Cardiac cell compositions

Abstract

The invention relates to compositions comprising cardiomyocytes, endothelial cells and fibroblasts, which are useful for studying cardiac biology and cardiotoxicity in vitro The invention also relates to cardiac spheroids obtained or obtainable from the compositions described herein, and their use in methods of drug screening and assessing cardiotoxicity.

Description

[0001] CARDIAC CELL COMPOSITIONS

[0002] FIELD OF THE INVENTION

[0003] The invention relates to compositions comprising cardiomyocytes, endothelial cells and fibroblasts, which are useful for studying cardiac biology and cardiotoxicity in vitro. The invention also relates to cardiac spheroids obtained or obtainable from the compositions described herein, and their use in methods of drug screening and assessing cardiotoxicity.

[0004] BACKGROUND

[0005] Cardiac tissue is composed of multiple cell types, including cardiomyocytes (contractile muscle cells, fibroblasts (connective / structural cells) and endothelial cells (cells that form blood vessels). Cardiotoxicity, defined as toxicity that affects the heart, is one of the most common adverse drug effects. Currently, cardiotoxicity is modelled and tested in vitro on cardiac cells derived from stem cells, combined with additional cell types obtained from cadaveric tissue. For example, some models utilise human embryonic stem cell derived cardiomyocytes combined with human primary endothelial cells and cardiac fibroblasts obtained from cadavers (Ravenscroft et al. (2016) Toxicol. Sci. 152:99-112). This admixture of cells makes it difficult to determine the cause (e.g. which cell type) of toxicity when studying adverse drug reactions. Other approaches use a single cell type to model cardiotoxicity, but such single cell populations are limited in function, often not displaying typical structural and functional characteristics of cardiac tissue. Consequently, such single cell models have poor predictive value.

[0006] Cardiac multicellular models have been described in WO2021186044 and WO2019174879. In such approaches, a population of stem cells is treated with a combination of factors to induce differentiation into multiple cell types (e.g. cardiomyocytes and endothelial cells), giving rise to an in vitro multicellular cardiac model. However, such models are inherently poorly reproducible, since the amount of each cell type present within a model can vary substantially, as can the extent of differentiation into each cell type. Consequently, such multicellular models are highly variable and are of limited use for reliably predicting cardiotoxicity and adverse drug events.

[0007] Thus, there is a need in the art for new in vitro research tools that permit the study of cardiac function and cardiotoxicity with high reproducibility, low variability and good predictive value.

[0008] SUMMARY OF THE INVENTION

[0009] In a first aspect, the invention provides a composition comprising cardiomyocytes, endothelial cells and fibroblasts, wherein the composition comprises the cardiomyocytes, endothelial cells and fibroblasts at a ratio of 2:2:1 to 6:3:1 , and wherein the composition is a frozen composition. In a second aspect, the invention provides a container comprising a composition as described herein.

[0010] In a third aspect, the invention provides an in vitro cardiac spheroid obtained or obtainable from the composition of any preceding claim.

[0011] In a fourth aspect, the invention provides an in vitro cardiac spheroid comprising cardiomyocytes, endothelial cells and fibroblasts, wherein at least 90% of the cardiomyocytes express MLC2v.

[0012] In a fifth aspect, the invention provides a kit comprising a composition as described herein, and a cell culture medium.

[0013] In a sixth aspect, the invention provides a method for preparing a composition as described herein, the method comprising: a) providing a plurality of iPSCs, b) independently differentiating the plurality of iPSCs into cardiomyocytes, endothelial cells and fibroblasts, c) dissociating the differentiated cardiomyocytes, endothelial cells and fibroblasts into single cells, and d) combining the cardiomyocytes, endothelial cells and fibroblasts at a ratio of 2:2:1 to 6:3:1.

[0014] In a seventh aspect, the invention provides a method for producing a cardiac spheroid, the method comprising: a) providing a composition as described herein, and b) culturing the cardiomyocytes, endothelial cells and fibroblasts.

[0015] In an eighth aspect, the invention provides an in vitro method for assessing toxicity of an agent, the method comprising: a) providing a composition as described herein, b) culturing the cells in the composition under conditions suitable to produce a cardiac spheroid, c) treating the cardiac spheroid with the agent, and determining the effect of the agent on one or more characteristics of the cardiac spheroid.

[0016] In a ninth aspect, the invention provides an in vitro method for drug screening, the method comprising: a) providing a composition as described herein, b) culturing the cells in the composition under conditions suitable to produce a cardiac spheroid, c) treating the cardiac spheroid with a candidate agent, and d) determining the effect of the candidate agent on one or more characteristics of the cardiac spheroid.

[0017] In a tenth aspect, the invention provides an in vitro method for drug screening, the method comprising: a) providing a composition as described herein, b) culturing the cells in the composition under conditions suitable to produce a cardiac spheroid, c) treating the cardiac spheroid with a candidate agent, and d) determining the effect of the candidate agent on one or more characteristics of the cardiac spheroid.

[0018] In an eleventh aspect, the invention provides an in vitro method for assessing toxicity of an agent, the method comprising: a) providing a cardiac spheroid as described herein, b) treating the cardiac spheroid with the agent, and c) determining the effect of the agent on one or more characteristics of the cardiac spheroid.

[0019] In a twelfth aspect, the invention provides an in vitro method for drug screening, the method comprising: a) providing a cardiac spheroid as described herein, b) treating the cardiac spheroid with a candidate agent, and c) determining the effect of the candidate agent on one or more characteristics of the cardiac spheroid.

[0020] In a thirteenth aspect, the invention provides an in vitro method for assessing cardiotoxicity of an agent, the method comprising: a) providing a cardiac spheroid as described herein, b) treating the cardiac spheroid with the agent, and c) determining the effect of the agent on one or more characteristics of the cardiac spheroid.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Brightfield and immunofluorescence images showing the consistent expression of lineage specific cell markers for cardiomyocytes (cTnT), endothelial cells (CD31) and fibroblasts (COL1) across four different iPS donor cell lines. Figure 2: Brightfield and immunostaining of known cardio toxins disrupting functional and structural components in iPS derived CM monolayers at 48hrs post exposure. (A) Mitochondrial staining (functional) (B) Phalloidin staining (structural)

[0023] Figure 3: Disrupted beat rate of i18F derived CM monolayers caused by known cardio toxins at 24hrs and 48hrs post exposure. (DOF - dofetilide, SUN - sunitinib, DOX - doxorubicin, ER - erythromycin).

[0024] Figure 4: Representative brightfield images showing cardiac spheroids generated from the compositions described herein. (A) brightfield image; (B) still from a video recording of a cardiac spheroid “beating” in vitro.

[0025] Figure 5: Representative immunofluorescence images showing formation of vascular networks in cardiac spheroids generated from the compositions described herein.

[0026] Figure 6: Cardioexcyte readings from cardiac spheroids of the invention treated with vandetanib at 10 pM, 1 pM, and with DMSO control.

[0027] Figure 7: Cardioexcyte readings from cardiac spheroids obtained from commercially available cardiomyocytes, primary endothelial cells and primary fibroblasts treated with vandetanib at 10 pM, 1 pM, and with DMSO control.

[0028] Figure 8: Immunofluorescence images showing expression of ventricular cardiac markers (MHC, IRX4 and MLC2v) by cardiomyocytes.

[0029] Figure 9: Flow cytometry analysis showing the expression of cardiac troponin T, MLC2v and MHC by cardiomyocytes obtained from 3 different starting iPSC lines (i2F, i3M, and i8M).

[0030] DETAILED DESCRIPTION

[0031] Abbreviations used:

[0032] BSA - bovine serum albumin

[0033] CD31 - cluster of differentiation 31

[0034] CE - cardiac endothelial cells

[0035] CF - cardiac fibroblasts

[0036] CM - cardiomyocyte

[0037] CMEF - cardiomyocytes, endothelial cells and fibroblasts

[0038] COL1 - type 1 collagen cTnT - cardiac muscle troponin T

[0039] DAPI - 4',6-diamidino-2-phenylindole DMSO - dimethyl sulfoxide

[0040] DOF - dofetilide

[0041] DOX - doxorubicin

[0042] ER - erythromycin

[0043] FGF - fibroblast growth factor

[0044] HAND1 - Heart And Neural Crest Derivatives Expressed 1

[0045] ICC - immunocytochemistry iPSCs - induced pluripotent stem cells

[0046] MACS - magnetic-activated cell sorting

[0047] MLC2v - ventricular myosin light chain 2

[0048] PECAM - Platelet endothelial cell adhesion molecule-1

[0049] ROCK - Rho kinase inhibitor

[0050] SUN - sunitinib

[0051] VEGF - vascular endothelial growth factor

[0052] The invention provides a new in vitro research tool for the study of cardiac function and cardiotoxicity. Described herein is a composition comprising three cell types present in cardiac tissue: cardiomyocytes, endothelial cells, and fibroblasts. Unlike in multicellular cardiac models, such as those described in WO2021186044, each cell type present in the compositions described herein has been independently differentiated from a separate starting population of cells (generally stem cells, such as iPSCs). In other words, each of the cardiomyocyte, fibroblast and endothelial cell types are separately differentiated. Once proper differentiation of each cell type has been established, the cells are dissociated to single cells, allowing each cell type to be combined in a precise ratio in the compositions described herein. This approach means that a composition of the invention comprises a specific, reproducible ratio of each cell type, wherein each cell type is appropriately differentiated.

[0053] The combination of the three cell types is capable of self-assembly into cardiac tissue. Consequently, the compositions described herein can be used to generate functional “mini-hearts”. Users of the composition of the invention can quickly, simply and reproducibly prepare in vitro cardiac models to screen the tolerability and toxicity of drugs on cardiac tissue. Since the compositions described herein contain defined cell populations in defined ratios, the cardiac models generated from such compositions are inherently less variable compared to previous approaches, as well as being quicker and simpler to use. The use of separately / independently differentiated cell types also enables greater control over the genetic background of the cells (and any resulting cardiac models), since the three cell types may be derived from iPSCs obtained from the same donor, or may be derived from iPSCs obtained from different donors. This in turn provides a more useful research tool that can be used to study the effects of multiple genetic drivers on cardiotoxicity, that would not be possible with multicellular cardiac models obtained from a single population of iPSCs, or with CMEF models derived from a mixture of iPSCs and primary cells, such as those described in Ravenscroft et al. (2016) Toxicol Sci. 152:99-112. Accordingly, provided herein is a composition comprising cardiomyocytes, endothelial cells and fibroblasts, wherein the composition comprises the cardiomyocytes, endothelial cells and fibroblasts at a ratio of 2:2:1 to 6:3:1 , and wherein the composition is frozen.

[0054] Generally, the cardiomyocytes, endothelial cells, and fibroblasts are dissociated cardiomyocytes, dissociated endothelial cells, and dissociated fibroblasts. This means that each cell type has been independently differentiated in culture and dissociated to single cells before being combined in the compositions described herein. For example, each of the cardiomyocytes, endothelial cells, and fibroblasts may be independently differentiated from stem cells, such as iPSCs, to generate separate populations of cardiomyocytes, endothelial cells, and fibroblasts. Once the separate populations of cardiomyocytes, endothelial cells, and fibroblasts have been obtained, each population may be dissociated to single cells. The single cells may then be combined in a composition as described herein, for example at a ratio of 2:2:1 to 6:3:1. Optionally, the dissociated cells may be counted prior to being combined in the composition. The compositions described herein may therefore be distinguished from multicellular cardiac models or other compositions in which a single population of stem cells is treated with various agents to induce differentiation into multiple cardiac cell types. Such cardiac models or compositions do not provide defined ratios of each cell type. Such cardiac models and compositions may also develop secondary structure or cellular networks as a result of all cell types being differentiated together. Such secondary structures and cellular networks are not typically present in the compositions described herein. The compositions described herein generally comprise cardiomyocytes, endothelial cells and fibroblasts, wherein the cardiomyocytes, endothelial cells and fibroblasts have been independently differentiated.

[0055] Cardiomyocytes are the contractile cells of the heart, responsible for generating the contractile force required to circulate blood round the body. The cardiomyocytes present in the compositions described herein may express or be positive for cardiac troponin T (cTnT), alpha actinin, or a combination thereof.

[0056] Endothelial cells are those cell types that form blood vessels and vascular tissue. The endothelial cells in the compositions described herein may be cardiac endothelial cells. The endothelial cells may express or be positive for CD31 .

[0057] Fibroblasts are supporting cells that produce connective tissue such as extracellular matrix, and can also produce and secrete growth factors, cytokines and other signalling molecules in cardiac tissue. The fibroblasts present in the compositions described herein may be cardiac fibroblasts. The fibroblasts present in the compositions described herein may express or be positive for type 1 collagen (COL1).

[0058] The cardiomyocytes, endothelial cells, and fibroblasts may be present in the composition at a ratio of 2:2:1 to 6:3:1 , in some cases 3:2:1 to 5:2:1 , preferably 4:2:1. Since each of the cell types are independently differentiated, the cardiomyocytes, endothelial cells, and fibroblasts can be dissociated, optionally counted, and combined in precise ratios to provide the composition of the invention. This in turn provides a research tool with greater consistency and repeatability, since cardiac models generated from the compositions as described herein are generated from a consistent number and ratio of starting cell lineages.

[0059] A composition as described herein is generally a frozen composition. The composition may be a cryopreserved composition. As such, any of the compositions described herein may further comprise a cryoprotectant. The cryoprotectant may be any cryoprotectant suitable for preserving and protecting the cells during freezing and storage at low temperatures (such as -70°C to -200°C). Suitable cryoprotectants are known in the art, and include CryoStor® CS10 (Stem Cell Technologies, catalog # 07930). The cryoprotectant may be free of animal components, such as animal serum. The cryoprotectant may comprise dimethyl sulfoxide (DMSO). The cryoprotectant may comprise glycerol. The cryoprotectant may comprise sucrose, glucose, ethylene glycol, and / or propylene glycol. The cryoprotectant may comprise a ROCK inhibitor (for example, the cryoprotectant may comprise a ROCK inhibitor at 10 pM). Freezing and / or cryopreservation allows the composition to be maintained as a mixture of dissociated cells until it is ready to be used, further enhancing the reproducibility of the resulting cardiac model.

[0060] Advantageously, the novel frozen 3-in-1 composition disclosed herein offers a significant advancement for automated and high-throughput drug screening. Through combining these key cardiac cell types into a single, ready-to-use vial, this technology provides a more physiologically relevant model of human heart tissue while also simplifying complex experimental workflows. The cells are pre-mixed and cryopreserved together, therefore researchers no longer need to thaw, count, and combine each cell type manually. This reduces human error, lowers the chance of technical variability, and saves valuable time and resources.

[0061] The frozen composition disclosed herein is highly compatible with large-scale automated platforms. Robotic systems can thaw the 3-in-1 cell mix, culture the cells in multi-well plates, and perform high- content imaging and analysis, all with minimal human intervention. The frozen composition makes for easy incorporation into fully automated, highly scalable workflows for drug screening. The standardisation and simplicity of using a frozen composition comprising cardiomyocytes, endothelial cells and fibroblasts at a ratio of 2:2:1 to 6:3:1 , supports significant scale-up, allowing hundreds or even thousands of drug candidates to be tested in parallel. A feat that would be hard to achieve with freshly grown and individual cell types that must be cultured and aligned ahead of combination. Freezing mature 'ready to assay' multicellular models also provides a huge advance in time saving, as they are ready immediately post thaw.

[0062] As a result, these models can be used not only to validate the safety and efficacy of known drugs but also to help identify and characterise novel compounds with therapeutic potential. The combination of physiological relevance, scalability, and automation makes this technology a powerful tool in modern drug discovery and development. In some cases, the compositions described herein consist of the cardiomyocytes, endothelial cells, fibroblasts, and cryoprotectant. For example, there may be no other cell types present in the composition. There may be no matrices (e.g. hydrogels), scaffold, support structure or connective tissue in the composition.

[0063] Generally, the cells of the composition are isolated in the sense that the each of the cell lineages (cardiomyocytes, endothelial cells, fibroblasts) have been produced via independent in vitro processes of differentiation from stem cells and dissociation into single cells. In other words, the invention described herein does not encompass the human body or its elements.

[0064] A composition as described herein may be a frozen composition. A composition as described herein may be a cryopreserved composition. Such compositions may be provided and / or stored at a temperature below freezing (0°C), such as at about -20°C, or about -70°C. A composition as described herein may be prepared by freezing the three cell lineages (preferably with cryoprotectant) using dry ice, liquid nitrogen or conventional freezer. The composition may then be stored in frozen form using any suitable frozen storage method (such as liquid nitrogen storage or ultralow (~ -150°C) freezer). Suitable freezing and cryopreservation techniques are known in the art.

[0065] Upon thawing the frozen compositions disclosed herein, each of the cardiomyocytes, endothelial cells and fibroblasts are viable cells, suitable for use in any of the methods disclosed herein. Cells can be counted post-thawing (e.g. in a haemocytometer, or any suitable automated device for cell counting) and the viability may be assessed.

[0066] Each of the cell lineages present in the compositions described herein (cardiomyocytes, endothelial cells, fibroblasts) are generally derived from stem cells, such as iPSCs. By “derived from”, it is meant that a population of stem cells (e.g. iPSCs) is treated with various differentiation factors to induce differentiation to the required cell lineage, such as a cardiomyocyte lineage or endothelial cell lineage. Suitable differentiation protocols for providing each cell lineage are known in the art. It will be understood that “derived from” in the context of the invention means “independently derived from”. In other words, each cell type within the composition has been derived separately from the other cell types. In general, the cell types of the composition are derived from iPSCs, although other suitable starting cells (such as embryonic stem cells) may be used (provided such approaches do not require the destruction of a human embryo).

[0067] Advantageously, the cardiomyocytes, endothelial cells and fibroblasts present in the compositions described herein may be derived from a single donor or may be derived from multiple donors. “Donor” in this context may refer to an individual or cell line from which a starting iPSC population is obtained. Where the starting iPSC populations are obtained from a single donor, the resulting cardiomyocytes, endothelial cells and fibroblasts will have the same genetic background. Where the starting iPSC populations are obtained from multiple, different donors, the resulting cardiomyocytes, endothelial cells and fibroblasts will have different genetic backgrounds. Because the cardiomyocytes, endothelial cells and fibroblasts present in the composition are generally independently differentiated from separate populations of iPSCs, the composition advantageously provides greater flexibility to combine multiple genetic backgrounds and potentially study multiple genetic sources of cardiotoxicity in a single cardiac model.

[0068] As described elsewhere herein, the cardiomyocytes, endothelial cells and fibroblasts present in the composition are capable of self-assembly into a cardiac spheroid and / or a cardiac model. The resulting cardiac spheroid may also be referred to as a “mini-heart”. The compositions described herein are not themselves cardiac organoids, cardiac models or cardiac spheroids, but can become a cardiac spheroid when cultured under appropriate conditions, as described in the examples. For example, a frozen composition as described herein may be thawed, plated into a multiwell plate, and cultured for a period of several days (such as 7 days) to allow the cells in the composition to self-assemble into the cardiac spheroid. By “self-assemble”, it is meant that no exogenously applied factors, supports or stimulants (such as growth factors, transcription factors or matrices) need to be applied to initiate or maintain assembly into the cardiac spheroid.

[0069] As noted above, the compositions described herein comprise dissociated cardiomyocytes, endothelial cells and fibroblasts and are not themselves cardiac organoids, cardiac spheroids, or cardiac models. A composition as described herein is generally a cell suspension (e.g. a suspension of the three dissociated cell types). Accordingly, the composition does not comprise any structural or functional characteristics typical of a cardiac model, in particular those multicellular models in which a single population of iPSCs is treated with differentiation factors to give rise to multiple cell types. The composition generally does not comprise cellular layers or cellular networks. In particular, there is generally no structural relationship or interaction between the cells in the composition, since they have been combined from dissociated single cells, rather than differentiated together in culture from one original starting stem cell population. The composition generally does not comprise cavities, cavity-like structures, or cardiac chamber-like structures. The composition can be considered a mixture of cells. For example, the composition may comprise a mixture of dissociated cardiomyocytes, dissociated endothelial cells and dissociated fibroblasts. Generally, the composition is an amorphous composition. Generally, the composition is an unstructured composition.

[0070] The compositions described herein may also be matrix-free. In other words, the composition generally does not comprise any exogenous extracellular matrix proteins, cellular supports or cellular scaffolds (such as hydrogels or alginate scaffolds).

[0071] Also provided herein is a container comprising a composition as described herein. The container may be a tube. The container may be any container that facilitates the storage and shipping of the composition, in particular in a frozen or cryopreserved format. The container may also be a multi-well plate. Advantageously, each well within the multi-well plate may comprise a composition as described herein. Such multi-well plates enable multiple cardiac spheroids to be obtained simultaneously in a format compatible with standard laboratory equipment. Each well within the multi-well plate may comprise a specific, consistent amount of a composition as described herein (including a defined ratio of each cell type), providing excellent reproducibility from well to well. The use of a multi-well plate enables automation or semi-automation of laboratory work, which in turn can support high-throughput applications such as high-throughput drug screening.

[0072] Also provided herein is a kit comprising a composition as described herein, and a cell culture medium.

[0073] Methods for preparing compositions

[0074] Described herein are methods for preparing a composition, in particular a composition of cardiac cells. Generally, such methods comprise (a) providing a plurality of iPSCs, (b) independently differentiating the plurality of iPSCs into cardiomyocytes, endothelial cells and fibroblasts, and (c) combining the cardiomyocytes, endothelial cells and fibroblasts at a ratio of 2:2:1 to 6:3:1. Described herein are method for preparing a composition, the method comprising: (a) providing a plurality of iPSCs, (b) independently differentiating the plurality of iPSCs into cardiomyocytes, endothelial cells and fibroblasts,

[0075] (c) dissociating the differentiated cardiomyocytes, endothelial cells and fibroblasts into single cells;, and

[0076] (d) combining the cardiomyocytes, endothelial cells and fibroblasts at a ratio of 2:2:1 to 6:3:1 to form a cell suspension; and (e) freezing the cell suspension. The method may further comprise dissociating the independently differentiated cardiomyocytes, endothelial cells and fibroblasts into single cells prior to step (c), and may optionally comprise counting the single cells. As used “independently differentiated” or “independently differentiating” means that each of the cell types (cardiomyocytes, endothelial cells and fibroblasts) are differentiated separately. This means each cell type can be obtained from a different starting cell line (such as a different iPSC line), if desired. This method can therefore be distinguished from a method in which a single population of iPSCs are treated with differentiation factors to produce all three cell types. Once each cell type has been independently (separately) differentiated, the cells may be dissociated to single cells. For example, iPSCs may be cultured under conditions that cause the iPSCs to differentiate into endothelial cells, and once differentiated, the endothelial cells may be dissociated (disaggregated) to separate the cells into single cells, before they are optionally counted and combined with cardiomyocytes and fibroblasts in the ratios described herein. As a further step, the combined cells may be frozen.

[0077] The cardiomyocytes, endothelial cells and fibroblasts may be combined at a ratio of 3:2:1 to 5:2:1. The cardiomyocytes, endothelial cells and fibroblasts may be combined at a ratio of 4:2:1. The plurality of iPSCs may be obtained from a single donor. The plurality of iPSCs may be obtained from multiple donors. Differentiation of each cell type may be performed according to suitable protocols known in the art. For example, cardiomyocyte differentiation is described in Burridge et al (2014) Nat. Methods. 11 :855-860. Commercially available kits may be used for differentiating each cell type, such as STEMdiff™ Ventricular Cardiomyocyte differentiation kit (StemCell Technologies, #05010), PSC Cardiomyocyte differentiation kit (ThermoFisher, #A2921201) and Cardiomyocyte Differentiation Medium (Sigma, #SCM102). The method may comprise contacting the combined cardiomyocytes, endothelial cells and fibroblasts with a cryoprotectant as described elsewhere herein. The method may comprise transferring the combined cardiomyocytes, endothelial cells and fibroblasts to a container, such as a tube or microwell plate. The method may comprise freezing the combined cardiomyocytes, endothelial cells and fibroblasts (preferably with a cryoprotectant). The method may comprise cryopreserving the combined cardiomyocytes, endothelial cells and fibroblasts (preferably with a cryoprotectant). The method may prepare any of the compositions as defined herein.

[0078] In vitro cardiac spheroids

[0079] The compositions as described herein can be used to provide in vitro cardiac spheroids. An in vitro cardiac spheroid is a three-dimensional cardiac model that can be used to study cardiac biology, disease, and toxicity. Accordingly, described herein is an in vitro cardiac spheroid obtained or obtainable from a composition as provided herein. The cardiac spheroid may be obtained or obtainable from a frozen composition as provided herein.

[0080] The compositions described herein and the cardiac spheroids obtainable from the compositions described herein comprise cardiomyocytes which have a more mature phenotype relative to cardiomyocytes in other cardiac models. For example, the cardiomyocytes express markers associated with a ventricular phenotype, such as MLC2v and / or HAND1. In contrast, cardiomyocytes in other cardiac models often have a fetal phenotype, affecting the expression of certain ion channels and the electrophysiological properties of such cardiac models, which in turn makes them less useful as a research tool. Accordingly, provided herein is an in vitro cardiac spheroid comprising cardiomyocytes, endothelial cells and fibroblasts, wherein at least 70% of the cardiomyocytes are ventricular cardiomyocytes. At least 75% of the cardiomyocytes may be ventricular cardiomyocytes. At least 80% of the cardiomyocytes may be ventricular cardiomyocytes. At least 85% of the cardiomyocytes may be ventricular cardiomyocytes. At least 90% of the cardiomyocytes may be ventricular cardiomyocytes. At least 95% of the cardiomyocytes may be ventricular cardiomyocytes. Provided herein is an in vitro cardiac spheroid comprising cardiomyocytes, endothelial cells and fibroblasts, wherein at least 70% of the cardiomyocytes express MLC2v. At least 75% of the cardiomyocytes may express MLC2v. At least 80% of the cardiomyocytes may express MLC2v. At least 85% of the cardiomyocytes may express MLC2v. At least 90% of the cardiomyocytes may express MLC2v. At least 95% of the cardiomyocytes may express MLC2v. At least 70% of the cardiomyocytes may express HAND1. At least 75% of the cardiomyocytes may express HAND1. At least 80% of the cardiomyocytes may express HAND1. At least 85% of the cardiomyocytes may express HAND1 . At least 90% of the cardiomyocytes may express HAND1 . At least 95% of the cardiomyocytes may express HAND1 .

[0081] In some embodiments, at least 90% of the cardiomyocytes of the compositions described herein express MLC2V and the composition is not a cardiac organoid / cardiac model. In some embodiments, at least 90% of the cardiomyocytes of the compositions described herein express MLC2V and the composition is a cell suspension.

[0082] In some embodiments, at least 90% of the cardiomyocytes of the compositions described herein are ventricular cardiomyocytes and the composition is not a cardiac organoid / cardiac model. In some embodiments, at least 90% of the cardiomyocytes of the compositions described herein ventricular cardiomyocytes and the composition is a cell suspension.

[0083] The cardiac spheroids described herein (which can be obtained from the compositions described herein) may comprise tubular structures, in particular blood vessel-like structures or pseudo-blood vessels. A portion of the endothelial cells of the cardiac spheroid may be arranged in tubular structures, blood vessel-like structures, and / or pseudo-blood vessels. Such tubular structures or blood vessel-like structures can be identified using methods known in the art, such as immunofluorescence to identify vascular markers (e.g. PECAM, CD31) within the cardiac spheroid. The formation of such vascular structures within the cardiac spheroid advantageously enables the study of endothelial function and cardiotoxicity of agents that may particularly affect cardiac vasculature.

[0084] Also provided herein is a method for producing a cardiac spheroid. In general, the method comprises providing a composition as described herein, optionally thawing the composition (where said composition is provided in a frozen or cryopreserved state), and culturing the cardiomyocytes, endothelial cells and fibroblasts. Generally, said culturing is in matrix-free conditions. The method may further comprise removing the cryoprotectant from the composition (if present), for example by centrifuging the composition, discarding the supernatant, and resuspending the pellet of cells in a suitable culture medium. The method may comprise contacting the cardiomyocytes, endothelial cells and fibroblasts with a suitable cell culture medium prior to said culturing step. The method may further comprise spinning or centrifuging the cells prior to said culturing step. Centrifugation can enhance the self-assembly of the cells into a cardiac spheroid.

[0085] Accordingly, a method for producing a cardiac spheroid may comprise (a) providing a composition as described herein, (b) removing the cryoprotectant from the composition, (c) contacting the cardiomyocytes, endothelial cells and fibroblasts with a cell culture medium, (d) centrifuging the cardiomyocytes, endothelial cells and fibroblasts, and (e) culturing the cardiomyocytes, endothelial cells and fibroblasts. A method for producing a cardiac spheroid may comprise (a) providing a composition as described herein, wherein the composition is frozen or cryopreserved, (b) thawing the composition, (c) removing the cryoprotectant from the composition, (d) contacting the cardiomyocytes, endothelial cells and fibroblasts with a cell culture medium, (e) centrifuging the cardiomyocytes, endothelial cells and fibroblasts, and (f) culturing the cardiomyocytes, endothelial cells and fibroblasts. A method for producing a cardiac spheroid may comprise: (a) thawing the product of any of the methods disclosed herein or the compositions disclosed herein, and (b) culturing the thawed cells under conditions suitable to produce a cardiac spheroid, whereby cardiac spheroids are formed. Any of the methods for producing cardiac spheroids as described herein may further comprise a step of counting the cardiomyocytes, endothelial cells and fibroblasts prior to said culturing step. Cell counting ensures an optimal seeding density can be used to generate the cardiac spheroids.

[0086] In some cases, a method for producing a cardiac spheroid comprises: a) providing a plurality of iPSCs, b) independently differentiating the plurality of iPSCs into cardiomyocytes, endothelial cells and fibroblasts, c) dissociating the differentiated cardiomyocytes, endothelial cells and fibroblasts into single cells, d) combining the cardiomyocytes, endothelial cells and fibroblasts at a ratio of 2:2:1 to 6:3:1 , and e) culturing the cardiomyocytes, endothelial cells and fibroblasts to produce the cardiac spheroid.

[0087] In some cases, the combined cardiomyocytes, endothelial cells and fibroblasts at a ratio of 2:2:1 to 6:3:1 are then frozen or cryopreserved, and subsequently thawed prior to step (e).

[0088] Also described herein is the use of a composition as provided herein in an in vitro method for producing a cardiac spheroid.

[0089] Uses of the cardiac spheroid

[0090] The cardiac spheroids described herein are useful in a variety of research applications, including assessing toxicity of an agent, drug screening, and assessing cardiotoxicity of an agent.

[0091] An in vitro method for assessing toxicity of an agent may comprise: a) providing a composition as described herein, b) culturing the cells in the composition under conditions suitable to produce a cardiac spheroid, c) treating the cardiac spheroid with the agent, and d) determining the effect of the agent on one or more characteristics of the cardiac spheroid.

[0092] In some cases, the composition is a frozen or cryopreserved composition and wherein the method comprises thawing the composition prior to step (b).

[0093] An in vitro method for assessing toxicity of an agent may also comprise: a) providing a cardiac spheroid as described herein, b) treating the cardiac spheroid with the agent, and c) determining the effect of the agent on one or more characteristics of the cardiac spheroid.

[0094] In some cases, the cardiac spheroid may be obtained or obtainable from a frozen composition as provided herein.

[0095] An in vitro method for drug screening may comprise: a) providing a composition as described herein, b) culturing the cells in the composition under conditions suitable to produce a cardiac spheroid, c) treating the cardiac spheroid with a candidate agent, and d) determining the effect of the candidate agent on one or more characteristics of the cardiac spheroid.

[0096] In some cases, the composition is a frozen or cryopreserved composition and wherein the method comprises thawing the composition prior to step (b).

[0097] An in vitro method for drug screening may comprise: a) providing a cardiac spheroid as described herein, b) treating the cardiac spheroid with a candidate agent, and c) determining the effect of the candidate agent on one or more characteristics of the cardiac spheroid.

[0098] In some cases, the cardiac spheroid may be obtained or obtainable from a frozen composition as provided herein.

[0099] An in vitro method for assessing cardiotoxicity of an agent may comprise: a) providing a composition as described herein, b) culturing the cells in the composition under conditions suitable to produce a cardiac spheroid, c) treating the cardiac spheroid with the agent, and d) determining the effect of the agent on one or more characteristics of the cardiac spheroid.

[0100] In some cases, the composition is a frozen or cryopreserved composition and wherein the method comprises thawing the composition prior to step (b).

[0101] An in vitro method for assessing cardiotoxicity of an agent may comprise: a) providing a cardiac spheroid as described herein, b) treating the cardiac spheroid with the agent, and c) determining the effect of the agent on one or more characteristics of the cardiac spheroid.

[0102] In some cases, the cardiac spheroid may be obtained or obtainable from a frozen composition as provided herein.

[0103] In some cases, the methods disclosed herein are automated. The frozen compositions disclosed herein are highly compatible with large-scale automated platforms, allowing for scalability. In some cases, the methods disclosed herein are high-throughput. In some cases, the methods disclosed herein are automated and high-throughput.

[0104] Determining the effect of the agent on one or more characteristics of the cardiac spheroid may comprise determining the effect of the agent on the beats per minute of the cardiac spheroid, on mitochondrial function of the cardiac spheroid, and / or on the structure of the cardiac spheroid. The structure of the cardiac spheroid may be assessed using a marker such as phalloidin, PECAM, CD31 , and / or MLC2v. Said determining step may comprise treating the cardiac spheroid with at least one selected from the group consisting of a plasma membrane label, a mitochondrial label, and a nuclear label, or a combination thereof. Said determining step may comprise determining the effect relative to the cardiac spheroid prior to said treating step, or may comprise determining the effect relative to an untreated cardiac spheroid or a cardiac spheroid treated with a control agent. Also described herein is the use of a composition or cardiac spheroid as described herein in an in vitro method of assessing drug toxicity, drug screening, or assessing cardiotoxicity.

[0105] Aspects and embodiments described herein with the term “comprising” may include other features or steps within the scope. It is also understood that aspects and embodiments described as “comprising” also describes aspect and embodiments wherein the term “comprising” is replaced by the term “consisting essentially of’ or “consisting of’.

[0106] The phrase "selected from the group comprising" may be substituted with the phrase "selected from the group consisting of and vice versa, wherever they occur herein.

[0107] It is also understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and / or optional features either singly or together with any of the other aspects, unless the context demands otherwise.

[0108] The invention will now be further described by way of the following Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention, with reference to the Figures.

[0109] EXAMPLES Example 1 - Generation of cardiac composition

[0110] Cardiomyocytes were differentiated from iPSCs according to the following protocol:

[0111] • Perform at least three passages of starting iPSCs. Commence differentiation on D3 post passage when the culture reaches 80% confluency.

[0112] • Prepare induction media according to Table 2 and supplement with 8 pM CHIR99021. Treat cells with this induction media for 24 hours. At 24 and 48 hours later, add induction media without CHIR99021 .

[0113] • Remove media from wells and replace with induction media supplemented with 4 pM of C59 Wnt inhibitor.

[0114] • Incubate for 48 hours at 37 C and 5% CO2 atmosphere.

[0115] • At day 5, feed cells with C1 media prepared according to Table 3. Incubate for 48 hours at 37 °C and 5% CO2 atmosphere.

[0116] • At day 7, feed cells with C1 media prepared according to Table 3. Incubate for 48 hours at 37 °C and 5% CO2 atmosphere.

[0117] • At day 9, exchange media to C2 media prepared according to Table 4. Incubate for 24 hours at 37 °C and 5% CO2 atmosphere.

[0118] • By day 10, cells should be spontaneously beating.

[0119] • For harvest, cells can be dissociated by adding 0.5 ml of dissociation solution (TrypLE™ Select Enzyme, Gibco) per well, and incubating for 5 minutes at 37 °C. Cells can be gently pipetted to generate a single cell suspension.

[0120] Table 1 : C18 media Table 2: Induction Media

[0121] Table 3: C1 media

[0122] Table 4: C2 media

[0123] Endothelial cells were differentiated from iPSCs according to the following protocol:

[0124] • Perform at least three passages of starting iPSCs. See cells in 2x6 well plates at a 1 :8 ratio in mTesRI media (StemCell Technologies, #85850). Incubate cells at 37 °C, 5% CO2. Maintain cells as iPSCs for 72 hours.

[0125] • Once cells are at 70-80% confluency, wash cells and replace media with 8 um CHIR supplemented induction media prepared according to Table 2. Incubate for 24 hours at 37 °C,

[0126] 5% CO2.

[0127] • Add 2ml induction media and incubate for 24 hours at 37 °C, 5% CO2.

[0128] • Add 2ml induction media and incubate for a further 24 hours at 37 °C, 5% CO2.

[0129] • Replace media with EGM2 media (Lonza CC-3162) supplemented with 100ng / mL VEGF, 25ng / mL FGF and 10uM SB-431542. Place back in incubator for 48 hours. Replace media with fresh and incubate for a further 24 hours.

[0130] • Dissociate cells using 1 mL of IxTrypLE Express (Gibco) per well as described above, count cells and isolate CD144 positive cells using microbeads and MACS column.

[0131] Fibroblasts were differentiated from iPSCs according to the following protocol:

[0132] • Culture iPSCs in mTESRI medium for 4-6 days until 80-100% confluency. • Change media to DO media (2.5 ml per 10cm2) (see Table 5) supplemented with 12 pM CHIR99021 . Incubate at 37°C / 5% CO2.

[0133] • After 24 hours, replace media with D1 media and incubate for another 24 hours at 37°C / 5% CO2.

[0134] • After day 2, but within 24 hours (D2.5) change the medium to 2.5mL of CFBM supplemented with 75ng / mL of FGFb.

[0135] • Replace medium with CFBM supplemented with 75 ng / mL of FGFb every other day until day 20.

[0136] • Dissociate cells using 1 mL of IxTrypLE Express (Gibco) per 10cm2 growth surface and incubate for 5 minutes at 37°C / 5% CO2.

[0137] • Perform a cell count, seed cells into wells at a density of 25,000-30,000 cells / cm2and change media every other day. Confluency should be reached within 4-6 days.

[0138] Table 5:

[0139] A harvest procedure was then performed to obtain cells of each lineage and combine them. After harvest, each cell type was dissociated to single cells and a cell count was performed. The cells were then combined in a 4:2:1 (cardiomyocytes:endothelial cells:fibroblasts) ratio. The combined cells were then centrifuged at 200G for 5 minutes and the supernatant was aspirated. The cells were resuspended in cryoprotectant (CryoStor supplemented with 10 pM ROCK inhibitor) to achieve a cell density of 2x106cells / mL. Cells were transferred to cryotubes, with 1 mL of the cell suspension in each tube. The cryotubes were placed in the Cool Cell box and transferred to the -80 °C freezer. After 48 hours, cells were transferred to liquid nitrogen storage or -150 °C ultralow temperature freezer.

[0140] Table 6 below shows a list of successfully differentiated lines into Cardiomyocytes (CMs), Cardiac Fibroblasts (CFs) and Cardiac Endothelial Cells (CEs), with sex and genetic background details.

[0141] Table 6:

[0142] Each of the cell lineages were assessed for their expression of various cell markers using immunofluorescence. As shown in Figure 1 , lineage specific cell markers were consistently shown across four different iPSC donor cell lines. Cardiomyocytes express cTnT, endothelial cells express CD31 , and fibroblasts express COL1 .

[0143] Example 2 - Generation of in vitro cardiac spheroid

[0144] Frozen compositions comprising cardiomyocytes, endothelial cells and fibroblasts (CMEFs) were generated as described above and were thawed in a 37 °C water bath for approximately 2 minutes. The CMEFs were transferred to a 50ml conical tube with 8 ml CMEF plating medium (1 :1 cardiac media (RPMI+B27) and endothelial media (EGMTM -2 Endothelial Cell Growth Medium BulletKit™ (Lonza, Catalog Number CC-3162, comprising basal media, heparin, hEGF, Hydrocortisone, Ascorbic Acid, Gentamicin, Amphotericin B and FBS), supplemented with 10% foetal bovine serum and 10 uM ROCK inhibitor) and centrifuged at 200xg for 5 minutes at room temperature. The supernatant was aspirated and discarded, and the pellet was resuspended in 1 ml of plating media (comprising 1 :1 cardiac media and endothelial media, but without supplementation with FBS and ROCK inhibitor). A cell count was performed to ensure optimal seeding density: 10 pl of cell suspension was removed, mixed with 10 pl of trypan blue and a triple count performed. Cells were then resuspended in plating medium to give the required plating density. The working volume per well of a 96 well plate was 200 pl. CMEFs were then cultured according to the following workflow:

[0145] Example 3 - Characterisation of cardiac spheroids

[0146] Cardiac spheroids were generated as described above. Spheroid formation was observed between 0 and 3 days in culture (see Figure 4 for representative images of cardiac spheroids).

[0147] Spheroids were assessed using Cardioexcyte. Spheroids were positioned onto human fibronectin treated (F0895, Sigma Aldrich) NSP-96 CardioExcyte96 0.6 mm sensor plates (cat # 201002, Nanion Technologies). Spheroids were attached at room temperature and then transferred to 37 °C / 5% CO2 for an overnight incubation. Impedance and extracellular field potential (EFP) signals were recorded periodically (10 to 30 min) in 30 s sweeps for the duration of the experiment, preceded by a half media change and a minimum of 1 hour equilibration period. More than 90% of cardiac spheroids showed beating at day 3, with a beat rate of approximately 30 beats per minute.

[0148] Cardiac spheroids were also assessed for markers of vascular structures. Spheroids were fixed with a 4% paraformaldehyde (PFA, Merck) solution and permeabilised with 5% BSA / 0.1 % Triton X-100 (Sigma Aldrich) blocking buffer for 1 hour at room temperature. Primary antibodies, collagen 1 (AF6220, R&D Systems), alpha actinin (MA1 -22863, Thermo Scientific) and CD31 (ab28364, Abeam), were diluted in blocking buffer and incubated with the spheroids overnight in the dark at 4 °C followed by three washes in 0.1 % PBS-Triton X-100. Directly conjugated secondary antibodies (A21448, A21202 and A10042, Thermo Fisher) were diluted in 0.1 % PBS-Triton X-100 and incubated with the spheroids in the dark at room temperature for 2 hours. Hoechst stain (62249, Thermo Fisher) solution diluted in 0.1 % PBS-Triton X-100 was used to label nuclei for 1 hour. Labelled spheroids were washed three times 5 minutes in 0.1 % PBS-Triton X-100. Spheroids were imaged in ULA plates or IBIDI angiogenesis slides using a Zeiss LSM 880 confocal microscope. The results are shown in Figure 5. As seen in Figure 5, formation of vascular structures occurs in the cardiac spheroids from day 3 onwards, with tubular, vessel like structures clearly observed at day 4 (see anti-CD31 staining in Figure 5).

[0149] This Example demonstrates that the frozen compositions described herein produce viable cell types upon thawing that are capable of and successful in producing cardiac spheroids. This represents a significant advancement in the field, allowing for freezing of mature “ready to assay” multicellular models.

[0150] Example 4 - Cardiotoxicity screening in cardiomyocyte monolayers

[0151] Cardiomyocyte monolayers were treated with known cardiological toxins (dofetilide, sunitib, doxorubicin and erythromycin) and compared to control. Cardiomyocyte monolayers show clear responses to cardiological toxins, with disrupted cardiomyocyte structure and cellular interactions (Figure 2) and reductions in spontaneous beating (Figure 3).

[0152] Cardiac spheroids were also assessed for their response to the known cardiotoxin vandetanib using Cardioexcyte. Spheroids were positioned onto human fibronectin treated (F0895, Sigma Aldrich) NSP-96 CardioExcyte96 0.6 mm sensor plates (cat # 201002, Nanion Technologies). Spheroids were attached at room temperature and then transferred to 37 °C / 5% CO2 for an overnight incubation. Impedance and extracellular field potential (EFP) signals were recorded periodically (10 to 30 min) in 30 s sweeps for the duration of the experiment, preceded by a half media change and a minimum of 1 hour equilibration period. Vandetanib was added at sweep 21 , at both 10 pM and 1 pM concentrations. As shown in Figure 6, addition of vandetanib at 10 pM caused beat rate shutdown, whilst addition at 1 pM had no effect. No effect on beat rate was observed in the control condition using DMSO. In contrast, spheroids generated from commercially available cardiomyocytes (Cellular Dynamics International), primary endothelial cells and primary fibroblasts did not show beat rate shutdown at 10 pM vandetanib. Some effects were observed at 1 pM, but response to the DMSO control was inconsistent (Figure 7). The results illustrate the utility of the cardiac spheroids described herein (and the compositions used to generate them) in assessing cardiotoxicity.

[0153] Example 5 - Ventricular phenotype

[0154] Cardiomyocytes generated as described herein show a ventricular phenotype, as demonstrated by immunohistochemistry for MHC, MLC2v and IRX4 shown in Figure 8. Briefly, cardiomyocytes were permeabilised, blocked, and treated with primary antibodies against cardiac troponin, myosin heavy chain (MHC), IRX4, and MLC2v. Cells were treated with fluorescent secondary antibodies, labelled with the nuclear stain DAPI, and imaged with confocal microscopy. The ventricular phenotype is obtained irrespective of the starting iPSC line (i2F, i3M and i8M all generate cardiomyocytes expressing MLC2V, MHC and IRX4), as demonstrated by the flow cytometry results in Figure 9. This ventricular phenotype makes the compositions described herein particularly useful as a research tool, as the cells respond to cardiotoxins in a similar manner to cardiac tissue, and as such can be used as a reliable and consistent tool for cardiotoxicity screening.

[0155] EMBODIMENTS

[0156] The invention may also be understood by reference to the following numbered embodiments.

[0157] 1 . A composition comprising cardiomyocytes, endothelial cells and fibroblasts, wherein the composition comprises the cardiomyocytes, endothelial cells and fibroblasts at a ratio of 2:2:1 to 6:3:1.

[0158] 2. The composition of embodiment 1 , wherein the mixture comprises the cardiomyocytes, endothelial cells and fibroblasts at a ratio of 3:2:1 to 5:2:1.

[0159] 3. The composition of embodiment 1 or 2, wherein the mixture comprises the cardiomyocytes, endothelial cells and fibroblasts at a ratio of 4:2:1.

[0160] 4. The composition of any preceding embodiment, further comprising a cryoprotectant.

[0161] 5. The composition of any preceding embodiment, wherein the composition consists of the cardiomyocytes, endothelial cells, fibroblasts and cryoprotectant.

[0162] 6. The composition of any preceding embodiment, wherein the composition is frozen.

[0163] 7. The composition of any preceding embodiment, wherein the composition is cryopreserved.

[0164] 8. The composition of any preceding embodiment, wherein the cardiomyocytes, endothelial cells and fibroblasts are derived from stem cells.

[0165] 9. The composition of embodiment 8, wherein the stem cells are iPSCs.

[0166] 10. The composition any preceding embodiment, wherein the cardiomyocytes, endothelial cells and fibroblasts are derived from a single donor.

[0167] 11. The composition of any of embodiments 1-9, wherein the cardiomyocytes, endothelial cells and fibroblasts are derived from multiple donors.

[0168] 12. The composition of any preceding embodiment, wherein the cardiomyocytes, endothelial cells and fibroblasts are independently differentiated from iPSCs. 13. The composition of any preceding embodiment, wherein the cardiomyocytes, endothelial cells and fibroblasts are capable of self-assembly into a cardiac spheroid and / or a cardiac model.

[0169] 14. The composition of any preceding embodiment, wherein the composition is not a cardiac organoid or cardiac model.

[0170] 15. The composition of any preceding embodiment, wherein the composition is a cell suspension.

[0171] 16. The composition of any preceding embodiment, wherein the composition is matrix-free.

[0172] 17. The composition of any preceding embodiment, wherein the cardiomyocytes express or are positive for cardiac troponin T (cTnT), alpha actinin, or a combination thereof.

[0173] 18. The composition of any preceding embodiment, wherein the endothelial cells express or are positive for CD31 .

[0174] 19. The composition of any preceding embodiment, wherein the endothelial cells are cardiac endothelial cells.

[0175] 20. The composition of any preceding embodiment, wherein the fibroblasts express or are positive for type 1 collagen (COL1).

[0176] 21. The composition of any preceding embodiment, wherein the fibroblasts are cardiac fibroblasts.

[0177] 22. A container comprising the composition of any preceding embodiment.

[0178] 23. The container of embodiment 22, wherein the container is a tube.

[0179] 24. The container of embodiment 22, wherein the container is a multi-well plate.

[0180] 25. The container of embodiment 24, wherein each well within the multi-well plate comprises a composition according to any of embodiments 1-21.

[0181] 26. An in vitro cardiac spheroid obtained or obtainable from the composition of any of embodiments 1-21 .

[0182] 27. An in vitro cardiac spheroid comprising cardiomyocytes, endothelial cells and fibroblasts, wherein at least 90% of the cardiomyocytes express MLC2v.

[0183] 28. The cardiac spheroid of embodiment 27, wherein at least 95% of the cardiomyocytes express MLC2v. 29. The cardiac spheroid of embodiment 27 or 28, wherein at least 90% of the cardiomyocytes are ventricular cardiomyocytes.

[0184] 30. The cardiac spheroid of any of embodiments 27-29, wherein at least 95% of the cardiomyocytes are ventricular cardiomyocytes.

[0185] 31. The cardiac spheroid of any of embodiments 27-30, comprising blood vessel-like structures.

[0186] 32. The cardiac spheroid of any of embodiments 27-31 , comprising tubular structures.

[0187] 33. The cardiac spheroid of any of embodiments 27-32, comprising pseudo-blood vessels.

[0188] 34. The cardiac spheroid of any of embodiments 27-33, wherein at least a portion of the endothelial cells are arranged in tubular structures, blood vessel-like structures, or pseudoblood vessels.

[0189] 35. A kit comprising the composition of any of embodiments 1-21 , and a cell culture medium.

[0190] 36. A method for preparing a composition according to any of embodiments 1-21 , the method comprising: a) providing a plurality of iPSCs, b) independently differentiating the plurality of iPSCs into cardiomyocytes, endothelial cells and fibroblasts, c) dissociating the differentiated cardiomyocytes, endothelial cells and fibroblasts into single cells, and d) combining the cardiomyocytes, endothelial cells and fibroblasts at a ratio of 2:2:1 to 6:3:1.

[0191] 37. The method of embodiment 36, wherein the cardiomyocytes, endothelial cells and fibroblasts are combined at a ratio of 3:2:1 to 5:2:1 .

[0192] 38. The method of embodiment 36 or 37, wherein the cardiomyocytes, endothelial cells and fibroblasts are combined at a ratio of 4:2:1 .

[0193] 39. The method of any of embodiments 36-38, wherein the plurality of iPSCs are obtained from a single donor.

[0194] 40. The method of any of embodiments 36-38, wherein the plurality of iPSCs are obtained from multiple donors.

[0195] 41. The method of any of embodiments 36-40, further comprising freezing the combined cardiomyocytes, endothelial cells and fibroblasts. 42. The method of any of embodiments 36-41 , further comprising cryopreserving the combined cardiomyocytes, endothelial cells and fibroblasts.

[0196] 43. A method for producing a cardiac spheroid, the method comprising: a) providing a composition according to any of embodiments 1-21 , and b) culturing the cardiomyocytes, endothelial cells and fibroblasts.

[0197] 44. The method of embodiment 43, wherein the composition is a frozen or cryopreserved composition and wherein the method comprises thawing the composition prior to step (b).

[0198] 45. The method of embodiment 43 or 44, wherein said culturing is in matrix-free conditions.

[0199] 46. The method of any of embodiments 43-45, further comprising spinning or centrifuging the cardiomyocytes, endothelial cells and fibroblasts prior to step (b).

[0200] 47. Use of the composition of any of embodiments 1-21 in an in vitro method for producing a cardiac spheroid.

[0201] 48. An in vitro method for assessing toxicity of an agent, the method comprising: a) providing a composition according to any of embodiments 1-21 , b) culturing the cells in the composition under conditions suitable to produce a cardiac spheroid, c) treating the cardiac spheroid with the agent, and d) determining the effect of the agent on one or more characteristics of the cardiac spheroid.

[0202] 49. An in vitro method for drug screening, the method comprising: a) providing a composition according to any of embodiments 1-21 , b) culturing the cells in the composition under conditions suitable to produce a cardiac spheroid, c) treating the cardiac spheroid with a candidate agent, and d) determining the effect of the candidate agent on one or more characteristics of the cardiac spheroid.

[0203] 50. An in vitro method for assessing cardiotoxicity of an agent, the method comprising: a) providing a composition according to any of embodiments 1-21 , b) culturing the cells in the composition under conditions suitable to produce a cardiac spheroid, c) treating the cardiac spheroid with the agent, and d) determining the effect of the agent on one or more characteristics of the cardiac spheroid.

[0204] 51. The method of any of embodiments 48-50, wherein step (d) comprises determining the effect of the agent on the beats per minute of the cardiac spheroid.

[0205] 52. The method of any of embodiments 48-51 , wherein step (d) comprises determining the effect of the agent on mitochondrial function of the cardiac spheroid.

[0206] 53. The method of any of embodiments 48-52, wherein step (d) comprises determining the effect of the agent on the structure of the cardiac spheroid.

[0207] 54. The method of any of embodiments 48-53, wherein step (d) comprises treating the cardiac spheroid with at least one selected from the group consisting of a plasma membrane label, a mitochondrial label, and a nuclear label, or a combination thereof.

[0208] 55. The method of any of embodiments 48-54, wherein step (d) comprises determining the effect relative to the cardiac spheroid prior to step (c).

[0209] 56. The method of any of embodiments 48-55, wherein step (d) comprises determining the effect relative to an untreated cardiac spheroid or a cardiac spheroid treated with a control agent.

[0210] 57. Use of the composition of any of embodiments 1-21 or the cardiac spheroid of any of embodiments 26-34 in an in vitro method for assessing drug toxicity.

[0211] 58. Use of the composition of any of embodiments 1-21 or the cardiac spheroid of any of embodiments 26-34 in an in vitro method for drug screening.

[0212] 59. Use of the composition of any of embodiments 1-21 or the cardiac spheroid of any of embodiments 26-34 in an in vitro method for assessing cardiotoxicity.

[0213] 60. An in vitro method for assessing toxicity of an agent, the method comprising: a) providing a cardiac spheroid according to any of embodiments 26-34, b) treating the cardiac spheroid with the agent, and c) determining the effect of the agent on one or more characteristics of the cardiac spheroid.

[0214] 61. An in vitro method for drug screening, the method comprising: a) providing a cardiac spheroid according to any of embodiments 26-34, b) treating the cardiac spheroid with a candidate agent, and c) determining the effect of the candidate agent on one or more characteristics of the cardiac spheroid.

[0215] 62. An in vitro method for assessing cardiotoxicity of an agent, the method comprising: a) providing a cardiac spheroid according to any of embodiments 26-34, b) treating the cardiac spheroid with the agent, and c) determining the effect of the agent on one or more characteristics of the cardiac spheroid.

[0216] 63. The method of any of embodiments 60-62, wherein step (c) comprises determining the effect of the agent on the beats per minute of the cardiac spheroid.

[0217] 64. The method of any of embodiments 60-63, wherein step (c) comprises determining the effect of the agent on mitochondrial function of the cardiac spheroid.

[0218] 65. The method of any of embodiments 60-64, wherein step (c) comprises determining the effect of the agent on the structure of the cardiac spheroid.

[0219] 66. The method of any of embodiments 60-65, wherein the structure of the cardiac spheroid is assessed using at least one marker selected from the group consisting of phalloidin, PECAM, CD31 , MLC2v.

[0220] 67. The method of any of embodiments 60-66, wherein step (c) comprises treating the cardiac spheroid with at least one selected from the group consisting of a plasma membrane label, a mitochondrial label, and a nuclear label, or a combination thereof.

[0221] 68. The method of any of embodiments 60-67, wherein step (c) comprises determining the effect relative to the cardiac spheroid prior to step (c).

[0222] 69. The method of any of embodiments 60-68, wherein step (c) comprises determining the effect relative to an untreated cardiac spheroid or a cardiac spheroid treated with a control agent.

[0223] 70. A method for preparing a composition, the method comprising: a) providing a plurality of iPSCs, b) independently differentiating the plurality of iPSCs into cardiomyocytes, endothelial cells and fibroblasts, c) dissociating the differentiated cardiomyocytes, endothelial cells and fibroblasts into single cells, and d) combining the cardiomyocytes, endothelial cells and fibroblasts at a ratio of 2:2:1 to 6:3:1.

[0224] 71. The method of embodiment 70, wherein the cardiomyocytes, endothelial cells and fibroblasts are combined at a ratio of 3:2:1 to 5:2:1 .

[0225] 72. The method of embodiment 70 or 71 , wherein the cardiomyocytes, endothelial cells and fibroblasts are combined at a ratio of 4:2:1 .

[0226] 73. The method of any of embodiments 70-72, wherein the plurality of iPSCs are obtained from a single donor.

[0227] 74. The method of any of embodiments 70-73, wherein the plurality of iPSCs are obtained from multiple donors.

[0228] 75. The method of any of embodiments 70-74, further comprising freezing the combined cardiomyocytes, endothelial cells and fibroblasts.

[0229] 76. The method of any of embodiments 70-75, further comprising cryopreserving the combined cardiomyocytes, endothelial cells and fibroblasts.

[0230] 77. A method for producing a cardiac spheroid, the method comprising: a) providing a composition comprising cardiomyocytes, endothelial cells and fibroblasts, wherein the composition comprises the cardiomyocytes, endothelial cells and fibroblasts at a ratio of 2:2:1 to 6:3:1 , and b) culturing the cardiomyocytes, endothelial cells and fibroblasts.

[0231] 78. The method of embodiment 76, wherein the composition is a frozen or cryopreserved composition and wherein the method comprises thawing the composition prior to step (b).

[0232] 79. An in vitro method for assessing toxicity of an agent, the method comprising: a) providing a composition comprising cardiomyocytes, endothelial cells and fibroblasts, wherein the composition comprises the cardiomyocytes, endothelial cells and fibroblasts at a ratio of 2:2:1 to 6:3:1 , b) culturing the cells in the composition under conditions suitable to produce a cardiac spheroid, c) treating the cardiac spheroid with the agent, and d) determining the effect of the agent on one or more characteristics of the cardiac spheroid. 80. An in vitro method for drug screening, the method comprising: a) providing a composition comprising cardiomyocytes, endothelial cells and fibroblasts, wherein the composition comprises the cardiomyocytes, endothelial cells and fibroblasts at a ratio of 2:2:1 to 6:3:1 , b) culturing the cells in the composition under conditions suitable to produce a cardiac spheroid, c) treating the cardiac spheroid with a candidate agent, and d) determining the effect of the candidate agent on one or more characteristics of the cardiac spheroid.

[0233] 81. An in vitro method for assessing cardiotoxicity of an agent, the method comprising: a) providing a composition comprising cardiomyocytes, endothelial cells and fibroblasts, wherein the composition comprises the cardiomyocytes, endothelial cells and fibroblasts at a ratio of 2:2:1 to 6:3:1 , b) culturing the cells in the composition under conditions suitable to produce a cardiac spheroid, c) treating the cardiac spheroid with the agent, and d) determining the effect of the agent on one or more characteristics of the cardiac spheroid.

[0234] 82. The method of any of embodiments 70-81 , wherein the composition is as defined in any of embodiments 1-21 .

[0235] 83. The method of any of embodiments 79 to 82, wherein the composition is a frozen or cryopreserved composition and wherein the method comprises thawing the composition prior to step (b).

[0236] 84. The method of any of embodiments 79 to 83 where the method is automated.

[0237] EQUIVALENTS AND SCOPE

[0238] Those skilled in the art will appreciate that the present invention is defined by the appended claims and not by the Examples or other description of certain embodiments included herein.

[0239] Similarly, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Unless defined otherwise above, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, genetics and protein and nucleic acid chemistry described herein are those well-known and commonly used in the art, or according to manufacturer’s specifications.

[0240] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

Claims

CLAIMS1 . A composition comprising cardiomyocytes, endothelial cells and fibroblasts, wherein the composition comprises the cardiomyocytes, endothelial cells and fibroblasts at a ratio of 2:2:1 to 6:3:1 , and wherein the composition is frozen.

2. The composition of claim 1 , wherein the mixture comprises the cardiomyocytes, endothelial cells and fibroblasts at a ratio of 3:2:1 to 5:2:1 .

3. The composition of claim 1 or 2, wherein the mixture comprises the cardiomyocytes, endothelial cells and fibroblasts at a ratio of 4:2:1.

4. The composition of any preceding claim, further comprising a cryoprotectant.

5. The composition of claim any preceding claim, wherein the composition consists of the cardiomyocytes, endothelial cells, fibroblasts and cryoprotectant.

6. The composition of any preceding claim wherein the composition is cryopreserved.

7. The composition of any preceding claim, wherein at least 90% of the cardiomyocytes express MLC2V, wherein the composition: a) is not a cardiac organoid or cardiac model; or b) is a cell suspension.

8. The composition of any preceding claim, wherein at least 90% of the cardiomyocytes are ventricular cardiomyocytes, wherein the composition: a) is not a cardiac organoid or cardiac model; or b) is a cell suspension.

9. A container comprising the composition of any preceding claim.

10. A kit comprising the composition of any of claims 1-8 and a cell culture medium.

11. A method for preparing a composition, the method comprising: a) providing a plurality of iPSCs, b) independently differentiating the plurality of iPSCs into cardiomyocytes, endothelial cells and fibroblasts, c) dissociating the differentiated cardiomyocytes, endothelial cells and fibroblasts into single cells; d) combining the cardiomyocytes, endothelial cells and fibroblasts at a ratio of 2:2:1 to 6:3:1 to form a cell suspension; and e) freezing the cell suspension.

12. The method of claim 11 , wherein the cardiomyocytes, endothelial cells and fibroblasts are combined at a ratio in the range of 3:2:1 to 5:2:1.

13. The method of claim 11 or 12 wherein the cardiomyocytes, endothelial cells and fibroblasts are combined at a ratio of 4:2:1.

14. The method of any of claims 11 to 13, wherein the plurality of iPSCs are obtained from a single donor.

15. The method of any of claims 11 to 13, wherein the plurality of iPSCs are obtained from multiple donors.

16. The method of any of claims 11 to 15, wherein in step e) the cell suspension is cryopreserved.

17. A method for producing a cardiac spheroid, the method comprising: a) thawing the product of any of the methods according to any of claims 11 to 16 or the composition of any of claims 1 to 8, and b) culturing the thawed cells under conditions suitable to produce a cardiac spheroid, whereby cardiac spheroids are formed.

18. The method of claim 17, wherein the product or composition is a cryopreserved product or composition prior to step (a).

19. An in vitro method for assessing toxicity of an agent, the method comprising: a) forming cardiac spheroids by a method according to claims 17 or 18, b) treating the cardiac spheroid with the agent, and c) determining the effect of the agent on one or more characteristics of the cardiac spheroid.

20. An in vitro method for drug screening, the method comprising: a) forming cardiac spheroids by a method according to claims 17 or 18, b) treating the cardiac spheroid with a candidate agent, and c) determining the effect of the candidate agent on one or more characteristics of the cardiac spheroid.

21. An in vitro method for assessing cardiotoxicity of an agent, the method comprising:a) forming cardiac spheroids by a method according to claims 17 or 18, b) treating the cardiac spheroid with the agent, and c) determining the effect of the agent on one or more characteristics of the cardiac spheroid.

22. The method of any of claims 19-21 , wherein said determining comprises determining the effect of the agent on: a) the beats per minute of the cardiac spheroid, b) the mitochondrial function of the cardiac spheroid, and / or c) the structure of the cardiac spheroid.

23. The method of any one of claims 19 to 22, wherein the method is automated.

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