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WO2025006637A1 - Milieux lipotoxiques - Google Patents

Milieux lipotoxiques Download PDF

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Publication number
WO2025006637A1
WO2025006637A1 PCT/US2024/035664 US2024035664W WO2025006637A1 WO 2025006637 A1 WO2025006637 A1 WO 2025006637A1 US 2024035664 W US2024035664 W US 2024035664W WO 2025006637 A1 WO2025006637 A1 WO 2025006637A1
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WIPO (PCT)
Prior art keywords
culture medium
cardiac tissue
tissue
palmitate
endothelin
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PCT/US2024/035664
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English (en)
Inventor
Stephen SOROTA
Nicole FERIC
Mark David Rekhter
Jamie GEARHART
Svetlana MARUKIAN
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Valo Health Inc
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Valo Health Inc
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Publication of WO2025006637A1 publication Critical patent/WO2025006637A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • a culture medium for cultivating cardiac tissue wherein the culture medium comprises: 100-300 ⁇ M palmitate; 100-300 ⁇ M 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.
  • 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.
  • 3D three-dimensional
  • culture medium 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.
  • basal culture medium for example, StemProTM-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.
  • 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.
  • BSA Bovine Serum Albumin
  • the palmitate is palmitic acid.
  • oleate as used herein, is intended to cover the salts and esters of oleic acid, including, for example, sodium oleate and calcium oleate.
  • the oleate is complexed with other proteins, such as Bovine Serum Albumin (BSA).
  • BSA Bovine Serum Albumin
  • the oleate is oleic acid.
  • Oleic acid may also be referred to as cis-9- octadecenoic acid.
  • Endothelin-1 is a vasoconstrictor peptide.
  • the endothelin-1 is human endothelin-1.
  • the endothelin-1 is preproendothelin-1 or proendothelin-1.
  • 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).
  • 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.
  • the culture medium is for cultivating a cardiac tissue (e.g., cardiac organoid) defined in WO 2016/183143 Al.
  • the cardiac tissue is generated from a cell line (e.g., a human cell line).
  • 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.
  • the proportion of cardiomyocytes to cardiac fibroblasts to endothelial cells is 1:3:6, 2:1:1, 4:1:2, or 2:2:1.
  • the culture medium is for inducing a diabetic cardiomyopathy disease phenotype in the cultivated cardiac tissue.
  • diabetes cardiomyopathy disease phenotype 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.
  • the culture medium comprises 120-280 ⁇ M palmitate.
  • the culture medium comprises 140-260 ⁇ M palmitate.
  • the culture medium comprises 160-240 ⁇ M palmitate.
  • the culture medium comprises 180-220 ⁇ M palmitate. In some embodiments, the culture medium comprises 190-210 ⁇ M palmitate. In some embodiments, the culture medium comprises 195-205 ⁇ M palmitate. In some embodiments, the culture medium comprises about 200 ⁇ M palmitate. In some embodiments, the culture medium comprises 200 ⁇ M palmitate. In some embodiments, the culture medium comprises 120-280 ⁇ M oleate. In some embodiments, the culture medium comprises 140-260 ⁇ M oleate. In some embodiments, the culture medium comprises 160-240 ⁇ M oleate. In some embodiments, the culture medium comprises 180-220 ⁇ M oleate. In some embodiments, the culture medium comprises 190-210 ⁇ M oleate.
  • the culture medium comprises 195-205 ⁇ M oleate. In some embodiments, the culture medium comprises about 200 ⁇ M oleate. In some embodiments, the culture medium comprises 200 ⁇ M 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.
  • the culture medium comprises 1 nM or 5 nM endothelin-1. In some embodiments, the culture medium comprises 100-250 ⁇ M palmitate; 100- 250 ⁇ M oleate; and 0.5-15 nM endothelin-1. In some embodiments, the culture medium comprises 100-225 ⁇ M palmitate; 100-225 ⁇ M oleate; and 0.5-15 nM endothelin-1. In some embodiments, the culture medium comprises:120-280 ⁇ M palmitate; 120- 280 ⁇ M oleate; and 0.5-15 nM endothelin-1.
  • the culture medium comprises:180-220 ⁇ M palmitate; 180-220 ⁇ M oleate; and 0.5-10 nM endothelin-1. In some embodiments, the culture medium comprises: 200 ⁇ M palmitate; 200 ⁇ M 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 ⁇ M glucocorticoid. In some embodiments, the culture medium comprises less than 0.1 ⁇ M glucocorticoid.
  • the culture medium comprises less than 0.2 ⁇ M cortisol or cortisone. In some embodiments, the culture medium comprises less than 0.1 ⁇ M cortisol or cortisone. In some embodiments, the culture medium does not comprise cortisol. In some embodiments, the culture medium further comprises StemProTM-34 serum- free base medium (e.g., supplied by GibcoTM). The skilled person will appreciate that any other suitable base medium could alternatively be used.
  • StemProTM-34 serum- free base medium e.g., supplied by GibcoTM. The skilled person will appreciate that any other suitable base medium could alternatively be used.
  • 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.
  • a sugar e.g., glucose
  • an amino acid e.g., L-glutamine
  • an antibiotic e.g., a vitamin, serum, a growth factor, a cytokine, sodium pyruvate, a recombinant protein, an iron transport protein, a combinations thereof.
  • the culture medium further comprises one or more of: (i) GlutaMAXTM Supplement (supplied by, e.g., GibcoTM), optionally 0.1-2% (v/v) or 1% (v/v) GlutaMAX; (ii) HEPES (supplied by, e.g., GibcoTM), optionally 10-30 mM or 20 mM HEPES; (iii) Penicillin-streptomycin (supplied by, e.g., GibcoTM), 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) StemProTM-34
  • the culture medium comprises: (i) 0.1-2% (v/v) GlutaMAXTM; (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) StemProTM-34 nutrient supplement.
  • 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.
  • 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.
  • the method comprises providing the cardiac tissue (e.g., cardiac organoid) defined in WO 2016/183143 Al.
  • the cardiac tissue comprises mature ultrastructures.
  • the ultrastructures are selected from the group consisting of: sarcomeres, mitochondria, T-tubules, sarcoplasmic reticulum, and combinations thereof.
  • the cardiac tissue comprises T-tubules.
  • the cardiac tissue exhibits a positive force-frequency relationship.
  • the force is about 0.25 to about 2 mN/mm 2 at 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.
  • 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 culture medium may be replenished/changed at intervals according to standard cell culture techniques.
  • 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 8TM Medium (Gibco), and StemFlexTM Medium (Gibco).
  • 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.
  • 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).
  • the method comprises a step of cultivating pluripotent stem cells in a differentiation medium to provide cardiomyocytes.
  • 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.
  • the differentiation step occurs after the cultivation of cells in a growth medium.
  • 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.
  • the stem cells are induced pluripotent stem cells (iPSC).
  • the pluripotent stem cells are human iPSC.
  • 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.
  • 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).
  • the differentiation medium comprises Gsk3 ⁇ 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.
  • 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.
  • any growth step occurs prior to any differentiation step.
  • 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.
  • the cells may be encapsulated in a hydrogel as described in WO 2016/183143 Al.
  • 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.
  • the method further comprises a step of electromechanically conditioning the cells.
  • a suitable conditioning regimen can be selected by one skilled in the art.
  • the cells may be electromechanically conditioned as described in WO 2016/183143 Al.
  • 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.
  • 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.
  • 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.
  • 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) .
  • the one or more parameters are measured before the cultivation step, during the cultivation step, and/or after the cultivation step.
  • the method further comprises: characterising the tissue morphology of the cardiac tissue.
  • the tissue morphology is characterised by microscopy with or without tissue staining.
  • the tissue morphology is characterised by microscopy of tissues stained with a lipid stain (e.g., HCS LipidTOXTM Deep Red Neutral Lipid Stain (Thermo Fisher Scientific, Cat. #H34477)).
  • the tissue stain is anti- ⁇ -Actinin (Sarcomeric) antibody (i.e., to stain sarcomeres) or anti- TOM20 antibody (i.e., to stain mitochondria).
  • the tissue morphology is characterised before the cultivation step, during the cultivation step, and/or after the cultivation step.
  • 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.
  • the one or more endpoints are characterised before the cultivation step, during the cultivation step, and/or after the cultivation step.
  • the method further comprises the step of incubating the disease phenotype cardiac tissue with a test agent.
  • 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
  • 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.
  • 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.
  • a diseased cardiac tissue comprising a population of cardiomyocytes in the culture medium according to the first aspect of the disclosure.
  • the cardiac tissue is generated using the system defined in WO 2015/061907 Al, WO 2021/158233 Al, and/or WO 2016/183143 Al.
  • the cardiac tissue further comprises two or more scaffold elements disposed within the cardiac tissue.
  • 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.
  • the cardiac tissue comprises two scaffold elements disposed at or near the opposing ends of the longitudinal axis of the cardiac tissue.
  • 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.
  • 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.
  • 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.
  • 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.
  • the cardiac tissue comprises mature ultrastructures.
  • the ultrastructures are selected from the group consisting of: sarcomeres, mitochondria, T-tubules, sarcoplasmic reticulum, and combinations thereof.
  • the cardiac tissue exhibits a positive force-frequency relationship.
  • the force is about 0.25 to about 2 mN/mm 2 at a frequency of about 0 to 6 Hz.
  • 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.
  • the bioreactor is as described in WO 2015/061907 Al.
  • the bioreactor is a multiwell plate.
  • the bioreactor is a multiwell plate with 12 wells, 96 wells, 384 wells or 1536 wells.
  • the bioreactor is comprised of a polymer.
  • the polymer is a biodegradable polymer.
  • 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.
  • 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.
  • the polymer is doped with a nanostructure.
  • the deformable scaffold elements are comprised of metal, silk, or a polymer.
  • the deformable scaffold elements are comprised of intestinal material, monocryl, polyglycolide, prolene, polyglactin, polydioxanone, polypropylene, nylon, or polyester.
  • the chamber is configured to be seeded by cardiomyocytes.
  • 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.
  • the deformable scaffold elements are configured to become embedded or partially embedded by the cardiac tissue upon the growth of the cardiac tissue.
  • 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.
  • the bioreactor further comprises a pair of electrodes configured to create an electrical current through the growth chamber of the bioreactor.
  • 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.
  • the cardiac tissue is generated using the system defined in WO 2015/061907 Al, WO 2021/158233 Al, and/or WO 2016/183143 Al.
  • 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.
  • 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: ⁇ twitch amplitude, ⁇ contraction time and ⁇ 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 ⁇ M of BSA- Control.
  • Figure 3 shows changes in contractility over time of cardiac tissues treated with different media.
  • Top panel shows relative change data: ⁇ twitch amplitude, ⁇ contraction time and ⁇ 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 ⁇ M of BSA-Control.
  • Figure 7 shows spontaneous beat rate and excitation threshold over time for the various treatment groups.
  • 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: ⁇ twitch amplitude, ⁇ contraction time and ⁇ 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: ⁇ twitch amplitude, ⁇ contraction time and ⁇ 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 + 200 ⁇ M Palm + 200 ⁇ M 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 ⁇ m.
  • Figure 14 shows changes in contractility over time of groups treated with a control defined medium (RPMI 1640 + 5 mM Glucose + 1.4 mM CaCl 2 + 50 ⁇ M Palmitate + 50 ⁇ M Oleate + B27 without insulin + 1 nM insulin) compared to a group treated with a lipotoxic medium (defined medium + 150 ⁇ M Palmitate + 150 ⁇ M Oleate + 1 nM ET-1).
  • Top panel shows relative change data: ⁇ twitch amplitude, ⁇ contraction time and ⁇ 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.
  • Figure 16 shows a tissue ruler detecting outline of tissue to compute tissue area.
  • Figure 18B shows APD90 measured from a cardiac action potential trace via MATLAB.
  • Figure 19B shows Time to 50% Calcium Transient Decay measured from a cardiac calcium transient trace via MATLAB.
  • WO 2015/061907 Al, WO 2021/158233 Al and/or WO 2016/183143 Al also known as the BiowireTM 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.
  • Example 1 As a general overview, development of the inventors’ lipotoxic cardiomyopathy model involved culturing healthy cardiac tissues in the BiowireTM 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.
  • Relaxation Time i.e., the time taken for a tissue to relax from a fully contracted state
  • Relaxation Time i.e., the time taken for a tissue to relax from a fully contracted state
  • Twitch Amplitude was also measured– the total force a tissue exerts during contraction –
  • Contraction Time the time it takes for a tissue to reach the peak of its contraction from a relaxed state ( Figure 1).
  • 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 first prepared base media by adding the following components to StemPro-34TM 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.
  • Cardiac tissue generation Cardiac tissues were generated by culturing hiPSC-derived cardiomyocytes in I3M media using the BiowireTM 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 inventors used two approaches: (1) the inventors treated the tissues with a lower concentration of palmitate (100, 150, or 200 ⁇ M) for 28 days and (2) the inventors started treatment with 250 ⁇ M palmitate for the first 10 days, then switched to a lower palmitate concentration (100 or 150 ⁇ M) afterwards.
  • the inventors found that the I3M + 200 ⁇ M Palmitate + 5nM Endothelin-1 treatment group caused a sustained increase in Relaxation Time (Figure 3).
  • 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.
  • treatment groups without Endothelin-1 – specifically: I3M + 200 ⁇ M Palmitate + 200 ⁇ M Oleate; and I3M + 250 ⁇ M Palmitate + 250 ⁇ M Oleate (for 10 days) down to I3M + 100 ⁇ M Palmitate + 100 ⁇ M Oleate – also increased Relaxation Time in tissues by 15% and 13% on average, respectively (Figure 6).
  • Figure 6 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 I3M + 250 ⁇ M Palmitate + 250 ⁇ M Oleate + 5nM Endothelin-1 (for 10 days) down to I3M + 100 ⁇ M Palmitate + 100 ⁇ M Oleate + 5nM Endothelin-1 treatment group exhibited on average a 12% increase in Relaxation Time
  • the I3M + 250 ⁇ M Palmitate + 250 ⁇ M Oleate (for 10 days) down to I3M + 100 ⁇ M Palmitate + 100 ⁇ M 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 + 200 ⁇ M Palmitate + 200 ⁇ M 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.
  • 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 + 200 ⁇ M Palmitate + 200 ⁇ M Oleate + 1nM Endothelin-1; and I3M + 200 ⁇ M Palmitate + 200 ⁇ M Oleate + 5nM Endothelin-1) caused the greatest increases in Relaxation Time at 15% and 8% on average, respectively (Figure 9).
  • the inventors added 4% (v/v) 5 mM BSA-Palmitate Saturated Fatty Acid Complex (Cayman Chemical, Cat. #29558, final concentration 200 ⁇ M), 4% (v/v) 5 mM BSA-Oleate Monounsaturated Fatty Acid Complex (Cayman Chemical, cat# 29557, final concentration 200 ⁇ M), and 0.1% (v/v) 1 ⁇ M Endothelin-1 (prepared by dissolving Endothelin-1 powder (Sigma-Aldrich, Cat. #E7764-10UG) in Cell Culture Grade Water (Corning, Cat.
  • Model Characterization Bright-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 200 ⁇ M Palmitate + 200 ⁇ M 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 LipidTOXTM Deep Red Neutral Lipid Stain (Thermo Fisher Scientific, Cat. #H34477). The resulting confocal images showed a greater amount of lipid droplets bigger than 10 ⁇ m 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 BiowireTM platform as described above and switched to Defined Media (RPMI 1640 + 5 mM Glucose + 1.4 mM CaCl2 + 50 ⁇ M Palmitate + 50 ⁇ M 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 ⁇ M Palmitate + 150 ⁇ M Oleate + 1 nM ET-1).
  • Control Media and Lipotoxic Culture Media I3M served as the base media for the Control and Lipotoxic Media formulations.
  • 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.
  • 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.
  • the Lipotoxic Media consisted of I3M + 200 ⁇ M Palmitate + 200 ⁇ M 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.
  • tissues were transferred from the incubator (5% CO 2 , 37°C) to an environmental chamber (5% CO 2 , 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 BiowireTM 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.
  • 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.
  • 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 BiowireTM 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 BiowireTM 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.
  • Excitation Threshold the minimum voltage required to generate tissue contraction at a set stimulation frequency
  • FIG. 18A displays APD 90 (action potential duration from peak to 90% repolarization) measured at the end of treatment in BiowireTM tissues treated with either Control Media or Lipotoxic Media. Tissues treated with Lipotoxic Media demonstrated significantly higher APD 90 values when compared to tissues treated with Control Media, which is consistent with the increase in Relaxation Time also observed in lipotoxic tissues.
  • 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.
  • metabolic disturbances e.g. hyperglycemia and insulin resistance
  • Figure 19A displays Time to 50% Calcium Transient Decay measured at the end of treatment in BiowireTM 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.

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Abstract

La présente invention concerne des milieux de culture pour la culture de tissus cardiaques, des procédés d'induction d'un phénotype de cardiomyopathie diabétique dans des tissus cardiaques, des tissus cardiaques malades et des kits. Un milieu de culture donné à titre d'exemple pour la culture du tissu cardiaque comprend 100 à 300 pM de palmitate, 100 à 300 pM d'oléate, et 0,5 à 15 nM d'endothéline-1.
PCT/US2024/035664 2023-06-27 2024-06-26 Milieux lipotoxiques Pending WO2025006637A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015061907A1 (fr) 2013-10-30 2015-05-07 Miklas Jason Dispositifs et procédés de culture de tissu tridimensionnel
WO2016183143A1 (fr) 2015-05-11 2016-11-17 The Trustees Of Columbia University Inthe City Of New York Tissu cardiaque humain de type adulte issu de l'ingénierie tissulaire
WO2021158233A1 (fr) 2020-02-07 2021-08-12 Tara Biosystems, Inc. Plate-forme microphysiologique à électrodes intégrées pour la culture de tissus en 3d

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015061907A1 (fr) 2013-10-30 2015-05-07 Miklas Jason Dispositifs et procédés de culture de tissu tridimensionnel
WO2016183143A1 (fr) 2015-05-11 2016-11-17 The Trustees Of Columbia University Inthe City Of New York Tissu cardiaque humain de type adulte issu de l'ingénierie tissulaire
WO2021158233A1 (fr) 2020-02-07 2021-08-12 Tara Biosystems, Inc. Plate-forme microphysiologique à électrodes intégrées pour la culture de tissus en 3d

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
FERIC ET AL.: "Engineered Cardiac Tissues Generated in the Biowire II: A Platform for Human-Based Drug Discovery", TOXICOL SCI., vol. 172, no. 1, 1 November 2019 (2019-11-01), pages 89 - 97
FERIC NICOLE T ET AL: "Engineered Cardiac Tissues Generated in the Biowire II: A Platform for Human-Based Drug Discovery", TOXICOLOGICAL SCIENCES, vol. 172, no. 1, 6 August 2019 (2019-08-06), pages 89 - 97, XP093210711, ISSN: 1096-6080, Retrieved from the Internet <URL:http://academic.oup.com/toxsci/article-pdf/172/1/89/30267732/kfz168.pdf> DOI: 10.1093/toxsci/kfz168 *
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, 2018, pages 1960 - 1967
GRAN?LI CECILIA ET AL: "Diabetic Cardiomyopathy Modelling Using Induced Pluripotent Stem Cell Derived Cardiomyocytes: Recent Advances and Emerging Models", STEM CELL REVIEWS AND REPORTS, HUMANA PRESS INC, US, vol. 15, no. 1, 20 October 2018 (2018-10-20), pages 13 - 22, XP036689728, ISSN: 1550-8943, [retrieved on 20181020], DOI: 10.1007/S12015-018-9858-1 *
GRANÉLI ET AL.: "Diabetic Cardiomyopathy Modelling Using Induced Pluripotent Stem", STEM CELL REVIEWS AND REPORTS, vol. 15, 2019, pages 13 - 22, XP036689728, DOI: 10.1007/s12015-018-9858-1
N. OZTURKS. USLUS. OZDEMIR: "Diabetes-induced changes in cardiac voltage-gated ion channels", WORLD JOURNAL OF DIABETES, vol. 12, no. 1, 18 January 2021 (2021-01-18)
PARKER A. ET AL: "The Mitochondria-Targeted Therapy AP39 Limits Injury in Induced Pluripotent Stem Cell (iPSC)-Derived Human Cardiomyocytes in a Type-2-Diabetic Milieu", HEART, LUNG AND CIRCULATION, vol. 32, 1 July 2023 (2023-07-01), AMSTERDAM, NL, pages S180, XP093210831, ISSN: 1443-9506, DOI: 10.1016/j.hlc.2023.06.132 *
PURNAMA UJANG ET AL: "Modelling Diabetic Cardiomyopathy: Using Human Stem Cell-Derived Cardiomyocytes to Complement Animal Models", METABOLITES, vol. 12, no. 9, 3 September 2022 (2022-09-03), pages 832, XP093210835, ISSN: 2218-1989, DOI: 10.3390/metabo12090832 *
R. H. RITCHIEE. D. ABEL: "Basic Mechanisms of Diabetic Heart Disease", CIRCULATION RESEARCH, vol. 126, no. 11, 2020, pages 1501 - 1525
S. VAN VLIERBERGHE ET AL.: "Biopolymer-Based Hydrogels As Scaffolds for Tissue Engineering Applications: A Review", BIOMACROMOLECULES, vol. 12, no. 5, 2011, pages 1387 - 1408, XP055084202, DOI: 10.1021/bm200083n
YIMU ZHAO ET AL: "A Platform for Generation of Chamber-Specific Cardiac Tissues and Disease Modeling", CELL, vol. 176, no. 4, 24 January 2019 (2019-01-24), Amsterdam NL, pages 913 - 927.e18, XP055667732, ISSN: 0092-8674, DOI: 10.1016/j.cell.2018.11.042 *
Z. LUY.-P. JIANGX.-H. XUL. M. BALLOUI. S. COHENR. Z. LIN: "Decreased 1-Type Ca2+ Current in Cardiac Myocytes of Type 1 Diabetic Akita Mice Due to Reduced Phosphatidylinositol 3-Kinase Signaling", DIABETES, vol. 56, no. 11, 2007, pages 2780 - 2789
ZHAO ET AL.: "A Platform for Generation of Chamber-Specific Cardiac Tissues and Disease Modeling.", CELL, vol. 176, no. 4, 7 February 2019 (2019-02-07), pages 913 - 927, XP055667732, DOI: 10.1016/j.cell.2018.11.042

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