Methods and assays for secretome activity analysis

Abstract

The present application provides methods and assays for evaluating a mesenchymal stem cell secretome for use of the MSC secretome in a method of treating an eye disease and / or disorder.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 359,743, filed on July 8, 2022, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Regenerative medicine is a field of medicine related to the replacement or regeneration of human cells, tissues, or organs in order to restore or establish normal functions. For example, stem cell therapy can be used to treat, prevent, or cure various diseases and disorders.

[0003] Stem cells are cells that have the ability to divide indefinitely and can differentiate into various different cell types under certain specific conditions. Totipotent stem cells are stem cells that have the potential to generate all of the cells and tissues that make up an embryo. Pluripotent stem cells are stem cells that give rise to cells of the mesoderm, endoderm, and ectoderm. Multipotent stem cells are stem cells that have the ability to differentiate into two or more cell types, while unipotent stem cells are stem cells that can differentiate into only one cell type. One such type of stem cell is mesenchymal stem cells. See, for example, U.S. Patent Application No. US20190046576.

[0004] However, it is difficult to manufacture and store live stem cell-based therapies on a clinically relevant scale (see Trayor et al., Nature Biotechnology 32(1)(2014)). Furthermore, the therapeutic efficacy and regenerative capacity of such therapies are often variable, and the cells can die before or during transplantation (see Newell, Seminars in Immunopathology 33(2):91(2011)). Transplanted stem cells are also susceptible to attack and / or rejection by the host immune system, and it is often difficult to assess efficacy and / or control “dosage”. Accordingly, there is a need in the art for additional regenerative therapies that can overcome the cost, storage, and manufacturing quality control limitations currently associated with cell-based regenerative medical therapies. In particular, in the context of eye diseases.

[0005] Blunt and blast injuries to the eye can cause a series of mechanical disruptions to the eye contents, including retinal concussion, traumatic cataract, disruption of the zonular attachments to the lens, angle recession, iridodialysis, and rupture of the pupillary sphincter, and disruption to the optic nerve. Treatment of these injuries has been limited to mechanical repair of the iris (when possible), replacement of the lens with a plastic lens implant, and repair of retinal detachment. There has been no treatment to repair the cellular structure of the retina or anterior chamber as a result of injury or disease or a genetic disease such as retinitis pigmentosa. Additionally, traumatic optic neuropathy and optic nerve avulsion wounds, incorporated herein by reference in their entirety, are among the six major types of eye injuries that required specialized ophthalmic care in "Operation Iraqi Freedom" (Cho and Savitsky in Combat Casualty Care: Lessons Learning from Oef and Oif by Brian Eastbridge and Eric Savitsky, "Ocular Trauma Chapter 7", pp. 299 - 342, Ft. Detrick, Md.: Borden Institute (US) Government Printing Office, 2012). Sixty percent of traumatic head injuries cause neuro - ocular system abnormalities (Van Stavern, et al., J Neuro - Ophthalmol 21(2):112 - 117, 2001) (incorporated herein by reference in their entirety), and half of those involve the optic nerve and visual pathways. Traumatic injury to neurons results in axonal damage and irreversible neuronal loss, leading to permanent deficits. Some potential neuroprotective therapies have been identified in animals, but these single agents generally have not translated well to therapies in human clinical trials (Turner, et al., J Neurosurg 118(5):1072 - 1085, 2013) (incorporated herein by reference in their entirety). Combinatorial therapies that affect several cellular targets may be required to prevent neuronal injury or restore neuronal function.

[0006] The cornea serves a protective role as the outermost tissue of the eye, but the cornea is very vulnerable to severe injuries and diseases. The lack of blood vessels allows for corneal transparency but also limits the ability to heal. Corneal injuries require prompt intervention and aggressive treatment due to their potential to cause irreversible blindness. The critical need for improved healing therapies for the ocular surface is particularly evident in chemical burns and severe corneal diseases such as acute and chronic graft-versus-host disease (GvHD), Stevens-Johnson syndrome, ocular mucous membrane pemphigoid, and other diseases that cause persistent corneal epithelial defects, collectively including an incidence rate of over 100,000 cases per year (see Dietrich-Ntoukas et al., Cornea. 2012, 31(3):299-310, Stevenson W, et al., Clin Ophthalmol. 2013, 7:2153-2158, White KD, et al., J Allergy Clin Immunol Pract. 2018:6(1):38-69, Tauber J. (2002) Autoimmune Diseases Affecting the Ocular Surface. In: Ocular Surface Disease Medical and Surgical Management. Springer, New York, NY. and Wirostko B, et al., Ocul Surf. 2015 Jul, 13(3), 204-21, and Haring, RS., et al., JAMA Ophthalmol. 2016 Oct 1, 134(10):1119-1124).

[0007] Furthermore, the development of topical eye drops is hindered by many anatomical constraints, including tear turnover and dilution, nasolacrimal drainage, and the blink reflex, and in many cases, less than 5% of the topical dose reaches the deeper eye tissues (Gaudana et al., 2009). In the case of corneal wounds, the initial injury causes cracks in the corneal epithelium, thereby allowing locally applied MSC-S to pass through and penetrate the epithelial layer.

[0008] Accordingly, there is a great unmet need in the art for ophthalmic therapies that can target the eye and deliver a therapeutic payload to sensory tissues that are difficult to reach and may have deteriorated due to inflammation associated with trauma (e.g., burns, acute inflammation, age, and / or oxidative stress, etc.). The present invention meets this need by providing a mesenchymal stem cell secretome composition for use in such therapies and a method of making such a composition. SUMMARY OF THE INVENTION

[0009] The present invention provides methods and assays for the analysis of the activity of a secretome derived from mesenchymal stem cells (MSCs).

[0010] In some embodiments, the present invention provides herein a method for characterizing an MSC secretome, the method comprising: (i) subjecting the MSC secretome to one or more characterization assays, wherein the characterization assays are selected from the group consisting of characterization of physical components, oxidative stress assay, misfolded protein response assay, ER stress assay, cell response assay, safety analysis, stability assay, proliferation assay, migration assay, adhesion assay, angiogenesis assay, differentiation / scarring assay, inflammation / immunity assay, gliosis assay, tissue explant survival and function assay, organoid development or survival / function assay, epithelial barrier integrity assay, retinal degeneration assay, and / or assay for hereditary retinal diseases including human and animal retinal explants, neuroprotection / neurotrophic assay, and the MSC secretome is optionally preconditioned; (ii) determining the results from one or more of the assays of (i); and comprising.

[0011] In some embodiments, the present invention provides herein a method for determining the biopotency and stability of an MSC secretome, the method comprising: (i) Subjecting the MSC secretome to one or more characterization assays, wherein the characterization assays are selected from the group consisting of characterization of physical components, oxidative stress assay, misfolded protein response assay, ER stress assay, cell response assay, safety analysis, stability assay, proliferation assay, migration assay, adhesion assay, angiogenesis assay, differentiation / scarring assay, inflammation assay, in vivo response to oxidative stress (e.g., retinal ischemia reperfusion), epithelial barrier integrity assay, retinal degeneration assay, and / or assay of hereditary retinal diseases including human and animal retinal explants, neuroprotection / neurotrophic assay, and the MSC secretome is optionally preconditioned, and subjecting; (ii) Determining a result from one or more of the assays of (i); comprising.

[0012] In some embodiments, the present invention provides, herein, a method for determining MSC secretome lot consistency among multiple MSC secretome lots, the method comprising: (i) Subjecting the MSC secretome to one or more characterization assays, wherein the characterization assays are selected from the group consisting of characterization of physical components, oxidative stress assay, misfolded protein response assay, ER stress assay, cell response assay, safety analysis, stability assay, proliferation assay, migration assay, adhesion assay, angiogenesis assay, differentiation / scarring assay, inflammation assay, epithelial barrier integrity assay, retinal degeneration assay, and / or assay of hereditary retinal diseases including human and animal retinal explants, neuroprotection / neurotrophic assay, and the MSC secretome is optionally preconditioned, and subjecting; (ii) Determining a result from one or more of the assays of (i); comprising.

[0013] In some embodiments, the result of (ii) of the characterization of physical components identifies an anti-angiogenic MSC secretome or a produced composition comprising the secretome.

[0014] In some embodiments, the result of (ii) of the safety analysis indicates blood compatibility and provides an MSC secretome that shows low pyrogen and / or endotoxin, and / or no pyrogen and / or endotoxin.

[0015] In some embodiments, the result of (ii) of the stability assay provides an MSC secretome that shows stability at -20°C, 4°C, and / or 20°C (e.g., room temperature) for at least 7 days or at least 14 days.

[0016] In some embodiments, the result of (ii) of the proliferation assay provides an MSC secretome that induces proliferation.

[0017] In some embodiments, the result of (ii) of the migration assay provides an MSC secretome that induces migration.

[0018] In some embodiments, the result of (ii) of the angiogenesis assay provides an MSC secretome that inhibits angiogenesis or does not promote angiogenesis.

[0019] In some embodiments, the result of (ii) of the differentiation / scarring assay provides an MSC secretome that inhibits differentiation and / or scarring.

[0020] In some embodiments, the result of (ii) of the inflammation assay provides an MSC secretome that inhibits inflammation or modifies the immune response.

[0021] In some embodiments, the method (iii) identifying an MSC secretome lot based on the result of (ii), further comprises.

[0022] In some embodiments, the method includes a preconditioning step for the secretome.

[0023] In some embodiments, the present invention provides a panel of tests and / or assays for characterizing the MSC secretome, the panel comprising at least two characterization assays, the characterization assays being selected from the group consisting of characterization of physical components, oxidative stress assay, misfolded protein response assay, ER stress assay, safety analysis, stability assay, proliferation assay, migration assay, adhesion assay, angiogenesis assay, differentiation / scarring assay, inflammation assay, epithelial barrier integrity assay, retinal degeneration assay, and / or assay of hereditary retinal diseases including human and animal retinal explants, neuroprotection / neurotrophic assay, and the secretome is optionally preconditioned.

[0024] In some embodiments, the present invention provides a panel of tests and / or assays for determining the consistency between MSC secretome lots, the panel comprising one or more characterization assays, the characterization assays being selected from the group consisting of characterization of physical components, oxidative stress assay, misfolded protein response assay, ER stress assay, safety analysis, stability assay, proliferation assay, migration assay, adhesion assay, angiogenesis assay, differentiation / scarring assay, inflammation assay, epithelial barrier integrity assay, retinal degeneration assay, and / or assay of hereditary retinal diseases including human and animal retinal explants, neuroprotection / neurotrophic assay, and the secretome is optionally preconditioned.

[0025] In some embodiments, the characterization of physical components identifies the MSC secretome characterized herein, or a composition made from the secretome.

[0026] In some embodiments, the result of (ii) of the safety analysis shows blood compatibility and provides an MSC secretome that shows low pyrogen and / or endotoxin, and / or non-pyrogen and / or endotoxin.

[0027] In some embodiments, the stability assay identifies an MSC secretome that exhibits stability at -20°C, 4°C, and / or 20°C (e.g., room temperature) for at least 7 days or at least 14 days.

[0028] In some embodiments, the proliferation assay identifies an MSC secretome that induces proliferation.

[0029] In some embodiments, the migration assay identifies an MSC secretome that induces migration.

[0030] In some embodiments, the angiogenesis assay identifies an MSC secretome that inhibits angiogenesis or does not promote angiogenesis.

[0031] In some embodiments, the differentiation / scarring assay identifies an MSC secretome that inhibits differentiation and / or scarring.

[0032] In some embodiments, the inflammation assay identifies an MSC secretome that inhibits inflammation or modifies the immune response.

[0033] In some embodiments, all of the following assays are performed: characterization of physical components, oxidative stress assay, misfolded protein response assay, ER stress assay, safety analysis, stability assay, proliferation assay, migration assay, adhesion assay, angiogenesis assay, differentiation / scarring assay, inflammation assay, and / or epithelial barrier integrity assay, retinal degeneration assay, and / or assay of hereditary retinal diseases including human and animal retinal explants, neuroprotection / neurotrophic assay.

[0034] In some embodiments, the test and / or assay identifies an MSC secretome.

[0035] In some embodiments, the MSC secretome is an anti-angiogenic MSC secretome and / or an anti-scarring MSC secretome.

[0036] In some embodiments, the MSC secretome is an anti-angiogenic MSC secretome and / or an anti-scarring MSC secretome.

[0037] In some embodiments, the panel of assays includes at least one migration assay.

[0038] In some embodiments, the panel of assays includes at least one adhesion assay.

[0039] In some embodiments, the migration assay is an in vitro wound closure assay.

[0040] In some embodiments, the in vitro wound closure assay is selected from the group consisting of a "scratch assay" (also referred to as a "scrape assay"), a circular scratch method, and a circular wound closure assay.

[0041] In some embodiments, the panel of assays includes measurement of reactive oxygen species (ROS).

[0042] In some embodiments, the ROS measurement assay is an in vitro assay that uses an antioxidant as a positive control. In some embodiments, the ROS measurement assay is an in vitro assay that uses an antioxidant as a positive control. In some embodiments, N-acetylcysteine (NAC) or ebselen is used as the positive control. Non-limiting examples of antioxidants include vitamins and their analogs, including vitamin A, vitamin B3 (e.g., niacin [nicotinic acid] and nicotinamide), vitamin C (ascorbic acid), vitamin E (including tocopherols [e.g., α-tocopherol] and tocotrienols), and vitamin E analogs (e.g., trolox [water-soluble]); carotenoids including carotene (e.g., β-carotene), xanthophylls (e.g., lutein, zeaxanthin, and meso-zeaxanthin), and carotenoids in saffron (e.g., crocin and crocetin); sulfur-containing antioxidants including glutathione (GSH), N-acetyl-L-cysteine (NAC), bucillamine, S-nitroso-N-acetyl-L-cysteine (SNAC), S-allyl-L-cysteine (SAC), S-adenosyl-L-methionine (SAM), α-lipoic acid, and taurine; carnosine, N-acetylcarnosine, curcuminoids (e.g., curcumin, demethoxycurcumin, and tetrahydrocurcumin), cysteamine, ebselen, glutathione, hydroxycinnamic acids, and their derivatives (e.g., esters and amides) (e.g., caffeic acid, rosmarinic acid, and tranilast), melatonin and its metabolites, nitrones (e.g., disodium disulfenton [NXY-059]), nitroxides (e.g., XJB-5-131), polyphenols (e.g., flavonoids [e.g., apigenin, genistein, luteolin, naringenin, and quercetin]), superoxide dismutase mimetics (described below), tirilazad, vitamin C, vitamin E and its analogs (e.g., α-tocopherol and trolox), and xanthine derivatives (e.g., pentoxifylline); scavengers of ROS and radicals including ubiquinone (coenzyme Q such as CoQ10), ubiquinol (the reduced, more biologically available form of ubiquinone such as ubiquinol-10),Mitochondrial antioxidants / "vitamins" containing ubiquinone / ubiquinol analogs (e.g., idebenone and mitoquinone) and derivatives; mitochondrial-targeted antioxidants including DMQ, DMMQ, MitoE, MitoQ, Mito-TEMPO, MitoVitE, and compounds of the SkQ class (e.g., SkQ1, SkQ2, SkQ3, SkQB, SkQR1, SkQT, SkQT1, SkQT1(m), SkQT1(p), SkQTK1, SkQTR1, SkQBerb, and SkQPalm); NADPH oxidase (NOX) inhibitors (e.g., apocynin, declomycin and angolensin of declomycinol [both inhibit the activity and expression of NOX-1, -2 and -4], diphenyleneiodonium, and GKT-831 [formerly GKT-137831, a dual NOX1 / 4 inhibitor]), NADH:ubiquinone oxidoreductase (Complex I) inhibitors (e.g., metformin and rotenone), xanthine oxidase inhibitors (e.g., allopurinol, oxypurinol, tiopurinol, febuxostat, topiroxostat, myo-inositol, phytic acid, and flavonoids [e.g., quercetin, myricetin, and kaempferol]), and myeloperoxidase inhibitors (e.g., azide, 4-aminobenzoic acid hydrazide and PF-06667272, and apoE mimetics such as AEM-28 and AEM-28-14) inhibitors of ROS-producing enzymes; superoxide dismutase (SOD) (e.g., manganese(III)- and zinc(III)-porphyrin complexes (e.g., MnTBAP, MnTMPyP, and ZnTBAP), manganese(II) pentacyclic ring complexes (e.g., M40401 and M40403), manganese(III)-salen complexes (e.g., those disclosed in U.S. Patent No. 7,122,537, which is incorporated herein by reference in its entirety) and OT-551 (a cyclopropyl ester prodrug of tempol hydroxylamine), and SOD mimetics such as apoA-I mimetics (both increase expression) such as resveratrol and 4F), catalase (e.g., manganese(III)-salen complexes [e.g., those disclosed in U.S. Patent No. 7,122,537], and catalase mimetics such as zinc [increases activity]),Substances that mimic or increase the activity or production of antioxidant enzymes, including glutathione peroxidase (GPx) (e.g., apomorphine and zinc [both increase activity], and beta-catenin, etoposide, and resveratrol [all three increase expression]), glutathione reductase (e.g., redox cofactor nucleotides such as 4-tert-butylcatechol and flavin adenine dinucleotide [FAD] and NADPH [all three enhance activity]), glutathione S-transferase (GST) (e.g., phenylalkyl isothiocyanate-cysteine complexes (e.g., S-[N-benzyl(thiocarbamoyl)]-L-cysteine), phenobarbital, rosemary extract, and carnosol [all enhance activity]), thioredoxin (Trx) (e.g., geranylgeranylacetone, prostaglandin E1, and sulforaphane [all increase expression]), NADPH-quinone oxidoreductase 1 (NQO1) (e.g., flavones [e.g., β-naphthoflavone (5,6-benzoflavone)] and triterpenoids [e.g., TP-151 (CDDO), TP-155 (CDDO methyl ester), TP-190, TP-218, TP-222, TP-223 (CDDO), TP-224 (CDDO monomethylamide), TP-225, TP-226 (CDDO dimethylamide), TP-230, TP-235 (CDDO imidazolid), TP-241, CDDO monoethylamide, CDDO mono(trifluoroethyl)amide, and (+)-TBE-B and other oleanolic acid analog carboxamides] (all of these increase expression by activating Nrf2), heme oxygenase 1 (HO-1) (e.g., curcuminoids (e.g., curcumin), triterpenoids (e.g., oleanolic acid analogs such as TP-225), and apoA-I mimetics (e.g., 4F), all of which increase expression), and paraoxonase 1 (PON-1) (e.g., apoE mimetics [e.g., AEM-28 and AEM-28-14] and apoA-I mimetics [e.g., 4F], both types increase activity); activators of nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or Nrf2) (e.g., bardoxolone methyl, OT-551,Activators of transcription factors that upregulate the expression of antioxidant enzymes, including fumarates (e.g., dimethyl and monomethyl fumarates), dithiothiones (e.g., orcipraz), flavones (e.g., β-naphthoflavone), isoflavones (e.g., genistein), sulforaphane, trichostatin A (which also upregulates glutathione synthesis), triterpenoids (e.g., oleanolic acid analogs [e.g., TP-225]), and melatonin (which increases Nrf2 expression); other types of antioxidants including anthocyanins, benzenediol avitan diterpenes (e.g., carnosic acid), cyclopentenone prostaglandins (such as 15d-PGJ2, which also upregulates glutathione synthesis), flavonoids (e.g., flavonoids from Ginkgo biloba leaves [e.g., quercetin and kaempferol, which increase the levels of GSH, SOD, catalase, GPx, and GST]), prenylflavonoids (e.g., isoxanthohumol), flavones (e.g., apigenin), isoflavones (e.g., genistein), flavanones (e.g., naringenin), and flavanols (e.g., catechin and epigallocatechin-3-gallate), omega-3 fatty acids and their esters (as described above), phenylethanoids (e.g., tyrosol and hydroxytyrosol), retinoids (e.g., all-trans retinol [vitamin A]), stilbenoids (e.g., resveratrol), uric acid, apoA-I mimetics (e.g., 4F), apoE mimetics (e.g., AEM-28 and AEM-28-14), and minerals (e.g., selenium and zinc [e.g., zinc monothioglycolate]); and their analogs, derivatives, and salts.

[0043] In some embodiments, the in vitro ROS measurement assay is evaluated using chloromethyl-modified H2DCFDA (CM-H2DCFDA) and / or MitoSOX Red.

[0044] In some embodiments, the assay panel includes a cell viability assay.

[0045] In some embodiments, the cell viability assay is a microtiter tetrazolium (MTT) assay.

[0046] In some embodiments, the cells are subjected to an oxidative stress assay.

[0047] In some embodiments, the oxidative stress assay comprises a) inducing oxidative stress in the cells; and b) evaluating the degree of oxidative stress.

[0048] In some embodiments, the oxidative stress is induced by one or more inducers selected from the group consisting of ketocholesterol, FeCl3-sodium nitrilotriacetate (Fe-NTA), H2O2, tert-butyl hydroperoxide (t-BHP), all-trans retinal, NaIO4, hydroquinone, and oxidized low-density lipoprotein (OxLDL).

[0049] In some embodiments, the oxidative stress is induced by culturing the cells in a hypoxic and / or anoxic condition.

[0050] In some embodiments, the oxidative stress is induced by administering ketocholesterol to the cells to be assayed at a concentration of about 0.1-1 mM, about 1-10 mM, or about 10-100 mM for about 1-24 hours, about 24-48 hours, or about 48-72 hours.

[0051] In some embodiments, the oxidative stress is induced by administering Fe-NTA to the cells to be assayed at a concentration of about 0.1-1 mM, about 1-10 mM, or about 10-100 mM for about 1-24 hours, about 24-48 hours, or about 48-72 hours.

[0052] ​In some embodiments, oxidative stress is induced by administering H2O2 to the cells being assayed at a concentration of about 0.01 - 0.1 μM, about 0.1 - 1 μM, about 1 - 10 μM, about 10 - 100 μM, or about 100 - 1000 μM for about 0.5 - 6 hours, about 6 - 12 hours, about 12 - 18 hours, or about 18 - 24 hours.

[0053] In some embodiments, oxidative stress is induced by administering t-BHB to the cells being assayed at a concentration of about 0.01 - 0.1 μM, about 0.1 - 1 μM, about 1 - 10 μM, about 10 - 100 μM, or about 100 - 1000 μM for about 0.5 - 6 hours, about 6 - 12 hours, about 12 - 18 hours, or about 18 - 24 hours.

[0054] In some embodiments, oxidative stress is induced by administering all-trans retinal to the cells being assayed at a concentration of about 0.01 - 0.1 μM, about 0.1 - 1 μM, about 1 - 10 μM, about 10 - 100 μM, or about 100 - 1000 μM for about 0.5 - 6 hours, about 6 - 12 hours, about 12 - 18 hours, or about 18 - 24 hours.

[0055] In some embodiments, oxidative stress is induced by administering NaIO4 to the cells being assayed at a concentration of about 0.01 - 0.1 μM, about 0.1 - 1 μM, about 1 - 10 μM, about 10 - 100 μM, or about 100 - 1000 μM for about 0.5 - 6 hours, about 6 - 12 hours, about 12 - 18 hours, or about 18 - 24 hours.

[0056] In some embodiments, oxidative stress is induced by administering OxLDL to the cells being assayed at a concentration of about 10 - 100 μg / mL, about 10 - 100 μg / mL, or about 100 - 500 μg / mL for about 0.5 - 6 hours, about 6 - 12 hours, about 12 - 18 hours, or about 18 - 24 hours.

[0057] In some embodiments, evaluating the degree of oxidative stress is as follows: i) Measuring DNA oxidation using 8-oxo-2'-deoxyguanosine (8-oxo-dG), ii) Measuring lipid oxidation using thiobarbituric acid reactive substances (TBARS), iii) Measuring cytoplasmic ROS using CM-H2 DCFDA, iv) Measuring mitochondrial ROS using Mitoxed, v) Measuring mitochondrial potential such as measuring JC-1 including one or more of the above.

[0058] In some embodiments, the cells to be assayed are assayed for one or more phenotypes selected from the group consisting of changes in epithelial polarity (e.g., Na / K ATPase polarity), tight junctions (e.g., ZO-1, trans-epithelial polarity, and trans-epithelial electrical resistance (TEER)), phagocytosis of the outer segment, degradation of the outer segment, phagosome composition and function, cathepsin D distribution activity, mitochondrial potential, inflammasome activation / activity, HIf-1 alpha activation / activity, apoptosis, ferroptosis, NRF-2 activation / activity, NOQ1 activation / activity, GPX4 activation / activity, SLC7AII activation / activity, annexin V activation / activity, propidium iodide staining, IRE1 / XBP1 activation / activity, PERK / ATF4 / CHOP activation / activity, UPR activation / activity, ACS24 activation / activity, complement activation / sensitivity, lipid deposition, apoptosis, and necrosis.

[0059] In some embodiments, the MSC secretome is preconditioned before oxidative stress is induced.

[0060] In some embodiments, the MSC secretome is preconditioned during the induction of oxidative stress.

[0061] In some embodiments, the MSC secretome is preconditioned after oxidative stress is induced.

[0062] In some embodiments, preconditioning the MSC secretome includes affecting the MSC secretory profile, hereinafter, by one or more of the following: changing the culture format (e.g., 2D planar vs. 3D bioreactor), different biomaterial scaffolds, co-culture, addition of pharmacological compounds, growth factors, chemokines, Toll-like receptor agonists, inflammatory cytokines, advanced glycation end products (AGEs), oxidized phospholipids, malondialdehyde, or carboxyethylpyrrole, agitation of the ECM, culturing under shear stress, agitation or suspension as aggregates or within a matrix, induced misfolded protein response, ER stress, induction of MSC differentiation, culturing in the presence of conditioned media, and hypoxia / anoxia.

[0063] In some embodiments, preconditioning the MSC secretome includes hypoxic preconditioning, which includes culturing the MSCs in a hypoxic culture environment.

[0064] In some embodiments, the oxygen level is from about 0% to 2%. In some embodiments, hypoxic preconditioning is performed for about 4 - 12 hours, about 12 - 24 hours, about 24 - 36 hours, about 36 - 48 hours, about 48 - 60 hours, or about 60 - 72 hours.

[0065] In some embodiments, preconditioning the MSC secretome includes treating the MSCs with one or more inflammatory cytokines.

[0066] In some embodiments, preconditioning the MSC secretome includes treating the MSCs with one or more of the following: IL-6, PGE2, IDO, IFNγ, SDF-1, TGF-α, H2O2, FGF-2, IGF-1, BMP-2, atorvastatin, oxytocin, curcumin, lipopolysaccharide, and nicotinamide (NIC), vasoactive intestinal peptide (VIP), and / or diazoxide.

[0067] In some embodiments, preconditioning the MSC secretome involves culturing the MSCs in 3D (e.g., by using a 3D bioreactor).

[0068] In some embodiments, the present invention provides a bone marrow-derived mesenchymal stem cell (MSC) secretome composition comprising HGF, pentraxin-3 (TSG-14), VEGF, TIMP-1, serpin E1, <5 ng / mL IL-8, and an osmotic modifier, and the MSC secretome is preconditioned.

[0069] In some embodiments, the MSC secretome composition further comprises i. at least one trophic factor / cytokine selected from the group consisting of TIMP-2 and VEGF-A, and ii. at least one additional factor selected from the group consisting of PEDF (serpin F1), IGFBP-2, IGFBP-3, SDF-1, kallikrein 3, MCP-1, angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, and PDGF, and iii. at least one further factor selected from the group consisting of DPPIV (dipeptidyl peptidase-4), uPA, angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, and thrombospondin-1. Including.

[0070] In some embodiments, the MSC secretome composition comprises at least one factor in the range of 1 ng / mL to 100 ng / mL selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), and serpin F1.

[0071] In some embodiments, the MSC secretome comprises at least one factor selected from the group consisting of angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, thrombospondin-1, angiogenin, DPPIV, IGFBP-3, and uPA, and the at least one factor is present at a level of 400 pg / mL to 3000 pg / mL.

[0072] In some embodiments, the MSC secretome composition further comprises at least one factor selected from the group consisting of apolipoprotein A1, factor D of complement, factor H of complement, factor I of complement, C1 esterase inhibitor (C1-INH), C4b-binding protein (C4BP), CD46, complement receptor type 1 (CR1), C-reactive protein, cystatin C, DKK-1, enppurin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15, and IFNγ.

[0073] In some embodiments, the MSC secretome composition comprises an anti-angiogenic to pro-angiogenic ratio, and the ratio is >2, >3, >4, or >5.

[0074] In some embodiments, the MSC secretome composition comprises VEGF at a level of 1 pg / mL to 400 pg / mL.

[0075] In some embodiments, the level of VEGF is 1 / 5 to 1 / 10 of the level of serpin E1.

[0076] In some embodiments, the composition comprises one or more anti-angiogenic factors, and the ratio of the total concentration of the one or more anti-angiogenic factors to the concentration of VEGF is >2, >3, >4, or >5.

[0077] In some embodiments, the MSC secretome comprises bFGF, PLGF, and PDGF at a level less than 1000 pg / mL.

[0078] In some embodiments, the MSC secretome composition has a pH of from about 4.7 to about 7.5.

[0079] In some embodiments, the MSC secretome composition is formulated in a buffer system selected from the group consisting of disodium monophosphate / sodium citrate, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and citric acid / disodium phosphate.

[0080] In some embodiments, the MSC secretome composition further comprises monosodium / disodium phosphate, mannitol, and trehalose, and the composition has a pH of about pH 7.4.

[0081] In some embodiments, the MSC secretome composition further comprises divalent cations.

[0082] In some embodiments, the divalent cation is selected from the group consisting of Mg2+, Ca2+, and Zn2+.

[0083] In some embodiments, the MSC secretome composition further comprises disodium phosphate / citric acid, mannitol, and trehalose, and the composition has a pH of about pH 6.4.

[0084] In some embodiments, the MSC secretome composition further comprises an agent that increases viscosity.

[0085] In some embodiments, the adhesive is selected from the group consisting of hypromellose, poloxamer 407, poloxamer 188, poloxamer 237, poloxamer 338, hypromellose (HPMC), polycarbophil, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyimide, sodium hyaluronate, gellan gum, poly(lactic-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethyl cellulose (CMC), or hydroxypropylmethyl cellulose (HPMC), hydroxymethyl cellulose, hydroxyethyl cellulose, sodium carboxymethyl cellulose, fibrin glue, polyethylene glycol, and GelCORE.

[0086] In some embodiments, the MSC secretome composition does not contain one or more components selected from the group consisting of foreign body components, phenol red, peptides and biomolecules <3 kDa, antibiotics, protein aggregates> 200 nm, cells, non-exosome / non-extracellular vesicle cell debris, hormones, and L-glutamine.

[0087] In some embodiments, the MSC secretome composition i. HGF at 0.3 to 4.5 ng / mL, and ii. pentraxin-3 (TSG-14) at 0.5 to 20 ng / mL, and iii. VEGF at 100 to 600 pg / mL, and iv. TIMP-1 at 10 to 200 ng / mL, and v. serpin E1 at 20 to 80 ng / mL, and vi. IL-8 <5 ng / mL, and and comprises.

[0088] In some embodiments, the MSC secretome composition comprises an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.

[0089] In some embodiments, the osmotic modifier is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin.

[0090] In some embodiments, the present invention i. MSC secretome at 2 μg to 20 μg per mL, and ii. monobasic sodium phosphate at 2 mg to 3 mg per mL, and iii. dibasic sodium phosphate at 11 mg to 12 mg per mL, and iv. mannitol at 11.5 mg to 13 mg per mL, and v. trehalose dihydrate at 23 mg to 24 mg, and vi. hypromellose at 0.5 mg to 2 mg per mL, and provides a stable bone marrow-derived mesenchymal stem cell (MSC) secretome formulation, the pH is from about 4.7 to about 7.5, and the MSC secretome is preconditioned.

[0091] In some embodiments, the present invention provides a stable bone marrow-derived mesenchymal stem cell (MSC) secretome formulation comprising i. 0.004% to 0.08% w / w MSC secretome, ii. 4% to 5% w / w monobasic sodium phosphate, iii. 21.5% to 23% w / w dibasic sodium phosphate, iv. 23% to 25% w / w mannitol, v. 46% to 48% trehalose dihydrate, and vi. 11% to 3% w / w hypromellose, the pH is from about 4.7 to about 7.5, and the MSC secretome is preconditioned.

[0092] In some embodiments, the present invention provides a method for treating an eye disease in a subject in need thereof, comprising administering to the subject a bone marrow-derived mesenchymal stem cell (MSC) secretome composition, the MSC secretome composition comprising HGF, pentraxin-3 (TSG-14), VEGF, TIMP-1, serpin E1, and <5 ng / mL IL-8, and the MSC secretome is preconditioned.

[0093] In some embodiments, the MSC secretome composition further comprises i. at least one trophic factor / cytokine selected from the group consisting of TIMP-2 and VEGF-A, and ii. at least one additional factor selected from the group consisting of PEDF (serpin F1), IGFBP-2, IGFBP-3, SDF-1, kallikrein 3, MCP-1, angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, and PDGF, and iii. at least one further factor selected from the group consisting of DPPIV (dipeptidyl peptidase-4), uPA, angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, and thrombospondin-1. Including.

[0094] In some embodiments, the MSC secretome composition comprises at least one factor in the range of 1 ng / mL to 100 ng / mL selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), and serpin F1.

[0095] In some embodiments, the MSC secretome composition comprises at least one factor in the range of 400 pg / mL to 3000 pg / mL selected from the group consisting of angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, thrombospondin-1, angiogenin, DPPIV, IGFBP-3, and uPA.

[0096] In some embodiments, the MSC secretome composition further comprises at least one factor selected from the group consisting of apolipoprotein A1, factor D of complement, factor H of complement, factor I of complement, C1 esterase inhibitor (C1-INH), C4b-binding protein (C4BP), CD46, complement receptor type 1 (CR1), C-reactive protein, cystatin C, DKK-1, enppin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15, and IFNγ.

[0097] In some embodiments, the MSC secretome composition comprises an anti-angiogenic to pro-angiogenic ratio, and the ratio is >2, >3, >4, or >5.

[0098] In some embodiments, the MSC secretome comprises VEGF at 1 pg / mL to 400 pg / mL.

[0099] In some embodiments, the level of VEGF is 1 / 5 to 1 / 10 of the level of serpin E1.

[0100] In some embodiments, the MSC secretome composition comprises one or more anti-angiogenic factors, and the sum of the concentrations of the one or more anti-angiogenic factors relative to the concentration of VEGF is >2, >3, >4, or >5.

[0101] In some embodiments, the MSC secretome comprises less than 1000 pg / mL of bFGF, PLGF, and PDGF.

[0102] In some embodiments, the MSC secretome composition has a pH of about 4.7 to about 7.5.

[0103] In some embodiments, the MSC secretome composition is formulated with a buffer system selected from the group consisting of disodium phosphate / sodium citrate, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and citric acid / disodium phosphate.

[0104] In some embodiments, the MSC secretome composition further comprises an osmotic modifier.

[0105] In some embodiments, the osmotic modifier is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin.

[0106] In some embodiments, the MSC secretome composition further comprises monosodium / disodium phosphate, mannitol, and trehalose, and the composition has a pH of about 7.4.

[0107] In some embodiments, the MSC secretome composition further comprises divalent cations.

[0108] In some embodiments, the divalent cations are selected from the group consisting of Mg2+, Ca2+, and Zn2+.

[0109] In some embodiments, the MSC secretome composition further comprises disodium phosphate / citric acid, mannitol, and trehalose, and the composition has a pH of about 6.4.

[0110] In some embodiments, the MSC secretome composition does not contain one or more components selected from the group consisting of foreign body components, phenol red, peptides and biomolecules <3 kDa, antibiotics, protein aggregates> 200 nm, cells, non-exosome / non-extracellular vesicle cell debris, hormones, and L-glutamine.

[0111] In some embodiments, the MSC secretome composition comprises an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.

[0112] In some embodiments, the MSC secretome composition further comprises an agent that increases viscosity.

[0113] In some embodiments, the adhesive is selected from the group consisting of hypromellose, poloxamer 407, poloxamer 188, poloxamer 237, poloxamer 338, hypromellose (HPMC), polycarbophil, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyimide, sodium hyaluronate, gellan gum, poly(lactic-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethyl cellulose (CMC), or hydroxypropyl methylcellulose (HPMC), hydroxymethyl cellulose, hydroxyethyl cellulose, sodium carboxymethyl cellulose, fibrin glue, polyethylene glycol, and GelCORE.

[0114] In some embodiments, the MSC secretome composition i. contains HGF at 0.3 to 4.5 ng / mL, and ii. contains pentraxin-3 (TSG-14) at 0.5 to 20 ng / mL, and iii. contains VEGF at 100 to 600 pg / mL, and iv. contains TIMP-1 at 10 to 200 ng / mL, and v. contains serpin E1 at 20 to 80 ng / mL, and vi. contains <5 ng / mL of IL-8, and and.

[0115] In some embodiments, the present invention provides a method for treating an eye disease in a subject in need thereof, comprising administering to the subject a bone marrow-derived mesenchymal stem cell (MSC) secretome composition, wherein the MSC secretome composition i. contains 2 μg to 20 μg of MSC secretome per mL, and ii. contains 2 mg to 3 mg of monobasic sodium phosphate per mL, and iii. contains 11 mg to 12 mg of dibasic sodium phosphate per mL, and iv. contains 11.5 mg to 13 mg of mannitol per mL, and v. contains 23 mg to 24 mg of trehalose dihydrate, and vi. 0.5 mg to 2 mg of hypromellose per 1 mL, and A stable bone marrow-derived mesenchymal stem cell (MSC) secretome formulation containing The pH is from about 4.7 to about 7.5 and the MSC secretome is preconditioned.

[0116] In some embodiments, the present invention provides a method for treating an eye disease in a subject in need thereof, comprising administering to the subject a bone marrow-derived mesenchymal stem cell (MSC) secretome composition, wherein the MSC secretome composition i. 0.004% to 0.08% w / w of MSC secretome, and ii. 4% to 5% w / w of monobasic sodium phosphate, and iii. 21.5% to 23% w / w of dibasic sodium phosphate, and iv. 23% to 25% w / w of mannitol, and v. 46% to 48% w / w of trehalose dihydrate, and vi. 1% to 3% w / w of hypromellose, and A stable mesenchymal stem cell (MSC) secretome formulation containing The pH is from about 4.7 to about 7.5 and the MSC secretome is preconditioned.

[0117] In some embodiments, the present invention provides a composition for inducing ocular wound healing comprising a mesenchymal stem cell (MSC) secretome and an osmotic modifier, wherein the MSC secretome is preconditioned, and the ability of the composition to promote ocular wound healing is a) providing a layer of corneal cells, and b) introducing a wound gap into the layer of corneal cells, and c) determining whether the wound gap heals in the presence of the composition, wherein the composition is administered to the corneal cells either before or after step b), and is shown by a wound healing assay comprising Suturing of the wound gap indicates the ability of the composition for inducing ocular wound healing.

[0118] In some embodiments, the corneal cells are corneal epithelial cells.

[0119] In some embodiments, the corneal cells are corneal keratocytes (or fibroblasts).

[0120] In some embodiments, the layer of corneal cells is a confluent monolayer.

[0121] In some embodiments, the wound gap is introduced by mechanically disrupting the layer of corneal cells.

[0122] In some embodiments, the wound gap is introduced by chemically disrupting the layer of corneal cells.

[0123] In some embodiments, the wound gap includes a linear gap.

[0124] In some embodiments, the wound gap includes a circular gap.

[0125] In some embodiments, determining whether the wound gap closes in step c) includes detecting and quantifying daily the migration and / or proliferation of corneal cells within the wound gap.

[0126] In some embodiments, the migration and / or proliferation of corneal cells is characterized as the number of corneal cells that have migrated and / or proliferated within the wound gap.

[0127] In some embodiments, determining whether the wound gap heals in step c) includes measuring daily the size of the wound gap, and the size of the wound gap is expressed as a percentage of the initial size of the wound gap measured immediately after the wound gap was introduced.

[0128] In some embodiments, the size of the wound gap is characterized as the surface area of the wound gap.

[0129] In some embodiments, the size of the wound gap is characterized as the width of the wound gap.

[0130] In some embodiments, step c) is performed within a period of 2 to 4 days after completion of step b).

[0131] In some embodiments, step c) is performed within a period of 2 days after completion of step b).

[0132] In some embodiments, step c) is performed within a period of 3 days after completion of step b).

[0133] In some embodiments, the wound healing assay further comprises concentrating the composition and optionally exchanging the buffer with the composition before administering the composition to the corneal cells.

[0134] In some embodiments, the wound healing assay further comprises diluting the composition and optionally exchanging the buffer with the composition before administering the composition to the corneal cells.

[0135] In some embodiments, the composition comprises at least 45 μg / ml of secretome protein.

[0136] In some embodiments, the present invention provides a composition for inducing ocular wound healing comprising a mesenchymal stem cell (MSC) secretome and an osmotic modifier, the MSC secretome being preconditioned, and the ability of the composition to promote ocular wound healing is a) providing a layer of corneal cells; b) introducing a wound gap into the layer of corneal cells; c) determining whether the wound gap heals in the presence of the composition, wherein the composition is administered to the corneal cells either before or after step b); as shown by a wound healing assay comprising Suturing of the wound gap indicates the ability of the composition to induce corneal wound healing.

[0137] In some embodiments, the corneal cells are corneal epithelial cells.

[0138] In some embodiments, the corneal cells are corneal keratocytes (or fibroblasts).

[0139] In some embodiments, the layer of corneal cells is a confluent monolayer.

[0140] In some embodiments, the wound gap is introduced by mechanically disrupting the layer of corneal cells.

[0141] In some embodiments, the wound gap is introduced by chemically disrupting the layer of corneal cells.

[0142] In some embodiments, the wound gap includes a linear gap.

[0143] In some embodiments, the wound gap includes a circular gap.

[0144] In some embodiments, determining whether the wound gap sutures in step c) includes detecting and quantifying daily the migration and / or proliferation of corneal cells within the wound gap.

[0145] In some embodiments, the migration and / or proliferation of corneal cells is characterized as the number of corneal cells that have migrated and / or proliferated within the wound gap.

[0146] In some embodiments, determining whether the wound gap heals in step c) includes measuring daily the size of the wound gap, and the size of the wound gap is expressed as a percentage of the initial size of the wound gap measured immediately after the wound gap was introduced.

[0147] In some embodiments, the size of the wound gap is characterized as the surface area of the wound gap.

[0148] In some embodiments, the size of the wound gap is characterized as the width of the wound gap.

[0149] In some embodiments, step c) is performed within a period of 2 to 4 days after completion of step b).

[0150] In some embodiments, step c) is performed within a period of 2 days after completion of step b).

[0151] In some embodiments, step c) is performed within a period of 3 days after completion of step b).

[0152] In some embodiments, the wound healing assay further includes a step of concentrating the composition and, optionally, a step of exchanging the buffer with the composition before administering the composition to the corneal cells.

[0153] In some embodiments, the wound healing assay further includes a step of diluting the composition and, optionally, a step of exchanging the buffer with the composition before administering the composition to the corneal cells.

[0154] In some embodiments, the composition contains at least 45 μg / ml of secretome protein.

[0155] In some embodiments, the present invention, hereinafter, changes in culture format (e.g., 2D planar vs. 3D bioreactor), different biomaterial scaffolds, co-culture, addition of pharmacological compounds, growth factors, chemokines, addition of Toll-like receptor agonists, inflammatory cytokines, advanced glycation end products (AGEs), oxidized phospholipids, malondialdehyde, or carboxyethylpyrrole, presence of agitation of the ECM, culture under shear stress, agitation or suspension as aggregates or within a matrix, induced misfolded protein response, ER stress, induction of MSC differentiation, culture in the presence of conditioned media, and hypoxia / anoxia, including one or more of the above, provides a method of preconditioning the MSC secretome to affect the MSC secretome profile.

[0156] In some embodiments, hypoxic preconditioning includes culturing MSCs in a hypoxic culture environment.

[0157] In some embodiments, the oxygen level is from about 0% to 2%.

[0158] In some embodiments, hypoxic preconditioning is performed for about 4 - 12 hours, about 12 - 24 hours, about 24 - 36 hours, about 36 - 48 hours, about 48 - 60 hours, or about 60 - 72 hours.

[0159] In some embodiments, preconditioning the MSC secretome includes treating the MSCs with one or more inflammatory cytokines.

[0160] In some embodiments, preconditioning the MSC secretome includes treating the MSCs with one or more of the following: IL-6, PGE2, IDO, IFNγ, SDF-1, TGF-α, H2O2, FGF-2, IGF-1, BMP-2, atorvastatin, oxytocin, curcumin, lipopolysaccharide, and nicotinamide (NIC), vasoactive intestinal peptide (VIP), and / or diazoxide.

[0161] In some embodiments, preconditioning the MSC secretome involves culturing the MSCs in 3D (e.g., by using a 3D bioreactor).

[0162] In some embodiments, preconditioning the MSC secretome involves inducing the unfolded protein response in the MSCs.

[0163] In some embodiments, preconditioning the MSC secretome involves culturing the MSCs under conditions appropriate to induce ER stress.

[0164] In some embodiments, preconditioning the MSC secretome involves culturing the MSCs under appropriate conditions to induce differentiation of the MSCs into mature retinal cell types or precursors thereof.

[0165] In some embodiments, the present invention provides a bone marrow-derived mesenchymal stem cell (MSC) secretome composition comprising differentiating MSCs into precursors of mature retinal cell types or mature retinal cell types. In some embodiments, the bone marrow-derived MSC secretome composition is obtained from MSC culture, and the MSCs are cultured under conditions suitable to induce differentiation of the MSCs into mature retinal cell types or precursors thereof.

[0166] In some embodiments, the present invention provides a method of treating an eye disease in a patient in need thereof, comprising administering to the patient the preconditioned MSC secretome disclosed herein.

[0167] In some embodiments, the present invention provides the use of the preconditioned MSC secretome disclosed herein for treating an eye disease in a patient in need thereof.

[0168] In some embodiments, the eye disease is selected from the group consisting of retinal diseases, macular diseases, chronic graft-versus-host disease (GvHD), Stevens-Johnson syndrome, ocular mucous membrane pemphigoid, persistent corneal epithelial defect (PCED), limbal stem cell deficiency (LSCD), dry eye, damage to the optic nerve tissue, and traumatic injury to the eye (such as traumatic injury, ocular contusion, or chemical burn).

DETAILED DESCRIPTION OF THE INVENTION

[0169] I. Introduction The present invention provides methods, assays, and protocols for the analysis of the activity of mesenchymal stem cell secretomes, as well as the use of such characterized compositions. Such methods and assays are described in more detail below.

[0170] Definitions The terms used in the claims and the specification are defined as described below, unless otherwise specified. In the event of a direct conflict with the terms used in the parent provisional patent application, the terms used in this specification shall prevail.

[0171] As used herein, "isolated" refers to a material that has been removed from its original environment and thus "manually" modified from its natural state.

[0172] As used herein, "enriched" means selectively enriching or increasing the amount of one or more materials by the exclusion of unwanted materials, or the selection and separation of desired materials from a mixture (e.g., separating cells having a specific cell marker from a heterogeneous cell population where not all cells in the population express the marker).

[0173] As used herein, the term "substantially purified" means a population of cells that is substantially homogeneous with respect to a particular marker or combination of markers. Substantially homogeneous means at least 90%, preferably 95%, homogeneous with respect to a particular marker or combination of markers. As used herein, the term "multipotent stem cell" is a true stem cell, but can differentiate into only a limited number of types. For example, bone marrow contains multipotent stem cells that can give rise to all the cells of the blood, but may not be able to differentiate into other cell types.

[0174] As used herein, the term "animal-free" when referring to the specific compositions, growth conditions, media, etc. described herein means that substances derived from non-human animals, such as bovine serum, proteins, lipids, carbohydrates, nucleic acids, vitamins, etc., are not used in the preparation, growth, culture, expansion, storage, or formulation of a particular composition or process. "Free of materials derived from non-human animals" means that the materials have never been in or contacted with the body or substances of non-human animals, so they are not contaminated with foreign substances. Generally, clinical-grade materials such as human proteins produced by recombinant technology are used in the preparation, growth, culture, expansion, storage, and / or formulation of such compositions and / or processes.

[0175] As used in reference to a cell composition, the term "expanded" means that the cell population constitutes a significantly higher concentration of cells than can be obtained using previous methods. For example, the cell level per gram of amniotic tissue in an expanded composition of AMP cells is at least 50-fold, up to 150-fold higher than the cell number in primary culture after 5 passages, compared to an approximately 20-fold increase in such cells using previous methods. In another example, the cell level per gram of amniotic tissue in an expanded composition of AMP cells is at least 30-fold, up to 100-fold higher than the cell number in primary culture after 3 passages. Thus, an "expanded" population has at least a 2-fold, up to 10-fold improvement in the number of cells per gram of amniotic tissue over previous methods. The term "expanded" means that it includes only situations where a person has intervened to increase the number of cells.

[0176] As used herein, "conditioned medium" is the medium in which a particular cell or population of cells has been cultured and then removed. When cells are cultured in a medium, the cells can secrete cytokines that can provide support for the behavior of other cells or affect the behavior of other cells. Such factors include, but are not limited to, hormones, cytokines, extracellular matrix (ECM), proteins, vesicles, antibodies, chemokines, receptors, inhibitors, and granules. A medium containing cytokines is a conditioned medium. An example of a method for preparing a conditioned medium is described in U.S. Patent No. 6,372,494, which is hereby incorporated by reference in its entirety. As used herein, "conditioned medium" also refers to a conditioned medium or a component such as a protein recovered and / or purified from, for example, MSC cells.

[0177] As used herein, the term "mesenchymal stem cell composition" or "MSC composition" means a conditioned medium derived from MSCs and, in some cases, further processed. In some embodiments, "MSC secretome" may refer to a crude conditioned medium derived from MSCs. In some embodiments, "MSC secretome" may refer to a composition obtained from a crude conditioned medium after being subjected to further processing as described herein.

[0178] As used herein, the term "suspension" means a liquid containing dispersed components such as, for example, cytokines. The dispersed components may be completely solubilized, partially solubilized, suspended, or dispersed in other ways in the liquid. Suitable liquids include, but are not limited to, water, osmotic solutions such as salt solutions and / or sugar solutions, cell culture media, and other aqueous or non-aqueous solutions.

[0179] "Amino acid" refers to naturally occurring amino acids, synthetic amino acids, and amino acid analogs and mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code and those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids, such as hydrogen, a carboxyl group, an amino group, and an α-carbon bonded to an R group, for example, homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. Such analogs have a modified R group (e.g., norleucine) or a modified peptide backbone but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids. Amino acids may be represented herein by either their generally known three-letter symbols or one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may likewise be represented by generally recognized one-letter abbreviations.

[0180] "Amino acid substitution" refers to the replacement of at least one existing amino acid residue in a given amino acid sequence (the amino acid sequence of the starting polypeptide) with a second, different "substituting" amino acid residue. "Amino acid insertion" refers to the incorporation of at least one additional amino acid into a given amino acid sequence. Insertions usually consist of the insertion of one or two amino acid residues, but larger "peptide insertions" can be made, such as the insertion of about 3 to about 5, or up to about 10, 15, or 20 amino acid residues. The residue(s) inserted may be naturally occurring or non-naturally occurring as disclosed above. "Amino acid deletion" refers to the removal of at least one amino acid residue from a given amino acid sequence.

[0181] "Polypeptide", "peptide", and "protein" are used interchangeably herein and refer to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0182] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, and polymers thereof. Unless otherwise specifically limited, the term encompasses nucleic acids having binding characteristics similar to those of the reference nucleic acid and known analogs of natural nucleotides that are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also encompasses not only the explicitly recited sequence, but also its variants (e.g., degenerate codon substitutions) that are implicitly and conservatively modified and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., Biol. Chem. 260:2605-2608, 1985; and Cassol et al., 1992; Rosssolini et al., Mol. Cell Probes 8:91-98, 1994). In the case of arginine and leucine, modifications at the second base may also be conservative. The term nucleic acid is used synonymously with gene, cDNA, and mRNA encoded by a gene. As used herein, polynucleotide may be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or more typically double-stranded or a mixture of single-stranded and double-stranded regions. In addition, polynucleotides can consist of triple-stranded regions containing RNA, or DNA, or both RNA and DNA. Polynucleotides can also contain one or more modified bases or a modified DNA or RNA backbone that has been modified for stability or other reasons. "Modified" bases include, for example, rare bases such as tritylated bases and inosine.Since various modifications can be added to DNA and RNA, "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms.

[0183] As used herein, the term "secretome composition" refers to a composition that includes one or more substances secreted from a cell. In certain embodiments, the secretome composition can include one or more cytokines, one or more exosomes, and / or one or more vesicles. The secretome composition can be purified or unpurified. In some embodiments, the secretome composition can further include one or more substances that are not secreted from the cell (e.g., medium, additives, growth factors, etc.). In some, the secretome composition includes only and / or does not include trace amounts of one or more substances that are not secreted from the cell (e.g., medium, additives, growth factors, etc.).

[0184] As used herein, the terms “treatment,” “treat,” or “treating” and the like include any treatment of a human or non-human mammal (e.g., rodents, cats, dogs, horses, cows, sheep, and primates, etc.), and include preventing the occurrence of a disease or disorder in a subject that is susceptible to, but not yet diagnosed as having, the disease or disorder. The terms also include inhibiting (preventing its onset), alleviating or improving (causing its regression), or curing (permanently halting its onset or progression) the disease, disorder, and / or any associated symptoms. As used herein, the terms “treatment,” “treat,” or “treating” include any treatment of a mammalian, particularly human, disease or disorder, and (a) preventing the occurrence of a disease or disorder in a subject that is susceptible to, but not yet diagnosed as having, the disease or disorder, (b) inhibiting the disease or disorder, e.g., preventing its manifestation, (c) reducing and / or improving the disease or disorder, e.g., causing regression of the disease or disorder, and (d) curing the disease or disorder, e.g., stopping its onset or progression. The population of subjects treated by the methods of the present invention includes subjects suffering from an undesirable disease or disorder and subjects at risk of developing a disease or disorder. In some embodiments, “treatment” (or “treat” or “treating”) refers to any administration of a therapy that partially or completely reduces, improves, alleviates, inhibits, delays the onset of, reduces the severity of, and / or decreases the incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be of a subject that exhibits no signs of the associated disease, disorder, and / or condition and / or exhibits only early signs of the disease, disorder, and / or condition. Alternatively and / or additionally, such treatment may be of a subject that exhibits one or more established signs of the associated disease, disorder, and / or condition.In some embodiments, the treatment may be for a subject diagnosed with a related disease, disorder, and / or illness. In some embodiments, the treatment may be for a subject known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing a related disease, disorder, and / or illness.

[0185] As used herein, "wound" is any disruption, for any reason, of a normal anatomical structure (internal and / or external anatomical structures), including but not limited to trauma such as mechanical injury (e.g., contusion, penetration), burns, chemical injury, electrical injury, radiation injury, blast injury, and incisional injury, elective injury such as surgery and resulting incisional hernia, fistula, etc., acute wounds, chronic wounds, infected wounds, and aseptic wounds, and wounds associated with disease states (e.g., ocular contusion). A wound is dynamic, and the healing process is a continuum that begins at the time of injury and requires a series of integrated and interconnected cellular processes that progress beyond the initial suture to a stable suture. These cellular processes are mediated or regulated by humoral substances including but not limited to cytokines, lymphokines, growth factors, and hormones. According to the subject invention, "wound healing" refers to improving the natural cellular processes and humoral substances of tissue repair by some form of intervention such that healing is faster and / or the scarring of the resulting healed area is less and / or the wound has a tissue strength closer to that of uninjured tissue and / or the wounded tissue achieves some degree of functional recovery.

[0186] As used herein, the term "a" or "an" means one or more or at least one.

[0187] As used herein, a "therapeutically effective" or "effective" dosage or amount of a composition is an amount sufficient to provide a positive effect against a given medical condition. If not immediate, a therapeutically effective or effective dosage or amount can provide a significant or measurable effect on the health and well-being of a patient over a period of time.

[0188] As used herein, "pharmaceutical composition" refers to an effective amount of the composition described herein in combination with a delivery component. The pharmaceutical composition may optionally include other components such as pharmaceutically suitable carriers and excipients, which may facilitate the administration of the composition and / or its individual components to a subject.

[0189] The term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the administered compound.

[0190] The term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of a compound.

[0191] As used herein, the terms "mix" and "mixing" refer to a mechanical process or treatment of components. For example, mixing can mean performing repeated cycles of pressing and folding, or equivalent processing steps leading to strong compression and mixing of a provided hydrophobic matrix.

[0192] Adult stem cells can be obtained from various adult tissues including bone marrow, adipose, and dental pulp tissue. All adult stem cells are self-renewable and considered pluripotent, but the therapeutic functions of somatic stem cells vary according to their origin. As a result, each type of adult stem cell has unique characteristics that make them suitable for specific diseases. Mesenchymal stem cells (MSCs) are typically derived from the mesoderm and are pluripotent non-hematopoietic (non-blood) stem cells that can be isolated (derived) from various tissues and are differentiable into various tissues including osteoblasts (e.g., bone cells), chondrocytes (cartilage cells), myocytes (e.g., muscle cells), and adipocytes (e.g., fat cells that give rise to bone marrow adipose tissue). As used herein, "isolated" refers to cells removed from their original environment. Stem cells regulate multiple biological processes or produce factors such as growth factors that are important for regulating them. Growth factors are agents such as naturally occurring substances that can stimulate cell growth and / or proliferation and / or cell differentiation. Typically, growth factors are proteins or steroid hormones. The terms "growth factor" and "factor" are used synonymously herein, but the term "biological factor" is not limited to growth factors.

[0193] Human mesenchymal stem cells (MSCs) can be characterized by a surface marker profile of CD45- / CD31- / CD73+ / CD90+ / CD105+ / CD44+ (or any suitable subset thereof). (See Bourin et al., Cytotherapy 15(6):641-648 (2013).) Additionally, suitable stem cells are CD34+ positive at the time of isolation but lose this marker during culture. Thus, a complete marker profile for one stem cell type that can be used in accordance with this application includes CD45- / CD31- / CD73+ / CD90+ / CD105+. In another embodiment utilizing mouse stem cells, the stem cells are characterized by the Sca-1 marker instead of CD34, defining what appears to be the homolog to the human cells described above, with the remaining markers being the same.

[0194] The phrase "conditioned medium" or "CM" refers to a medium containing biological factors secreted by MSCs. This may also be referred to herein as "secretome", "MSC-CM", "MSC secretome" and / or "MSC-derived secretome". Also provided is a processed "conditioned medium" that contains biological factors secreted by MSCs and has been further processed, for example, by filtration, purification, and / or concentration procedures. The "conditioned medium" is obtained, as described in detail herein, by culturing stem cells in a medium and separating the resulting medium, which contains hepatocytes and their secreted stem cell products (secretome), into a conditioned medium that contains biological factors and fewer stem cells than were present prior to separation. The conditioned medium can be used in the methods described herein and is substantially free of hepatocytes (may contain a low percentage of stem cells) or is stem cell-free. Biological factors that may be present in the conditioned medium include, but are not limited to, proteins (e.g., cytokines, chemokines, growth factors, enzymes), nucleic acids (e.g., miRNAs), lipids (e.g., phospholipids), polysaccharides, and / or combinations thereof. Any combination(s) of these biological factors may be either bound within or on the surface of extracellular vesicles (e.g., exosomes) or separated from extracellular vesicles.

[0195] The terms "cell preconditioning", "MSC preconditioning", or "secretome preconditioning" refer to any operation performed on MSC or secretome-producing cells, or secretome-producing cells prior to secretome collection. Such operations include, but are not limited to, changes in culture format (e.g., 2D planar vs. 3D bioreactor), different biomaterial scaffolds, co-culture, addition of pharmacological compounds, growth factors, chemokines, Toll-like receptor agonists, inflammatory cytokines, advanced glycation end products (AGEs), oxidized phospholipids, malondialdehyde, or carboxyethylpyrrole, agitation of the ECM, culturing under shear stress, agitation or suspension as aggregates or within a matrix, induced misfolded protein response, ER stress, induction of MSC differentiation, culturing in the presence of conditioned media, and / or one or more of hypoxia / anoxia.

[0196] As used herein, the term "mature retinal cell" refers to cells that may be included in human adult retinal tissue. Specific examples of mature retinal cells include differentiated cells such as photoreceptor cells (rod and cone photoreceptor cells), bipolar cells, horizontal cells, amacrine cells, interneurons, retinal ganglion cells (ganglion cells), bipolar cells (rod bipolar cells, cone bipolar cells), Müller glial cells, retinal pigment epithelial (RPE) cells, and ciliary marginal zone cells.

[0197] As used herein, the term "retinal precursor cell" or "retinal progenitor cell" means a progenitor cell that has been confirmed to differentiate into mature retinal cells. Non-limiting examples of retinal progenitor cells include photoreceptor progenitor cells, bipolar progenitor cells, retinal progenitor cells, horizontal progenitor cells, amacrine progenitor cells, retinal ganglion progenitor cells, Müller glial progenitor cells, and retinal pigment epithelial progenitor cells.

[0198] Compositions and Formulations According to the present specification, a composition is provided herein that includes a mesenchymal stem cell (MSC) secretome, and / or a conditioned medium containing mesenchymal stem cells (MSCs) (including the processed MSC secretome).

[0199] In some embodiments, the MSC secretome is generally low for angiogenic factors. In some embodiments, the MSC secretome does not promote angiogenesis. In some embodiments, the MSC secretome exhibits an anti-angiogenic effect. In some embodiments, the MSC secretome results in a reduction of angiogenesis compared to other secretomes. In some embodiments, the MSC secretome results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% reduction of angiogenesis. In some embodiments, the MSC secretome results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% reduction of angiogenesis compared to another secretome. In some embodiments, the MSC secretome results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% reduction of angiogenesis compared to the conditioned medium before processing into the MSC secretome. In some embodiments, the MSC secretome has low angiogenic induction. In some embodiments, the MSC secretome has a reduced angiogenic response. In some embodiments, the MSC secretome has reduced angiogenic activity. In some embodiments, the MSC secretome impairs and / or reduces the normal formation of blood vessels in the presence of a medium that supports angiogenesis. In some embodiments, the MSC secretome has reduced angiogenic activity when the MSC secretome is compared to an untreated control. In some embodiments, the MSC secretome has reduced angiogenic activity compared to a sample treated with serum-containing medium. In some embodiments, the MSC secretome attenuates the angiogenic response. In some embodiments, the MSC secretome reduces the angiogenic response induced by serum-containing medium. In some embodiments, when the secretome supplemented with serum-containing medium (reduced or no angiogenic response) is compared to serum-containing medium (angiogenic response), the reduction of the angiogenic response is induced by the MSC secretome. In some embodiments, the angiogenic response is indicated by tube formation in a cell-based assay.In some embodiments, the angiogenesis response is indicated by tube formation in an endothelial cell tube formation assay. In some embodiments, the angiogenesis response is indicated by angiogenesis in a CAM (chick chorioallantoic membrane) assay. In some embodiments, the angiogenesis response is indicated by angiogenesis in any angiogenesis assay known in the art.

[0200] In some embodiments, the mesenchymal stem cell (MSC) secretome composition i. IDO (indoleamine-2,3-dioxygenase) enzyme activity and ii. the "threshold" ppm level of at least one trophic factor / cytokine selected from the group consisting of HGF, FGF-7, TIMP-1, TIMP-2, PAI-1 (serpin E1), VEGF-A, and b-NGF, iii. the "threshold" ppm level of at least one additional factor selected from the group consisting of sFLT-1, PEDF (serpin F1), IGFBP-2, IGFBP-3, SDF-1, TSG-14, kallikrein 3, MCP-1, bFGF, angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, PDGF, SOD1, SOD2, SOD3, and HO-1, iv. the "threshold" ppm level of at least one further factor selected from the group consisting of DPPIV (dipeptidyl peptidase-4), uPA, angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, and thrombospondin-1, and comprises.

[0201] In some embodiments, the mesenchymal stem cell (MSC) secretome composition i. less than about 250 μM IDO (indoleamine-2,3-dioxygenase) enzyme activity and ii. At least one trophic factor / cytokine selected from the group consisting of HGF, FGF-7, TIMP-1, TIMP-2, PAI-1 (serpin E1), VEGF-A, and / or b-NGF, iii. At least one additional factor selected from the group consisting of sFLT-1, PEDF (serpin F1), IGFBP-2, IGFBP-3, SDF-1, TSG-14, kallikrein 3, MCP-1, bFGF, angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, PDGF, SOD1, SOD2, SOD3, and / or HO-1, iv. At least one further factor selected from the group consisting of DPPIV (dipeptidyl peptidase-4), uPA, angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, and / or thrombospondin-1, and comprising.

[0202] The mesenchymal stem cell (MSC) secretome composition i. At least one trophic factor / cytokine selected from the group consisting of HGF, TIMP-1, TIMP-2, PAI-1 (serpin E1), VEGF-A, and b-NGF, ii. At least one additional factor selected from the group consisting of PEDF (serpin F1), IGFBP-2, IGFBP-3, SDF-1, TSG-14, kallikrein 3, MCP-1, angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, and PDGF, iii. At least one further factor selected from the group consisting of DPPIV (dipeptidyl peptidase-4), uPA, angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, and thrombospondin-1, and comprising.

[0203] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor including, but not limited to, apolipoprotein A1, factor D of complement, factor H of complement, factor I of complement, C1 esterase inhibitor (C1-INH), C4b-binding protein (C4BP), CD46, complement receptor type 1 (CR1), C-reactive protein, cystatin C, DKK-1, enppurin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15, and / or IFNγ.

[0204] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one factor selected from the group consisting of apolipoprotein A1, factor D of complement, factor H of complement, factor I of complement, C1 esterase inhibitor (C1-INH), C4b-binding protein (C4BP), CD46, complement receptor type 1 (CR1), C-reactive protein, cystatin C, DKK-1, enppurin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15, and IFNγ.

[0205] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor including, but not limited to, serine protease inhibitors, i.e., serpin family members including serpin F1, serpin E1, serpin A1, serpin G1, serpin H1, serpin B6, serpin E2, serpin A3, serpin C1, serpin F2, serpin I1), serpin B1, serpin B7, serpin D1, serpin B3, serpin B8, serpin B2, serpin B12, serpin A7, serpin A4, and / or serpin A6. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor including, but not limited to, serpin F1 (also known as PEDF), serpin E1, and serpin A1. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises serpin F1 (also known as PEDF). In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises serpin E1. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises serpin A1.

[0206] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor, including but not limited to proteins involved in antioxidant activity. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor, including but not limited to catalase, protein disulfide-isomerase, protein disulfide-isomerase A3, protein disulfide-isomerase A4, protein disulfide-isomerase A6, peroxiredoxin-6, peroxiredoxin-1, peroxiredoxin-2, and / or peroxiredoxin-4. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises catalase. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises protein disulfide-isomerase. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises protein disulfide-isomerase A3. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises protein disulfide-isomerase A4. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises protein disulfide-isomerase A6. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises peroxiredoxin-6. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises peroxiredoxin-1. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises peroxiredoxin-2. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises peroxiredoxin-4.

[0207] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor, including but not limited to matrix metalloproteinases. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor, including but not limited to MMP2, MMP1, and / or MMP14. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises MMP2. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises MMP1. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises MMP14.

[0208] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor, including but not limited to soluble scavenger receptor cysteine-rich domain-containing protein SSC5D, tumor necrosis factor-inducible gene 6 protein (also known as TSG-6), serum albumin, and latent transforming growth factor-binding proteins (LTGFBP-1) including various isoforms, LTGFBP-2, LTGFBP-3, and LTGFBP-4. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises soluble scavenger receptor cysteine-rich domain-containing protein SSC5D. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises tumor necrosis factor-induced gene 6 protein (also known as TSG-6). In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises serum albumin. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises LTGFBP-1. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises LTGFBP-2. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises LTGFBP-3. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises LTGFBP-4.

[0209] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises pentraxin-3, TIMP-1, serpin E1, TSP-1, and HGF.

[0210] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises pentraxin-3 at 2-16 ng / mL, or 9.8 + / - 0.5 ng / ml.

[0211] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises TIMP-1 at 10-200 ng / mL, or 90 + / - 21.5 ng / ml.

[0212] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises serpin E1 at 10-100 ng / mL, or 49.2 + / - 9.8 ng / ml.

[0213] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises HGF at 0.1-10 ng / mL, or 2.0 + / - 0.3 ng / ml.

[0214] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises VEGF at 100-800 pg / mL, or 304 + / - 44 ng / ml.

[0215] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises IL-8 at 0.1-100 pg / mL, or <1 ng / ml.

[0216] In some embodiments, the IDO (indoleamine-2,3-dioxygenase) enzyme activity is less than about 250 μM. In some embodiments, the IDO (indoleamine-2,3-dioxygenase) enzyme activity is from about 0 μM to 250 μM. In some embodiments, the IDO (indoleamine-2,3-dioxygenase) enzyme activity is 50 μM to about 250 μM of L-kynurenine per million MSCs. In some embodiments, the IDO (indoleamine-2,3-dioxygenase) enzyme activity is 50 μM to about 200 μM of L-kynurenine per million MSCs. In some embodiments, the IDO (indoleamine-2,3-dioxygenase) enzyme activity is 100 μM to about 250 μM of L-kynurenine per million MSCs. In some embodiments, the IDO (indoleamine-2,3-dioxygenase) enzyme activity is 100 μM to about 200 μM of L-kynurenine per million MSCs. In some embodiments, the IDO (indoleamine-2,3-dioxygenase) enzyme activity is about 0 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 110 μM, about 120 μM, about 130 μM, about 140 μM, about 150 μM, about 160 μM, about 170 μM, about 180 μM, about 190 μM, about 200 μM, about 210 μM, about 220 μM, about 230 μM, about 240 μM, or about 250 μM of L-kynurenine per million MSCs.

[0217] In some embodiments, the MSC secretome comprises a “threshold” ppm level of at least one additional factor including, but not limited to, sFLT-1, PEDF (serpin F1), IGFBP-2, IGFBP-3, SDF-1, TSG-14, kallikrein 3, MCP-1, bFGF, angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, PDGF, SOD1, SOD2, SOD3, and / or HO-1. In some embodiments, the MSC secretome comprises a “threshold” ppm level of at least one additional factor selected from the group consisting of sFLT-1, PEDF (serpin F1), IGFBP-2, IGFBP-3, SDF-1, TSG-14, kallikrein 3, MCP-1, bFGF, angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, PDGF, SOD1, SOD2, SOD3, and HO-1. In some embodiments, the MSC secretome comprises one additional factor in a concentration range of 200 pg / mL to 5000 pg / mL, and the one additional factor includes, but is not limited to, sFLT-1, PEDF (serpin F1), IGFBP-2, IGFBP-3, SDF-1, TSG-14, kallikrein 3, MCP-1, bFGF, angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, PDGF, SOD1, SOD2, SOD3, and / or HO-1. In some embodiments, the MSC secretome further comprises 1000 to 3000 pg / mL of sFLT-1. In some embodiments, the MSC secretome further comprises 400 to 800 pg / mL of sTSG-6.

[0218] In some embodiments, the MSC secretome further comprises PEDF at 2000 - 8000 pg / mL. In some embodiments, the MSC secretome further comprises PEDF at 2000 - 7000 pg / mL. In some embodiments, the MSC secretome further comprises PEDF at 2000 - 6000 pg / mL. In some embodiments, the MSC secretome further comprises PEDF at 2000 - 5000 pg / mL. In some embodiments, the MSC secretome further comprises PEDF at 2000 - 4000 pg / mL. In some embodiments, the MSC secretome further comprises PEDF at 2000 - 3000 pg / mL. In some embodiments, the MSC secretome further comprises PEDF at 150 - 300 ng / mL. In some embodiments, the MSC secretome further comprises PEDF at 200 - 300 ng / mL. In some embodiments, the MSC secretome further comprises PEDF at 200 - 275 ng / mL. In some embodiments, the MSC secretome further comprises PEDF at 225 - 275 ng / mL. In some embodiments, the MSC secretome further comprises PEDF at 150 - 300 ng / mL. In some embodiments, the MSC secretome further comprises PEDF at 273 ± 27 ng / mL.

[0219] In some embodiments, the MSC secretome composition further comprises at least one factor at a "higher" level selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome further comprises serpin E1 at a "higher" level. In some embodiments, the MSC secretome further comprises serpin A1 at a "higher" level. In some embodiments, the MSC secretome further comprises TIMP-1 at a "higher" level. In some embodiments, the MSC secretome further comprises thrombospondin-1 at a "higher" level. In some embodiments, the MSC secretome further comprises pentraxin-3 (TSG-14) at a "higher" level. In some embodiments, the MSC secretome further comprises platelet factor 4 at a "higher" level. In some embodiments, the MSC secretome further comprises serpin F1 at a "higher" level. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 1 ng / mL to 20 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 1 ng / mL to 8 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 2 ng / mL to 8 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 3 ng / mL to 8 ng / mL.In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 4 ng / mL to 8 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 5 ng / mL to 8 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 6 ng / mL to 8 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 2 ng / mL to 7 ng / mL.

[0220] In some embodiments, the MSC secretome composition further comprises at least one factor at a "mid-range" level, including but not limited to angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, and thrombospondin-1, angiogenin, DPPIV (dipeptidyl peptidase-4), IGFBP-3, and / or uPA. In some embodiments, the MSC secretome composition further comprises at least one factor at a "mid-range" level selected from the group consisting of angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2 and thrombospondin-1, angiogenin, DPPIV, IGFBP-3, and uPA. In some embodiments, the MSC secretome composition comprises at least one factor at a "mid-range" level selected from the group consisting of angiogenin, DPPIV, IGFBP-3, and uPA. In some embodiments, the MSC secretome composition comprises at least one factor at a level of about 200 pg / mL to about 800 pg / mL selected from the group consisting of angiogenin, DPPIV, IGFBP-3, and uPA. In some embodiments, the MSC secretome composition further comprises at least one factor at a level of about 200 pg / mL to about 700 pg / mL, about 300 pg / mL to about 800 pg / mL, about 200 pg / mL to about 500 pg / mL, or about 300 pg / mL to about 500 pg / mL selected from the group consisting of angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, and thrombospondin-1, angiogenin, DPPIV, IGFBP-3, and uPA.In some embodiments, the MSC secretome composition further comprises at least one factor selected from the group consisting of angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, and thrombospondin-1, angiogenin, DPPIV, IGFBP-3, and uPA, at about 200 pg / mL to about 300 pg / mL, about 400 pg / mL, about 500 pg / mL, about 600 pg / mL, about 700 pg / mL, or about 800 pg / mL. In some embodiments, the MSC secretome composition comprises angiogenin at about 200 pg / mL to about 800 pg / mL, about 300 pg / mL to 800 pg / mL, about 200 pg / mL to about 500 pg / mL, or about 300 pg / mL to about 500 pg / mL. In some embodiments, the MSC secretome composition further comprises DPPIV at about 200 pg / mL to about 800 pg / mL, about 300 pg / mL to about 800 pg / mL, about 200 pg / mL to about 500 pg / mL, or about 300 pg / mL to about 500 pg / mL. In some embodiments, the MSC secretome composition comprises IGFBP-3 at about 200 pg / mL to about 800 pg / mL, about 300 pg / mL to about 800 pg / mL, about 200 pg / mL to 500 pg / mL, or about 300 pg / mL to about 500 pg / mL. In some embodiments, the MSC secretome composition comprises uPA at 200 pg / mL to about 800 pg / mL, about 300 pg / mL to 800 pg / mL, about 200 pg / mL to 500 pg / mL, or about 300 pg / mL to about 500 pg / mL.

[0221] In some embodiments, the MSC secretome further comprises a "low" level of VEGF. In some embodiments, the MSC secretome further comprises about 1 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 10 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 20 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 30 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 40 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 50 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 60 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 70 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 80 pg / mL of VEGF.

[0222] In some embodiments, the MSC secretome further comprises about 90 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 100 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 125 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 150 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 175 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 1 pg / mL to about 400 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 10 pg / mL to about 400 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 50 pg / mL to about 350 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 50 pg / mL to about 300 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 10 pg / mL to about 300 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 100 pg / mL to about 300 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises less than about 200 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises less than about 200 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 0 pg / mL to about 200 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 0 pg / mL to about 200 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 10 pg / mL to about 200 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 20 pg / mL to about 200 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 30 pg / mL to about 200 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 40 pg / mL to about 200 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 50 pg / mL to about 200 pg / mL of VEGF.In some embodiments, the MSC secretome further comprises VEGF at about 60 pg / mL to about 200 pg / mL. In some embodiments, the MSC secretome further comprises VEGF at about 70 pg / mL to about 200 pg / mL. In some embodiments, the MSC secretome further comprises VEGF at about 80 pg / mL to about 200 pg / mL. In some embodiments, the MSC secretome further comprises VEGF at about 90 pg / mL to about 200 pg / mL. In some embodiments, the MSC secretome further comprises VEGF at about 100 pg / mL to about 200 pg / mL. In some embodiments, the MSC secretome further comprises VEGF at about 10 pg / mL to about 150 pg / mL. In some embodiments, the MSC secretome further comprises VEGF at about 20 pg / mL to about 150 pg / mL. In some embodiments, the MSC secretome further comprises VEGF at about 30 pg / mL to about 150 pg / mL. In some embodiments, the MSC secretome further comprises VEGF at about 40 pg / mL to about 150 pg / mL. In some embodiments, the MSC secretome further comprises VEGF at about 50 pg / mL to about 150 pg / mL. In some embodiments, the MSC secretome further comprises VEGF at about 60 pg / mL to about 150 pg / mL. In some embodiments, the MSC secretome further comprises VEGF at about 70 pg / mL to about 150 pg / mL. In some embodiments, the MSC secretome further comprises VEGF at about 80 pg / mL to about 150 pg / mL. In some embodiments, the MSC secretome further comprises VEGF at about 90 pg / mL to about 150 pg / mL. In some embodiments, the MSC secretome further comprises VEGF at about 100 pg / mL to about 150 pg / mL. In some embodiments, the MSC secretome further comprises VEGF at about 10 pg / mL to about 100 pg / mL. In some embodiments, the MSC secretome further comprises VEGF at about 20 pg / mL to about 100 pg / mL. In some embodiments, the MSC secretome further comprises VEGF at about 30 pg / mL to about 100 pg / mL.In some embodiments, the MSC secretome further comprises VEGF at about 40 pg / mL to about 100 pg / mL. In some embodiments, the MSC secretome further comprises VEGF at about 50 pg / mL to about 100 pg / mL. In some embodiments, the MSC secretome further comprises VEGF at about 60 pg / mL to about 100 pg / mL. In some embodiments, the MSC secretome further comprises VEGF at about 70 pg / mL to about 100 pg / mL. In some embodiments, the MSC secretome further comprises VEGF at about 80 pg / mL to about 100 pg / mL. In some embodiments, the MSC secretome further comprises VEGF at about 90 pg / mL to about 100 pg / mL. In some embodiments, the MSC secretome further comprises VEGF at about 100 pg / mL to about 100 pg / mL.

[0223] In some embodiments of the MSC secretome composition, the level of VEGF is one-fifth to one-tenth of the level of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is one-sixth to one-tenth of the level of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is one-seventh to one-tenth of the level of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is one-eighth to one-tenth of the level of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is one-ninth to one-tenth of the level of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is one-fifth of the level of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is one-sixth of the level of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is one-seventh of the level of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is one-eighth of the level of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is one-ninth of the level of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is one-tenth of the level of serpin E1.

[0224] In some embodiments, the MSC secretome composition does not contain and / or contains very low levels of bFGF, PLGF, and PDGF. In some embodiments, the MSC secretome composition contains less than about 200 pg / mL, less than about 150 pg / mL, less than about 100 pg / mL, less than about 75 pg / mL, less than about 50 pg / mL, or less than about 25 pg / mL of bFGF, PLGF, and / or PDGF. In some embodiments, the MSC secretome composition contains less than about 200 pg / mL, less than about 150 pg / mL, less than about 100 pg / mL, less than about 75 pg / mL, less than about 50 pg / mL, or less than about 25 pg / mL of bFGF, PLGF, and PDGF. In some embodiments, the MSC secretome composition does not contain bFGF, PLGF, and / or PDGF. In some embodiments, the MSC secretome composition does not contain bFGF, PLGF, and PDGF. In some embodiments, the MSC secretome composition contains less than about 200 pg / mL, less than about 150 pg / mL, less than about 100 pg / mL, less than about 75 pg / mL, less than about 50 pg / mL, or less than about 25 pg / mL of bFGF. In some embodiments, the MSC secretome composition does not contain bFGF. In some embodiments, the MSC secretome composition contains less than about 200 pg / mL, less than about 150 pg / mL, less than about 100 pg / mL, less than about 75 pg / mL, less than about 50 pg / mL, or less than about 25 pg / mL of PLGF. In some embodiments, the MSC secretome composition does not contain PLGF. In some embodiments, the MSC secretome composition contains less than about 200 pg / mL, less than about 150 pg / mL, less than about 100 pg / mL, less than about 75 pg / mL, less than about 50 pg / mL, or less than about 25 pg / mL of PDGF. In some embodiments, the MSC secretome composition does not contain PDGF. In some embodiments, the MSC secretome composition does not contain bFGF. In some embodiments, the MSC secretome composition does not contain PLGF. In some embodiments, the MSC secretome composition does not contain PDGF. In some embodiments, the MSC secretome composition contains very low levels of bFGF, PLGF, and PDGF.In some embodiments, the MSC secretome composition comprises very low levels of bFGF. In some embodiments, the MSC secretome composition comprises very low levels of PLGF. In some embodiments, the MSC secretome composition comprises very low levels of PDGF.

[0225] In some embodiments, the MSC secretome composition comprises apolipoprotein A1, factor D of complement, factor H of complement, factor I of complement, C1 esterase inhibitor (C1-INH), C4b-binding protein (C4BP), CD46, complement receptor type 1 (CR1), C-reactive protein, cystatin C, DKK-1, enppurin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15, and / or IFNγ.

[0226] In some embodiments, the MSC secretome composition further comprises at least one factor at a "higher" level selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at 1 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at 1 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at 1 ng / mL to 200 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at 1 ng / mL to 100 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at 10 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at 10 ng / mL to 300 ng / mL.In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 10 ng / mL to 200 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 10 ng / mL to 100 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 20 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 20 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 20 ng / mL to 200 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 20 ng / mL to 100 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 30 ng / mL to 400 ng / mL.In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 30 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 30 ng / mL to 200 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 30 ng / mL to 100 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 4 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 40 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 40 ng / mL to 200 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 40 ng / mL to 100 ng / mL.In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 50 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 50 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 50 ng / mL to 200 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 50 ng / mL to 100 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 60 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 60 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 60 ng / mL to 200 ng / mL.In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 60 ng / mL to 100 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 70 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 70 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 70 ng / mL to 200 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 70 ng / mL to 100 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 80 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 80 ng / mL to 300 ng / mL.In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 80 ng / mL to 200 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 80 ng / mL to 100 ng / mL. In some embodiments, the MSC secretome composition comprises serpin E1, serpin A1, TIMP-1, thrombospondin-1, pen. It contains at least one factor selected from the group consisting of thrombospondin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 90 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition contains at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 90 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition contains at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 90 ng / mL to 200 ng / mL. In some embodiments, the MSC secretome composition contains at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 90 ng / mL to 100 ng / mL. In some embodiments, the MSC secretome composition contains at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 100 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition contains at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 100 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition contains at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 100 ng / mL to 200 ng / mL.In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 110 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 110 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 110 ng / mL to 200 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 120 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 120 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 120 ng / mL to 200 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 130 ng / mL to 400 ng / mL.In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 130 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 130 ng / mL to 200 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 140 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 140 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 140 ng / mL to 200 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 150 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 150 ng / mL to 300 ng / mL.In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 150 ng / mL to 200 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 160 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 160 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 160 ng / mL to 200 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 170 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 170 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 170 ng / mL to 200 ng / mL.In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, and the concentration of the at least one factor is 180 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, and the concentration of the at least one factor is 180 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, and the concentration of the at least one factor is 180 ng / mL to 200 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, and the concentration of the at least one factor is 190 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, and the concentration of the at least one factor is 190 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, and the concentration of the at least one factor is 190 ng / mL to 200 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, and the concentration of the at least one factor is 200 ng / mL to 400 ng / mL.In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 200 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 210 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelets. It contains at least one factor selected from the group consisting of Factor 4 and Serpin F1, with a concentration of 210 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition contains at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1, with a concentration of 220 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition contains at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1, with a concentration of 220 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition contains at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1, with a concentration of 230 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition contains at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1, with a concentration of 230 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition contains at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1, with a concentration of 240 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition contains at least one factor selected from the group consisting of Serpin E1, Serpin A1, TIMP-1, Thrombospondin-1, Pentraxin-3 (TSG-14), Platelet Factor 4, and Serpin F1, with a concentration of 240 ng / mL to 300 ng / mL.In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 250 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 250 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 260 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 260 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 270 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 270 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 280 ng / mL to 400 ng / mL.In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 280 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 290 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 290 ng / mL to 300 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 310 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 320 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 330 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, in an amount of 340 ng / mL to 400 ng / mL.In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 350 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 360 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 370 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 380 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 390 ng / mL to 400 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 10 ng / mL to 90 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 10 ng / mL to 80 ng / mL.In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 20 ng / mL to 100 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 30 ng / mL to 100 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 40 ng / mL to 100 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 50 ng / mL to 100 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 10 ng / mL to 70 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 10 ng / mL to 60 ng / mL. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a concentration of 10 ng / mL to 50 ng / mL.

[0227] In some embodiments, the MSC secretome composition comprises i. HGF at 0.3 to 4.5 ng / mL, and ii. pentraxin-3 (TSG-14) at 0.5 to 20 ng / mL, and iii. VEGF at 100 to 600 pg / mL, and iv. TIMP-1 at 10 to 200 ng / mL, and v. serpin E1 at 20 to 80 ng / mL, and vi. IL-8 at <5 ng / mL. It comprises.

[0228] In some embodiments, the MSC secretome composition comprises i. HGF at 1.5 to 3.5 ng / mL, and ii. pentraxin-3 (TSG-14) at 5 to 15 ng / mL, and iii. VEGF at 200 to 400 pg / mL, and iv. TIMP-1 at 50 to 120 ng / mL, and v. serpin E1 at 30 to 70 ng / mL, and vi. IL-8 at <3 ng / mL. It comprises.

[0229] In some embodiments, the MSC secretome composition comprises i. HGF at 1.5 to 2.5 ng / mL, and ii. pentraxin-3 (TSG-14) at 8 to 12 ng / mL, and iii. VEGF at 250 to 350 pg / mL, and iv. TIMP-1 at 70 to 110 ng / mL, and v. serpin E1 at 30 to 70 ng / mL, and vi. IL-8 at <2 ng / mL. It comprises.

[0230] In some embodiments, the MSC secretome composition comprises i. HGF at 2.0 + / - 0.3 ng / mL, and ii. pentraxin-3 (TSG-14) at 9.8 + / - 0.5 ng / mL, and iii. VEGF of 304 + / - 44 pg / mL, and iv. TIMP-1 of 90 + / - 20 ng / mL, and v. Serpin E1 of 49.2 + / - 10 ng / mL, and vi. IL-8 of < 1 ng / mL, and contain.

[0231] In some embodiments, the MSC secretome composition is formulated at a pH of about pH 4.5 to about pH 8. In some embodiments, the MSC secretome composition is formulated at a pH of about pH 4.7 to about pH 7.8. In some embodiments, the MSC secretome composition is formulated at a pH of about 5.0 to about 7.5. In some embodiments, the MSC secretome composition is formulated at a pH of about 5.5 to about 7.5. In some embodiments, the MSC secretome composition is formulated at a pH of about 6 to about 7.5.

[0232] In some embodiments, the MSC secretome composition is formulated at a pH of about pH 4.5, about pH 5.0, about pH 5.5, about pH 6.0, about pH 6.5, about pH 7.0, about pH 7.4, about pH 8.0. In some embodiments, the MSC secretome composition is formulated at a pH of about pH 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.

[0233] In some embodiments, the MSC secretome composition does not contain specific components. In some embodiments, the MSC secretome composition does not contain specific components detected in the cell culture medium. In some embodiments, the MSC secretome composition does not contain one or more components selected from the group consisting of foreign biological components (e.g., animal serum), phenol red, peptides and biomolecules <3 kDa, antibiotics, protein aggregates (e.g., protein aggregates >200 nm), cells, cell debris (cell debris does not include exosomes / extracellular vesicles (EVs). For example, non-exosomal, non-EV cell debris), hormones (e.g., hormones include but are not limited to insulin and / or hydrocortisone), and / or L-glutamine. In some embodiments, the MSC secretome composition does not contain foreign biological components. In some embodiments, the MSC secretome composition does not contain phenol red. In some embodiments, the MSC secretome composition does not contain peptides and biomolecules <3 kDa. In some embodiments, the MSC secretome composition does not contain antibiotics. In some embodiments, the MSC secretome composition does not contain protein aggregates (e.g., protein aggregates >200 nm). In some embodiments, the MSC secretome composition does not contain cells. In some embodiments, the MSC secretome composition does not contain cell debris (cell debris does not include exosomes / Ev, e.g., non-exosomal, non-EV cell debris). In some embodiments, the MSC secretome composition does not contain hormones (e.g., hormones include but are not limited to insulin and / or hydrocortisone). In some embodiments, the MSC secretome composition does not contain L-glutamine.

[0234] In some embodiments, the MSC secretome further comprises mannitol, lactose, sorbitol, xylitol, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, dextrose, and / or combinations thereof. In some embodiments, the MSC secretome comprises a phosphate. In some embodiments, the phosphate source is sodium phosphate or potassium phosphate. In some embodiments, the phosphate source is sodium phosphate. In some embodiments, the phosphate source is potassium phosphate. In some embodiments, the MSC secretome further comprises monosodium / disodium phosphate, mannitol, and trehalose, and the composition has a pH of about 7.4.

[0235] In some embodiments, the MSC secretome composition can include one or more additional agents including, but not limited to, glycine, glycerol, sodium chloride, potassium chloride, and / or dextrose. In some embodiments, the MSC secretome composition can include one or more additional agents selected from the group consisting of glycine, glycerol, sodium chloride, potassium chloride, and dextrose. In some embodiments, the MSC secretome composition can include one or more additional agents selected from the group consisting of glycine, glycerol, and dextrose. In some embodiments, the MSC secretome composition can include one or more additional agents selected from the group consisting of sodium chloride and potassium chloride.

[0236] In some embodiments, the MSC secretome composition is formulated in a buffer system. In some embodiments, the MSC secretome composition is formulated in a buffer system including, but not limited to, disodium phosphate / monosodium phosphate, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and / or citric acid / disodium phosphate. In some embodiments, the MSC secretome composition is formulated in a buffer system selected from the group consisting of disodium phosphate / monosodium phosphate, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and / or citric acid / disodium phosphate. In some embodiments, the MSC secretome composition is formulated in a disodium phosphate buffer system. In some embodiments, the MSC secretome composition is formulated in a sodium citrate / citric acid buffer system. In some embodiments, the MSC secretome composition is formulated in a boric acid / sodium citrate buffer system. In some embodiments, the MSC secretome composition is formulated in a boric acid / sodium tetraborate buffer system. In some embodiments, the MSC secretome composition is formulated in a citric acid / disodium phosphate buffer system.

[0237] In some embodiments, the phosphate source is sodium phosphate or potassium phosphate. In some embodiments, the phosphate source is sodium phosphate. In some embodiments, the phosphate source is potassium phosphate. In some embodiments, the MSC secretome composition includes disodium phosphate / citric acid, mannitol, and trehalose, and the composition has a pH of about 6.4.

[0238] In some embodiments, the MSC secretome composition further comprises an osmotic pressure regulator or an osmotic pressure modifier. In some embodiments, the osmotic pressure regulator or osmotic pressure modifier includes, but is not limited to, NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and / or glycerin. In some embodiments, the osmotic pressure regulator or osmotic pressure modifier is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and / or glycerin.

[0239] In some embodiments, the MSC secretome composition further comprises an adhesive. In some embodiments, the MSC secretome composition further comprises an adhesive including, but not limited to, hypromellose, poloxamer 407, poloxamer 188, poloxamer 237, poloxamer 338, hypromellose (HPMC), HEC, polycarbophil, polyvinylpyrrolidone (PVP), PVA (polyvinyl alcohol), polyimide, sodium hyaluronate, gellan gum, poly(lactic-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethylcellulose (CMC), or hydroxypropylmethylcellulose (HPMC), hydroxymethylcellulose, hydroxyethylcellulose, sodium carboxymethylcellulose, fibrin glue, polyethylene glycol, and GelCORE. In some embodiments, the adhesive is hypromellose. In some embodiments, the adhesive is fibrin glue. In some embodiments, the adhesive is polyethylene glycol. In some embodiments, the adhesive is GelCORE (see Sani, et al., Science Advances, Vol. 5, no. 3 (2019)).

[0240] In some embodiments, the MSC secretome composition comprises (a) a processed conditioned medium comprising an MSC secretome produced by any one of the methods described herein, and (b) a polymer. In some embodiments, the MSC secretome composition comprises a processed conditioned medium comprising an MSC secretome produced as described herein, and a polymer. In some embodiments, the MSC secretome composition comprises a conditioned medium comprising an MSC secretome produced as described herein, and a polymer. In some embodiments, the polymer may be a biodegradable polymer capable of releasing the MSC secretome and / or the processed MSC secretome composition. In some embodiments, the polymer enables sustained release (controlled release) of the MSC secretome components.

[0241] In some embodiments, the MSC secretome composition provided herein is in the form of a therapeutic dressing (e.g., a polymer impregnated with the MSC secretome composition). The therapeutic dressing can be configured as needed depending on the application. In some embodiments, the dressing is in the form of a patch or configured as a mesh.

[0242] In some embodiments, the MSC secretome composition exhibits bio-penetrance, such as, for example, intraocular penetration, corneal penetration, and / or corneal permeability. In some embodiments, the MSC secretome composition exhibits the ability to be absorbed by the eye. In some embodiments, the MSC secretome composition exhibits inherent bio-penetrance. In some embodiments, the MSC secretome composition exhibits bio-penetrance enabled by excipients. In some embodiments, the MSC secretome composition exhibits bio-penetrance due to upregulation of smaller factors. In some embodiments, the MSC secretome composition exhibits bio-penetrance due to the presence of a biopreservative. In some embodiments, the MSC secretome composition exhibits bio-penetrance due to the presence of the biopreservative benzalkonium chloride.

[0243] In some embodiments, the MSC secretome composition exhibits a long half-life and / or has increased stability compared to other therapies. In some embodiments, the MSC secretome composition provided herein enables upregulation of proteins that allow for increased stability of the MSC secretome. In some embodiments, the MSC secretome composition provided herein enables upregulation of chaperone proteins to improve the stability of other proteins in the MSC secretome.

[0244] In some embodiments, the MSC secretome composition exhibits superpotency when administered to a subject in need thereof. In some embodiments, the MSC secretome composition enables a therapeutic effect with one drop or one administration per day.

[0245] Generation / Manufacturing Method According to the present invention, the conditioned medium (and thus the mesenchymal stem cell secretome) can be obtained from mesenchymal stem cells obtained from a patient or an individual to be treated (the patient in need thereof), or from another (donor) individual such as a young donor and / or a healthy donor, and / or from commercially obtained mesenchymal stem cells. For example, MSCs obtained from an individual to be treated (autologous stem cells) or from a donor (allogeneic stem cells) can be used to generate the conditioned medium described herein, which can then be further processed into the MSC secretome composition as described herein. In some embodiments, MSCs can also be obtained from private suppliers. In some embodiments, commercially obtained MSCs can be used for the production of the MSC secretome.

[0246] According to the present invention, a method for producing an anti-angiogenic mesenchymal stem cell (MSC) secretome composition is i. culturing mesenchymal stem cells (MSCs) in a first medium; ii. removing the first medium of step (i) from the MSCs; iii. washing the MSCs of step (ii); iv. Add the second medium and culture for about 1 to 5 days, and v. Collect the second medium from step (iv) as the acclimation medium, and vi. Process the acclimation medium of step (v) into an MSC secretome composition as described herein, and include.

[0247] In some embodiments, culturing can be performed using a bioreactor system for culturing cells. In some embodiments, culturing can be performed using a bioreactor system for culturing stem cells. In some embodiments, culturing can be performed using a bioreactor system for culturing mesenchymal stem cells. In some embodiments, culturing can be performed using a medium mixing technique. In some embodiments, culturing can be performed using a PBS Vertical Wheel (trademark) mixing technique.

[0248] In some embodiments, in step (iv), processing the acclimation medium of step (v) into a secretome composition a) Filter the collected acclimation medium of step (v) to remove cell microparticles, and b) Concentrate the filtered acclimation medium of step (a), and c) Perform buffer exchange with a formulation buffer, and include.

[0249] In some embodiments, step c) includes performing buffer exchange with a buffer system selected from the group consisting of disodium phosphate / sodium citrate, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and citric acid / disodium phosphate.

[0250] In some embodiments, step (a) of filtering comprises the use of a 0.45 μm filter, a 0.22 μm filter, an 0.8 μm filter, and a 0.65 micron, low protein binding PVDF membrane, and / or PES (polyethersulfone). In some embodiments, step (a) of filtering comprises the use of a 0.45 μm filter. In some embodiments, step (a) of filtering comprises the use of a 0.22 μm filter. In some embodiments, step (a) of filtering comprises the use of an 0.8 μm filter. In some embodiments, step (a) of filtering comprises the use of a 0.65 micron. In some embodiments, step (a) of filtering comprises the use of a low protein binding PVDF membrane. In some embodiments, step (a) of filtering comprises the use of PES (polyethersulfone).

[0251] In some embodiments, step (b) of concentrating comprises using a hollow fiber filter, a tangential flow filtration system, or a centrifugation-based size exclusion technique. In some embodiments, step (b) of concentrating comprises using a hollow fiber filter technique. In some embodiments, step (b) of concentrating comprises using a tangential flow filtration system. In some embodiments, step (b) of concentrating comprises using a centrifugation-based size exclusion technique.

[0252] In some embodiments, the centrifugation-based size exclusion technique uses an MW cut-off of 3 to 10 kDa. In some embodiments, the centrifugation-based size exclusion technique uses an MW cut-off of at least 3 kDa, at least 4 kDa, at least 5 kDa, at least 6 kDa, at least 7 kDa, at least 8 kDa, at least 9 kDa, at least 10 kDa, at least 11 kDa, at least 12 kDa, at least 13 kDa, at least 14 kDa, at least 15 kDa, at least 16 kDa, at least 17 kDa, at least 18 kDa, at least 19 kDa, at least 20 kDa, at least 21 kDa, at least 22 kDa, at least 23 kDa, at least 24 kDa, at least 25 kDa, at least 26 kDa, at least 27 kDa, at least 28 kDa, at least 29 kDa, and / or at least 30 kDa.

[0253] In some embodiments, the method manufactures an MSC secretome composition and / or formulation as described above herein. In some embodiments, the first medium and / or the second medium is an MSC medium and / or MSC-XF.

[0254] MSCs, i.e., cells differentiated from MSCs, can be generated, for example, to produce a conditioned medium containing a desired secretome, such as a desired cytokine and / or desired therapeutic properties as described herein. For example, the secretome can be produced from MSCs of a super donor cell line. The secretome can also be produced from commercially available MSCs. In some embodiments, allogeneic MSCs (and / or cells derived therefrom) and / or allogeneic MSC-derived secretome compositions can be prepared and stored for large populations. Allogeneic MSCs (and / or cells derived therefrom) and / or MSC-derived secretome compositions can be prepared in advance so that they are ready when people need them. In certain embodiments, MSCs (and / or cells derived therefrom) and / or MSC-derived secretome compositions can be processed to produce a more concentrated solution or composition (e.g., a mesenchymal stem cell-derived secretome composition as described herein, or an MSC secretome composition).

[0255] In some embodiments, the initial medium and the first medium are different. In some embodiments, the initial medium and the first cell culture medium are the same. Non-limiting examples of one or more cell culture media useful for culturing MSCs to produce a conditioned medium containing the MSC secretome according to the present invention include hMSC Media Booster XFM, hMSC High Performance Basal Media, Minimum Essential Medium Eagle (MEME), ADC-1, LPM (without bovine serum albumin), F10 (HAM), F12 (HAM), DCCM1, DCCM2, RPMI 1640, BGJ Medium (with and without Fitton-Jackson modification), StemPro, MSCGro, MesenCult, NutriStem, Basal Medium Eagle (with addition of Earles' salt base), Dulbecco’s Modified Eagle Medium (DMEM - with or without serum), Yamane, IMM-20, Glasgow Modification Eagle Medium (GMEM), Leibovitz L-15 Medium, McCoy’s 5A Medium, Medium M199 (with M199E - Earles' salt base), Medium M199 (with M199H - Hank's salt base), Minimum Essential Medium Alpha (MEM-alpha), Minimum Essential Medium Eagle (with MEM-E - Earles' salt base), Minimum Essential Medium Eagle (with MEM-H - Hank's salt base), and Medium 199, CMRL1415, CMRL1969, CMRL1066, NCTC135, MB75261, MAB8713, DM145, Williams’G, Neuman&Tytell, Higuchi, MCDB301, MCDB202, MCDB501, MCDB401, MCDB411, MDBC153, and among many other amino acids, especially including Minimum Essential Medium Eagle (with MEM-NAA non-essential amino acids). A preferred medium for use in the present invention is MEM-alpha.These media and other useful media are available, among other sources, from GIBCO, Grand Island, NY, USA, and Biological Industries, Bet HaEmek, Israel. Some of these media are summarized in Methods in Enzymology, Volume LVIII, "Cell Culture", pp. 62 - 72, edited by William B. Jakoby and Ira H. Pastan and published by Academic Press, Inc.

[0256] In some embodiments, the cell culture medium for mesenchymal stem cells may be a serum - free medium. In some embodiments, the cell culture medium for mesenchymal stem cells can be supplemented with serum. In some embodiments, the cell culture medium for mesenchymal stem cells can be supplemented with human platelet lysate. In some embodiments, the serum can include fetal bovine serum (FBS). In some embodiments, the cell culture medium for mesenchymal stem cells can be supplemented with serum such as fetal serum from cows or other species. In some embodiments, the cell culture medium for mesenchymal stem cells can be supplemented with other components such as mercaptoethanol and / or antibiotics to promote cell growth and / or to promote the health of the cells. In some embodiments, the cell culture medium for mesenchymal stem cells is not supplemented with antibiotics.

[0257] In some embodiments, the oxygen percentage is varied to promote cell growth and / or to promote the health of the cells. In some embodiments, the oxygen is at a volume of 5%, 10%, 15%, 20%, or 25% to promote cell growth and / or to promote the health of the cells. In some embodiments, mesenchymal stem cells are grown under an oxygen partial pressure to promote cell growth and / or to promote the health of the cells. In some embodiments, mesenchymal stem cells are grown in a hypoxic environment to promote cell growth and / or to promote the health of the cells.

[0258] In one aspect, the present invention is directed to a conditioned medium (CM) containing biological factors secreted by mesenchymal stem cells, which may be referred to as a conditioned medium containing the MSC secretome. The conditioned medium, as described herein, is obtained by culturing mesenchymal stem cells in a medium and separating the resulting medium (including) into component parts of the conditioned medium containing the secretome and mesenchymal stem cells grown in the conditioned medium, which contains mesenchymal stem cells and their secreted mesenchymal stem cell products (referred to as biological factors and / or secretome). Once separated, the conditioned medium contains the mesenchymal stem cell secretome and can be further processed and / or used according to the methods described herein, and may contain substantially no mesenchymal stem cells (may contain a small number of stem cells and / or trace amounts of stem cells) or no mesenchymal stem cells. The MSC secretome contains various biological factors including hormones, cytokines, extracellular matrix, proteins, vesicles, antibodies, chemokines, receptors, inhibitors, and granules. As described herein, the conditioned medium (s) containing the MSC secretome (CM containing the MSC secretome, i.e., the conditioned medium) can be further processed to produce a concentrated conditioned medium (pCM or concentrated MSC secretome).

[0259] In some embodiments, the conditioned medium containing the MSC secretome or the concentrated MSC secretome is produced by culturing mesenchymal stem cells in a medium and replacing the medium in which the mesenchymal stem cells were cultured. In some embodiments, the resulting conditioned medium containing the MSC secretome is collected and then processed to produce the concentrated MSC secretome. In certain embodiments, the processing of the collected conditioned medium containing the MSC secretome includes the removal of some, most, or essentially all of the medium, or some, most, or essentially all of the selected components of the conditioned medium.

[0260] In some embodiments, the harvested conditioned medium containing the MSC secretome is filtered to produce a concentrated MSC secretome. In some embodiments, the harvested conditioned medium containing the MSC secretome is ultrafiltered to produce a concentrated MSC secretome.

[0261] In one aspect, provided herein is a method for generating a processed conditioned medium, comprising: (a) culturing stem cells in a cell culture medium to thereby generate a conditioned medium containing factors secreted by mesenchymal stem cells (e.g., a conditioned medium containing a mesenchymal stem cell secretome); (b) harvesting the conditioned medium to thereby generate a harvested conditioned medium (e.g., a harvested mesenchymal stem cell secretome); and (c) filtering the harvested conditioned medium (e.g., the harvested mesenchymal stem cell secretome) to generate a processed conditioned medium (mesenchymal stem cell secretome). In some embodiments, the stem cells in (a) are cultured (grown) in a growth medium before being cultured in a growth factor-free medium. Thus, in some embodiments, the method comprises: (a) culturing mesenchymal stem cells in a first growth medium; (b) replacing the first growth medium with a second growth medium and culturing the stem cells in the second growth medium to thereby generate a conditioned medium containing a mesenchymal stem cell secretome; (c) harvesting the conditioned medium containing the mesenchymal stem cell secretome to thereby generate a harvested conditioned medium containing the mesenchymal stem cell secretome; and (d) filtering the harvested conditioned medium to generate a processed conditioned medium containing the mesenchymal stem cell secretome.

[0262] In some embodiments, the MSC secretome of the present invention is further processed. In some embodiments, the MSC secretome of the present invention is subjected to extraction, freeze-thaw, homogenization, filtration, centrifugation, density gradient centrifugation, CsCl gradient centrifugation, iodixanol gradient centrifugation, ultracentrifugation, fractionation, precipitation, SDS-PAGE, native PAGE, size exclusion chromatography, liquid chromatography, gas chromatography, hydrophobic interaction chromatography, ion exchange chromatography, anion exchange chromatography, cation exchange chromatography, affinity chromatography, heparin sulfate affinity chromatography, sialic acid affinity chromatography, immunoaffinity chromatography, metal binding chromatography, nickel column chromatography, epitope tag purification, or lyophilization, or any combination thereof, and is further processed using techniques known in the art, but not limited thereto.

[0263] In some embodiments, the MSC secretome of the present invention is concentrated by one or more of the following methods, namely, affinity-based concentration, size-based concentration, cation or anion-based concentration, and concentration by one or more of the fractions for concentration of favorable properties.

[0264] In some embodiments, the stem cells are mesenchymal stem cells. Mesenchymal stem cells (MSCs) are multipotent (able to differentiate into multiple cell lineages, although not all) non-hematopoietic (non-blood) stem cells isolated (derived) from various adult tissues including bone marrow and adipose tissue. In certain embodiments, the mesenchymal stem cells are isolated from bone marrow. "Isolated" refers to cells removed from their original environment. MSCs can differentiate into mesodermal cells such as adipocytes, osteoblasts, and chondrocytes. MSCs have a small cell body with few elongated cell processes. The cell body contains a large round nucleus with prominent nucleoli surrounded by finely dispersed chromatin particles, giving the nucleus a distinct appearance. The rest of the cell body contains a small amount of Golgi apparatus, rough endoplasmic reticulum, mitochondria, and polyribosomes. The elongated cells are widely dispersed, and there are a few reticular fibers in the adjacent extracellular matrix, but other types of collagen fibers are absent [Brighton, et al. 1991 The Journal of Bone and Joint Surgery 73(6):832-47]. The MSCs described herein can express the following molecular marker (protein molecule characteristic of the plasma membrane of a cell or cell type) profile, namely, bone morphogenetic protein receptor "1" (BMPR+), CD34+Scal+Lin", CD44+, c-kit+, Sca-1+, Thy-1+, NOTCH3, JAG1, ITGA11. MSCs can also express other cell type-specific markers (see World Wide Web (stemcells.nih.gov, Kaltz, et al. 2010 Exp Cell Res Oct 1, 316(16):2609-17, which is incorporated herein by reference). The MSCs described herein can be identified based on a colony-forming unit assay to detect the potential for universal differentiation of MSCs (what cell types MSCs give rise to). However, somewhat differentiated cells (progenitor cells) can also be used.

[0265] MSC Secretome - Preconditioning In some embodiments, in vitro preconditioning of secretome-producing MSCs can be used to enhance the therapeutic capacity / therapeutic potential of MSCs. In some embodiments, this enhancement occurs by affecting the secretion profile of MSCs. In some embodiments, such preconditioning can include, but is not limited to, changing the culture format (e.g., 2D flat vs. 3D bioreactor), different biomaterial scaffolds, co-culture, addition of pharmacological compounds, growth factors, chemokines, Toll-like receptor agonists, inflammatory cytokines, advanced glycation end products (AGEs), oxidized phospholipids, malondialdehyde, or carboxyethylpyrrole, agitation in the presence of ECM, culture under shear stress, agitation or suspension as aggregates or within a matrix, induced misfolded protein response, ER stress, induction of MSC differentiation, culture in the presence of conditioned media, and hypoxia / anoxia. See Ferreira et al., 2018 Frontiers in Immunol Vol.9, Art.2387, which is incorporated herein by reference.

[0266] The physiological oxygen pressure within tissues varies from 1% in cartilage and bone marrow to 12% in peripheral blood. Thus, the 21% oxygen level routinely used in cell culture incubators is much higher than the physiological state. In some embodiments, hypoxic preconditioning of MSCs can improve their regenerative and cytoprotective effects and / or proliferation rate, and generally increase the levels of cytoprotective molecules and exosome secretion. In some embodiments, the ability of MSCs to switch from aerobic to anaerobic metabolism in vivo can enable cell adaptation. In some embodiments, MSCs can adapt to very low oxygen pressures in vitro. In some embodiments, the hypoxic values can range from anoxia (0% O2) to 2% O2. In some embodiments, the hypoxic values can range from anoxia (0% O2) to 2% O2 for between 4 hours and 72 hours, including 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 60 hours, or 72 hours. In some embodiments, hypoxic preconditioning can result in MSCs that express factors associated with angiogenesis. In some embodiments, hypoxic preconditioning can result in MSCs that express higher levels of HIF-1α, GDNF, BDNF, VEGF, Ang-1, SDF-1 and its receptor CXCR4, and EPO and its receptor EPOR. In some embodiments, hypoxic preconditioning can result in MSCs that express higher levels of neuroprotective factors and angiogenesis-promoting factors.

[0267] In some embodiments, MSCs are stimulated with inflammatory cytokines. In some embodiments, MSCs are stimulated with inflammatory cytokines that promote the production of a secretome that includes factors involved in the regulation of the immune response. In some embodiments, MSCs are stimulated with inflammatory cytokines that can give rise to a secretome that includes factors that promote chemoattraction of immune cells, regulation of inflammation, and / or enhancement of migration / homing of other cells. Other immunomodulatory capabilities of the MSC secretome can include inhibition of NK cells, inhibition of complement system activation, monocyte differentiation into M2 macrophages, suppression of cytotoxic T cell proliferation, and increase in the number of regulatory T cells. In some embodiments, effector cytokines in these processes can include, but are not limited to, IL-6, PGE2, and IDO. In some embodiments, pretreatment with IFNγ gives rise to an MSC secretome with enhanced immunosuppressive capacity. In some embodiments, pretreatment with IFNγ gives rise to an MSC secretome with enhanced immunosuppressive capacity through IDO. In some embodiments, SDF-1 preconditioning can be used as part of the preconditioning process. In some embodiments, preconditioning with SDF-1 provides an MSC secretome that results in increased angiogenesis and decreased fibrosis. In some embodiments, TGF-α preconditioned MSCs provide a secretome with increased VEGF production. In some embodiments, melatonin preconditioned MSCs provide an MSC secretome that results in improved cell survival under oxidative stress, unfolded protein response, or ER stress. In some embodiments, H2O2 preconditioned MSCs give rise to an MSC secretome that results in increased angiogenesis, higher survival rate, and decreased inflammation in a rat model of ischemia / reperfusion injury. MSCs can also be preconditioned with a cocktail of growth factors, namely, FGF-2, IGF-1, and BMP-2, to produce an MSC secretome having these activities.

[0268] In some embodiments, the preconditioning method is mechanical in nature and involves mimicking the MSC microenvironment via a 3D culture method. In some embodiments, a 3D cell culture vessel / bioreactor is used. In some embodiments, a 3D culture of a prolate ellipsoid is used, enabling mimicking of the physiological state within the bone marrow. In some embodiments, the prolate ellipsoid culture creates a microenvironment where the inner layer is exposed to much lower levels of oxygen and nutrients, creating a hypoxic environment. In such embodiments, MSCs express higher levels of TSG-6, SCT-1, LIF, IL-24, TRAIL, and CXCR4, thus enabling the production of an MSC secretome with higher concentrations of these factors. In such embodiments, MSC prolate ellipsoid culture shows increased survival, proliferation, and angiogenesis. In such embodiments, MSCs produce an MSC secretome with enhanced immunomodulatory, angiogenic, antifibrotic, and anti-apoptotic activities.

[0269] A variety of drugs can also be used to precondition MSCs. In some embodiments, MSCs can be preconditioned with atorvastatin, oxytocin, curcumin, lipopolysaccharide, nicotinamide (NIC), vasoactive intestinal peptide (VIP), and / or diazoxide. In some embodiments, it is possible to perform preconditioning of MSCs with nicotinamide (NIC) and / or vasoactive intestinal peptide (VIP) to protect against oxidative stress. In some embodiments, pharmaceutical compositions based on the MSC secretome from MSCs preconditioned with the neuroprotective drugs VIP, NIC, or both enhance the proliferative and neuroprotective effects of the secretome. See Alonso-Alonso et al., (2020) Stem Cell Int Vol.2020, Article ID9463548, which is incorporated herein by reference.

[0270] In some embodiments, preconditioning the MSC secretome includes inducing a misfolded protein response in the MSCs.

[0271] In some embodiments, preconditioning the MSC secretome includes culturing the MSCs under conditions appropriate to induce ER stress.

[0272] In some embodiments, preconditioning the MSC secretome includes culturing the MSCs under conditions appropriate to induce differentiation of the MSCs into mature retinal cell types or their precursors.

[0273] In some embodiments, retinal progenitor cells include, but are not limited to, photoreceptor progenitor cells, bipolar progenitor cells, retinal progenitor cells, horizontal progenitor cells, amacrine progenitor cells, retinal ganglion progenitor cells, Müller glia progenitor cells, and retinal pigment epithelial progenitor cells.

[0274] The present invention relates to i. an IDO (indoleamine-2,3-dioxygenase) enzyme activity of less than about 250 μM, and ii. at least one trophic factor / cytokine selected from the group consisting of HGF, FGF-7, TIMP-1, TIMP-2, PAI-1 (serpin E1), VEGF-A, and / or b-NGF, and iii. at least one additional factor selected from the group consisting of sFLT-1, PEDF (serpin F1), serpin A1, IGFBP-2, IGFBP-3, SDF-1, TSG-14, kallikrein 3, MCP-1, bFGF, angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, PDGF, SOD1, SOD2, SOD3, and / or HO-1, and iv. at least one additional factor selected from the group consisting of DPPIV (dipeptidyl peptidase-4), uPA, angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, and / or thrombospondin-1, to provide a preconditioned mesenchymal stem cell (MSC) secretome composition comprising

[0275] In some embodiments, the preconditioned MSC secretome composition further comprises at least one factor at a "higher" level selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and / or serpin F1, optionally at 1 ng / mL to 8 ng / mL.

[0276] In some embodiments, the preconditioned MSC secretome further comprises at least one factor at a "mid-range" level selected from the group consisting of angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2 and thrombospondin-1, angiogenin, DPPIV, IGFBP-3, and / or uPA, optionally at 400 pg / mL to 3000 pg / mL.

[0277] In some embodiments, the preconditioned MSC secretome further comprises at least one factor selected from the group consisting of apolipoprotein A1, complement factor D, complement factor H, complement factor I, C1 esterase inhibitor (C1-INH), C4b-binding protein (C4BP), CD46, complement receptor type 1 (CR1), C-reactive protein, cystatin C, DKK-1, enppurin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15, and / or IFNγ.

[0278] In some embodiments, the preconditioned MSC secretome comprises an anti-angiogenic to pro-angiogenic ratio, and the ratio is >2, >3, >4, or >5.

[0279] In some embodiments, the preconditioned MSC secretome further comprises a "low" level of VEGF, optionally from 0 pg / mL to 200 pg / mL.

[0280] In some embodiments, the level of VEGF is 1 / 5 to 1 / 10 of the level of serpin E1.

[0281] In some embodiments, the composition comprises one or more anti-angiogenic factors, and the total concentration of the one or more anti-angiogenic factors relative to the concentration of VEGF is >2, >3, >4, or >5.

[0282] In some embodiments, the preconditioned MSC secretome does not contain and / or contains very low levels of bFGF, PLGF, and PDGF, optionally less than 1000 pg / mL.

[0283] In some embodiments, the preconditioned MSC secretome composition has a pH of about 4.7 to about 7.5.

[0284] In some embodiments, the preconditioned MSC secretome is formulated in a buffer system selected from the group consisting of disodium phosphate / sodium citrate, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and citric acid / disodium phosphate.

[0285] In some embodiments, the preconditioned MSC secretome composition further comprises an osmotic modifier.

[0286] In some embodiments, the osmotic modifier is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin.

[0287] In some embodiments, the preconditioned MSC secretome further comprises monosodium / disodium phosphate, mannitol, and trehalose, and the composition has a pH of about 7.4.

[0288] In some embodiments, the preconditioned MSC secretome further comprises divalent cations.

[0289] In some embodiments, the divalent cations are selected from the group consisting of Mg2+, Ca2+, and Zn2+.

[0290] In some embodiments, the preconditioned MSC secretome further comprises disodium phosphate / citric acid, mannitol, and trehalose, and the composition has a pH of about 6.4.

[0291] In some embodiments, the composition further comprises an adhesive.

[0292] In some embodiments, the adhesive is selected from the group consisting of hypromellose, poloxamer 407, poloxamer 188, poloxomer 237, poloxomer 338, hypromellose (HPMC), polycarbophil, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyimide, sodium hyaluronate, gellan gum, poly(lactic-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethyl cellulose (CMC), or hydroxypropylmethyl cellulose (HPMC), hydroxymethyl cellulose, hydroxyethyl cellulose, sodium carboxymethyl cellulose, fibrin glue, polyethylene glycol, and GelCORE.

[0293] The present invention also i. Culturing mesenchymal stem cells (MSCs) in a first medium; ii. Removing the first medium of step (i) from the MSCs; iii. Washing the MSCs of step (ii); iv. Adding a second medium and culturing for about 1 - 5 days; v. Collecting the second medium from step (iv) as a conditioning medium; vi. Processing the conditioning medium of step (v) into a preconditioned MSC secretome composition as described herein; To provide a method for preparing a preconditioned mesenchymal stem cell (MSC) secretome composition comprising:

[0294] In some embodiments, the preconditioned MSC secretome composition is the secretome composition described herein.

[0295] In some embodiments, processing the conditioning medium of step (v) into a secretome composition in step (iv) comprises: a) Filtering the collected conditioning medium of step (v) to remove cell microparticles; b) Concentrating the filtered conditioning medium of step (a); c) Performing buffer exchange with a formulation buffer; and comprising.

[0296] In some embodiments, step c) comprises performing buffer exchange with a buffer system selected from the group consisting of disodium phosphate / sodium citrate, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and citric acid / disodium phosphate.

[0297] In some embodiments, step (a) of filtering comprises the use of a 0.45 μm filter, a 0.22 μm filter, a 0.8 μm filter, and a 0.65 μm filter, a low protein binding PVDF membrane, and / or PES (polyethersulfone).

[0298] In some embodiments, the concentration step (b) includes using a hollow fiber filter, a tangential flow filtration system, or a centrifugation-based size exclusion technique.

[0299] In some embodiments, the centrifugation-based size exclusion technique uses a MW cut-off of 3 - 10 kDa.

[0300] In some embodiments, the present invention provides a method for treating an eye disease, comprising administering to a patient in need of treatment for an eye disease a therapeutically effective amount of a mesenchymal stem cell secretome composition as described herein, or a composition prepared according to the method described herein.

[0301] In some embodiments, the composition is administered to a target region.

[0302] The present invention also provides a method for treating visual dysfunction following trauma to the eye structure, comprising administering to a patient in need thereof a therapeutically effective amount of a mesenchymal stem cell secretome composition or a composition prepared according to the method described herein.

[0303] The present invention also provides a method for inducing and / or promoting wound healing of the eye, comprising administering to a patient in need thereof a therapeutically effective amount of a mesenchymal stem cell secretome composition or a composition prepared according to the method described herein.

[0304] The present invention also provides methods for reducing and / or inhibiting neovascularization, reducing and / or inhibiting scarring, promoting and / or maintaining visual acuity, and / or increasing the wound suture rate (e.g., reducing the wound suture time), comprising administering to patients in need thereof a therapeutically effective amount of a mesenchymal stem cell secretome composition or a composition prepared according to the method described herein.

[0305] The present invention also provides a method for reducing and / or inhibiting neovascularization and / or reducing scarring to promote visual maintenance, which includes administering to a patient a therapeutically effective amount of a mesenchymal stem cell secretome composition or a composition prepared according to the methods described herein, to patients in need thereof.

[0306] In some embodiments, the mesenchymal stem cell secretome composition is formulated for topical administration.

[0307] In some embodiments, the mesenchymal stem cell secretome composition is formulated for subconjunctival injection.

[0308] In some embodiments, the mesenchymal stem cell secretome composition is formulated for intravitreal injection.

[0309] The present invention also provides a method for characterizing an MSC secretome, the method comprising: (i) subjecting the MSC secretome to one or more characterization assays, wherein the characterization assays are selected from the group consisting of characterization of physical components, oxidative stress assay, misfolded protein response assay, ER stress assay, safety analysis, stability assay, proliferation assay, migration assay, adhesion assay, neovascularization assay, differentiation / scarring assay, inflammation assay, epithelial barrier integrity assay, retinal degeneration assay, and / or assay of hereditary retinal diseases including human and animal retinal explants, neuroprotection / neurotrophic assay, and the MSC secretome is optionally preconditioned; (ii) determining the results from one or more of the assays of (i); and comprising.

[0310] Exemplary disclosures of property evaluation assays are described in Oslowski CM et al., Methods Enzymol. 2011;490:71-92, Wagstaff PE, et al., Int J Mol Sci. 2021 Jun 30;22(13):7081, Bandyopadhyay M, et al., Mol Vis. 2013 May 29;19:1149-57, Murali A, et al., Clin Exp Ophthalmol. 2019 Mar;47(2):274-285, Srinivasan B, et al., J Lab Autom. 2015;20(2):107-26, Slijkerman RW, et al., Prog Retin Eye Res. 2015;48:137-59, ARTO Castro A, et al., Stem Cells. 2019 Dec;37(12):1496-1504, and Weigle S, et al., J Biol Methods. 2019 Jun 3;6(2):e115, each of which is incorporated herein by reference in its entirety.

[0311] The present invention also provides a method for determining the biopotency and stability of the MSC secretome, the method comprising: (i) subjecting the MSC secretome to one or more property evaluation assays, wherein the property evaluation assays are selected from the group consisting of property evaluation of physical components, oxidative stress assay, misfolded protein response assay, ER stress assay, safety analysis, stability assay, proliferation assay, migration assay, adhesion assay, angiogenesis assay, differentiation / scarring assay, inflammation assay, epithelial barrier integrity assay, retinal degeneration assay, and / or assay of hereditary retinal diseases including human and animal retinal explants, neuroprotection / neurotrophic assay, and the MSC secretome is optionally preconditioned; (ii) determining the results from the one or more assays of (i); and comprising.

[0312] The present invention provides a method for determining MSC secretome lot consistency among multiple MSC secretome lots, the method comprising: (i) subjecting the MSC secretome to one or more characterization assays, wherein the characterization assays are selected from the group consisting of characterization of physical components, oxidative stress assay, misfolded protein response assay, ER stress assay, safety analysis, stability assay, proliferation assay, migration assay, adhesion assay, angiogenesis assay, differentiation / scarring assay, inflammation assay, epithelial barrier integrity assay, retinal degeneration assay, and / or assay for hereditary retinal diseases including human and animal retinal explants, neuroprotection / neurotrophic assay, and the MSC secretome is optionally preconditioned; (ii) determining a result from one or more of the assays of (i); and comprising.

[0313] In some embodiments, the result of (ii) of the characterization of physical components identifies an anti-angiogenic MSC secretome, as described herein.

[0314] In some embodiments, the result of (ii) of the safety analysis indicates blood compatibility and provides an MSC secretome that is pyrogen-free and / or endotoxin-free and / or non-pyrogenic and / or endotoxin-free.

[0315] In some embodiments, the result of (ii) of the stability assay provides an MSC secretome that is stable at 4°C, 20°C, and / or 25°C (e.g., room temperature) for at least 7 days.

[0316] In some embodiments, the result of (ii) of the proliferation assay provides an MSC secretome that induces proliferation.

[0317] In some embodiments, the result of (ii) of the migration assay provides an MSC secretome that induces migration.

[0318] In some embodiments, the result of (ii) of the adhesion assay provides an MSC secretome that induces cell adhesion. The adhesion assay can be performed using techniques known in the art. Exemplary disclosures of adhesion assays are shown in U.S. Patent Publication Nos. 20170067061 A1 and 20150050325 A1, and Blue et al., Blood 2008, 111,1248, each of which is incorporated herein by reference in its entirety.

[0319] In some embodiments, the result of (ii) of the angiogenesis assay provides an MSC secretome that inhibits or does not promote angiogenesis.

[0320] In some embodiments, the result of (ii) of the differentiation / scarring assay provides an MSC secretome that inhibits differentiation and / or scarring.

[0321] In some embodiments, the result of (ii) of the inflammation assay provides an MSC secretome that inhibits inflammation or modifies the immune response.

[0322] In some embodiments, the method (iii) identifying an MSC secretome lot based on the result of (ii), further comprises.

[0323] The present invention also provides a panel of tests and / or assays for characterizing an MSC secretome, the panel comprising at least two property evaluation assays, the property evaluation assays being selected from the group consisting of property evaluation of physical components, oxidative stress assay, misfolded protein response assay, ER stress assay, safety analysis, stability assay, proliferation assay, migration assay, adhesion assay, angiogenesis assay, differentiation / scarring assay, and / or inflammation assay.

[0324] The present invention also provides a panel of tests and / or assays for determining consistency between MSC secretome lots, the panel including one or more characterization assays, the characterization assays being selected from the group consisting of characterization of physical components, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analyses, stability assays, proliferation assays, migration assays, adhesion assays, angiogenesis assays, differentiation / scarring assays, and / or inflammation assays.

[0325] In some embodiments, the characterization of physical components identifies the MSC secretome described herein.

[0326] In some embodiments, the result of (ii) of the safety analysis indicates blood compatibility and provides an MSC secretome that shows low pyrogens and / or endotoxins, and / or no pyrogens and / or endotoxins.

[0327] In some embodiments, the stability assay identifies an MSC secretome that is stable at 4°C, 20°C, and / or 25°C (e.g., room temperature) for at least 7 days.

[0328] In some embodiments, the proliferation assay identifies an MSC secretome that induces proliferation.

[0329] In some embodiments, the migration assay identifies an MSC secretome that induces migration.

[0330] In some embodiments, the angiogenesis assay identifies an MSC secretome that inhibits or does not promote angiogenesis.

[0331] In some embodiments, the differentiation / scarring assay identifies an MSC secretome that inhibits differentiation and / or scarring.

[0332] In some embodiments, the inflammation assay identifies an MSC secretome that inhibits inflammation or modifies the immune response.

[0333] In some embodiments, an assessment of the properties of physical components, an oxidative stress assay, a misfolded protein response assay, an ER stress assay, a safety analysis, a stability assay, a proliferation assay, a migration assay, an adhesion assay, an angiogenesis assay, a differentiation / scarring assay, an inflammation assay, an epithelial barrier integrity assay, a retinal degeneration assay, and / or an assay for hereditary retinal diseases including human and animal retinal explants, a neuroprotection / neurotrophic assay are all performed.

[0334] In some embodiments, the panel of tests and / or assays described herein identifies the MSC secretome described herein.

[0335] In some embodiments, the panel of tests and / or assays described herein includes at least one migration assay. In some embodiments, the migration assay is an in vitro wound closure assay. In some embodiments, the in vitro wound closure assay is selected from the group consisting of a "scratch assay" (also referred to as a "scrape assay"), a circular scrape method, and a circular wound closure assay. In some embodiments, the preconditioned MSC secretome is an anti-angiogenic MSC secretome and / or an anti-scarring MSC secretome.

[0336] In some embodiments, the preconditioned MSC secretome is an anti-angiogenic MSC secretome and / or an anti-scarring MSC secretome.

[0337] In some embodiments, the preconditioned MSC secretome is an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.

[0338] The present invention also provides i. 1 to 20 μg, optionally 2 μg to 8 μg of MSC secretome per mL, and ii. 2 to 3 mg of sodium monophosphate per mL, and iii. 11 to 12 mg of disodium phosphate per mL, and iv. 11.5 to 13 mg of mannitol per mL, and v. 23 to 24 mg of trehalose dihydrate, and vi. 0.5 to 2 mg of hypromellose per mL, and comprising a stable preconditioned mesenchymal stem cell (MSC) secretome formulation having a pH of from about 4.7 to about 7.5.

[0339] The present invention also provides i. 0.004% to 0.0375%, optionally 0.008% to 0.015% w / w of MSC secretome, and ii. 4% to 5% w / w of sodium monophosphate, and iii. 21.5% to 23% w / w of disodium phosphate, and iv. 23% to 25% w / w of mannitol, and v. 46% to 48% w / w of trehalose dihydrate, and vi. 1% to 3% w / w of hypromellose, and comprising a stable preconditioned mesenchymal stem cell (MSC) secretome formulation having a pH of from about 4.7 to about 7.5.

[0340] The present invention also provides i. 6 μg of MSC secretome per mL, and ii. 2.28 mg of sodium monophosphate per mL, and iii. 11.45 mg of disodium phosphate per mL, and iv. 12.2 mg of mannitol per mL, and v. 24 mg of trehalose dihydrate per mL, and vi. 1 mg of hypromellose per 1 mL, and comprising a stable preconditioned mesenchymal stem cell (MSC) secretome formulation having a pH of about 7.4 is provided.

[0341] The present invention also provides i. 6 μg of MSC secretome per 1 mL, and ii. 1.31 mg of monobasic sodium phosphate per 1 mL, and iii. 5.73 mg of dibasic sodium phosphate per 1 mL, and iv. 12.2 mg of mannitol per 1 mL, and v. 24 mg of trehalose dihydrate per 1 mL, and vi. 1 mg of hypromellose per 1 mL, and vii. 0.2 mg of magnesium chloride per 1 mL, and viii. 1.17 mg of sodium chloride per 1 mL, and comprising a stable preconditioned mesenchymal stem cell (MSC) secretome formulation having a pH of about 7.4 is provided.

[0342] The present invention also provides i. 0.012% w / w of MSC secretome, and ii. 4.5% w / w of monobasic sodium phosphate, and iii. 22.4% w / w of dibasic sodium phosphate, and iv. 24% w / w of mannitol, and v. 47.1% w / w of trehalose dihydrate, and vi. 2.0% w / w of hypromellose, and comprising a stable preconditioned mesenchymal stem cell (MSC) secretome formulation having a pH of about 7.4 is provided.

[0343] The present invention also provides i. 10 mM of sodium phosphate, and ii. 10 mM of histidine HCL, and iii. 10% trehalose dihydrate, and iv. 0.01% polysorbate 20, and comprising a stable preconditioned mesenchymal stem cell (MSC) secretome formulation having a pH of about 5.5 is provided.

[0344] The present invention also provides i. 10 mM sodium phosphate, and ii. 5% sucrose, and iii. 10 mM sodium chloride, and comprising a stable preconditioned mesenchymal stem cell (MSC) secretome formulation having a pH of about 6.2 is provided.

[0345] The present invention also provides i. 10 mM sodium phosphate, and ii. 5.8% sucrose, and iii. 0.02% polysorbate 80, and comprising a stable preconditioned mesenchymal stem cell (MSC) secretome formulation having a pH of about 6.2 is provided.

[0346] The present invention also provides i. at least one trophic factor / cytokine selected from the group consisting of HGF, TIMP-1, TIMP-2, PAI-1 (serpin E1), VEGF-A, and b-NGF, and ii. at least one additional factor selected from the group consisting of PEDF (serpin F1), serpin A1, IGFBP-2, IGFBP-3, SDF-1, TSG-14, kallikrein 3, MCP-1, angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, and PDGF, and iii. At least one additional factor selected from the group consisting of DPPIV (dipeptidyl peptidase-4), uPA, angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, and thrombospondin-1, to provide a preconditioned mesenchymal stem cell (MSC) secretome composition comprising

[0347] In some embodiments, the preconditioned MSC secretome composition further comprises at least one factor at a high level selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1.

[0348] In some embodiments, the preconditioned MSC secretome composition comprises at least one factor at 1 ng / mL to 400 ng / mL selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1.

[0349] In some embodiments, the preconditioned MSC secretome composition further comprises at least one factor at a medium level selected from the group consisting of angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, thrombospondin-1, angiogenin, DPPIV, IGFBP-3, and uPA.

[0350] In some embodiments, the preconditioned MSC secretome composition comprises at least one factor at 400 pg / mL to 3000 pg / mL selected from the group consisting of angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, thrombospondin-1, angiogenin, DPPIV, IGFBP-3, and uPA.

[0351] In some embodiments, the preconditioned MSC secretome composition further comprises at least one factor selected from the group consisting of apolipoprotein A1, factor D of complement, factor H of complement, factor I of complement, C1 esterase inhibitor (C1-INH), C4b-binding protein (C4BP), CD46, complement receptor type 1 (CR1), C-reactive protein, cystatin C, DKK-1, enppurin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15, and IFNγ.

[0352] In some embodiments, the preconditioned MSC secretome composition comprises an anti-angiogenic to pro-angiogenic ratio, and the ratio is >2, >3, >4, or >5.

[0353] In some embodiments of the preconditioned MSC secretome composition, the anti-angiogenic factors include one or more factors selected from the group consisting of PEDF, low levels of VEGF, and serpin E1, and the pro-angiogenic factors include one or more factors selected from the group consisting of VEGF, angiogenin, IGFBP-3, uPA, Angio-1, Angio-2, and endothelin-1.

[0354] In some embodiments, the preconditioned MSC secretome composition further comprises low levels of VEGF.

[0355] In some embodiments, the preconditioned MSC secretome composition comprises VEGF at 1 pg / mL to 400 pg / mL.

[0356] In some embodiments of the preconditioned MSC secretome composition, the level of VEGF is 1 / 5 to 1 / 10 of the level of serpin E1.

[0357] In some embodiments, the preconditioned MSC secretome composition comprises one or more anti-angiogenic factors, and the sum of the concentrations of the one or more anti-angiogenic factors relative to the concentration of VEGF is >2, >3, >4, or >5.

[0358] In some embodiments, the preconditioned MSC secretome composition does not contain and / or contains very low levels of bFGF, PLGF, and PDGF.

[0359] In some embodiments, the preconditioned MSC secretome composition contains less than 1000 pg / mL of bFGF, PLGF, and PDGF.

[0360] In some embodiments, the preconditioned MSC secretome composition has a pH of about 4.7 to about 7.5.

[0361] In some embodiments, the preconditioned MSC secretome composition is formulated with a buffer system selected from the group consisting of disodium monophosphate / sodium citrate, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and citric acid / disodium phosphate.

[0362] In some embodiments, the preconditioned MSC secretome composition further comprises an osmotic modifier.

[0363] In some embodiments of the preconditioned MSC secretome composition, the osmotic modifier is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin.

[0364] In some embodiments, the preconditioned MSC secretome composition further comprises monosodium / disodium phosphate, mannitol, and trehalose, and the composition has a pH of about pH 7.4.

[0365] In some embodiments, the preconditioned MSC secretome composition further comprises a divalent cation.

[0366] In some embodiments, the divalent cation of the preconditioned MSC secretome composition is selected from the group consisting of Mg2+, Ca2+, and Zn2+.

[0367] In some embodiments, the preconditioned MSC secretome composition further comprises disodium phosphate / citric acid, mannitol, and trehalose, and the composition has a pH of about 6.4.

[0368] In some embodiments, the preconditioned MSC secretome composition further comprises an adhesive.

[0369] In some embodiments of the preconditioned MSC secretome composition, the adhesive is selected from the group consisting of hypromellose, poloxamer 407, poloxamer 188, poloxomer 237, poloxomer 338, hypromellose (HPMC), polycarbophil, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyimide, sodium hyaluronate, gellan gum, poly(lactic-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethylcellulose (CMC), or hydroxypropylmethylcellulose (HPMC), hydroxymethylcellulose, hydroxyethylcellulose, sodium carboxymethylcellulose, fibrin glue, polyethylene glycol, and GelCORE.

[0370] In some embodiments, the preconditioned MSC secretome composition does not contain one or more components selected from the group consisting of foreign body components, phenol red, peptides and biomolecules <3 kDa, antibiotics, protein aggregates> 200 nm, cells, non-exosome / non-extracellular vesicle cell debris, hormones, and L-glutamine.

[0371] In some embodiments, the preconditioned MSC secretome composition comprises HGF, pentraxin-3 (TSG-14), VEGF, TIMP-1, serpin E1, and <5 ng / mL of IL-8.

[0372] In some embodiments, the preconditioned MSC secretome composition i. 0.3 to 4.5 ng / mL of HGF, ii. 0.5 to 20 ng / mL of pentraxin-3 (TSG-14), iii. 100 to 600 pg / mL of VEGF, iv. 10 to 200 ng / mL of TIMP-1, v. 20 to 80 ng / mL of serpin E1, vi. <5 ng / mL of IL-8, and

[0373] In some embodiments, the preconditioned MSC secretome composition comprises an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.

[0374] The present invention also provides i. 2 μg to 20 μg of MSC secretome per mL, ii. 2 mg to 3 mg of monobasic sodium phosphate per mL, iii. 11 mg to 12 mg of dibasic sodium phosphate per mL, iv. 11.5 mg to 13 mg of mannitol per mL, v. 23 mg to 24 mg of trehalose dihydrate, vi. 0.5 mg to 2 mg of hypromellose per mL, and provides a stable preconditioned mesenchymal stem cell (MSC) secretome formulation having a pH of from about 4.7 to about 7.5.

[0375] The present invention also provides i. 0.004% to 0.08% w / w of MSC secretome, and ii. 4% to 5% w / w of monobasic sodium phosphate, and iii. 21.5% to 23% w / w of dibasic sodium phosphate, and iv. 23% to 25% w / w of mannitol, and v. 46% to 48% w / w of trehalose dihydrate, and vi. 1% to 3% w / w of hypromellose, and comprising, to provide a stable preconditioned mesenchymal stem cell (MSC) secretome formulation having a pH of about 4.7 to about 7.5.

[0376] The present invention further provides a method for treating an eye disease in a subject in need thereof, comprising administering to the subject a preconditioned mesenchymal stem cell (MSC) secretome composition, wherein the preconditioned MSC secretome composition comprises i. at least one trophic factor / cytokine selected from the group consisting of HGF, TIMP-1, TIMP-2, PAI-1 (serpin E1), VEGF-A, and b-NGF, and ii. at least one additional factor selected from the group consisting of PEDF (serpin F1), serpin A1, IGFBP-2, IGFBP-3, SDF-1, TSG-14, kallikrein 3, MCP-1, angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, and PDGF, and iii. at least one further factor selected from the group consisting of DPPIV (dipeptidyl peptidase-4), uPA, angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, and thrombospondin-1. comprising.

[0377] In some embodiments, the preconditioned MSC secretome composition further comprises a high level of at least one factor selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1.

[0378] In some embodiments, the preconditioned MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1, at a level of 1 ng / mL to 100 ng / mL.

[0379] In some embodiments, the preconditioned MSC secretome composition further comprises at least one factor selected from the group consisting of angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, thrombospondin-1, angiogenin, DPPIV, IGFBP-3, and uPA, at a medium level.

[0380] In some embodiments, the preconditioned MSC secretome composition comprises at least one factor selected from the group consisting of angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, thrombospondin-1, angiogenin, DPPIV, IGFBP-3, and uPA, at a level of 400 pg / mL to 3000 pg / mL.

[0381] In some embodiments, the preconditioned MSC secretome composition further comprises at least one factor selected from the group consisting of apolipoprotein A1, factor D of complement, factor H of complement, factor I of complement, C1 esterase inhibitor (C1-INH), C4b-binding protein (C4BP), CD46, complement receptor type 1 (CR1), C-reactive protein, cystatin C, DKK-1, enppurin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15, and IFNγ.

[0382] In some embodiments, the preconditioned MSC secretome composition comprises an anti-angiogenic to pro-angiogenic ratio, the ratio being >2, >3, >4, or >5.

[0383] In some embodiments, the anti-angiogenic factors include PEDF, low levels of VEGF, and serpin E1, and one or more factors selected from the group consisting of pro-angiogenic VEGF, angiogenin, IGFBP-3, uPA, Angio-1, Angio-2, and endothelin-1.

[0384] In some embodiments, the preconditioned MSC secretome composition further comprises low levels of VEGF.

[0385] In some embodiments, the preconditioned MSC secretome comprises VEGF at 1 pg / mL to 400 pg / mL.

[0386] In some embodiments, the level of VEGF is 1 / 5 to 1 / 10 of the level of serpin E1.

[0387] In some embodiments, the preconditioned MSC secretome composition comprises one or more anti-angiogenic factors, and the sum of the concentrations of the one or more anti-angiogenic factors relative to the concentration of VEGF is >2, >3, >4, or >5.

[0388] In some embodiments, the preconditioned MSC secretome composition does not contain, or contains, very low levels of bFGF, PLGF, and PDGF.

[0389] In some embodiments, the preconditioned MSC secretome composition contains less than 1000 pg / mL of bFGF, PLGF, and PDGF.

[0390] In some embodiments, the preconditioned MSC secretome composition has a pH of from about 4.7 to about 7.5.

[0391] In some embodiments, the preconditioned MSC secretome composition is formulated in a buffer system selected from the group consisting of disodium / monosodium phosphate, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and citric acid / disodium phosphate.

[0392] In some embodiments, the preconditioned MSC secretome composition further comprises an osmotic modifier.

[0393] In some embodiments, the osmotic modifier is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin.

[0394] In some embodiments, the preconditioned MSC secretome composition further comprises monosodium / disodium phosphate, mannitol, and trehalose, and the composition has a pH of about pH 7.4.

[0395] In some embodiments, the preconditioned MSC secretome composition further comprises divalent cations.

[0396] In some embodiments, the divalent cations are selected from the group consisting of Mg2+, Ca2+, and Zn2+.

[0397] In some embodiments, the preconditioned MSC secretome composition further comprises disodium phosphate / citric acid, mannitol, and trehalose, and the composition has a pH of about 6.4.

[0398] In some embodiments, the preconditioned MSC secretome composition further comprises an adhesive.

[0399] In some embodiments, the adhesive is selected from the group consisting of hypromellose, poloxamer 407, poloxamer 188, poloxomer 237, poloxomer 338, hypromellose (HPMC), polycarbophil, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyimide, sodium hyaluronate, gellan gum, poly(lactic-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethyl cellulose (CMC), or hydroxypropylmethyl cellulose (HPMC), hydroxymethyl cellulose, hydroxyethyl cellulose, sodium carboxymethyl cellulose, fibrin glue, polyethylene glycol, and GelCORE.

[0400] In some embodiments, the preconditioned MSC secretome composition does not contain one or more components selected from the group consisting of foreign body components, phenol red, peptides and biomolecules <3 kDa, antibiotics, protein aggregates> 200 nm, cells, non-exosome / non-extracellular vesicle cell debris, hormones, and L-glutamine.

[0401] In some embodiments, the preconditioned MSC secretome composition comprises HGF, pentraxin-3 (TSG-14), VEGF, TIMP-1, serpin E1, and <5 ng / mL of IL-8.

[0402] In some embodiments, the preconditioned MSC secretome composition i. 0.3 to 4.5 ng / mL of HGF and ii. 0.5 to 20 ng / mL of pentraxin-3 (TSG-14), and iii. 100 to 600 pg / mL of VEGF, and iv. 10 to 200 ng / mL of TIMP-1, and v. 20 to 80 ng / mL of serpin E1, and vi. <5 ng / mL of IL-8, and comprises.

[0403] In some embodiments, the preconditioned MSC secretome composition comprises an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.

[0404] The present invention also provides a method of treating an eye disease in a subject in need thereof, comprising administering to the subject a preconditioned mesenchymal stem cell (MSC) secretome composition, wherein the preconditioned MSC secretome preparation i. 2 μg to 20 μg of MSC secretome per mL, and ii. 2 mg to 3 mg of monobasic sodium phosphate per mL, and iii. 11 mg to 12 mg of dibasic sodium phosphate per mL, and iv. 11.5 mg to 13 mg of mannitol per mL, and v. 23 mg to 24 mg of trehalose dihydrate, and vi. 0.5 mg to 2 mg of hypromellose per mL, and comprises, and is a stable preconditioned mesenchymal stem cell (MSC) secretome preparation having a pH of about 4.7 to about 7.5.

[0405] The present invention also provides a method of treating an eye disease in a subject in need thereof, comprising administering to the subject a preconditioned mesenchymal stem cell (MSC) secretome composition, wherein the preconditioned MSC secretome preparation i. 0.004% to 0.08% w / w of MSC secretome, and ii. 4% to 5% w / w of monobasic sodium phosphate, and iii. 21.5% to 23% w / w of dibasic sodium phosphate, and iv. 23% to 25% w / w of mannitol, and v. 46% to 48% w / w of trehalose dihydrate, and vi. 1% to 3% w / w of hypromellose, and comprising a stable preconditioned mesenchymal stem cell (MSC) secretome formulation having a pH of about 4.7 to about 7.5.

[0406] The present invention also relates to i. 1 to 20 μg, optionally 2 μg to 8 μg of MSC secretome per mL, and ii. 2 mg to 3 mg of monobasic sodium phosphate per mL, and iii. 11 mg to 12 mg of dibasic sodium phosphate per mL, and iv. 11.5 mg to 13 mg of mannitol per mL, and v. 23 mg to 24 mg of trehalose dihydrate, and vi. optionally 0.5 mg to 2 mg of hypromellose per mL, and comprising a stable preconditioned mesenchymal stem cell (MSC) secretome formulation having a pH of about 4.7 to about 7.5.

[0407] The present invention also relates to i. 0.004% to 0.0375%, optionally 0.008% to 0.015% w / w of MSC secretome, and ii. 4% to 5% w / w of monobasic sodium phosphate, and iii. 21.5% to 23% w / w of dibasic sodium phosphate, and iv. 23% to 25% w / w of mannitol, and v. 46% to 48% w / w of trehalose dihydrate, and vi. optionally 1% to 3% w / w of hypromellose, and comprising Provided is a stable preconditioned mesenchymal stem cell (MSC) secretome formulation having a pH of from about 4.7 to about 7.5.

[0408] The present invention also i. 6 μg of MSC secretome per 1 mL, and ii. 2.28 mg of monobasic sodium phosphate per 1 mL, and iii. 11.45 mg of dibasic sodium phosphate per 1 mL, and iv. 12.2 mg of mannitol per 1 mL, and v. 24 mg of trehalose dihydrate, and vi. optionally 1 mg of hypromellose per 1 mL, and comprising Provided is a stable preconditioned mesenchymal stem cell (MSC) secretome formulation having a pH of about 7.4.

[0409] The present invention also i. 6 μg of MSC secretome per 1 mL, and ii. 1.31 mg of monobasic sodium phosphate per 1 mL, and iii. 5.73 mg of dibasic sodium phosphate per 1 mL, and iv. 12.2 mg of mannitol per 1 mL, and v. 24 mg of trehalose dihydrate per 1 mL, and vi. 1 mg of hypromellose per 1 mL, and vii. 0.2 mg of magnesium chloride per 1 mL, and viii. 1.17 mg of sodium chloride per 1 mL, and comprising Provided is a stable preconditioned mesenchymal stem cell (MSC) secretome formulation having a pH of about 7.4.

[0410] The present invention also i. 0.012% w / w of MSC secretome, and ii. 4.5% w / w of monobasic sodium phosphate, and iii. 22.4% w / w of disodium phosphate, and iv. 24% w / w of mannitol, and v. 47.1% w / w of trehalose dihydrate, and vi. optionally 2.0% w / w of hypromellose, and comprising a stable preconditioned mesenchymal stem cell (MSC) secretome formulation having a pH of about 7.4.

[0411] The present invention also provides i. 2 μg to 20 μg of MSC secretome per mL, and ii. 2 mg to 3 mg of monosodium phosphate per mL, and iii. 11 mg to 12 mg of disodium phosphate per mL, and iv. 11.5 mg to 13 mg of mannitol per mL, and v. 23 mg to 24 mg of trehalose dihydrate, and vi. optionally 0.5 mg to 2 mg of hypromellose per mL, and comprising a stable preconditioned mesenchymal stem cell (MSC) secretome formulation having a pH of about 4.7 to about 7.5.

[0412] The present invention also provides i. 0.004% to 0.08% w / w of MSC secretome, and ii. 4% to 5% w / w of monosodium phosphate, and iii. 21.5% to 23% w / w of disodium phosphate, and iv. 23% to 25% w / w of mannitol, and v. 46% to 48% w / w of trehalose dihydrate, and vi. optionally 1% to 3% w / w of hypromellose, and comprising a stable preconditioned mesenchymal stem cell (MSC) secretome formulation having a pH of about 4.7 to about 7.5.

[0413] In some embodiments of a stable preconditioned mesenchymal stem cell (MSC) secretome formulation, the formulation does not contain hypromellose.

[0414] The present invention also provides, in a subject in need thereof, a method of treating an eye disease comprising administering to the subject a preconditioned mesenchymal stem cell (MSC) secretome composition, wherein the preconditioned MSC secretome formulation i. 2 μg to 20 μg of MSC secretome per 1 mL, and ii. 2 mg to 3 mg of monobasic sodium phosphate per 1 mL, and iii. 11 mg to 12 mg of dibasic sodium phosphate per 1 mL, and iv. 11.5 mg to 13 mg of mannitol per 1 mL, and v. 23 mg to 24 mg of trehalose dihydrate, and vi. optionally 0.5 mg to 2 mg of hypromellose per 1 mL, and comprises a stable preconditioned mesenchymal stem cell (MSC) secretome formulation having a pH of about 4.7 to about 7.5.

[0415] The present invention also provides, in a subject in need thereof, a method of treating an eye disease comprising administering to the subject a preconditioned mesenchymal stem cell (MSC) secretome composition, wherein the preconditioned MSC secretome formulation i. 0.004% to 0.08% w / w of MSC secretome, and ii. 4% to 5% w / w of monobasic sodium phosphate, and iii. 21.5% to 23% w / w of dibasic sodium phosphate, and iv. 23% to 25% w / w of mannitol, and v. 46% to 48% w / w of trehalose dihydrate, and vi. optionally 1% to 3% w / w of hypromellose, and comprising a stable preconditioned mesenchymal stem cell (MSC) secretome formulation having a pH of from about 4.7 to about 7.5.

[0416] In some embodiments of methods of treating eye diseases, the preconditioned MSC secretome composition and / or formulation used in the treatment method does not contain hypromellose.

[0417] In some embodiments of the methods described herein, the preconditioned MSC secretome composition and / or formulation does not contain hypromellose.

[0418] In some embodiments of the preconditioned MSC secretome composition and / or formulation, the composition and / or formulation does not contain hypromellose.

[0419] II. MSC Secretome - Treatment

[0420] The conditioned medium containing the MSC secretome described herein can, in some embodiments, be harvested, filtered, and / or purified to remove cell microparticles and / or other harmful components. For example, as described above in step (v), the second medium from step (iv) is taken as the conditioned medium. The filtration membrane used herein can be selected from any of those known in the art having an appropriate membrane and configuration such that the filtration membrane can retain the desired MSC secretome components while allowing cell microparticles and / or other harmful components to pass through. Thus, any suitable membrane can be used that allows harmful components to pass through for removal while allowing retention of cells under the selected hydrodynamic conditions. In some embodiments, an upper pore size limit of about 5 microns and a lower limit of about 0.1 micron would be appropriate. In some embodiments, filtration can be performed using a microporous filter. In some embodiments, filtration can be performed using a filter with a pore size of 0.5 μm to 0.2 μm. In some embodiments, filtration can be performed using 0.5 μm, 0.45 μm, 0.4 μm, 0.35 μm, 0.3 μm, 0.25 μm, 0.22 μm, and / or 0.2 μm filters. In some embodiments, filtration can be performed using a 0.45 μm filter. In some embodiments, filtration can be performed using a 0.22 μm filter. In some embodiments, filtration / purification can be performed using a low protein binding polyvinylidene difluoride (PVDF) membrane. In some embodiments, filtration / purification can be performed using polyethersulfone (PES).

[0421] In some embodiments, filtration is by ultrafiltration. In some embodiments, the conditioned medium is filtered using a filter size of 3 kD (to achieve purification, desalting, and concentration of the conditioned medium of molecules larger than the filter size). In some embodiments, a filter size of less than 3 kD is used to filter the conditioned medium, while in other embodiments, a filter size greater than 3 kD is used depending on the use for which the processed conditioned medium is to be used. In other embodiments, ultrafiltration of the harvested conditioned medium is performed using filters of different pore sizes (e.g., 2 kD, <2 kD, or >2 kD) selected to determine the size of the components of the resulting processed conditioned medium, including the MSC secretome.

[0422] In some embodiments, harmful components in the growth-supporting medium are removed by medium exchange, preferably via "cross-flow filtration". Cross-flow filtration refers to a mode of filtration in which a suspension of MSC secretome cells flows substantially parallel to a filter that is permeable to components of the suspension other than the cells. The cross-flow filtration process is characterized by a set of hydrodynamic parameters including Re = Reynolds number, γw = wall shear rate, ΔP = pressure drop, and TMP = transmembrane pressure difference. Re, γw, and ΔP depend on the shape of the filtration system, the flow conditions, and the properties of the fluid. Such cross-flow processes may, in some embodiments, also include hollow fiber filtration systems. See, for example, U.S. Patent No. 5,053,334, which is incorporated herein by reference in its entirety.

[0423] In some embodiments, the MSC secretome may be further subjected to concentration without filtration and / or after filtration. In some embodiments, the MSC secretome can be concentrated using hollow fiber tangential flow technology or

[0424] In some embodiments, the MSC secretome can be concentrated using centrifugation-based size exclusion techniques, for example, Amicon and / or Centricon can be used during the concentration step. In some embodiments, the size cut-off is a MW cut-off of 3-10 kDa. In some embodiments, the molecular weight cut-off for use during centrifugation-based size exclusion technique concentration methods is at least about 3 kDa, at least about 4 kDa, at least about 5 kDa, at least about 6 kDa, at least about 7 kDa, at least about 8 kDa, at least about 9 kDa, or at least about 10 kDa.

[0425] In some embodiments, the MSC secretome is concentrated about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, or about 100-fold. In some embodiments, the MSC secretome is concentrated about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, or about 100-fold compared to the conditioned medium before concentration.

[0426] In some embodiments, the MSC secretome is further buffer exchanged after the concentration step into the final formulation buffer. In some embodiments, the MSC secretome is further buffer exchanged after the concentration step into the final formulation buffer without an adhesive. In some embodiments, buffer exchange includes modifying the buffer components of the MSC secretome. In some embodiments, the MSC secretome is not diluted during the buffer exchange step. In some embodiments, the MSC secretome is diluted less than 1%, less than 5%, less than 10%, less than 15%, less than 20%, or less than 25% during the buffer exchange step.

[0427] In some embodiments, the MSC secretome is buffer exchanged after concentration such that all trace amounts of media components are removed. In some embodiments, the MSC secretome is buffer exchanged after concentration such that less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%, or about 0% of the media components remain.

[0428] III. MSC Secretome - Formulation In some embodiments, the MSC secretome is formulated in a preparation containing about 2 μg to 20 μg of MSC secretome per mL. In some embodiments, the MSC secretome is formulated in a preparation containing 0.004% to 0.0375% of MSC secretome per mL.

[0429] In some embodiments, the MSC secretome is formulated in a preparation containing about 2 μg to 8 μg of MSC secretome per mL. In some embodiments, the MSC secretome is formulated in a preparation containing 0.008% to 0.015% of MSC secretome per mL.

[0430] In some embodiments, the MSC secretome is formulated in a preparation containing 2 mg to 3 mg of sodium monophosphate per mL. In some embodiments, the MSC secretome is formulated in a preparation containing 4% to 5% of sodium monophosphate per mL.

[0431] In some embodiments, the MSC secretome is formulated in a preparation containing 11 mg to 12 mg of sodium dibasic phosphate per mL. In some embodiments, the MSC secretome is formulated in a preparation containing 21.5% to 23% of sodium dibasic phosphate per mL.

[0432] In some embodiments, the MSC secretome is prepared in a formulation containing 11.5 mg to 13 mg of mannitol per mL. In some embodiments, the MSC secretome is prepared in a formulation containing 23% to 25% of mannitol per mL.

[0433] In some embodiments, the MSC secretome is prepared in a formulation containing 23 mg to 25 mg of trehalose dihydrate per mL. In some embodiments, the MSC secretome is prepared in a formulation containing 46% to 48% of trehalose dihydrate per mL.

[0434] In some embodiments, the MSC secretome is prepared in a formulation that does not contain hypromellose. In some embodiments, the MSC secretome is prepared in a formulation optionally containing hypromellose. In some embodiments, the MSC secretome is prepared in a formulation containing 0.5 mg to 2 mg of hypromellose per mL. In some embodiments, the MSC secretome is prepared in a formulation containing 1% to 3% of hypromellose per mL.

[0435] In some embodiments, the MSC secretome is prepared in a formulation containing hydrochloric acid and / or sodium hydroxide. In some embodiments, the MSC secretome is prepared in a formulation containing hydrochloric acid. In some embodiments, the MSC secretome is prepared in a formulation containing sodium hydroxide. In some embodiments, hydrochloric acid and / or sodium hydroxide are used to obtain the desired pH.

[0436] In some embodiments, the MSC secretome is prepared in a formulation containing the components shown in Tables 1 to 4 below.

[0437] [Table 1]

[0438] [Table 2]

[0439] [Table 3]

[0440] [Table 4]

[0441] In some embodiments, the MSC secretome formulation shown in Table 2 has a pH of about 5.5.

[0442] [Table 5]

[0443] In some embodiments, the MSC secretome formulation shown in Table 3 has a pH of about 6.2.

[0444] [Table 6]

[0445] In some embodiments, the MSC secretome formulation shown in Table 4 has a pH of about 7.2. In some embodiments, the MSC secretome is formulated with water for injection according to USP standards.

[0446] D. Assay Methods / Therapeutic Characteristics In some embodiments of the present invention, the MSC secretome is processed to achieve specific component ratios / concentrations and the characteristics of the MSC secretome.

[0447] In some embodiments, the MSC secretome composition comprises an anti-angiogenic to pro-angiogenic ratio, and the ratio is >1. In some embodiments, the MSC secretome composition comprises an anti-angiogenic to pro-angiogenic ratio, and the ratio is >2, >3, >4, or >5. In some embodiments, the MSC secretome composition comprises an increased concentration of pro-angiogenic factors (compared to the concentration of pro-angiogenic factors in the conditioned medium from which the MSC secretome composition is produced). In some embodiments, the MSC secretome composition comprises the sum of several anti-angiogenic factors that exceed the level of VEGF. In some embodiments, the MSC secretome composition comprises the sum of several anti-angiogenic factors such that the ratio of the plurality of anti-angiogenic factors to VEGF is >2, >3, >4, or >5. In some embodiments, the MSC secretome composition comprises one or more anti-angiogenic factors, and the sum of the concentrations of the one or more anti-angiogenic factors relative to the concentration of VEGF is >2, >3, >4, or >5. In some embodiments, the pro-angiogenic factor includes, but is not limited to, serpin E1 relative to VEGF-A. In some embodiments, the pro-angiogenic factor is serpin E1. In some embodiments, the pro-angiogenic factor is VEGF-A.

[0448] In some embodiments of the present invention, the MSC secretome is processed to achieve certain potency performance criteria. In some embodiments, the buffer exchange step facilitates obtaining a potent MSC secretome.

[0449] Extracellular vesicles are membrane-bound particles that carry the cargo of the soluble and insoluble substances described above. The term "extracellular vesicles" refers to a group of vesicles secreted or discharged by various species. These are generally divided into the following subtypes: 1) microvesicles or shed microvesicles typically showing a size range of 50-1500 nm, 2) exosomes usually showing a size range of 30-120 nm, and 3) vesicles typically showing a size range of less than 500 nm (i.e., <500 nm). (See, e.g., WO2019016799, which is incorporated herein by reference in its entirety). In some embodiments, the MSC secretome can be analyzed to quantify, in terms of particle number, and / or the extracellular vesicles (EVs) present in the secretome.

[0450] In some embodiments, EVs are present at a concentration of about 2.5×10^5 / uL, 2.6×10^5 / uL, 2.7×10^5 / uL, 2.8×10^5 / uL, 2.9×10^5 / uL, 3.0×10^5 / uL, 3.1×10^5 / uL, 3.2×10^5 / uL, 3.3×10^5 / uL, 3.4×10^5 / uL, 3.5×10^5 / uL, 3.6×10^5 / uL, 3.7×10^5 / uL, 3.8×10^5 / uL, 3.9×10^5 / uL, 4.0×10^5 / uL, 4.1×10^5 / uL, 4.2×10^5 / uL, 4.3×10^5 / uL, 4.4×10^5 / uL, 4.5×10^5 / uL, 4.6×10^5 / uL, 4.7×10^5 / uL, 4.8×10^5 / uL, 4.9×10^5 / uL, or about 5.0×10^5 / uL. In some embodiments, EVs are present at a concentration of about 3.8×10^5 / uL + / - 0.8×10^5.

[0451] In some embodiments, Ev is present at a concentration of about 2.5×10^5 / uL, 2.6×10^5 / uL, 2.7×10^5 / uL, 2.8×10^5 / uL, 2.9×10^5 / uL, 3.0×10^5 / uL, 3.1×10^5 / uL, 3.2×10^5 / uL, 3.3×10^5 / uL, 3.4×10^5 / uL, 3.5×10^5 / uL, 3.6×10^5 / uL, 3.7×10^5 / uL, 3.8×10^5 / uL, 3.9×10^5 / uL, 4.0×10^5 / uL, 4.1×10^5 / uL, 4.2×10^5 / uL, 4.3×10^5 / uL, 4.4×10^5 / uL, 4.5×10^5 / uL, 4.6×10^5 / uL, 4.7×10^5 / uL, 4.8×10^5 / uL, 4.9×10^5 / uL, or about 5.0×10^5 / uL, and has an average diameter of 110 - 120 nm. In some embodiments, Ev is present at a concentration of about 2.5×10^5 / uL, 2.6×10^5 / uL, 2.7×10^5 / uL, 2.8×10^5 / uL, 2.9×10^5 / uL, 3.0×10^5 / uL, 3.1×10^5 / uL, 3.2×10^5 / uL, 3.3×10^5 / uL, 3.4×10^5 / uL, 3.5×10^5 / uL, 3.6×10^5 / uL, 3.7×10^5 / uL, 3.8×10^5 / uL, 3.9×10^5 / uL, 4.0×10^5 / uL, 4.1×10^5 / uL, 4.2×10^5 / uL, 4.3×10^5 / uL, 4.4×10^5 / uL, 4.5×10^5 / uL, 4.6×10^5 / uL, 4.7×10^5 / uL, 4.8×10^5 / uL, 4.9×10^5 / uL, or about 5.0×10^5 / uL, and has an average diameter of 112 - 116 nm.In some embodiments, Ev is present at a concentration of about 2.5×10^5 / uL, 2.6×10^5 / uL, 2.7×10^5 / uL, 2.8×10^5 / uL, 2.9×10^5 / uL, 3.0×10^5 / uL, 3.1×10^5 / uL, 3.2×10^5 / uL, 3.3×10^5 / uL, 3.4×10^5 / uL, 3.5×10^5 / uL, 3.6×10^5 / uL, 3.7×10^5 / uL, 3.8×10^5 / uL, 3.9×10^5 / uL, 4.0×10^5 / uL, 4.1×10^5 / uL, 4.2×10^5 / uL, 4.3×10^5 / uL, 4.4×10^5 / uL, 4.5×10^5 / uL, 4.6×10^5 / uL, 4.7×10^5 / uL, 4.8×10^5 / uL, 4.9×10^5 / uL, or about 5.0×10^5 / uL and has an average diameter of 114 nm. In some embodiments, Ev is present at a concentration of about 3.8×10^5 / uL + / - 0.8×10^5 and has an average diameter of 114 nm.

[0452] i. MSC secretome - Therapeutic properties The MSC secretome of the present disclosure exhibits various therapeutic properties including, for example, anti - angiogenic effects (blood vessels and / or lymphatic vessels), anti - fibrotic properties, anti - inflammatory properties, properties that promote cell migration and proliferation, properties that promote mitosis, and anti - oxidative stress / damage properties.

[0453] In some embodiments, the anti - angiogenic (blood vessels and / or lymphatic vessels) properties can be determined by the presence and / or levels of one or more factors in the MSC secretome. In some embodiments, the anti - angiogenic factors include, but are not limited to, one or more of PEDF, sFLT - 1, low levels of VEGF, and / or serpin E1. In some embodiments, the anti - angiogenic factors include, but are not limited to, one or more of PEDF, low levels of VEGF, and / or serpin E1. In some embodiments, the anti - angiogenic factor is PEDF. In some embodiments, the anti - angiogenic factor is sFLT - 1. In some embodiments, the anti - angiogenic factor corresponds to low levels of VEGF. In some embodiments, the anti - angiogenic factor is serpin E1.

[0454] In some embodiments, the angiogenesis promoting (vascular and / or lymphatic vessel) properties can be determined by the presence and / or levels of one or more factors in the MSC secretome. In some embodiments, the angiogenesis promoting factors include one or more factors selected from the group consisting of VEGF, angiogenin, IGFBP-3, uPA, Angio-1, Angio-2, and endothelin-1. In some embodiments, the angiogenesis promoting factor is VEGF. In some embodiments, the angiogenesis promoting factor is angiogenin. In some embodiments, the angiogenesis promoting factor is IGFBP-3. In some embodiments, the angiogenesis promoting factor is uPA. In some embodiments, the angiogenesis promoting factor is Angio-1. In some embodiments, the angiogenesis promoting factor is Angio-2. In some embodiments, the angiogenesis promoting factor is endothelin-1.

[0455] In some embodiments, the MSC secretome exhibits anti-fibrotic properties. In some embodiments, such anti-fibrotic properties can be assayed using standard assays. In some embodiments, the presence of various factors and / or activities associated with the MSC secretome exhibits anti-fibrotic properties. In some embodiments, the factors exhibiting anti-fibrotic properties include, but are not limited to, FGF7 and / or FGF10. In some embodiments, the factor exhibiting anti-fibrotic properties is FGF7. In some embodiments, the factor exhibiting anti-fibrotic properties is FGF10. In some embodiments, the factor exhibiting anti-fibrotic properties is HGF. In some embodiments, the activities exhibiting anti-fibrotic properties include, but are not limited to, activation of SMAD, inhibition of the TGFβ pathway, inhibition of myofibroblast differentiation, and / or inhibition of excessive ECM deposition. In some embodiments, the activity exhibiting anti-fibrotic properties includes activation of SMAD. In some embodiments, the activity exhibiting anti-fibrotic properties includes inhibition of the TGFβ pathway. In some embodiments, the activity exhibiting anti-fibrotic properties includes inhibition of myofibroblast differentiation. In some embodiments, the activity exhibiting anti-fibrotic properties includes inhibition of excessive ECM deposition.

[0456] In some embodiments, the MSC secretome exhibits anti-inflammatory properties. In some embodiments, the MSC secretome inhibits inflammation. In some embodiments, the MSC secretome inhibits inflammation by 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (e.g., complete reduction of inflammation). In some embodiments, the MSC secretome prevents mast cell degranulation.

[0457] In some embodiments, the MSC secretome promotes cell migration and proliferation, including, for example, mitotic and motility-inducing activities. In some embodiments, the MSC secretome promotes mitotic activity. In some embodiments, the MSC secretome promotes motility-inducing activity. In some embodiments, the MSC secretome provides FGF7, which provides cell migration and proliferation activity of the MSC secretome.

[0458] In some embodiments, the MSC secretome provides FGF7, which provides cell migration and proliferation activity of the MSC secretome.

[0459] In some embodiments, the MSC secretome provides HGF, which provides cell migration and proliferation activity of the MSC secretome.

[0460] In some embodiments, the MSC secretome comprises anti-apoptotic agents that provide cell migration and proliferation activity of the MSC secretome. In some embodiments, the MSC secretome includes, but is not limited to, FGF-2, HGF, and IGF-1, which provide anti-apoptotic agents that provide cell migration and proliferation activity of the MSC secretome. In some embodiments, the MSC secretome is selected from the group consisting of FGF-2, HGF, and IGF-1 and comprises anti-apoptotic agents that provide cell migration and proliferation activity of the MSC secretome.

[0461] In some embodiments, the MSC secretome includes NGF, which provides cell migration and proliferation activities of the MSC secretome.

[0462] In some embodiments, the MSC secretome provides reduction of oxidative stress and / or cell damage. In some embodiments, the MSC secretome includes reduction of oxidative stress and / or cell damage factors. In some embodiments, the reduction of oxidative stress and cell damage factors includes, but is not limited to, SOD-1, SOD-2, SOD-3, HO-1. In some embodiments, the reduction of oxidative stress and cell damage factors is selected from the group consisting of SOD-1, SOD-2, SOD-3, HO-1.

[0463] ii. MSC secretome - Biophysical properties / Biochemical properties Biochemical property evaluation and biophysical property evaluation: In some embodiments, the present invention provides a method for characterizing an MSC secretome. In some embodiments, MSC secretome characterization includes: 1) comprehensive and / or quantitative mapping of molecular entities in the MSC secretome; 2) measuring the contribution of selected factors to biopotency; and 3) measuring biophysical parameters. In some embodiments, various potency assays can be performed on the MSC secretome as described herein to determine the characteristics of the MSC secretome. In some embodiments, the MSC secretome can be subjected to: 1) comprehensive and / or quantitative mapping of molecular entities in the MSC secretome; 2) measuring the contribution of selected factors to biopotency; and 3) measuring biophysical parameters. In some embodiments, the characterization assays include, but are not limited to, biophysical assays, biochemical assays, and bioassays. In some embodiments, the characterization assays include, but are not limited to, characterization of physical components, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analysis, stability assays, proliferation assays, migration assays, adhesion assays, angiogenesis assays, differentiation / scarring assays, inflammation assays, immune assays, gliosis assays, survival and function assays of tissue explants, development or survival / function assays of organoids, epithelial barrier integrity assays, retinal degeneration assays, and / or assays of hereditary retinal diseases including human and animal retinal explants, neuroprotection / neurotrophic assays. In some embodiments, the characterization assays are selected from the group consisting of characterization of physical components, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analysis, stability assays, proliferation assays, migration assays, adhesion assays, angiogenesis assays, differentiation / scarring assays, inflammation assays, immune assays, gliosis assays, survival and function assays of tissue explants, development or survival / function of organoids, epithelial barrier integrity assays, retinal degeneration assays, and / or assays of hereditary retinal diseases including human and animal retinal explants, neuroprotection / neurotrophic assays.

[0464] Characteristics of physical components: In some embodiments, the characterization of the MSC secretome includes methods using a combination of biological analysis techniques. In some embodiments, the characterization of the MSC secretome includes determining the physical components of the MSC secretome. In some embodiments, the characterization of the MSC secretome includes using protein arrays, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, and immunoblotting. In some embodiments, MSC secretome characterization can be used to identify molecules in the MSC secretome. In some embodiments, protein arrays can be used to identify factors in the MSC secretome. In some embodiments, mass spectrometry can be used to determine the presence of one or more factors in the MSC secretome. In some embodiments, quantification techniques can be used to measure the levels of one or more factors. In some embodiments, quantification techniques such as ELISA can be used to measure the levels of each factor.

[0465] In some embodiments, the secretome includes protein factors and extracellular vesicles (EVs). In some embodiments, the MSC secretome includes trophic factors. In some embodiments, the protein factors of the MSC secretome include pentraxin-3, TIMP-1, serpin E1, TSP-1, and HGF. In some embodiments, the MSC secretome includes EVs. In some embodiments, the MSC secretome is analyzed for simple lipid content to quantitatively measure total lipids. In some embodiments, the EV fraction of the MSC secretome can be evaluated for EV markers. In some embodiments, the EV fraction of the MSC secretome can be evaluated for EV markers including, but not limited to, AUX, TSG101, CD63, CD9, and CD8.

[0466] In some embodiments, the secretome is 30 - 200 nm and 1×10 8 ~5×109 It includes extracellular vesicles (EVs) within the size range of EVs of EVs.

[0467] In some embodiments, depletion studies can be performed to extract the individual contributions of important factors. In some embodiments, an antibody-based pulldown method can be used to remove a defined factor from the MSC secretome. In some embodiments, depletion can be verified by Western blotting and then evaluated by one or more bioassays, as described herein below. In some embodiments, depletion studies can be performed to evaluate the contributions of the protein fraction and the EV fraction. In some embodiments, TIMP1 and / or serpin E1 may be depleted. In some embodiments, TIMP1 and / or serpin E1 may be depleted.

[0468] Oxidative stress: In some embodiments, oxidative stress prevention assays can be performed on the MSC secretome. In some embodiments, the MSC secretome prevents damage to corneal epithelium. In some embodiments, the MSC secretome reduces the presence of inflammation. In some embodiments, the MSC secretome reduces the presence of inflammation as determined by an increase in the presence of anti-inflammatory markers. In some embodiments, the MSC secretome reduces the presence of inflammation as determined by an increase in the presence of anti-inflammatory markers such as, for example, IL-8.

[0469] Safety property evaluation: In some embodiments, the MSC secretome can be evaluated for performing tests on levels of pyrogens and endotoxins, as well as blood compatibility and sterility. In some embodiments, the MSC secretome can be evaluated for blood compatibility. In some embodiments, evaluating blood compatibility includes assays for hemolysis and erythrocyte aggregation. In some embodiments, the MSC secretome shows no harmful effects upon systemic exposure. In some embodiments, the MSC secretome shows no harmful effects upon systemic exposure, such as severe ocular burns. In some embodiments, the MSC secretome shows no erythrocyte aggregation activity. In some embodiments, the MSC secretome does not induce hemolysis. In some embodiments, the MSC secretome does not induce hemolytic activity.

[0470] In some embodiments, the MSC secretome may be sterile so as to be administrable as part of a pharmaceutical formulation. In some embodiments, the MSC secretome may be endotoxin-free or substantially endotoxin-free. In some embodiments, the MSC secretome may be microbe-free or substantially microbe-free.

[0471] Stability: In some embodiments, the biophysical properties of the MSC secretome can be evaluated and / or determined. In some embodiments, fluorescence, static light scattering, and dynamic light scattering for characterizing protein stability criteria. In some embodiments, to further characterize the secretome, the following parameters can be measured: thermal melting, thermal aggregation, delta G, and / or viscosity. In some embodiments, the thermal melting assay is used to determine the stability of the MSC secretome. In some embodiments, the thermal aggregation assay is used to determine the stability of the MSC secretome. In some embodiments, delta G is used as a measure to determine the stability of the MSC secretome. In some embodiments, viscosity is measured as an MSC secretome property. In some embodiments, viscosity is for determining the stability of the MSC secretome.

[0472] In some embodiments, the biophysical measurement criteria can be used to establish stability parameters for characterizing different MSC secretome formulations.

[0473] In some embodiments, the MSC secretome is stable at -20°C, 4°C, and room temperature (20°C) for at least 7 days. In some embodiments, the MSC secretome is stable at -20°C, 4°C, and room temperature (20°C) for at least 14 days. In some embodiments, the MSC secretome is stable for at least 7 days, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 1 month. In some embodiments, the MSC secretome is stable at approximately -20°C for at least 7 days, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 1 month, at least 2 months, or at least 3 months. In some embodiments, the MSC secretome is stable at approximately 4°C for at least 7 days, at least 14 days, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 1 month. In some embodiments, the MSC secretome is stable at approximately 20°C (or room temperature) for at least 7 days, at least 14 days, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 1 month.

[0474] In some embodiments, the MSC secretome is stable at approximately -20°C for at least 7 days. In some embodiments, the MSC secretome is stable at approximately 4°C for at least 7 days. In some embodiments, the MSC secretome is stable at approximately 20°C for at least 7 days. In some embodiments, the MSC secretome is stable at approximately 25°C (room temperature) for at least 7 days.

[0475] In some embodiments, the MSC secretome is stable at approximately -20°C for at least 14 days. In some embodiments, the MSC secretome is stable at approximately 4°C for at least 14 days. In some embodiments, the MSC secretome is stable at approximately 20°C (or room temperature) for at least 14 days. In some embodiments, the MSC secretome is stable at approximately 25°C (room temperature) for at least 14 days.

[0476] Epithelial barrier integrity assay The corneal epithelium, and more precisely the apical surface of the epithelium, makes a major contribution to the overall barrier properties of the cornea, and changes to the corneal barrier function as a highly sensitive factor in biocompatibility analysis. In some embodiments, the biophysical properties of the MSC secretome can be evaluated and / or determined by, for example, an epithelial barrier wall integrity assay. In some embodiments, the epithelial barrier wall integrity assay is transepithelial electrical resistance (TEER). In some embodiments, transepithelial electrical resistance (TEER) can be evaluated to measure overall barrier properties. In some embodiments, the 3D tissue can be transferred into a 24-well plate containing 2 mL of TEER buffer and incubated for 10 minutes. In some embodiments, TEER can be measured using an epithelial volt-ohmmeter EVOMO and an EndOhm-12 chamber (World Precision, Sarasota, FL). In some embodiments, at the end of the procedure, the tissue can be used for tissue viability assessment using the following formula. Barrier integrity % = 100 × [TEER (treated tissue) / TEER (placebo control)]

[0477] In some embodiments, TEER can be used to evaluate the effect of the MSC secretome on barrier integrity following topical application. In some embodiments, TEER can be used to evaluate the effect of the MSC secretome on barrier integrity following topical application after corneal epithelial injury caused by topical exposure to nitrogen mustard (NM) using an EpiCorneal tissue model (MatTek Corp). In some embodiments, the MSC secretome can be topically applied, for example, at 6 μg / ml (diluted in placebo solution) as described in Example 6. In some embodiments, the corneal tissue was cultured in 5 ml of medium for 24 hours under standard culture conditions.

[0478] Bioassay In some embodiments, the bioassay can be used to characterize the MSC secretome. In some embodiments, the bioassay can be associated with corneal wound healing, namely, epithelial cell migration and proliferation, stromal cell differentiation (e.g., scarring), neovascularization, and inflammation. In some embodiments, the bioassay can be used to evaluate the ability of the MSC secretome to mediate corneal wound healing, namely, epithelial cell migration and proliferation, stromal cell differentiation (e.g., scarring), neovascularization, and inflammation.

[0479] Migration and proliferation In some embodiments, the MSC secretome can be evaluated for its ability to promote proliferation and migration. In some embodiments, the MSC secretome can be evaluated for its ability to promote proliferation. In some embodiments, the MSC secretome can be evaluated for its ability to promote migration. In some embodiments, the MSC secretome promotes proliferation and / or migration. In some embodiments, the MSC secretome promotes proliferation. In some embodiments, the MSC secretome promotes migration. In some embodiments, the MSC secretome can be evaluated using a transwell migration assay to determine its proliferation-promoting ability.

[0480] In some embodiments, the migration assay can be used to evaluate the ability of the MSC secretome to promote migration. In some embodiments, the migration assay can be used to evaluate the ability of the MSC secretome to promote migration, and the migration assay is an in vitro wound healing assay. In some embodiments, the migration assay can include a "scratch assay" (also referred to as a "scrape assay"). In some embodiments, the MSC secretome promotes migration, and this promotion of migration is determined and / or examined using a "scratch assay". Generally, the scratch assay method is based on when an artificial gap, also called a "scratch", occurs on a confluent cell monolayer. The "scratch" can be monitored for cells on the edge of a newly created gap that migrates towards the opening to "heal" / cover the "scratch". (See, e.g., Liang, C., Park, A. & Guan, J. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2, 329 - 333 (2007).)

[0481] In some embodiments, the migration assay can include a transwell migration assay using corneal epithelial cells (or other cell surrogates after confirmation), and (e.g., wound healing) can be performed on the MSC secretome. In some embodiments, a transwell migration assay using corneal epithelium as a test of the wound healing efficacy of the MSC secretome. In some embodiments, the MSC secretome promotes wound healing as determined using a transwell migration assay.

[0482] In some embodiments, the in vitro wound healing assay includes, but is not limited to, a "scratch assay" (also referred to as a "scrape assay"), or a circular scraping method, or a circular scraping assay, or a circular wound healing assay.

[0483] In some embodiments, the human corneal epithelial cell proliferation assay can be performed on the MSC secretome. In some embodiments, the human corneal epithelial cell proliferation assay demonstrates the wound closure properties of the MSC secretome. In some embodiments, the MSC secretome promotes wound closure as determined using the human corneal epithelial cell proliferation assay.

[0484] In some embodiments, a circular scratch method or a circular scratch assay or a circular wound closure assay can be used. In some embodiments, the Oris™ Cell Migration Assay platform can be used (see also reference as described in Example 6 herein).

[0485] In some embodiments, an endothelial cell tube formation assay can be performed on the MSC secretome. In some embodiments, the endothelial cell tube formation assay may indicate that the MSC secretome is not angiogenic promoting. In some embodiments, the endothelial cell tube formation assay provides a criterion for the angiogenic potential of the MSC secretome. In some embodiments, the MSC secretome exhibits an anti-angiogenic effect. In some embodiments, the MSC secretome has an anti-angiogenic effect. In some embodiments, the endothelial cell tube formation assay provides a ratio of anti-angiogenic signals to angiogenic promoting signals. In some embodiments, a negative result of the endothelial cell tube formation assay confirms that the anti:promoting ratio is high and ensures that the MSC secretome does not promote neovascularization. In some embodiments, a negative result of the endothelial cell tube formation assay confirms that the anti:promoting ratio is high and ensures that the MSC secretome does not generally promote CNV (choroidal neovascularization) or neovascularization. In some embodiments, inhibition of the TGFb-induced myofibroblast differentiation assay can be performed on the MSC secretome. In some embodiments, inhibition of the TGFb-induced myofibroblast differentiation assay can be performed with the MSC secretome to show that the MSC secretome prevents scarring. In some embodiments, the MSC secretome prevents scarring. In some embodiments, the MSC secretome prevents scarring corneal opacity. In some embodiments, the MSC secretome has low angiogenic induction. In some embodiments, the MSC secretome has a reduced angiogenic response. In some embodiments, the MSC secretome has reduced angiogenic activity. In some embodiments, the MSC secretome impairs and / or reduces the normal formation of blood vessels in the presence of a medium that supports angiogenesis. In some embodiments, the MSC secretome has reduced angiogenic activity when the MSC secretome is compared to an untreated control. In some embodiments, the MSC secretome has reduced angiogenic activity compared to a sample treated with serum-containing medium. In some embodiments, the MSC secretome attenuates the angiogenic response.In some embodiments, the MSC secretome reduces the angiogenic response induced by serum-free medium. In some embodiments, when the secretome supplemented with serum-containing medium (reduced or no angiogenic response) is compared with serum-containing medium (angiogenic response), the reduction of the angiogenic response is induced by the MSC secretome. In some embodiments, the angiogenic response is indicated by tube formation in a cell-based assay. In some embodiments, the angiogenic response is indicated by tube formation in an endothelial cell tube formation assay.

[0486] Differentiation / Scarring: In some embodiments, the MSC secretome can be evaluated for its ability to prevent differentiation and its ability to prevent scarring. In some embodiments, the MSC secretome prevents and / or impairs scarring. In some embodiments, the MSC secretome prevents scarring. In some embodiments, the MSC secretome reduces scarring compared to other standard treatments. In some embodiments, the MSC secretome prevents and / or impairs differentiation. In some embodiments, the MSC secretome prevents and / or impairs the differentiation of myofibroblasts. In some embodiments, the MSC secretome reduces the loss of corneal transparency. In some embodiments, the MSC secretome reduces the loss of corneal transparency by preventing and / or impairing the differentiation of myofibroblasts.

[0487] In some embodiments, the MSC secretome can be evaluated for its ability to regulate factors involved in differentiation. In some embodiments, the MSC secretome can be evaluated for its ability to regulate factors involved in differentiation, including but not limited to TGFB2, collagen I, collagen III (which are typically upregulated during differentiation), TFGB3, MMP-2, and MMP-9 (which are typically downregulated during differentiation). In some embodiments, the MSC secretome regulates a factor selected from the group consisting of TGFB2, collagen I, collagen III (which are typically upregulated during differentiation), TFGB3, MMP-2, and MMP-9 (which are typically downregulated during differentiation). In some embodiments, the MSC secretome induces a decrease in factors that are upregulated during normal differentiation. In some embodiments, the MSC secretome induces an increase in factors that are downregulated during normal differentiation. In some embodiments, the MSC secretome induces a decrease in the expression of factors such as SMA. In some embodiments, the MSC secretome induces a decrease in the expression of factors such as SMA that indicate the efficacy of the MSC secretome.

[0488] Angiogenesis: In some embodiments, the MSC secretome can be evaluated for its ability to prevent angiogenesis. In some embodiments, the MSC secretome prevents, impairs, inhibits, and / or reduces angiogenesis. In some embodiments, the MSC secretome inhibits or does not promote angiogenesis. In some embodiments, the MSC secretome can be evaluated for its ability to prevent neovascularization. In some embodiments, the MSC secretome prevents, impairs, inhibits, and / or reduces neovascularization. In some embodiments, the MSC secretome inhibits neovascularization.

[0489] In some embodiments, the MSC secretome can be further evaluated using depletion assays. In some embodiments, specific factors can be depleted from the MSC secretome. In some embodiments, specific factors including, but not limited to, TIMP1 and / or serpin E1 can be depleted from the MSC secretome. In some embodiments, TIMP1 and / or serpin E1 can be depleted from the MSC secretome. In some embodiments, TIMP1 can be depleted from the MSC secretome. In some embodiments, serpin E1 can be depleted from the MSC secretome.

[0490] Inflammation: In some embodiments, the MSC secretome can be evaluated for its ability to prevent, impair, inhibit, and / or reduce inflammation. In some embodiments, the MSC secretome prevents, impairs, inhibits, and / or reduces inflammation. In some embodiments, the MSC secretome inhibits inflammation. In some embodiments, the MSC secretome is characterized in vitro and / or in vivo to determine its ability to prevent, impair, inhibit, and / or reduce inflammation. In some embodiments, the MSC secretome prevents, impairs, inhibits, and / or reduces inflammation in vitro and / or in vivo. In some embodiments, the MSC secretome prevents, impairs, inhibits, and / or reduces inflammation in vitro. In some embodiments, the MSC secretome prevents, impairs, inhibits, and / or reduces inflammation in vivo. In some embodiments, tissue models can be used to characterize their ability to prevent, impair, inhibit, and / or reduce inflammation in vitro. In some embodiments, 3D tissue models can be used to characterize their ability to prevent, impair, inhibit, and / or reduce inflammation in vitro. In some embodiments, the nitrogen mustard (NM) gas burn model can be used to evaluate its ability to prevent, impair, inhibit, and / or reduce inflammation in vitro. In some embodiments, the nitrogen mustard (NM) gas burn model can be used to evaluate its ability to prevent, impair, inhibit, and / or reduce inflammation in vitro and as an alternative to the in vivo state. In some embodiments, the cytokine profile in response to treatment with the MSC secretome and / or administration of the MSC secretome can be determined. In some embodiments, the levels of specific cytokines can be determined. In some embodiments, the levels of IL-8 can be determined. In some embodiments, the expression level of IL-8 can be reduced in tissues treated with the MSC secretome.In some embodiments, the expression level of IL-8 is reduced in tissues treated with the MSC secretome, indicating that it prevents, impairs, inhibits, and / or reduces inflammation.

[0491] Oxidative stress: In some embodiments, cell survival is evaluated under oxidative stress conditions or under an induced misfolded protein response, for example, by tunicamycin treatment or by ER stress induced by thapsigargin and / or brefeldin A treatment. In some embodiments, the oxidative stressor is selected from the group consisting of iron(III) chloride-sodium nitrilotriacetate (Fe-NTA), sodium periodate (NaIO4), 7-ketocholesterol (7-KC), hydrogen peroxide (H2O2), all-trans retinoic acid (ATRA), and tert-butyl hydroperoxide (t-BHP). In some embodiments, the misfolded protein response is induced by tunicamycin treatment. In some embodiments, the ER stress response is induced by thapsigargin and brefeldin A treatment.

[0492] In some embodiments, the positive controls include vitamins and their analogs including vitamin A, vitamin B3 (e.g., niacin [nicotinic acid] and nicotinamide), vitamin C (ascorbic acid), vitamin E (including tocopherols [e.g., α-tocopherol] and tocotrienols), and vitamin E analogs (e.g., trolox [water-soluble]); carotenoids including carotenes (e.g., β-carotene), xanthophylls (e.g., lutein, zeaxanthin, and mesozeaxanthin), and carotenoids in saffron (e.g., crocin and crocetin); sulfur-containing antioxidants including glutathione (GSH), N-acetyl-L-cysteine (NAC), bucillamine, S-nitroso-N-acetyl-L-cysteine (SNAC), S-allyl-L-cysteine (SAC), S-adenosyl-L-methionine (SAM), α-lipoic acid, and taurine; carnosine, N-acetyl carnosine, curcuminoids (e.g., curcumin, demethoxycurcumin, and tetrahydrocurcumin), cysteamine, ebselen, glutathione, hydroxycinnamic acids, and derivatives (e.g., esters and amides) thereof (e.g., caffeic acid, rosmarinic acid, and tranilast), melatonin and its metabolites, nitrones (e.g., disodium diethyldithiocarbamate [NXY-059]), nitroxides (e.g., XJB-5-131), polyphenols (e.g., flavonoids [e.g., apigenin, genistein, luteolin, naringenin, and quercetin]), superoxide dismutase mimetics (described below), tirilazad, vitamin C, vitamin E and its analogs (e.g., α-tocopherol and trolox), and xanthine derivatives (e.g., pentoxifylline), which are scavengers of ROS and radicals; mitochondrial antioxidants / “vitamins” including ubiquinone (coenzyme Q such as CoQ10), ubiquinol (the reduced, more biologically available form of ubiquinone such as ubiquinol-10), ubiquinone / ubiquinol analogs (e.g., idebenone and mitoquinone) and derivatives; DMQ, DMMQ, MitoE, MitoQ, Mito-TEMPO, MitoVitE, and compounds of the SkQ class (e.g., SkQ1, SkQ2, SkQ3, SkQB, SkQR1, SkQT, SkQT1,Mitochondrial-targeted antioxidants including SkQT1(m), SkQT1(p), SkQTK1, SkQTR1, SkQBerb, and SkQPalm; NADPH oxidase (NOX) inhibitors (e.g., apocynin, declomycin and the angerebate of declomycinol [both of which inhibit the activity and expression of NOX-1, -2 and -4], diphenyleneiodonium, and GKT-831 [formerly GKT-137831, a dual NOX1 / 4 inhibitor]), NADH:ubiquinone oxidoreductase (Complex I) inhibitors (e.g., metformin and rotenone), xanthine oxidase inhibitors (e.g., allopurinol, oxypurinol, tiopurinol, febuxostat, topiroxostat, myo-inositol, phytic acid, and flavonoids [e.g., quercetin, myricetin, and kaempferol]), and myeloperoxidase inhibitors (e.g., azide, 4-aminobenzoic acid hydrazide and PF-06667272, and apoE mimetics such as AEM-28 and AEM-28-14) inhibitors of enzymes that produce ROS; superoxide dismutase (SOD) (e.g., manganese(III)- and zinc(III)-porphyrin complexes (e.g., MnTBAP, MnTMPyP, and ZnTBAP), manganese(II) pentacyclic ring complexes (e.g., M40401 and M40403), manganese(III)-salen complexes (e.g., those disclosed in U.S. Patent No. 7,122,537, which is incorporated herein by reference in its entirety), and OT-551 (a cyclopropyl ester prodrug of temmol hydroxylamine), and SOD mimetics such as apoA-I mimetics like resveratrol and 4F [both of which increase expression]), catalase (e.g., manganese(III)-salen complexes [e.g., those disclosed in U.S. Patent No. 7,122,537], and catalase mimetics such as zinc [which increases activity]), glutathione peroxidase (GPx) (e.g., apomorphine and zinc [both of which increase activity], and beta-catenin, etoposide, and resveratrol [all three of which increase expression]), glutathione reductase (e.g.,Substances that mimic or increase the activity or production of antioxidant enzymes, including redox cofactor nucleotides such as 4-tert-butylcatechol and flavin adenine dinucleotide [FAD] and NADPH (all three enhance activity), glutathione S-transferase (GST) (e.g., phenylalkyl isothiocyanate-cysteine complexes (e.g., S-[N-benzyl(thiocarbamoyl)]-L-cysteine), phenobarbital, rosemary extract, and carnosol [all enhance activity]), thioredoxin (Trx) (e.g., geranylgeranylacetone, prostaglandin E1, and sulforaphane [all increase expression]), NADPH-quinone oxidoreductase 1 (NQO1) (e.g., flavones [e.g., β-naphthoflavone (5,6-benzoflavone)] and triterpenoids [e.g., TP-151 (CDDO), TP-155 (CDDO methyl ester), TP-190, TP-218, TP-222, TP-223 (CDDO), TP-224 (CDDO monomethylamide), TP-225, TP-226 (CDDO dimethylamide), TP-230, TP-235 (CDDO imidazolid), TP-241, CDDO monoethylamide, CDDO mono(trifluoroethyl)amide, and oleanolic acid analog carboxamides such as (+)-TBE-B] (all of these increase expression by activating Nrf2), heme oxygenase 1 (HO-1) (e.g., curcuminoids (e.g., curcumin), triterpenoids (e.g., oleanolic acid analogs such as TP-225), and apoA-I mimetics (e.g., 4F), all of which increase expression), and paraoxonase 1 (PON-1) (e.g., apoE mimetics [e.g., AEM-28 and AEM-28-14] and apoA-I mimetics [e.g., 4F], both types increase activity); activators of nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or Nrf2) (e.g., bardoxolone methyl, OT-551, fumarates (e.g., dimethyl and monomethyl fumarate), dithiothione (e.g., oripraz), flavones (e.g., β-naphthoflavone), isoflavones (e.g., genistein), sulforaphane,Activators of transcription factors that up-regulate the expression of antioxidant enzymes, including trichostatin A (which also up-regulates glutathione synthesis), triterpenoids (e.g., oleanolic acid analogs [e.g., TP-225]), and melatonin (which increases Nrf2 expression)); anthocyanins, benzenediol abitan diterpenes (e.g., carnosic acid), cyclopentenone prostaglandins (such as 15d-PGJ2, which also up-regulates glutathione synthesis), flavonoids (e.g., ginkgo leaf flavonoids [e.g., quercetin and kaempferol, which increase the levels of GSH, SOD, catalase, GPx, and GST]), prenylflavonoids (e.g., isoxanthohumol), flavones (e.g., apigenin), isoflavones (e.g., genistein), flavanones (e.g., naringenin), and flavanols (e.g., catechin and epigallocatechin-3-gallate)), omega-3 fatty acids and their esters (as above), phenylethanoids (e.g., tyrosol and hydroxytyrosol), retinoids (e.g., all-trans retinol [vitamin A]), stilbenoids (e.g., resveratrol), uric acid, apoA-I mimetics (e.g., 4F), apoE mimetics (e.g., AEM-28 and AEM-28-14), and minerals (e.g., selenium and zinc [e.g., zinc monothioglycolate]); and analogs, derivatives, and salts thereof, selected from the group consisting of.,

[0493] In some embodiments, the positive control comprises N-acetylcysteine (NAC) or ebselen.

[0494] In some embodiments, the MSC secretome of the present invention induces a response that is about 5% to over 100% of the response induced by a positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 10% of the response induced by a positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 15% of the response induced by a positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 20% of the response induced by a positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 25% of the response induced by a positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 30% of the response induced by a positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 35% of the response induced by a positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 40% of the response induced by a positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 45% of the response induced by a positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 50% of the response induced by a positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 55% of the response induced by a positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 60% of the response induced by a positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 65% of the response induced by a positive control in an oxidative stress assay.In some embodiments, the MSC secretome of the present invention induces a response that is about 70% of the response induced by a positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 75% of the response induced by a positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 80% of the response induced by a positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 85% of the response induced by a positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 90% of the response induced by a positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 95% of the response induced by a positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 100% of the response induced by a positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is 1 to 100 times or more the response induced by a positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 1 times the response induced by a positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 2 times the response induced by a positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 3 times the response induced by a positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 4 times the response induced by a positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 5 times the response induced by a positive control in an oxidative stress assay.In some embodiments, the MSC secretome of the present invention induces a response that is about 6 times the response induced by the positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 7 times the response induced by the positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 8 times the response induced by the positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 9 times the response induced by the positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 10 times the response induced by the positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 20 times the response induced by the positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 30 times the response induced by the positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 40 times the response induced by the positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 50 times the response induced by the positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 60 times the response induced by the positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 70 times the response induced by the positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 80 times the response induced by the positive control in an oxidative stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 90 times the response induced by the positive control in an oxidative stress assay.In some embodiments, the MSC secretome of the present invention induces a response that is about 100-fold or more of the response induced by the positive control in the oxidative stress assay.

[0495] In some embodiments, positive controls for the misfolded protein response assay include, but are not limited to, GSK2606414 and KIRA6. See Mahameed, M. et al. Cell death & disease vol. 10,4 300. 1 Apr. 2019, which is incorporated herein by reference in its entirety.

[0496] In some embodiments, the MSC secretome of the present invention induces a response that is about 5% to 100% or more of the response induced by the positive control in the misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 10% of the response induced by the positive control in the misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 15% of the response induced by the positive control in the misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 20% of the response induced by the positive control in the misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 25% of the response induced by the positive control in the misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 30% of the response induced by the positive control in the misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 35% of the response induced by the positive control in the misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 40% of the response induced by the positive control in the misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 45% of the response induced by the positive control in the misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 50% of the response induced by the positive control in the misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 55% of the response induced by the positive control in the misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 60% of the response induced by the positive control in the misfolded protein response assay.In some embodiments, the MSC secretome of the present invention induces a response that is about 65% of the response induced by the positive control in a misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 70% of the response induced by the positive control in a misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 75% of the response induced by the positive control in a misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 80% of the response induced by the positive control in a misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 85% of the response induced by the positive control in a misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 90% of the response induced by the positive control in a misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 95% of the response induced by the positive control in a misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 100% of the response induced by the positive control in a misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 1 to 100 times or more of the response induced by the positive control in a misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 1 times the response induced by the positive control in a misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 2 times the response induced by the positive control in a misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 3 times the response induced by the positive control in a misfolded protein response assay.In some embodiments, the MSC secretome of the present invention induces a response that is about 4 times the response induced by the positive control in a misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 5 times the response induced by the positive control in a misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 6 times the response induced by the positive control in a misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 7 times the response induced by the positive control in a misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 8 times the response induced by the positive control in a misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 9 times the response induced by the positive control in a misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 10 times the response induced by the positive control in a misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 20 times the response induced by the positive control in a misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 30 times the response induced by the positive control in a misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 40 times the response induced by the positive control in a misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 50 times the response induced by the positive control in a misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 60 times the response induced by the positive control in a misfolded protein response assay.In some embodiments, the MSC secretome of the present invention induces a response that is about 70 times the response induced by the positive control in a misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 80 times the response induced by the positive control in a misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 90 times the response induced by the positive control in a misfolded protein response assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 100-fold or more the response induced by the positive control in a misfolded protein response assay.

[0497] In some embodiments, the positive control for the ER stress assay includes, but is not limited to, 4-phenylbutyric acid. See Zeng, M. et al. Toxicology Letters vol. 271 (2017): 26-37, which is incorporated herein by reference in its entirety.

[0498] In some embodiments, the MSC secretome of the present invention induces a response that is about 5% to more than 100% of the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 10% of the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 15% of the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 20% of the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 25% of the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 30% of the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 35% of the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 40% of the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 45% of the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 50% of the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 55% of the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 60% of the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 65% of the response induced by the positive control in an ER stress assay.In some embodiments, the MSC secretome of the present invention induces a response that is about 70% of the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 75% of the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 80% of the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 85% of the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 90% of the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 95% of the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 100% of the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is 1 to 100 times or more the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 1 times the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 2 times the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 3 times the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 4 times the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 5 times the response induced by the positive control in an ER stress assay.In some embodiments, the MSC secretome of the present invention induces a response that is about 6 times the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 7 times the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 8 times the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 9 times the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 10 times the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 20 times the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 30 times the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 40 times the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 50 times the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 60 times the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 70 times the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 80 times the response induced by the positive control in an ER stress assay. In some embodiments, the MSC secretome of the present invention induces a response that is about 90 times the response induced by the positive control in an ER stress assay.In some embodiments, the MSC secretome of the present invention induces a response that is at least about 100 times greater than the response induced by a positive control in an ER stress assay.

[0499] In some embodiments, the cells used to evaluate cell survival under oxidative stress, misfolded protein response, or ER stress are selected from the group consisting of ARPE-19, primary human RPE, 661W, and MIO-M1 human Müller cell lines.

[0500] In some embodiments, the cells are seeded at a density of 30% in a 96-well plate and allowed to adhere overnight, after which the cells are incubated with the chloromethyl derivative of H2DCFDA (CM-H2DCFDA) and Mitosoxred for 30 minutes. In some embodiments, the cells are washed before being treated with various concentrations of NaIO4, H2O2, 7-KC, ATRA, or t-BHP for 1 hour in the absence / presence of NAC. In some embodiments, the relative levels of reactive oxygen species (ROS) production are measured 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours or more after treatment, in parallel with cell viability. In some embodiments, the levels of ROS production increase 10- to 1000-fold by oxidative stressors. In some embodiments, the levels of ROS production increase at least 10-fold by oxidative stressors. In some embodiments, the levels of ROS production increase at least 100-fold by oxidative stressors. In some embodiments, the levels of ROS production increase at least 1000-fold by oxidative stressors. In some embodiments, the dose-dependent increase in ROS is evaluated using CM-H2DCFDA and MitoSOX Red and then correlated with cell viability 24 hours after injury. In some embodiments, the dose-dependent increase in ROS is compared to that inhibited by NAC (antioxidant positive control).

[0501] In some embodiments, the MSC secretome provides a prophylactic effect.

[0502] In some embodiments, the advantages of secretome therapy are evaluated based on the results and findings described herein. In some embodiments, the efficacy or activity of the secretome is evaluated based on the results and findings described herein. In some embodiments, one, two, or three conditions of oxidative stress are selected to test secretome activity. In most embodiments, the positive control is NAC. In some embodiments, initial studies are performed in parallel to observe the ROS levels achieved in the study. In some embodiments, the evaluation item is cell viability by microtiter tetrazolium (MTT) assay. In some embodiments, cells exposed to the selected oxidative damage are treated with various concentrations of the secretome, and their viability is evaluated 24 hours after the damage. In some embodiments, the cells are treated with various concentrations of the secretome before being exposed to the selected oxidative damage, and their viability 24 hours after the damage is evaluated. In some embodiments, the negative control consists of a basal medium containing 1% serum condition and heat-denatured secretome condition, and the positive control is NAC. In some embodiments, the effect of the secretome on cellular ROS levels is evaluated under protective conditions.

[0503] Oxidative stress inducer In some embodiments, to induce oxidative stress and / or cellular ROS production, one or more oxidative stress inducers are administered to the assayed cells, and the degree of oxidative stress and / or ROS production in the assayed cells is evaluated.

[0504] In some embodiments, the assayed cells are RPE cells including, but not limited to, ARPE-19, primary human RPE, 661W cells, and MIO-M1 human Müller cells, extraocular explant tissues including, but not limited to, retina, cornea, iris, ciliary body, and / or ciliary body.

[0505] In some embodiments, the oxidative stress inducer includes, but is not limited to, ketocholesterol, FeCl3-sodium nitrilotriacetate (Fe-NTA), H2O2, T-BHP, all-trans retinal, NaIO4, hydroquinone, and oxidized cholesterol (OxLDL).

[0506] In some embodiments, oxidative stress is induced by culturing cells in a hypoxic / oxygen-deprived state.

[0507] In some embodiments, inducing oxidative stress in the assayed cells comprises treating the cells with one or more inducers for about 0.5 to 96 hours. In some embodiments, inducing oxidative stress in the assayed cells comprises treating the cells with one or more inducers for about 0.5 to 72 hours. In some embodiments, inducing oxidative stress in the assayed cells comprises treating the cells with one or more inducers for about 0.5 to 48 hours. In some embodiments, inducing oxidative stress in the assayed cells comprises treating the cells with one or more inducers for about 0.5 to 24 hours. In some embodiments, inducing oxidative stress in the assayed cells comprises treating the cells with one or more inducers for about 0.5 to 12 hours. In some embodiments, inducing oxidative stress in the assayed cells comprises treating the cells with one or more inducers for about 0.5 to 6 hours. In some embodiments, inducing oxidative stress in the assayed cells comprises treating the cells with one or more inducers for about 6 to 96 hours. In some embodiments, inducing oxidative stress in the assayed cells comprises treating the cells with one or more inducers for about 12 to 96 hours. In some embodiments, inducing oxidative stress in the assayed cells comprises treating the cells with one or more inducers for about 24 to 96 hours. In some embodiments, inducing oxidative stress in the assayed cells comprises treating the cells with one or more inducers for about 36 to 96 hours. In some embodiments, inducing oxidative stress in the assayed cells comprises treating the cells with one or more inducers for about 48 to 96 hours. In some embodiments, inducing oxidative stress in the assayed cells comprises treating the cells with one or more inducers for about 60 to 96 hours. In some embodiments, inducing oxidative stress in the assayed cells comprises treating the cells with one or more inducers for about 72 to 96 hours. In some embodiments, inducing oxidative stress in the assayed cells comprises treating the cells with one or more inducers for about 84 to 96 hours.

[0508] In some embodiments, inducing oxidative stress in the assayed cells comprises treating the cells with one or more inducers for about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 60, 72, 84, 96 hours or more.

[0509] In some embodiments, inducing oxidative stress in the assayed cells comprises culturing the cells in a hypoxic / anoxic condition for about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 60, 72, 84, 96 hours or more.

[0510] In some embodiments, one or more oxidative stress inducers are administered to the assayed cells at a concentration of about 0.01 μM to about 100 mM. In some embodiments, one or more oxidative stress inducers are administered to the assayed cells at a concentration of about 0.01 μM to about 0.1 μM. In some embodiments, one or more oxidative stress inducers are administered to the assayed cells at a concentration of about 0.1 μM to about 1 μM. In some embodiments, one or more oxidative stress inducers are administered to the cells being assayed at a concentration of about 1 μM to about 10 μM. In some embodiments, one or more oxidative stress inducers are administered to the cells being assayed at a concentration of about 10 μM to about 100 μM. In some embodiments, one or more oxidative stress inducers are administered to the cells being assayed at a concentration of about 100 μM to about 1 mM. In some embodiments, one or more oxidative stress inducers are administered to the cells being assayed at a concentration of about 1 mM to about 10 mM. In some embodiments, one or more oxidative stress inducers are administered to the cells being assayed at a concentration of about 10 mM to about 100 mM.

[0511] In some embodiments, the oxidative stress inducer is ketocholesterol nitrilotriacetic acid (Fe-NTA) administered at a concentration of 0.1 to 100 mM for 1 to 72 hours. In some embodiments, oxidative stress is induced by administering Fe-NTA to the cells being assayed at a concentration of about 0.1 to 1 mM, about 1 to 10 mM, or about 10 to 100 mM for about 1 to 24 hours, about 24 to 48 hours, or about 48 to 72 hours.

[0512] In some embodiments, the oxidative stress inducer is sodium nitrilotriacetate iron(III) chloride (Fe-NTA) administered at a concentration of 0.1 to 100 mM for 1 to 72 hours. In some embodiments, oxidative stress is induced by administering Fe-NTA to the cells being assayed at a concentration of about 0.1 to 1 mM, about 1 to 10 mM, or about 10 to 100 mM for about 1 to 24 hours, about 24 to 48 hours, or about 48 to 72 hours.

[0513] In some embodiments, the oxidative stress inducer is H2O2 administered at a concentration of 0.01 to 1000 μM for an exposure of 0.5 to 24 hours. In some embodiments, oxidative stress is induced by administering H2O2 to the cells being assayed at a concentration of about 0.01 to 0.1 μM, about 0.1 to 1 μM, about 1 to 10 μM, about 10 to 100 μM, or about 100 to 1000 μM for about 0.5 to 6 hours, about 6 to 12 hours, about 12 to 18 hours, or about 18 to 24 hours.

[0514] In some embodiments, the oxidative stress inducer is tert-butyl hydroperoxide (T-BHP) at a concentration of 0.01 to 1000 μM for an exposure of 0.5 to 24 hours. In some embodiments, oxidative stress is induced by administering t-BHB to the cells being assayed at a concentration of about 0.01 to 0.1 μM, about 0.1 to 1 μM, about 1 to 10 μM, about 10 to 100 μM, or about 100 to 1000 μM for about 0.5 to 6 hours, about 6 to 12 hours, about 12 to 18 hours, or about 18 to 24 hours.

[0515] In some embodiments, the oxidative stress inducer is all-trans retinol administered at a concentration of 0.01 - 1000 μM for an exposure of 0.5 - 24 hours. In some embodiments, oxidative stress is induced by administering all-trans retinol to the cells being assayed for about 0.5 - 6 hours, about 6 - 12 hours, about 12 - 18 hours, or about 18 - 24 hours at a concentration of about 0.01 - 0.1 μM, about 0.1 - 1 μM, about 1 - 10 μM, about 10 - 100 μM, or about 100 - 1000 μM.

[0516] In some embodiments, the oxidative stress inducer is NaIO4 administered at a concentration of 0.01 - 1000 μM for an exposure of 0.5 - 24 hours. In some embodiments, oxidative stress is induced by administering NaIO4 to the cells being assayed for about 0.5 - 6 hours, about 6 - 12 hours, about 12 - 18 hours, or about 18 - 24 hours at a concentration of about 0.01 - 0.1 μM, about 0.1 - 1 μM, about 1 - 10 μM, about 10 - 100 μM, or about 100 - 1000 μM.

[0517] In some embodiments, the oxidative stress inducer is oxidized cholesterol / OxLDL at a concentration of 0.5 - 24 hours, 10 - 500 ug / ml. In some embodiments, the oxidative stress inducer is glucose oxidase (GOx) at a concentration of about 24 hours, 1 - 100 mU / ml. In some embodiments, oxidative stress is induced by administering OxLDL to the cells being assayed for about 0.5 - 6 hours, about 6 - 12 hours, about 12 - 18 hours, or about 18 - 24 hours at a concentration of about 10 - 100 μg / mL, about 10 - 100 μg / mL, or about 100 - 500 μg / mL.

[0518] Evaluation of the degree of oxidative stress In some embodiments, oxidative stress and / or ROS production is evaluated based on the measurement of DNA oxidation by anti-8-oxo-2'-deoxyguanosine (8-oxo-dG).

[0519] In some embodiments, oxidative stress and / or ROS production is evaluated based on the measurement of lipid oxidation by thiobarbituric acid reactive substances (TBARS).

[0520] In some embodiments, oxidative stress and / or ROS production is evaluated based on the measurement of cytoplasmic ROS using CM-H2DCFDA.

[0521] In some embodiments, oxidative stress and / or ROS production is evaluated based on the measurement of mitochondrial ROS using Mitoxed.

[0522] In some embodiments, oxidative stress and / or ROS production is evaluated based on the measurement of mitochondrial potential (including, but not limited to, evaluating JC-1).

[0523] In some embodiments, oxidative stress is induced by one or more of 7-ketocholesterol, iron(III) nitrilotriacetate sodium (Fe-NTA), hydrogen peroxide (H2O2), tert-butyl hydroperoxide (t-BHP), all-trans retinal, sodium periodate (NaIO4), hydroquinone, oxidized low-density lipoprotein (OxLDL), and the degree of induced oxidative stress and / or ROS production is evaluated based on one or more of DNA oxidation (anti-8-oxo-2'-deoxyguanosine (8-oxo-dG)), lipid oxidation (thiobarbituric acid reactive substances, TBARS), cytoplasmic ROS (CM-H2DCFDA), mitochondrial (ROS) (Mitoxed), and / or mitochondrial potential (JC-1).

[0524] In some embodiments, oxidative stress is induced by ketocholesterol, and the degree of induced oxidative stress and / or ROS production is evaluated based on DNA oxidation. In some embodiments, oxidative stress is induced by ketocholesterol, and the degree of induced oxidative stress and / or ROS production is evaluated based on lipid oxidation. In some embodiments, oxidative stress is induced by ketocholesterol, and the degree of induced oxidative stress and / or ROS production is evaluated based on cytoplasmic ROS. In some embodiments, oxidative stress is induced by ketocholesterol, and the degree of induced oxidative stress and / or ROS production is evaluated based on mitochondrial (ROS). In some embodiments, oxidative stress is induced by ketocholesterol, and the degree of induced oxidative stress and / or ROS production is evaluated based on mitochondrial potential.

[0525] In some embodiments, oxidative stress is induced by Fe-NTA, and the degree of induced oxidative stress and / or ROS production is evaluated based on DNA oxidation. In some embodiments, oxidative stress is induced by Fe-NTA, and the degree of induced oxidative stress and / or ROS production is evaluated based on lipid oxidation. In some embodiments, oxidative stress is induced by Fe-NTA, and the degree of induced oxidative stress and / or ROS production is evaluated based on cytoplasmic ROS. In some embodiments, oxidative stress is induced by Fe-NTA, and the degree of induced oxidative stress and / or ROS production is evaluated based on mitochondrial (ROS). In some embodiments, oxidative stress is induced by Fe-NTA, and the degree of induced oxidative stress and / or ROS production is evaluated based on mitochondrial potential.

[0526] In some embodiments, oxidative stress is induced by H2O2, and the degree of induced oxidative stress is evaluated based on DNA oxidation. In some embodiments, oxidative stress is induced by H2O2, and the degree of induced oxidative stress and / or ROS production is evaluated based on lipid oxidation. In some embodiments, oxidative stress is induced by H2O2, and the degree of induced oxidative stress and / or ROS production is evaluated based on cytoplasmic ROS. In some embodiments, oxidative stress is induced by H2O 2に and is induced by H2O2, and the degree of induced oxidative stress and / or ROS production is evaluated based on mitochondrial (ROS). In some embodiments, oxidative stress is induced by H2O2, and the degree of induced oxidative stress and / or ROS production is evaluated based on mitochondrial potential.

[0527] In some embodiments, oxidative stress is induced by T-BHP, and the degree of induced oxidative stress and / or ROS production is evaluated based on DNA oxidation. In some embodiments, oxidative stress is induced by T-BHP, and the degree of induced oxidative stress and / or ROS production is evaluated based on lipid oxidation. In some embodiments, oxidative stress is induced by T-BHP, and the degree of induced oxidative stress and / or ROS production is evaluated based on cytoplasmic ROS. In some embodiments, oxidative stress is induced by T-BHP, and the degree of induced oxidative stress and / or ROS production is evaluated based on mitochondrial (ROS). In some embodiments, oxidative stress is induced by T-BHP, and the degree of induced oxidative stress and / or ROS production is evaluated based on mitochondrial potential.

[0528] In some embodiments, oxidative stress is induced by all - trans retinal, and the degree of induced oxidative stress and / or ROS production is evaluated based on DNA oxidation. In some embodiments, oxidative stress is induced by all - trans retinal, and the degree of induced oxidative stress and / or ROS production is evaluated based on lipid oxidation. In some embodiments, oxidative stress is induced by all - trans retinal, and the degree of induced oxidative stress and / or ROS production is evaluated based on cytoplasmic ROS. In some embodiments, oxidative stress is induced by all - trans retinal, and the degree of induced oxidative stress and / or ROS production is evaluated based on mitochondrial (ROS). In some embodiments, oxidative stress is induced by all - trans retinal, and the degree of induced oxidative stress and / or ROS production is evaluated based on mitochondrial membrane potential.

[0529] In some embodiments, oxidative stress is induced by NaIO4, and the degree of induced oxidative stress and / or ROS production is evaluated based on DNA oxidation. In some embodiments, oxidative stress is induced by NaIO4, and the degree of induced oxidative stress and / or ROS production is evaluated based on lipid oxidation. In some embodiments, oxidative stress is induced by NaIO4, and the degree of induced oxidative stress and / or ROS production is evaluated based on cytoplasmic ROS. In some embodiments, oxidative stress is induced by NaIO4, and the degree of induced oxidative stress and / or ROS production is evaluated based on mitochondrial (ROS). In some embodiments, oxidative stress is induced by NaIO4, and the degree of induced oxidative stress and / or ROS production is evaluated based on mitochondrial membrane potential.

[0530] In some embodiments, oxidative stress is induced by OxLDL, and the degree of induced oxidative stress and / or ROS production is evaluated based on DNA oxidation. In some embodiments, oxidative stress is induced by OxLDL, and the degree of induced oxidative stress and / or ...

Claims

1. A method for characterizing an MSC secretome, comprising: (i) subjecting the MSC secretome to one or more characterization assays, wherein the characterization assays are selected from the group consisting of characterization of physical components, oxidative stress assay, misfolded protein response assay, ER stress assay, cell response assay, safety analysis, stability assay, proliferation assay, migration assay, adhesion assay, angiogenesis assay, differentiation / scarring assay, inflammation assay, survival and function assay of tissue explants, development or survival / function assay of organoids, epithelial barrier integrity assay, retinal degeneration assay, and / or assay of hereditary retinal diseases including human and animal retinal explants, neuroprotection / neurotrophic assay, and wherein the MSC secretome is optionally preconditioned; and the subjecting; (ii) determining results from the one or more assays of (i); The method comprising the above steps.

2. A method for determining the biopotency and stability of an MSC secretome, comprising: (i) subjecting the MSC secretome to one or more characterization assays, wherein the characterization assays are selected from the group consisting of characterization of physical components, oxidative stress assay, misfolded protein response assay, ER stress assay, cell response assay, safety analysis, stability assay, proliferation assay, migration assay, adhesion assay, angiogenesis assay, differentiation / scarring assay, inflammation assay, in vivo response to oxidative stress (e.g., retinal ischemia-reperfusion), epithelial barrier integrity assay, retinal degeneration assay, and / or assay of hereditary retinal diseases including human and animal retinal explants, neuroprotection / neurotrophic assay, and wherein the MSC secretome is optionally preconditioned; and the subjecting; (ii) determining results from the one or more assays of (i); The method comprising the above steps.

3. A method for determining MSC secretome lot consistency among multiple MSC secretome lots, comprising: (i) providing the MSC secretome to one or more property evaluation assays, wherein the property evaluation assay is selected from the group consisting of property evaluation of physical components, oxidative stress assay, misfolded protein response assay, ER stress assay, cell response assay, safety analysis, stability assay, proliferation assay, migration assay, adhesion assay, angiogenesis assay, differentiation / scarring assay, inflammation assay, epithelial barrier integrity assay, retinal degeneration assay, and / or assay of hereditary retinal diseases including human and animal retinal explants, neuroprotection / neurotrophic assay, and the MSC secretome is optionally preconditioned; the providing; (ii) determining results from the one or more assays of (i); The method comprising the above.

4. The method according to any one of claims 1 to 3, wherein the result of (ii) of the property evaluation of physical components identifies an anti-angiogenic MSC secretome or a prepared composition comprising the secretome.

5. The method according to any one of claims 1 to 3, wherein the result of (ii) of the safety analysis indicates blood compatibility and provides an MSC secretome that shows low pyrogen and / or endotoxin, and / or non-pyrogen and / or endotoxin.

6. The method according to any one of claims 1 to 3, wherein the result of (ii) of the stability assay provides an MSC secretome that shows stability at -20°C, 4°C, and / or 20°C (e.g., room temperature) for at least 7 days or at least 14 days.

7. The method according to any one of claims 1 to 3, wherein the result of (ii) of the proliferation assay provides an MSC secretome that induces proliferation.

8. The method according to any one of claims 1 to 3, wherein the result of (ii) of the migration assay provides an MSC secretome that induces migration.

9. The method according to any one of claims 1 to 3, wherein the result of (ii) of the angiogenesis assay provides an MSC secretome that inhibits or does not promote angiogenesis.

10. The method according to any one of claims 1 to 3, wherein the result of (ii) of the differentiation / scarring assay provides an MSC secretome that inhibits differentiation and / or scarring.

11. The method according to any one of claims 1 to 3, wherein the result of (ii) of the inflammation assay provides an MSC secretome that inhibits inflammation or modifies the immune response.

12. (iii) identifying an MSC secretome lot based on the result of (ii) above, further comprising the method.

13. The method according to any one of claims 1 to 3, further comprising a preconditioning step for the secretome.

14. A panel of tests and / or assays for characterizing the MSC secretome, wherein the panel includes at least two characterization assays, the characterization assay is selected from the group consisting of characterization of physical components, oxidative stress assay, misfolded protein response assay, ER stress assay, safety analysis, stability assay, proliferation assay, migration assay, adhesion assay, angiogenesis assay, differentiation / scarring assay, inflammation assay, epithelial barrier integrity assay, retinal degeneration assay, and / or assay for hereditary retinal diseases including human and animal retinal explants, neuroprotection / neurotrophic assay, the secretome is optionally preconditioned, the panel of tests and / or assays.

15. A panel of tests and / or assays for determining consistency between MSC secretome lots, wherein the panel includes one or more characterization assays, the characterization assay is selected from the group consisting of characterization of physical components, oxidative stress assay, misfolded protein response assay, ER stress assay, safety analysis, stability assay, proliferation assay, migration assay, adhesion assay, angiogenesis assay, differentiation / sscarring assay, inflammation assay, epithelial barrier integrity assay, retinal degeneration assay, and / or assay for hereditary retinal diseases including human and animal retinal explants, neuroprotection / neurotrophic assay, the secretome is optionally preconditioned, the panel of tests and / or assays.

16. The panel of tests and / or assays according to any one of claims 14 to 15, wherein the characterization of the physical components identifies the MSC secretome characterized according to any one of claims 1 to 3, or a composition prepared from the secretome.

17. The panel of tests and / or assays according to any one of claims 14 to 15, wherein the result of (ii) of the safety analysis shows blood compatibility and provides an MSC secretome showing low pyrogen and / or endotoxin, and / or non-pyrogen and / or endotoxin.

18. The panel of tests and / or assays according to any one of claims 14 to 15, wherein the stability assay identifies an MSC secretome that is stable at -20°C, 4°C, and / or 20°C (e.g., room temperature) for at least 7 days or at least 14 days.

19. The panel of tests and / or assays according to any one of claims 14 to 15, wherein the proliferation assay identifies an MSC secretome that induces proliferation.

20. The panel of tests and / or assays according to any one of claims 14 to 15, wherein the migration assay identifies an MSC secretome that induces migration.

21. The panel of tests and / or assays according to any one of claims 14 to 15, wherein the angiogenesis assay identifies an MSC secretome that inhibits angiogenesis or does not promote it.

22. The panel of tests and / or assays according to any one of claims 14 to 15, wherein the differentiation / scarring assay identifies an MSC secretome that inhibits differentiation and / or scarring.

23. The panel of tests and / or assays according to any one of claims 14 to 15, wherein the inflammation assay identifies an MSC secretome that inhibits inflammation or modifies the immune response.

24. The panel of tests and / or assays according to any one of claims 14 to 15, wherein all of the physical component property evaluation, oxidative stress assay, misfolded protein response assay, ER stress assay, safety analysis, stability assay, proliferation assay, migration assay, adhesion assay, angiogenesis assay, differentiation / scarring assay, inflammation assay, and / or epithelial barrier integrity assay, retinal degeneration assay, and / or assay of hereditary retinal diseases including human and animal retinal explants, neuroprotection / neurotrophic assay are performed.

25. The panel of tests and / or assays according to claim 24, wherein the tests and / or assays identify the MSC secretome according to claims 15 to 23.

26. The panel of tests and / or assays according to any one of claims 14 to 25, wherein the MSC secretome is an anti-angiogenic MSC secretome and / or an anti-scarring MSC secretome.

27. The panel of tests and / or assays according to any one of claims 1 to 13, wherein the MSC secretome is an anti-angiogenic MSC secretome and / or an anti-scarring MSC secretome.

28. The panel of tests and / or assays according to any one of claims 14 to 15, wherein the assay panel includes at least one migration assay.

29. The panel of tests and / or assays according to claim 28, wherein the migration assay is an in vitro wound closure assay.

30. The panel of tests and / or assays according to claim 29, wherein the in vitro wound closure assay is selected from the group consisting of a "scratch assay" (also called a "scraping assay"), a circular scraping method, and a circular wound closure assay.

31. The panel of tests and / or assays according to any one of claims 14 to 15, wherein the assay panel includes measurement of reactive oxygen species (ROS).

32. The panel of tests and / or assays according to claim 31, wherein the ROS measurement assay is an in vitro assay using N-acetylcysteine (NAC) or ebselen as a positive control.

33. The in vitro ROS measurement assay is evaluated using chloromethyl-modified H ２ DCFDA (CM-H ２ DCFDA) and / or MitoSOX Red, a panel of tests and / or assays according to claim 32.

34. The panel of tests and / or assays according to any one of claims 14 to 15, wherein the assay panel includes a cell viability assay.

35. The panel of tests and / or assays according to claim 34, wherein the cell viability assay is a microtiter tetrazolium (MTT) assay.

36. The panel of tests and / or assays according to claim 14 or 15, wherein the cells are subjected to an oxidative stress assay.

37. The oxidative stress assay is a) inducing oxidative stress in the cells; and b) evaluating the degree of the oxidative stress. The panel of tests and / or assays according to claim 36, including the above.

38. wherein the oxidative stress is induced by one or more inducers selected from the group consisting of ketocholesterol, FeCl ３ -sodium nitrilotriacetate (Fe-NTA), H ２ O ２ , tert-butyl hydroperoxide (t-BHP), all-trans retinal, NaIO ４ , hydroquinone, and oxidized cholesterol (OxLDL), the panel of tests and / or assays according to claim 37.

39. The oxidative stress is induced by administering ketocholesterol to the cells to be assayed at a concentration of about 0.1 to 1 mM, about 1 to 10 mM, or about 10 to 100 mM for about 1 to 24 hours, about 24 to 48 hours, or about 48 to 72 hours. The panel of tests and / or assays according to claim 37 or 38.

40. The oxidative stress is induced by administering Fe-NTA to the cells to be assayed at a concentration of about 0.1 to 1 mM, about 1 to 10 mM, or about 10 to 100 mM for about 1 to 24 hours, about 24 to 48 hours, or about 48 to 72 hours. The panel of tests and / or assays according to any one of claims 37 to 39.

41. The oxidative stress is induced by administering H ２ O ２ to the cells to be assayed at a concentration of about 0.5 to 6 hours, about 6 to 12 hours, about 12 to 18 hours, or about 18 to 24 hours, about 0.01 to 0.1 μM, about 0.1 to 1 μM, about 1 to 10 μM, about 10 to 100 μM, or about 100 to 1000 μM, the test and / or assay panel according to any one of claims 37 to 40.

42. The oxidative stress is induced by administering t-BHB to the cells to be assayed at a concentration of about 0.01 to 0.1 μM, about 0.1 to 1 μM, about 1 to 10 μM, about 10 to 100 μM, or about 100 to 1000 μM for about 0.5 to 6 hours, about 6 to 12 hours, about 12 to 18 hours, or about 18 to 24 hours. The panel of tests and / or assays according to any one of claims 37 to 41.

43. The oxidative stress is induced by administering all-trans retinol to the cells to be assayed at a concentration of about 0.01 to 0.1 μM, about 0.1 to 1 μM, about 1 to 10 μM, about 10 to 100 μM, or about 100 to 1000 μM for about 0.5 to 6 hours, about 6 to 12 hours, about 12 to 18 hours, or about 18 to 24 hours. The panel of tests and / or assays according to any one of claims 37 to 42.

44. The oxidative stress is induced by administering NaIO ４ to the cells to be assayed at a concentration of about 0.5 to 6 hours, about 6 to 12 hours, about 12 to 18 hours, or about 18 to 24 hours, about 0.01 to 0.1 μM, about 0.1 to 1 μM, about 1 to 10 μM, about 10 to 100 μM, or about 100 to 1000 μM, the test and / or assay panel according to any one of claims 37 to 43.

45. The oxidative stress is induced by administering OxLDL to the cells to be assayed at a concentration of about 10 to 100 μg / mL, about 10 to 100 μg / mL, or about 100 to 500 μg / mL for about 0.5 to 6 hours, about 6 to 12 hours, about 12 to 18 hours, or about 18 to 24 hours. The panel of tests and / or assays according to any one of claims 37 to 44.

46. Evaluating the degree of the oxidative stress is as follows: i) Measuring DNA oxidation using anti-8-oxo-2'-deoxyguanosine (8-oxo-dG); ii) Measuring lipid oxidation using thiobarbituric acid-reactive substances (TBARS); iii) Measuring cytoplasmic ROS using CM-H2DCFDA, iv) Measuring mitochondrial ROS using MitoXred, v) Measuring mitochondrial potential such as measuring JC-1, A panel of tests and assays according to any one of claims 37 to 45, comprising one or more of the above.

47. The cells to be assayed are Changes in epithelial polarity (e.g., Na / K ATPase polarity), tight junctions (e.g., ZO-1, transepithelial polarity, and transepithelial electrical resistance (TEER)), phagocytosis of the outer segment, degradation of the outer segment, phagosome composition and function, cathepsin D distribution activity, mitochondrial potential, inflammasome activation / activity, Hif-1 alpha activation / activity, apoptosis, ferroptosis, NRF-2 activation / activity, NOQ1 activation / activity, GPX4 activation / activity, SLC7AII activation / activity, annexin V activation / activity, propidium iodide staining, IRE1 / XBP1 activation / activity, PERK / ATF4 / CHOP activation / activity, UPR activation / activity, ACS24 activation / activity, complement activation / sensitivity, lipid deposition, apoptosis, and necrosis A panel of tests and / or assays according to any one of claims 36 to 46, wherein the panel is evaluated for one or more phenotypes selected from the group consisting of

48. The panel of tests and / or assays according to any one of claims 36 to 47, wherein the MSC secretome is preconditioned before the oxidative stress is induced.

49. The panel of tests and / or assays according to any one of claims 36 to 47, wherein the MSC secretome is preconditioned during the induction of the oxidative stress.

50. The panel of tests and / or assays according to any one of claims 36 to 47, wherein the MSC secretome is preconditioned after the oxidative stress is induced.

51. Preconditioning the MSC secretome includes affecting the secretory profile of the MSCs as described in any one of the preceding claims, including one or more of the following: changing the culture format (e.g., 2D plane vs. 3D bioreactor), different biomaterial scaffolds, co-culture, addition of pharmacological compounds, growth factors, chemokines, addition of Toll-like receptor agonists, inflammatory cytokines, advanced glycation end products (AGEs), oxidized phospholipids, malondialdehyde, or carboxyethylpyrrole, presence of agitation of the ECM, culture under shear stress, agitation or suspension as aggregates or within a matrix, induction of misfolded protein response, ER stress, induction of MSC differentiation, culture in the presence of conditioned media, and hypoxia / anoxia.

52. Preconditioning the MSC secretome includes hypoxic preconditioning, which includes culturing the MSCs in a hypoxic culture environment, of the method or panel of tests and / or assays according to any one of the preceding claims.

53. The method or panel of tests and / or assays according to any one of the preceding claims, wherein the oxygen level is from about 0% to 2%.

54. The method or panel of tests and / or assays according to any one of the preceding claims, wherein the hypoxic preconditioning is carried out for about 4 to 12 hours, about 12 to 24 hours, about 24 to 36 hours, about 36 to 48 hours, about 48 to 60 hours, or about 60 to 72 hours.

55. Preconditioning the MSC secretome includes treating the MSCs with one or more inflammatory cytokines, of the method or panel of tests and / or assays according to any one of the preceding claims.

56. Preconditioning the MSC secretome involves treating the MSC with one or more of the following, namely IL-6, PGE2, IDO, IFNγ, SDF-1, TGF-α, H ２ O ２ , FGF-2, IGF-1, BMP-2, atorvastatin, oxytocin, curcumin, lipopolysaccharide, and nicotinamide (NIC), vasoactive intestinal peptide (VIP), and / or diazoxide, a method or test and / or assay panel according to any one of the preceding claims.

57. Preconditioning the MSC secretome includes culturing the MSCs in 3D (e.g., using a 3D bioreactor), of the method or panel of tests and / or assays according to any one of the preceding claims.

58. Preconditioning the MSC secretome includes inducing a misfolded protein response in the MSCs, of the method or panel of tests and / or assays according to any one of the preceding claims.

59. The method or test and / or assay panel according to any one of the preceding claims, wherein preconditioning the MSC secretome comprises culturing the MSCs under conditions appropriate for inducing ER stress.

60. The method or test and / or assay panel according to any one of the preceding claims, wherein preconditioning the MSC secretome comprises culturing the MSCs under appropriate conditions to induce differentiation of the MSCs into mature retinal cell types or their precursors.

61. A bone marrow-derived mesenchymal stem cell (MSC) secretome composition comprising HGF, pentraxin-3 (TSG-14), VEGF, TIMP-1, serpin E1, <5 ng / mL IL-8, and an osmotic modifier, wherein the MSC secretome is preconditioned.

62. The MSC secretome composition, wherein i. at least one trophic factor / cytokine selected from the group consisting of TIMP-2 and VEGF-A, ii. at least one additional factor selected from the group consisting of PEDF (serpin F1), IGFBP-2, IGFBP-3, SDF-1, kallikrein 3, MCP-1, angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, and PDGF, and / or iii. at least one further factor selected from the group consisting of DPPIV (dipeptidyl peptidase-4), uPA, angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, and thrombospondin-1 is further included. The MSC secretome composition according to claim 61.

63. The MSC secretome composition according to claim 62, wherein the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), and serpin F1, at a concentration of 1 ng / mL to 100 ng / mL.

64. The MSC secretome composition according to claim 62, wherein the MSC secretome contains at least one factor selected from the group consisting of angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, thrombospondin-1, angiogenin, DPPIV, IGFBP-3, and uPA, and has a concentration of 400 pg / mL to 3000 pg / mL.

65. The MSC secretome composition according to claim 62, further comprising at least one factor selected from the group consisting of apolipoprotein A1, complement factor D, complement factor H, complement factor I, C1 esterase inhibitor (C1-INH), C4b-binding protein (C4BP), CD46, complement receptor type 1 (CR1), C-reactive protein, cystatin C, DKK-1, enppurin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15, and IFNγ.

66. The MSC secretome composition according to claim 62, wherein the MSC secretome composition includes a ratio of anti-angiogenic to pro-angiogenic, and the ratio is >2, >3, >4, or >5.

67. The MSC secretome composition according to claim 62, wherein the MSC secretome composition contains VEGF at a concentration of 1 pg / mL to 400 pg / mL.

68. The MSC secretome composition according to claim 62, wherein the level of VEGF is 1 / 5 to 1 / 10 of the level of serpin E1.

69. The MSC secretome composition according to claim 62, wherein the composition contains one or more anti-angiogenic factors, and the ratio of the total concentration of the one or more anti-angiogenic factors to the concentration of VEGF is >2, >3, >4, or >5.

70. The MSC secretome composition according to claim 62, wherein the MSC secretome contains bFGF, PLGF, and PDGF at a concentration of less than 1000 pg / mL.

71. The MSC secretome composition according to claim 60, wherein the MSC secretome composition has a pH of about 4.7 to about 7.

5.

72. The MSC secretome composition according to claim 62, wherein the MSC secretome composition is formulated in a buffer system selected from the group consisting of disodium monophosphate / sodium monophosphate, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and citric acid / disodium phosphate.

73. The MSC secretome composition according to claim 62, wherein the MSC secretome composition further comprises monosodium / disodium phosphate, mannitol, and trehalose, and the composition has a pH of about 7.

4.

74. The MSC secretome composition according to claim 62, wherein the MSC secretome composition further comprises a divalent cation.

75. The MSC secretome composition according to claim 74, wherein the divalent cation is selected from the group consisting of Mg2+, Ca2+, and Zn2+.

76. The MSC secretome composition according to claim 62, wherein the MSC secretome composition further comprises disodium phosphate / citric acid, mannitol, and trehalose, and the composition has a pH of about 6.

4.

77. The MSC secretome composition according to claim 62, wherein the MSC secretome composition further comprises an agent that increases viscosity.

78. The MSC secretome composition according to claim 77, wherein the adhesive is selected from the group consisting of hypromellose, poloxamer 407, poloxamer 188, poloxomer 237, poloxomer 338, hypromellose (HPMC), polycarbophil, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyimide, sodium hyaluronate, gellan gum, poly(lactic-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethyl cellulose (CMC), or hydroxypropylmethyl cellulose (HPMC), hydroxymethyl cellulose, hydroxyethyl cellulose, sodium carboxymethyl cellulose, fibrin glue, polyethylene glycol, and GelCORE.

79. The MSC secretome composition according to claim 62, wherein the MSC secretome composition does not contain one or more components selected from the group consisting of foreign body components, phenol red, peptides and biomolecules <3 kDa, antibiotics, protein aggregates> 200 nm, cells, non-exosome / non-extracellular vesicle cell debris, hormones, and L-glutamine.

80. The MSC secretome composition, i. 0.3 to 4.5 ng / mL of HGF, ii. 0.5 to 20 ng / mL of pentraxin-3 (TSG-14), iii. 100 to 600 pg / mL of VEGF, iv. 10 to 200 ng / mL of TIMP-1, v. 20 to 80 ng / mL of serpin E1, vi. <5 ng / mL of IL-8, and the MSC secretome composition according to claim 62.

81. The MSC secretome composition according to claim 62, wherein the MSC secretome composition contains an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.

82. The MSC secretome composition according to claim 61, wherein the osmotic pressure modifier is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin.

83. A stable bone marrow-derived mesenchymal stem cell (MSC) secretome preparation, i. 2 μg to 20 μg of MSC secretome per mL, ii. 2 mg to 3 mg of monobasic sodium phosphate per mL, iii. 11 mg to 12 mg of dibasic sodium phosphate per mL, iv. 11.5 mg to 13 mg of mannitol per mL, v. 23 mg to 24 mg of trehalose dihydrate, vi. 0.5 mg to 2 mg of hypromellose per mL, comprising, having a pH of about 4.7 to about 7.5, and the MSC secretome is preconditioned, the stable bone marrow-derived mesenchymal stem cell (MSC) secretome preparation.

84. A stable bone marrow-derived mesenchymal stem cell (MSC) secretome preparation, i. 0.004% to 0.08% w / w of MSC secretome, ii. 4% to 5% w / w of monobasic sodium phosphate, iii. 21.5% to 23% w / w of dibasic sodium phosphate, iv. 23% to 25% w / w of mannitol, v. 46% to 48% w / w of trehalose dihydrate, vi. 1% to 3% w / w of hypromellose, comprising a stable bone marrow-derived mesenchymal stem cell (MSC) secretome preparation, having a pH of about 4.7 to about 7.5 and wherein the MSC secretome is preconditioned. **Claim 85** A stable bone marrow-derived mesenchymal stem cell (MSC) secretome preparation comprising: i. 10 mM sodium phosphate; ii. 10 mM histidine HCl; iii. 10% trehalose dihydrate; iv. 0.01% polysorbate 20; comprising a stable bone marrow-derived mesenchymal stem cell (MSC) secretome preparation, having a pH of about 5.5 and wherein the MSC secretome is preconditioned. **Claim 86** A stable bone marrow-derived mesenchymal stem cell (MSC) secretome preparation comprising: i. 10 mM sodium phosphate; ii. 5% sucrose; iii. 10 mM sodium chloride; comprising a stable bone marrow-derived mesenchymal stem cell (MSC) secretome preparation, having a pH of about 6.2 and wherein the MSC secretome is preconditioned. **Claim 87** A stable bone marrow-derived mesenchymal stem cell (MSC) secretome preparation comprising: i. 10 mM sodium phosphate; ii. 5.8% sucrose; iii. 0.02% polysorbate 80; comprising a stable bone marrow-derived mesenchymal stem cell (MSC) secretome preparation, having a pH of about 6.2 and wherein the MSC secretome is preconditioned. **Claim 88** A method for treating an eye disease in a subject in need thereof, comprising administering to the subject a bone marrow-derived mesenchymal stem cell (MSC) secretome composition, wherein the MSC secretome composition comprises HGF, pentraxin-3 (TSG-14), VEGF, TIMP-1, serpin E1, and <5 ng / mL IL-8, and wherein the MSC secretome is preconditioned. **Claim 89** The MSC secretome composition comprises: i. at least one trophic factor / cytokine selected from the group consisting of TIMP-2 and VEGF-A; ii. at least one additional factor selected from the group consisting of PEDF (serpin F1), IGFBP-2, IGFBP-3, SDF-1, kallikrein 3, MCP-1, angiotensinogen, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, and PDGF; iii. at least one further factor selected from the group consisting of DPPIV (dipeptidyl peptidase-4), uPA, angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, and thrombospondin-1; The treatment method according to claim 88, further comprising the above.

90. The treatment method according to claim 89, wherein the MSC secretome composition contains at least one factor selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), and serpin F1, in an amount of 1 ng / mL to 100 ng / mL.

91. The treatment method according to claim 89, wherein the MSC secretome composition contains at least one factor selected from the group consisting of angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, thrombospondin-1, angiotensinogen, DPPIV, IGFBP-3, and uPA, in an amount of 400 pg / mL to 3000 pg / mL.

92. The treatment method according to claim 89, wherein the MSC secretome composition further contains at least one factor selected from the group consisting of apolipoprotein A1, complement factor D, complement factor H, complement factor I, C1 esterase inhibitor (C1-INH), C4b-binding protein (C4BP), CD46, complement receptor type 1 (CR1), C-reactive protein, cystatin C, DKK-1, enmprin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15, and IFNγ.

93. The treatment method according to claim 89, wherein the MSC secretome composition contains a ratio of anti-angiogenic to pro-angiogenic, and the ratio is >2, >3, >4, or >5.

94. The treatment method according to claim 89, wherein the MSC secretome contains VEGF in an amount of 1 pg / mL to 400 pg / mL.

95. The treatment method according to claim 89, wherein the level of said VEGF is 1 / 5 to 1 / 10 of the level of said serpin E1.

96. The treatment method according to claim 89, wherein the MSC secretome composition contains one or more anti-angiogenic factors, and the total of the concentrations of the one or more anti-angiogenic factors relative to the concentration of said VEGF is >2, >3, >4, or >5.

97. The treatment method according to claim 89, wherein the MSC secretome composition contains bFGF, PLGF, and PDGF at less than 1000 pg / mL.

98. The treatment method according to claim 89, wherein the MSC secretome composition has a pH of about 4.7 to about 7.

5.

99. The treatment method according to claim 89, wherein the MSC secretome composition is formulated with a buffer system selected from the group consisting of disodium monophosphate / sodium citrate, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and citric acid / disodium phosphate.

100. The treatment method according to claim 89, wherein the MSC secretome composition further contains an osmotic pressure modifier.

101. The treatment method according to claim 100, wherein the osmotic pressure modifier is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin.

102. The treatment method according to claim 89, wherein the MSC secretome composition further contains monosodium / disodium phosphate, mannitol, and trehalose, and the composition has a pH of about pH 7.

4.

103. The treatment method according to claim 89, wherein the MSC secretome composition further contains divalent cations.

104. The treatment method according to claim 103, wherein the divalent cation is selected from the group consisting of Mg2+, Ca2+, and Zn2+.

105. The treatment method according to claim 89, wherein the MSC secretome composition further contains disodium phosphate / citric acid, mannitol, and trehalose, and the composition has a pH of about pH 6.

4.

106. The treatment method according to claim 89, wherein the MSC secretome composition does not contain one or more components selected from the group consisting of foreign body components, phenol red, peptides and biomolecules <3 kDa, antibiotics, protein aggregates> 200 nm, cells, non-exosome / non-extracellular vesicle cell debris, hormones, and L-glutamine.

107. The treatment method according to claim 89, wherein the MSC secretome composition contains an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.

108. The treatment method according to claim 88, wherein the MSC secretome composition further contains an agent that increases viscosity.

109. The treatment method according to claim 108, wherein the adhesive is selected from the group consisting of hypromellose, poloxamer 407, poloxamer 188, poloxamer 237, poloxamer 338, hypromellose (HPMC), polycarbophil, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyimide, sodium hyaluronate, gellan gum, poly(lactic-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethyl cellulose (CMC), or hydroxypropyl methylcellulose (HPMC), hydroxymethyl cellulose, hydroxyethyl cellulose, sodium carboxymethyl cellulose, fibrin glue, polyethylene glycol, and GelCORE.

110. The MSC secretome composition is i. 0.3 to 4.5 ng / mL of HGF, ii. 0.5 to 20 ng / mL of pentraxin-3 (TSG-14), iii. 100 to 600 pg / mL of VEGF, iv. 10 to 200 ng / mL of TIMP-1, v. 20 to 80 ng / mL of serpin E1, vi. <5 ng / mL of IL-8, and the treatment method according to claim 88.

111. A method for treating an eye disease in a subject in need of treatment for an eye disease, comprising administering a bone marrow-derived mesenchymal stem cell (MSC) secretome composition to the subject, wherein the MSC secretome composition is i. 2 μg to 20 μg of MSC secretome per mL, ii. 2 mg to 3 mg of monobasic sodium phosphate per mL, iii. 11 mg to 12 mg of dibasic sodium phosphate per mL. iv. Mannitol at 11.5 mg to 13 mg per 1 mL, and v. Trehalose dihydrate at 23 mg to 24 mg, and vi. Hypromellose at 0.5 mg to 2 mg per 1 mL, A stable bone marrow-derived mesenchymal stem cell (MSC) secretome formulation, with a pH of about 4.7 to about 7.5, and the MSC secretome being preconditioned, The treatment method.

112. A method for treating an eye disease in a subject in need of treatment for the eye disease, comprising administering to the subject a bone marrow-derived mesenchymal stem cell (MSC) secretome composition, wherein the MSC secretome composition comprises i. 0.004% to 0.08% w / w of MSC secretome, and ii. Monobasic sodium phosphate at 4% to 5% w / w, and iii. Dibasic sodium phosphate at 21.5% to 23% w / w, and iv. Mannitol at 23% to 25% w / w, and v. Trehalose dihydrate at 46% to 48% w / w, and vi. Hypromellose at 1% to 3% w / w, A stable mesenchymal stem cell (MSC) secretome formulation, with a pH of about 4.7 to about 7.5, and the MSC secretome being preconditioned, The treatment method.

113. A method for treating an eye disease in a subject in need of treatment for the eye disease, comprising administering to the subject a bone marrow-derived mesenchymal stem cell (MSC) secretome composition, wherein the MSC secretome composition comprises i. Sodium phosphate 10 mM, and ii. Histidine HCl 10 mM, and iii. Trehalose dihydrate 10%, and iv. Polysorbate 20 0.01%, A stable mesenchymal stem cell (MSC) secretome formulation, with a pH of about 5.5, and the MSC secretome being preconditioned, The treatment method.

114. A method for treating an eye disease in a subject in need of treatment for the eye disease, comprising administering to the subject a bone marrow-derived mesenchymal stem cell (MSC) secretome composition, wherein the MSC secretome composition comprises i. Sodium phosphate 10 mM, and ii. Sucrose 5%, and iii. Sodium chloride 10 mM, A stable mesenchymal stem cell (MSC) secretome formulation, with a pH of about 6.2, and the MSC secretome being preconditioned, The treatment method.

115. A method for treating an eye disease in a subject in need of treatment for the eye disease, comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition derived from bone marrow, wherein the MSC secretome composition comprises i. 10 mM sodium phosphate, and ii. 5.8% sucrose, and iii. 0.02% polysorbate 80, and is a stable mesenchymal stem cell (MSC) secretome formulation, having a pH of about 6.2 and wherein the MSC secretome is preconditioned, said treatment method.

116. A composition for inducing eye wound healing, comprising a mesenchymal stem cell (MSC) secretome and an osmotic pressure modifier, wherein the MSC secretome is preconditioned, and the ability of the composition to promote eye wound healing is a) providing a layer of corneal cells, and b) introducing a wound gap into the layer of corneal cells, and c) determining whether the wound gap heals in the presence of the composition, wherein the composition is administered to the corneal cells either before or after step b), as shown by a wound healing assay, wherein suturing of the wound gap indicates the ability of the composition to induce eye wound healing, said composition.

117. The composition according to claim 116, wherein the corneal cells are corneal epithelial cells.

118. The composition according to claim 116, wherein the corneal cells are corneal keratocytes (or fibroblasts).

119. The composition according to claim 116, wherein the layer of corneal cells is a confluent monolayer.

120. The composition according to claim 116, wherein the wound gap is introduced by mechanically disrupting the layer of corneal cells.

121. The composition according to claim 116, wherein the wound gap is introduced by chemically disrupting the layer of corneal cells.

122. The composition according to claim 116, wherein the wound gap comprises a linear gap.

123. The composition according to claim 114, wherein the wound gap comprises a circular gap.

124. The composition according to claim 116, wherein determining whether the wound gap sutures in step c) comprises detecting and quantifying daily the migration and / or proliferation of the corneal cells within the wound gap.

125. The composition according to claim 124, wherein the migration and / or proliferation of the corneal cells is characterized as the number of corneal cells that have migrated and / or proliferated within the wound gap.

126. Determining whether the wound gap heals in step c) includes measuring the size of the wound gap daily, and the size of the wound gap is expressed as a percentage of the initial size of the wound gap measured immediately after the wound gap was introduced. The composition according to claim 116.

127. The composition according to claim 126, wherein the size of the wound gap is characterized as the surface area of the wound gap.

128. The composition according to claim 126, wherein the size of the wound gap is characterized as the width of the wound gap.

129. The composition according to claim 116, wherein step c) is performed within a period of 2 to 4 days after completion of step b).

130. The composition according to claim 116, wherein step c) is performed within a period of 2 days after completion of step b).

131. The composition according to claim 116, wherein step c) is performed within a period of 3 days after completion of step b).

132. The composition according to claim 116, wherein the wound healing assay further includes a step of concentrating the composition and, optionally, a step of exchanging a buffer with the composition before administering the composition to the corneal cells.

133. The composition according to claim 116, wherein the wound healing assay further includes a step of diluting the composition and, optionally, a step of exchanging a buffer with the composition before administering the composition to the corneal cells.

134. The composition according to claim 116, wherein the composition contains at least 45 μg / ml of secretome protein.

135. A composition for inducing ocular wound healing comprising a mesenchymal stem cell (MSC) secretome and an osmotic modifier, wherein the MSC secretome is preconditioned, and the ability of the composition to promote ocular wound healing is a) providing a layer of corneal cells; b) introducing a wound gap into the layer of corneal cells; c) determining whether the wound gap heals in the presence of the composition, wherein the composition is administered to the corneal cells either before or after step b). Indicated by a wound healing assay, including The suturing of the wound gap indicates the ability of the composition to induce corneal wound healing. Said composition.

136. The composition according to claim 135, wherein the corneal cells are corneal epithelial cells.

137. The composition according to claim 135, wherein the corneal cells are corneal keratocytes (or fibroblasts).

138. The composition according to claim 135, wherein the layer of corneal cells is a confluent monolayer.

139. The composition according to claim 135, wherein the wound gap is introduced by mechanically disrupting the layer of corneal cells.

140. The composition according to claim 135, wherein the wound gap is introduced by chemically disrupting the layer of corneal cells.

141. The composition according to claim 135, wherein the wound gap includes a linear gap.

142. The composition according to claim 135, wherein the wound gap includes a circular gap.

143. Determining whether the wound gap closes in step c) includes detecting the migration and / or proliferation of the corneal cells within the wound gap and quantifying it daily. The composition according to claim 135.

144. The composition according to claim 143, wherein the migration and / or proliferation of the corneal cells is characterized as the number of corneal cells that have migrated and / or proliferated within the wound gap.

145. Determining whether the wound gap heals in step c) includes measuring the size of the wound gap daily, and the size of the wound gap is expressed as a percentage of the initial size of the wound gap measured immediately after the wound gap was introduced. The composition according to claim 135.

146. The composition according to claim 145, wherein the size of the wound gap is characterized as the surface area of the wound gap.

147. The composition according to claim 145, wherein the size of the wound gap is characterized as the width of the wound gap.

148. The composition according to claim 135, wherein step c) is performed within a period of 2 to 4 days after completion of step b).

149. The composition according to claim 135, wherein step c) is performed within a period of 2 days after completion of step b).

150. The composition according to claim 135, wherein step c) is carried out within a period of 3 days after completion of step b).

151. The composition according to claim 135, wherein the wound healing assay further comprises a step of concentrating the composition and optionally a step of exchanging a buffer solution for the composition before administering the composition to the corneal cells.

152. The composition according to claim 135, wherein the wound healing assay further comprises a step of diluting the composition and optionally a step of exchanging a buffer solution for the composition before administering the composition to the corneal cells.

153. The composition according to claim 135, wherein the composition contains at least 45 μg / ml of secretome protein.

154. The following methods for preconditioning the MSC secretome, including one or more of the following: changing the culture format (e.g., 2D plane vs. 3D bioreactor), different biomaterial scaffolds, co-culture, addition of pharmacological compounds, growth factors, chemokines, Toll-like receptor agonists, inflammatory cytokines, advanced glycation end products (AGEs), oxidized phospholipids, malondialdehyde, or carboxyethylpyrrole, agitation of the ECM, culturing under shear stress, agitation or suspension as aggregates or within a matrix, induced misfolded protein response, ER stress, induction of MSC differentiation, culturing in the presence of conditioned media, and hypoxia / anoxia, which affect the secretion profile of the MSC.

155. The method according to claim 154, including hypoxia preconditioning by culturing the MSC in a hypoxic culture environment.

156. The method according to claim 155, wherein the oxygen level is about 0% to 2%.

157. The method according to claim 155 or 156, wherein the hypoxia preconditioning is carried out for about 4 to 12 hours, about 12 to 24 hours, about 24 to 36 hours, about 36 to 48 hours, about 48 to 60 hours, or about 60 to 72 hours.

158. The method according to claim 154, wherein preconditioning the MSC secretome includes treating the MSC with one or more inflammatory cytokines.

159. Preconditioning the MSC secretome involves treating the MSC with one or more of the following, namely IL-6, PGE2, IDO, IFNγ, SDF-1, TGF-α, H ２ O ２ , FGF-2, IGF-1, BMP-2, atorvastatin, oxytocin, curcumin, lipopolysaccharide, and nicotinamide (NIC), vasoactive intestinal peptide (VIP), and / or diazoxide, according to the method of claim 154.

160. The method according to claim 154, wherein preconditioning the MSC secretome comprises culturing the MSCs in 3D (for example, using a 3D bioreactor).

161. The method according to claim 154, wherein preconditioning the MSC secretome comprises inducing an unfolded protein response in the MSCs.

162. The method according to claim 154, wherein preconditioning the MSC secretome comprises culturing the MSCs under conditions appropriate for inducing ER stress.

163. The method according to claim 154, wherein preconditioning the MSC secretome comprises culturing the MSCs under appropriate conditions to induce differentiation of the MSCs into mature retinal cell types or their precursors.

164. A method of treating an eye disease in a patient in need thereof, the method comprising administering to the patient the preconditioned MSC secretome according to any one of the preceding claims.

165. Use of the preconditioned MSC secretome according to any one of the preceding claims for treating an eye disease in a patient in need of treatment of an eye condition.

166. The method according to claim 164, or the use according to claim 165, wherein the eye disease is selected from the group consisting of retinal diseases, macular diseases, chronic graft-versus-host disease (GvHD), Stevens-Johnson syndrome, ocular mucous membrane pemphigoid, persistent corneal epithelial defect (PCED), limbal stem cell deficiency (LSCD), dry eye, damage to optic nerve tissue, and traumatic injury to the eye (such as concussion injury, ocular contusion, or chemical burn).

167. Use of the preconditioned MSC secretome according to any one of the preceding claims by the method according to any one of the preceding claims.

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