Scalable generation of human induced pluripotent stem cell derived cardiac organoids

Abstract

A method (100) of generation of embryoid bodies (EBs) and differentiation to form human cardiac organoids, including: preparing (110) a cell mixture, allowing (120) centrifugation of the cell mixture, incubating (130) the mixture to form embryoid bodies (EBs), transferring (140) the EBs into a well, developing (150) the EBs to mesodermal germ layer, exchanging (160) the medium completely and inducing CO fate in medium containing growth factors, inducing (170) CO development by a specific growth factor composition, and characterizing (180) the COs

Description

SCALABLE GENERATION OF HUMAN INDUCED PLURIPOTENT STEM CELLDERIVED CARDIAC ORGANOIDSFIELD OF THE INVENTION

[0001] Embodiments of the present invention relates to the medicine and healthcare industry. Particularly, present disclosure relates to a method of scalable generation of human cardiac organoids from human induced pluripotent stem cells.BACKGROUND OF THE INVENTION

[0002] The subject matter discussed in the background section should not be assumed to be prior art merely as a result of it being mentioned in the background section. Similarly, a problem mentioned in the background section or associated with the subject matter of the background section should not be assumed to have been previously recognized in the prior art. The subject matter in the background section merely represents different approaches, which in and of themselves may also correspond to implementations of the claimed technology.

[0003] One of the biggest causes of death in the globe is cardiovascular disease. Cardiomyocytes generated from human pluripotent stem cells (hPSC-CMs) are important cell sources for disease models, regenerative treatments, and drug discovery. To increase the effectiveness, purity, maturity, and even specification of these cardiomyocytes, scientists have experimented with a number of techniques, including gene editing, delta-like, and the addition of tiny chemicals.

[0004] One of the most well-known and significant 3D culture developments in the fields of biological and medical research is organoid technology. Organoids are multicellular structures that resemble organs and are thought to form spontaneously or systematically using stem cells' natural capacity for self-assembly. Organoids can imitate the salient features of the organs that serve as their models. Cardiovascular organoids (COs) are employed in the creation of new pharmaceutical medications via in-vitro drug validations. They allow researchers to study specific physiological pathways during phases of embryogenesis. Their application lowers costs and substitutes human test systems for animal cells.

[0005] COs make it easier to conduct experimental transplantation experiments to repair the heart after a myocardial infarction, and they improve tissue engineering's 3-D bioprinting of human cardiac tissue. It remains a problem, nonetheless, to produce human cardiac organoids on a large scale in suspension culture. Such COs should ideally be made up entirely of cardiac fibroblasts,endothelial cells, and cardiomyocytes and not contain any cells from other organs, including the primitive intestine.

[0006] The development of human heart organoid models for cardiovascular disease investigations has made only modest progress, despite the significance of comprehending human cardiovascular illnesses for treatment and prevention. With the use of human pluripotent stem cells (hPSCs), we are able to easily mass-produce specific cardiac cell types with high purity and recapitulate significant developmental stages in vitro. Over the past ten years, significant efforts have been undertaken to overcome the dearth of pertinent cardiac organoid models. Although these methods give the user a high degree of control over the final product, they are frequently pricy, labor-intensive, and difficult to scale. Furthermore, they typically do not accurately reflect the composition and arrangement of the heart's original cells.

[0007] Available solution for the above problem lack scalable system to support industrial production and as well as the production of COs is rather complex and costly and increases workload. Some prior art also offer Matrigel dependent methods for COs formation and lack a first heart field development. In the present invention, for the first time, up to 70 COs per mL from human induced pluripotent stem cells were produced in a suspension bioreactor operated with 100 mL cell culture medium (i.e., 70000 COs are produced per litre in the suspension stirred bioreactor). Differentiation was performed under serum- free conditions. The protocol was successfully tested in 96-well plates, six well-plates, six cm and ten cm dishes and a stirring vessel bioreactor as well.OBJECT OF THE INVENTION

[0008] An object of the invention is to produce EBs in large numbers in a bioreactor.

[0009] An object of the invention is to produce EBs by centrifugation method in 96-well plates, and different size dishes.

[0010] Another object of the invention is to omit Matrigel and animal derived components to facilitate the development of GMP conform protocols in preparation of pre-clinical and clinical trials.

[0011] Yet another object of the present invention is to produce COs with high quality cardiac cells, less workload and lower cost.

[0012] Yet another object of the present invention is to generate COs containing pluripotent stem cells derived cardiomyocytes, endothelial cells, smooth muscle cells, and cardiac fibroblasts with mature cardiomyocytes action potential characteristics within 20 days.SUMMARY OF THE INVENTION

[0013] According to an aspect of the present invention, method of generation embryoid bodies (EBs) by centrifugation and differentiation to form human cardiac organoids is provided. The method for generating EBs by centrifugation, comprises the steps of: preparing a cell mixture; allowing centrifugation of the cell mixture; incubating the mixture to form embryoid bodies (EBs) transferring the EBs into a well; develop the EBs to mesodermal germ layer; exchange the medium completely and induce CO fate in medium containing growth factors inducing CO development by a specific growth factor composition; and characterize the COs.

[0014] In accordance with an embodiment of the present invention, hiPSCs are dissociated into single cells when the culture reaches confluency in the range of 70-80%.

[0015] In accordance with an embodiment of the present invention, predetermined quantity of hiPSCs is added in the range of 0.01 X 106to 0.03 X 106in 100-120 pl E8 medium.

[0016] In accordance with an embodiment of the present invention, the mixture is centrifuged at a temperature in the range of 15 °C to 30 °C for predetermined time in the range of 5 to 15 minutes.

[0017] In accordance with an embodiment of the present invention, the mixture is incubated at a predetermined temperature in the range of 30 °C- 37 °C for 24 to 72 hours.

[0018] In accordance with an embodiment of the present invention, embryoid bodies or singular spheroids are transferred into a single or multiwell plate containing a predetermined quantity of the medium.

[0019] In accordance with an embodiment of the present invention, a well of a U- shape ultra-low attachment 96 well plate is used in centrifugation.

[0020] In accordance with an embodiment of the present invention, CO induction by biphasic wnt- signalling inhibition through addition of wnt- signalling inhibitors such as IWP2 and XAV939 with simultaneous addition of bFGF, VEGF, and protein kinase A (PKA) stimulator.

[0021] In accordance with an embodiment of the present invention, inducing early COs by combined addition of bFGF and VEGF.

[0022] In accordance with an embodiment of the present invention, maturation induction of COs by M-medium supplemented with insulin, VEGF, bFGF, dexamethasone, elevated concentration of calcium ions, creatine, taurine, L-carnitine, oleic acid, palmic acid, T3 thyroid hormone, penicilline-streptomycine, B-mecarpoethanol, L-Glutamin, Heparin, sodium selenium, and transferrin.

[0023] According to another aspect of the present invention, method of generation embryoid bodies (EBs) directly inside a bioreactor and differentiation to form human cardiac organoids is provided. The method for generating EBs using by inside a bioreactor, comprises the steps of: culturing a predetermined quantity of hiPSC cells in a medium; washing the hiPSC cells with E8 medium; dissociating and filtering the cells; counting number of cells by an automatic cell counter; suspending the cells in the bioreactor; agitating the culture to obtain EBs; inducing mesodermal differentiation in a culture containing EBs; supplementing a medium without insulin and repeatedly incubating the culture for a pre-determined time period; exchanging the medium and inducing wnt- signalling; inducing development of COs; allowing the cardiac cells to be mature to obtain high quality COs; harvesting COs in a tube and allowing COs to settle at the bottom of the tube; and characterizing COs using an active reagent.

[0024] In accordance with an embodiment of the present invention, the COs are characterized using an activated papain reagent for dissociation.

[0025] In accordance with an embodiment of the present invention, the mixture is stimulated by human vascular endothelial growth factor namely VEGF and bFGF and protein kinase A (PKA) stimulator.

[0026] In accordance with an embodiment of the present invention, Dulbecco's Phosphate Buffered Saline (DPBS) is used to wash cells.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] So that the manner in which the above recited features of the present invention can be understood in detail, a more particular to the description of the invention, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typicalembodiments of this invention and are therefore not to be considered limiting of its scope, the invention may admit to other equally effective embodiments. These and other features, benefits and advantages of the present invention will become apparent by reference to the following text figure, with like reference numbers referring to like structures across the views, wherein:

[0028] Fig. 1A illustrates a method of generation of embryoid bodies (EBs) by centrifugation and differentiation to form cardiac organoids (COs), in accordance with an embodiment of the present invention;

[0029] Fig. IB illustrates a method of generation of embryoid bodies in a bioreactor and differentiation to form cardiac organoids, in accordance with an embodiment of the present invention;

[0030] Fig. 2 illustrates experimental data, in accordance with an embodiment of the present invention; and

[0031] Fig. 3 illustrates a diagram of single cell RNA sequencing of cardiac organoids at day 20, and selective markers, in accordance with an embodiment of the present invention.DETAILED DESCRIPTION OF THE DRAWINGS

[0032] The present invention is described hereinafter by various embodiments with reference to the accompanying drawing, wherein reference numerals used in the accompanying drawing correspond to the like elements throughout the description.

[0033] While the present invention is described herein by way of example using embodiments and illustrative drawings, those skilled in the art will recognize that the invention is not limited to the embodiments of drawing or drawings described and are not intended to represent the scale of the various components.

[0034] This invention is not limited to the embodiments of drawing or drawings described and are not intended to represent the scale of the various components. Further, some components that may form a part of the invention may not be illustrated in certain figures, for ease of illustration, and such omissions do not limit the embodiments outlined in any way. It should be understood that the drawings and detailed description thereto are not intended to limit the invention to the particular form disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the scope of the present invention as defined by the appended claims.As used throughout this description, the word "may" is used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense, (i.e., meaning must). Further, the words "a" or "an" mean "at least one” and the word “plurality” means “one or more” unless otherwise mentioned. Furthermore, the terminology and phraseology used herein is solely used for descriptive purposes and should not be construed as limiting in scope. Language such as "including," "comprising," "having," "containing," or "involving," and variations thereof, is intended to be broad and encompass the subject matter listed thereafter, equivalents, and additional subject matter not recited, and is not intended to exclude other additives, components, integers or steps. Likewise, the term "comprising" is considered synonymous with the terms "including" or "containing" for applicable legal purposes. Any discussion of documents, acts, materials, devices, articles and the like is included in the specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters form part of the prior art base or are common general knowledge in the field relevant to the present invention.

[0035] This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiment set forth herein. Rather, the embodiment is provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art. In the following detailed description, numeric values and ranges are provided for various aspects of the implementations described. These values and ranges are to be treated as examples only and are not intended to limit the scope of the claims. In addition, a number of materials are identified as suitable for various facets of the implementations. These materials are to be treated as exemplary and are not intended to limit the scope of the invention.

[0036] In accordance with an embodiment of the present invention, human cardiac organoids (hCO) may be defined as, but not limited to, organ structures that may be scaled down and made simpler. An organoid is a self-organized 3D tissue that resembles the essential functional, structural, and biological complexity of an organ and is often produced from stem cells (pluripotent cells). Organoids may be made of cells taken from tissue, including normal stem / progenitor cells, differentiated cells, and cancer cells, or from induced pluripotent stem cells. Cardiac organoids may be multicellular, three-dimensional, in vitro self-organizing structures made of different types of cardiac cells (cardio myocytes, endothelial cells, cardiac fibroblasts, etc.), with or without biological scaffolding. Organoids may be used to explore how diseases affect human tissues, producing knowledge and tools that may be employed in preclinical research, such as drug testing.They may be utilized in regenerative medicine, cardiac cell replacement therapy, drug discovery, disease modelling, and heart development biology.

[0037] Human-induced pluripotent stem cells (hiPSCs) may be defined as, but not limited to, reprogrammed cells that may have characteristics with embryonic stem cells, such as the ability to self-renew and differentiate into cardiac myocytes. The creation of hiPSC begins with somatic cells. By overexpressing important reprogramming genes, adult somatic cells may be converted into induced pluripotent stem cells (iPSCs). Cardiac myocytes (CMs) produced from hiPSC mimic phenotypic variations brought on by genetic variants, making them appealing illness models for human beings as well as helpful tools for drug discovery and toxicology testing. Because they can multiply endlessly, pluripotent stem cells show promise in the field of regenerative medicine. iPSC are made from skin or blood cells that have undergone reprogramming to return to their pluripotent stage, which is similar to that of an embryo and allows for the creation of an infinite source of any kind of human cell. The benefits of hiPSCs include their human origin, ease of access, expandability, ability to produce nearly any type of cell required, avoidance of ethical difficulties connected with human ESCs, and the possibility to create donor -specific iPSCs for personalised treatment.

[0038] In accordance with an embodiment of the present invention, figure 1A illustrates a method (100) of generation of embryoid bodies (EBs) by centrifugation and differentiation to form cardiac organoids (COs). As shown in Figure 1A, for generating EBs by centrifugation technique, human induced pluripotent stem cells (hiPSCs) are placed in a well containing a medium. The method (100) starts at step 110, a cell mixture is prepared by adding a predetermined quantity of human induced pluripotent stem cells or hiPSCs, for example 0.01 X 106to 0.03 X 106hiPSCs in a medium poured in a 96 well plate. For smaller plate formats cell counts and medium volume are adapted. hiPSCs are used for cell differentiation. HiPSCs cells have the ability to develop into any form of a body cell. This property of hiPSCs makes them a fit for generation and regeneration of human cells. Stem cells further have the ability to self-renew and have the ability to differentiate into different types of tissue or cells for example cardiac cells, hepatic cells, gastrointestinal cells, neural cells. In a preferred embodiment, hiPSCs is dissociated into single cells when the culture reaches confluency in the range of 70-80%. Further, maintaining the hiPSC cultures utilized for CO generation at higher levels of pluripotency for example, over 90-99% pluripotency for repeatability.

[0039] HiPSCs may be differentiated to form cardiac tissue mimicking micro-tissues in vitro also called as cardiac organoids (COs). The hiPSCs may be added in 5-15pL of E8 medium inside awell of a U-shape ultra-low attachment 96-well plate containing E8 medium as explained above. In the present embodiment, the hiPSCs are added in 5-15 pL of E8 medium, which may be added on top of 90- 110 pL of E8 medium supplement with 5 pM ROCK inhibitor inside the well of a 96 well plate. E8 medium may be defined as, but not limited to, a medium used to culture induced pluripotent stem cell (iPSC). E8 medium is a xenogen-free medium, which may be used for both clinical and academic purposes. This offers a simpler medium for the cultivation of pluripotent stem cells because it may contain the necessary elements for maintaining embryonic stem cells and induced pluripotent stem cells. (E8 medium may chemically be defined as a medium containing Dulbecco's modified Eagle's medium (DMEM) / F12, 64 mg / 1 L-ascorbic acid-2- phosphate magnesium, 14 pg / 1 sodium selenium, 100 pg / 1 fibroblast growth factor (FGF)2, 20 mg / 1 insulin, 543 mg / 1 NaHCCE, and 10.7 mg / 1 transferrin, 2 pg / 1 transforming growth factor (TGF)pl or 100 pg / 1 Nodal).

[0040] Then, in step 120, the cell mixture in the plate may be centrifuged at up to 2400g for a predetermined time in the range of 3 to 15 minutes at a temperature in the range of 15°C to 30°C. By this step, cells are collected into aggregates. Followed by, in step 130, the cell aggregates may be incubated at a predetermined temperature in the range of 30°C- 37°C for about 24-48 hours to form one embryoid body (EB) per individual well of the multiwell plate. Incubation is carried out in the presence of carbon dioxide in the range of about 2-5%. In a preferred embodiment, the carbon dioxide is 5%. After about 24- 48 hours, one or more single spheroids or embryoid bodies or EBs are formed. Embryoid bodies may be defined as, but not limited to, three-dimensional aggregates of pluripotent stem cells (PSC), including embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC). The process of EB differentiation is frequently used to produce particular cell lineages from PSCs.

[0041] In step 140, the EBs formed may be transferred using a cut tip of let’s say, a 1000 pl pipette into a single or multiwell plate containing predetermined quantity, let’s say for example 0.3 ml to 2 ml E8 medium. As an alternative, EBs may be combined into a dish with roughly 3.5 cm diameter with 1-5 mL of medium. As a further alternative to generate very uniform COs, the EBs are continuously maintained in individual wells of ultralow adhesive multiwell plates initially used in step 130 such as 96-wells or smaller formats. Plates may be cultured on a rocking table at a rate of 30-60 rotations per minute (rpm) at about 37 °C with 2-5% CO2 supply. Well plates such as, but not limited to, 12 well plates, 24 well plates, 48 well plates may also be used.

[0042] Alternatively, to steps 130 and 140, for greater scale production of COs, embryoid bodies are generated from suspension instead by the centrifugation method. In this method, pluripotentstem cells are diluted in an appropriate amount of medium. Let’s say, for example, 0.1 X 106- 0.5 X 106hiPSCs may be introduced to approximately 1 mL of E8 medium supplemented with ROCK inhibitor and transferred into a single or multiwell plate. The amount of cell suspension is scaled up depending on the size of the single or multiwell plate. For example, the cells are cultivated in non-adhesive 6 or 10 cm plates and the cell suspension is agitated for 40-50 hours at 40 rpm at a temperature of about 37 °C, and 2-5% CO2. From the starting day that counted as day -2 to mesodermal induction that counted a day 0, E8 medium was supplemented with rho kinase inhibitor (ROCK inhibitor), and the plates or dishes were incubated inside a humidified incubator at 37°C and 5% CO2. E8 medium may be supplemented with 2-10 pM ROCK inhibitor (an inhibitor to rho kinase, a kinase belonging to the AGC family of serine-threonine specific protein kinases). It is involved mainly in regulating the shape and movement of cells by acting on the cytoskeleton).

[0043] Further, step 150 involves developing the EBs to mesodermal germ layer. The mesoderm is the middle layer of the three germ layers that develops during gastrulation in the very early development of the embryo of most animals. The outer layer is the ectoderm, and the inner layer is the endoderm. Cardiac tissues are derived from mesoderm. Density of EBs at day 0 may vary depending on cell line and medium agitation. In our hand 75-100 EBs per mL are a good starting point. Lower densities of EBs do not impair differentiation while too high numbers negatively affect the process. Further, the embryoid bodies may be suspended on day 0, into a mesodermal induction medium composed of RPMI 1640 with a lx B27 supplement without insulin and a 24- hour addition of CHIR99021 (small molecule to inhibit canonical Wnt signalling pathway) on day 0. Concentration of CHIR99021 may be optimized for individual stem cell lines. A typical concentration of CHIR99021 is 12pM. On day 1 (step 150), the medium may be changed into RPMI 1640 with a lx B27 supplement without insulin.

[0044] On day 2 (step 160), the medium was completely exchanged with RPMI 1640, lx B27 supplement (without insulin), 2-10 pM IWP2, 2-10 pM XAV939, 10 ng / mL bFGF, 10 ng / mL VEGF, and 0.2 mM 8 -Bromoadenosine 3', 5 '-cyclic monophosphate sodium salt monohydrate (8Bro), further inducing CO fate in medium containing growth factors. The Wnt signalling pathway, a critical signalling route involved in numerous biological processes, including cell proliferation, differentiation, and development, is specifically known for its function as an inhibitor of this pathway. XAV939 is frequently used to control Wnt signalling in cell culture and molecular biology investigations to examine its impact on cell behaviour and gene expression.Later, cells may be kept on a rocking table at 30-60 rpm about 48 hours without having their medium changed.

[0045] On day 4, inducing mesodermal induction is completed. The entire medium may switch to RPMI 1640 with a lx B27 supplement without insulin. Then, in step 170, the culture is stimulated with a specific combination of growth factors, 5-15 ng / mL VEGF (Vascular endothelial growth factor) growth factor and 5-15 ng / mL of bFGF (Basic fibroblast growth factor) growth factor may be added in the media for approximately 72 hours. Growth factor may be defined as, but not limited to, a class of proteins that encourage the development of particular tissues. Growth factors are crucial in encouraging cell division and differentiation. For example, VEGF growth factor is a powerful angiogenic factor identified as being crucial for the proliferation of vascular endothelial cells. VEGF is crucial for cell proliferation and differentiation and bFGF is a crucial member of the family of heparin-binding proteins.

[0046] On day 7 and onwards every three days, the medium may entirely change into maturation medium (M-Medium). M-medium (maturation medium) may be defined as, but not limited to, a medium designed to provide oxidative substrates adapted to the metabolic needs of human induced pluripotent stem cell derived cardiomyocytes. The medium is used to support the maturation of cardiomyocytes in vitro. The medium may even receive constant additions, for example, lOOpg / mL ascorbate (ascorbic acid). Ascorbate may be defined as, but not limited to, a compound that may be used for the development and upkeep of healthy cells both in vivo and in vitro. Esterified and non-esterified unsaturated fatty acids may be shielded against peroxidation by the water-soluble antioxidant ascorbic acid. Antioxidant efficacy of ascorbate is well known. Glutathione, tocopherol, carotenoids, flavonoids, cysteine, methionine, polyamines, and dehydrins and annexins are among the other antioxidants that may be used in a cell culture media. The medium is further supplemented with: Insulin, VEGF, bFGF, dexamethasone, elevated concentration of calcium ions, creatine, taurine, L-carnitine, oleic acid, palmic acid, T3 thyroid hormone, pencilline-streptomycine, B-mecarpoethanol, L-Glutamin, Heparin, 14 pg / 1 sodium selenium, and 10.7 mg / 1 transferrin. Cultivation in M-medium is performed till day 10, or 20 or longer depending on the demand and desired degree of maturation.

[0047] Further in step 180, the cells may be matured and characterized to obtain high quality COs by continuous culture in M-medium. Maturation of a cell may be defined as, but not limited to, a process of gaining profound phenotypic and functional changes as the cells convert from the processing to the costimulatory stages. A mature cell refers to a cell that has differentiated, which means it has acquired a specific rather than a generalized function. COs are used for any desiredapplication usually from day 10 (early stage of cardiac electrophysiology) or day 20 (mature action potentials characteristics) onwards. The cells are then isolated and allowed to settle in a container. To perform single cell analysis of cells isolated from COs, organoids may be dissociated by enzymatic digest. In the present invention, for example, Papain reagent may be used to dissociate the cells. Briefly, 2-10pL of 0.2 M L-cysteine may be added let’s say in 15pL of papain solution into 1 mL of DPBS and incubated at 37 °C for at least 15 minutes to activate the papain reagent. DPBS also known as Dulbecco's Phosphate Buffered Saline may be a buffering agent, used to keep cell culture medium within the physiological range of 7.2-7.6 pH. Potassium chloride, monobasic potassium phosphate, sodium chloride, and dibasic sodium phosphate are all present in this well-balanced salt solution.

[0048] In accordance with an embodiment of the present invention, figure IB illustrates a method (200) of generation of embryoid bodies (EBs) directly inside a bioreactor and differentiation to form cardiac organoids (COs). The CO differentiation process may be enhanced for scaling up. For this, a share stress free bioreactor of the clinostat type for example, ClinoStar (provided by Celvivo, Maryland, USA) may be used. Typically, the process of CO differentiation and scaling up was carried out in a suspension bioreactor, Applikon, equipped with mass flow controllers and at least 250 mL multi-use glass vessel. The growing and synthesis of various biological materials, such as cells, bacteria, or tissue cultures, in a suspended condition inside a liquid medium, may take place in a suspension bioreactor, a type of bioreactor often used in biotechnology and bioprocessing.

[0049] As shown in Figure IB, for generating EBs inside a bioreactor, protocol was optimized for scale up via two different types of bioreactors. The method starts at step 202, culturing a predetermined quantity of hiPSC cells in a medium. Prior to bioreactor inoculation, the hiPSCs may be cultured on Matrigel-coated 100 mm dishes (Coming Life Science, # 353003) and incubated in a humidified incubator at 35-37°C, with 2-10% CO2. Matrigel may be replaced by xenogen-free matrices such as GMP qualified vitronectin. The hiPSCs were cultured in E8 medium for about one week with daily medium change until approximately 85% cell confluency is reached. Then, washing the cells with a salt solution (step 204). In the present embodiment, DPBS or Dulbecco's Phosphate Buffered Saline (DPBS) is used which may provide a buffer system for maintaining cell culture media in the physiological range of 7.2-7.6. The usage of DPBS may be, but not limited to, as an irrigating, transporting or diluting fluid while it DPBS may also help in maintaining cell tonicity and viability for a limited period of time in a cell culture.

[0050] In step 206, dissociating the cells and filtering the cell culture. For dissociation, cells were incubated with 1-2 mL ReLeSR™ (Stemcell Technologies, # 05872) per plate for 5-10 minutes at room temperature. Dissociation may be done my methods such as, but not limited to, enzymatic dissociation, chemical dissociation and mechanical dissociation. In enzymatic dissociation, cells may be treated with the enzymes such as, but not limited to, Accutase, TrypLE, or trypsin / EDTA, these enzymes followed by trituration, or a combination one of these enzymes followed by incubation with another enzyme, including DNase I. Chemical approaches for dissociation of cells may include, but not limited to, in vitro and in vivo cell dissociation techniques and methods. Mechanical dissociation refers to, but not limited to, the use of filters, chopping techniques, microfluidic devices, and various trituration strategies using a variety of pipettes. After cell dissociation, the dissociated cells may be passed through a 30-40 pm filter (Greiner Bio-One, # 542040) and may be collected in a 50 mL Falcon tube with 5-15 mL E8 medium containing 5-15 pM ROCK inhibitor. ROCK inhibitor may enhance survival of human pluripotent stem cells when they are dissociated to single cells by preventing dissociation-induced apoptosis, thus increasing their cloning efficiency.

[0051] Next step is to count (208) number of cells by an automatic cell counter. Cells are counted by an automatic cell counter (NaNoEnTek, Korea). Prior to single cell inoculation the bioreactor glass vessel was placed under laminar airflow, for sterilizing (210) the bioreactor and equilibration E8 medium was discarded. Next, suspending 15 million single cells in 50 mL E8 medium supplemented with 5-15 pM of ROCK inhibitor and this suspension may be inoculated into the bioreactor vessel. Further, agitating (212) the culture to obtain EBs at a propeller speed of 120- 150 rpm for a standard impeller or about 50 rpm for a membrane impeller (Biothrust, Germany), gassed with approximately 95% air, and 2-10% CO2 followed by incubation at 37 °C for 24-48 hours in E8 medium supplemented with ROCK inhibitor.

[0052] Differentiation starts with the step of inducing (214) mesodermal differentiation in a culture containing EBs to COs fate. At day 0, the mesodermal differentiation was induced in a medium containing EBs. At day 0, the medium may be changed into to RPMI 1640 supplemented with lx B27 without insulin along with 80-100 pg / mL ascorbate and 10-15 pM CHIR9902L Ascorbic acid is a vital compound for cell growth and maintenance of healthy cells in vivo and in vitro. It is a water soluble antioxidant that may protect esterified and non-esterified unsaturated fatty acids from peroxidation. Then, after 24 hours (216), the complete medium was changed into RPMI 1640 containing lx B27 without insulin and 80-100 pg / mL ascorbate, and cells were incubated for another 24 hours. At day 2 (218), 100 mL fresh RPMI 1640 supplemented with lxB27 without insulin, 80-100 pg / mL ascorbate, 10 pM IWP2, 10 pM XAV939, 10 ng / mL VEGF, 10 ng / mL bFGF, and 0.2 mM 8Bro were added into the vessel and culture was continued for another 48 hours. At day 4 (220) the medium was replaced by 100 mL fresh RPMI 1640 supplemented with lx B27 without insulin and 80-100 pg / mL ascorbate, 10 ng / mL VEGF, and 10 ng / mL bFGF. From day 7 onward, every three days the medium was refreshed with 100 mL M- Medium.

[0053] In step 222, the cells may be mature enough to obtain high quality COs. Maturation of a cell may be defined as, but not limited to, a process of gaining profound phenotypic and functional changes as the cells convert from the processing to the costimulatory stages. A mature cell refers to a cell that has differentiated, which means it has acquired a specific rather than a generalized function. Suspension bioreactors allow cells to grow freely in the liquid medium, typically swirled or agitated to give nutrients and maintain a uniform environment, in contrast to traditional adherent cell cultures, where cells grow attached to a surface (e.g., a petri dish or flask). Since no particular attachment surfaces are required, suspension bioreactors may be able to sustain a greater range of species and function on a much larger scale than immobilised cultures. Suspension bioreactors may commonly be used in large-scale bioproduction processes, such as the production of biopharmaceuticals, vaccines, organoids etc. Further, step 224, harvesting COs in one or more 50 mL Falcon tube(s) and allowing COs to settle at the bottom of the tube by gravity. Cell harvesting may be done by, but not limited to, tools including pipettes, and centrifuges. Centrifuges are used for pelleting cells and separating them from culture media.

[0054] To dissociate COs into single cells, a reagent may be used for example trypsin, versene solution, papain, and collagenase. After harvesting, it's important to characterize (226) the harvested COs to confirm their identity and functionality. COs were characterized at day 10, 20, and 40. Characterization of stem cells may be done by techniques such as, but not limited to, immunocytochemistry, flow cytometry, gene expression analysis, and functional assays. In the present invention, for example, Papain reagent may be used to dissociate the cells. Briefly, 2-10pL of 0.2 M L-cysteine may be added let’s say in 15pL of papain into 1 mL of DPBS and incubated at 37 °C for at least 15 minutes to activate the papain reagent. DPBS also known as Dulbecco's Phosphate Buffered Saline may be a buffering agent, used to keep cell culture medium within the physiological range of 7.2-7.6 pH. Potassium chloride, monobasic potassium phosphate, sodium chloride, and dibasic sodium phosphate are all present in this well-balanced salt solution.

[0055] Large-scale production of cardiac organoids in suspension bioreactors offers several advantages, including the potential for higher yields, better scalability, and the ability to controlculture conditions more precisely compared to traditional static cultures. This approach has the potential to support various applications in cardiac research and regenerative medicine. However, it may also requires careful optimization and monitoring to ensure the production of functional cardiac organoids suitable for the intended purpose.

[0056] Figure 2 illustrates experimental data, in accordance with an embodiment of the present invention. As shown in figure 2, in section A. Schematic illustration of differentiation; in section B. Bright field of cardiac organoids; in section C. Representative flow cytometric scatter plots analysis of dissociated cardiac organoids at day 10 of single cells stained for TNNT2-FITC cardiac maker: VE-Cadherin-PE endothelial marker; in section D. and E. Whole mount immunostaining of cardiac organoids where cardiac organoids were stained without prior dissociation with antibodies against a- actinin, CX43, VE-Cadherin, vWF; in section F. Electron microscopic images of cardiac organoid; in section G. Sharp electrode electrical activity of cardiac organoid.

[0057] Figure 3 illustrates a diagram of single cell RNA sequencing of cardiac organoids at day 20. Determination of high selective markers expression per cluster base logarithmic differential data, in accordance with an embodiment of the present invention.

[0058] In accordance with the present invention, the advantage of the present invention is that it is a robust method of generation of human cardiac organoids. The method does not utilize any matrix including Matrigel during the process of differentiation of human induced pluripotent cells into cardiac organoids. Matrigel is a widely used matrix which is often difficult to handle and expensive. It also uses animal rather than human material as base material and is considered of having ethical concerns about its production. Thus, in the present invention usage of Matrigel is strictly avoided. Matrigel used in the culture of pluripotent stem cells prior to the beginning of the differentiation method described here can be replaced by standardized and GMP conform recombinant matrices including recombinant human vitronectin and laminin.

[0059] The present invention further avoids animal derived components including serum in any step of the entire process. Further, the present method of generation of human cardiac organoids COs is applicable in a wide range of culture systems for example multi-well plates, suspension bioreactors and even rotating bioreactors. Production of CO usually requires a large investment, this is overcome by the present invention. The present invention provides a low cost CO production method. The process also offers no growth factor utilization during differentiation from hiPSC to mesodermal cells which helps to increase reproducibility. Furthermore, durability of CO cells have improved. It can be easily shipped at 4°C to 20°C without losing quality for at least 2 days.

[0060] Various modifications to these embodiments are apparent to those skilled in the art from the description and the accompanying drawings. The principles associated with the various embodiments described herein may be applied to other embodiments. Therefore, the description is not intended to be limited to the embodiments shown along with the accompanying drawings but is to be providing broadest scope of consistent with the principles and the novel and inventive features disclosed or suggested herein. Accordingly, the invention is anticipated to hold on to all other such alternatives, modifications, and variations that fall within the scope of the present invention and the appended claims

Claims

Claims1. A method (100) of generation of embryoid bodies (EBs) by centrifugation and differentiation to form human cardiac organoids (COs), the method comprising steps of: preparing (110) a cell mixture by adding a predetermined quantity of human induced pluripotent stem cells or hiPSCs in a medium poured in a well plate; allowing (120) centrifugation of the cell mixture for a predetermined time; incubating (130) the mixture at a predetermined temperature to form one or more embryoid bodies (EBs); and transferring (140) the EBs into a well of a well plate containing the medium, developing (150) the EBs to the mesodermal germ layer in a mesodermal induction medium; exchanging (160) the medium completely and inducing CO fate by a biphasic wnt-signalling inhibitory medium containing specific growth factors and incubating the EBs for a predetermined time period; inducing (170) CO development by a specific growth factor composition; and stimulating (180) the maturation of COs by a specific maturation medium.

2. The method (100) of generation of embryoid bodies (EBs) by centrifugation and differentiation to form human cardiac organoids as claimed in claim 1, wherein hiPSCs is dissociated into single cells when the culture reaches confluency in the range of 70- 80%.

3. The method (100) of generation of embryoid bodies (EBs) by centrifugation and differentiation to form human cardiac organoids as claimed in claim 1, wherein predetermined quantity of hiPSCs is added to produce EB by centrifugation in the range of 0.01 X 106to 0.03 X 106.

4. The method (100) of generation of embryoid bodies (EBs) by centrifugation and differentiation to form human cardiac organoids as claimed in claim 1, wherein the mixture is centrifuged at a temperature in the range of 15°C to 30°C for predetermined time in the range of 5 to 15 minutes.

5. The method (100) of generation of embryoid bodies (EBs) by centrifugation and differentiation to form human cardiac organoids as claimed in claim 1, wherein repeatedly incubating the EBs for a predetermined time period of 24-72 hours.

6. The method (100) of generation of embryoid bodies (EBs) by centrifugation and differentiation to form human cardiac organoids as claimed in claim 1, wherein a well of a U-shape ultra-low attachment 96 well plate is used in centrifugation.

7. The method (100) of generation of embryoid bodies (EBs) by centrifugation and differentiation to form human cardiac organoids as claimed in claim 1, wherein CO induction by biphasic wnt- signalling inhibition through addition of wnt- signalling inhibitors such as IWP2 and XAV939 with simultaneous addition of bFGF, VEGF, and protein kinase A (PKA) stimulator.

8. The method (100) of generation of embryoid bodies (EBs) by centrifugation and differentiation to form human cardiac organoids as claimed in claim 1, wherein of inducing early COs by combined addition of bFGF and VEGF.

9. The method (100) of generation of embryoid bodies (EBs) by centrifugation and differentiation to form human cardiac organoids as claimed in claim 1, wherein of maturation induction of COs by M-medium supplemented with insulin, VEGF, bFGF, dexamethasone, elevated concentration of calcium ions, creatine, taurine, L-carnitine, oleic acid, palmic acid, T3 thyroid hormone, pencilline-streptomycine, B- mecarpoethanol, L-Glutamin, Heparin, sodium selenium, and transferrin.

10. A method (200) of generation of embryoid bodies (EBs) directly inside a bioreactor and differentiation to form human cardiac organoids, the method comprising steps: culturing (202) a predetermined quantity of hiPSC cells in a medium; washing (204) the hiPSC cells with a salt solution; dissociating (206) and filtering the cells; counting (208) number of cells by an automatic cell counter; suspending (210) the cells in the bioreactor; and agitating (212) the culture to obtain EBs. differentiating COs, steps including: inducing (214) mesodermal differentiation in a culture containing EBs toCOs fate;supplementing (216) a medium without insulin and repeatedly incubating the culture for a predetermined time period; exchanging (218) the medium and inducing CO fate by a biphasic wnt- signalling inhibitory medium containing specific growth factors and incubating the EBs for a predetermined time period inducing (220) CO further development by a specific growth factor combination maturation (222) of COs by replacing the medium for a specific maturation medium. harvesting (224) COs in a tube and allowing COs to settle at the bottom of the tube; and characterizing (226) COs using an active reagent;11. The method (200) of generation of embryoid bodies (EBs) directly inside a bioreactor and differentiation to form human cardiac organoids as claimed in claim 10, wherein the COs are characterized using an activated papain reagent.

12. The method (200) of generation of embryoid bodies (EBs) directly inside a bioreactor and differentiation to form human cardiac organoids as claimed in claim 10, wherein the mixture is stimulated by human vascular endothelial growth factor namely VEGF and bFGF and protein kinase A (PKA) stimulator.

13. The method (200) of generation of embryoid bodies (EBs) directly inside a bioreactor and differentiation to form human cardiac organoids as claimed in claim 10, wherein CO induction by biphasic wnt-signalling inhibition through addition of wnt-signalling inhibitors such as IWP2 and XAV939 with simultaneous addition of bFGF, VEGF, and protein kinase A (PKA) stimulator.

14. The method (200) of generation of embryoid bodies (EBs) directly inside a bioreactor and differentiation to form human cardiac organoids as claimed in claim 10, wherein inducing early COs by combined addition of bFGF and VEGF.

15. The method (200) of generation of embryoid bodies (EBs) directly inside a bioreactor and differentiation to form human cardiac organoids as claimed in claim 10, wherein maturation induction of COs by M-medium supplemented with insulin, VEGF, bFGF, dexamethasone, elevated concentration of calcium ions, creatine, taurine, E-carnitine,oleic acid, palmic acid, T3 thyroid hormone, pencilline-streptomycine, B- mecarpoethanol, L-Glutamin, Heparin, sodium selenium, and transferrin.

16. The method (200) of generation of embryoid bodies (EBs) directly inside a bioreactor and differentiation to form human cardiac organoids as claimed in claim 10, wherein Dulbecco's Phosphate Buffered Saline (DPBS) is used to wash cells.

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