Generation of secretome-containing compositions, and methods of using and analyzing the same

Abstract

To provide a method for generating secretomes using a serum-free medium.SOLUTION: A method for generating a secretome comprises: (a) culturing one or more progenitor cells in a first serum-free culture medium, the first serum-free culture medium comprising a basal medium, human serum albumin, and one or more growth factors; (b) removing the first serum-free culture medium from the one or more progenitor cells; (c) culturing the one or more progenitor cells in a second serum-free culture medium, the second serum-free culture medium comprising a basal medium but not comprising human serum albumin or growth factors; and (d) recovering the second serum-free culture medium after the culturing the step (c), thereby obtaining a conditioned medium comprising the secretome of one or more progenitor cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure generally relates to the generation, purification, isolation, and / or enrichment of secretomes from cells (including, but not limited to, progenitor cells); secretome-containing compositions containing such generated, purified, isolated, and / or enriched secretomes; and methods for analyzing one or more activities, properties, and / or characteristics of such secretome-containing compositions. The present disclosure also relates to therapeutic uses of secretome-containing compositions containing secreted bioactive molecules produced, purified, isolated, and / or enriched by one or more methods disclosed herein. The present disclosure further relates to good manufacturing practices (GMP)-compliant, scalable culture protocols for the release, purification, isolation, and / or enrichment of clinical-ready secretomes. [Background technology]

[0002] Cells, including those in vitro or ex vivo culture, secrete a wide variety of molecules and biological factors (collectively known as the secretome) into the extracellular space. See Vlassov et al. (Biochim Biophys Acta, 2012; 940-948). As part of the secretome, various bioactive molecules are secreted from cells into membrane-bound extracellular vesicles such as exosomes. Extracellular vesicles have the ability to alter the biology of other cells through signal transduction or by delivering their cargo (e.g., including proteins, lipids, and nucleic acids). The membrane-enclosed cargo of extracellular vesicles allows, among other things, specific targeting (e.g., targeting cells) via specific markers on the membrane; and increased stability during transport in biological fluids, such as through the bloodstream or across the blood-brain barrier (BBB).

[0003] Exosomes fulfill a wide variety of important physiological functions, for example, by acting as molecular messengers communicating between different cell types. For example, exosomes deliver proteins, lipids, and soluble factors, including RNA and microRNA, that participate in signaling pathways that can affect apoptosis, metastasis, angiogenesis, tumor progression, thrombosis, immunity by directing T cells toward immune activation, immunosuppression, growth, division, survival, differentiation, stress response, and apoptosis, depending on their source. See Vlassov et al. (Biochim Biophys Acta, 2012; 940-948). Extracellular vesicles can contain combinations of molecules that can act in concert to exert specific biological effects. Exosomes incorporate a wide range of cytoplasmic and membrane components that reflect the properties of the parent cell. Therefore, in some instances, the terminology applied to the cell of origin can be used simply to refer to secreted exosomes.

[0004] The ability of progenitor cells to proliferate and differentiate into mature cells makes them attractive for therapeutic applications, such as regenerative medicine in the treatment of myocardial infarction and congestive heart failure. Extracellular vesicles secreted by stem cell-derived cardiovascular progenitor cells have been reported to produce therapeutic effects similar to those of the cells that secrete them in a mouse model of chronic heart failure (see Kervadec et al. (J. Heart Lung Transplant, 2016;35:795-807)). This suggests that a significant mechanism of action of transplanted progenitor cells is the release of biological factors after transplantation (e.g., stimulating endogenous regenerative or repair pathways). This raises the possibility of effective cell-free therapies (with benefits such as simplicity, stability, and improved operator accessibility). See El Harane et al. (Eur. Heart J., 2018;39:1835-1847). However, there is currently a need for improved production methods for generating, purifying, isolating, and / or enriching extracellular vesicles.

[0005] For example, regulatory approval for the production of drugs and biological substances requires strict adherence to promulgated laws and regulations designed to establish safe and effective manufacturing facilities and products. As a non-limiting example, with respect to drugs and biologicals, "Good Manufacturing Practices" (GMP) and "Good Laboratory Practices" (GLP) have been established regulatory standards and are enforced by the FDA (US Food and Drug Administration), CDER (Center for Drug Evaluation and Research), and CBER (Center for Biologics Evaluation and Research). Similar GMP and / or GLP legislation is in place around the world, for example, in the EMEA.

[0006] However, established extracellular vesicle production techniques typically use reagents and / or conditions that are not suitable for clinical or therapeutic use or comply with GMP standards. For example, the use of serum in culture protocols raises reliability and biosafety concerns, especially when serum obtained from animals may be contaminated with infectious agents such as viruses or prions. Fetal bovine serum (FBS) is a growth supplement widely used in cell and tissue culture media; however, for these reasons, FBS is unsuitable for clinical or therapeutic use.

[0007] In contrast, the use of serum-free media has many advantages, including formulation consistency and safety. However, the use of serum-free media alone can adversely affect cell metabolism and growth, and there is a need for good manufacturing practices (GMP) compliant compositions and methods for producing, purifying, isolating, and / or enriching secretome compositions. Summary of the Invention [Problem to be solved by the invention]

[0008] The present disclosure addresses the above-mentioned limitations in the art by providing methods for producing, purifying, isolating, and / or enriching secretomes using serum-free media, thus enabling quality-controlled, GMP-compliant, scalable culture protocols for release of clinical-ready secretomes. [Means for solving the problem]

[0009] The present disclosure also provides methods for producing, purifying, isolating, and / or enriching secretomes, extracellular vesicles, and fractions thereof from cells (including, but not limited to, progenitor cells); and compositions containing such produced, purified, isolated, and / or enriched secretomes, extracellular vesicles, and fractions thereof. The present disclosure further provides methods for analyzing one or more activities, properties, and / or characteristics of such secretomes, extracellular vesicles, and fractions thereof, as well as therapeutic uses of secretomes, extracellular vesicles, and fractions thereof.

[0010] Non-limiting embodiments of the present disclosure include:

[0011] [1] A method for producing a secretome, the method comprising: (a) culturing one or more progenitor cells in a first serum-free culture medium, the first serum-free culture medium comprising a basal medium, human serum albumin, and one or more growth factors; (b) removing the first serum-free culture medium from the one or more progenitor cells; (c) culturing the one or more progenitor cells in a second serum-free culture medium, the second serum-free culture medium comprising a basal medium but not comprising human serum albumin or a growth factor; and (d) recovering the second serum-free culture medium after the culturing of step (c), thereby obtaining a conditioned medium comprising the secretome of one or more progenitor cells.

[0012] [2] The method of [1], wherein one of the one or more growth factors is fibroblast growth factor 2 (FGF-2).

[0013] [3] The method of [1] or [2], wherein the first and second serum-free media are supplemented with a carbohydrate source.

[0014] [4] The method of [3], wherein the carbohydrate source is glucose.

[0015] [5] Any one of the methods [1] to [4], wherein the first and second serum-free media are supplemented with an antibiotic.

[0016] [6] The method of [5], wherein the antibiotic is gentamicin.

[0017] [7] Any one of the methods [1] to [6], wherein the first serum-free medium further contains one or more selected from the group consisting of glutamine; biotin; DL-α-tocopherol acetate; DL-α-tocopherol; vitamin A; catalase; insulin; transferrin; superoxide dismutase; corticosterone; D-galactose; ethanolamine, glutathione; L-carnitine; linoleic acid; progesterone; putrescine; sodium selenite; triiodo-I-thyronine; amino acids; sodium pyruvate; lipoic acid; vitamin B12; nucleosides; and ascorbic acid.

[0018] [8] The method according to any one of [1] to [7], wherein the basal medium is a minimum essential medium (MEM).

[0019] [9] The method of [8], wherein the MEM is α-MEM.

[0020]

[10] Any one of the methods [1] to [9], wherein the culturing in step (a) lasts for 6 to 96 hours.

[0021]

[11] The method of

[10] , wherein the culturing in step (a) lasts for 12 to 96 hours.

[0022]

[12] The method of

[11] , wherein the culture in step (a) lasts for 36 to 84 hours.

[0023]

[13] The method of

[12] , wherein the culturing in step (a) is for about 72 hours.

[0024]

[14] Any one of the methods [1] to

[13] , wherein the culturing in step (c) lasts for 6 to 96 hours.

[0025]

[15] The method of

[14] , wherein the culturing in step (c) lasts for 12 to 72 hours.

[0026]

[16] The method of

[15] , wherein the culture in step (c) lasts for 36 to 60 hours.

[0027]

[17] The method of

[16] , wherein the culturing in step (c) lasts for about 48 hours.

[0028]

[18] The method of

[14] , in which the final 12 to 36 hours of culture in step (c) is carried out under hypoxic conditions.

[0029]

[19] The method according to

[18] , wherein the culture conditions include culturing in an atmosphere having 1 to 21% oxygen.

[0030]

[20] The method of any one of [1] to

[19] , wherein after step (b) but before step (c), the one or more progenitor cells are washed.

[0031]

[21] Any one of the methods [1] to

[20] , wherein the one or more progenitor cells include progenitor cells selected from the group consisting of cardiomyocyte progenitor cells, cardiac progenitor cells, and cardiovascular progenitor cells.

[0032]

[22] Any one of the methods [1] to

[21] , wherein the one or more progenitor cells are obtained from induced pluripotent stem cells (iPSCs).

[0033]

[23] The method according to any one of [1] to [4] and [7] to

[22] , wherein the first and second serum-free media do not contain antibiotics.

[0034]

[24] Any one of the methods [1] to

[23] , wherein the culture in one or more of steps (a) and (c) is a two-dimensional cell culture.

[0035]

[25] The method of

[24] , wherein the two-dimensional cell culture comprises culturing the one or more progenitor cells on the surface of a culture vessel.

[0036]

[26] The method according to

[25] , wherein the surface of the culture vessel is coated with a substance that promotes cell adhesion.

[0037]

[27] The method according to

[26] , wherein the substance that promotes cell adhesion is vitronectin or fibronectin.

[0038]

[28] Any one of the methods [1] to

[23] , wherein the culture in one or more of steps (a) and (c) is a three-dimensional cell culture.

[0039]

[29] The method of

[28] , wherein the three-dimensional cell culture comprises culturing cell aggregates in suspension in a bioreactor, spinner flask, or stirred culture vessel, or comprises culturing cells in a microcarrier culture system.

[0040]

[30] Any one of the methods [1] to

[29] , further comprising pre-clarifying the medium recovered in step (d) by centrifugation, filtration, or a combination of centrifugation and filtration.

[0041]

[31] Any one of the methods [1] to

[30] , further comprising freezing the medium collected in step (d).

[0042]

[32] Any one of the methods [1] to

[31] , wherein the one or more progenitor cells cultured in step (a) have been frozen in advance.

[0043]

[33] Any one of the methods [1] to

[32] , further comprising concentrating and / or enriching small extracellular vesicle enriched fractions (sEVs) from the medium collected in step (d).

[0044]

[34] The method of

[33] , wherein the sEVs are concentrated and / or enriched from the collected medium by at least one process selected from the group consisting of ultracentrifugation, filtration, ultrafiltration, tangential flow filtration, size exclusion chromatography, and affinity capture.

[0045]

[35] The accumulation accumulates extracellular vesicles having one or more of the following characteristics: (a) CD63 + , CD81 + and / or CD9 + (b) have a diameter of 50-200 nm; (c) are positive for one or more of CD49e, ROR1 (receptor tyrosine kinase-like orphan receptor 1), SSEA-4 (stage-specific fetal antigen 4), MSCP (mesenchymal stem cell-like protein), CD146, CD41b, CD24, CD44, CD236, CD133 / 1, CD29, and CD142; and / or (d) are negative for one or more of CD19, CD4, CD209, HLA-ABC (human leukocyte antigen ABC), CD62P, CD42a, and CD69.

[0046]

[36] The method of

[33] , wherein the sEVs comprise one or more of exosomes, microparticles, extracellular vesicles, and secreted peptides / proteins.

[0047]

[37] A secretome-containing composition obtained by any one of the methods [1] to

[32] .

[0048]

[38] An sEV-containing composition obtained by any one of the methods

[33] to

[36] .

[0049]

[39] A method for preparing a therapeutic composition suitable for administration to a patient, the method comprising preparing a secretome-containing composition according to any one of the methods [1] to

[32] .

[0050]

[40] The method of

[39] , further comprising purifying, concentrating, isolating, and / or enriching the secretome-containing composition by one or more purification, enrichment, isolation, and / or enrichment steps.

[0051]

[41] The method of

[39] , further comprising adding a pharmaceutically acceptable excipient or carrier to the secretome-containing composition.

[0052]

[42] A method for preparing a therapeutic composition suitable for administration to a patient, the method comprising preparing an sEV-containing composition according to any one of methods

[33] to

[36] .

[0053]

[43] The method of

[42] , further comprising purifying, concentrating, isolating, and / or enriching the sEV-containing composition by one or more purification, concentration, isolation, and / or enrichment steps.

[0054]

[44] The method of

[42] , further comprising adding a pharmaceutically acceptable excipient or carrier to the sEV-containing composition.

[0055]

[45] A therapeutic composition comprising the secretome-containing composition of

[37] and a pharmaceutically acceptable excipient or carrier.

[0056]

[46] A therapeutic composition comprising the sEV-containing composition of

[38] and a pharmaceutically acceptable excipient or carrier.

[0057]

[47] A secretome-containing composition obtainable by the method of [1], wherein the one or more progenitor cells include progenitor cells selected from the group consisting of cardiomyocyte progenitor cells, cardiac progenitor cells, and cardiovascular progenitor cells.

[0058]

[48] ​​An sEV-containing composition obtained by the method of

[33] , wherein the one or more progenitor cells include progenitor cells selected from the group consisting of cardiomyocyte progenitor cells, cardiac progenitor cells, and cardiovascular progenitor cells.

[0059]

[49] A therapeutic composition comprising the composition of

[47] and a pharmaceutically acceptable excipient or carrier.

[0060]

[50] A therapeutic composition comprising the composition of

[48] and a pharmaceutically acceptable excipient or carrier.

[0061]

[51] A method for treating acute myocardial infarction or heart failure, comprising administering to a subject in need thereof the therapeutic composition of

[49] or

[50] .

[0062]

[52] A method for improving angiogenesis, comprising administering to a subject in need thereof the therapeutic composition of

[49] or

[50] .

[0063]

[53] A method for improving cardiac workload, comprising administering to a subject in need thereof the therapeutic composition of

[49] or

[50] .

[0064]

[54] The method of

[11] , wherein the culture in step (a) lasts for 60 to 84 hours.

[0065]

[55] The method of

[14] , in which the final 12–36 h of culture in step (c) is carried out under normoxic conditions.

[0066]

[56] The method of

[55] , wherein the normoxic conditions include culturing in an atmosphere containing 20 to 21% oxygen.

[0067]

[57] The method of

[29] , wherein the bioreactor is a vertical wheel bioreactor.

[0068]

[0069]

[58] The method of

[39] , further comprising cryopreserving, freezing, or lyophilizing the secretome-containing composition.

[0070]

[59] The method of

[42] , further comprising cryopreserving, freezing, or lyophilizing the sEV-containing composition.

[0071]

[60] The method of [2], wherein the first serum-free medium contains 0.1 to 10 μg / mL FGF-2.

[0072]

[61] The method of

[60] , wherein the first serum-free medium contains 0.5 to 5 μg / mL FGF-2.

[0073]

[62] The method of

[61] , wherein the first serum-free medium contains 0.5 to 2.5 μg / mL FGF-2.

[0074]

[63] The method of

[62] , wherein the first serum-free medium contains about 1 μg / mL FGF-2.

[0075]

[64] Any one of the methods [1]-

[36] ,

[39] -

[44] and

[54] -

[63] that comply with Good Manufacturing Practices (GMP).

[0076]

[65] A secretome-containing composition of

[37] that is GMP-compliant.

[0077]

[66] GMP-compliant sEV-containing composition from

[38] .

[0078]

[67] The method of

[14] , in which the final 12–36 h of culture in step (c) is carried out under normoxic conditions.

[0079]

[68] The method of

[67] , wherein the normoxic conditions include culturing in an atmosphere containing 20 to 21% oxygen.

[0080]

[69] The method of

[30] , wherein the preliminary clarification comprises at least three filtration steps.

[0081]

[70] The method of

[34] , wherein the separation of the sEVs from the collected medium comprises tangential flow filtration.

[0082]

[71] The secretome-containing composition of

[37] , comprising trehalose, and optionally L-histidine.

[0083]

[72] The sEV-containing composition of

[38] , comprising trehalose, and optionally L-histidine.

[0084]

[73] A secretome-containing composition of

[37] or

[65] that is capable of promoting wound scratch healing in an in vitro wound scratch healing assay and / or promoting cardiomyocyte viability in an in vitro cardiomyocyte viability assay.

[0085]

[74] An sEV-containing composition of

[38] or

[66] that is capable of promoting wound scratch healing in an in vitro wound scratch healing assay and / or promoting cardiomyocyte viability in an in vitro cardiomyocyte viability assay.

[0086]

[75] At least one of the following: a composition enriched for extracellular vesicles having a diameter of about 50-200 nm or 50-200 nm, preferably about 50-150 nm or 50-150 nm; a composition that is substantially free of or contains no whole cells; and / or a composition that is substantially free of one or more culture medium components, the secretome-containing composition of

[37] or

[65] .

[0087]

[76] At least one of the following: a composition enriched for extracellular vesicles having a diameter of about 50-200 nm or 50-200 nm, preferably about 50-150 nm or 50-150 nm; a composition that is substantially free of or free of whole cells; and / or a composition that is substantially free of one or more culture medium components, the sEV-containing composition of

[38] or

[66] .

[0088]

[77] The method of

[51] , wherein the heart failure is acute heart failure, chronic heart failure, ischemic heart failure, non-ischemic heart failure, heart failure with ventricular dilation, heart failure without ventricular dilation, heart failure with reduced left ventricular ejection fraction, or heart failure with preserved left ventricular ejection fraction.

[0089]

[78] The method of

[77] , wherein the heart failure is selected from the group consisting of ischemic heart disease, cardiomyopathy, myocarditis, hypertrophic cardiomyopathy, diastolic hypertrophic cardiomyopathy, dilated cardiomyopathy, and post-chemotherapy-induced heart failure.

[0090] Incorporation by Reference All patents, publications, and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual patent, publication, or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0091] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0092] [Figure 1] Figure 1 illustrates the process flow diagram from iPSCs to CPCs, describing the generation of cardiovascular progenitor cells from hiPSCs (steps 1–4). After CPC generation, cells were either maintained as fresh aggregates (5a) or dissociated into single cells (step 5b) for the vesicle formation process. Single cells were either plated fresh or cryopreserved and plated after thawing for the vesicle formation process (steps 6–7).

[0093] [Figure 2]Figure 2 illustrates a flowchart showing the materials generated in Example 1. As shown in Figure 2, two batches of CPCs (CPC1 and CPC2) were generated, each divided into three vesicle formation conditions: aggregate vesicle formation, plate vesicle formation of fresh CPCs, and plate vesicle formation of thawed CPCs. Conditioned medium from each condition was collected, pre-clarified, and frozen (MC1-6). At the end of the 4-day vesicle formation process (day +4), cells were also collected and analyzed (C+4 #1-6). Small vesicle fractions were isolated by ultracentrifugation (UC) of the conditioned medium (sEV1-6). For MC5, three separate UC rounds were performed on separate aliquots of MC5. In parallel, vessels containing medium but without cells were "cultured" and unused medium was collected (unused medium 1-3), and MV controls were generated using the same UC protocol (MV1.1-3).

[0094] [Figure 3] Figure 3 illustrates a heatmap of relative gene expression of 48 relevant genes for CPC differentiation and potential off-targets. Data were generated using a custom Fluidigm qPCR panel. Data from CPCs at the end of the differentiation process (CPC) and 4 days into the vesicle formation process (C+4) are shown, along with iPSC and cardiomyocyte (CM) controls. Under these conditions, CPCs cluster and separate from C+4 cells, which are more mature than CPCs but less mature than CMs. Day 4 vesicle formation aggregates (Agg+4) differ from day 4 cells plated in HyperFlask (HF+4). Both conditions show increased cTNT (cardiac troponin T) and α-MHC (α-myosin heavy chain) expression compared to CPCs. This supports the idea that CPCs during the vesicle formation process remain in the cardiac lineage but do not reach a CM state, as indicated by the persistence of CPC marker expression, such as PDGFRa, ISL-1, and KDR.

[0095] [Figure 4] FIG. 4 illustrates a process flow diagram for the production of conditioned media and virgin media controls.

[0096] [Figure 5] Figure 5 illustrates the process flow diagram for isolation of sEVs or mock (unused medium) control samples.

[0097] [Figure 6] Figure 6 illustrates representative particle size distribution curves from two sEV and two control MV samples. Suspension cultures yielded higher particle concentrations than plate cultures, and both were significantly higher than the controls. The mode particle diameters of sEVs (74 nm, 99 nm) are consistent with exosomes or small microparticles.

[0098] [Figure 7] Figure 7 illustrates the ELISA results for CD-63 detection. sEV and MV controls were analyzed using the FUJIFILM Wako ELISA kit for the detection of CD-63, a protein found on the surface of EVs, particularly exosomes. The results show that for a given protein input, MVs do not contain a CD-63 signal, whereas sEVs from aggregate and plate cultures do. Aggregate sEVs generated a higher CD-63 / protein signal than sEVs from the plate vesicle formation protocol. Replicate preparations of sEVs from the same MC (5.1, 5.2, and 5.3) generated comparable CD63 signals. Furthermore, sEVs isolated from different MCs generated from separate lots also generated comparable CD-63 / µg protein (sEV2 vs. sEV5.1 / .2 / .3).

[0099] [Figure 8]Figure 8 illustrates the relative scratch wound closure in a HUVEC scratch wound healing assay. sEVs from the floating and flat vesicle formation processes and their corresponding mock EV controls (MVs) were tested in a HUVEC scratch wound healing assay. Controls included complete HUVEC medium (positive), nutrient-poor HUVEC medium (no supplements, negative), and sEVs isolated from fetal bovine serum (FBS-EVs, positive control) in nutrient-poor medium plus UC. sEVs from the floating and flat vesicle formation processes demonstrated improved wound healing compared to the negative and MV controls.

[0100] [Figure 9] Figure 9 illustrates the results of the H9c2 viability assay. The results of the H9c2 cell viability assay demonstrate that sEVs from suspension and plate cultures improve H9c2 viability in a serum deprivation assay. MVs showed minimal to no positive effect in this assay. sEVs generated from the suspension vesicle formation method showed improved cell numbers compared to the positive control, suggesting increased cell proliferation in addition to sustained survival.

[0101] [Figure 10] Figure 10 illustrates the time course of cardiomyocyte viability in a staurosporine-induced cardiotoxicity assay. sEVs from plate and aggregate cultures improve CM viability in this staurosporine assay. MV had little to no effect on CM viability. Arrows connect each sEV to its corresponding MV control.

[0102] [Figure 11] 11A and 11B illustrate a flow chart describing the production steps (vesicle formation, conditioned medium clarification, and TFF, FIG. 11A; followed by final formulation, FIG. 11B) of the first GMP-compliant process described in Example 5. The final formulation in this example was made with and without the addition of trehalose prior to sterile filtration. The various steps that underwent quality control testing are indicated with an "*" (e.g., *1, *2, *3, etc.).

[0103] [Figure 12] Figure 12 illustrates the analysis of cell marker expression profiles of CPCs by flow cytometry experiments at various time points (D+0, D+3, and D+5) during the vesicle formation process. iPSCs and cardiomyocytes (CMs) were used as control cells and analyzed separately. Values ​​shown are mean values.

[0104] [Figure 13] Figure 13 illustrates the results of transcriptome analysis of CPCs at various time points (D+0, D+3, and D+5) during the vesicle formation process. RNA was extracted from CPCs on D+0 and from cells on D+3 and D+5 during the vesicle formation process. RNA was also extracted from iPSCs (pluripotent cell control) and iPSC-derived cardiomyocytes (differentiated cardiomyocyte control; CM). Total RNA was sequenced on the Illumina NovaSeq 6000 platform, and differential gene expression was determined on normalized data. Heatmaps were generated based on hierarchical clustering analysis using the UPGMA clustering method with correlation distance metric in TIBCO Spotfire software v11.2.0.

[0105] [Figure 14] Figure 14 illustrates the morphology of CPCs during the vesicle formation process as observed under a light microscope. Cell morphology was analyzed for cells in both T75 flasks and selected CS10 flasks. The image on the left is a representative image showing the typical D+3 morphology observed in all vessels analyzed on D+3. The image on the right is a representative image showing the typical D+5 morphology observed in all vessels analyzed on D+5. For clarity, T75 flasks were used for image acquisition.

[0106] [Figure 15]Figures 15A and 15B illustrate the results of analyzing EV particle concentration and size distribution. Figure 15A illustrates the particle concentration and size distribution of EVs in the clarified conditioned medium before tangential flow filtration (TFF) (*5) and in the final formulation with and without trehalose (*7) using nanoparticle tracking analysis. Figure 15B illustrates the particle concentration and size distribution of EVs in the clarified conditioned medium before TFF (*5) and in the post-storage retentate samples (with and without trehalose or histidine) ("*6", samples a-c) that were not sterile filtered. As shown in Figures 15A and 15B, TFF increased the particle concentration by approximately 32-fold.

[0107] [Figure 16] Figures 16A-16D illustrate the results of MACSPlex analysis. Figures 16A and 16B illustrate the results of analyzing small EV-enriched secretome final preparations with and without trehalose for the expression of extracellular vesicle tetraspanins (CD9, CD81, and CD63) frequently expressed on the surface of extracellular vesicles (Figure 16A); and for various additional markers that showed little or no expression (Figure 16B). Figures 16C and 16D illustrate the results of analyzing non-sterilized filter-storage retentate samples (with and without trehalose or histidine) (see Figure 11B, *6, samples a-c) for the expression of extracellular vesicle tetraspanins (CD9, CD81, and CD63) frequently expressed on the surface of extracellular vesicles (Figure 16C); and for various additional markers that showed little or no expression (Figure 16D).

[0108] [Figure 17] Figures 17A and 17B illustrate the analysis of EVs for the presence of cardiac-related markers. Figure 17A illustrates the results for the expression of cardiac-related markers for the final small-EV-enriched secretome preparation with and without trehalose. Figure 17B illustrates the results for the expression of cardiac-related markers for the non-sterilized storage retentate samples (with and without trehalose or histidine).

[0109] [Figure 18] Figure 18 illustrates relative scratch wound healing in a HUVEC scratch wound healing assay. The final small EV-enriched secretome formulations with and without trehalose were tested in a HUVEC scratch wound healing assay. The positive control (+ve) consisted of culturing scratched wells in complete HUVEC cell medium (Comp) + PBS "treated," and the negative control (-ve) consisted of culturing scratched wells in basal medium (Poor) + PBS "treated." FBS-derived EVs served as the EV control (EV Ctl). 1x is equivalent to secretomes derived from 150,000 cells. Values ​​are normalized to the positive control after baseline subtraction (negative control).

[0110] [Figure 19] Figure 19 illustrates cardiomyocyte viability in a staurosporine-induced cardiotoxicity assay. The final small EV-enriched secretome preparations with and without trehalose were tested in a cardiomyocyte viability assay. 1x is equivalent to secretomes derived from 150,000 cells. PBS controls with and without staurosporine served as negative (-ve) and positive (+ve) controls, respectively. Mesenchymal stem cell (MSC)-derived EVs served as an EV control (EV Ctl). Plated cells were either stressed with staurosporine (+) or not stressed with staurosporine (-) for 4 hours prior to treatment.

[0111] [Figure 20] FIG. 20 illustrates an exemplary secretome / extracellular vesicle process / product test panel.

[0112] [Figure 21] FIG. 21 illustrates the secretome / extracellular vesicle process / product test panel associated with Examples 5-17.

[0113] [Figure 22]FIG. 22 illustrates the results for certain criteria shown in the test panel of FIG. 21, relating to Examples 5-11.

[0114] [Figure 23] FIG. 23 illustrates the enrichment (as calculated by the increase in particles per unit protein) for the retentate and final formulations made in Example 6 compared to the conditioned medium after clarification.

[0115] [Figure 24] 24A and 24B illustrate flow charts describing the production steps (vesicle formation, conditioned medium clarification, and TFF, FIG. 24A; and final formulation, FIG. 24B) in the second GMP-compliant process described in Example 12. The final formulation in this example was made with and without the addition of trehalose prior to sterile filtration. The various steps that underwent quality control testing are indicated with an "*" (e.g., *6, *7, etc.).

[0116] [Figure 25] Figure 25 illustrates the analysis of cell marker expression profiles of CPCs by flow cytometry experiments at various time points (D+0, D+3, and D+5) during the vesicle formation process. iPSCs and cardiomyocytes (CMs) were used as control cells and analyzed separately. Values ​​shown are mean values.

[0117] [Figure 26] Figure 26 illustrates the morphology of CPCs during the vesicle formation process as observed under a light microscope. Cell morphology was analyzed for cells in both T75 flasks and selected CS10 flasks. The image on the left is a representative image showing the typical D+3 morphology observed in all vessels analyzed on D+3. The image on the right is a representative image showing the typical D+5 morphology observed in all vessels analyzed on D+5. For clarity, T75 flasks were used for image acquisition.

[0118] [Figure 27]Figures 27A and 27B illustrate the results of analyzing EV particle concentration and size distribution. Figure 27A illustrates the particle concentration and size distribution of EVs in the conditioned medium (pre- and post-clarification) before tangential flow filtration (TFF) (*4 and *5) and in the final formulation with and without trehalose (*7) using nanoparticle tracking analysis. Figure 27B illustrates the particle concentration and size distribution of EVs in the retentate (*6) and in the pre-frozen, filter-sterilized final formulation without trehalose (*7).

[0119] [Figure 28] Figures 28A-28B illustrate the results of analyzing the final small EV-enriched secretome preparations with and without trehalose for expression of extracellular vesicle tetraspanins (CD9, CD81, and CD63), which are often expressed on the surface of extracellular vesicles (Figure 28A); and for various other markers that showed little or no expression (Figure 28B).

[0120] [Figure 29] Figure 29 illustrates the results regarding the expression of cardiac-related markers for the final small EV-enriched secretome preparations with and without trehalose.

[0121] [Figure 30] Figures 30A and 30B illustrate relative scratch wound healing in a HUVEC scratch wound healing assay. Results for samples a and b (illustrated in Figure 24B) are shown in Figure 30A. Results for samples c and d (illustrated in Figure 24B) are shown in Figure 30B. The positive control (+ve) consisted of culturing scratched wells in complete HUVEC cell medium (Comp) + PBS "treatment," and the negative control (-ve) consisted of culturing scratched wells in basal medium (Poor) + PBS "treatment." FBS-derived EVs served as the EV control (EV Ctl). 1x is equivalent to secretomes from 150,000 cells. Values ​​are normalized to the positive control after baseline subtraction (negative control).

[0122] [Figure 31] Figures 31A and 31B illustrate cardiomyocyte viability in a staurosporine-induced cardiotoxicity assay. The results for samples a and b (illustrated in Figure 24B) are shown in Figure 31A. The results for samples c and d (illustrated in Figure 24B) are shown in Figure 31B. 1x is equivalent to secretomes derived from 150,000 cells. PBS controls with and without staurosporine served as negative (-ve) and positive (+ve) controls, respectively. Mesenchymal stem cell (MSC)-derived EVs served as an EV control (EV Ctl). Plated cells were either stressed with staurosporine (+) or not stressed with staurosporine (-) for 4 hours before treatment.

[0123] [Figure 32] FIG. 32 illustrates results for certain criteria shown in the test panel of FIG. 21, relating to Examples 12-17.

[0124] [Figure 33] FIG. 33 illustrates the enrichment (as calculated by the increase in particles per unit protein) for the retentate and final formulation made in Example 12 compared to the conditioned medium after clarification.

[0125] [Figure 34] Figure 34 illustrates the results of echocardiography after administration of CPC EV ("sEV5.3") or PBS (as a control) to mice with induced chronic heart failure. The data illustrate absolute changes in left ventricular end-systolic volume (LVESV); left ventricular end-diastolic volume (LVEDV); and ejection fraction (EF). DETAILED DESCRIPTION OF THE INVENTION

[0126] It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, the reference to a "cell" includes one or more cells.

[0127] Unless otherwise defined, 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 pertains. Although other methods and materials similar or equivalent to those described herein can be useful in the present invention, the preferred materials and methods are described herein.

[0128] As used herein, the terms "subject," "individual," or "patient" are used interchangeably herein and refer to any member of the phylum Chordata, including, without limitation, humans and other primates, including non-human primates such as rhesus monkeys, chimpanzees, and other monkey and ape species; agricultural animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals including rabbits, mice, rats, and guinea pigs; and birds, including poultry, wild birds, and game birds such as chickens, turkeys, and other pheasants, ducks, and geese. The term does not denote a particular age or sex. Thus, the term includes adult, juvenile, and newborn individuals, as well as males and females. In some embodiments, cells (e.g., stem cells, including pluripotent stem cells, progenitor cells, or tissue-specific cells) are derived from a subject. In some embodiments, the subject is a non-human subject.

[0129] As used herein, "differentiation" refers to the process by which an unspecialized cell (such as a pluripotent stem cell or other stem cell), or for example, a multipotent or oligopotent cell, acquires specialized structural and / or functional characteristics characteristic of a more mature or fully mature cell. "Transdifferentiation" is the process by which one differentiated cell type transforms into another differentiated cell type.

[0130] As used herein, "embryoid body" refers to a three-dimensional aggregate of pluripotent stem cells that can undergo differentiation into cells of the three germ layers: endoderm, mesoderm, and ectoderm. This three-dimensional structure allows for differentiation and morphogenesis, including the establishment of complex cell adhesion bodies and paracrine signaling within the embryoid body microenvironment.

[0131] As used herein, "stem cell" refers to a cell that has the capacity to self-renew, i.e., undergo numerous cell division cycles, while remaining non-terminally differentiated. Stem cells can be totipotent, pluripotent, multipotent, oligopotent, or unipotent. Stem cells can be, for example, embryonic stem cells, fetal stem cells, amniotic stem cells, adult stem cells, or induced pluripotent stem cells.

[0132] As used herein, "pluripotent stem cells" (PSCs) refer to cells that have the ability to replicate themselves indefinitely and differentiate into any other cell type in an adult organism. Generally, pluripotent stem cells are stem cells that are capable of inducing teratomas when transplanted into immunodeficient (SCID) mice; have the potential to differentiate into cell types of all three germ layers (e.g., can differentiate into ectodermal, mesodermal, and endodermal cell types); and express one or more markers characteristic of PSCs. Examples of such markers expressed by PSCs, including embryonic stem cells (ESCs) and iPSCs, include Oct 4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, nanog, TRA-1-60, TRA-1-81, SOX2, and REX1.

[0133] As used herein, "induced pluripotent stem cells" (iPSCs) refer to a type of pluripotent stem cell that is artificially derived from a non-pluripotent cell, typically a somatic cell. In some embodiments, the somatic cell is a human somatic cell. Examples of somatic cells include, but are not limited to, skin fibroblasts, bone marrow-derived mesenchymal cells, cardiomyocytes, keratinocytes, hepatocytes, gastric cells, neural stem cells, lung cells, kidney cells, spleen cells, and pancreatic cells. Further examples of somatic cells include cells of the immune system, including, but not limited to, B cells, dendritic cells, granulocytes, innate lymphoid cells, megakaryocytes, monocytes / macrophages, myeloid-derived suppressor cells, natural killer (NK) cells, T cells, thymocytes, and hematopoietic stem cells.

[0134] iPSCs may be generated by reprogramming somatic cells by expressing or inducing the expression of one or a combination of factors (referred to herein as reprogramming factors) in the somatic cells. iPSCs can be generated using embryonic, postnatal, neonatal, juvenile, or adult somatic cells. In some examples, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, OCT4 (OCT3 / 4), SOX2, c-MYC, and KLF4, NANOG, and LIN28. In some examples, somatic cells may be reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or at least four reprogramming factors to reprogram the somatic cells into pluripotent stem cells. Cells may be reprogrammed by introducing the reprogramming factors using vectors, including, for example, lentivirus, retrovirus, adenovirus, and Sendai virus vectors. Alternatively, non-viral techniques for introducing reprogramming factors include, for example, mRNA transfection, miRNA infection / transfection, piggyback, minicircle vectors, and episomal plasmids. iPSCs can also be generated by introducing reprogramming factors or activating endogenous programming genes using, for example, CRISPR-Cas9-based techniques.

[0135] As used herein, "embryonic stem cells" are embryonic cells derived from embryonic tissue, preferably the inner cell mass or morula of a blastocyst, optionally serially passaged as a cell line. The term includes cells isolated from one or more blastomeres of an embryo, preferably without destroying the remainder of the embryo. The term also includes cells produced by somatic cell nuclear transfer. ESCs can be produced or derived from mammalian embryos at the zygote, blastomere, or blastocyst stage, for example, produced by sperm and egg fusion, nuclear transfer, or parthenogenesis. Human ESCs include, without limitation, MAO1, MAO9, ACT-4, No. 3, H1, H7, H9, H14, and ACT30 embryonic stem cells. Exemplary pluripotent stem cells include embryonic stem cells derived from the inner cell mass (ICM) of a blastocyst-stage embryo and embryonic stem cells derived from one or more blastomeres of a cleavage- or morula-stage embryo. These embryonic stem cells can be generated from embryonic material produced by fertilization or by asexual means, including somatic cell nuclear transfer (SCNT), parthenogenesis, and androgenesis. PSCs alone cannot develop into fetuses or adult animals when transplanted in utero because they lack the ability to contribute to all extraembryonic tissues (e.g., placenta in vivo or trophoblast in vitro).

[0136] As used herein, the term "progenitor cell" refers to the descendants of stem cells that have the potential to further differentiate into one or more specialized cell types but cannot divide and replicate indefinitely. That is, unlike stem cells (which have unlimited self-renewal potential), progenitor cells have only limited self-renewal potential. Progenitor cells may be multipotent, oligopotent, or unipotent and are typically classified based on the type of specialized cell they can differentiate into. For example, a "cardiomyocyte progenitor cell" is a progenitor cell derived from a stem cell that has the potential to differentiate into a cardiomyocyte. Similarly, a "cardiac progenitor cell" can differentiate into multiple specialized cells that constitute cardiac tissue, including, for example, cardiomyocytes, smooth muscle cells, and endothelial cells. Additionally, a "cardiovascular progenitor cell" has the potential to differentiate into cells of the cardiac and vascular lineages, for example.

[0137] As used herein, "expanding" or "growing" can refer to the process by which the number of cells in a cell culture increases due to cell division.

[0138] "Multipotent" implies that a cell has the ability, through its progeny, to give rise to several different cell types found in the adult animal.

[0139] "Pluripotent" implies that a cell has the ability to give rise, through its progeny, to all cell types contained in an adult animal, including germ cells. Embryonic stem cells, induced pluripotent stem cells, and embryonic germ cells are pluripotent cells under this definition.

[0140] The term "autologous cells," as used herein, refers to donor cells that are genetically identical to the recipient.

[0141] As used herein, the term "allogeneic cells" refers to cells derived from another genetically non-identical individual of the same species.

[0142] The term "totipotent," as used herein, can refer to a cell that gives rise to a live-born animal. The term "totipotent" can also refer to a cell that gives rise to every cell of a particular animal. A totipotent cell can give rise to every cell of an animal when it is utilized in a procedure to develop an embryo from one or more nuclear transfer steps.

[0143] As used herein, the term "extracellular vesicles" refers collectively to biological nanoparticles derived from cells, examples of which include, but are not limited to, exosomes, ectosomes, exovesicles, microparticles, microvesicles, nanovesicles, blebbing vesicles, budding vesicles, exosome-like vesicles, matrix vesicles, membrane vesicles, shedding vesicles, membrane particles, shedding microvesicles, oncosomes, exomers, and apoptotic bodies.

[0144] Extracellular vesicles can be classified, for example, based on size. For example, as used herein, the term "small extracellular vesicles" refers to extracellular vesicles having a diameter of about 50-200 nm. In contrast, extracellular vesicles having a diameter greater than about 200 nm and less than about 400 nm may be referred to as "intermediate extracellular vesicles," and extracellular vesicles having a diameter greater than about 400 nm may be referred to as "large extracellular vesicles." As used herein, the term "small extracellular vesicle fraction" ("sEVs") refers to a portion, extract, or fraction of a secretome or conditioned medium that is enriched and / or enriched in small extracellular vesicles having a diameter of about 50-200 nm. Such enrichment and / or enrichment may be achieved using one or more of the purification, isolation, enrichment, and / or enrichment techniques disclosed herein. In some alternative embodiments herein, enrichment may not be performed, realized, or possible.

[0145] The term "exosomes," as used herein, refers to extracellular vesicles that are released from cells when multivesicular bodies (MVBs), intermediate endocytic compartments, fuse with the plasma membrane.

[0146] "Exosome-like vesicles," which share a common origin with exosomes, are typically described as having size and sedimentation properties that distinguish them from exosomes, and in particular, as lacking lipid raft microdomains. "Ectosomes," as used herein, are typically neutrophil- or monocyte-derived microvesicles.

[0147] "Microparticles," as used herein, are typically about 100-1000 nm in diameter and originate from cell membranes. "Extracellular membrane structures" also include linear or folded membrane fragments, e.g., from necrosis, as well as membrane structures from other cellular sources, including secreted lysosomes and nanotubes.

[0148] As used herein, an "apoptotic bleb or body" is typically about 1-5 μm in diameter and is released as a bleb of cells undergoing apoptosis, i.e., diseased, unwanted and / or abnormal cells.

[0149] Within the class of extracellular vesicles, a key component is the "exosome" itself, which can be membrane vesicles of endocytic origin, i.e., vesicles surrounded by a phospholipid bilayer, with a diameter of between about 40-50 nm and about 200 nm, and arise from the exocytic fusion of multivesicular bodies (MVBs), i.e., "exocytosis." In some cases, exosomes can be between about 40-50 nm and up to about 200 nm in diameter, such as between 60 nm and 180 nm.

[0150] As used herein, the terms "secretome" and "secretome composition" refer synonymously to one or more molecules and / or biological factors secreted by cells into the extracellular space (e.g., into culture medium). Secretomes or secretome compositions can include, without limitation, extracellular vesicles (e.g., exosomes, microparticles, etc.), proteins, nucleic acids, cytokines, and / or other molecules secreted by cells into the extracellular space (e.g., into culture medium). Secretomes or secretome compositions can remain unpurified or can be further processed (e.g., components of the secretome or secretome composition can be present in culture medium, such as in conditioned medium; or alternatively, components of the secretome or secretome composition can be purified, isolated, and / or enriched from culture medium or an extract, portion, or fraction thereof). Secretomes or secretome compositions can further include one or more substances not secreted by cells (e.g., culture medium, additives, nutrients, etc.). Alternatively, the secretome or secretome composition does not contain (or contains only trace amounts of) one or more substances that are not secreted by the cell (eg, culture medium, additives, nutrients, etc.).

[0151] As used herein, the term "conditioned medium" refers to a culture medium (or an extract, part, or fraction thereof) in which one or more cells of interest have been cultured. Preferably, the conditioned medium is separated from the cultured cells before use and / or further processing. When cells are cultured in a culture medium, one or more molecules and / or biological factors (including, but not limited to, extracellular vesicles (e.g., exosomes, microparticles, etc.), proteins, nucleic acids, cytokines, and / or other molecules secreted by cells into the extracellular space) may be secreted and / or accumulated; a medium containing such one or more molecules and / or biological factors is a conditioned medium. An example of a method for preparing a conditioned medium is described, for example, in U.S. Pat. No. 6,372,494, which is incorporated herein by reference in its entirety.

[0152] As used herein, the term "cell culture" refers to cells grown under one or more controlled conditions outside the cells' natural environment. For example, cells can be grown completely outside their natural environment (in vitro) or removed from their natural environment and cultured (ex vivo). During cell culture, cells can survive in a non-replicating state or can replicate and grow in number, depending, for example, on the specific culture medium, culture conditions, and cell type. The in vitro environment can be any medium known in the art suitable for maintaining cells in vitro, such as a suitable liquid medium or agar.

[0153] The term "cell line," as used herein, may refer to a culture of cells that can be passaged at least once without being terminated.

[0154] The term "suspension," as used herein, may refer to a cell culture condition in which the cells are not attached to a solid support. Cells grown in suspension can be agitated during growth using equipment well known to those skilled in the art.

[0155] The term "monolayer," as used herein, may refer to cells that are attached to a solid support while growing under suitable culture conditions. A small portion of cells growing in a monolayer under suitable growth conditions may be attached to the cells in the monolayer rather than to the solid support.

[0156] The terms "plated" or "plating," as used herein with respect to cells, can refer to the establishment of a cell culture in vitro. For example, cells can be diluted in cell culture medium and then added to a cell culture plate, dish, or flask. Cell culture plates are commonly known to those skilled in the art. Cells can be plated at various concentrations and / or cell densities.

[0157] The term "cell plating" can also be extended to the term "cell passaging." Cells can be passaged using cell culture techniques well known to those skilled in the art. The term "cell passaging" can refer to a technique involving (1) the release of cells from a solid support or substrate and dissociation of those cells, and (2) the dilution of the cells into a medium suitable for further cell growth. Cell passaging can also refer to removing a portion of the liquid medium containing the cultured cells and adding the liquid medium to the original culture vessel to dilute the cells for further cell growth. Additionally, the cells can also be added to a new culture vessel supplemented with a medium suitable for further cell growth.

[0158] As used herein, the terms "culture medium," "growth medium," or "culture medium" are used interchangeably and refer to a composition intended to support the growth and survival of an organism. Culture media are often in liquid form, although other physical forms may also be used, such as, for example, solids, semi-solids, gels, suspensions, etc.

[0159] As used herein, the term "serum-free," in the context of a culture or growth medium, refers to a culture or growth medium that is free of serum. Serum typically refers to the liquid component of clotted blood after clotting factors (e.g., fibrinogen and prothrombin) have been removed by clot formation. Serum, such as fetal bovine serum, is routinely used in the art as a component of cell culture media because the various proteins and growth factors present therein are particularly useful for cell survival, growth, and division.

[0160] As used herein, the term "basal medium" refers to an unsupplemented synthetic medium that may contain a buffer, one or more carbon sources, amino acids, and salts. Depending on the application, the basal medium may be supplemented with growth factors and supplements, including, but not limited to, additional buffers, amino acids, antibiotics, proteins, and growth factors useful, for example, for promoting the growth of a particular cell type or for maintaining or altering the differentiation state (e.g., fibroblast growth factor-basic (bFGF), also known as fibroblast growth factor 2 (FGF-2)).

[0161] As used herein, the terms "wild-type," "naturally occurring," and "unmodified" are used herein to refer to the typical (or most common) form, appearance, phenotype, or strain found in nature; e.g., the typical form of a cell, organism, polynucleotide, protein, macromolecular complex, gene, RNA, DNA, or genome as it appears in, and can be isolated from, a natural source. The wild-type form, appearance, phenotype, or strain serves as the original parent prior to intentional modification. Thus, mutants, variants, engineered, recombinant, and modified forms are not wild-type forms.

[0162] As used herein, the term "isolated" refers to material that has been removed from its original environment and has thus been altered "by the hand of man" from its natural state.

[0163] As used herein, the term "enriched" means to selectively concentrate or increase the amount of one or more components in a composition relative to one or more other components. For example, enrichment may include reducing or diminishing (e.g., removing or eliminating) the amount of undesirable material; and / or may include specifically selecting or isolating a desired material from a composition.

[0164] The terms "engineered," "genetically engineered," "genetically modified," "recombinant," "modified," "non-naturally occurring," and "non-naturally occurring" refer to the deliberate human manipulation of the genome of an organism or cell. These terms encompass genome modification methods, including genome editing as defined herein, as well as techniques that alter gene expression or inactivation, enzyme engineering, directed evolution, knowledge-based design, random mutagenesis, gene shuffling, codon optimization, and the like. Genetic engineering methods are known in the art.

[0165] As used herein, the terms "nucleic acid sequence," "nucleotide sequence," and "oligonucleotide" all refer to a polymeric form of nucleotides. As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides that, when linear, has one 5' end and one 3' end and can contain one or more nucleic acid sequences. The nucleotides may be deoxyribonucleotides (DNA), ribonucleotides (RNA), analogs thereof, or combinations thereof, and may be of any length. Polynucleotides may perform any function and may have a variety of secondary and tertiary structures. The term encompasses known analogs of natural nucleotides as well as nucleotides modified in the base, sugar, and / or phosphate moieties. Analogs of a particular nucleotide have the same base-pairing specificity (e.g., an analog of A base pairs with T). A polynucleotide may contain one modified nucleotide or multiple modified nucleotides. Examples of modified nucleotides include fluorinated nucleotides, methylated nucleotides, and nucleotide analogs. The nucleotide structure may be modified before or after assembly of the polymer. After polymerization, polynucleotides may be further modified, for example, by conjugation with a labeling moiety or a target-binding moiety. Nucleotide sequences may incorporate non-nucleotide components. These terms also encompass nucleic acids containing synthetic, naturally occurring, and / or non-naturally occurring modified backbone residues or groups that have similar binding properties to a reference polynucleotide (e.g., DNA or RNA). Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acid (PNA), locked nucleic acid (LNA™) (Exiqon, Inc., Woburn, MA) nucleosides, glycol nucleic acid, bridged nucleic acid, and morpholino structures. Peptide nucleic acid (PNA) is a synthetic nucleic acid homolog in which the polynucleotide phosphate-sugar backbone is replaced with a flexible pseudo-peptide polymer. Nucleobases are linked to the polymer. PNAs have the ability to hybridize with high affinity and specificity to complementary sequences of RNA and DNA.Polynucleotide sequences are presented herein in conventional 5' to 3' orientation unless otherwise indicated.

[0166] As used herein, "sequence identity" generally refers to the percent identity of nucleotide bases or amino acids comparing a first polynucleotide or polypeptide to a second polynucleotide or polypeptide using algorithms with various weighting parameters. Sequence identity between two polynucleotides or two polypeptides can be determined using sequence alignment by various methods and computer programs (e.g., Exonerate, BLAST, CS-BLAST, FASTA, HMMER, L-ALIGN, etc.) available at sites on the World Wide Web, including, but not limited to, GENBANK (www.ncbi.nlm.nih.gov / genbank / ) and EMBL-EBI (www.ebi.ac.uk). Sequence identity between two polynucleotide or two polypeptide sequences is generally calculated using standard default parameters of the various methods or computer programs. A high degree of sequence identity between two polynucleotides or two polypeptides is often about 90% to 100% identity over the length of the reference polynucleotide or polypeptide or query sequence, for example, about 90% or more identity, about 91% or more identity, about 92% or more identity, about 93% or more identity, about 94% or more identity, about 95% or more identity, about 96% or more identity, about 97% or more identity, about 98% or more identity, or about 99% or more identity over the length of the reference polynucleotide or polypeptide or query sequence. Sequence identity can also be calculated for regions of overlap between two sequences where only a portion of the two sequences can be aligned.

[0167] Moderate sequence identity between two polynucleotides or two polypeptides is often between about 80% identity and about 90% identity over the length of the reference polynucleotide or polypeptide or query sequence, e.g., about 80% or more identity, about 81% or more identity, about 82% or more identity, about 83% or more identity, about 84% or more identity, about 85% or more identity, about 86% or more identity, about 87% or more identity, about 88% or more identity, or about 89% or more identity over the length of the reference polynucleotide or polypeptide or query sequence, but less than 90%.

[0168] A low degree of sequence identity between two polynucleotides or two polypeptides is often between about 50% identity and 75% identity over the length of the reference polynucleotide or polypeptide or query sequence, for example, about 50% or more identity, about 60% or more identity, about 70% or more identity but less than 75% identity over the length of the reference polynucleotide or polypeptide or query sequence.

[0169] As used herein, "binding" refers to a non-covalent interaction between macromolecules (e.g., between a protein and a polynucleotide, between a polynucleotide and a polynucleotide, or between a protein and a protein, etc.). Such non-covalent interactions are also referred to as "associating" or "interacting" (e.g., when a first macromolecule interacts with a second macromolecule, the first macromolecule binds to the second macromolecule in a non-covalent manner). A portion of the binding interaction may be sequence-specific (the terms "sequence-specific binding," "sequence-specific binding," "site-specific binding," and "site-specific binding" are used interchangeably herein). A binding interaction can be characterized by a dissociation constant (Kd). "Binding affinity" refers to the strength of the binding interaction. Increased binding affinity correlates with a lower Kd.

[0170] "Gene," as used herein, refers to a polynucleotide sequence comprising exons and associated regulatory sequences. A gene may further comprise introns and / or untranslated regions (UTRs).

[0171] As used herein, "expression" refers to transcription of a polynucleotide from a DNA template to give, for example, messenger RNA (mRNA) or other RNA transcripts (e.g., non-coding RNA, such as structural or scaffolding RNA). The term also refers to the process by which the transcribed mRNA is translated into a peptide, polypeptide, or protein. The transcript and encoded polypeptide are sometimes collectively referred to as the "gene product." Expression can also include splicing of the mRNA in eukaryotic cells if the polynucleotide is derived from genomic DNA.

[0172] A "coding sequence" or a sequence "encoding" a selected polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide when placed under the control of appropriate regulatory sequences in vitro or in vivo. The boundaries of the coding sequence are determined by a start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. A transcription termination sequence may be located 3' to the coding sequence.

[0173] As used herein, a "different" or "altered" level, for example, a characteristic or property, is measurably different and preferably statistically significant (e.g., cannot be attributed to the standard error of the assay). In some embodiments, the difference, for example, when compared with a control or reference sample, can be, for example, greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, such as greater than 2-fold difference; greater than 5-fold difference; greater than 10-fold difference; greater than 20-fold difference; greater than 50-fold difference; greater than 75-fold difference; greater than 100-fold difference; greater than 250-fold difference; greater than 500-fold difference; greater than 750-fold difference; or greater than 1,000-fold difference.

[0174] As used herein, the term "between" includes the endpoints of a given range (e.g., between about 1 and about 50 nucleotides in length includes 1 nucleotide and 50 nucleotides).

[0175] As used herein, the term "amino acid" refers to natural and synthetic (unnatural) amino acids, including amino acid analogs, modified amino acids, peptidomimetics, glycine, and D or L optical isomers.

[0176] As used herein, the terms "peptide," "polypeptide," and "protein" are synonymous and refer to a polymer of amino acids. A polypeptide can be of any length. It can be branched or linear, it can be interrupted by non-amino acids, and it can contain modified amino acids. These terms also refer to amino acid polymers that have been modified, for example, by acetylation, disulfide bond formation, glycosylation, lipidation, phosphorylation, pegylation, biotinylation, cross-linking, and / or conjugation (e.g., with a labeling moiety or ligand). Polypeptide sequences are presented herein in the conventional N-terminal to C-terminal orientation unless otherwise indicated. Polypeptides and polynucleotides can be made using routine techniques in the field of molecular biology.

[0177] "Moiety," as used herein, refers to a portion of a molecule. A moiety may be a functional group or may describe a portion of a molecule that has multiple functional groups (e.g., shares a common structural aspect). The terms "moiety" and "functional group" are typically used interchangeably; however, "functional group" can more specifically refer to a portion of a molecule that includes some common chemical behavior. "Moiety" is often used as a structural description.

[0178] The term "effective amount" or "therapeutically effective amount" of a composition or agent, such as a therapeutic composition as provided herein, refers to a sufficient amount of the composition or agent to bring about a desired response, which will depend on the particular disease at issue.

[0179] "Transformation," as used herein, refers to the insertion of an exogenous polynucleotide into a host cell, regardless of the method used for the insertion. For example, transformation can be by direct uptake, transfection, infection, etc. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, an episome, or alternatively, may be integrated into the host genome.

[0180] As used herein, the terms "hypoxic condition" or "hypoxia" refer to a condition in which the oxygen (O2) concentration is below atmospheric O2 concentration (typically 20-21%). In some embodiments, hypoxic condition refers to a condition in which the O2 concentration is 0%-19%, 2%-18%, 3%-17%, 4%-16%, 5%-15%, 5%-10%, or less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

[0181] As used herein, the term "normoxia" refers to normal atmospheric oxygen concentrations, typically about 20% to 21% O2.

[0182] Generation of progenitor cells from stem cells

[0183] The present disclosure relates, in part, to methods for generating secretomes containing extracellular vesicles (EVs) from progenitor cells. In certain embodiments herein, progenitor cells may be isolated from a subject or tissue and used in the methods of the present disclosure. In other embodiments, progenitor cells may be generated from pluripotent stem cells, such as from embryonic stem (ES) cells or induced pluripotent stem cells (iPSCs).

[0184] Generation of iPSC cells

[0185] iPSC cells may be obtained from somatic cells, including, for example, human somatic cells. The somatic cells may be derived from human or non-human animals, including, for example, humans and other primates, including non-human primates such as rhesus monkeys, chimpanzees, and other monkey and ape species; agricultural animals such as cows, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals including rabbits, mice, rats, and guinea pigs; and birds, including poultry, wild birds, and game birds, such as chickens, turkeys, and other pheasants, ducks, and geese.

[0186] In some embodiments, the somatic cells are selected from keratinized squamous epithelial cells, mucosal epithelial cells, exocrine epithelial cells, endocrine cells, hepatocytes, epithelial cells, endothelial cells, fibroblasts, muscle cells, cells of the blood and immune systems, cells of the nervous system including neurons and glial cells, pigment cells, and progenitor cells including hematopoietic stem cells. Somatic cells may be fully differentiated (specialized) or less than fully differentiated. For example, undifferentiated progenitor cells that are not PSCs, including somatic stem cells, and terminally differentiated mature cells can be used. Somatic cells may be from animals of any age, including adult and fetal cells.

[0187] The somatic cells may be of mammalian origin. For example, when secretomes (or extracellular vesicles) from progenitor cells thereof are used for in vivo administration, allogeneic or autologous stem cells can be used. In some embodiments, the iPSCs are not MHC- / HLA-matched to the subject. In some embodiments, the iPSCs are MHC- / HLA-matched to the subject. In embodiments, for example, when the iPSCs are to be used to generate PSC-derived progenitor cells (to obtain secretomes or extracellular vesicles for therapeutic use in the subject), the somatic cells may be obtained from the subject to be treated or from another subject of the same or substantially the same HLA type as the subject. The somatic cells can be cultured before nuclear reprogramming, or can be reprogrammed without culture, e.g., after isolation.

[0188] To introduce reprogramming factors into somatic cells, viral vectors can be used, including vectors derived from viruses such as SV40, adenovirus, vaccinia virus, adeno-associated virus, herpesviruses including HSV and EBV, Sindbis virus, alphavirus, human herpesvirus vectors (HHV) such as HHV-6 and HHV-7, and retroviruses. Lentiviruses include, but are not limited to, human immunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type 2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), bovine immunodeficiency virus (BIV), ovine visna virus (VISNA), and caprine arthritis-encephalitis virus (CAEV). Lentiviral vectors have the ability to infect non-dividing cells and can be used for gene transfer and expression of nucleic acid sequences both in vivo and in vitro. Viral vectors can be targeted to specific cell types by linking viral proteins, such as envelope proteins, to binding agents such as antibodies or to specific ligands (e.g., for targeting to receptors or proteins on or within specific cell types).

[0189] In some embodiments, viral vectors, such as lentiviral vectors, may integrate into the genome of a host cell. This transferred genetic material is then transcribed and, in some cases, translated into proteins within the host cell. In other embodiments, viral vectors are used that do not integrate into the genome of the host cell.

[0190] Viral gene delivery systems can be RNA-based or DNA-based viral vectors. Episomal gene delivery systems can be, for example, plasmids, Epstein-Barr virus (EBV)-based episomal vectors, yeast-based vectors, adenovirus-based vectors, Simian virus 40 (SV40)-based episomal vectors, bovine papillomavirus (BPV)-based vectors, or lentiviral vectors.

[0191] Somatic cell can be reprogrammed by methods known to those skilled in the art to produce induced pluripotent stem cell (iPSC).Those skilled in the art can easily make induced pluripotent stem cell, for example, see US Patent Application Publication No. 2009 / 0246875, US Patent Application Publication No. 2010 / 0210014; US Patent Application Publication No. 2012 / 0276636; US Patent No. 8,058,065; US Patent No. 8,129,187; and US Patent No. 8,268,620 (all of which are incorporated herein by reference).

[0192] Generally, reprogramming factors that can be used to generate induced pluripotent stem cells include, but are not limited to, one or more of the following genes: Oct4 (Oct3 / 4, Pou5f1), Sox (e.g., Sox1, Sox2, Sox3, Sox18, or Sox15), Klf (e.g., Klf4, Klf1, Klf3, Klf2, or Klf5), Myc (e.g., c-myc, N-myc, or L-myc), nanog, or LIN28, either alone, in combination, or as a fusion with a transactivation domain. As examples of these gene and protein sequences, the following accession numbers are provided: mouse MyoD: M84918, NM_010866; mouse Oct4 (POU5F1): NM_013633; mouse Sox2: NM_011443; mouse Klf4: NM_010637; mouse c-Myc: NM_001177352, NM_001177353, NM_001177354 Mouse Nanog: NM_028016; mouse Lin28: NM_145833; human MyoD: NM_002478; human Oct4 (POU5F1): NM_002701, NM_203289, NM_001173531; human Sox2: NM_003106; human Klf4: NM_004235; human c-Myc: NM_002467; human Nanog: NM_024865; and / or human Lin28: NM_024674. Also contemplated are sequences similar thereto, including those having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity. In some embodiments, at least three or at least four of Klf4, c-Myc, Oct3 / 4, Sox2, Nanog, and Lin28 are utilized. In other embodiments, Oct3 / 4, Sox2, c-Myc, and Klf4 are utilized.

[0193] Exemplary reprogramming factors for the generation of iPSCs include: (1) Oct3 / 4, Klf4, Sox2, L-Myc (Sox2 can be replaced with Sox1, Sox3, Sox15, Sox17, or Sox18; Klf4 can be replaced with Klf1, Klf2, or Klf5); (2) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, SV40 large T antigen (SV40LT); (3) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, human papillomavirus (HPV) 16 E6; (4) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, HPV16 E7; (5) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, HPV16 E6, HPV16 E7;(6)Oct3 / 4, Klf4, Sox2, L-Myc, TERT, Bmi1;(7)Oct3 / 4, Klf4, Sox2, L-Myc, Lin28;(8)Oct3 / 4, Klf4, Sox2, L-Myc, Lin28, SV40LT;(9)Oct3 / 4, Klf4, Sox2, L-Myc, Lin28, TERT, SV40LT;(10)Oct3 / 4, Klf4, Sox2, L-Myc, SV40LT; (11) Oct3 / 4, Esrrb, Sox2, L-Myc (Esrrb can be replaced by Esrrg); (12) Oct3 / 4, Klf4, Sox2; (13) Oct3 / 4, Klf4, Sox2, TERT, SV40LT; (14) Oct3 / 4, Klf4, Sox2, TERT, HPV16 (15) Oct3 / 4, Klf4, Sox2, TERT, HPV16 E7; (16) Oct3 / 4, Klf4, Sox2, TERT, HPV16 E6, HPV16 E7; (17) Oct3 / 4, Klf4, Sox2, TERT, Bmil; (18) Oct3 / 4, Klf4, Sox2, Lin28; (19) Oct3 / 4, Klf4, Sox2, Lin28, SV40LT; (20) Oct3 / 4, Klf4, Sox2, Lin28, TERT, SV40LT; (21) Oct3 / 4, Klf4, Sox2, SV40LT; or (22) Oct3 / 4, Esrrb, Sox2 (Esrrb can be replaced with Esrrg).

[0194] iPSCs typically exhibit the characteristic morphology of human embryonic stem cells (hESCs) and express the pluripotency factor, NANOG. Embryonic stem cell-specific surface antigens (SSEA-3, SSEA-4, TRA1-60, TRA1-81) can also be used to identify fully reprogrammed human cells. In addition, at the functional level, PSCs, such as ESCs and iPSCs, also demonstrate the ability to differentiate into lineages from all three embryonic germ layers and to form teratomas in vivo (e.g., in SCID mice).

[0195] Generation of progenitor cells by differentiating PSCs

[0196] The present disclosure further contemplates differentiating PSCs, including ESCs and iPSCs, into progenitor cells, which can then be used to generate the secretomes (and extracellular vesicles) of the present disclosure.

[0197] Progenitor cells of the present disclosure include, for example, hematopoietic progenitor cells, myeloid progenitor cells, neural progenitor cells; pancreatic progenitor cells, cardiac progenitor cells, cardiomyocyte progenitor cells, cardiovascular progenitor cells, renal progenitor cells, skeletal myoblasts, satellite cells, intermediate progenitor cells formed in the subventricular zone, radial glial cells, bone marrow stromal cells, periosteal cells, endothelial progenitor cells, blast cells, boundary cap cells, and mesenchymal stem cells. Methods for differentiating pluripotent stem cells into progenitor cells, and methods for culturing and maintaining progenitor cells are known in the art, such as those described in U.S. Provisional Patent Application No. 63 / 243,606, entitled "Methods for the Production of Committed Cardiac Progenitor Cells," which is incorporated herein by reference in its entirety.

[0198] Culturing progenitor cells for secretome / extracellular vesicle production

[0199] The present disclosure encompasses culturing progenitor cells for secretome / extracellular vesicle production under GMP-compliant and / or GMP-compatible conditions, e.g., to produce a GMP-compliant and / or GMP-compatible product. The present disclosure also encompasses culturing progenitor cells for secretome / extracellular vesicle production under non-GMP-compliant and / or non-GMP-compatible conditions, e.g., to produce a non-GMP-compliant and / or non-GMP-compatible product.

[0200] In the disclosed methods for producing secretomes or extracellular vesicles, progenitor cells are typically subjected to two or more culture steps in a serum-free culture medium.

[0201] In a first culture step, one or more progenitor cells are cultured in a first serum-free culture medium containing a basal medium, human serum albumin, and one or more growth factors. This first serum-free culture medium is then replaced with a second serum-free culture medium containing the basal medium but not containing human serum albumin or one or more growth factors. In a second culture step, one or more progenitor cells are then cultured in the second serum-free culture medium. After the second culture step, the second serum-free culture medium is collected, thereby obtaining a conditioned medium containing the secretome of one or more progenitor cells.

[0202] The one or more progenitor cells may be, for example, newly isolated (e.g., from stem cells) or newly differentiated progenitor cells. Alternatively, in some embodiments, the culture methods of the present disclosure may use progenitor cells that have been previously refrigerated, frozen, and / or cryopreserved. In some embodiments, the progenitor cells are thawed from a cryopreserved state (e.g., at -80°C or below) prior to use. In some such embodiments, the cells are thawed in a thawing medium. In some embodiments, the thawing medium may include a liquid medium containing one or more supplements (e.g., α-MEM, STEMdiff™ Cardiomyocyte Support Medium (StemCell, Reference: 05027)). In some embodiments, the supplements in the thawing medium may be one or more of a carbon source (e.g., glucose), albumin, B-27, insulin, FGF-2, FGF, and an antibiotic (e.g., gentamicin). In some embodiments, the cells may be thawed in a thawing device, such as, for example, a water bath or a waterless thawing system (e.g., the ThawSTAR™ Automated Thawing System, Biolife Solutions®). The cells may be thawed in a bag (e.g., an ethyl vinyl acetate (EVA) bag), such as, for example, a tube or bottle (e.g., plastic, glass), or a bag of 500-1000 mL capacity (e.g., Corning, Refs. 91-200-41, 91-200-42).

[0203] The one or more growth factors may be selected based on, for example, the type of progenitor cell. In some embodiments, the one or more growth factors are adrenomedullin, angiopoietin, autocrine motility stimulating factor, bone morphogenetic protein (BMP), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), epidermal growth factor (EGF), ephrin A1, ephrin A2, ephrin A3, ephrin A4, ephrin A5, ephrin B1, ephrin B2, ephrin B3, erythropoietin (EPO), fibroblast growth factor 1 (FGF-1), fibroblast growth factor 2 (FGF-2), fibroblast growth factor 3 (FGF-3), fibroblast growth factor 4 (FGF-4), fibroblast growth factor 5 (FGF-5), fibroblast growth factor 6 (FGF- 6), fibroblast growth factor 7 (FGF-7), fibroblast growth factor 8 (FGF-8), fibroblast growth factor 9 (FGF-9), fibroblast growth factor 10 (FGF-10), fibroblast growth factor 11 (FGF-11), fibroblast growth factor 12 (FGF-12), fibroblast growth factor 13 (FGF-13), fibroblast growth factor 14 (FGF-14), fibroblast growth factor 15 (FGF-15), Fibroblast growth factor 16 (FGF-16), fibroblast growth factor 17 (FGF-17), fibroblast growth factor 18 (FGF-18), fibroblast growth factor 19 (FGF-19), fibroblast growth factor 20 (FGF-20), fibroblast growth factor 21 (FGF-21), fibroblast growth factor 22 (FGF-F22), fibroblast growth factor 23 (FGF-23), fetal bovine somatotropin (Foetal Bovine Somatotropin)Somatotrophin (FBS), glial cell line-derived neurotrophic factor (GDNF), neurturin, persephin, artemin, growth differentiation factor-9 (GDF-9), hepatocyte growth factor (HGF), hepatocellular carcinoma-derived growth factor (HDGF), insulin, insulin-like growth factor-1 (IGF-1), insulin-like growth factor-2 (IGF-2), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, keratinocyte growth factor (KGF), migration-stimulating factor (MSF), macrophage-stimulating protein (MSP), myostatin (GDF-8), neuregulin 1 (NRG1), neuregulin 2 (NRG2), neuregulin 3 (NRG3), neuregulin 4 (NRG4), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), placental growth factor (PGF), platelet-derived growth factor (PDGF), renalase (RNLS), T-cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF).

[0204] The amount of growth factor may be adjusted depending on desired culture conditions and / or needs. In some embodiments, one or more growth factors may each independently be present in an amount of 0.001 μg / mL to 1000 μg / mL, 0.01 μg / mL to 100 μg / mL, 0.1 μg / mL to 10 μg / mL, 0.05 μg / mL to 5 μg / mL, 0.5 μg / mL to 2.5 μg / mL, or about 0.5 μg / mL, about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, or about 5 μg / mL.

[0205] In some embodiments, the one or more growth factors comprise FGF-2. In some embodiments, the one or more growth factors consist of FGF-2.

[0206] The basal medium may be any basal culture medium suitable for the cell type to be cultured, including, for example, Dulbecco's Modified Eagle's Medium (DMEM), DMEM F12 medium, Eagle's Minimum Essential Medium (MEM), α-MEM, F-12K medium, Iscove's Modified Dulbecco's Medium, Knockout DMEM, or RPMI-1640 medium, or variants, combinations, or modifications thereof.

[0207] Additional supplements may also be added to the basal medium to provide cells with trace elements for optimal growth and expansion, including, for example, insulin, transferrin, sodium selenium, Hank's balanced salt solution, Earle's salt solution, antioxidant supplement, MCDB-201, phosphate buffered saline (PBS), N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid (HEPES), nicotinamide, ascorbic acid and / or ascorbic acid-2-phosphate, and additional amino acids, and combinations thereof. Such amino acids include, but are not limited to, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine, L-cysteine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-inositol, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.

[0208] Optionally, hormones can also be used in cell culture, including, but not limited to, D-aldosterone, diethylstilbestrol (DES), dexamethasone, β-estradiol, hydrocortisone, insulin, prolactin, progesterone, somatostatin / human growth hormone (HGH), thyrotropin, thyroxine, and L-thyronine. β-Mercaptoethanol can also be supplemented to the cell culture medium.

[0209] Depending on the cell type, lipids and lipid carriers may also be used to supplement the cell culture medium, including, but not limited to, cyclodextrin, cholesterol, albumin-conjugated linoleic acid, albumin-conjugated linoleic acid and oleic acid, unconjugated linoleic acid, albumin-conjugated linoleic-oleic-arachidonic acid, and albumin-unconjugated and conjugated oleic acid, among others.

[0210] In certain embodiments, albumin, such as human serum albumin, is present in the first serum-free culture medium. The albumin may be, for example, isolated, synthetic, recombinant, and / or modified, including human serum albumin. The amount of albumin may be adjusted depending on the desired culture conditions and / or needs. In some embodiments, albumin may be present in an amount of 0.1 μg / mL to 50 mg / mL, 1 μg / mL to 25 mg / mL, 10 μg / mL to 20 mg / mL, 100 μg / mL to 10 mg / mL, 0.5 mg / mL to 5 mg / mL, 1 mg / mL to 3 mg / mL, or about 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, or 5 mg / mL.

[0211] In some embodiments, the serum-free medium further comprises one or more selected from the group consisting of glutamine; biotin; DL alpha tocopherol acetate; DL alpha-tocopherol; vitamin A; catalase; insulin; transferrin; superoxide dismutase; corticosterone; D-galactose; ethanolamine, glutathione; L-carnitine; linoleic acid; progesterone; putrescine; sodium selenite; triodo-I-thyronine; amino acids; sodium pyruvate; lipoic acid; vitamin B12; nucleosides; and ascorbic acid.

[0212] The basal medium may also be supplemented with one or more carbon sources, which may be selected from carbon sources such as glycerol, glucose, galactose, sucrose, fructose, mannose, lactose, or maltose.

[0213] The first and second culturing steps may be carried out for various lengths of time, for example, the first and second culturing steps may each independently be carried out for between 6 and 96 hours, 12 and 72 hours, 36 and 60 hours, 42 and 56 hours, or for about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours, about 66 hours, about 72 hours, about 78 hours, about 84 hours, about 90 hours, or about 96 hours.

[0214] In some embodiments, the first culturing step is carried out for between 42 and 56 hours, such as about 48 hours, hi some embodiments, the second culturing step is carried out for between 42 and 56 hours, such as about 48 hours.

[0215] In some embodiments, the first culturing step is carried out for between 42 and 96 hours, such as about 72 hours, hi some embodiments, the second culturing step is carried out for between 42 and 56 hours, such as about 48 hours.

[0216] In some embodiments, all or part of the first and / or second culture steps are performed under hypoxic conditions. In some embodiments, all or part of the second culture step is performed under hypoxic conditions. In some embodiments, the last 6-72 hours, the last 10-48 hours, or the last 12-36 hours of the second culture step are performed under hypoxic conditions. In some embodiments, the hypoxic conditions are an O2 concentration of 0%-15%, 0%-10%, or less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

[0217] In some embodiments, all or part of the first and / or second culturing step is performed under normoxic conditions. In some embodiments, all or part of the second culturing step is performed under normoxic conditions. In some embodiments, at least the last 6-72 hours, the last 10-48 hours, or the last 12-36 hours of the second culturing step are performed under normoxic conditions. In some embodiments, normoxic conditions are an O2 concentration between 20% and 21%.

[0218] In some embodiments, all or part of the first and / or second culture steps are performed in the presence of insulin. In some embodiments, all or part of the first culture step is performed in the presence of insulin. In some embodiments, the first culture step comprises culturing in the presence of insulin for at least 24 hours, at least 48 hours, or at least 72 hours. In some embodiments, all or part of the second culture step is performed in the presence of insulin. In some embodiments, the second culture step comprises culturing in the presence of insulin for at least 24 hours, at least 48 hours, or at least 72 hours.

[0219] In some embodiments, the one or more progenitor cells are washed using one or more washing steps between the first and second culture steps. In some embodiments, the washing medium may comprise a liquid medium (e.g., α-MEM, DMEM) optionally containing one or more supplements. In some embodiments, the supplement is a carbon source (e.g., glucose). In some embodiments, the one or more progenitor cells are not washed between the first and second culture steps (e.g., the first culture medium is removed and then the second culture medium is added).

[0220] The first and / or second culturing steps can be carried out in suspension or attached to a solid support. The culturing can be a two-dimensional or three-dimensional cell culture.

[0221] For example, in some embodiments, the culture vessel used for culturing may be, for example, a flask, a tissue culture flask (e.g., T25, T75), a HyperFlask (e.g., CellBind surface HYPERFlask®; Corning, Reference: 10024) or a HyperStack (e.g., 12 or 36 chamber, HYPERStacks®, Corning, References: 10012, 10036, 10013, 10037), a dish, a Petri dish, a tissue culture dish, a multi-dish, a microplate, a microwell plate, a multi-plate, a multiwell plate, a microslide, a chamber slide, a tube, a tray, a CellSTACK® chamber (e.g., 1ST, 2ST, 5ST, 10ST; Corning, References: 3268, 3269, 3313, 3319), a culture bag, a roller bottle, a bioreactor, a stirred culture vessel, a spinner flask, a microcarrier, or a vertical wheel bioreactor. The one or more progenitor cells may be cultured in a volume of, for example, at least or about 0.2, 0.5, 1, 2, 5, 10, 15, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, 1 L, 5 L, 10 L, 50 L, 100 L, 1000 L, 5000 L, or 10,000 L.

[0222] In embodiments in which the culture comprises a two-dimensional cell culture, such as on the surface of a culture vessel, the culture surface (to which the cells are intended to adhere) may be coated with one or more substances that promote cell adhesion. Such substances useful for enhancing attachment to a solid support include, for example, types I, II, and IV collagen, concanavalin A, chondroitin sulfate, fibronectin, fibronectin-like polymers, gelatin, laminin, poly-D and poly-L-lysine, Matrigel, thrombospondin, osteopontin, poly-D-lysine, human extracellular matrix, Corning® Cell-Tak™ cell and tissue adhesive, Corning® PuraMatrix® peptide hydrogel, and / or vitronectin.

[0223] In some embodiments, where the culturing of cells is carried out as an adherent culture, e.g., the cells are attached to a solid support, the cells are grown in a medium containing 1 cm 2 25,000 to 250,000 cells per cm 2 50,000 to 200,000 cells per 1cm 2 75,000 to 175,000 cells per cm 2 The cells may be seeded at a density of 100,000 to 150,000 cells per well.

[0224] In some embodiments, where the cell culture is performed as an adherent culture, e.g., where the cells are attached to a solid support, the cells may be seeded onto the solid support under gravity. In other embodiments, the cells may be seeded onto the solid support under centrifugation.

[0225] In some embodiments, after the second culturing step, the second serum-free culture medium used in the second culturing step is collected to obtain a conditioned medium containing the secretome of one or more progenitor cells.

[0226] The recovered conditioned medium may, in some embodiments, be subjected to one or more further processing steps. After the second culture step, the second serum-free culture medium used in the second culture step may be removed, analyzed, collected, concentrated, accumulated, isolated, purified, refrigerated, frozen, cryopreserved, lyophilized, sterilized, etc.

[0227] In some embodiments, the harvested conditioned medium may be pre-clarified or clarified to remove particulate matter above a certain particle size, for example, the harvested conditioned medium may be pre-clarified or clarified by one or more centrifugation and / or filtration techniques.

[0228] In some embodiments, the recovered conditioned medium is further processed to obtain a specific extract or fraction of the recovered conditioned medium. For example, the recovered conditioned medium may be further processed to separate a small extracellular vesicle-enriched fraction (sEV) therefrom. The sEV fraction may be separated from the recovered conditioned medium (or from a previously processed extract or fraction thereof) by one or more techniques, such as centrifugation, ultracentrifugation, filtration, ultrafiltration, gravity, sonication, density gradient ultracentrifugation, tangential flow filtration, size exclusion chromatography, ion exchange chromatography, affinity capture, polymer-based precipitation, or organic solvent precipitation.

[0229] In some embodiments, the conditioned medium is subjected to clarification by one or more filtration steps, in some embodiments, one or more of which utilize a filter membrane having a specific pore size. In some embodiments, filters having a pore size of 0.1 μm to 500 μm, or 0.2 μm to 200 μm; or filters having a pore size of 500 μm, 400 μm, 300 μm, 200 μm, 100 μm, 50 μm, 40 μm, 30 μm, 20 μm, 15 μm, 10 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm, 0.9 μm, 0.8 μm, 0.7 μm, 0.6 μm, 0.5 μm, 0.4 μm, 0.3 μm, 0.2 μm, or 0.1 μm or less are used.

[0230] In some embodiments, clarification comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 filtration steps. In some embodiments, clarification comprises 4 filtration steps. In some embodiments, the successive filtration steps utilize filters with progressively smaller pore sizes.

[0231] In some embodiments thereof, as illustrated in Example 5 and FIG. 11A , the first filtration step comprises use of an approximately 200 μm filter (e.g., a 200 μm drip chamber filter; Gravity Blood set, BD careFusion, Reference: VH-22-EGA); the second filtration step comprises use of an approximately 15 μm filter (e.g., DIDACTIC, Reference: PER1FL25); the third filtration step comprises use of an approximately 0.2 μm filter (e.g., Sartoguard PES XLG MidiCaps, pore size: 1.2 μm+0.2 μm, Sartorius, Reference: 5475307F7--OO--A), optionally including a prefilter, for example, an approximately 1.2 μm prefilter; and the fourth filtration step comprises use of an approximately 0.22 μm filter (e.g., a vacuum filter / storage bottle system, 0.22 μm pores, 33.2 cm 2 This involves the use of a PES membrane (Corning, ref: 431097).

[0232] In other embodiments, as illustrated in Example 12 and FIG. 24A, the first filtration step includes the use of an approximately 5 μm filter (e.g., Sartopure PP3 MidiCaps, pore size: 5 μm, Sartorius, ref: 5055342P9--OO--A); the second filtration step optionally includes the use of a prefilter, e.g., an approximately 0.2 μm filter with an approximately 1.2 μm prefilter (e.g., Sartoguard PES MidiCaps, pore size: 1.2 μm+0.2 μm, Sartorius, ref: 5475307F9--OO--A); and the third filtration step optionally includes the use of a prefilter, e.g., an approximately 0.2 μm filter with an approximately 0.45 μm prefilter (e.g., Sartopure 2 This involves the use of MidiCaps, pore size: 0.45 μm + 0.2 μm, Sartorius, reference: 5445307H8--OO--A).

[0233] In some embodiments, the conditioned medium may be subjected to clarification by one or more centrifugation steps, hi some embodiments, the conditioned medium may be subjected to clarification by a combination of one or more centrifugation and filtration steps.

[0234] In some embodiments, one or more additives are added to the conditioned medium, such as before and / or after clarification. In some embodiments, an additive that reduces aggregation is added. In some embodiments, the additive is one or more selected from trehalose, histidine (e.g., L-histidine), arginine (e.g., L-arginine), citrate-dextrose solution, DNase (e.g., DNase I), iron citrate, or an anti-aggregating agent (Gibco / Life technologies, Reference: 01-0057; Lonza, Reference: BE02-058E).

[0235] In some embodiments, the conditioned medium or sEVs may be subjected to one or more isolation, enrichment, and / or concentration steps using tangential flow filtration (TFF). In some embodiments, the conditioned medium or sEVs are subjected to TFF after clarification using one or more clarification steps (e.g., after one or more filtration and / or centrifugation steps). TFF is a rapid and efficient method for separating, enriching, and purifying biomolecules. In some embodiments, TFF can be used, for example, for concentration (e.g., enrichment of small extracellular vesicles from conditioned medium); diafiltration; and concentration and diafiltration. Diafiltration is a type of ultrafiltration process in which the retentate (the fraction that does not pass through the membrane) is diluted with a buffer and ultrafiltered again to reduce the concentration of soluble permeant components and further increase the concentration of retained components.

[0236] In some embodiments, TFF is used for the accumulation, concentration, and diafiltration of conditioned medium or sEVs (e.g., for the concentration and diafiltration of EV secretomes). In some embodiments, TFF is first used to concentrate conditioned medium or sEVs, followed by diafiltration. In some embodiments, the TFF process may include an additional step of concentration after diafiltration. In some embodiments, TFF is used for diafiltration but not for concentration. In some embodiments, TFF is used for concentration but not for diafiltration.

[0237] In some embodiments, the TFF membrane has a cutoff value of 10 kDa or less, 20 kDa or less, 30 kDa or less, 40 kDa or less, 50 kDa or less, 60 kDa or less, 70 kDa or less, 80 kDa or less, 90 kDa or less, 100 kDa or less, or 150 kDa or less. In some embodiments, the TFF membrane has a cutoff value of about 10 kDa, about 30 kDa, about 100 kDa, or about 500 kDa. In some embodiments, the TFF membrane has a cutoff value of 30 kDa or about 30 kDa.

[0238] In some embodiments, the TFF membrane comprises cellulose. In some embodiments, the TFF membrane comprises regenerated cellulose. In some embodiments, the TFF membrane comprises a polyethersulfone (PES) membrane.

[0239] In some embodiments, the conditioned medium or sEVs subjected to TFF can be further purified, isolated, and / or enriched (after TFF) using one or more purification, isolation, and / or enrichment techniques. For example, the resulting product from TFF can be subjected to a chromatography step, such as an ion exchange chromatography step or a steric exclusion chromatography step, to further purify the small extracellular vesicles. In some embodiments, the conditioned medium subjected to TFF, with or without further purification, isolation, and / or enrichment, can be further concentrated, such as by ultracentrifugation.

[0240] Any of the above processing techniques can be performed, for example, on harvested conditioned medium (or pre-processed extracts or fractions thereof) that is fresh or that has been previously frozen and / or refrigerated.

[0241] In some embodiments, the secretome-containing, extracellular vesicle-containing, and sEV-containing compositions produced by the methods herein may contain at least one additive to prevent aggregation. The additive may be one or more selected from trehalose, histidine (e.g., L-histidine), arginine (e.g., L-arginine), citrate-dextrose solution, DNase (e.g., DNase I), iron citrate, or an anti-aggregating agent (Gibco / Life technologies, Reference: 01-0057; Lonza, Reference: BE02-058E). In some embodiments, trehalose is added. In some embodiments, trehalose or L-histidine is added.

[0242] In some embodiments, the sEV fraction is CD63 + , CD81 +, and / or CD9 + The sEV fraction may contain one or more types of extracellular vesicles, such as one or more of exosomes, microparticles, and extracellular vesicles. The sEV fraction may also contain secreted proteins (enveloped and / or non-enveloped). The extracellular vesicles present in the conditioned medium or sEV fraction of the present disclosure may contain one or more components selected from, for example, tetraspanins (e.g., CD9, CD63, and CD81), ceramide, MHC class I, MHC class II, integrins, adhesion molecules, phosphatidylserine, sphingomyelin, cholesterol, cytoskeletal proteins (e.g., actin, gelsolin, myosin, tubulin), enzymes (e.g., catalase, GAPDH, nitric oxide synthase, LT synthase), nucleic acids (e.g., RNA, miRNA), heat shock proteins (e.g., HSP70 and HSP90), exosome biogenesis proteins (ALIX, Tsg101), LT, prostaglandins, and S100 proteins.

[0243] In some embodiments, the presence of a desired type of extracellular vesicles in a fraction may be determined, for example, by nanoparticle tracking analysis (determining the size of particles in the fraction); and / or by confirming the presence of one or more markers associated with the desired type of extracellular vesicles. For example, a fraction of the collected conditioned medium may be analyzed for the presence of the desired type of extracellular vesicles by detecting the presence of one or more markers in the fraction, such as, for example, CD9, CD63, and / or CD81.

[0244] In some embodiments, the sEV preparation or composition is positive for CD9, CD63, and CD81 (canonical EV markers) and positive for the cardiac-related markers CD49e, ROR1, SSEA-4, MSCP, CD146, CD41b, CD24, CD44, CD236, CD133 / 1, CD29, and CD142. In some embodiments, the sEV preparation or composition contains a reduced amount of one or more markers selected from the group consisting of CD3, CD4, CD8, HLA-DRDPDQ, CD56, CD105, CD2, CD1c, CD25, CD40, CD11c, CD86, CD31, CD20, CD19, CD209, HLA-ABC, CD62P, CD42a, and CD69 relative to the amount of CD9, CD63, and / or CD81 in the sEV preparation or composition. In some embodiments, the sEV preparation or composition contains an undetectable amount of, or is negative for, one or more markers selected from the group consisting of CD19, CD209, HLA-ABC, CD62P, CD42a, and CD69 (e.g., by MACSPlex assay, by immunoassay, etc.).

[0245] In some embodiments, the sEV preparation or composition is at least one of the following: an sEV preparation or composition enriched in extracellular vesicles having a diameter of about 50-200 nm or 50-200 nm; an sEV preparation or composition enriched in extracellular vesicles having a diameter of about 50-150 nm or 50-150 nm; an sEV preparation or composition that is substantially free of or free of whole cells; and an sEV preparation or composition that is substantially free of one or more culture medium components (e.g., phenol red).

[0246] In some embodiments, such as in some GMP-compliant processes, a test panel is performed to analyze and / or determine one or more characteristics of the process, its products, intermediate products, etc.

[0247] For example, during the vesicle formation stage (e.g., including thawing, plating, culturing, and / or recovery steps), one or more characteristics of the cells (including, for example: viable cell count, cell viability; cell morphology; cell identity; cell karyotype; and / or cell transcriptome) can be examined.

[0248] Additionally or alternatively, one or more properties of the secretome and / or extracellular vesicle-containing fraction, extract, or composition may be analyzed using one or more tests to determine one or more properties of the secretome / extracellular vesicles (e.g., particle concentration and / or particle size distribution; protein concentration; protein profile concentration; RNA profile; potency; marker identity; host cell protein evaluation; residual DNA quantification and / or characterization; sterility; mycoplasma; endotoxin; appearance; pH; osmolality; extractable volume; hemolytic activity; complement activation; platelet activation; and / or genotoxicity). For example, one or more of these properties may be assessed in the conditioned medium before clarification; in the conditioned medium after clarification; in the isolated and / or concentrated secretome / extracellular vesicles; and / or in the final formulation. In some embodiments, the final formulation may be tested immediately after production and / or after 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, or several years after formulation.

[0249] An exemplary process / product testing panel is shown in FIG.

[0250] Therapeutic Compositions and Applications

[0251] The present disclosure contemplates the production of secretome-containing, extracellular vesicle-containing, and sEV-containing compositions useful as therapeutic agents. In some embodiments, the methods of the present disclosure include administering an effective amount of a secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition to a subject in need thereof.

[0252] Tissues that may be treated by the methods of the present disclosure include, without limitation, cardiac tissue, brain or other nervous tissue, skeletal muscle tissue, lung tissue, arterial tissue, capillary tissue, kidney tissue, liver tissue, gastrointestinal tissue, epithelial tissue, connective tissue, urinary tract tissue, etc. The tissue to be treated may be damaged or completely or partially non-functional due to, for example, injury, age-related degeneration, acute or chronic disease, cancer, or infection. Such tissue may be treated, for example, by intravenous administration of a secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition.

[0253] In some embodiments, the compositions of the present disclosure may be used to treat diseases such as myocardial infarction, stroke, heart failure, and critical limb ischemia. In some embodiments, the compositions of the present disclosure may be used to treat heart failure having one or more of the following characteristics: acute, chronic, ischemic, non-ischemic, with ventricular dilation, without ventricular dilation, with reduced left ventricular ejection fraction, or with preserved left ventricular ejection fraction. In some embodiments, the compositions of the present disclosure may be used to treat heart failure selected from the group consisting of ischemic heart disease, cardiomyopathy, myocarditis, hypertrophic cardiomyopathy, diastolic hypertrophic cardiomyopathy, dilated cardiomyopathy, and post-chemotherapy-induced heart failure. In some embodiments, the compositions of the present disclosure may be used to treat diseases such as congestive heart failure, heart disease, ischemic heart disease, valvular heart disease, connective tissue disease, viral or bacterial infection, myopathy, dystrophinopathy, liver disease, kidney disease, sickle cell disease, diabetes, eye disease, and neurological disease. It will be appreciated that the type or types of suitable progenitor cells may be selected depending on the disease to be treated or the tissue to be targeted.

[0254] For example, in some embodiments, a subject with a cardiac disease, such as acute myocardial infarction or heart failure, can be treated with a secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition made from cardiomyocyte progenitor cells, cardiac progenitor cells, and / or cardiovascular progenitor cells.

[0255] Additionally, secretome-, extracellular vesicle-, and / or sEV-containing compositions made from appropriate progenitor cell types can also be used to improve tissue function or performance. For example, improved angiogenesis or cardiac performance can be achieved by delivering secretome-, extracellular vesicle-, and / or sEV-containing compositions made from cardiomyocyte progenitor cells, cardiac progenitor cells, and / or cardiovascular progenitor cells to a subject in need thereof.

[0256] In some embodiments, administration includes administration at a tissue or organ site that is the same as the target tissue. In some embodiments, administration includes administration at a tissue or organ site that is different from the target tissue. Such administration may include, for example, intravenous administration.

[0257] Secretome-, extracellular vesicle-, and / or sEV-containing compositions may contain or be administered with a pharmaceutically acceptable diluent, carrier, or excipient. In some embodiments, such compositions may also contain one or more pharmaceutically acceptable concentrations of salts, buffers, preservatives, or other therapeutic agents. Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; buffers such as magnesium hydroxide and aluminum hydroxide; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solution; and other non-toxic substances compatible with use in pharmaceutical formulations. For example, in some embodiments, secretome-, extracellular vesicle-, and / or sEV-containing compositions may be formulated with a biomaterial, such as an injectable biomaterial. Exemplary injectable biomaterials are described, for example, in WO 2018 / 046870, which is incorporated by reference in its entirety.

[0258] The secretome-containing, extracellular vesicle-containing, and / or sEV-containing compositions of the present disclosure may be administered in an effective amount, such as a therapeutically effective amount, depending on the purpose. The effective amount will depend on various factors, including the material selected for administration, whether administration is in a single dose or multiple doses, and individual patient parameters, including age, health, size, weight, and stage of disease. These factors are well known to those skilled in the art.

[0259] Any suitable route of administration may be used, for example, administration may be parenteral, intravenous, intraarterial, subcutaneous, intratumoral, intramuscular, intracranial, intraorbital, intraocular, intraventricular, intrahepatic, intracapsular, intrathecal, intracisternal, intraperitoneal, intranasal, intramyocardial, intracoronary, aerosol, suppository, epicardial patch, oral administration, or by perfusion. For example, therapeutic compositions for parenteral administration may be in the form of a liquid solution or suspension; for oral administration, the formulation may be in the form of a tablet or capsule; and for intranasal formulations, the formulation may be in the form of a powder, nasal drops, or aerosol. For example, in some embodiments, a subject with a cardiac disease such as acute myocardial infarction or heart failure can be treated with a secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition made from cardiomyocyte progenitor cells, cardiac progenitor cells, and / or cardiovascular progenitor cells, wherein the composition is administered intravenously.

[0260] In some embodiments, a single dose of a secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition may be administered. In other embodiments, multiple doses are administered to a subject over one or more daily, weekly, or monthly periods. In some embodiments, single or repeated administrations of a secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition may be administered, including two, three, four, five, or more administrations. In some embodiments, a secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition may be administered continuously. Repeated or continuous administration may occur over a period of several hours (e.g., 1-2, 1-3, 1-6, 1-12, 1-18, or 1-24 hours), several days (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, or 1-7 days), or several weeks (e.g., 1-2 weeks, 1-3 weeks, or 1-4 weeks), depending on the nature and / or severity of the condition being treated. When administration is repeated but not continuous, the time between administrations can be several hours (e.g., 4 hours, 6 hours, or 12 hours), several days (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days), or several weeks (e.g., 1 week, 2 weeks, 3 weeks, or 4 weeks). The time between administrations can be the same or different. For example, if symptoms worsen or do not improve, the secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition may be administered more frequently. Conversely, if symptoms stabilize or become milder, the secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition may be administered less frequently.

[0261] In some embodiments, the secretome-containing, extracellular vesicle-containing, and / or sEV-containing compositions are administered intravenously in several doses, e.g., three doses, spaced several days, weeks, or months or so apart, e.g., two weeks apart. In some embodiments, the compositions may be diluted, formulated with, and / or administered with a carrier, diluent, or suitable material (e.g., saline).

[0262] Assays for determining the activity, function, and / or potency of secretomes and extracellular vesicles

[0263] The present disclosure also encompasses methods for analyzing the activity, function, and / or efficacy of conditioned media; or secretome-containing, extracellular vesicle-containing, and / or sEV-containing compositions.

[0264] The activity, function, and / or efficacy of conditioned medium; or secretome-containing, extracellular vesicle-containing, and / or sEV-containing compositions, can be assessed by a variety of techniques, depending, for example, on the type of progenitor cells used to generate the conditioned medium or composition and the desired use of the conditioned medium or composition.

[0265] For example, the activity, function, and / or efficacy of the conditioned medium; or secretome-, extracellular vesicle-, and / or sEV-containing compositions may be assessed by administering the conditioned medium, secretome-, extracellular vesicle-, and / or sEV-containing compositions to target cells in vitro, ex vivo, or in vivo. One or more characteristics of the target cells, such as, for example, cell viability, hypertrophy, cell health, cell adhesion, cell physiology, ATP content, cell number, and cell morphology, can then be analyzed to determine the activity, function, and / or efficacy of the conditioned medium; or secretome-, extracellular vesicle-, and / or sEV-containing compositions.

[0266] In some embodiments, assays known in the art can be used to determine the activity, function, and / or potency of conditioned medium; or secretome-containing, extracellular vesicle-containing, and / or sEV-containing compositions.

[0267] For example, for conditioned medium obtained from cardiovascular progenitor cells or cardiomyocyte progenitor cells; or for secretome-containing, extracellular vesicle-containing, and / or sEV-containing compositions, the activity, function, and / or efficacy may be measured using known cardiomyocyte viability assays, such as those described in El Harane et al. (Eur. Heart J., 2018, 39(20):1835-1847).

[0268] Specifically, serum-depleted cardiac myoblasts (e.g., H9c2 cells) may be contacted with conditioned medium or a secretome-, extracellular vesicle-, and / or sEV-containing composition, and then cell viability may be measured. In some embodiments of this assay, the cells are serum-depleted before administering the conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing composition. In other embodiments, the cells are serum-depleted after administering the conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing composition. In some embodiments, the cells are serum-depleted before and after administering the conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing composition.

[0269] In other embodiments, the angiogenic activity of the conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing composition can be measured, for example, using a HUVEC scratch wound healing assay. In the HUVEC scratch wound healing assay, HUVEC cells are cultured on a culture surface, and then one or more cultured cell layers are scratched; the angiogenic activity of the conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing composition can then be determined by the ability of the conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing composition to produce wound closure under serum-free conditions.

[0270] Cell viability (in a cell viability assay) may be measured, for example, using DNA labeling dyes or nuclear staining dyes, which may be used in conjunction with live cell imaging.

[0271] The activity, function, and / or efficacy of the conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing composition may also be determined relative to one or more control samples. For example, the control cells can be one or more of serum-depleted control cells that are not administered with conditioned medium or a secretome-, extracellular vesicle-, and / or sEV-containing composition; non-serum-depleted control cells; or serum-depleted control cells that are administered with mock conditioned medium or a mock secretome-, extracellular vesicle-, and / or sEV-containing composition.

[0272] In some methods of the present disclosure, the activity, function, and / or efficacy of a conditioned medium; or a secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition, can be assessed by a method comprising administering the conditioned medium or secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition to target cells cultured under at least one stress-inducing condition and analyzing at least one characteristic of the cells. The one or more characteristics of the target cells that can be analyzed may be selected from, for example, cell migration, cell viability, cell growth, cell health, cell adhesion, cell physiology, ATP content, cell number, and cell morphology. In some embodiments, the at least one characteristic measured is cell adhesion, cell number, cell growth, and / or cell morphology, and wherein cell adhesion, cell number, cell growth, and / or cell morphology are determined by measuring the electrical impedance of a culture vessel surface in the culture.

[0273] In the first method, target cells are cultured in a preconditioning medium under at least one stress-inducing condition, followed by administration of a conditioned medium or a secretome-, extracellular vesicle-, and / or sEV-containing composition to the cell culture. The target cells are then cultured in the presence of the conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing composition, and at least one characteristic of the cultured cells is measured one or more times during the culture. In some embodiments, the at least one characteristic is measured multiple times during the culture in the presence of the conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing composition (e.g., 5 minutes to 10 hours apart; 10 minutes to 4 hours apart; or 30 minutes to 2 hours apart).

[0274] In some embodiments of this first method, culturing in the presence of the conditioned medium or secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition is performed in the presence of at least one stress-inducing condition. In other embodiments of this first method, culturing in the presence of the conditioned medium or secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition is performed in the absence of at least one stress-inducing condition.

[0275] In some embodiments of this first method, the preconditioning medium is removed from the cells prior to culturing in the presence of the conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing composition. Thus, in embodiments of the first method in which at least one stress-inducing condition is imparted by the preconditioning medium (e.g., by a stress-inducing agent present in the preconditioning medium), culturing in the presence of the conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing composition is performed in the absence of the at least one stress-inducing condition.

[0276] In other embodiments of this first method, the preconditioning medium is not removed from the cells prior to culturing in the presence of the conditioned medium or the secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition. Thus, in embodiments of the first method in which at least one stress-inducing condition is imparted by the preconditioning medium (e.g., by a stress-inducing agent present in the preconditioning medium), culturing in the presence of the conditioned medium or the secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition is performed in the presence of at least one stress-inducing condition.

[0277] In a second method, target cells are cultured in a preconditioning medium, followed by administration of conditioned medium or a secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition (and optionally thereafter, the target cells are cultured in the presence of the conditioned medium or secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition). The target cells are then cultured under at least one stress-inducing condition, and at least one property of the cultured cells is measured one or more times during culture under the at least one stress-inducing condition (which is also performed in the presence of the conditioned medium or secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition). In some embodiments, the at least one property is measured multiple times during culture under the at least one stress-inducing condition (and in the presence of the conditioned medium or secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition), e.g., 5 minutes to 10 hours apart; 10 minutes to 4 hours apart; or 30 minutes to 2 hours apart.

[0278] In some embodiments of this second method, the target cells are cultured in the presence of conditioned medium or a secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition before being cultured under at least one stress-inducing condition. In other embodiments of this second method, the target cells are not cultured in the presence of conditioned medium or a secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition before being cultured under at least one stress-inducing condition. In some embodiments of this second method, the conditioned medium or secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition is removed from the target cells before being cultured under at least one stress-inducing condition.

[0279] In some embodiments of the first and second methods described above, the stress-inducing condition is culturing in the presence of a cell stress agent. In some embodiments of the second method, the cell stress agent is co-administered to the target cells with the conditioned medium or the secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition.

[0280] In some embodiments of the first and second methods above, the cell stress agent is one or more apoptosis-inducing agents.

[0281] The one or more apoptosis inducers may be selected from, for example, doxorubicin, staurosporine, etoposide, camptothecin, paclitaxel, vinblastine, gambogic acid, daunorubicin, tyrphostins, thapsigargin, okadaic acid, mifepristone, colchicine, ionomycin, 24(S)-hydroxycholesterol, cytochalasin D, brefeldin A, raptinal, carboplatin, C2 ceramide, actinomycin D, rosiglitazone, kaempferol, berberine chloride, bioimifi, betulinic acid, tamoxifen, embelin, phytosphingosine, mitomycin C, birinapant, anisomycin, genistein, cycloheximide, and the like.

[0282] In some embodiments, the apoptosis inducer is indolocarbazole. In some embodiments, the apoptosis inducer is indolo(2,3-a)pyrrole(3,4-c)carbazole. In some embodiments, the apoptosis inducer is staurosporine or a derivative thereof. In other embodiments, the apoptosis inducer is doxorubicin or a derivative thereof.

[0283] In some embodiments of the first and second methods, the at least one characteristic measured is viability of the cultured cells. Viability may be measured, for example, using a DNA labeling dye or a nuclear staining dye. In some embodiments thereof, the DNA labeling dye or the nuclear staining dye is a fluorescent dye, such as a near-infrared fluorescent dye.

[0284] In some embodiments of the first and second methods, one or more of the following may be performed in the absence of serum: (a) preconditioning medium; (b) conditioned medium or secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition; and (c) culturing the target cells with at least one stress-inducing condition. In some embodiments, the target cells may be serum-depleted before administering the conditioned medium or secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition. In other embodiments, the target cells may be serum-depleted after administering the conditioned medium or secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition. In some embodiments, the cells may be serum-depleted before and after administering the conditioned medium or secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition.

[0285] In embodiments of the first and second methods, the target cells can be cultured in the pretreatment medium for various lengths of time, for example, the target cells can be cultured in the pretreatment medium for 30 minutes to 10 hours, for 1 hour to 5 hours, or for more than, less than, or about 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours.

[0286] In embodiments of the first and second methods, the target cells are cultured with the conditioned medium or the secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition for at least 30 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, or at least 48 hours.

[0287] In some embodiments of the first and second methods, the target cells are cultured in vitro before culturing in the pretreatment medium. For example, the target cells may be cultured in vitro for 1 to 21 days, 3 to 17 days, 5 to 14 days, or less than 20 days, less than 18 days, less than 16 days, less than 14 days, less than 12 days, less than 10 days, less than 8 days, less than 6 days, less than 4 days, or less than 2 days before culturing in the pretreatment medium. In certain embodiments in which the target cells are cultured in vitro before culturing in the pretreatment medium, the target cells are fed fresh culture medium before culturing in the pretreatment medium. For example, the target cells may be fed fresh culture medium for 6 to 72 hours, 8 to 60 hours, 10 to 48 hours, or 12 to 36 hours before culturing in the pretreatment medium.

[0288] In the first and second method embodiments, the culture of target cells may be a two-dimensional or three-dimensional cell culture. For example, in some embodiments, the culture vessel used for the culture may be, for example, a flask, tissue culture flask, hyperflask, dish, Petri dish, tissue culture dish, multi-dish, microplate, microwell plate, multi-plate, multiwell plate, microslide, chamber slide, tube, tray, CellSTACK® chamber, culture bag, roller bottle, bioreactor, stirred culture vessel, spinner flask, microcarrier, or vertical wheel bioreactor.

[0289] In embodiments in which the culture comprises a two-dimensional cell culture, such as on the surface of a culture vessel, the culture surface (to which the cells are intended to adhere) may be coated with one or more substances that promote cell adhesion. Such substances useful for enhancing attachment to a solid support include, for example, types I, II, and IV collagen, concanavalin A, chondroitin sulfate, fibronectin, fibronectin-like polymers, gelatin, laminin, poly-D and poly-L-lysine, Matrigel, thrombospondin, and / or vitronectin.

[0290] In the first and second method embodiments, the at least one property may also be analyzed relative to one or more control samples.

[0291] For example, the first and second methods may further include culturing positive control cells in parallel, where the positive control cells are not administered conditioned medium or the secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition and are not cultured under at least one stress-inducing condition. Thus, in embodiments where the stress-inducing condition is the presence of an apoptosis-inducing agent, the positive control cells are not administered the apoptosis-inducing agent.

[0292] The first and second methods may include culturing negative control cells in parallel, where the negative control cells are not administered conditioned medium or secretome-containing, extracellular vesicle-containing, and / or sEV-containing compositions. In some embodiments, the negative control cells include negative control cells that are subjected to the same steps as the target cells, except that they are not administered secretome.

[0293] In certain embodiments, the negative control cells comprise negative control cells cultured in a pre-conditioning medium under at least one stress-inducing condition, and then the at least one property measured in the target cells can also be measured in the negative control cells either while they are being cultured in a pre-conditioning medium under at least one stress-inducing condition or thereafter.

[0294] In some embodiments, negative control cells include negative control cells to which mock conditioned medium or a mock secretome-, extracellular vesicle-, and / or sEV-containing composition is added, hi specific embodiments thereof, the mock conditioned medium or mock secretome-, extracellular vesicle-, and / or sEV-containing composition is produced by omitting cells from a process for producing a conditioned medium or secretome-, extracellular vesicle-, and / or sEV-containing composition, such as a process of the present disclosure.

[0295] The use of such one or more negative controls allows for the determination of the activity, function, and / or efficacy of the conditioned medium or secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition. For example, if at least one property being measured is the viability of cultured cells, the conditioned medium or secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition can be determined to have activity, function, efficacy (and / or exhibit a therapeutic effect) if the viability of the target cells is greater than the viability of the negative control cells.

[0296] Alternatively, for example, if at least one property measured is cell adhesion, cell growth, and / or cell number, and cell adhesion, cell growth, and / or cell number are determined by measuring the electrical impedance of the culture vessel surface in the culture, the conditioned medium or secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition may be determined to have activity, function, efficacy (and / or exhibit a therapeutic effect) when the electrical impedance of the culture vessel surface in the culture is higher than the electrical impedance of the culture vessel surface in a culture of negative control cells.

[0297] Any one or more samples, and / or any one or more positive and / or negative controls may be performed in replicates, e.g., in duplicate, triplicate, etc. In some embodiments where cell viability is measured and replicate cultures are performed, the number of positive control cells in the replicate cultures may be averaged to determine an average maximum cell number (and cell viability may be calculated by normalizing the number of target cells in each replicate test culture by this average maximum cell number).

[0298] To more accurately compare the activity, function, and / or potency between different conditioned medium or secretome-containing, extracellular vesicle-containing, and / or sEV-containing compositions, it may be beneficial to determine the amount of conditioned medium or secretome-containing, extracellular vesicle-containing, and / or sEV-containing composition to add to target cells, which can be determined based on, for example, one or more of the amount of secretory cells that produced the secretome, the protein content of the secretome, the RNA content of the secretome, the exosome content of the secretome, and particle number. [Example]

[0299] The following examples illustrate non-limiting embodiments of the present invention. While efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., amounts, concentrations, rates of change, etc.), some experimental error and deviation must be accounted for. It should be understood that these examples are provided as illustrations only and are not intended to limit the scope of what the inventors regard as various embodiments of the present invention. Not all of the following steps shown in each example are required, nor are the steps in each example necessarily in the exact order presented.

[0300] Example 1 Generation of cardiovascular progenitor cells from iPSCs Using the process illustrated in Figure 1, human iPS cells (iPSCs) were expanded in suspension culture in PBS minivessels (PBS MINI 0.5L Bioreactor Single-Use Vessels; PBS Biotech Reference: 1A-0.5-D-001) and differentiated into cardiovascular progenitor cells (CPCs). At the end of the CPC differentiation period, cells were counted as follows: a small sample (5–10 mL) of the cell aggregates in suspension was removed from the suspension culture vessel, the cell aggregates were allowed to settle by gravity, the supernatant was removed, and the aggregates were resuspended in 3–5 mL of room-temperature TrypLE Select (Invitrogen Reference: 12563029) and incubated at 37°C for 3–10 minutes. Digestion was stopped using two volumes of RPMI-B27 Quench Medium (RPMI 1640 Medium (Gibco Reference: 118875-085) supplemented with B-27 XenoFree, CTS Grade 50x (Gibco Reference: A14867-01, fc = 1x), filter-sterilized using a 0.2 μm filter (ThermoScientific Reference: 567-0020). The cell suspension was then centrifuged at 300 × g for 5 min, and the resulting supernatant was discarded. The remaining cell pellet was carefully loosened, and the cells were resuspended in 5–10 mL of MEMα medium base (MEMα, GlutaMAX™, nucleoside-free, Gibco Reference: 32561-037). One or two 500 μL samples of these resuspended cells were counted using a ViCell XR Cell Viability Analyzer (Beckman Coulter) according to the manufacturer's instructions. Viable cells / mL were recorded. As illustrated in Figure 2, two separate differentiation runs were performed that achieved similar yields of CPCs per input iPSC.

[0301] To confirm that the resulting cells were indeed CPCs, we analyzed RNA expression by the resulting cells. Specifically, 1–2 million cells were removed from the cell sample and lysed in RLT plus buffer (Qiagen 1030963) for RNA extraction. RNA was extracted from this lysate using the Qiasymphony RNA Kit (Qiagen, Ref: 931636) according to the manufacturer's instructions. The mRNA levels of 48 custom-selected genes were determined using the Fluidigm platform. Unsupervised hierarchical clustering was performed on the raw data using the Fluidigm package. RNA expression by the resulting cells was compared with that of iPSC and cardiomyocyte control cells, confirming that the gene expression by the resulting cells was consistent with their identification as CPCs (Figure 3).

[0302] To dissociate CPC aggregates into single cells, 300–800 mL of CPC aggregate suspension was collected from differentiated suspension cultures and allowed to settle for approximately 5 minutes in a 500 mL conical tube. The spent medium was then removed, and the cell aggregates were washed with DPBS- / -. The washed cell aggregates were then resuspended in room temperature TrypLE (approximately 25 mL TrypLE per 100 mL of original disaggregated suspension volume) and allowed to dissociate for 10 minutes at 37°C. Dissociation of the cell aggregates was quenched with an equal volume of RPMI-B27 quench medium, and the dissociated cells were spun at 400 × g for 5 minutes. The resulting cell pellet was resuspended in RPMI-B27 quench medium and then filtered (Falcon 100 μm cell strainer, Corning Ref: 352360) into a conical tube and counted using a ViCell XR cell viability analyzer (Beckman Coulter).

[0303] A portion of these cells was spun again at 300 × g for 5 min and resuspended in α-MEM complete medium (MEM α medium base (MEM α, GlutaMAX™, without nucleosides, Gibco reference 32561-0.37); gentamicin (Gibco reference 15750060, final concentration (fc) = 0.025 mg / mL); glucose supplement (Gibco reference A2494001, ratio 1:200); Flexbumin (containing 25% w / vol human serum albumin, Baxter reference: NDC0944-0493-02 code 2G0012, fc HSA = 2 mg / mL); B27 (without insulin) (50×, Gibco reference A1895601, fc = 1×); human FGF-2 Premium grade (Miltenyi Biotec The cells were resuspended in a 0.2 μm filter (reference: A12873-01, fc=1 μg / mL; filter sterilized using a 0.2 μm filter (ThermoScientific reference 567-0020); medium was used the same day). Freshly harvested single cells were counted again using a ViCell XR cell viability analyzer (Beckman Coulter) and plated (see Example 2 below). The remaining single cell suspension was spun at 400×g for 5 minutes, and the cells were resuspended at 25 million cells / mL in cryopreservation medium (CryoStor CS-10, Biolife Solutions reference: 210102), frozen at −80°C, and then stored in liquid nitrogen for later use in CPC plate vesicle formation cultures after thawing.

[0304] Example 2 Vesicle-forming culture of cardiovascular progenitor cells In the vesicle formation process, CPCs were cultured as fresh aggregates in suspension culture, as fresh single cells seeded in HyperFlasks, or as thawed single cells seeded in HyperFlasks after cryopreservation and maintaining at or below -80°C until use. Specifically, the CPCs prepared in Example 1 were used in suspension vesicle formation culture and adherent vesicle formation culture in HyperFlasks as described below.

[0305] For suspension vesicle-forming cultures, the volume of the aggregates in the PBS minivessels at the end of the CPC differentiation process was recorded (300–400 mL per vessel; "day +0" volume). Cell aggregates were subjected to a 100% medium exchange according to the following steps: (1) the cell aggregates were transferred from the PBS minivessels to conical tubes and allowed to settle for approximately 15 minutes; (2) the PBS minivessels were rinsed three times with MEMα medium base (MEMα, GlutaMAX™, nucleoside-free, Gibco reference 32561-.37); (3) the spent medium was removed from the settled cell aggregates; (4) the cell aggregates were washed three times with the appropriate volume of MEMα medium base; and (5) the washed cell aggregates were reseeded in their original (washed) PBS minivessels in α-MEM complete medium (as described above) at their day +0 volume to maintain cell density.

[0306] The seeded cell aggregates were then cultured (37°C, 5% CO2, atmospheric oxygen) in suspension with agitation at 40 rpm for 2 days (until "Day +2"). On Day +2, the cell aggregates were rinsed three times with MEM alpha medium base, followed by a 100% medium change. For this Day +2 medium change, the cell aggregates were reseeded in their original PBS minivessels at the same volume as their Day +0 volume in alpha-MEM nutrient-poor medium (MEM alpha, GlutaMAX™, nucleoside-free, Gibco Reference 32561-.37) supplemented with gentamicin (Gibco Reference 15750060, final concentration (fc) = 0.025 mg / mL) and glucose supplement (Gibco Reference A2494001, ratio 1:200, filter-sterilized using a 0.2 μm filter (ThermoScientific Reference 567-0020)). The cell aggregates were then cultured (37°C, 5% CO2, atmospheric oxygen) in suspension with agitation at 40 rpm for an additional 2 days until the end of the vesicle formation period ("day +4").

[0307] For HyperFlask adherent culture, fresh single-cell CPCs were plated at 100,000 cells / cm in α-MEM complete medium in vitronectin-coated HyperFlasks. 2("Day +0"). Additionally, cryopreserved CPCs were thawed at 37°C for 3 minutes, transferred to an empty conical tube, and then resuspended (dropwise) in α-MEM complete medium. The thawed cell suspension was centrifuged, and the cell pellet was resuspended in α-MEM complete medium. Thawed CPCs were plated at 100,000 cells / cm in α-MEM complete medium in vitronectin-coated HyperFlasks. 2 Cells were seeded at 37°C, 5% CO2, and atmospheric oxygen for 2 days (until "Day +2") for both fresh and thawed CPCs. On Day +2, the spent medium was removed and the flasks were rinsed three times with 50-100 mL of prewarmed MEM α medium base. The culture vessels were then filled with α-MEM nutrient-poor medium according to the manufacturer's instructions and incubated at 37°C, 5% CO2, and atmospheric oxygen for an additional 2 days until the end of the vesicle formation period ("Day +4").

[0308] On days +2 and +4, cells in suspension culture were counted as described in Example 1 above. On day +4, cells in adherent culture were harvested by: 1 / rinsing the cells with DPBS; 2 / incubating the cells with 100 mL of pre-warmed 0.05% trypsin-EDTA (Gibco, 15400-054, diluted in DPBS) for 2-3 minutes at room temperature; 3 / quenching the harvest with 100 mL aMEM+glutamax supplemented with B27 (insulin-free) (fc1x); 4 / collecting the bulk cell suspension into a 500 mL conical centrifuge tube; 5 / rinsing the harvested flask with basal aMEM medium to recover any remaining cells, and adding this rinse to the bulk cell suspension. The concentration of cells in the suspension was determined using a ViCell automated cell counter, and 1 cm of the harvested vessel was removed. 2 The number of cells per well was back-calculated.

[0309] In addition to the CPC adherent and floating vesicle-forming cultures, unused medium controls were also performed for the adherent and floating cultures.

[0310] For the floating vesicle culture (unused medium control), a new 0.5 L PBS minivessel was filled with 400 mL α-MEM complete medium ("Day +0") and incubated for 2 days (37°C, 5% CO2, atmospheric oxygen) with agitation at 40 rpm. After 2 days ("Day +2"), the spent culture medium was removed and the vessel was thoroughly rinsed (3 times with 50-100 mL of pre-warmed MEM α-medium base). The PBS minivessel was then filled with 400 mL α-MEM nutrient-poor medium and incubated for an additional 2 days (37°C, 5% CO2, atmospheric oxygen) until "Day +4".

[0311] For the adherent vesicle formation culture unused medium control, vitronectin-coated HyperFlasks were filled with α-MEM complete medium and incubated for 2 days (37°C, 5% CO2, atmospheric oxygen). After these 2 days ("Day +2"), the spent culture medium was removed and the vessel was thoroughly rinsed (3 times with 50-100 mL of pre-warmed MEM α-medium base). The HyperFlasks were then filled with α-MEM nutrient-poor medium and incubated for an additional 2 days (37°C, 5% CO2, atmospheric oxygen), until "Day +4".

[0312] Day +4 medium from suspension and adherent cell cultures (conditioned medium, MC) and day +4 medium from unused control vessels (virgin medium, MV) were collected and pre-clarified by sequential centrifugation (400×g for 10 minutes at 4° C., followed by 2000×g for 30 minutes at 4° C.). This pre-clarified medium was then aliquoted into conical tubes and frozen at −80° C. Figure 4 illustrates the process flow diagram for the generation of conditioned medium and virgin medium controls.

[0313] Example 3 Preparation of small extracellular vesicle-enriched fraction (sEV) To validate the vesicle formation process, samples of conditioned and control media were subjected to ultracentrifugation to generate sEV and MV preparations for molecular characterization and in vitro functional analysis. Two biological replicates were prepared for each sample type. Figure 5 illustrates the process flow diagram for the isolation of sEV or mock (unused media) control samples.

[0314] MC and MV were thawed for 1–4 hours at room temperature or overnight at 4°C. After thawing, MC and MV were ultracentrifuged at 100,000 × g for 16 hours at 4°C (wX+ Ultra series centrifuge, ThermoScientific; rotor: F50L-8x39; acceleration: 9; deceleration: 9), and the resulting supernatant was removed. The bottom of each tube was rinsed twice with 100 μL of DPBS (0.1 μm PES filter unit, ThermoFisher 565-0010) after passing through a 0.1 μm filter, without disturbing the pellet. Each pellet was then resuspended in DPBS by gently stirring with a sterile glass stir bar after passing through a 0.1 μm filter. The sEV preparation was harvested and the tubes were rinsed with DPBS − / − after passing through a 0.1 μm filter to maximize recovery of the product (to a target volume of total resuspension + rinse, calculated based on the number of secreting cells giving rise to conditioned medium): 1.4 × 10 as calculated by the following formula: 6 Target 45 μL per day +4 secreting cells:

[0315] Target sEV resuspension volume = (total number of live cells on day +4 ÷ total volume of conditioned medium on day +4) × MC volume after centrifugation × (45 μL ÷ 1.4 × 10 6 living cells).

[0316] The target resuspension volume for MV controls matched the relevant MC target resuspension volume. For MC and MV generated in PBS minivessels, sEV preparations were filtered through 0.65 μm (Ultrafree 0.65 μm DV Durapore, Millipore ref: UFC30DV05) to remove large particulate matter. sEV and MV control preparations were aliquoted and frozen at -80°C.

[0317] The sEV and MV control preparations were further analyzed as described below.

[0318] First, the particle concentration and size distribution of sEV and MV control preparations were determined by nanoparticle tracking analysis (NTA; NanoSight). Nanoparticle tracking analysis confirmed the presence of exosome- and microparticle-sized particles in sEV prepared from CPC-conditioned medium, but not in the MV control. Figure 6 illustrates representative particle size distribution curves from two sEV and two control MV samples. Observable particle sizes ranged from approximately <30 nm to approximately 300 nm, with a peak generally between 50 and 150 nm, corresponding to the size of exosomes or small microparticles.

[0319] Second, the presence of the exosome-associated vesicle surface marker CD63 was also analyzed using a PS Capture Exosome ELISA kit (Wako Chemicals, Reference: 293-77601) with anti-CD63 antibody (Wako Chemicals, Reference: 292-79251) as the primary antibody and HRP-conjugated anti-mouse IgG antibody (Wako Chemicals, Reference: 299-79261) as the secondary antibody. The input volume was set so that 400 ng of protein from the sEV and MV control preparations was added to each well. This anti-CD63 ELISA confirmed the presence of the exosome-associated CD63 surface antigen in each of the sEV samples but not in any of the MV controls (Figure 7). The CD63 signal was higher in the aggregate samples than in the plate samples, but the CD63 signal was consistent between replicates of the plate samples. The protein content of sEV and MV control preparations was determined by BCA analysis using the Pierce Micro BCA kit (ThermoScientific reference: 23235).

[0320] Example 4 In vitro analysis of sEV function To analyze the functionality of the sEV preparations, three in vitro assays were used: a HUVEC scratch wound healing assay; a cardiomyocyte viability assay using serum-starved H9c2 cells; and a cardiomyocyte viability assay using staurosporine-treated human cardiomyocytes.

[0321] For the HUVEC scratch wound healing assay, a scratch wound healing assay (developed for Incucyte by Essen BioSciences) was used according to the manufacturer's instructions. Briefly, HUVEC cells were expanded using HUVEC complete medium: Endothelial Cell Basal Medium (PromoCell, Reference: C-22210) supplemented with Endothelial Cell Growth Medium Supplement Pack (PromoCell, Reference: C-39210). After expansion, cells were plated in CS10 (Cryostore, Reference: 210102) at 1–2 × 10 per aliquot. 6 Cells (enough to fill half to the full volume of a 96-well plate) were cryopreserved. Two days before the assay, an aliquot of HUVECs was thawed and seeded at 10,000 cells / well into ImageLock 96-well plates (EssenBio, Ref: 4379) and grown in complete HUVEC medium for 2 days. Cultures were maintained at 37°C (atmospheric oxygen, 5% CO2) throughout the maintenance and assay process. Scratches were made in the wells using a Wound Maker (EssenBio, Ref: 4493) according to the manufacturer's instructions, and the cells were then rinsed with endothelial cell basal medium and cultured overnight (either in complete HUVEC medium alone as a positive control; in endothelial cell basal medium alone as a negative control; or in endothelial cell basal medium supplemented with sEV or MV preparations). Plates were imaged every 3 hours for a total of 18 hours using an Incucyte with a scratch wound healing module. Wound closure was determined using the manufacturer's software, and values ​​were subtracted from the baseline (negative control) and normalized to the positive control. Figure 8 illustrates that the sEV preparation promoted wound healing, whereas the control MV preparation did not, demonstrating that the sEV preparation was functional.

[0322] For the cardiomyocyte viability assay using serum-starved H9c2 cells, the assay was performed essentially as described by El Harane et al. (Eur. Heart J., 2018;39:1835-1847). In this assay, H9c2 cardiomyocytes are proliferative when the culture medium is serum-rich (e.g., cultured in H9c2 complete medium), but when serum is deprived (e.g., cultured in H9c2 nutrient-poor medium), they cease proliferation and lose viability. The ability of sEV and MV preparations to promote H9c2 cardiomyocyte viability was determined by supplementing H9c2 nutrient-poor medium with increasing concentrations of sEV and MV control preparations. Figure 9 illustrates that the sEV preparation, but not the control MV preparation, improved H9c2 cardiomyocyte viability in the absence of serum, demonstrating that the sEV preparations are functional.

[0323] For cardiomyocyte viability assays using staurosporine-treated human cardiomyocytes, see iCell Cardiomyocytes. 2(Fujifilm Cellular Dynamics, Inc., Reference: CMC-100-012-001) were plated at 50,000 cells per well of a fibronectin-coated 96-well plate in iCell Cardiomyocyte Plating Medium (Fujifilm Cellular Dynamics, Inc., Reference: M1001) and cultured for 4 hours. The medium was then replaced with iCell Cardiomyocyte Maintenance Medium (iCMM, Fujifilm Cellular Dynamics, Inc., Reference: M1003), and the cells were cultured for up to 7 days, with complete medium changes every 2–3 days. After a minimum of 4 days, cells were exposed to iCMM containing NucSpot Live 650 dye (Biotium, ref. 40082) (which served as a live cell control) or iCMM containing NucSpot Live 650 dye and staurosporine (Abcam, ref. ab146588) at a final well concentration of 2 μM (which served as an apoptotic cell control). The dye, PBS, and DMSO concentrations, as well as the final well volume, were equal in all wells. Cells were cultured in these preincubation media for 4 hours. After this incubation, the preincubation media was removed, and the wells were rinsed with iCMM. Cells were then fed with iCMM containing NucSpot Live 650 dye and PBS, or iCMM containing NucSpot Live 650 supplemented with increasing concentrations of sEV or MV control preparations, maintaining the final PBS volume. Wells were imaged every hour for 24 hours in an Incucyte and nuclei counts were determined. Figure 10 illustrates that the sEV preparation, but not the control MV preparation, improved cardiomyocyte viability, demonstrating that the sEV preparation is functional.

[0324] Example 5 First exemplary Good Manufacturing Practices (GMP) compliant small extracellular vesicle (sEV) formulation production process We developed a first exemplary GMP-compliant sEV-containing formulation production process. This production process included four major steps: vesicle formation, conditioned medium clarification, small EV-enriched secretome enrichment and concentration, and production of the final sEV formulation. A flow diagram outlining the GMP-compliant process performed is shown in Figures 11A and 11B.

[0325] Vesicle formation

[0326] For the vesicle formation step, cardiovascular progenitor cells (CPCs) that had been cryopreserved and stored under vapor-phase liquid nitrogen (or in a −150°C freezer) were first thawed in EVA bags (Corning) in thawing medium (MEMα (MEMα, GlutaMAX™ supplement, no nucleosides; Gibco / Life Technologies; ref. 32561-029); glucose (30%) supplement (Macopharma ref. CARELIDE, to a final total glucose concentration of 2 mg / mL; Ydralbum® (LFB), final concentration 20 mg / mL; B-27™ supplement (50×, Life Technologies ref. 17504001, final concentration 1×); and Rock Inhibitor H1152 (Sigma ref. 555550, final concentration 0.392 μg / mL) for 2 min at 37°C. 18 mL of thawing medium was used per mL of CPCs.

[0327] After thawing, 0.2 mL / cm of vitronectin (Life Tech Reference: VTN-N; recombinant human protein, truncated (Reference: A31804); 5 μg / mL, sterilized using a 0.22 μm filter (syringe filter 0.2 μm polyethersulfone (PES) membrane) coated culture flasks (8 x 10 CellStack culture chambers, tissue culture (TC) treated (Corning Reference: 3271); and 2 x TC-treated vitronectin-coated T75 flasks) were added. 2Complete medium (MEMα, GlutaMAX™ supplement, no nucleosides; Gibco / Life technologies; ref: 32561-029; glucose (30%) supplement (Macopharma reference: CARELIDE, to a final total glucose concentration of 2 mg / mL; Ydralbum® (LFB; 200 g / L); B-27™ supplement (50×, Life Tech reference: 17504001 or 17504044, final concentration 1×); gentamicin (Panpharma, final concentration 25 μg / mL); and human FGF-2 Premium grade (Miltenyi Biotec reference: A12873-01, final concentration 1 μg / mL)) was used to grow CPCs to a density of 1 cm. 2 The cells were seeded at a seeding density of approximately 100,000 cells per well. Seeding was performed without prior centrifugation of the cell suspension. The seeded CPCs were then cultured in complete medium at 37°C for 3 days in the presence of 5% CO2 and atmospheric oxygen.

[0328] Immediately prior to seeding ("D+0"), cells were analyzed to determine the number and percentage of viable cells by DAPI / AO staining (Ph.Eur.2.7.29) using a NucleoCounter NC-200 (Chemometec) (see Figure 22, column 1 ("D+0 cells")); their identity was determined by flow cytometry using a MACSQuant 10 flow cytometer (see Figure 12 and Example 7); and their transcriptome was analyzed (see Figure 13 and Example 8).

[0329] After 3 days of culture ("D+3"), cells were harvested from one of the cultured T75 flasks. These harvested cells were analyzed to determine the number and percentage of viable cells with DAPI / AO staining (Ph.Eur.2.7.29) using a NucleoCounter NC-200 (Chemometec) (see Figure 22, column 2 ("D+3 Material")); their identity was determined by flow cytometry using a MACSQuant 10 flow cytometer (see Figure 12 and Example 7); and their transcriptome was analyzed (see Figure 13 and Example 8). Spent medium from the 10ST CellStack culture chamber was also tested for sterility and the presence of mycoplasma and endotoxin.

[0330] For the remaining flasks (8 x 10ST CellStack culture chambers; and 1 x T75), cells were visualized by microscopy to determine their morphology (see Figure 14) and washed twice with wash medium (MEMα (Macopharma ref: BC0110021); glucose (30%) supplement (Macopharma ref: CARELIDE, to a final total glucose concentration of 2 mg / mL)) before being cultured in starvation medium (nutrient-poor medium) (MEMα (1000 mL Macopharma ref: BC0110021); glucose (30%) supplement (Macopharma ref: CARELIDE, to a final total glucose concentration of 2 mg / mL)) at 37 °C for 2 days in the presence of 5% CO. After this 2-day incubation ("D+5"), the culture medium (conditioned medium) was collected, and cells were harvested from the 10ST CellStack culture chambers and the remaining T75 flasks.

[0331] Similar to the D+3 cells, the D+5 cells were also visualized by microscopy to determine their morphology (see FIG. 14); and the cells collected on D+5 were further analyzed to determine the number and percentage of viable cells (see FIG. 22, column 3 (“D+5 cells”)); their identity was determined by flow cytometry using a MACSQuant 10 flow cytometer (see FIG. 12 and Example 7); and their transcriptome was analyzed (see FIG. 13 and Example 8). The collected conditioned medium was tested for sterility and the presence of mycoplasma and endotoxin before further processing.

[0332] Conditioned Medium Clarification

[0333] The conditioned medium was clarified through a series of four filtration steps. First, filtration was performed using a 200 μm drip chamber filter (Gravity Blood Set, BD careFusion, reference: VH-22-EGA). Next, the obtained filtrate was infused using a 15 μm filter (DIDACTIC, reference: PER1FL25). Next, the obtained filtrate was infused using Sartoguard PES XLG MidiCaps (pore size (prefilter + filter): 1.2 μm + 0.2 μm, size 7 (0.065 μm)). 2 The resulting filtrate was then filtered using a vacuum filter / storage bottle system (0.22 μm, 33.2 cm pore size). 2 Further filtration was performed using a PES membrane (Corning ref: 431097).

[0334] Accumulation and concentration

[0335] After clarification of the conditioned medium, the conditioned medium was subjected to enrichment and concentration of small EV secretomes.

[0336] First, the clarified conditioned medium was subjected to tangential flow filtration (TFF) using a TFF Allegro™ CM150 (PALL / Sartorius). The TFF manifold used a sterile, single-use flow-line manual valve P&F (PALL / Sartorius, ref. 744-69N) in conjunction with a 5 L retentate assembly (sterile, single-use; PALL / Sartorius ref. 744-69L). The TFF cassette contained a sterile, single-use regenerated cellulose filter (30 kDa cutoff; 0.14 m 2 Sartorius Reference: Opta Filter Assembly + 3D51445901MFFSG) was used. For collection of the retentate (i.e. what remains in the TFF) a benchtop TFF 1 L bag was used (PALL / Sartorius, Reference: 7442-0303P).

[0337] Before starting the TFF device, it was first washed with 10 L of HO and 1 L of 1x PBS (sterilized by filtration using a 0.2 μm filter). The clarified conditioned medium was then administered to the TFF device, after which the retentate was concentrated (until 500 mL; pressure did not exceed 3 bar). After this initial concentration step, the retentate was subjected to diafiltration (6 diafiltration volumes; 1x DPBS sterilized by filtration using a 0.2 μM filter was used). After diafiltration, the retentate was further concentrated to produce a total volume of at least 100 mL. The parameters of the TFF process were as follows: feed manifold pressure (PT01) - 0.86~2.1 bar; retentate manifold pressure (PT02) - 0.11~0.14 bar; retentate manifold flow rate (FT01) - 0.03~0.32 L / min; transmembrane pressure (TMP01) - 0.4~1.1 bar; and quattroflow pump (P01) - 18~23%.

[0338] Example 6 Formulations / Compositions After TFF enrichment and concentration, the retentate was processed as illustrated in Figure 11B. Briefly, the retentate alone, the retentate containing 25 mM trehalose, and the retentate containing 5 g / L L-histidine were each stored in glass vials (2 mL, bromobutyl cap; Adelphi reference: VCDIN2RDLS1) and stored at -80°C. These samples were subjected to quality control checks (the various stages where quality control checks were performed are described, e.g., in the table below). * 6. * 7th prize, “ * "). In addition, the final sEV formulations were also prepared by filter-sterilizing the retentate (with or without 25 mM trehalose) using a 0.22 μm filter (Sterivex™-GP Pressure Filter Unit, 0.22 μm, Millipore, ref: SVGPL10RC). After the sterilization step, the final formulations (with or without the addition of 25 mM trehalose) were filled into glass vials (2 mL, bromobutyl cap; Adelphi ref: VCDIN2RDLS1). The final formulations were stored at -80°C for later use or testing.

[0339] Thus, the final formulation was in PBS (with or without trehalose) and was positive for CD9, CD63, and CD81 (canonical EV markers) as well as the cardiac-related markers CD49e, ROR1, SSEA-4, MSCP, CD146, CD41b, CD24, CD44, CD236, CD133 / 1, CD29, and CD142 when detected by MACSPlex (as shown in Figures 16A, 16C, 17A, and 17B).

[0340] Example 7 Characterization of CPC identity during vesicle formation in a GMP-compliant process To assess cell identity during the vesicle formation process in Example 5, D+0 CPCs and cells collected on D+3 and D+5 were analyzed by flow cytometry. iPSC and cardiomyocyte (CM) cells were included as controls. As shown in Figure 12, flow cytometry analysis of iPSC, CPC, and cardiac markers using a MACSQuant 10 flow cytometer demonstrated further CPC maturation over the 5-day vesicle formation period. Specifically, CPCs maintained little to no NANOG or SOX2 protein expression and continued to exhibit increased CD56, cTNT, and aMHC protein expression (however, they never reached CD56, cTNT, and aMHC expression levels similar to those of cardiomyocytes, indicating that they remained progenitor cells throughout the process). iPSC and CM control cells were analyzed separately, and the average values ​​are presented in Figure 12 for comparison.

[0341] Example 8 Transcriptome analysis of CPC during vesicle formation in a GMP-compliant process To assess the cellular transcriptome during the vesicle formation process in Example 5, RNA was extracted from CPCs on D+0 and from cells harvested on D+3 and D+5 of the vesicle formation process. RNA was also extracted from iPSCs (pluripotent cell control) and iPSC-derived cardiomyocytes (differentiated cardiomyocyte control). Total RNA was sequenced on an Illumina NovaSeq 6000 platform, and differential gene expression was determined on normalized data.

[0342] The heatmap shown in Figure 13 was generated based on hierarchical clustering analysis using the UPGMA clustering method with correlation distance metric in TIBCO Spotfire software v11.2.0. The genes included in the panel included genes expressed at various stages of differentiation (from iPSCs to beating cardiomyocytes) as well as related off-target cells. Therefore, the gene expression analysis results shown in Figure 13 confirmed that the cells retained cardiovascular progenitor characteristics throughout the vesicle formation process.

[0343] Example 9 Analysis of EV particle concentration and EV size distribution in a GMP-compliant process To assess the particle concentration and size distribution of the EVs produced in Example 5, the clarified conditioned medium (before TFF) and the final formulation (with and without trehalose) were analyzed by nanoparticle tracking analysis (NTA; NanoSight). Figure 15A illustrates a representative particle size distribution curve for each sample. The overall particle size distribution, mean, and mode were similar between samples. A peak was generally observed between 50 and 150 nm, which corresponds to the size of exosomes or small microparticles. The TFF step resulted in an approximately 32-fold increase in particle concentration. Similar experiments were also performed on the post-storage retentate samples (with and without trehalose or histidine) depicted in Figure 11B that were not filter-sterilized (see " * 6", Samples a-c). The results of these experiments are shown in Figure 15B.

[0344] Example 10 Analysis of EV markers in EVs produced by a GMP-compliant process To assess the presence of EV markers in the clarified conditioned medium (pre-TFF) and final formulations (with and without trehalose) from Example 5, the MACSPlex Exosome Kit Human (Miltenyi Reference: 130-108-813) was used to identify and quantify the presence of EV markers. As shown in Figure 16A, this analysis confirmed the presence of extracellular vesicle tetraspanins (CD9, CD81, and CD63) in both the conditioned medium (pre-TFF) and the final formulations (with and without trehalose). Furthermore, as shown in FIG. 16B, MACSPlex analysis also revealed various markers that were either present in low amounts (e.g., CD3, CD4, CD8, HLA-DRDPDQ, CD56, CD105, CD2, CD1c, CD25, CD40, CD11c, CD86, CD31, and CD20) or were virtually absent (CD19, CD209, HLA-ABC, CD62P, CD42a, and CD69) in the conditioned medium (pre-TFF) and / or in the final formulations (with and without trehalose). Similar experiments were also performed on post-storage retentate samples (with and without trehalose or histidine) depicted in FIG. 11B that were not filter-sterilized (see " * 6", Samples a-c). The results of these experiments are shown in Figures 16C and 16D.

[0345] In addition, as shown in Figure 17A, additional cardiac-related markers were also observed in the conditioned medium (before TFF) and the final formulation (with and without trehalose). Similar experiments were also performed to confirm the presence of these additional cardiac-related markers in the post-storage retentate samples (with and without trehalose or histidine) depicted in Figure 11B, which were not filter-sterilized (see " * 6", Samples a-c). The results of these experiments are shown in Figure 17B.

[0346] Example 11 In vitro analysis of the potency of EVs produced by a GMP-compliant process To analyze the functionality and efficacy of the final formulation produced by the GMP-compliant process in Example 5, two in vitro assays using human cardiomyocytes after staurosporine treatment were used: a HUVEC scratch wound healing assay; and a cardiomyocyte viability assay.

[0347] For the HUVEC scratch wound healing assay, a scratch wound healing assay (developed for Incucyte by Essen BioSciences) was used according to the manufacturer's instructions. Briefly, HUVEC cells were expanded using HUVEC complete medium: Endothelial Cell Basal Medium (PromoCell, Reference: C-22210) supplemented with Endothelial Cell Growth Medium Supplement Pack (PromoCell, Reference: C-39210). After expansion, cells were plated in CS10 (Cryostore, Reference: 210102) at 1–2 × 10 per aliquot. 6 Cells (enough to fill half to the full volume of a 96-well plate) were cryopreserved. Two days before the assay, an aliquot of HUVECs was thawed and seeded at 10,000 cells / well into ImageLock 96-well plates (EssenBio, Ref. 4379) and grown in complete HUVEC medium for two days. Throughout the maintenance and assay process, cultures were maintained at 37°C (atmospheric oxygen, 5% CO2). Scratches were made in the wells using a Wound Maker (EssenBio, Ref. 4493) according to the manufacturer's instructions, and the cells were then rinsed with endothelial cell basal medium and cultured overnight (either HUVEC complete medium and PBS as a positive control; endothelial cell basal medium and PBS as a negative control; or endothelial cell basal medium supplemented with sEV preparation in PBS). Plates were imaged 21 hours after treatment using an Incucyte with the scratch wound healing module. Wound closure was determined using the manufacturer's software, and values ​​were subtracted from the baseline (negative control) and normalized to the positive control. Figure 18 illustrates that the final formulations with and without trehalose (samples b and a, respectively) promoted wound healing.

[0348] For cardiomyocyte viability assays using human cardiomyocytes after staurosporine treatment, see iCell Cardiomyocytes 2 (Fujifilm Cellular Dynamics, Inc., Reference: CMC-100-012-001) were plated at 50,000 cells per well of a fibronectin-coated 96-well plate in iCell Cardiomyocyte Plating Medium (Fujifilm Cellular Dynamics, Inc., Reference: M1001) and cultured for 4 hours. The medium was then replaced with iCell Cardiomyocyte Maintenance Medium (iCMM, Fujifilm Cellular Dynamics, Inc., Reference: M1003), and the cells were cultured for up to 7 days, with complete medium changes every 2–3 days. After a minimum of 4 days, cells were exposed to iCMM containing NucSpot Live 650 dye (Biotium, ref. 40082) (which served as a live cell control) or iCMM containing NucSpot Live 650 dye and staurosporine (Abcam, ref. ab146588) at a final well concentration of 2 μM (which served as an apoptotic cell control). The dye, PBS, and DMSO concentrations, as well as the final well volume, were equal in all wells. Cells were cultured in these preincubation media for 4 hours. After this incubation, the preincubation media was removed, and the wells were rinsed with iCMM. Next, while maintaining the final PBS volume, cells were fed with iCMM containing NucSpot Live 650 dye and PBS, or iCMM containing NucSpot Live 650 supplemented with increasing concentrations of sEV preparations (samples a and b). At 24 hours, wells were imaged in an Incucyte and nuclei counts were determined. Figure 19 illustrates that the final formulations with and without trehalose promoted cardiomyocyte survival.

[0349] The test panel used for the process / product of Example 5 and as embodied in Examples 6-11, for example, is shown in Figure 21. The results are therefore shown in Figure 22. Additionally, Figure 23 illustrates the enrichment for the retentate and final formulation made in Example 6 compared to the conditioned medium after clarification.

[0350] Example 12 A second exemplary Good Manufacturing Practices (GMP)-compliant small extracellular vesicle-enriched fraction (sEV) formulation production process A second exemplary GMP-compliant sEV-containing formulation production process was developed. This production process included four major steps: vesicle formation, conditioned medium clarification, small EV-enriched secretome enrichment and concentration, and production of the final sEV formulation. A flow diagram outlining the GMP-compliant process performed is illustrated in Figures 24A and 24B.

[0351] Vesicle formation

[0352] For the vesicle formation step, cardiovascular progenitor cells (CPCs) that had been cryopreserved and stored under vapor-phase liquid nitrogen (or in a −150°C freezer) were first thawed in EVA bags (Corning) for 2.5 min at 37°C in thawing medium (MEMα (1000 mL Macopharma ref. BC0110021); glucose (30%) supplement (Macopharma ref. CARELIDE, to a final total glucose concentration of 2 mg / mL; Ydralbum® (LFB), final concentration 20 mg / mL; B-27™ supplement (50×, LifeTech ref. 17504001, final concentration 1×); and Rock Inhibitor H1152 (Sigma ref. 555550, final concentration 0.392 μg / mL, sterilized using a 0.2 μm cellulose acetate (CA) membrane syringe filter). 18 mL of thawing medium was used per mL of CPCs.

[0353] After thawing, 0.2 mL / cm of the solution was added to vitronectin (Life Tech Reference: VTN-N; recombinant human protein, truncated (Reference: A31804); 5 μg / mL, sterilized using a cellulose acetate (CA) membrane syringe filter) coated culture flasks (12 x 10ST CellStack culture chambers, tissue culture (TC) treated (Corning Reference: 3271); and 2 x TC treated vitronectin coated T75 flasks). 2 Complete medium (MEMα (1000 mL Macopharma reference: BC0110021); glucose (30%) supplement (Macopharma reference: CARELIDE, to a final total glucose concentration of 2 mg / mL; Ydralbum® (LFB; 200 g / L); B-27™ supplement (50×, Life Tech reference: 17504001 or 17504044, to a final concentration of 1×); gentamicin (Panpharma, to a final concentration of 25 μg / mL); and human FGF-2 Premium grade (Miltenyi Biotec reference: A12873-01, to a final concentration of 1 μg / mL, sterilized using a 0.2 μm cellulose acetate (CA) membrane syringe filter)) was used to culture CPCs to a density of 1 cm. 2 The cells were seeded at a seeding density of approximately 100,000 cells per well. Seeding was performed without prior centrifugation of the cell suspension. The seeded CPCs were then cultured in complete medium at 37°C for 3 days in the presence of 5% CO2 and atmospheric oxygen.

[0354] Immediately prior to seeding ("D+0"), cells were analyzed to determine the number and percentage of viable cells by DAPI / AO staining (Ph.Eur.2.7.29) using a NucleoCounter NC-200 (Chemometec) (see Figure 32, column 1 ("D+0 cells")); their identity was determined by flow cytometry using a MACSQuant 10 flow cytometer (see Figure 25 and Example 14).

[0355] After 3 days of culture ("D+3"), cells were harvested from one of the cultured T75 flasks. These harvested cells were analyzed to determine the number and percentage of viable cells by DAPI / AO staining (Ph.Eur.2.7.29) using a NucleoCounter NC-200 (Chemometec) (see Figure 32, column 2 ("D+3 Material")); and their identity was determined by flow cytometry using a MACSQuant 10 flow cytometer (see Figure 25 and Example 14). Spent medium from the 10ST CellStack culture chamber was also tested for sterility and the presence of mycoplasma and endotoxin.

[0356] For the remaining flasks (12 x 10ST CellStack culture chambers; and 1 x T75), cells were visualized by microscopy to determine their morphology (see Figure 26) and washed twice with wash medium (MEM alpha (1000 mL Macopharma ref: BC0110021); glucose (30%) supplement (Macopharma ref: CARELIDE, to a final total glucose concentration of 2 mg / mL)) before being cultured for 2 days at 37°C in the presence of 5% CO and atmospheric oxygen in starvation medium (nutrient-poor medium) (MEM alpha (1000 mL Macopharma ref: BC0110021); glucose (30%) supplement (Macopharma ref: CARELIDE, to a final total glucose concentration of 2 mg / mL). After this 2-day incubation ("D+5"), the culture medium (conditioned medium) was collected, and cells were harvested from the 10ST CellStack culture chambers and the remaining T75 flasks.

[0357] Similar to the D+3 cells, the D+5 cells were also visualized by microscopy to determine their morphology (see Figure 26); and the cells collected on D+5 were further analyzed to determine the number and percentage of viable cells (see Figure 32, column 3 ("D+5 cells")); and their identity was determined by flow cytometry using a MACSQuant 10 flow cytometer (see Figure 25 and Example 14). The collected conditioned media was tested for sterility and the presence of mycoplasma and endotoxin before further processing.

[0358] Conditioned Medium Clarification

[0359] The conditioned medium was clarified through a series of three filtration steps. First, filtration was performed using a Sartopure PP3 MidiCaps 5 μm PES filter (Sartorius, Reference: 5055342P9--OO--A (Sartorius)). Next, the resulting filtrate was filtered using a Sartoguard PES MidiCaps filter (pore size (prefilter + filter): 1.2 μm + 0.2 μm; Sartorius Reference: 5475307F9--OO--A). Next, the resulting filtrate was filtered using a Sartopure 2 MidiCaps filter (pore size (prefilter + filter): 0.45 μm + 0.2 μm; Sartorius Reference: 5445307H8--OO--A).

[0360] Accumulation and concentration

[0361] After clarification of the conditioned medium, the conditioned medium was subjected to enrichment and concentration of small EV secretomes.

[0362] First, the clarified conditioned medium was subjected to tangential flow filtration (TFF) using a TFF Allegro™ CM150 (PALL / Sartorius). The TFF manifold used a sterile, single-use flow-line manual valve P&F (PALL / Sartorius, ref. 744-69N) in conjunction with a 10 L retentate assembly (sterile, single-use; PALL / Sartorius ref. 744-69M). The TFF cassette contained a sterile, single-use regenerated cellulose filter (30 kDa cutoff; 0.14 m 2 Sartorius Reference: Opta Filter Assembly + 3D51445901MFFSG) was used. For collection of the retentate (i.e. what remains in the TFF) a benchtop TFF 1 L bag was used (PALL / Sartorius, Reference: 7442-0303P).

[0363] The TFF device was first washed with 10 L of HO and 2 L of 1x PBS before operation. The clarified conditioned medium was then dosed into the TFF device, after which the retentate was concentrated (to 500 mL; pressure not to exceed 3 bar). After this initial concentration step, the retentate was subjected to diafiltration (6 diafiltration volumes; using 1x DPBS). After diafiltration, the retentate was further concentrated to a total volume of at least 100 mL. The TFF process parameters were as follows: feed manifold pressure (PT01)—0.94 to 2.1 bar; retentate manifold pressure (PT02)—0.12 to 0.13 bar; retentate manifold flow rate (FT01)—0.012 to 0.58 L / min; transmembrane pressure (TMP01)—0.53 to 1.11 bar; and quattroflow pump (P01)—14 to 20%.

[0364] Example 13 Formulations / Compositions After enrichment and concentration by TFF, the resulting retentate was then sterile filtered using a 0.22 μm filter (Sterivex™-GP Pressure Filter Unit, 0.22 μm, Millipore, ref. SVGPL10RC) to prepare the final sEV formulation. In some experiments, 25 mM trehalose was added before this sterilization step to prevent aggregation. After the sterilization step, the final formulation (with or without the addition of 25 mM trehalose) was filled into glass vials (2 mL, bromobutyl cap; Adelphi ref. VCDIN2RDLS1). The final product formulation was then stored at -80°C for later use or testing. Additionally, final formulations were also tested, where the retentate was first frozen and stored at −80° C., followed by sterile filtration using either a 0.22 μm filter (Sterivex™-GP Pressure Filter Unit, 0.22 μm, Millipore, Ref: SVGPL10RC), or a Sartopure 2 filter (pore size (prefilter+filter): 0.45 μm+0.2 μm; Sartorius Ref: 5441307H4--OO--B), as shown in FIG. 24B.

[0365] Thus, the final formulation was in PBS (with or without trehalose) and was positive for CD9, CD63, and CD81 (canonical EV markers) as well as the cardiac-related markers CD49e, ROR1, SSEA-4, MSCP, CD146, CD41b, CD24, CD44, CD236, CD133 / 1, CD29, and CD142 when detected by MACSPlex (as shown in Figure 28A and Figure 29).

[0366] Example 14 Characterization of CPC identity during vesicle formation in a GMP-compliant process To assess cell identity during the vesicle formation process in Example 12, D+0 CPCs and cells collected on D+3 and D+5 were analyzed by flow cytometry. iPSC and cardiomyocyte (CM) cells were included as controls. As shown in Figure 25, flow cytometry analysis of iPSC, CPC, and cardiac markers using a MACSQuant 10 flow cytometer demonstrated further CPC maturation over the 5-day vesicle formation period. Specifically, CPCs maintained little to no Nanog or SOX2 protein expression and continued to exhibit increased CD56, cTNT, and aMHC protein expression (however, they never reached CD56, cTNT, and aMHC expression levels similar to those of cardiomyocytes, indicating that they remained progenitor cells throughout the process). iPSC and CM control cells were analyzed separately, and mean values ​​are presented in Figure 25 for comparison.

[0367] Example 15 Analysis of EV particle concentration and EV size distribution in a GMP-compliant process To evaluate the particle concentration and particle size distribution of the EVs prepared in Example 12, * 4) and after clarification ( * The conditioned medium from 5) and the final formulation (with and without trehalose, samples b and a, respectively) were analyzed by nanoparticle tracking analysis (NTA; NanoSight). Figure 27A illustrates a representative particle size distribution curve for each sample. The overall particle size distribution, mean, and mode were similar between samples. A peak was generally observed between 50 and 150 nm, which corresponds to the size of exosomes or small microparticles. The TFF step resulted in an approximately 32-fold increase in particle concentration. Similar experiments were performed on the pre-frozen retentate and final formulation samples (filtered through STerivex-GP or Sartopore 2) (Figure 24B). * 6”, sample a; and *7, samples c and d). The results of these experiments are shown in Figure 27B. Even though particles were lost during the final sterile filtration (particularly for the final formulation made from the thawed retentate), the TFF step resulted in an approximately 20-fold increase in particle concentration.

[0368] Example 16 Analysis of EV markers in EVs produced by a GMP-compliant process To assess the presence of EV markers in the clarified conditioned medium (pre-TFF) and final formulations (with and without trehalose) of Example 12, the MACSPlex Exosome Kit Human (Miltenyi Reference: 130-108-813) was used to identify and quantify the presence of EV markers. As shown in Figure 28A, this analysis confirmed the presence of extracellular vesicle tetraspanins (CD9, CD81, and CD63) in both the conditioned medium (pre-TFF) and the final formulations (with and without trehalose). Furthermore, as shown in Figure 28B, MACSPlex analysis also revealed various markers that were either present in low amounts (e.g., CD3, CD4, CD8, HLA-DRDPDQ, CD56, CD105, CD2, CD1c, CD25, CD40, CD11c, CD86, CD31, and CD20) or were virtually absent (CD19, CD209, HLA-ABC, CD62P, CD42a, and CD69) in the conditioned medium (pre-TFF) and / or in the final formulations (with and without trehalose).

[0369] In addition, as shown in Figure 29, additional cardiac-related markers were also observed in the conditioned medium (pre-TFF) and the final formulations (with and without trehalose).

[0370] Example 17 In vitro analysis of the potency of EVs produced by a GMP-compliant process To analyze the functionality and efficacy of the final formulation produced by the GMP-compliant process in Example 12, two in vitro assays using human cardiomyocytes after staurosporine treatment were used: a HUVEC scratch wound healing assay; and a cardiomyocyte viability assay.

[0371] For the HUVEC scratch wound healing assay, a scratch wound healing assay (developed for Incucyte by Essen BioSciences) was used according to the manufacturer's instructions. Briefly, HUVEC cells were expanded using HUVEC complete medium: Endothelial Cell Basal Medium (PromoCell, Reference: C-22210) supplemented with Endothelial Cell Growth Medium Supplement Pack (PromoCell, Reference: C-39210). After expansion, cells were plated in CS10 (Cryostore, Reference: 210102) at 1–2 × 10 per aliquot. 6 Cells (enough to fill half to the full volume of a 96-well plate) were cryopreserved. Two days before the assay, an aliquot of HUVECs was thawed and seeded at 10,000 cells / well into ImageLock 96-well plates (EssenBio, Ref. 4379) and grown for 2 days in complete HUVEC medium. Throughout the maintenance and assay process, cultures were maintained at 37°C (atmospheric oxygen, 5% CO2). Scratches were made in the wells using a Wound Maker (EssenBio, Ref. 4493) according to the manufacturer's instructions, and the cells were then rinsed with endothelial cell basal medium and cultured overnight (either in PBS-containing complete HUVEC medium as a positive control; PBS-containing endothelial cell basal medium as a negative control; or in endothelial cell basal medium supplemented with sEV preparation in PBS). Plates were imaged 18 hours after treatment using an Incucyte with the scratch wound healing module. Wound closure was determined using the manufacturer's software, and values ​​were subtracted from the baseline (negative control) and normalized to the positive control. Figure 30A shows the results of the final formulation ( * 7, samples b and a), respectively, promoted wound healing. Figure 30B shows that the pre-frozen final formulation ( * 7, illustrates that samples c and d) promoted wound healing.

[0372] For cardiomyocyte viability assays using human cardiomyocytes after staurosporine treatment, see iCell Cardiomyocytes 2 (Fujifilm Cellular Dynamics, Inc., Reference: CMC-100-012-001) were plated at 50,000 cells per well of a fibronectin-coated 96-well plate in iCell Cardiomyocyte Plating Medium (Fujifilm Cellular Dynamics, Inc., Reference: M1001) and cultured for 4 hours. The medium was then replaced with iCell Cardiomyocyte Maintenance Medium (iCMM, Fujifilm Cellular Dynamics, Inc., Reference: M1003), and the cells were cultured for up to 7 days, with complete medium changes every 2–3 days. After a minimum of 4 days, cells were exposed to iCMM containing NucSpot Live 650 dye (Biotium, ref. 40082) (which served as a live cell control) or iCMM containing NucSpot Live 650 dye and staurosporine (Abcam, ref. ab146588) at a final well concentration of 2 μM (which served as an apoptotic cell control). The dye, PBS, and DMSO concentrations, as well as the final well volume, were equal in all wells. Cells were cultured in these preincubation media for 4 hours. After this incubation, the preincubation media was removed, and the wells were rinsed with iCMM. Next, while maintaining the final PBS volume, cells were fed with iCMM containing NucSpot Live 650 dye and PBS, or iCMM containing NucSpot Live 650 supplemented with increasing concentrations of sEV preparations. At 24 hours, wells were imaged in an Incucyte and nuclei counts were determined. Figure 31A shows the final formulation ( * 7, samples b and a), respectively, promoted cardiomyocyte survival. Figure 31B shows that the pre-frozen final formulation ( * 7, samples c and d) promoted cardiomyocyte survival.

[0373] The test panel used for the process / product of Example 12 and as embodied in Examples 13-17, for example, is shown in Figure 21. The results are therefore shown in Figure 32. Additionally, Figure 33 illustrates the enrichment (as calculated by the increase in particles per unit protein) for the retentate and final formulations made in Example 12 as compared to the conditioned medium after clarification.

[0374] Example 18 Analysis of the effects of cardiovascular progenitor cell (CPC) EVs on cardiac function in a mouse heart failure model To analyze the in vivo function and efficacy of sEV preparations generated by the methods described in this disclosure, a mouse model was used to determine the effect of sEV preparations on cardiac function (in mice in which heart failure had been induced).

[0375] Heart failure was induced in C57BL / 6 mice essentially as described in Kervadec et al. (J. Heart Lung Transplant, 2016, 35(6):795-807; incorporated herein by reference in its entirety). Briefly, a total of 42 mice underwent surgical occlusion of the left coronary artery to induce chronic heart failure (CHF). Three weeks after occlusion, 22 of the mice were treated with either PBS vehicle control (60 μL, n=11) or sEV (60 μL, n=11) delivered via percutaneous injection into the peri-infarct myocardium under echocardiographic guidance (as described in Kervadec et al.). The administered sEVs were generated according to the "sEV5.3" scheme illustrated in Figure 2 (the sEVs were therefore prepared by ultracentrifugation from clarified "MC5"), and the resulting EVs were resuspended in half the typical PBS volume (to generate a 2x concentrated sEV preparation, containing secretomes from 6.22E+04 cells per μL of sEV preparation).

[0376] Four weeks after occlusion, cardiac function was assessed by echocardiography. The results are shown in Figure 34. Among CHF mice, significantly fewer sEV-treated mice (compared to PBS-treated mice) had severe, progressive heart failure (defined here as a greater than 14% increase in left ventricular end-systolic volume, LVESV) (p<0.05). Furthermore, although not statistically significant, the mean ejection fraction of the PBS group worsened by more than 2.5-fold compared to the sEV-treated group (-4% vs. -1.6%, respectively; ns). These results confirmed the ability of sEV preparations to improve cardiac function in vivo.

Claims

1. 1. A method for producing a secretome, comprising: (a) culturing one or more progenitor cells in a first serum-free culture medium, the first serum-free culture medium comprising a basal medium, human serum albumin, and one or more growth factors; (b) removing the first serum-free culture medium from the one or more progenitor cells; (c) culturing the one or more progenitor cells in a second serum-free culture medium, the second serum-free culture medium comprising a basal medium but not comprising human serum albumin or a growth factor; and (d) recovering the second serum-free culture medium after culturing in step (c), thereby obtaining a conditioned medium comprising the secretome of one or more progenitor cells.

2. The method of claim 1, wherein one of said one or more growth factors is fibroblast growth factor 2 (FGF-2).

3. 3. The method of claim 1 or 2, wherein the first and second serum-free media are supplemented with a carbohydrate source.

4. 4. The method of claim 3, wherein the carbohydrate source is glucose.

5. 5. The method of any one of claims 1 to 4, wherein the first and second serum-free media are supplemented with an antibiotic.

6. 6. The method of claim 5, wherein the antibiotic is gentamicin.

7. 7. The method of any one of claims 1 to 6, wherein the first serum-free medium further comprises one or more selected from the group consisting of glutamine; biotin; DL-alpha tocopherol acetate; DL-alpha-tocopherol; vitamin A; catalase; insulin; transferrin; superoxide dismutase; corticosterone; D-galactose; ethanolamine, glutathione; L-carnitine; linoleic acid; progesterone; putrescine; sodium selenite; triiodo-I-thyronine; amino acids; sodium pyruvate; lipoic acid; vitamin B12; nucleosides; and ascorbic acid.

8. The method of any one of claims 1 to 7, wherein the basal medium is Minimum Essential Medium (MEM).

9. The method of claim 8, wherein the MEM is α-MEM.

10. 10. The method of any one of claims 1 to 9, wherein the culturing in step (a) is for 6 to 96 hours.

11. 11. The method of claim 10, wherein the culturing in step (a) is for 12 to 96 hours.

12. 12. The method of claim 11, wherein the culturing in step (a) is for 36 to 84 hours.

13. 13. The method of claim 12, wherein the culturing in step (a) is for about 72 hours.

14. 14. The method of any one of claims 1 to 13, wherein the culturing in step (c) is for 6 to 96 hours.

15. 15. The method of claim 14, wherein the culturing in step (c) is for 12 to 72 hours.

16. 16. The method of claim 15, wherein the culturing in step (c) is for 36 to 60 hours.

17. 17. The method of claim 16, wherein the culturing in step (c) is for about 48 hours.

18. 15. The method of claim 14, wherein the final 12 to 36 hours of culturing in step (c) is carried out under hypoxic conditions.

19. 19. The method of claim 18, wherein the culture conditions include culturing in an atmosphere having 1 to 21% oxygen.

20. 20. The method of any one of claims 1 to 19, wherein after step (b) but before step (c), the one or more progenitor cells are washed.

21. 21. The method of any one of claims 1 to 20, wherein the one or more progenitor cells comprise progenitor cells selected from the group consisting of cardiomyocyte progenitor cells, cardiac progenitor cells, and cardiovascular progenitor cells.

22. 22. The method of any one of claims 1 to 21, wherein the one or more progenitor cells are obtained from induced pluripotent stem cells (iPSCs).

23. 23. The method of any one of claims 1 to 4 and 7 to 22, wherein the first and second serum-free media do not contain antibiotics.

24. 24. The method of any one of claims 1 to 23, wherein the culture in one or more of steps (a) and (c) is a two-dimensional cell culture.

25. 25. The method of claim 24, wherein the two-dimensional cell culture comprises culturing the one or more progenitor cells on a surface of a culture vessel.

26. 26. The method of claim 25, wherein the culture vessel surface is coated with a substance that promotes cell adhesion.

27. 27. The method of claim 26, wherein the substance that promotes cell adhesion is vitronectin or fibronectin.

28. 24. The method of any one of claims 1 to 23, wherein the culture in one or more of steps (a) and (c) is a three-dimensional cell culture.

29. 30. The method of claim 28, wherein the three-dimensional cell culture comprises culturing cell aggregates in suspension in a bioreactor, spinner flask, or stirred culture vessel, or comprises culturing cells in a microcarrier culture system.

30. 30. The method of any one of claims 1 to 29, further comprising pre-clarifying the medium collected in step (d) by centrifugation, filtration, or a combination of centrifugation and filtration.

31. 31. The method of any one of claims 1 to 30, further comprising freezing the medium collected in step (d).

32. The method of any one of claims 1 to 31, wherein the one or more progenitor cells cultured in step (a) have been previously frozen.

33. The method of any one of claims 1 to 32, further comprising concentrating and / or enriching small extracellular vesicle-enriched fractions (sEVs) from the medium collected in step (d).

34. 34. The method of claim 33, wherein the sEVs are concentrated and / or enriched from the harvested medium by at least one process selected from the group consisting of ultracentrifugation, filtration, ultrafiltration, tangential flow filtration, size exclusion chromatography, and affinity capture.

35. The accumulation accumulates extracellular vesicles having one or more of the following characteristics: (a) CD63 ＋ , CD81 ＋ and / or CD9 ＋ (b) are 50-200 nm in diameter; (c) are positive for one or more of CD49e, ROR1 (receptor tyrosine kinase-like orphan receptor 1), SSEA-4 (stage-specific embryonic antigen 4), MSCP (mesenchymal stem cell-like protein), CD146, CD41b, CD24, CD44, CD236, CD133 / 1, CD29, and CD142; and / or (d) are negative for one or more of CD19, CD4, CD209, HLA-ABC (human leukocyte antigen ABC), CD62P, CD42a, and CD69.

36. 34. The method of claim 33, wherein the sEVs comprise one or more of exosomes, microparticles, extracellular vesicles, and secreted peptides / proteins.

37. A secretome-containing composition obtainable by the method of any one of claims 1 to 32.

38. 37. An sEV-containing composition obtainable by the method of any one of claims 33 to 36.

39. 33. A method of making a therapeutic composition suitable for administration to a patient, comprising making a secretome-containing composition according to the method of any one of claims 1 to 32.

40. 40. The method of claim 39, further comprising purifying, concentrating, isolating, and / or enriching the secretome-containing composition by one or more purification, enrichment, isolation, and / or enrichment steps.

41. 40. The method of claim 39, further comprising adding a pharmaceutically acceptable excipient or carrier to the secretome-containing composition.

42. A method of making a therapeutic composition suitable for administration to a patient, the method comprising making an sEV-containing composition according to any one of claims 33 to 36.

43. 43. The method of claim 42, further comprising purifying, concentrating, isolating, and / or enriching the sEV-containing composition by one or more purification, concentration, isolation, and / or enrichment steps.

44. 43. The method of claim 42, further comprising adding a pharmaceutically acceptable excipient or carrier to the sEV-containing composition.

45. 40. A therapeutic composition comprising the secretome-containing composition of claim 37 and a pharmaceutically acceptable excipient or carrier.

46. A therapeutic composition comprising the sEV-containing composition of claim 38 and a pharmaceutically acceptable excipient or carrier.

47. 2. The secretome-containing composition obtainable by the method of claim 1, wherein the one or more progenitor cells comprise progenitor cells selected from the group consisting of cardiomyocyte progenitor cells, cardiac progenitor cells, and cardiovascular progenitor cells.

48. 34. The sEV-containing composition obtainable by the method of claim 33, wherein the one or more progenitor cells comprise progenitor cells selected from the group consisting of cardiomyocyte progenitor cells, cardiac progenitor cells, and cardiovascular progenitor cells.

49. 48. A therapeutic composition comprising the composition of claim 47 and a pharmaceutically acceptable excipient or carrier.

50. 49. A therapeutic composition comprising the composition of claim 48 and a pharmaceutically acceptable excipient or carrier.

51. 51. A method for treating acute myocardial infarction or heart failure, comprising administering to a subject in need thereof the therapeutic composition of claim 49 or 50.

52. 51. A method for improving angiogenesis, comprising administering to a subject in need thereof the therapeutic composition of claim 49 or 50.

53. 51. A method for improving cardiac performance, comprising administering to a subject in need thereof the therapeutic composition of claim 49 or 50.

54. 12. The method of claim 11, wherein the culturing in step (a) is for 60 to 84 hours.

55. 15. The method of claim 14, wherein the final 12 to 36 hours of culturing in step (c) is conducted under normoxic conditions.

56. 56. The method of claim 55, wherein said normoxic conditions comprise culturing in an atmosphere containing 20-21% oxygen.

57. 30. The method of claim 29, wherein the bioreactor is a vertical wheel bioreactor.

58. 40. The method of claim 39, further comprising cryopreserving, freezing, or lyophilizing the secretome-containing composition.

59. 43. The method of claim 42, further comprising cryopreserving, freezing, or lyophilizing the sEV-containing composition.

60. The method of claim 2, wherein the first serum-free medium contains 0.1 to 10 μg / mL FGF-2.

61. 61. The method of claim 60, wherein the first serum-free medium comprises 0.5 to 5 μg / mL FGF-2.

62. 62. The method of claim 61, wherein the first serum-free medium comprises 0.5 to 2.5 μg / mL FGF-2.

63. 63. The method of claim 62, wherein the first serum-free medium comprises about 1 μg / mL FGF-2.

64. 64. The method of any one of claims 1 to 36, 39 to 44, and 54 to 63, which is Good Manufacturing Practices (GMP) compliant.

65. 38. The secretome-containing composition of claim 37, which is GMP compliant.

66. 39. The sEV-containing composition of claim 38, which is GMP-compliant.

67. 15. The method of claim 14, wherein the final 12 to 36 hours of culturing in step (c) is conducted under normoxic conditions.

68. 68. The method of claim 67, wherein said normoxic conditions comprise culturing in an atmosphere containing 20-21% oxygen.

69. 31. The method of claim 30, wherein the pre-clarification comprises at least three filtration steps.

70. 35. The method of claim 34, wherein separating the sEVs from the harvested medium comprises tangential flow filtration.

71. 38. The secretome-containing composition of claim 37, comprising trehalose, and optionally L-histidine.

72. 39. The sEV-containing composition of claim 38, comprising trehalose, and optionally L-histidine.

73. 66. The secretome-containing composition of claim 37 or 65, which is capable of promoting wound scratch healing in an in vitro wound scratch healing assay and / or is capable of promoting cardiomyocyte viability in an in vitro cardiomyocyte viability assay.

74. 67. The sEV-containing composition of claim 38 or claim 66, which is capable of promoting wound scratch healing in an in vitro wound scratch healing assay and / or is capable of promoting cardiomyocyte viability in an in vitro cardiomyocyte viability assay.

75. The secretome-containing composition of claim 37 or claim 65, which is at least one of the following: a composition enriched for extracellular vesicles having a diameter of about 50-200 nm or 50-200 nm, preferably having a diameter of about 50-150 nm or 50-150 nm; a composition that is substantially free of or free of whole cells; and / or a composition that is substantially free of one or more culture medium components.

76. 67. The sEV-containing composition of claim 38 or claim 66, which is at least one of the following: a composition enriched for extracellular vesicles having a diameter of about 50-200 nm or 50-200 nm, preferably having a diameter of about 50-150 nm or 50-150 nm; a composition that is substantially free of or free of whole cells; and / or a composition that is substantially free of one or more culture medium components.

77. 52. The method of claim 51, wherein the heart failure is acute heart failure, chronic heart failure, ischemic heart failure, non-ischemic heart failure, heart failure with ventricular dilation, heart failure without ventricular dilation, heart failure with reduced left ventricular ejection fraction, or heart failure with preserved left ventricular ejection fraction.

78. 78. The method of claim 77, wherein the heart failure is selected from the group consisting of ischemic heart disease, cardiomyopathy, myocarditis, hypertrophic cardiomyopathy, diastolic hypertrophic cardiomyopathy, dilated cardiomyopathy, and post-chemotherapy induced heart failure.

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