Lipotoxic media

Abstract

The invention provides culture media for cultivating cardiac tissue, methods of inducing a diabetic cardiomyopathy disease phenotype in cardiac tissue, diseased cardiac tissue, and kits. An exemplary culture medium for cultivating cardiac tissue includes 100-300 pM palmitate, 100-300 pM oleate, and 0.5-15 nM endothelin-1.

Description

[0001]P603581PC00 Lipotoxic media Field of the Invention The present invention relates to a culture medium for cultivating cardiac tissue, a method of inducing a diabetic cardiomyopathy disease phenotype in cardiac tissue, diseased cardiac tissue produced by the method, a diseased cardiac tissue, and a kit. Background to the Invention Heart failure currently affects more than 64 million people globally, and it only seems to be increasing in prevalence. In the United States alone, health experts are projecting a 46% increase in the prevalence of heart failure by 2030. Among all cases of heart failure, heart failure with preserved ejection fraction (HFpEF) accounts for around 50% of cases and the percentage of heart failure patients with HFpEF has been increasing recently. Due to its status as a global epidemic, along with the heavy burden of symptoms imposed by HFpEF, there is a pressing need to identify drug candidates that may treat and prevent HFpEF. HFpEF is typically associated with other comorbidities such as diabetes, obesity, and metabolic syndrome, with approximately 65% of HFpEF patients having at least one of these diseases. A key feature of metabolic disorders that are presumed to lead to HFpEF is hyperlipidemia, a condition in which abnormally high amounts of lipids and fatty acids circulate in the blood. Hyperlipidemia induces a negative effect on 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. In addition, lipids such as diacylglycerols and ceramides accumulate in cardiac cells over time, impacting cell morphology and function, which can lead to a condition called diabetic cardiomyopathy. Diabetic cardiomyopathy may be formally defined as cardiac dysfunction in the absence of any other risk factors, like 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. This disease may later progress to include left ventricular hypertrophy, worsening diastolic dysfunction, and HFpEF. In extreme cases, heart failure with reduced ejection fraction (HFrEF) may be observed as well. 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 a good representation of diabetic cardiomyopathy. There is a need to provide improved models of diabetic cardiomyopathy which can be used to better understand lipotoxic mechanisms and for drug discovery and development. Summary of the Disclosure According to a first aspect of the disclosure, there is provided a culture medium for cultivating cardiac tissue, wherein the culture medium comprises: 100-300 µM palmitate; 100-300 µM oleate, and 0.5-15 nM endothelin-1. The inventors have developed a lipotoxic culture medium that can be used to cultivate 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 that recapitulates key features of diabetic cardiomyopathy and can therefore be used to better understand lipotoxic mechanisms and can be used in drug discovery and development. It could not have been expected or anticipated from the prior art that the combination of palmitate, oleate and endothelin-1, and particularly at the concentrations recited, could be used to induce a diabetic cardiomyopathy phenotype in cardiac tissue. In addition, it could not have been expected or anticipated from the prior art that endothelin-1, when combined with palmitate and oleate, would induce a diabetic cardiomyopathy phenotype in cardiac tissue. The term “culture medium”, as used herein, refers to solutions that contain factors and nutrients including, for example, growth factors, energy sources, amino acids, and organic and inorganic salts, which are used for the maintenance and growth of cells in ex vivo or in vitro culture. Culture media are often buffered to an approximately neutral pH (e.g., a pH from about pH 6.6 to about pH 7.8) and can be supplemented with one or more antibiotics to prevent the growth of a bacterial and / or fungal contaminants. Any suitable basal culture medium may be used, for example, 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 growth medium, cardiomyocyte maintenance medium. The term “lipotoxic”, as used herein, is intended to mean that the culture medium causes the accumulation of lipids and lipid intermediates in the cultured cardiac tissue which results in deleterious effects such as cellular dysfunction (e.g., manifesting as increased tissue relaxation time) and / or cell death. The terms “cultivating” or “culturing” refer to the in vitro or ex vivo incubation, maintenance, growth, differentiation or maturation of cells or tissues on or in culture media to obtain a desired phenotype, structure and / or functionality. The term “palmitate”, as used herein, is intended to cover the salts and esters of palmitic acid, including, for example, sodium palmitate and calcium palmitate. In some embodiments, the palmitate is complexed with other proteins, such as Bovine Serum Albumin (BSA). In some embodiments, the palmitate is palmitic acid. The term “oleate”, as used herein, is intended to cover the salts and esters of oleic acid, including, for example, sodium oleate and calcium oleate. In some embodiments, the oleate is complexed with other proteins, such as Bovine Serum Albumin (BSA). In some embodiments, the oleate is oleic acid. Oleic acid may also be referred to as cis-9- octadecenoic acid. “Endothelin-1” is a vasoconstrictor peptide. In some embodiments, the endothelin-1 is human endothelin-1. In some embodiments, the endothelin-1 is preproendothelin-1 or proendothelin-1. The term “cardiac tissue”, as used herein, is intended to cover ex vivo cardiac tissue and in vitro engineered tissues, including both 2D and 3D cardiac cell cultures. The cardiac tissue may be healthy or diseased and / or modified in some manner (e.g., by a test agent, therapeutic agent, genetic modification etc). In some embodiments, the culture medium is for cultivating a cardiac tissue generated using the system 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 cultivating a cardiac tissue (e.g., cardiac organoid) defined in WO 2016 / 183143 Al. 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 iPSC). In some embodiments, the cardiac tissue is a human cardiac tissue. In some embodiments, the cardiac tissue comprises cardiomyocytes that have been genetically modified to give a desired phenotype. In some embodiments, in addition to comprising a population of cardiomyocytes, the cardiac tissue comprises a population of cardiac fibroblasts. The proportion of cardiomyocytes to cardiac fibroblasts may be between about 1:3 and 15:1. The proportion of cardiomyocytes to cardiac fibroblasts may be between about 1:1 and 10:1. In some embodiments, the proportion of cardiomyocytes to cardiac fibroblasts is about 4:1. In some embodiments, the proportion of cardiomyocytes to cardiac fibroblasts is about 10:1. In some embodiments, in addition to comprising a population of cardiomyocytes (and optionally cardiac fibroblasts), the cardiac tissue comprises a population of endothelial cells. The proportion of cardiomyocytes to endothelial cells may be between about 1:10 and 4:1. The proportion of cardiomyocytes to endothelial cells may be between about 1:6 and 2:1. In some embodiments, the proportion of cardiomyocytes to cardiac fibroblasts to endothelial cells is 1:3:6, 2:1:1, 4:1:2, or 2:2:1. In some embodiments, the culture medium is for inducing a diabetic cardiomyopathy disease phenotype in the cultivated cardiac tissue. The term “diabetic cardiomyopathy disease phenotype”, as used herein, is intended to mean an increased relaxation time of the cardiac tissue, which is analogous to delayed relaxation of the left ventricle of the heart. In some embodiments, the culture medium comprises 120-280 µM palmitate. In some embodiments, the culture medium comprises 140-260 µM palmitate. In some embodiments, the culture medium comprises 160-240 µM palmitate. In some embodiments, the culture medium comprises 180-220 µM palmitate. In some embodiments, the culture medium comprises 190-210 µM palmitate. In some embodiments, the culture medium comprises 195-205 µM palmitate. In some embodiments, the culture medium comprises about 200 µM palmitate. In some embodiments, the culture medium comprises 200 µM palmitate. In some embodiments, the culture medium comprises 120-280 µM oleate. In some embodiments, the culture medium comprises 140-260 µM oleate. In some embodiments, the culture medium comprises 160-240 µM oleate. In some embodiments, the culture medium comprises 180-220 µM oleate. In some embodiments, the culture medium comprises 190-210 µM oleate. In some embodiments, the culture medium comprises 195-205 µM oleate. In some embodiments, the culture medium comprises about 200 µM oleate. In some embodiments, the culture medium comprises 200 µM oleate. In some embodiments, the culture medium comprises 0.5-15 nM endothelin-1. In some embodiments, the culture medium comprises 0.6-12 nM endothelin-1. In some embodiments, the culture medium comprises 0.7-10 nM endothelin-1. In some embodiments, the culture medium comprises 0.8-8 nM endothelin-1. In some embodiments, the culture medium comprises 0.9-6 nM endothelin-1. In some embodiments, the culture medium comprises 1-5 nM endothelin-1. In some embodiments, the culture medium comprises 1 nM or 5 nM endothelin-1. In some embodiments, the culture medium comprises 100-250 µM palmitate; 100- 250 µM oleate; and 0.5-15 nM endothelin-1. In some embodiments, the culture medium comprises 100-225 µM palmitate; 100-225 µM oleate; and 0.5-15 nM endothelin-1. In some embodiments, the culture medium comprises:120-280 µM palmitate; 120- 280 µM oleate; and 0.5-15 nM endothelin-1. In some embodiments, the culture medium comprises:180-220 µM palmitate; 180-220 µM oleate; and 0.5-10 nM endothelin-1. In some embodiments, the culture medium comprises: 200 µM palmitate; 200 µM oleate; and 1-5 nM endothelin-1. In some embodiments, the culture medium does not comprise a glucocorticoid, such as cortisol, cortisone, or hydrocortisone. In some embodiments, the culture medium comprises less than 0.2 µM glucocorticoid. In some embodiments, the culture medium comprises less than 0.1 µM glucocorticoid. In some embodiments, the culture medium comprises less than 0.2 µM cortisol or cortisone. In some embodiments, the culture medium comprises less than 0.1 µM cortisol or cortisone. In some embodiments, the culture medium does not comprise cortisol. In some embodiments, the culture medium further comprises StemPro™-34 serum- free base medium (e.g., supplied by Gibco™). The skilled person will appreciate that any other suitable base medium could alternatively be used. 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, serum, a growth factor, a cytokine, sodium pyruvate, a recombinant protein, an iron transport protein, a combinations thereof. In some embodiments, the culture medium further comprises one or more of: (i) GlutaMAX™ Supplement (supplied by, e.g., Gibco™), optionally 0.1-2% (v / v) or 1% (v / v) GlutaMAX; (ii) HEPES (supplied by, e.g., Gibco™), optionally 10-30 mM or 20 mM HEPES; (iii) Penicillin-streptomycin (supplied by, e.g., Gibco™), optionally 0.1-2% or 1% Penicillin-streptomycin; (iv) Transferrin solution (supplied by, e.g., Sigma-Aldrich), optionally 0.05-0.25 mg / mL or 0.15 mg / mL Transferrin solution; (v) Ascorbic Acid solution (supplied by, e.g., Sigma-Aldrich), optionally 0.1-0.4 mg / mL or 0.256 mg / mL Ascorbic Acid solution; and (vi) StemPro™-34 nutrient supplement (supplied by, e.g., Gibco™), optionally 1-4% (v / v) or 2.6% (v / v) StemPro™-34 nutrient supplement. In some embodiments, the culture medium comprises: (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; and (vi) 1-4% (v / v) StemPro™-34 nutrient supplement. In some embodiments, the culture medium comprises a cardiac tissue. According to a second aspect of the disclosure, there is provided a method of inducing a diabetic cardiomyopathy disease phenotype in cardiac tissue, wherein the method comprises cultivating cardiac tissue in the culture medium as described with respect to the first aspect of the disclosure. In some embodiments, the method comprises generating the cardiac tissue using the system defined in WO 2015 / 061907 Al, WO 2021 / 158233 Al, and / or WO 2016 / 183143 Al. In some embodiments, the method comprises providing the cardiac tissue (e.g., cardiac organoid) defined in WO 2016 / 183143 Al. In some embodiments, the 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, the cardiac tissue comprises T-tubules. In some embodiments, the cardiac tissue exhibits a positive force-frequency relationship. In some embodiments, the force is about 0.25 to about 2 mN / mm2at a frequency of about 0 to 6 Hz. The cardiac tissue may be cultivated in the culture medium for any suitable length of time. In some embodiments, the cardiac tissue is cultivated in the culture medium for 3-100 days. In some embodiments, the cardiac tissue is cultivated in the culture medium for 4-90 days. In some embodiments, the cardiac tissue is cultivated in the culture medium for 5-80 days. In some embodiments, the cardiac tissue is cultivated in the culture medium for 6-70 days. In some embodiments, the cardiac tissue is cultivated in the culture medium for 7-60 days. In some embodiments, the cardiac tissue is cultivated in the culture medium for 9-50 days. In some embodiments, the cardiac tissue is cultivated in the culture medium for 14-45 days. In some embodiments, the cardiac tissue is cultivated in the culture medium for 18-40 days. In some embodiments, the cardiac tissue is cultivated in the culture medium for 21-35 days. In some embodiments, the cardiac tissue is cultivated in the culture medium for about 7 days. In some embodiments, the cardiac tissue is cultivated in the culture medium for about 14 days. In some embodiments, the cardiac tissue is cultivated in the culture medium for about 21 days. In some embodiments, the cardiac tissue is cultivated in the culture medium for about 28 days. In some embodiments, the cardiac tissue is cultivated in the culture medium for about 35 days. The skilled person will appreciate that the culture medium may be replenished / changed at intervals according to standard cell culture techniques. In some embodiments, the method comprises a step of cultivating cells in a growth medium to expand the cells. The cells may be ex vivo primary cardiomyocytes, cell line cells or stem cells (e.g., iPSC, such as human iPSC). The growth medium may be any suitable medium that promotes expansion of the cells. Exemplary growth media are mTeSRl (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 cultivation of the cardiac tissue in the culture medium as defined with respect to the first aspect of the disclosure. The cells may be cultivated in the growth medium for any suitable length of time. In some embodiments, the cells are cultivated in the growth medium for 1 day -75 weeks. In some embodiments, the cells are cultivated in the growth medium for 1-24 days. In some embodiments, the cells are cultivated in the growth medium for 2-21 days. In some embodiments, the cells are cultivated in the growth medium for 3-18 days. In some embodiments, the cells are cultivated in the growth medium for 4-15 days. In some embodiments, the cells are cultivated in the growth medium for 5-12 days. In some embodiments, the cells are cultivated in the growth medium for 6-11 days. In some embodiments, the cells are cultivated in the growth medium for 7-10 days. In some embodiments, the cells are cultivated in the growth medium for 8-9 days. In some embodiments, the method comprises a step of cultivating cells in a differentiation medium to differentiate the cells into cardiomyocytes for the formation of the cardiac tissue. The cells may be cell line cells or stem cells (e.g., iPSC, such as human iPSC). In some embodiments, the method comprises a step of cultivating pluripotent stem cells in a differentiation medium to provide cardiomyocytes. In these embodiments, the differentiation step occurs prior to the cultivation of the cardiac tissue in the medium as defined with respect to the first aspect of the disclosure. Typically, the differentiation step occurs after the cultivation of cells in a growth medium. As an example, the stem cells may be embryonic stem cells (ESC), fetal stem cells (FSC), and / or adult (or somatic) stem cells (SSC). The stem cells, in terms of potency potential, can be totipotent (also referred to as omnipotent) (stem cells that can differentiate into embryonic and extra-embryonic cell types), pluripotent stem cells (stem cells that can differentiate into nearly all cells), multipotent stem cells (stem cells that can differentiate into a number of cell types), oligopotent stem cells (stem cells that can differentiate into only a few cell types), or unipotent cells (stem cells that can produce only one cell type). Stem cells can be obtained commercially, or obtained / isolated directly from patients, or from any other suitable source. In some embodiments, the stem cells are induced pluripotent stem cells (iPSC). In some embodiments, the pluripotent stem cells are human iPSC. In some embodiments, the pluripotent stem cells are autologous. The differentiation medium may be any suitable medium that results in the differentiation of the stem cells into the cardiac lineage. In some embodiments, the differentiation medium comprises 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, the differentiation medium comprises Gsk3β inhibitor and Wnt inhibitor. Exemplary differentiation media are described in WO 2016 / 183143 A1. The cells may be cultivated in the differentiation medium for any suitable length of time. In some embodiments, the cells are cultivated in the differentiation medium for 1- 30 days. In some embodiments, the cells are cultivated in the differentiation medium for 2-27 days. In some embodiments, the cells are cultivated in the differentiation medium for 3-24 days. In some embodiments, the cells are cultivated in the differentiation medium for 4-21 days. In some embodiments, the cells are cultivated in the differentiation medium for 5-18 days. In some embodiments, the cells are cultivated in the differentiation medium for 6-15 days. In some embodiments, the cells are cultivated in the differentiation medium for 7-12 days. In some embodiments, the cells are cultivated in the differentiation medium for 8-10 days. Typically, the cells are cultivated in the differentiation medium for 21-24 days. Typically, a growth step and / or a differentiation step occurs prior to the cultivation step in the culture medium according to the first aspect of the disclosure. Typically, any growth step occurs prior to any differentiation step. In some embodiments, the method comprises a step of encapsulating the cells in a hydrogel. This encapsulation step may occur after 10-30 days of growing the cells in the growth and / or differentiation medium. This encapsulation step may occur after 15-25 days of growing the cells in the growth and / or differentiation medium. Typically, the encapsulation step occurs after about 20 days of growing the cells in the growth and / or differentiation medium. As an example, the cells may be encapsulated in a hydrogel as described in WO 2016 / 183143 Al. As used herein, the term “hydrogel” refers to a physically or chemically cross-linked polymer network that is able to absorb large amounts of water and is a common material for forming tissue engineering scaffolds. They can be classified into different categories depending on various parameters including the preparation method, the charge, and the mechanical and structural characteristics. Reference can be made to 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 polyvinyl alcohol, sodium polyacrylate, acrylate polymers and copolymers with an abundance of hydrophilic groups, among other materials. Natural hydrogel materials include agarose, methylcellulose, hyaluronan, and other naturally derived polymers. In some embodiments, the method further comprises a step of electromechanically conditioning the cells. A suitable conditioning regimen can be selected by one skilled in the art. As an example, the cells may be electromechanically conditioned as described in WO 2016 / 183143 Al. In some embodiments, the method further comprises a step of electromechanically conditioning the cardiomyocytes by exposing the cardiomyocytes to electromechanical stimuli that increases in intensity over a period of time, such that a cardiac tissue having molecular, structural and functional properties that mimic native adult cardiac tissue is formed. This provides the advantage of inducing maturation of the cells. Typically, the electromechanical conditioning step is performed prior to the cultivation step in the culture medium according to the first aspect of the disclosure. In alternative embodiments, the electromechanical conditioning step is performed simultaneously with the cultivation step in the culture medium according to the first aspect of the disclosure. In some embodiments, the period of time is 1-6 weeks. In some embodiments, the period of time is 2-5 weeks. In some embodiments, the period of time is 3-4 weeks. In some embodiments, the method further comprises: measuring one or more parameters of contractility of the cardiac tissue. In some embodiments, the one of more parameters of contractility is selected from the group consisting of relaxation time, twitch amplitude, contraction time, and combinations thereof. The 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). The twitch amplitude is the total force a tissue exerts during contraction (peak amplitude). The contraction time is the time taken for a 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, which are incorporated herein by reference). In some embodiments, the one or more parameters are measured before the cultivation step, during the cultivation step, and / or after the cultivation step. In some embodiments, the method further comprises: characterising the tissue morphology of the cardiac tissue. In some embodiments, the tissue morphology is characterised by microscopy with or without tissue staining. In some embodiments, the tissue morphology is characterised by microscopy of tissues stained with a lipid stain (e.g., HCS LipidTOX™ Deep Red Neutral Lipid Stain (Thermo Fisher Scientific, Cat. #H34477)). In some embodiments the tissue stain is anti-α-Actinin (Sarcomeric) antibody (i.e., to stain sarcomeres) or anti- TOM20 antibody (i.e., to stain mitochondria). In some embodiments, the tissue morphology is characterised before the cultivation step, during the cultivation step, and / or after the cultivation step. In some embodiments, the diabetic cardiomyopathy disease phenotype is induced in a cardiac tissue, and the method further comprises characterising one or more endpoints of the cardiac tissue selected from the group consisting of: lipidomics, phosphorylated Akt Western Blotting, RNA sequencing, proteomics, intracellular calcium transients analysis, and combinations thereof. In some embodiments, the one or more endpoints are characterised before the cultivation step, during the cultivation step, and / or after the cultivation step. In some embodiments, the method further comprises the step of incubating the disease phenotype cardiac tissue with a test agent. A “test agent” is any substance that is evaluated for its ability to diagnose, cure, mitigate, treat, prevent, modify or promote a disease in a subject. A test agent in an embodiment can be a "drug" as that term is defined under the Food Drug and Cosmetic Act, Section 321(g)(1). Test agents include, but are not limited to, chemical compounds, biologic agents, proteins, peptides, antibodies, nucleic acids, lipids, polysaccharides, supplements, diagnostic agents and immune modulators and may also be referred to as “pharmacologic agents.” Any suitable test agent may be tested, including opioid analgesics, anti-inflammatory drugs such as antihistamines and non-steroidal anti-inflammatory drugs (NSAIDs), diuretics such as carbonic anhydrase inhibitors, loop diuretics, high-ceiling diuretics, thiazide and thiazide- like agents, and potassium-sparing diuretics, agents that impinge on the cardiovascular system such as angiotensin converting enzyme inhibitors, cardiac drugs such as organic nitrates, calcium channel blockers, sympatholytic agents, vasodilators, beta-adrenergic receptor agonists and antagonists, alpha-adrenergic receptor agonists and antagonists, cardiac glycosides, anti-arrhythmic drugs, agents that affect hyperlipoproteinemias such as 3- hydroxymethylglutaryl-coenzyme A (HMG-CoA) inhibitors, anti-neoplastic agents such as alkylating agents, antimetabolites, natural products, antibiotics, and other drugs, immunomodulators, antidiabetic agents, and anti-microbial agents such as antibacterial agents, antiviral agents, antifungal agents, antiprotozoal agents, and antihelminthic agents. In general, a test agent can be incubated with the disease phenotype cardiac tissue in a dosage range estimated to cause an effect and for a duration sufficient to produce an effect (e.g., metabolic effects or effects indicating toxicity or efficacy). The incubation time can range between about 1 hour to 24 hours, or can be extended as necessary for several days or even weeks. The incubation conditions typically involve standard culture conditions known in the art, including culture temperatures of about 37 degrees Celsius. The disease phenotype cardiac tissue can be used to determine the range of effective dosimetry of a test agent. The effect of increasing concentrations of the test agent (i.e., dose) on the cardiac tissue can be monitored to detect efficacy. Various doses of individual test agents and combinations of test agents may be screened in panels comprised of cardiac tissues having diverse genetic backgrounds (e.g., derived from different donors and / or comprising genetically modified cells) to determine the pharmacogenetic efficacy profile of the test agents. For example, multiple doses of, or combinations with, test agents will be screened for efficacy, or the lack thereof, specific to one or more genetic backgrounds. According to a third aspect of the disclosure, there is provided diseased cardiac tissue produced by the method according to the second aspect of the disclosure, wherein the diseased cardiac tissue displays a diabetic cardiomyopathy disease phenotype. In some embodiments, the diseased cardiac tissue is directly produced by the method described herein. According to a fourth aspect of the disclosure, there is provided a diseased cardiac tissue comprising a population of cardiomyocytes in the culture medium according to the first aspect of the disclosure. In some embodiments, the cardiac tissue is generated using the system defined in WO 2015 / 061907 Al, WO 2021 / 158233 Al, and / or WO 2016 / 183143 Al. 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 opposing (which can be formed from a single element or separate elements) and function to form anchor points for the cardiac tissue formed therebetween. The cardiac tissue is not limited to having two scaffold elements, but may include 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 so long as there is the ability to form a 3D tissue that forms around each of the scaffold elements and becomes joined therebetween such that the cardiac tissue becomes disposed between the scaffold elements. In some embodiments, the cardiac tissue comprises two scaffold elements disposed at or near the opposing ends of the longitudinal axis of the cardiac tissue. In some embodiments, the scaffold elements are deflectable, deformable, bendable, or the like, which are further configured to allow the measurement of contractile forces exerted by the cardiac tissue on the scaffold elements. The shape, thickness, length, orientation, and surface topographical properties of the scaffold elements can vary any number of suitable ways so long as the scaffold elements are capable of deforming, bending, or otherwise changing shape in response to the contractile action or activity of the cardiac tissue connected therebetween, and that such deforming, bending, or otherwise shape changing can be reliably measured. In some embodiments, the scaffold elements have an elasticity from about 20 kPa to 0.5 MPa. The scaffold elements may be made from any suitable material, including, for example, poly(dimethysiloxane) (PDMS), poly(methylmethacrylate) (PMMA), polystyrene. The scaffold elements may be made of a biodegradable material. Other suitable materials may include poly(glycerol sebacate), POMaC without citric acid, poly(lactic acid), poly(glycolic acid), poly(e-caprolactone), various polyurethanes as well as co-polymers thereof, silk, microstructured, nanofabricated materials, and / or materials doped with nanostructures such as nanorods or quantum dots, among others. Optionally in certain embodiments, the scaffold element material can be perfusable to allow exchange and / or passage of water and molecules, including proteins, drugs, nutrients, and metabolic waste materials. In certain other embodiments, perfusability may be implemented through the formation of pores in the scaffold element material. In still other embodiments, the scaffold elements may be fabricated by any suitable means, including microfabrication, soft lithography processes (including, but not limited to step- and-flash imprint lithography (STIL), 3D printing (i.e., additive manufacturing), hot embossing, extrusion, injection molding, phase-shifting edge lithography, and nanoskiving. In some embodiments, the 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, the cardiac tissue exhibits a positive force-frequency relationship. In some embodiments, the force is about 0.25 to about 2 mN / mm2at a frequency of about 0 to 6 Hz. According to a fifth aspect of the disclosure, there is provided a kit comprising the culture medium according to the first aspect of the disclosure and a bioreactor for cultivation of a cardiac tissue, wherein the bioreactor comprises a plurality of wells, wherein each well comprises a chamber configured for growing a cardiac tissue therein and one or more deformable scaffold elements affixed to each chamber. In some embodiments, the bioreactor is as described in WO 2015 / 061907 Al. In some embodiments, the bioreactor is a multiwell plate. In some embodiments, the bioreactor is a multiwell plate with 12 wells, 96 wells, 384 wells or 1536 wells. In some embodiments, the bioreactor is comprised 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), polyglycolide, polylactide, polyhydroxobutyrate, polyhydroxyalcanoic acids, chitosan, hyaluronic acid, hydrogels, poly(2-hydroxyethyl- methacrylate), poly(ethylene glycol), poly(L-lactide) (PLA), or any combination thereof. In some embodiments, the polymer is poly(dimethysiloxane) (PDMS), poly(methylmethacrylate) (PMMA), polystyrene, poly(glycerol sebacate), POMaC without citric acid, poly(e-caprolactone), polyurethane, silk, or nanofabricated materials, or a co-polymer or blended polymer thereof. In some embodiments, the polymer is doped with a nanostructure. In some embodiments, the deformable scaffold elements are comprised of metal, silk, or a polymer. In some embodiments, the deformable scaffold elements are comprised of intestinal material, monocryl, polyglycolide, prolene, polyglactin, polydioxanone, polypropylene, nylon, or polyester. In some embodiments, the chamber is configured to be seeded by cardiomyocytes. In some embodiments, the deformable scaffold elements are affixed to each chamber are in a substantially perpendicular orientation, a substantially parallel orientation, or a substantially diagonal orientation relative to the orientation of the longitudinal axis of the chamber. In some embodiments, the deformable scaffold elements are configured to become embedded or partially embedded by the cardiac tissue upon the growth of the cardiac tissue. In some embodiments, the deformable scaffold elements are configured to be encapsulated or partially encapsulated by the cardiac tissue and attached thereto such that the cardiac tissue moves in conjunction with the movement of the deformable scaffold elements. In some embodiments, the bioreactor further comprises a pair of electrodes configured to create an electrical current through the growth chamber of the bioreactor. According to a sixth aspect of the disclosure, there is provided a method for evaluating the safety and / or efficacy of a test agent on a cardiac tissue, comprising: (a) cultivating a cardiac tissue in the culture medium described herein; (b) contacting the cardiac tissue with a test agent; (c) measuring the effect on one or more physiological parameters indicative of safety and / or efficacy; (d) comparing (c) to the same physiological parameter measured from a control cardiac tissue not exposed to the test agent, wherein a statistically significant change in the physiological parameter in (c) as compared to (d) indicates that the test agent lacks safety and / or efficacy. In some embodiments, the cardiac tissue is generated using the system defined in WO 2015 / 061907 Al, WO 2021 / 158233 Al, and / or WO 2016 / 183143 Al. Where applicable or not specifically disclaimed, any one of the embodiments described herein are contemplated to be able to combine with any other one or more embodiments, even though the embodiments are described under different aspects of the disclosure. The foregoing and other objects, features and advantages of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures. The disclosure will now be described in detail by way of example only with reference to the figures in which: Figure 1 shows a schematic of contractile force over time, identifying the twitch 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 in between peaks). Figure 2 shows changes in contractility over time of cardiac tissues treated with different media. Top panel shows relative change data: Δ twitch amplitude, Δ contraction time and Δ relaxation time for different treatment groups. Data presented as Median (circle) change relative to response with no treatment (Day 0). Bottom panel shows raw data: twitch amplitude, contraction time and relaxation time for different treatment groups. Data presented as Median (circle). Tissues were switched from I3M medium to treatment groups on day 3. The I3M + BSA-Control group received 250 µM of BSA- Control. Figure 3 shows changes in contractility over time of cardiac tissues treated with different media. Top panel shows relative change data: Δ twitch amplitude, Δ contraction time and Δ relaxation time for different treatment groups. Data presented as Median (circle) change relative to response with no treatment (Day 0). Bottom panel shows raw data: twitch amplitude, contraction time and relaxation time for different treatment groups. Data presented as Median (circle). Tissues were switched from I3M medium to treatment groups on day 2. N=3 tissues per group; shading denotes 95% confidence interval. The I3M (Control) group received 250 µM of BSA-Control. Figure 4 shows Δ twitch amplitude for various treatment groups. Tissues were switched from I3M medium to treatment groups on day 2. Data presented as median change relative to response with no treatment (Day 0) ± 95% CI. N=3 tissues per group. Figure 5 shows Δ contraction time for various treatment groups. Tissues were switched from I3M medium to treatment groups on day 2. Data presented as median change relative to response with no treatment (Day 0) ± 95% CI. N=3 tissues per group. Figure 6 shows Δ relaxation time for various treatment groups. Tissues were switched from I3M medium to treatment groups on day 2. Data presented as median change relative to response with no treatment (Day 0) ± 95% CI. N=3 tissues per group. Figure 7 shows spontaneous beat rate and excitation threshold over time for the various treatment groups. Figure 8 shows Δ relaxation time for various treatment groups. Tissues were switched from I3M medium to treatment groups on day 2. Data presented as median change relative to response with no treatment (Day 0) ± 95% CI. N=6 tissues per group. Figure 9 shows changes in contractility over time of cardiac tissues treated with different media whenET-1 was added to media on Day 0. The top panel shows relative change data: Δ twitch amplitude, Δ contraction time and Δ relaxation time for different treatment groups. Data presented as Median (circle) change relative to response with no treatment (Day 0). The bottom panel shows raw data: twitch amplitude, contraction time and relaxation time for different treatment groups. Data presented as Median (circle). Tissues were switched from I3M medium to treatment groups on day 2 and washout starts on day 30. N=3 tissues per group; shading denotes 95% CI. Figure 10 shows changes in contractility over time of cardiac tissues treated with different media whenET-1 was added to media at each media change. The top panel shows relative change data: Δ twitch amplitude, Δ contraction time and Δ relaxation time for different treatment groups. Data presented as Median (circle) change relative to response with no treatment (Day 0). The bottom panel shows raw data: twitch amplitude, contraction time and relaxation time for different treatment groups. Data presented as Median (circle). Tissues were switched from I3M medium to treatment groups on day 2 and washout starts on day 30. N=3 tissues per group; shading denotes 95% CI. Figure 11 shows images of tissues at Day 0 of treatment (top panel) and tissues at day 30 of treatment (bottom panel) for different treatment groups. Figure 12 shows images of tissues at Day 0 of treatment (top panel) and tissues at day 30 of treatment (bottom panel) for the I3M + 200µM Palm + 200µM Ol + 5nM ET-1 treatment group. Figure 13 shows 40x confocal stack images of different treatment groups with lipid drop staining (red) and nuclei staining (blue). The circled features are lipid droplets > 10 µm. Figure 14 shows changes in contractility over time of groups treated with a control defined medium (RPMI 1640 + 5 mM Glucose + 1.4 mM CaCl2+ 50 µM Palmitate + 50 µM Oleate + B27 without insulin + 1 nM insulin) compared to a group treated with a lipotoxic medium (defined medium + 150 µM Palmitate + 150 µM Oleate + 1 nM ET-1). Top panel shows relative change data: Δ twitch amplitude, Δ contraction time and Δ relaxation time for different treatment groups. Data presented as Median (circle) change relative to response with no treatment (Day 0). Bottom panel shows raw data: twitch amplitude, contraction time and relaxation time for different treatment groups. Data presented as Median (circle). N = 3 tissues in the Defined Media group; N = 2 tissues in the Defined Media + 150 µM Palm + 150 µM Ol + 1 nM ET-1 group (one tissue broke during the experiment). Shading denotes 95% confidence interval. Tissues were switched from I3M medium to defined media on Day 2. Tissues were treated with defined media for 10 days before being switched to their respective treatment groups on Day 12. Figure 15A shows measurements of the change in Passive Tension from Day 0 to Day 14 or Day 15. Bars represent Median. N=27 tissues per group across 3 independent experiments. Figure 15B shows measurements of the change in Tissue Area from Day 0 to Day 14 or Day 15. Bars represent Median. N=27 tissues per group across 3 independent experiments. Figure 16 shows a tissue ruler detecting outline of tissue to compute tissue area. Figure 17 shows measurements of the change in Excitation Threshold (ET) from Day 0 to Day 14 or Day 15. Bars represent Median. N=27 tissues per group across 3 independent experiments. Figure 18A shows APD90(action potential duration from peak to 90% repolarization) measured at the end of treatment. Bars represent Median. N=6 tissues per group across 2 independent experiments. Figure 18B shows APD90 measured from a cardiac action potential trace via MATLAB. Figure 19A shows Time to 50% Calcium Transient Decay measured at the end of treatment. Bars represent Median ± 95% Confidence Interval. N=9 tissues per group across 3 independent experiments. Figure 19B shows Time to 50% Calcium Transient Decay measured from a cardiac calcium transient trace via MATLAB. Detailed Description of the Invention Due to their unique ability to visualize and quantify contractile parameters of cardiac tissues 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, the inventors were able to measure more robust metrics of contractility such as increased Relaxation Time, which serves as an analogous manifestation of diastolic dysfunction – a feature of lipotoxic diabetic cardiomyopathy which other groups have not demonstrated in their models. The 3D in vitro lipotoxic cardiomyopathy model is the first metabolically-driven in vitro model of diastolic dysfunction. The concentration of each lipotoxic component added to a base medium – namely palmitate, oleate, and endothelin-1 – was rigorously and specifically optimized to produce the desired phenotype of lipotoxic diabetic cardiomyopathy in cardiac tissues. The following examples are provided to illustrate particular features and / or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described. Example 1 As a general overview, development of the inventors’ lipotoxic cardiomyopathy model involved culturing healthy cardiac tissues in the Biowire™ II platform and exposing the tissues to a lipotoxic medium for 28 days in order to induce a lipotoxic cardiomyopathy phenotype in the tissues. During treatment with the lipotoxic media, the inventors recorded changes in tissues contractility over time. One key parameter of tissue contractility measured was Relaxation Time (i.e., the time taken for a tissue to relax from a fully contracted state) since increased Relaxation Time in cardiac tissues is analogous to delayed relaxation of the left ventricle of the heart (Figure 1). In addition to Relaxation Time, Twitch Amplitude was also measured– the total force a tissue exerts during contraction – and Contraction Time – the time it takes for a tissue to reach the peak of its contraction from a relaxed state (Figure 1). At the end of the course of treatment, the inventors harvested the tissues to perform a more detailed model characterization to confirm that the tissues exhibited other key characteristics of lipotoxic cardiomyopathy. The inventors found that they could achieve a consistent contractile phenotype in cardiac tissues treated with their lipotoxic culture media consistent with lipotoxic diabetic cardiomyopathy. The inventors first prepared base media by adding the following components to StemPro-34™ Base Media (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 0.15mg / mL), 0.4% (v / v) 64 mg / mL Ascorbic Acid solution (prepared by dissolving 2-phospho-L-ascorbic acid trisodium salt (Sigma-Aldrich, Cat. #49752) in PBS (Sigma-Aldrich, Cat. #D8537), final concentration 0.256 mg / mL), and 2.6% (v / v) StemPro™-34 nutrient supplement (Life Technologies, Cat. #10639011). This base media is referred to as “I3M”. It will be apparent to the skilled person that other media may be suitable as the base medium. The inventors then prepared various test media by adding different components to the base media at varying concentrations as summarised in the table below. Cardiac tissue generation Cardiac tissues were generated by culturing hiPSC-derived cardiomyocytes in I3M media using the Biowire™ system and a seven week maturation protocol as described in 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 matured tissues into a chronic testing chamber, which separated the tissues into 6 wells (with 3 tissues assigned per well). The tissues were exposed to a test lipotoxic medium for 28 days. Results Preliminary experiments in which the inventors added cortisol to their lipotoxic medium demonstrated that 1 µM cortisol induced a large and sustained increase in Twitch Amplitude in Biowire™ tissues, inconsistent with the expected phenotype. Therefore, supplementation with 1 µM cortisol was excluded from all future iterations of the lipotoxic media. The inventors demonstrated that the I3M + 250µM Palmitate + 10nM Endothelin-1 group induced a decrease in Twitch Amplitude, along with increases in Contraction Time and Relaxation Time, after 14 days of treatment. The inventors also found that a reduced concentration of Endothelin-1 of 5 nM (I3M + 250µM Palmitate + 5nM Endothelin-1) had a comparable effect on tissue contractility (Figure 2). Thus, future experiments used the lower concentration of Endothelin-1 of 5 nM. However, the skilled person would recognise that higher concentrations of Endothelin-1 would also be suitable. All tissues treated with I3M + 250µM Palmitate + either 5nM or 10nM Endothelin-1 stopped beating within 24 days of the time course (Figure 2). This was likely due to toxic effects from the high concentration of palmitate added to the medium. To address this observed toxicity, the inventors used two approaches: (1) the inventors treated the tissues with a lower concentration of palmitate (100, 150, or 200µM) for 28 days and (2) the inventors started treatment with 250µM palmitate for the first 10 days, then switched to a lower palmitate concentration (100 or 150µM) afterwards. The inventors found that the I3M + 200µM Palmitate + 5nM Endothelin-1 treatment group caused a sustained increase in Relaxation Time (Figure 3). However, the tissues treated with this media, despite surviving longer than the tissues in the previous experiment, had still stopped beating by Day 30 of treatment. Therefore, the inventors sought an additional approach to combat the observed toxic effects of treatment with high concentrations of palmitate. In the next experiment, the inventors started adding Oleate (Ol) in a 1:1 ratio with Palmitate. The inventors considered that a 1:1 ratio of oleate to palmitate would help to reduce the toxicity of the palmitate without dramatically increasing the overall fatty acid content in the media, as that could potentially induce other negative effects in the tissues and prevent them from exhibiting the desired phenotype. The inventors found that the tissues treated with oleate and palmitate in a 1:1 ratio for the duration of the time course neither stopped beating nor displayed a substantial drop in Twitch Amplitude after 28 days of treatment (Figure 4). These same groups – namely, I3M + 150µM Palmitate + 150µM Oleate + 5nM Endothelin-1; I3M + 200µM Palmitate + 200µM Oleate + 5nM Endothelin-1; and I3M + 250µM Palmitate + 250µM Oleate + 5nM Endothelin-1 (for 10 days) down to I3M + 100µM Palmitate + 100µM Oleate + 5nM Endothelin-1 – increased Relaxation Time in tissues by 5%, 13%, and 15% on average, respectively, without negatively impacting other metrics of tissue health and maturity, like excitation threshold (the minimum voltage required for the tissues to respond to external stimulation) and spontaneous beating (beats initiated without electrical stimulation) (Figure 6, Figure 7). A substantial increase in either excitation threshold or spontaneous beating would indicate an extreme decline in tissue health, and in this particular experiment, the tissues that were not treated with oleate for the duration of the time course exhibited these characteristics (Figure 7). At the end of the time course, the inventors performed a washout of the lipotoxic media with standard base media for 7 days and observed either a partial or full reversal of the contractile effects in the tissues that were treated with both palmitate and oleate for the duration of the time course, showing that the lipotoxic media containing both palmitate and oleate does not cause irreversible damage to the tissues. This provides support for the notion that the lipotoxic phenotype in cardiac tissues may potentially be reversed with the addition of compounds that mechanistically inhibit lipid production. The inventors also found that treatment groups without Endothelin-1 – specifically: I3M + 200µM Palmitate + 200µM Oleate; and I3M + 250µM Palmitate + 250µM Oleate (for 10 days) down to I3M + 100µM Palmitate + 100µM Oleate – also increased Relaxation Time in tissues by 15% and 13% on average, respectively (Figure 6). These amounts are comparable to the increases achieved by the treatment groups that contained both fatty acids and Endothelin-1. Thus, the inventors hypothesized that the increase in Relaxation Time observed in tissues over the course of treatment is an effect primarily caused by the fatty acids (rather than Endothelin-1). The inventors repeated the treatment groups from the prior experiment which (1) had the greatest impact on Relaxation Time and (2) did not negatively affect excitation threshold, spontaneous beating, or twitch amplitude. Of those groups, I3M + 200µM Palmitate + 200µM Oleate + 5nM Endothelin-1 and I3M + 250µM Palmitate + 250µM Oleate + 5nM Endothelin-1 (for 10 days) down to I3M + 100µM Palmitate + 100µM Oleate + 5nM Endothelin-1 induced the largest increases in Relaxation Time by the end of treatment at 18% and 12% on average, respectively (Figure 8). This effect decayed toward baseline values in both groups by implementing a washout with standard base media at the end of the time course. For future iterations of the lipotoxic media, the inventors used concentrations of 200µM Palmitate + 200µM Oleate since these concentrations consistently increased Relaxation Time by over 10% in two independent experiments, and they also provide the added simplicity of remaining the same for the duration of the time course (i.e., the concentrations of palmitate and oleate do not have to be changed to lower concentrations after 10 days of treatment). However, the skilled person will recognise that variations in these concentrations are possible. This experiment once again proved that treatment with fatty acids alone is capable of increasing Relaxation Time in cardiac tissues. However, the increase does tend to be more pronounced when tissues receive both fatty acids and Endothelin-1. This becomes clear when the inventors compared treatment groups containing both fatty acids and Endothelin-1 to their counterparts excluding Endothelin-1. For example, in this experiment, the I3M + 200µM Palmitate + 200µM Oleate + 5nM Endothelin-1 treatment group displayed on average an 18% increase in Relaxation Time by the end of the time course, whereas the I3M + 200µM Palmitate + 200µM Oleate treatment group achieved only on average a 10% increase. Similarly, the I3M + 250µM Palmitate + 250µM Oleate + 5nM Endothelin-1 (for 10 days) down to I3M + 100µM Palmitate + 100µM Oleate + 5nM Endothelin-1 treatment group exhibited on average a 12% increase in Relaxation Time, while the I3M + 250µM Palmitate + 250µM Oleate (for 10 days) down to I3M + 100µM Palmitate + 100µM Oleate treatment group displayed only an 8% increase on average. The inventors probed the individual contributions of fatty acids and Endothelin-1 to the contractile effects observed in cardiac tissues by including a treatment group containing only Endothelin-1 (I3M + 5nM Endothelin-1) and a treatment group containing only fatty acids (I3M + 200µM Palmitate + 200µM Oleate). The inventors also aimed to determine if the Endothelin-1 receptors in the tissues were becoming desensitized over time due to repeated exposure to high concentrations of Endothelin-1, which could possibly account for the waning effect on contractile parameters over time. Therefore, the inventors included two additional treatment groups: one group consisted of a lower concentration of Endothelin-1 at 1nM (I3M + 200µM Palmitate + 200µM Oleate + 1 nM Endothelin-1), and another group consisted of adding Endothelin-1 to the tissues every ~96 hours, as opposed to every ~48 hours (I3M + 200µM Palmitate + 200µM Oleate + 5 nM Endothelin-1 (alternating)). In this experiment, the inventors found that treatment groups without Endothelin-1 (I3M + BSA Control; and I3M + 200µM Palmitate + 200µM Oleate) did not seem to impact contractility much, while treatment groups with Endothelin-1 (I3M + 5nM Endothelin-1; I3M + 200µM Palmitate + 200µM Oleate + 1nM Endothelin-1; I3M + 200µM Palmitate + 200µM Oleate + 5nM Endothelin-1; and I3M + 200µM Palmitate + 200µM Oleate + 5nM Endothelin-1 (Alternating)) caused a sustained increase in Contraction Time and an initial increase in Twitch Amplitude that subsided by the end of treatment (Figure 9). Importantly, the 5nM Endothelin-1 group did not increase Relaxation Time, leading the inventors to conclude that treatment with Endothelin-1 alone was not capable of achieving a phenotype consistent with lipotoxic cardiomyopathy. The inventors also found that the groups in which fatty acids and Endothelin-1 were added at every media change (I3M + 200µM Palmitate + 200µM Oleate + 1nM Endothelin-1; and I3M + 200µM Palmitate + 200µM Oleate + 5nM Endothelin-1) caused the greatest increases in Relaxation Time at 15% and 8% on average, respectively (Figure 9). The inventors once again confirmed that after a 7 day washout at the end of the time course the effects of all treatment groups on Contraction Time and Relaxation Time waned, while Twitch Amplitude decreased with time irrespective of the washout. Since the I3M + 200µM Palmitate + 200µM Oleate + 5nM Endothelin-1 group consistently caused an increase in Relaxation Time across three independent experiments, the inventors determined that this group and the 1nM Endothelin-1 group are particularly effective lipotoxic culture media. Since media containing I3M + 200µM Palmitate + 200µM Oleate + 1nM Endothelin-1 induced the largest increase in Relaxation Time in this experiment, this medium was used in subsequent model characterization. I3M + 200µM Palmitate + 200µM Oleate + 1nM Endothelin-1 Formulation To I3M media, the inventors added 4% (v / v) 5 mM BSA-Palmitate Saturated Fatty Acid Complex (Cayman Chemical, Cat. #29558, final concentration 200 µM), 4% (v / v) 5 mM BSA-Oleate Monounsaturated Fatty Acid Complex (Cayman Chemical, cat# 29557, final concentration 200 µM), and 0.1% (v / v) 1µM 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).Model CharacterizationBright-field Imaging In addition to measuring changes in contractility induced by the lipotoxic media, the inventors also characterized changes in tissue morphology by taking bright-field images of the tissues at the beginning and end of the time course. These images showed that tissues treated with both Palmitate and Oleate displayed rough, bumpy edges along their peripheries that were not present prior to treatment with the lipotoxic media (Figure 11). These jagged edges appeared consistently across three independent experiments in tissues that were treated with media containing 200µM Palmitate + 200µM Oleate + 5nM Endothelin-1 (Figure 12). 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, Cat. #H34477). The resulting confocal images showed a greater amount of lipid droplets bigger than 10µm present in tissues treated with both Palmitate and Oleate (Figure 13), which were the same tissues that displayed rough, bumpy edges along their peripheries in the bright-field images (Figure 11). Example 2 The inventors further demonstrated that the basal culture medium is not restricted to I3M medium by carrying out an experiment using an alternative basal culture medium. Cardiac tissues were generated in I3M media in the Biowire™ platform as described above and switched to Defined Media (RPMI 1640 + 5 mM Glucose + 1.4 mM CaCl2 + 50 µM Palmitate + 50 µM Oleate + B27 without insulin + 1 nM insulin) on Day 2. Cardiac tissues were treated with Defined Media for 10 days before being switched to their respective treatment groups: Defined Media (control group) or Lipotoxic Defined Media (Defined Media + 150 µM Palmitate + 150 µM Oleate + 1 nM ET-1). As shown in Figure 14 and Tables 1 and 2 below, treatment with Lipotoxic Defined Media increased relaxation time compared to the control group, demonstrating that the lipotoxic components are capable of inducing the disease phenotype in other basal media. Data presented as Mean ± Standard Deviation. Example 3 Control Media and Lipotoxic Culture Media I3M served as the base media for the Control and Lipotoxic Media formulations. To prepare I3M media, the inventors added the following components to StemPro-34 Base Media (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 20mM), 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 0.15mg / mL), 0.4% (v / v) 64 mg / mL Ascorbic Acid solution (prepared by dissolving 2-phospho-L-ascorbic acid trisodium salt (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). In all experiments, the Control Media consisted of I3M + 64uM BSA-control. The media was prepared by adding 8% (v / v) 0.8 mM BSA Control for Fatty Acid Complexes (Cayman Chemical Company, Cat# 29556) to I3M Media. In all experiments, the Lipotoxic Media consisted of I3M + 200µM Palmitate + 200µM Oleate + 1nM Endothelin-1. The media was prepared by adding the following components to I3M: 4% (v / v) 5mM BSA-Palmitate Saturated Fatty Acid Complex (Cayman Chemical, Cat. #29558, final concentration 200µM), 4% (v / v) 5mM BSA- Oleate Monounsaturated Fatty Acid Complex (Cayman Chemical, cat# 29557, final concentration 200µM), and 0.1% (v / v) 1µM 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 1nM). Methods Chronic Force Experiments During the chronic force experiments, tissues were treated with either Control or Lipotoxic Media for 14-15 days. A 100% media change was performed every 2-3 days for the duration of treatment. Tissues were assessed every 2-5 days in order to measure changes in contractility parameters. During the assessments, tissues were transferred from the incubator (5% CO2, 37°C) to an environmental chamber (5% CO2, 37°C) housing the objectives of the microscope. Electrical stimulation was initiated at 1Hz (2 ms pulse duration, monophasic, at 3V). The tissues were equilibrated in the chamber for 30 min prior to video acquisition. During video acquisition, the polymer wire in the Biowire™ II platform was exposed to an excitation wavelength of 405 nm, causing the wire to fluoresce. Ten second videos of the wire were captured while the tissues were stimulating at 1Hz. Once videos were acquired for all wires, the tissues were returned to the incubator to continue stimulation until the next assessment. This process was repeated until the end of treatment. Videos were analyzed using a custom MATLAB code that tracks polymer wire position to measure the amount of wire displacement in each video frame. The displacement values were converted to 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 maximal peak amplitude, and relaxation time was calculated by measuring the time from maximal peak amplitude to 90% relaxation (or 10% of the peak height). Passive tension was calculated by measuring the amount of force in the wire in between contractions (i.e. the flat line in between peaks). Tissue Area Measurements For the tissue area measurements, bright-field images of the tissues were acquired at 2X magnification during each assessment. These images were processed using a custom tissue measurement software, Tissue Ruler, which determined the outline of each tissue by contrasting the dark areas comprising the tissue against the light background of the platform (see Figure 16). This outline was then used to compute the area of the tissue. Excitation Threshold Measurements The excitation threshold of each tissue was determined by observing the tissue under 2X bright-field magnification, setting the stimulation frequency to 2Hz, and then decreasing the stimulation voltage gradually from a starting voltage of 3V until the tissue stopped contracting at a frequency of 2Hz. The minimum voltage required to keep the tissue beating at 2Hz was recorded as the excitation threshold. Action Potential Measurements Action potentials were measured by incubating the tissues with a voltage-sensitive dye, causing the cells to fluoresce based on their membrane potential. During the cardiac action potential, cell membrane potential fluctuates, causing the fluorescence of the cells to fluctuate as well. The voltage-sensitive dye used in the experiments caused the cells to fluoresce brighter at hyperpolarized membrane potentials and become dimmer at depolarized membrane potentials. After incubating the tissues in the dye, 10-second videos of the tissues were acquired at a stimulation frequency of 1Hz. A custom MATLAB program was then used to generate action potential traces based on the measured fluorescence. The same MATLAB program was also used to compute APD90– the time required to get from the peak of the action potential to 90% repolarization (see Figure 18B). Calcium Transient Measurements Calcium transients were measured by incubating the tissues with a calcium- sensitive dye, which enters into the cells and fluoresces upon binding to calcium. During a cardiac calcium transient, the calcium dye fluorescence increases as the intracellular calcium increases (during contraction), and the fluorescence decreases as the intracellular calcium decreases (during relaxation). After incubating the tissues in the dye, 7-second videos of the tissues were acquired at a stimulation frequency of 1Hz. A custom MATLAB program was then used to generate calcium transient traces based on the measured fluorescence. The same MATLAB program was also used to compute Time to 50% Calcium Transient Decay – the time required to get from the peak of the transient to 50% transient decay (see Figure 19B). Results Figure 15A displays measurements of the change in Passive Tension (i.e. tension in the polymer wire when the tissue is not actively contracting) from Day 0 to the end of treatment in Biowire™ tissues treated with either Control Media or Lipotoxic Media. Tissues treated with Lipotoxic Media demonstrated a significant increase in Passive Tension by Day 14 / 15 when compared to tissues treated with Control Media. The increase in Passive Tension in the lipotoxic tissues could be evidence of increased ECM cross- linking, fibrosis, and / or increased fibroblast contractile activity, which is consistent with the increased myocardial stiffness that is observed in patients with lipotoxic cardiomyopathy. Figure 15B displays measurements of the change in Tissue Area from Day 0 to the end of treatment in Biowire tissues treated with either Control Media or Lipotoxic Media. Tissues treated with Lipotoxic Media demonstrated a significant increase in Tissue Area by Day14 / 15 when compared to tissues treated with Control Media, which could be evidence of hypertrophy – a symptom commonly associated with lipotoxic cardiomyopathy. Figure 17 displays measurements of the change in Excitation Threshold (the minimum voltage required to generate tissue contraction at a set stimulation frequency) from Day 0 to the end of treatment in Biowire™ tissues treated with either Control Media or Lipotoxic Media. Tissues treated with Lipotoxic Media demonstrated a significant increase in Excitation Threshold by Day14 / 15 when compared to tissues treated with Control Media. Since cardiac cells rely on cell-to-cell interactions via gap junctions and adhesion complexes for effective electrical conduction, the increase in Excitation Threshold observed in the lipotoxic tissues could be indicative of disrupted cell-to-cell communication and / or declining cell health, which without wishing to be bound by theory, the inventors hypothesize could play a role in the increased Relaxation Time also observed in the lipotoxic tissues. Figure 18A displays APD90(action potential duration from peak to 90% repolarization) measured at the end of treatment in Biowire™ tissues treated with either Control Media or Lipotoxic Media. Tissues treated with Lipotoxic Media demonstrated significantly higher APD90values when compared to tissues treated with Control Media, which is consistent with the increase in Relaxation Time also observed in lipotoxic tissues. Without wishing to be bound by theory, the increase in action potential duration observed in the lipotoxic tissues could be the result of impaired ion channel function that is downstream of pathways activated by metabolic disturbances (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, L. M. Ballou, I. S. Cohen and R. Z. 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, p. 2780–2789, 2007). Figure 19A displays Time to 50% Calcium Transient Decay measured at the end of treatment in Biowire™ tissues treated with either Control Media or Lipotoxic Media. Tissues treated with Lipotoxic Media demonstrated significantly longer Time to 50% Transient Decay when compared to tissues treated with Control Media. This observation is consistent with the inventors’ hypothesis that the increase in Relaxation Time in lipotoxic tissues is caused by impaired calcium handling – specifically, longer decay in calcium transients – a known mechanism leading to diastolic dysfunction (see R. H. Ritchie and E. D. Abel, "Basic Mechanisms of Diabetic Heart Disease," Circulation Research, vol.126, no.11, p.1501–1525, 2020).

Claims

Claims 1. A culture medium for cultivating cardiac tissue, wherein the culture medium comprises: 100-300 µM palmitate; 100-300 µM oleate, and 0.5-15 nM endothelin-1.

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

3. A culture medium according to any preceding claim, wherein the culture medium comprises 180-220 µM palmitate.

4. A culture medium according to claim 3, wherein the culture medium comprises 200 µM palmitate.

5. A culture medium according to any preceding claim, wherein the culture medium comprises 180-220 µM oleate.

6. A culture medium according to claim 5, wherein the culture medium comprises 200 µM oleate.

7. A culture medium according to any preceding claim, wherein the culture medium comprises 1-5 nM endothelin-1.

8. A culture medium according to any preceding claim, wherein the culture medium comprises 1 nM or 5 nM endothelin-1.

9. A culture medium according to claim 1, wherein the culture medium comprises: 180-220 µM palmitate; 180-220 µM oleate, and 0.5-10 nM endothelin-1.

10. A culture medium according to claim 1, wherein the culture medium comprises:200 µM palmitate; 200 µM oleate, and 1-5 nM endothelin-1.

11. A culture medium according to any preceding claim, wherein the culture medium does not comprise cortisol.

12. A culture medium according to any preceding claim, further comprising StemPro-34 serum-free base medium.

13. A culture medium according to any preceding claim, further comprising one or more of: (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 nutrient supplement, optionally 1-4% (v / v) StemPro-34 nutrient supplement, and optionally 2.6% (v / v) StemPro-34 nutrient supplement.

14. A 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 nutrient supplement.

15. A method of inducing a diabetic cardiomyopathy disease phenotype in cardiac tissue, wherein the method comprises: cultivating cardiac tissue in the culture medium according to any one of claims 1-14.

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

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

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

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

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

21. A method according to any one of claims 18-20, further comprising a step of electromechanically conditioning the cardiomyocytes by exposing the cardiomyocytes to electromechanical stimuli that increases in intensity over a period of time, such that a cardiac tissue having molecular, structural and functional properties that mimic native adult cardiac tissue is formed.

22. A method according to claim 21, wherein the cardiac tissue comprises T-tubules.

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

24. A method according to any one of claims 15-23, wherein the method further comprises: measuring one or more parameters of contractility of the cardiac tissue.

25. A method according to claim 24, wherein the one of more parameters of contractility is selected from the group consisting of relaxation time, twitch amplitude, contraction time, and combinations thereof.

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

27. A method according to any one of claims 15-26, wherein the method further comprises: characterising the tissue morphology of the cardiac tissue.

28. A method according to claim 27, wherein the tissue morphology is characterised by microscopy with or without tissue staining.

29. A method according to claim 27 or claim 28, wherein the tissue morphology is characterised before the cultivation step, during the cultivation step, and / or after the cultivation step.

30. A method according to any one of claims 15-29, wherein the method further comprises: characterising one or more endpoints of the cardiac tissue selected from the group consisting of: lipidomics, phosphorylated Akt Western Blotting, RNA sequencing, proteomics, intracellular calcium transients analysis, and combinations thereof.

31. A method according to claim 30, wherein the one or more endpoints are characterised before the cultivation step, during the cultivation step, and / or after the cultivation step.

32. Diseased cardiac tissue produced by the method according to any one of 15-31, wherein the diseased cardiac tissue displays a diabetic cardiomyopathy disease phenotype.

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

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

35. A diseased cardiac tissue according to claim 33 or claim 34, wherein the cardiac tissue comprises mature ultrastructures.

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

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

38. A diseased cardiac tissue according to claim 37, wherein the force is about 0.25 to about 2 mN / mm2at a frequency of about 0 to 6 Hz.

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

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

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

42. A kit according to any one of claims 39-41, wherein the bioreactor is comprised of a polymer.

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

44. A kit according to claim 43, wherein the biodegradable polymer is polylactic acid, poly(lactic- co-glycolic) acid, or poly(caprolactone), polyglycolide, polylactide, polyhydroxobutyrate, polyhydroxyalcanoic acids, chitosan, hyaluronic acid, hydrogels, poly(2- hydroxyethyl-methacrylate), poly(ethylene glycol), poly(L-lactide) (PLA), or any combination thereof.

45. A kit according to claim 44, wherein the polymer is poly(dimethysiloxane) (PDMS), poly(methylmethacrylate) (PMMA), polystyrene, poly(glycerol sebacate), POMaC withoutcitric acid, poly(e-caprolactone), polyurethane, silk, or nanofabricated materials, or a co- polymer or blended polymer thereof.

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

47. A kit according to any one of claims 39-46, wherein the deformable scaffold elements are comprised of metal, silk, or a polymer.

48. A kit according to any one of claims 39-46, wherein the deformable scaffold elements are comprised of intestinal material, monocryl, polyglycolide, prolene, polyglactin, polydioxanone, polypropylene, nylon, or polyester.

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

50. A kit according to any one of claims 39-49, wherein the deformable scaffold elements are affixed to each chamber are in a substantially perpendicular orientation, a substantially parallel orientation, or a substantially diagonal orientation relative to the orientation of the longitudinal axis of the chamber.

51. A kit according to any one of claims 39-50, wherein the deformable scaffold elements are configured to become embedded or partially embedded by the cardiac tissue upon the growth of the cardiac tissue.

52. A kit according to any one of claims 39-51, wherein the deformable scaffold elements are configured to be encapsulated or partially encapsulated by the cardiac tissue and attached thereto such that the cardiac tissue moves in conjunction with the movement of the deformable scaffold elements.

53. A kit according to any one of claims 39-52, wherein the bioreactor further comprises a pair of electrodes configured to create an electrical current through the growth chamber of the bioreactor.

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