Cardiac cell maturation platforms and related methods

Abstract

Methods and systems for cardiomyocyte maturation are generally described. In some embodiments, immature cardiomyocytes may be incubated with a three-dimensional matrix in an incubation medium and permitted to mature over a period of several weeks. In some embodiments, the three-dimensional matrix may include at least one population of microcarriers. The systems for cardiomyocyte maturation are evaluated for their scalability to meet large-scale global demand for cardiac cell therapies.

Description

CARDIAC CELL MATURATION PLATFORMS AND RELATED METHODSRELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 664,399, filed June 26, 2024, entitled “CARDIAC CELL MATURATION PLATFORMS AND RELATED METHODS,” the entire disclosure of which is hereby incorporated by reference herein in its entirety.FIELD

[0002] Disclosed embodiments are related to cardiac cell therapies and related methods, and more specifically, to scalable platforms for high-throughput cardiomyocyte maturation.BACKGROUND

[0003] Cardiac conditions, such as heart disease and heart failure are some of the most common causes of death and illness around the world. For example, myocardial infarctions typically result in a loss of myocardial tissue, which is naturally replaced with scar tissue. The reduction in contractile tissue reduces the capacity of the myocardium, leading to negative health outcomes. A major challenge in treating cardiac conditions is the slow pace of cardiac tissue regeneration, requiring the need for major surgical intervention, such as heart transplantation. Heart failure procedures are costly, carry great risk, and limited due to organ availability.SUMMARY

[0004] Cardiac cell therapies, such as cardiomyocyte grafts, are useful to treat subjects having injured cardiac tissue (e.g., as the result of heart disease or failure). However, administration of immature cardiomyocyte grafts can sometimes induce arrythmias with severe consequences. Surprisingly, the inventors of the present disclosure have identified methods and compositions which promote maturation of immature cardiomyocytes in a scalable manner, which may be useful for industrial production of mature cardiomyocytes.

[0005] Described herein, in some aspects, is a method that includes providing an incubation medium comprising at least one three-dimensional carrier matrix, and incubating apopulation of immature cardiomyocytes with the at least one three-dimensional carrier matrix under incubation conditions to obtain a population of mature cardiomyocytes.

[0006] In some embodiments, the at least one three-dimensional carrier matrix includes a plurality of microcarriers.

[0007] In some embodiments, the plurality of microcarriers have an average transverse dimension between 100 microns and 300 microns.

[0008] In some embodiments, at least 75 percent of the plurality of microcarriers are substantially spherical.

[0009] In some embodiments, the plurality of microcarriers is substantially optically transparent.

[0010] In some embodiments, the at least one three-dimensional carrier matrix includes at least one of the following materials: silica, polystyrene, hydrogel, cellulose, dextran, gelatin.

[0011] In some embodiments, a concentration of the at least one three-dimensional carrier matrix is at least 5 wt% of the incubation medium.

[0012] In some embodiments, a surface chemistry of the at least one three- dimensional carrier matrix is configured to adhere to at least a portion of the population of immature cardiomyocytes.

[0013] In some embodiments, the at least one three-dimensional carrier matrix is at least partially coated with a high-molecular weight glycoprotein.

[0014] In some embodiments, the high-molecular weight glycoprotein is laminin.

[0015] In some embodiments, the incubation conditions comprise at least one of the following: an incubation duration of at least 1 week, and an incubation temperature below 37°C.

[0016] In some embodiments, the step of incubating the population of immature cardiomyocytes with the at least one three-dimensional carrier matrix includes agitating the population of immature cardiomyocytes and the at least one three-dimensional carrier matrix with a stir tank bioreactor.

[0017] In some embodiments, a substantial portion of the population of immature cardiomyocytes are disposed on the surface of the at least one three-dimensional carrier matrix.

[0018] In some embodiments, the at least one three-dimensional carrier matrix is suspended in the incubation medium.

[0019] In some embodiments, the method further includes extracting the population of mature cardiomyocytes from the at least one three-dimensional carrier matrix, exposing the population of mature cardiomyocytes with a cryoprotective agent, storing the population of mature cardiomyocytes at a cryogenic temperature, and implanting the mature cardiomyocytes into a subject.

[0020] In some embodiments, at least 60% of the population of mature cardiomyocytes expresses cTNT.

[0021] In some embodiments, the incubation conditions comprise at least 10 days of incubating.

[0022] In some embodiments, the yield of the population of mature cardiomyocytes relative to the population of immature cardiomyocytes is 0.1.

[0023] In some embodiments, the immature cardiomyocytes were produced by differentiating induced pluripotent stem cells for 14 days.Described herein, in some aspects, is a cell culture composition that includes a plurality of three-dimensional carrier matrices dispersed in an incubation medium, and a population of immature cardiomyocytes, wherein incubation of the population of immature cardiomyocytes with the plurality of three-dimensional carrier matrices under incubation conditions yields a population of mature cardiomyocytes.

[0024] In some embodiments, the plurality of three-dimensional carrier matrices includes a plurality of microcarriers.

[0025] In some embodiments, the incubation conditions comprise at least one of the following: an incubation duration of at least 1 week, and an incubation temperature below 37 °C.

[0026] In some embodiments, a bioreactor comprising the cell culture composition is provided.

[0027] Described herein, in some aspects, is a method that includes administering a population of mature cardiomyocytes to a subject, wherein the population of mature cardiomyocytes has been produced by incubating a population of immature cardiomyocytes with at least one three-dimensional carrier matrix.

[0028] In some embodiments, the population of immature cardiomyocytes has been incubated on the surface of the at least one three-dimensional carrier matrix.

[0029] In some embodiments, the at least one three-dimensional carrier matrix includes a plurality of microcarriers.

[0030] In some embodiments, the population of immature cardiomyocytes has been incubated under incubation conditions comprising at least one of the following: an incubation duration of at least 1 week, and an incubation temperature below 37 °C.

[0031] In some embodiments, the plurality of microcarriers have at least one of the following properties: an average transverse dimension between 100 microns and 300 microns, at least 75 percent of the plurality of microcarriers being substantially spherical, formed of a substantially optically transparent material, formed of at least one of a silica, polystyrene, hydrogel, cellulose, dextran, gelatin material, a concentration of at least at least 5 wt% of an incubation medium, and a high-molecular weight glycoprotein.

[0032] Described herein, in some aspects, is a composition that includes a population of mature cardiomyocytes, wherein the population of mature cardiomyocytes has been produced by incubating a population of immature cardiomyocytes with at least one three- dimensional carrier matrix.

[0033] In some embodiments, the population of immature cardiomyocytes has been incubated on the surface of the at least one three-dimensional carrier matrix.

[0034] In some embodiments, the at least one three-dimensional carrier matrix includes a plurality of microcarriers.

[0035] In some embodiments, the plurality of microcarriers have at least one of the following properties: an average transverse dimension between 100 microns and 300 microns, at least 75 percent of the plurality of microcarriers being substantially spherical, formed of a substantially optically transparent material, formed of at least one of a silica, polystyrene, hydrogel, cellulose, dextran, and / or gelatin material, a concentration of at least 5 wt% of an incubation medium, and a high-molecular weight glycoprotein.

[0036] In some embodiments, a bioreactor comprising the composition is provided.

[0037] Described herein, in some aspects, is a composition that includes a population of mature cardiomyocytes, wherein the population of mature cardiomyocytes has been extracted from at least one three-dimensional carrier matrix.

[0038] Described herein, in some aspects, is a method that includes providing an incubation medium, and incubating a population of immature cardiomyocytes with the incubation medium under incubation conditions to obtain a population of mature cardiomyocytes.

[0039] It should be appreciated that the foregoing concepts, and additional concepts discussed below, may be arranged in any suitable combination, as the present disclosure is not limited in this respect. Further, other advantages and novel features of the presentdisclosure will become apparent from the following detailed description of various nonlimiting embodiments when considered in conjunction with the accompanying figures.BRIEF DESCRIPTION OF DRAWINGS

[0040] The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures may be represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

[0041] FIG. 1 shows a flow chart of a method of maturing and administering a population of cardiomyocytes to a subject according to one illustrative embodiment;

[0042] FIG. 2 shows an image of a population of cardiomyocytes arranged on the surface of microcarriers within a stir tank bioreactor system;

[0043] FIG. 3 shows an image of a population of cardiomyocytes maturing over time within a stir tank bioreactor system;

[0044] FIG. 4 shows another image of a population of cardiomyocytes maturing over time within a stir tank bioreactor system according to another illustrative embodiment;

[0045] FIG. 5 shows a cell count of the illustrative experiment of FIG. 4;

[0046] FIG. 6 shows an image of a cardiomyocyte maturation platform employing the use of a fixed-bed bioreactor;

[0047] FIG. 7 shows an image of a cardiomyocyte maturation platform employing the use of cell aggregates; and

[0048] FIG. 8 shows the comparative viability performance of systems depicted in FIGs. 3-7.DETAILED DESCRIPTION

[0049] Described herein, in some aspects, are methods and compositions useful for preparing cardiac cell therapies. Cardiac cell therapies generally refer to stem-cell derived exogenous cells that are administered to the heart of a subject (e.g., in the form of a graft) in order to repair cardiac tissue in need thereof. Though cardiac cell therapies hold great promise for treating cardiac injuries, their use in large animal test subjects (e.g., pigs) has faced a number of obstacles, including the emergence of graft-induced arrythmias. Large animal test subjects to which cardiac grafts are administered often experience arrythmias of varying frequency and severity, some of which cannot be treated with antiarrhythmic drugs orcardioversion. In many instances, cardiac cell therapies comprising immature cardiomyocytes are more likely to induce graft-induced arrythmias as compared to mature cardiomyocytes.

[0050] While use of pluripotent stem-cell derived cardiomyocytes that have sufficiently undergone a maturation process prior to introduction to a subject can reduce the likelihood of negative outcomes, large-scale production of mature cardiomyocytes has been challenging . In some aspects, the inventors have recognized the benefits associated with scalable platforms for stem-cell derived cardiomyocyte maturation to meet patient demand. Such platforms may facilitate the maturation of a large number of stem-cell derived cardiomyocytes in parallel, which may lower the cost of operation through scale while ensuring substantially uniform and consistent cardiomyocyte functionality across the platform. In some instances, such platforms may be substantially automated, further reducing the costs of operation, and therefore dose production. Of course, instances in which different benefits are offered by the systems and methods disclosed herein are also possible.

[0051] Described herein, in some aspects, are methods and compositions useful for large-scale preparation of cardiac cell therapies. In some aspects, systems described herein comprise a three-dimensional carrier matrix incubated along with a population of immature cardiomyocytes in solution. In some embodiments, the three-dimensional carrier matrix can be used to mimic the natural maturation physiology. Cardiomyocytes may then be harvested from the solution and prepared for injection into the patient for cardiac cell therapy. The systems of the present disclosure may be capable of producing large numbers of mature cardiomyocytes for cell therapies in a uniform manner, with reduced costs due to economies of scale, and benefits of automation. In some embodiments, the systems of the present disclosure may enable sampling of the cells during the maturation process to test for proper maturation.

[0052] In some embodiments, a population of immature stem-cell derived cardiomyocytes may be matured with one or more maturation platforms. The immature cardiomyocytes may be derived from an induced pluripotent stem cell (iPSC). The immature cardiomyocytes may be introduced into the maturation platforms and incubated with a three- dimensional carrier matrix. In some embodiments, the three-dimensional carrier matrix may include a population of microcarrier particles suspended in solution. In some embodiments, the three-dimensional carrier matrix may include a fixed-bed grid. In some embodiments, the three-dimensional carrier matrix may include a cell aggregate of cardiomyocytes and / or cardiac fibroblasts. The three-dimensional carrier matrix may facilitate the maturation process by mimicking the natural maturation physiology.Microcarriers

[0053] In some embodiments, a maturation platform may employ the use of a three- dimensional carrier matrix in the form of a population of microcarriers. In some instances, the microcarriers are suspended in solution and / or are statically or otherwise bound to a fixed- bed surface. In some embodiments, cardiomyocytes may adhere to (either through a particular binding mechanism, through static binding, and / or through any other means, as described in further detail below) to the microcarriers. The microcarriers may retain the cardiomyocytes in any suitable manner known in the art.

[0054] In some embodiments, cardiomyocytes may be situated on the surfaces of the microcarriers. In other embodiments, cardiomyocytes may be situated within interstitial surfaces of the microcarriers and / or the three-dimensional carrier matrices. In other embodiments, cardiomyocytes may be situated on both the surface and within interstitial surfaces of the microcarriers, it should be appreciated that the cardiomyocytes may populate any surface relative to the microcarriers that may facilitate the maturation process.

[0055] In embodiments where suspended microcarriers are employed, the microcarriers may be free to migrate throughout the incubation solution. This migration, which may be a result of Brownian noise and / or agitation of the solution (e.g., in a stir tank bioreactor), may improve the access of the cardiomyocytes to compounds in the solution to help the maturation process. As microcarriers flow through the incubation solution, they may reduce the likelihood of depletion zones within the fluid and ensure sufficient nutrient delivery to the cardiomyocytes.

[0056] Each of the microcarriers of the population may have three-dimensional geometry, such that the population of microcarriers may have an overall enhanced surface area compared to a flat surface. This increased surface area may enable a greater yield of mature cardiomyocytes in a reduced volumetric footprint, which may in turn, enhance the density and efficiency of the maturation platform. In some embodiments, the use of a population of three-dimensional microcarriers may promote maturation through the development of synchronous beating behavior. The morphology (e.g., spherical) of the microcarriers may facilitate the maturation of the cardiomyocytes.

[0057] In some embodiments, the microcarriers may be arranged at a particular concentration of the incubation solution. The concentration may be optimized to facilitate maturation of a large number of cardiomyocytes while still maintaining suitable maturation conditions. The concentration of microcarriers may be dependent on a variety of factors, suchas size of microcarriers, material composition of microcarriers, porosity and / or hygroscopicity, and / or any other factor. Thus, although examples of microcarrier concentrations are described herein, it should be appreciated that the microcarriers may be employed at any suitable concentration to facilitate maturation, as the present disclosure is not so limited.

[0058] In some illustrative embodiments, the microcarriers may consist of greater than or equal to 2 wt%, 5 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, and / or less than or equal to 25 wt%, 20 wt%, 15 wt%, 10 wt%, 5 wt%, 2 wt%, and / or combinations of ranges, including, but not limited to, between 2 wt% and 25 wt%, between 5 wt% and 15 wt%, and / or any other suitable range or combination of ranges of weight percent of the incubation solution. Of course, microcarrier concentrations greater than and less than the non-limiting ranges noted above are also contemplated, as the present disclosure is not so limited.

[0059] In some embodiments, one or more of the microcarriers may be formed of a core material and a coating material. The coating material may serve to induce binding or adhesion between the microcarrier surface (and / or interstitial surfaces between microcarriers, and / or interstitial spaces of a microcarrier) and the cardiomyocytes. In some embodiments, the microcarriers may not include any coating material when the core carrier material may itself induce binding with the microcarrier. In some embodiments, microcarriers may be formed of a polymeric material. In other embodiments, microcarriers may be formed of a silica-based material. In some embodiments, microcarriers may be formed of both a polymer and a silica-based material. Microcarriers may be formed of core materials including, but not limited to, polystyrene, cellulose, dextran, silica, titanium dioxide, gelatin, polyethylene, collagen, polyesters, latex, hydrogels, and / or any other suitable material or combination of materials. It should be appreciated that microcarriers may be formed of any suitable material or combination of materials to facilitate the cardiomyocyte maturation process in solution.

[0060] In some embodiments, the microcarriers may have a coating with surface features that facilitate binding with the cardiomyocytes. The coatings may range from a monolayer in thickness (e.g., a self-assembled monolayer) to a thicker coating for retaining the cardiomyocytes. In non-limiting embodiments, microcarriers may have surface features including, but not limited to, cationic amine groups, cationic charge groups, collagen, silica, recombinant fibronectin, hydroxyl groups, positive charge groups, positively charged resin groups (e.g., diethylaminoethyl), gelatin, negative charge groups, glycoproteins, neutral charge groups, combinations thereof, and / or any other suitable surface features to help retain the cardiomyocytes in place during the maturation process. In one non-limiting example,microcarriers are coated with a high-molecular weight glycoprotein such as laminin (e.g., LN521) at a concentration of approximately 10 pg / mL, although other concentrations are also contemplated. It should be appreciated that in some embodiments, the entire population of microcarriers may substantially have the same coating, while in other embodiments, different sub-populations of the microcarriers may have different coatings. In some embodiments, microcarriers may be partially coated, while in other embodiments, microcarriers may be entirely coated.

[0061] In some embodiments, the material composition of the microcarriers may enable visualization of the microcarriers and the cardiomyocytes. For example, in some embodiments, the microcarriers may be substantially optically transparent, such that they may be readily viewable through optical imaging methodologies. In other embodiments, the microcarriers may have one or more optical tags (e.g., fluorescent tags) to enable their visualization. Thus, in some embodiments, the microcarriers may not be optically transparent. Microcarriers having opaque and / or translucent optical properties are also contemplated.

[0062] The microcarriers of the present disclosure may be any suitable size to facilitate maturation of the cardiomyocyte. The microcarrier size may be tuned in accordance with other properties of the maturation platform, such as the concentration of microcarriers, the incubation conditions, the surface chemistry of microcarriers, and / or any other suitable property. It should be appreciated that the size of the microcarriers (along with the concentration, as described above) may be selected through a calculation of the surface area needed for a desired output yield. Additionally, the microcarrier size may be selected to best mimic natural physiology. It should be appreciated that certain ranges of curvatures (in the case of rounded microcarriers) may induce more accurate maturation and beating behavior compared to others. For examples, microcarriers with curvatures significantly larger than the size of the cardiomyocyte may seem to be flat surfaces to the cardiomyocytes. Thus, the size of the microcarriers may be determined through a number of factors. Accordingly, the sizes disclosed herein are non-limiting, such that other suitable microcarrier sizes may be employed to facilitate and support maturation.

[0063] In some embodiments, a population of microcarriers may have an average transverse dimension (e.g., an average diameter for rounded microcarrier) greater than or equal to approximately 20 microns, 50 microns, 100 microns, 150 microns, 200 microns, 250 microns, 300 microns, 400 microns, 500 microns, 750 microns, 1 mm, 2 mm, and / or any other suitable size. The population of microcarriers may have an average transverse dimension less than or equal to approximately 2 mm, 1 mm, 500 microns, 400 microns, 300microns, 250 microns, 200 microns, 150 microns, 100 microns, 50 microns, 20 microns, and / or any other suitable size. Combinations of the foregoing, including microcarriers with an average transverse dimension between approximately 100 microns and 500 microns, 20 microns and 2 mm, 100 microns and 300 microns and / or others, are also contemplated. As noted earlier, the microcarrier size may be dependent on a variety of factors and parameters of the maturation system, such that the present disclosure is not limited by the microcarrier size. Thus, ranges of microcarrier sizes both greater than and less than those noted above are contemplated. It should be appreciated that the size of the microcarriers may be compatible with the optical methods employed to sample the maturation process.

[0064] It should also be appreciated that the size of the microcarriers may also be selected in accordance with the external forces used for operation of the system. In embodiments where the microcarriers are driven by gravity or other body forces through the incubation solution, the microcarriers may be sufficiently sized to react to the external force in order to move through fluid and expose the cardiomyocytes to nutrients in the solution. In some embodiments, the size of the microcarriers may be determined by the viscosity of the testing sample fluid.

[0065] In some embodiments, the microcarriers may have a three-dimensional geometry. Non-limiting examples of geometries include spherical, cylindrical, elliptical, conical, ovoid, cubical, tube-like, flat- sided, irregular, oblate, football- shaped, discoid, slender, and polyhedral, combinations thereof, and / or any other appropriate shape. In some embodiments, the microcarriers may be formed of a combination of geometric shapes. The microcarriers of the present disclosure may be symmetric or asymmetric. In some embodiments, the microcarriers may be uniform in shape, whereas in others, the microcarriers population may include more than one shape. It should be appreciated that any of the microcarriers of the present disclosure may have any suitable shape or combination of shapes, compositions, or patterns as the present disclosure is not limited by microcarrier shape or composition.

[0066] It should be appreciated that the microcarrier population(s) may be formed of any material, size, shape, type, configuration, and / or other appropriate property of a desired microcarrier to facilitate maturation, as the systems described herein are not limited to any type of microcarrier.

[0067] In some embodiments, the microcarriers may be substantially monodisperse in their size distribution. In other embodiments, the maturation platforms may employ more than one population of microcarriers with differing microcarrier sizes. In other embodimentsstill, a single population of microcarriers may have a broad range of particle sizes. It should be appreciated that any suitable size distribution conducive to cardiomyocyte maturation may be employed as the present disclosure is not so limited.

[0068] In some embodiments, the microcarriers may be nonporous, microporous, or macroporous. In some embodiments, the pores of the microcarriers may be situated to permit the passage of cardiomyocytes through, whereas in other embodiments, the cardiomyocytes may not pass through the pores of the microcarriers. Solid, nonporous microcarriers are also contemplated.

[0069] In some embodiments, the microcarriers of the present disclosure may be employed in a fixed-bed bioreactor system. In such embodiments, the cardiomyocytes may be oriented within a polymeric mesh within a well-plate, arranged within a fixed-bed bioreactor. Conventional fixed-bed bioreactor systems are formed of known polymeric surfaces which facilitate retention of cardiomyocytes while enabling the monitoring of oxygen and pH during the maturation process.

[0070] In some embodiments, the microcarriers of the present disclosure may be employed in a stir tank bioreactor system. In such embodiments, the microcarriers may be freely mobile through the incubation fluid and able to deliver nutrients to the maturing cardiomyocytes. Such systems enable in situ monitoring of oxygen and pH levels, and additionally, enable in-process sampling to inspect the maturation process. The stir tank bioreactor may serve to agitate the solution for more effective fluid transport and a reduced risk of nutrient depletion zones.Incubation Conditions

[0071] It should be appreciated that the cardiomyocytes and three-dimensional carrier matrix of the present disclosure may be incubated within a maturation solution (also referred to as an incubation medium) to facilitate their maturation process. Such a solution may be aqueous and composed of materials and nutrients necessary for the maturation process. The solution may include any suitable component, including, but not limited to, glucose, pH indicator(s), buffer solutions, hormones, and / or one of more growth materials. In one nonlimiting example, an incubation solution may include GW7647, dexamethasone, thyroid hormone T3, a palmitate, and glucose. In some embodiments, the incubation medium may include shear stress protectants to reduce the likelihood of cell damage through shear stress.

[0072] In some embodiments, the cardiomyocytes and three-dimensional carrier matrix of the present disclosure may be agitated during the maturation process. The agitation may be carried out with any conventional agitation mechanisms, such as a roller bottle apparatus, a stir tank bioreactor, an orbital mixer, and / or any other suitable system. The cardiomyocytes may be agitated at any suitable rate compatible with the maturation process. For example, the agitation rate may be low enough to substantially reduce the risk of shear stress on the cardiomyocytes while being high enough to permit the microcarriers (and cardiomyocytes loaded on the microcarriers) to flow through the incubation medium.

[0073] In some embodiments, the cardiomyocytes may be incubated for the duration of the maturation process. The cardiomyocytes may be incubated for greater than, less than, or equal to approximately 1 week, 10 days, 2 weeks, and / or 3 weeks. The incubation duration can be selected based on a variety of other factors, and thus is a non-limiting parameter of the incubation conditions.

[0074] It should be appreciated that any suitable rotation rate and incubation duration may be employed as the present disclosure is not limited by the incubation conditions. The cardiomyocytes of the present disclosure may be incubated at physiologically relevant temperatures (e.g., 37 °C), although other temperatures are also contemplated.Cardiomyocytes

[0075] In some embodiments, cardiomyocytes are cells having cardiomyocyte lineage, including, but not limited to ventricular cardiomyocytes, atrial cardiomyocytes, and / or cardiac smooth muscle cells. Cardiomyocytes can be understood to be cells at any stage of cardiomyocyte development without restriction, unless stated otherwise.

[0076] In some embodiments, cardiomyocytes are cardiomyocyte precursors. In some embodiments, a cardiomyocyte precursor is any precursor (e.g., progenitor) cell with the capacity to differentiate into a cardiomyocyte cell. In some embodiments, a cardiomyocyte precursor is or is derived from a stem cell (e.g., a human stem cell). In some embodiments, a cardiomyocyte precursor is or is derived from an induced pluripotent stem cell (iPSC). In some embodiments, a cardiomyocyte precursor is or is derived from a human pluripotent stem cell (hPSC). In some embodiments, a cardiomyocyte precursor is or is derived from an embryonic stem cell (ESC) (e.g., a human embryonic stem cell). As described herein, cardiomyocyte precursors may have the following characteristics: (1) positive / high expression of one or more of the following markers: stage- specific embryonic antigen 3and / or 4 (SSEA3 / 4), podocalyxin (TRA-l-60). octamer-binding transcription 3 and / or 4 (OCT3 / 4). homeobox protein NANOG (NANOG). and sex-determining region Y-box 2 (.8'0X2); (2) capable of self-replication. In some embodiments, cardiomyocyte precursors are capable of differentiation into all three germ layers (e.g., endoderm, mesoderm, ectoderm) or derivatives thereof. In some embodiments, cardio myocyte precursors are mesoderm cells induced from stem cells which have been contacted by induction media (e.g., a Wnt agonist). In some embodiments, cardiomyocyte precursors are cardiac progenitors differentiated from mesoderm cells which have been contacted by cardiomyocyte differentiation media (e.g., contacted by a Wnt antagonist). In some embodiments, cardiomyocyte precursor cells are not excitable.

[0077] In some embodiments, cardiomyocytes are immature cardiomyocytes. In some embodiments, an immature cardiomyocyte is any cell which is in any stage of differentiation to become a mature cardiomyocyte and comparable to cardiac muscle cells in early stages of fetal development. In some embodiments, an immature cardiomyocyte is an immature human cardiomyocyte. In some embodiments, an immature cardiomyocyte is derived from a stem cell (e.g., a human stem cell). In some embodiments, an immature cardiomyocyte has been contacted with one or more differentiation agents (e.g., RPMI-1640 supplemented with B27). In some embodiments, an immature cardiomyocyte has been contacted with a maturation media (e.g., RPMI-1640 supplemented with B27 without insulin]). Immature cardiomyocytes are generally not quiescent (e.g., have not entered cell cycle arrest). In some embodiments, cardiomyocytes are immature ventricular cardiomyocytes. In some embodiments, cardiomyocytes are immature atrial cardiomyocytes. In some embodiments, cardiomyocytes are immature cardiac smooth muscle cells. As described herein, immature cardiomyocytes may exhibit the following characteristics within about 14 days from mesoderm induction: (1) cluster of differentiation 36 (CD36) negative / low (relative to a mature cardiomyocyte), myosin regulatory light chain 2v (MLC2v, also known as MYL2) negative / low (relative to a mature cardiomyocyte), and myosin regulatory light chain 2a MLC2a, also known as MYL7) positive / high (relative to a mature cardiomyocyte), e.g., using flow cytometric analysis; and (2) capable of spontaneous action potentials and contraction. In some embodiments, an immature cardiomyocyte expresses NK2 homeobox 5 (NKX2-5). In some embodiments, an immature cardiomyocyte has positive / high expression of one or more positive / high genetic or molecular markers provided in Table 1 and has negative / low expression of one or more negative genetic or molecular markers provided in Table 1. In some embodiments, animmature cardiomyocyte has one or more electrophysiological properties according to Table 1.

[0078] In some embodiments, cardiomyocytes are mature cardiomyocytes. In some embodiments, a mature cardiomyocyte is any terminally differentiated (e.g., quiescent) cardiac muscle cell. In some embodiments, a mature cardiomyocyte is derived from an immature cardiomyocyte which has been contacted by a maturation cocktail (e.g., RPMI- 1640 supplemented with b27 minus insulin). In some embodiments, a mature cardiomyocyte is derived from a stem cell (e.g., a human stem cell). In some embodiments, the cardiomyocytes are matured in vivo (e.g., in a subject). In some embodiments, the cardiomyocytes are matured in vitro. In some embodiments, cardiomyocytes are mature ventricular cardiomyocytes. In some embodiments, cardiomyocytes are mature atrial cardiomyocytes. In some embodiments, cardiomyocytes are mature cardiac smooth muscle cells. In some embodiments, mature cardiomyocytes exhibit the following characteristics within about 51 days from mesoderm induction: (1) cardiac muscle troponin T (cTNT, also referred to synonymously herein as TNNT2) positive / high, MLC2v positive / high, and downregulated MLC2a (relative to an immature cardiomyocytes), e.g., using flow cytometric analysis; and (2) capable of contraction with application of an external stimulus (e.g., electrical stimulus). In some embodiments, mature cardiomyocytes are TNI positive / high. In some embodiments, a mature cardiomyocyte has positive / high expression of one or more positive / high genetic or molecular markers provided in Table 1 and has negative / low expression of one or more negative genetic or molecular markers provided in Table 1. In some embodiments, a mature cardiomyocyte has one or more electrophysiological properties according to Table 1.Table 1. Non-limiting characteristics of cardiomyocytes throughout development

[0079] Non-limiting examples of cardiomyocyte differentiation and maturation procedures can be found in PCT Publication No.: WO 2014 / 200339, PCT Publication No.: WO 2017 / 039445, PCT Publication No.: WO 2020 / 227232, U.S. Publication No.: US 2020 / 0407687, each of which are incorporated herein by reference.

[0080] Expression of a marker by a cell can be measured any number of ways, but generally refers to quantification of the presence (or absence) of a distinct signal corresponding to the marker in or on the cell, compared to a control and / or baseline. In some instances, expression of a marker is measured as an absolute quantity (e.g., weight, molar amount, concentration). In some instances, expression of a marker is expressed as a relative quantity (e.g., fold-increase, fold-decrease, percent). In some instances, expression of a marker in a cell is measured in a semi-quantitative manner (e.g., “high” or “low” fluorescence, as used in flow cytometry).

[0081] Cells can be understood to be “positive” or have “high expression” of one or more markers if they express the marker at or above a baseline level. For example, a cell may be understood to be positive (+) for a marker which is detected in the cell. A cell may beunderstood to have “high” expression for a marker when the quantity (e.g., absolute, relative, semi-quantitative) of the marker in the cell is above some baseline or threshold (e.g., population average, control cell). Cells can be understood to be “negative” or have “low expression” of one or more markers if they do not express the marker at or above a baseline level. For example, a cell may be understood to be negative (-) for a marker which is not detected in the cell. A cell may be understood to have “low” expression for a marker when the quantity (e.g., absolute, relative, semi-quantitative) of the marker in the cell is below some baseline or threshold (e.g., population average, control cell).

[0082] Methods of determining expression of a marker by a cell are known to those of skill in the art and are dependent on the marker of interest and / or its form (e.g., surface protein encoded by a gene, transcript of a gene). In a non-limiting example, presence of a marker is determined using flow cytometry, immunohistochemistry, immunofluorescence, polymerase chain reaction (e.g., reverse transcription PCR), fluorescence-activated cell sorting (FACS), microarrays, and / or ribonucleic acid (RNA) sequencing, such as single cell RNA sequencing (scRNA-seq).Cardiac Cell Therapies

[0083] Cells prepared (e.g., matured) by a method disclosed herein may be used for cardiac cell therapy. Cardiac cell therapies (e.g., cardiac grafts) of the disclosure include, in some embodiments, cellular compositions comprising a plurality of cardiomyocytes and physiologically acceptable medium. Cardiac cell therapies may comprise one or more types of cardiomyocytes (e.g., ventricular cardiomyocytes, atrial cardiomyocytes, and / or smooth muscle cells) at one or more stages of development (e.g., mature cardiomyocytes and / or immature cardiomyocytes). In some embodiments, the cardiomyocytes are ventricular cardiomyocytes. In some embodiments, cardiomyocytes are immune evading or hypoimmune. In some embodiments, cardiac cell therapies (e.g., cardiac grafts) further comprise vascular cells and / or cardiac cells. In some embodiments, cardiac cell therapies (e.g., cardiac grafts) further comprise endothelial cells, conduction cells, pacemaker cells, and / or fibroblasts. Cardiac cell therapies may comprise any physiologically acceptable medium helpful for administering, adhering, growing, and / or maintaining the cardiac cell therapy (e.g., cardiac graft) in a subject. Physiologically acceptable media for administration of tissues are known in the art.

[0084] In some embodiments, cardiac cell therapies are administered to a subject having experienced cardiac injury, including, but not limited to, damage to cells and tissue of the heart (e.g., cardiomyocytes). Cardiac injury may be caused by a number of factors, including, but not limited to, heart disease (e.g., coronary heart disease, cardiomyopathy, endocarditis, congenital cardiovascular defects, congestive heart failure), medications, and non-cardiac diseases (e.g., high blood pressure, diabetes, viruses). In some embodiments, cardiac injury is caused by heart failure (e.g., HFrEF). In some embodiments, cardiac injury is caused by myocardial infarction. In some embodiments, cardiac injury is caused by ischemia. In some embodiments, cardiac injury comprises an injured ventricle (e.g., left ventricle). In some embodiments, a cardiac cell therapy (e.g., a cardiac graft) is administered to a subject in need thereof, such as a subject having experienced cardiac injury. A subject may be any mammal, including, but not limited to, mice, rats, guinea pigs, hamsters, pigs, cows, sheep, goats, horses, and primates, including humans.

[0085] In some embodiments, a cardiac graft is administered to a subject which may have or may receive mechanical circulatory support before, after, or at the time of receiving the cardiac graft. In some embodiments the mechanical circulatory support may be a Left Ventricular Assist Device (LVAD), a Right Ventricular Assist Device (RVAD), a Bi Ventricular Assist Device (BiVAD), Extra Corporeal Membrane Oxygenation (ECMO), or Implantable Cardiac Defibrillator (ICD), or a combination thereof. In certain embodiments, the ICD is linked by a biventricular pacer. In some embodiments, mechanical circulatory support may be an intravascular, microaxial blood pump (such as Impella 5.0). In some embodiments, this mechanical circulatory support is ceased after the subject receives a cardiac graft. In some embodiments, the mechanical circulatory support is used or implanted on or to the subject prior to the administration of the cardiac graft.

[0086] In some embodiments, a cardiac cell therapy is a cardiac graft. In some embodiments, a cardiac graft comprises about 100 million to about 1 billion cardiomyocytes. In some embodiments, a cardiac graft comprises about 200 million to about 1 billion cardiomyocytes. In some embodiments, a cardiac graft comprises about 300 million to about 1 billion cardiomyocytes. In some embodiments, a cardiac graft comprises about 400 million to about 1 billion cardiomyocytes. In some embodiments, a cardiac graft comprises about 500 million to about 1 billion cardiomyocytes. In some embodiments, a cardiac graft comprises about 600 million to about 1 billion cardiomyocytes. In some embodiments, a cardiac graft comprises about 700 million to about 1 billion cardiomyocytes. In some embodiments, acardiac graft comprises about 800 million to about 1 billion cardiomyocytes. In some embodiments, a cardiac graft comprises about 900 million to about 1 billion cardiomyocytes.

[0087] It should be appreciated that the maturation platforms described herein may employ any concentration of cardiomyocyte precursors and / or immature cardiomyocyte. In some embodiments, the seeding conditions prior to maturation may be any suitable density associated with the maturation process, including, but not limited to, greater than or equal to approximately 10k cells / cm2, 30k cells / cm2, 50k cells / cm2, 100k cells / cm2, 150k cells / cm2, 200k cells / cm2, and / or less than or equal to approximately 200k cells / cm2, 150k cells / cm2, 100k cells / cm2, 50k cells / cm2, 30k cells / cm2, 10k cells / cm2, and / or any other suitable seeding concentration. Combinations of the foregoing ranges, including cell seeding concentrations between 10k cells / cm2and 200k cells / cm2are also contemplated, as well as ranges above and below the aforementioned ranges, as the present disclosure is not limited by the cell seeding concentration of the maturation process.Sampling

[0088] In some embodiments, the cardiomyocyte solution may be inspected during the maturation process to evaluate the process and alter conditions if needed. Thus, the cardiomyocytes and the three-dimensional carrier matrix may be tagged with one or more markers (e.g., fluorescent markers or tags) for visualization. In some embodiments, the sampling process may involve the removal of a small portion (e.g., 5 mL) of the incubation medium for imaging and evaluation.Verification of Maturation & Yield

[0089] In some embodiments, the cardiomyocytes may exhibit properties that indicate the stage of their maturation. Such properties may be evaluated while sampling throughout the maturation process, and / or may be evaluated at the terminus of the maturation process to verify the process. In some embodiments, mature cardiomyocytes may be evaluated through the expression of specific biomarkers. Non-limiting markers of mature cardiomyocytes include CD36. LDLR. FABP3. ACSLE COX6A2. ATP5A1 , COX7A1, CKMT2, SOD2, ASB2, FGF12, CPT1B, MLYCD, PDK4. TCAP. KCNJ2. ATP2A2. ADRB1, UCP2. UCP3.TP53INP2. NMRK2, NPPB, HSPB6, KLF9, CEBPB, MASP1, HRC, ACSL1, ESRRA, and / or SCD, among other biomarkers. In some embodiment, mature cardiomyocytes are evaluated for the positive / high expression of any one or more of potassium voltage-gated channel,shaker-related subfamily, member 5 (KCNA5; also known as Kvl.5), potassium inwardly rectifying channel subfamily J Member 3 KCNJ3', also known as G1RK-1 gap junction alpha-5 protein (GVA5; also known as Cx40y and nuclear receptor subfamily 2, group F, member 2 NR2F2', also known as COUP-TFHy. and / or negative / low expression of MLC2v biomarkers. In one embodiment, mature cardiomyocytes are evaluated for the positive / high expression of KCNA5. KCNJ3. GJA5. and NR2F2'. and negative / low expression of MLC2v. In some embodiments, cardiomyocytes may be evaluated based on their expressed of cTNT (i.e., TNNT2).

[0090] In some embodiments, successful maturation may be determined by a minimum percentage of cells expressing one or more biomarkers. For example, a population of cells with 85% expressing cTNT may be considered to have undergone successful maturation. Other ranges of cTNT expression, including, but not limited to, greater than or equal to approximately 50%, 60%, 70%, 80%, 90%, and / or less than or equal to approximately 95%, 90%, 80%, 70%, 60%, 50%, and / or any other suitable percentage of cells expressing cTNT may also be considered to indicate successful maturation.

[0091] In some embodiments, the yield of the maturation process may be evaluated by comparing the size of the population of mature cardiomyocytes to the original immature cardiomyocytes. In some embodiments, cell counting processes may be used to identify population size. It should be appreciated that the yield may depend on a variety of conditions, including incubation medium composition, incubation conditions, immature cardiomyocyte conditions, among many others. In one non-limiting embodiment, a microcarrier maturation platform using a stir tank bioreactor has a yield of 0.1. Embodiments with yields greater than or less than approximately 0.1 are also contemplated. For example, a microcarrier maturation platform, using any of the aforementioned systems, may have a yield of greater than or equal to approximately 0.01, 0.05, 0.1, 0.15, 0.2, 0.3, 0.5, 0.7, 0.8, 0.9, 1, and / or less than or equal to approximately 1, 0.9, 0.8, 0.7, 0.5, 0.3, 0.2, 0.15, 0.1, 0.05, 0.01, and / or any other suitable yield.Purification and Administration

[0092] Cardiac cell therapies (e.g., cardiac grafts) are typically administered directly to the heart of a subject, and may be administered to a subject through any suitable method; non-limiting examples include open surgical approaches (e.g., direct administration to theheart during open heart surgery), minimally invasive approaches (e.g., administration via a cardiac catheter or needle), or percutaneous / intravascular approaches.

[0093] In some embodiments, mature cardiomyocytes may be disengaged from the three-dimensional carrier matrix (e.g., a population of microcarriers) and purified in preparation to be administered to the subject. Any suitable means of purifying the cardiomyocytes may be employed. In some embodiments, cardiomyocytes may be matured for a select duration, after which they may be purified through a process by which they are disengaged from the carrier matrix. In some embodiments, a filtration process may be employed to separate the matrix population from the cardiomyocyte population. The mature cardiomyocyte population may then be treated for administration and loaded on a suitable method for injection into the subject. In some embodiments, the cardiomyocytes may be stored (e.g., either prior to maturation and / or after maturation) at cryogenic temperatures for storage. In such embodiments, a cryoprotective agent may be employed to protect the cells.Scaling Viability

[0094] In some embodiments, it may be desirable for the maturation platform to be scaled up to meet patient demand. Such increases in scale may help reduce costs by increasing yields and enhancing levels of automation. The viability of scale can be measured through a variety of metrics.

[0095] In some embodiments, the maturation platform may include a manual roller bottle process with automatic features. In some embodiments, while the current standard roller bottle methods are viable for cardiomyocyte maturation, there is a need to enhance their commercial viability and scalability. For example, Table 2 below outlines various parameters associated with operating conventional roller bottle processes.Table 2. Operational parameters for conventional roller bottle platforms according to some embodiments.

[0096] To objectively evaluate a number of methods of cardiomyocyte maturation at large yield scales, the inventors have recognized the benefits associated with a viability score. Table 3 below outlines a variety of categories, weights, and scores associated with a minimum viable process (“MVP”) evaluation. In some embodiments, the MVP defines a bioprocess with the minimum requirement ready and in accordance with regulatory compliance standards (e.g., Good Manufacturing Practice (GMP)) technology transfer. Table 3 below may be used to prospectively compare different platforms based on potential and to aid program-related platform selection. Bioprocesses are scored from various weighted categories, scored from 0-3. The higher the MVP score, the better the likelihood for scaling viability. It should be appreciated that the MVP method is a method of evaluating scalability according to some embodiments, and that other methods and measurements may also be employed.Table 3. Non-limiting minimum viability process evaluation table.

[0097] In some embodiments, the maturation platforms described herein may be scaled up to a 2000 L stir tank bioreactor.

[0098] Turning to the figures, specific non-limiting embodiments are described in further detail. It should be understood that the various systems, components, features, and methods described relative to these embodiments may be used either individually and / or in any desired combination as the disclosure is not limited to only the specific embodiments described herein.

[0099] FIG. 1 depicts a method of maturing a population of immature cardiomyocytes, according to some embodiments. In block 100, a population of induced pluripotent stem cell (iPSC) were differentiated into immature cardiomyocytes. It should be appreciated that other forms of stem cells may also be employed. In block 110, a population of immature cardiomyocytes were extracted from the differentiation medium. In block 121, the population of immature cardiomyocytes were introduced into a maturation medium including microcarriers. This solution of microcarriers and cardiomyocytes may be incubated together in a fixed-bed bioreactor, as shown in block 131. Alternatively, the population of immature cardiomyocytes and microcarriers may be incubated together in a stir tankbioreactor with agitation, as shown in block 132. In some embodiments, the population of immature cardiomyocytes may be introduced into a maturation medium, as shown in block 122, and subsequently left to incubate either in a fixed-bed bioreactor, as shown in block 131, or a stir tank bioreactor, as shown in block 132. In other embodiments, the population of immature cardiomyocytes may be introduced into a TideXcell maturation medium, as shown in block 123.

[0100] Regardless of the incubation environment, the cardiomyocytes may be permitted to mature into a population of mature cardiomyocytes under maturation conditions, as shown in block 140. The mature cardiomyocytes may then be extracted from the maturation solution, as shown in block 150, and purified and prepared for administration, as shown in block 160. In some embodiments, the extraction and purification steps may include the filtering out of the three-dimensional carrier matrix. The mature cardiomyocytes may then be administered to the subject to ameliorate heart function, as shown in block 170.

[0101] It should be appreciated that the method shown in FIG. 1 is not liming, and that alternative maturation and administration processes are also contemplated.

[0102] FIG. 2 shows an image of a maturation system 200 with a population of microcarriers in an incubation medium, according to one illustrative embodiment. In FIG. 2, a population of cardiomyocytes 216 are arranged on the surface of a population of microcarriers 214. In some embodiments, the microcarriers 214 may be free to flow through an incubation medium 212. In some embodiments, the system shown in FIG. 2 may employ the use of a stir tank bioreactor, which helps agitate the incubation medium and subsequently, the microcarriers.EXAMPLESExample 1 - Microcarriers in a Stir Tank Bioreactor

[0103] FIG. 3 shows an image of a population of immature cardiomyocytes using microcarriers with a stir tank bioreactor, according to one illustrative embodiment. FIG. 3 depicts a population of cardiomyocytes five days after thawing (following differentiation) loaded on the surface of a population of microcarriers. Here, the immature cardiomyocytes were thawed for three days and incubated at a cell density of approximately 180 cells / cm2with Dextran-based microcarriers at a concentration of approximately 0.9 g / L and glass-based microcarriers at a concentration of approximately 11.1 g / L. The microcarriers were coated through exposure to approximately 10 pg / mL solution of LN521. Thus, the microcarriers ofFIG. 3 include a LN521 coating and are arranged within a well plate within a stir tank bioreactor system. The stir tank bioreactor agitated the medium, permitting greater yields and growth of cardiomyocyte multilayer. The cardiomyocytes of FIG. 3 exhibit initial indications of beating behavior.Example 2 - Microcarriers in a Stir Tank Bioreactor

[0104] FIG. 4 shows a maturation experiment using microcarriers in a stir tank reactor, according to one illustrative embodiment. Here, a population of both plastic microcarriers and Dextran-based microcarriers were prepared through water soaking (plastic) and soaking and rinsing with buffer (Dextran) and subsequently autoclaving. The sterilized microcarriers were then transferred to a 250 mL centrifuge tube, where they were aspirated and washed with buffer (PBS) and exposed to LN521 at a concentration of 1 pg / mL solution. The microcarriers were left in solution overnight at 10 RPM at 4 °C to form coatings. Meanwhile, immature cardiomyocytes were thawed, pelleted, and introduced into a solution with cryoprotectant in media and a rho-kinase inhibitor (ROCKi) to reduce the risk of cell death prior to maturation.

[0105] Following the coating process, the microcarriers were washed with media and ROCKi and introduced into a stir tank reactor through a harvest line for a final concentration of approximately 0.9 g / L. The immature cells were added at a concentration of approximately 110k cells / cm2. The mixture was topped up with media and ROCKi to arrive at a final volume of approximately 220 mL. The cells were allowed to adhere to the microcarriers for approximately four hours, at which time their agitation rate was increased slightly to initiate maturation. FIG. 4 shows the cardiomyocytes at 19 days after thaw.

[0106] FIG. 5 is a measurement of the total live cell number of the experiment from FIG. 4 over a 38-day period. Cell counts were taken by removing 5 mL of culture volume every media exchange up to maturation day 16 (Ml 6). Since cell counts were near the limit of detection and gradually dropping after an apparent peak at M10, sampling was stopped to preserve remaining cells. On maturation day 35, cardiomyocytes were harvested by dissociation with TryLE 10X for 20 minutes. The harvest cell count reflects the total cells in suspension with microcarriers, showing a yield of 0.1. The cardiomyocytes were further processed by straining with a mesh filter to remove microcarriers. Flow cytometry for cardiomyocyte purity marker cTNT was done on the final cell solution, showing a 70%cTNT-positive population. Thus, it was shown that cardiomyocytes may successfully adhere to microcarriers for more than 37 days.Example 3 - Maturation on a Fixed-Bed Bioreactor

[0107] FIG. 6 shows an image of stained cardiomyocytes 330 matured on a fixed-bed bioreactor, according to one illustrative embodiment. The cells are arranged within a polymer mesh 335 inside of a well-plate 325 and operated by the fixed-bed bioreactor to reduce the risk of damage through shear stress.Example 4 - Maturation through Cell Aggregates

[0108] FIG. 7 shows an image of a system 400 wherein cardiomyocytes were grown through a cell aggregate method, according to one illustrative embodiment. The cardiomyocytes may self-aggregate 420 within an incubation medium 410 and subsequently mature.Example 5 - MVP Comparison of Maturation Platforms

[0109] FIG. 8 shows a comparative MVP score (see Table 3 above) for a series of maturation platforms. The size of the points on the graph scale with improved process scalability due to development time and ease of scalability for the cell aggregate model (embodiment from FIG. 5), the microcarrier model (embodiment from FIGs. 4-5), the fixed- bed bioreactor model (embodiment from FIG. 6), and a conventional TideXcell model. The scores are plotted on an x-axis that represents the estimated time to achieve the MVP for the platform.

[0110] As shown in FIG. 8, in some embodiments, the microcarrier embodiment may outperform the manual roller bottle method in terms of the MVP score. Similarly, in some embodiments, cell aggregate embodiments may score higher on the MVP scale compared to the manual roller bottle method.EQUIVALENTS AND SCOPE

[0111] In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, orotherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes aspects in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes aspects in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0112] Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is / are referred to as comprising particular elements and / or features, certain aspects of the invention or aspects of the invention consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those aspects have not been specifically set forth in haec verba herein.

[0113] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one aspect, to A only (optionally including elements other than B); in another aspect, to B only (optionally including elements other than A); in yet another aspect, to both A and B (optionally including other elements); etc.

[0114] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or”1 as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0115] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one aspect, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another aspect, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another aspect, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0116] The embodiments described herein may be embodied as a method, of which an example has been provided. The acts performed as part of the method may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments. It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0117] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that aspects described in this document using an open-endedtransitional phrase (e.g., “comprising”) are also contemplated, in alternative aspects, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the application describes “a composition comprising A and B,” the application also contemplates the alternative aspects “a composition consisting of A and B” and “a composition consisting essentially of A and B.”

[0118] Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different aspects of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0119] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular aspect of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such aspects are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular aspect of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[0120] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific aspects described herein. The scope of the present aspects described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

[0121] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an aspect for a variable herein includes that aspect as any single aspect or in combination with any other aspects or portions thereof. The recitation of an aspect herein includes that aspect as any single aspect or in combination with any other aspects or portions thereof.

[0122] Further, some actions are described as taken by a “user.” It should be appreciated that a “user” need not be a single individual, and that in some embodiments,actions attributable to a “user” may be performed by a team of individuals and / or an individual in combination with computer-assisted tools or other mechanisms.

[0123] While the present teachings have been described in conjunction with various embodiments and examples, it is not intended that the present teachings be limited to such embodiments or examples. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art. Accordingly, the foregoing description and drawings are by way of example only.

[0124] While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present invention is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the scope of the present invention.

Claims

CLAIMSWhat is claimed is:

1. A method comprising: providing an incubation medium comprising at least one three-dimensional carrier matrix; and incubating a population of immature cardiomyocytes with the at least one three- dimensional carrier matrix under incubation conditions to obtain a population of mature cardiomyocytes.

2. The method of claim 1, wherein the at least one three-dimensional carrier matrix comprises a plurality of microcarriers.

3. The method of claim 2, wherein the plurality of microcarriers have an average transverse dimension between 100 microns and 300 microns.

4. The method of any one of claims 2-3, wherein at least 75 percent of the plurality of microcarriers are substantially spherical.

5. The method of any one of claims 2-4, wherein the plurality of microcarriers is substantially optically transparent.

6. The method of any one of claims 1-5, wherein the at least one three-dimensional carrier matrix comprises at least one of the following materials: silica, polystyrene, hydrogel, cellulose, dextran, gelatin.

7. The method of any one of claims 1-6, wherein a concentration of the at least one three-dimensional carrier matrix is at least 5 wt% of the incubation medium.

8. The method of any one of claims 1-7, wherein a surface chemistry of the at least one three-dimensional carrier matrix is configured to adhere to at least a portion of the population of immature cardiomyocytes.

9. The method of any one of claims 1-8, wherein the at least one three-dimensional carrier matrix is at least partially coated with a high-molecular weight glycoprotein.

10. The method of claim 9, wherein the high-molecular weight glycoprotein is laminin.

11. The method of any one of claims 1-10, wherein the incubation conditions comprise at least one of the following: an incubation duration of at least 1 week, and an incubation temperature below 37°C.

12. The method of any one of claims 1-11, wherein the step of incubating the population of immature cardiomyocytes with the at least one three-dimensional carrier matrix comprises agitating the population of immature cardiomyocytes and the at least one three-dimensional carrier matrix with a stir tank bioreactor.

13. The method of any one of claims 1-12, wherein a substantial portion of the population of immature cardiomyocytes are disposed on the surface of the at least one three-dimensional carrier matrix.

14. The method of any one of claims 1-13, wherein the at least one three-dimensional carrier matrix is suspended in the incubation medium.

15. The method of any one of claims 1-14, further comprising: extracting the population of mature cardiomyocytes from the at least one three- dimensional carrier matrix; exposing the population of mature cardiomyocytes with a cryoprotective agent; storing the population of mature cardiomyocytes at a cryogenic temperature; and implanting the mature cardiomyocytes into a subject.

16. The method of any one of claims 1-15, wherein at least 60% of the population of mature cardiomyocytes expresses cTNT.

17. The method of any one of claims 1-16, wherein the incubation conditions comprise at least 10 days of incubating.

18. The method of any one of claims 1-17, wherein the yield of the population of mature cardiomyocytes relative to the population of immature cardiomyocytes is 0.1.

19. The method of any one of claims 1-18, wherein the immature cardiomyocytes were produced by differentiating induced pluripotent stem cells for 14 days.

20. A cell culture composition comprising: a plurality of three-dimensional carrier matrices dispersed in an incubation medium; and a population of immature cardiomyocytes, wherein incubation of the population of immature cardiomyocytes with the plurality of three-dimensional carrier matrices under incubation conditions yields a population of mature cardiomyocytes.

21. The cell culture composition of claim 20, wherein the plurality of three-dimensional carrier matrices comprises a plurality of microcarriers.

22. The cell culture composition of claim 20 or 21, wherein the incubation conditions comprise at least one of the following: an incubation duration of at least 1 week; and an incubation temperature below 37 °C.

23. A bioreactor comprising the cell culture composition of any one of claims 20-22.

24. A method comprising administering a population of mature cardiomyocytes to a subject, wherein the population of mature cardiomyocytes has been produced by incubating a population of immature cardiomyocytes with at least one three-dimensional carrier matrix.

25. The method of claim 24, wherein the population of immature cardiomyocytes has been incubated on the surface of the at least one three-dimensional carrier matrix.

26. The method of claim 24 or 25, wherein the at least one three-dimensional carrier matrix comprises a plurality of microcarriers.

27. The method of any one of claims 24-26, wherein the population of immature cardiomyocytes has been incubated under incubation conditions comprising at least one of the following: an incubation duration of at least 1 week, and an incubation temperature below 37 °C.

28. The method of claim 26 or claim 28, wherein the plurality of microcarriers have at least one of the following properties: an average transverse dimension between 100 microns and 300 microns; at least 75 percent of the plurality of microcarriers being substantially spherical; formed of a substantially optically transparent material; formed of at least one of a silica, polystyrene, hydrogel, cellulose, dextran, gelatin material; a concentration of at least at least 5 wt% of an incubation medium; and a high-molecular weight glycoprotein.

29. A composition comprising a population of mature cardiomyocytes, wherein the population of mature cardiomyocytes has been produced by incubating a population of immature cardiomyocytes with at least one three-dimensional carrier matrix.

30. The composition of claim 29, wherein the population of immature cardiomyocytes has been incubated on the surface of the at least one three-dimensional carrier matrix.

31. The composition of any one of claims 29-30, wherein the at least one three- dimensional carrier matrix comprises a plurality of microcarriers.

32. The composition of claim 31, wherein the plurality of microcarriers have at least one of the following properties: an average transverse dimension between 100 microns and 300 microns; at least 75 percent of the plurality of microcarriers being substantially spherical; formed of a substantially optically transparent material; formed of at least one of a silica, polystyrene, hydrogel, cellulose, dextran, and / or gelatin material;a concentration of at least 5 wt% of an incubation medium; and a high-molecular weight glycoprotein.

33. A bioreactor comprising the composition of any one of claims 29-32.

34. A composition comprising a population of mature cardiomyocytes, wherein the population of mature cardiomyocytes has been extracted from at least one three-dimensional carrier matrix.

35. A method comprising: providing an incubation medium; and incubating a population of immature cardiomyocytes with the incubation medium under incubation conditions to obtain a population of mature cardiomyocytes.

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