There were N = 9 tissues in each of the 3 independent trials. Figure 19 B shows the time to 50% calcium transient decay, measured from cardiac calcium transient trajectory via MATLAB. Detailed Implementation

[0118] Due to its unique ability to visualize and quantify systolic parameters of cardiac tissue using the systems described in WO 2015 / 061907 A1, WO 2021 / 158233 A1, and / or WO 2016 / 183143 A1 (also known as the Biowire™ system), the inventors were able to measure more robust systolic parameters, such as increased relaxation time, which acts as a similar manifestation of diastolic dysfunction – a characteristic of lipotoxic diabetic cardiomyopathy that has not yet been confirmed in other research groups in their models. The 3D in vitro lipotoxic cardiomyopathy model is the first in vitro model of metabolically driven diastolic dysfunction.

[0119] The concentrations of each lipotoxic component (i.e., palmitic acid / salt / ester, oleic acid / salt / ester, and endothelin-1) added to the basal medium were rigorously and specifically optimized to produce the desired lipotoxic diabetic cardiomyopathy phenotype in cardiac tissue.

[0120] 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.

[0121] Example 1

[0122] In general, the inventors developed a lipotoxic cardiomyopathy model involving culturing healthy cardiac tissue in the Biowire™ II platform and exposing the tissue to a lipotoxic medium for 28 days to induce a lipotoxic cardiomyopathy phenotype in the tissue. During treatment with the lipotoxic medium, the inventors recorded changes in tissue contractility over time. A key parameter of the measured tissue contractility was relaxation time (i.e., the time it takes for the tissue to relax from a fully contracted state), because the increased relaxation time in cardiac tissue is analogous to the delayed relaxation of the left ventricle of the heart. Figure 1 In addition to relaxation time, twitching amplitude (the total force exerted by the tissue during contraction) and contraction time (the time it takes for the tissue to return from a relaxed state to its contraction peak) were also measured. Figure 1 At the end of the processing, the inventors harvested tissue for more detailed model characterization to confirm that these tissues exhibited other key features of lipotoxic cardiomyopathy.

[0123] The inventors discovered that they could achieve a consistent contractile phenotype in cardiac tissue treated with their lipotoxic culture medium, consistent with lipotoxic diabetic cardiomyopathy.

[0124] Lipid-toxic culture medium treatment group

[0125] The inventors first prepared the basal medium by adding the following components to StemPro-34™ basal medium (LifeTechnologies, Cat. #10639011): 1% (v / v) GlutaMAX (LifeTechnologies, Cat. #35050-061), 2% (v / v) 1M HEPES (Life Technologies, Cat. #15630-080, final concentration 20 mM), 1% (v / v) penicillin-streptomycin (Gibco, Cat. #15140-122), 0.5% (v / v) 30 mg / mL transferrin solution (prepared by dissolving transferrin powder (Sigma-Aldrich, Cat. #T8158) in IMDM (Gibco, Cat. #12440-053, final concentration 0.15 mg / mL), and 0.4% (v / v) 64 A 0.256 mg / mL ascorbic acid solution (prepared by dissolving trisodium 2-phosphate-L-ascorbate (Sigma-Aldrich, Cat. #49752) in PBS (Sigma-Aldrich, Cat. #D8537) to a final concentration of 0.256 mg / mL) and 2.6% (v / v) StemPro™-34 nutritional supplement (Life Technologies, Cat. #10639011) were used. This basal medium is referred to as "I3M". It will be apparent to those skilled in the art that other media may be suitable as basal media.

[0126] The inventors then prepared various test media by adding different components to the basal culture medium at different concentrations, as summarized in the table below.

[0127]

[0128] Cardiac tissue formation

[0129] Cardiac tissue was generated through the following steps: hiPSC-derived cardiomyocytes were cultured in I3M medium using the Biowire™ system and a seven-week maturation protocol as described by Feric et al. (“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), which is incorporated herein by reference. The inventors then transferred the mature tissues to a chronic testing chamber that aliquoted the tissues into six wells (three tissues per well). The tissues were exposed to a lipotoxicity testing medium for 28 days.

[0130] result

[0131] Preliminary experiments (in which the inventors added cortisol to their lipotoxic medium) showed that 1 µM cortisol induced a significant and sustained increase in the amplitude of convulsions in Biowire™ tissues, which was inconsistent with the expected phenotype. Therefore, supplementation with 1 µM cortisol was excluded in all subsequent iterations of the lipotoxic medium.

[0132] The inventors demonstrated that, after 14 days of treatment, the I3M + 250 µM palmitic acid / salt / ester + 10 nM endothelin-1 group induced a decrease in convulsive amplitude and an increase in contraction and relaxation times. The inventors also found that a reduced endothelin-1 concentration of 5 nM (I3M + 250 µM palmitic acid / salt / ester + 5 nM endothelin-1) had a significant effect on tissue contractility. Figure 2 Therefore, a lower concentration of 5 nM endothelin-1 was used in subsequent experiments. However, those skilled in the art will recognize that higher concentrations of endothelin-1 would also be appropriate.

[0133] All patients who stopped pulsating within a 24-day timeframe were treated with I3M + 250 µM palmitate / salt / ester + 5 nM or 10 nM endothelin-1. Figure 2This may be due to the toxicity caused by the high concentration of palmitic acid / salt / ester added to the culture medium. To address this observed toxicity, the inventors used two methods: (1) the inventors treated tissues with lower concentrations of palmitic acid / salt / ester (100 µM, 150 µM, or 200 µM) for 28 days; (2) the inventors started treatment with 250 µM palmitic acid / salt / ester for the first 10 days, and then switched to lower concentrations of palmitic acid / salt / ester (100 µM or 150 µM). The inventors found that the I3M + 200 µM palmitic acid / salt / ester + 5 nM endothelin-1 treatment group resulted in a sustained increase in relaxation time. Figure 3 However, although the tissues treated with this medium survived longer than those in previous experiments, they still stopped pulsating on day 30 of treatment.

[0134] Therefore, the inventors sought an additional method to counteract the toxic effects observed with high concentrations of palmitic acid / salt / ester treatment. In subsequent experiments, the inventors began adding oleic acid / salt / ester (Ol) to palmitic acid / salt / ester at a 1:1 ratio. The inventors believed that the 1:1 ratio of oleic acid to palmitic acid would help reduce the toxicity of palmitic acid without significantly increasing the total fatty acid content in the culture medium, as this could induce other negative effects in tissues and prevent them from exhibiting the desired phenotype.

[0135] The inventors discovered that, over a period of time, tissues treated with a 1:1 ratio of oleic acid / salt / ester and palmitic acid / salt / ester neither ceased pulsating nor showed a significant decrease in twitching amplitude after 28 days of treatment. Figure 4 These are the same groups - that is,

[0136] I3M + 150 µM palmitic acid / salt / ester + 150 µM oleic acid / salt / ester + 5 nM endothelin-1;

[0137] I3M + 200 µM palmitic acid / salt / ester + 200 µM oleic acid / salt / ester + 5 nM endothelin-1; and

[0138] I3M + 250 µM palmitic acid / salt / ester + 250 µM oleic acid / salt / ester + 5 nM endothelin-1 (lasting 10 days) decreased to I3M + 100 µM palmitic acid / salt / ester + 100 µM oleic acid / salt / ester + 5 nM endothelin-1

[0139] – The relaxation time of the tissues increased by an average of 5%, 13%, and 15%, respectively, without negatively impacting other indicators of tissue health and maturity, such as the ignition threshold (the minimum voltage required for a tissue to respond to an external stimulus) and spontaneous pulsation (pulsations that begin without electrical stimulation). Figure 6 , Figure 7 A significant increase in the excitation threshold or spontaneous pulsation indicates an extreme decline in tissue health, and in certain experiments, tissues not treated with oleic acid / salt / ester during the time course exhibited these characteristics. Figure 7 ).

[0140] At the end of the time period, the inventors rinsed the lipotoxic medium with standard basal medium for 7 days and observed that the shrinkage effect was partially or completely reversed in tissues treated with both palmitic acid / salt / ester and oleic acid / salt / ester during the time period, indicating that the lipotoxic medium containing both palmitic acid / salt / ester and oleic acid / salt / ester does not cause irreversible tissue damage. This provides support for the view that the addition of compounds that mechanically inhibit lipid production can potentially reverse the lipotoxic phenotype in cardiac tissue.

[0141] The inventors also discovered that the treatment group without endothelin-1 – specifically:

[0142] I3M + 200 µM palmitic acid / salt / ester + 200 µM oleic acid / salt / ester; and

[0143] I3M + 250 µM palmitic acid / salt / ester + 250 µM oleic acid / salt / ester (lasting 10 days) decreased to I3M + 100 µM palmitic acid / salt / ester + 100 µM oleic acid / salt / ester

[0144] – This also increased the organization's relaxation time by an average of 15% and 13%, respectively. Figure 6 These amounts are comparable to the increases achieved in treatment groups containing both fatty acids and endothelin-1. Therefore, the inventors hypothesize that the increase in tissue relaxation time observed during treatment is primarily caused by fatty acids (rather than endothelin-1).

[0145] The inventors replicated the treatment groups from previous experiments, which (1) had the greatest effect on relaxation time and (2) did not negatively affect the excitation threshold, spontaneous pulsation, or convulsion amplitude. Among these groups, I3M + 200 µM palmitate / salt / ester + 200 µM oleic acid / salt / ester + 5 nM endothelin-1 and I3M + 250 µM palmitate / salt / ester + 250 µM oleic acid / salt / ester + 5 nM endothelin-1 (lasting 10 days) decreased to I3M + 100 µM palmitate / salt / ester + 100 µM oleic acid / salt / ester + 5 nM endothelin-1, inducing a maximum mean increase of 18% and 12% in relaxation time, respectively, at the end of treatment. Figure 8 At the end of the time period, the effect in both groups decreased to baseline by washing with standard basal medium.

[0146] For future iterations of the lipotoxic culture medium, the inventors used a concentration of 200 µM palmitic acid / salt / ester + 200 µM oleic acid / salt / ester because these concentrations consistently increased the relaxation time by more than 10% in two independent experiments, and they also provided the added convenience of remaining constant over time (i.e., the concentrations of palmitic acid / salt / ester and oleic acid / salt / ester do not need to become lower after 10 days of treatment). However, those skilled in the art will recognize that variations in these concentrations are possible.

[0147] This experiment further demonstrates that treatment with fatty acids alone can increase the relaxation time of cardiac tissue. However, the increase tends to be more pronounced when tissue receives both fatty acids and endothelin-1. This becomes clear when the inventors compare the treatment group containing both fatty acids and endothelin-1 with the treatment group excluding endothelin-1. For example, in this experiment, the I3M + 200 µM palmitic acid / salt / ester + 200 µM oleic acid / salt / ester + 5 nM endothelin-1 treatment group showed an average increase of 18% in relaxation time at the end of the time period, while the I3M + 200 µM palmitic acid / salt / ester + 200 µM oleic acid / salt / ester treatment group only achieved an average increase of 10%. Similarly, the treatment group receiving I3M + 250 µM palmitic acid / salt / ester + 250 µM oleic acid / salt / ester + 5 nM endothelin-1 (lasting 10 days) showed an average increase of 12% in relaxation time when reduced to the treatment group receiving I3M + 100 µM palmitic acid / salt / ester + 100 µM oleic acid / salt / ester + 5 nM endothelin-1, while the treatment group receiving I3M + 250 µM palmitic acid / salt / ester + 250 µM oleic acid / salt / ester (lasting 10 days) showed an average increase of only 8% in relaxation time when reduced to the treatment group receiving I3M + 100 µM palmitic acid / salt / ester + 100 µM oleic acid / salt / ester.

[0148] The inventors demonstrated the individual contributions of fatty acids and endothelin-1 to the contractile effects observed in cardiac tissue through treatments containing only endothelin-1 (I3M + 5 nM endothelin-1) and treatments containing only fatty acids (I3M + 200 µM palmitic acid / salt / ester + 200 µM oleic acid / salt / ester). The inventors also aimed to determine whether endothelin-1 receptors in tissue become desensitized over time due to repeated exposure to high concentrations of endothelin-1, which may explain the weakening effect of contractile parameters over time. Therefore, the inventors included two additional treatment groups: one consisting of a lower concentration of 1 nM endothelin-1 (I3M + 200 µM palmitic acid / salt / ester + 200 µM oleic acid / salt / ester + 1 nM endothelin-1), and the other consisting of adding endothelin-1 to the tissue every approximately 96 hours instead of every approximately 48 hours (I3M + 200 µM palmitic acid / salt / ester + 200 µM oleic acid / salt / ester + 5 nM endothelin-1 (alternating)). In this experiment, the inventors found that the treatment groups without endothelin-1 (I3M + BSA control; and I3M + 200 µM palmitic acid / salt / ester + 200 µM oleic acid / salt / ester) appeared to have little effect on contractility, while the treatment groups with endothelin-1 (I3M + 5 nM endothelin-1; I3M + 200 µM palmitic acid / salt / ester + 200 µM oleic acid / salt / ester + 1 nM endothelin-1; I3M + 200 µM palmitic acid / salt / ester + 200 µM oleic acid / salt / ester + 5 nM endothelin-1; and I3M + 200 µM palmitic acid / salt / ester + 200 µM oleic acid / salt / ester + 5 nM endothelin-1 (alternating)) resulted in a sustained increase in contraction time and an initial increase in twitching amplitude, which subsided at the end of treatment. Figure 9 Importantly, the 5 nM endothelin-1 group did not increase relaxation time, leading the inventors to conclude that endothelin-1 treatment alone could not achieve a phenotype consistent with lipotoxic cardiomyopathy. The inventors also found that the groups supplemented with fatty acids and endothelin-1 each time the culture medium was changed (I3M + 200 µM palmitic acid / salt / ester + 200 µM oleic acid / salt / ester + 1 nM endothelin-1; and I3M + 200 µM palmitic acid / salt / ester + 200 µM oleic acid / salt / ester + 5 nM endothelin-1) increased relaxation time by an average of 15% and 8%, respectively. Figure 9 ).

[0149] The inventors further confirmed that, at the end of the time process, after 7 days of washing, the effects of all treatment groups on contraction and relaxation times were reduced, while the twitching amplitude decreased over time, independent of washing. Since the I3M + 200 µM palmitic acid / salt / ester + 200 µM oleic acid / salt / ester + 5 nM endothelin-1 group consistently led to an increase in relaxation time in three independent experiments, the inventors determined that this group, along with the 1 nM endothelin-1 group, were particularly effective lipotoxic media. Because the medium containing I3M + 200 µM palmitic acid / salt / ester + 200 µM oleic acid / salt / ester + 1 nM endothelin-1 caused the largest increase in relaxation time in this experiment, this medium was used in subsequent model characterization.

[0150] I3M + 200 µM palmitic acid / salt / ester + 200 µM oleic acid / salt / ester + 1 nM endothelin-1 formulation

[0151] The inventors added 4% (v / v) 5 mM BSA-palmitic acid / salt / ester saturated fatty acid complex (Cayman Chemical, Cat.#29558, final concentration 200 µM), 4% (v / v) 5 mM BSA-oleic acid / salt / ester monounsaturated fatty acid complex (Cayman Chemical, cat# 29557, final concentration 200 µM), and 0.1% (v / v) 1 µM endothelin-1 (prepared by dissolving endothelin-1 powder (Sigma-Aldrich, Cat.#E7764-10UG) in cell culture grade water (Corning, Cat.#25-055-CI, final concentration 1 nM) to I3M medium.

[0152] Model representation

[0153] Bright field imaging

[0154] In addition to measuring the shrinkage changes induced by the lipotoxic culture medium, the inventors also characterized changes in tissue morphology by taking bright-field images of the tissue at the beginning and end of the time process. These images show that tissues treated with both palmitic acid / salt / ester and oleic acid / salt / ester exhibited rough, uneven edges along their periphery, whereas these edges were absent before treatment with the lipotoxic culture medium. Figure 11 These serrated edges were consistently observed in three independent experiments conducted on tissues treated with a medium containing 200 µM palmitic acid / salt / ester + 200 µM oleic acid / salt / ester + 5 nM endothelin-1. Figure 12 ).

[0155] Lipid staining

[0156] The inventors also used HCS LipidTOX™ deep red neutral lipid staining agent (Thermo Fisher Scientific, Cat. #H34477) to... Figure 11 The same group of tissues shown was stained with lipids. The resulting confocal images show a large number of lipid droplets larger than 10 µm in tissues treated with both palmitic acid / salt / ester and oleic acid / salt / ester. Figure 13 ), which is in the bright field image ( Figure 11 The same tissue shows rough, uneven edges along its periphery.

[0157] Example 2

[0158] The inventors further demonstrated through experiments using alternative basal media that the basal medium is not limited to I3M medium. As described above, heart tissue was generated in I3M medium on the Biowire™ platform and transferred to composition-determined medium (RPMI 1640 + 5 mM glucose + 1.4 mM CaCl2 + 50 µM palmitic acid / salt / ester + 50 µM oleic acid / salt / ester + insulin-free B27 + 1 nM insulin) on day 2. Heart tissue was treated with composition-determined medium for 10 days before being transferred to their respective treatment groups: composition-determined medium (control group) or lipotoxicity-determined medium (composition-determined medium + 150 µM palmitic acid / salt / ester + 150 µM oleic acid / salt / ester + 1 nM ET-1). Figure 14 As shown in Tables 1 and 2 below, treatment with lipotoxic components in the culture medium increased relaxation time compared to the control group, indicating that lipotoxic components can induce disease phenotypes in other basal media.

[0159] Table 1

[0160]

[0161] Table 2

[0162]

[0163] The data are presented as mean ± standard deviation.

[0164] Example 3

[0165] Control culture medium and lipotoxic culture medium

[0166] I3M serves as the basal medium for both the control and lipotoxicity-prone medium formulations. To prepare I3M medium, the inventors added the following components to StemPro-34 basal medium (Life Technologies, Cat.#10639011): 1% (v / v) GlutaMAX (Life Technologies, Cat.#35050-061), 2% (v / v) 1M HEPES (Life Technologies, Cat.#15630-080, final concentration 20 mM), 1% (v / v) penicillin-streptomycin (Gibco, Cat.#15140-122), 0.5% (v / v) 30 mg / mL transferrin solution (prepared by dissolving transferrin powder (Sigma-Aldrich, Cat.#T8158) in IMDM (Gibco, Cat.#12440-053), final concentration 0.15 mg / mL), and 0.4% (v / v) 64 mg / mL ascorbic acid solution (prepared by dissolving trisodium 2-phosphate-L-ascorbate (Sigma-Aldrich, Cat.#49752) in PBS (Sigma-Aldrich, Cat.#D8537, final concentration 0.256 mg / mL) and 2.6% (v / v) StemPro supplement (Life Technologies, Cat.#10639011).

[0167] In all experiments, the control medium consisted of I3M + 64 uM BSA. The medium was prepared by adding 8% (v / v) 0.8 mM BSA fatty acid complex control (Cayman Chemical Company, Cat# 29556) to the I3M medium.

[0168] In all experiments, the lipotoxic medium consisted of I3M + 200 µM palmitic acid / salt / ester + 200 µM oleic acid / salt / ester + 1 nM endothelin-1. The medium was prepared by adding the following components to I3M: 4% (v / v) 5 mM BSA-palmitic acid / salt / ester saturated fatty acid complex (Cayman Chemical, Cat.#29558, final concentration 200 µM), 4% (v / v) 5 mM BSA-oleic acid / salt / ester monounsaturated fatty acid complex (Cayman Chemical, cat# 29557, final concentration 200 µM), and 0.1% (v / v) 1 µM endothelin-1 (prepared by dissolving endothelin-1 powder (Sigma-Aldrich, Cat.#E7764-10UG) in cell culture grade water (Corning, Cat.#25-055-CI, final concentration 1 nM).

[0169] method

[0170] Chronic force experiment

[0171] During the chronic force experiment, tissues were treated with control or lipotoxic medium for 14–15 days. The medium was changed 100% every 2–3 days during treatment. Tissues were assessed every 2–5 days to measure changes in shrinkage parameters. During assessment, tissues were transferred from the incubator (5% CO2, 37°C) to an environmental chamber equipped with microscope objectives (5% CO2, 37°C). Electrical stimulation was initiated at 1 Hz (2 ms pulse duration, monophasic, at 3 V). Tissues were equilibrated in the chamber for 30 min prior to video acquisition. During video acquisition, polymer wires in the Biowire™ II platform were exposed to an excitation wavelength of 405 nm, causing the wires to fluoresce. Ten-second videos of the wires were captured while the tissues were stimulated at a frequency of 1 Hz. After video acquisition of all wires, the tissues were returned to the incubator for continued stimulation until the next assessment. This process was repeated until the end of treatment.

[0172] Video analysis was performed using custom MATLAB code that tracked the position of the polymer conductor to measure the amount of linear displacement in each video frame. The displacement values ​​were converted into force values ​​using an experimentally derived force-displacement equation. Twitch amplitude was calculated as the height of the force peak. Contraction time was calculated by measuring the time from 10% of the peak height to the maximum peak amplitude, and relaxation time was calculated by measuring the time from the maximum peak amplitude to 90% relaxation (or 10% of the peak height). Passive tension was calculated by measuring the amount of force on the line between contractions (i.e., the horizontal line between peaks).

[0173] Tissue area measurement

[0174] For tissue area measurements, bright-field images of the tissue were acquired at 2x magnification during each evaluation period. These images were processed using custom tissue measurement software, Tissue Ruler, which determines the contour of each tissue by contrasting the dark areas containing the tissue against the bright background of the platform (see [link to Tissue Measurement]). Figure 16 Then use this profile to calculate the area of ​​the tissue.

[0175] Excitation threshold measurement

[0176] The excitation threshold for each tissue was determined by observing the tissue at 2x bright-field magnification. The stimulation frequency was set to 2 Hz, and then the stimulation voltage was gradually decreased from an initial voltage of 3V until the tissue stopped contracting at a frequency of 2 Hz. The lowest voltage required to maintain tissue pulsation at a frequency of 2 Hz was recorded as the excitation threshold.

[0177] Action potential measurement

[0178] Action potentials were measured by incubating tissue with a voltage-sensitive dye, causing cells to fluoresce based on their membrane potential. During cardiac action potentials, cell membrane potential fluctuates, resulting in fluctuations in cell fluorescence. The voltage-sensitive dye used in the experiment caused cells to fluoresce brighter at hyperpolarized membrane potentials and darker at depolarized membrane potentials.

[0179] After incubating the tissue in the dye, 10-second videos of the tissue were acquired at a stimulation frequency of 1 Hz. Action potential trajectories were then generated based on measured fluorescence using a custom MATLAB program. The same MATLAB program was also used to calculate the action potential trajectories (APDs). 90 – The time required for the action potential peak to reach 90% repolarization (see [reference]). Figure 18 B).

[0180] Calcium transient measurement

[0181] Calcium transients were measured by incubating tissue with a calcium-sensitive dye that enters cells and fluoresces upon binding with calcium. During cardiac calcium transients, the calcium dye fluorescence increased with increasing intracellular calcium (during contraction) and decreased with decreasing intracellular calcium (during relaxation).

[0182] After incubating the tissue in the dye, 7-second videos of the tissue were acquired at a stimulation frequency of 1 Hz. The calcium transient trajectory was then generated based on the measured fluorescence using a custom MATLAB program. The same MATLAB program was also used to calculate the time for the calcium transient to decay to 50% – i.e., the time required for the transient peak to reach 50% transient decay (see [link to MATLAB]). Figure 19 B).

[0183] result

[0184] Figure 15 A shows measurements of changes in passive tension (i.e., tension in the polymer wires when the tissue does not actively contract) in Biowire™ tissue treated with control or lipotoxic medium from day 0 to the end of treatment. Tissue treated with lipotoxic medium showed a significant increase in passive tension at day 14 / 15 compared to tissue treated with control medium. This increase in passive tension in lipotoxic tissue may be evidence of increased ECM crosslinking, fibrosis, and / or increased fibroblast contractile activity, consistent with increased myocardial stiffness observed in patients with lipotoxic cardiomyopathy.

[0185] Figure 15 B shows measurements of tissue area changes in Biowire tissue treated with control or lipotoxic medium from day 0 to the end of treatment. Tissue treated with lipotoxic medium showed a significant increase in tissue area at day 14 / 15 compared to tissue treated with control medium, which may be evidence of hypertrophy – a symptom commonly associated with lipotoxic cardiomyopathy.

[0186] Figure 17 Measurements show the change in the excitation threshold (the minimum voltage required to induce tissue contraction at the set stimulation frequency) in Biowire™ tissue treated with either control or lipotoxic medium from day 0 to the end of treatment. Tissue treated with lipotoxic medium exhibited a significant increase in the excitation threshold at day 14 / 15 compared to tissue treated with control medium. Since cardiac cells rely on intercellular interactions via gap junctions and adhesion complexes for efficient electrical conduction, the increased excitation threshold observed in lipotoxic tissue could indicate disruption of intercellular communication and / or decreased cellular health. Unbound by theory, the inventors hypothesize that this may play a role in the increased relaxation time also observed in lipotoxic tissue.

[0187] Figure 18 A shows the APD measured at the end of treatment in Biowire™ tissues treated with either control or lipotoxic medium. 90 (Duration of action potential from peak to 90% repolarization). Tissues treated with lipotoxic medium showed significantly higher APD compared to tissues treated with control medium. 90This value is consistent with the increased relaxation time also observed in lipotoxic tissues. Unbound by theory, the action potential duration observed in lipotoxic tissues may be a result of impaired ion channel function downstream of pathways activated by metabolic disorders (e.g., hyperglycemia and insulin resistance; 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, LM Ballou, IS Cohen and RZ Lin, "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).

[0188] Figure 19 A shows the time to 50% calcium transient decay measured at the end of treatment in Biowire™ tissues treated with either control or lipotoxic medium. Tissues treated with lipotoxic medium exhibited a significantly longer time to 50% calcium transient decay compared to tissues treated with control medium. This observation is consistent with the inventors' hypothesis that the increased relaxation time in lipotoxic tissues is caused by impaired calcium treatment, specifically a longer decay of calcium transients – a known mechanism leading to diastolic dysfunction (see RH Ritchie and ED Abel, "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 comprises: 100-300 µM palmitic acid / salt / ester; 100-300 µM oleic acid / salt / ester, and 0.5-15 nM endothelin-1.

2. The culture medium according to claim 1, wherein the culture medium is used to induce a diabetic cardiomyopathy phenotype in cultured cardiac tissue.

3. The culture medium according to any of the preceding claims, wherein the culture medium comprises 180-220 µM palmitic acid / salt / ester.

4. The culture medium according to claim 3, wherein the culture medium comprises 200 µM palmitic acid / salt / ester.

5. The culture medium according to any of the preceding claims, wherein the culture medium comprises 180-220 µM oleic acid / salt / ester.

6. The culture medium according to claim 5, wherein the culture medium comprises 200 µM oleic acid / salt / ester.

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

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

9. The culture medium according to claim 1, wherein the culture medium comprises: 180-220 µM palmitic acid / salt / ester; 180-220 µM oleic acid / salt / ester, and 0.5-10 nM endothelin-1.

10. The culture medium according to claim 1, wherein the culture medium comprises: 200 µM palmitic acid / salt / ester; 200 µM oleic acid / salt / ester, and 1-5 nM endothelin-1.

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

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

13. The culture medium according to any of the preceding claims, further comprising one or more of the following: (i) GlutaMAX, optionally 0.1%-2% (v / v) GlutaMAX, and optionally 1% (v / v) GlutaMAX; (ii) HEPES, optionally 10-30 mM HEPES, and optionally 20 mM HEPES; (iii) Penicillin-streptomycin, optionally 0.1%-2% penicillin-streptomycin, and optionally 1% penicillin-streptomycin; (iv) Transferrin solution, optionally 0.05-0.25 mg / mL transferrin solution, and optionally 0.15 mg / mL transferrin solution; (v) Ascorbic acid solution, optionally 0.1-0.4 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.

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

15. A method for inducing a diabetic cardiomyopathy phenotype in cardiac tissue, wherein the method comprises: Heart tissue was cultured in the culture medium according to any one of claims 1 to 14.

16. The method of claim 15, wherein the heart tissue is cultured in the culture medium for 3-80 days.

17. The method of claim 16, wherein the heart tissue is cultured in the culture medium for 21-35 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 of claim 18, wherein the pluripotent stem cell is a human induced pluripotent stem cell.

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

21. The method according to any one of claims 18 to 20, further comprising the following steps: The cardiomyocytes are electromechanically modulated by exposing them to electromechanical stimulation of increasing intensity over a period of time, thereby forming heart tissue with molecular, structural, and functional characteristics that mimic natural adult heart tissue.

22. The method of claim 21, wherein the cardiac tissue comprises T-tubules.

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

24. The method according to any one of claims 15 to 23, wherein the method further comprises: Measure one or more contractility parameters of the heart tissue.

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

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

27. The method according to any one of claims 15 to 26, wherein the method further comprises: Characterizes the tissue morphology of the heart tissue.

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

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

30. The method according to any one of claims 15 to 29, wherein the method further comprises: Characterizing one or more endpoints of the cardiac tissue, the one or more endpoints being selected from the group consisting of: lipidomics, phosphorylated Akt Western blot, RNA sequencing, proteomics, intracellular calcium transient analysis, and combinations thereof.

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

32. A diseased cardiac tissue produced by the method according to any one of 15 to 31, wherein the diseased cardiac tissue exhibits a diabetic cardiomyopathy phenotype.

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

34. The diseased cardiac tissue of 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 diseased cardiac tissue of claim 37, wherein the force is about 0.25 to about 2 mN / mm at a frequency of about 0 to 6 Hz. 2 .

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

40. The kit according to claim 39, wherein the bioreactor is a multi-well plate.

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

42. The kit according to any one of claims 39 to 41, wherein the bioreactor is made 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-co-glycolic acid), or poly(caprolactone), polyglycolic acid, polylactide, polylactide, polyhydroxybutyrate, polyhydroxyalkanoic acid, chitosan, hyaluronic acid, hydrogel, poly(2-hydroxyethyl-methacrylate), poly(ethylene glycol), poly(L-lactide) (PLA), or any combination thereof.

45. The kit according to claim 44, wherein the polymer is poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PMMA), polystyrene, poly(glycerol sebacate), citric acid-free POMaC, poly(e-caprolactone), polyurethane, silk, or nanomaterials, or copolymers or blends thereof.

46. ​​The kit of 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 made of intestinal material, monocryl, polyglycolic acid, prolene, polyglactin, 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 with cardiomyocytes.

50. The kit according to any one of claims 39 to 49, wherein the deformable support element fixed to each chamber is oriented substantially perpendicularly, substantially parallelly, or substantially diagonally relative to 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 in the heart tissue during the growth of the heart 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 heart tissue and attached to the heart tissue such that movement of the heart tissue coincides with movement of the deformable scaffold element.

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

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