Methods and assays for secretory proteome activity analysis

Abstract

The present application provides methods and assays for assessing mesenchymal stem cell secretory proteomes for use of MSC secretory proteomes in methods of treating ocular disorders and / or conditions.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 359,743, filed on July 8, 2022, which is incorporated herein by reference in its entirety. Background Art

[0003] Regenerative medicine is a field of medicine concerned with the replacement or regeneration of human cells, tissues, or organs to restore or establish normal function. For example, stem cell therapy can be used to treat, prevent, or cure a variety of diseases and conditions.

[0004] Stem cells are cells with unlimited division ability, and under certain specific conditions, can differentiate into a variety of different cell types. Totipotent stem cells are stem cells with the potential to produce all cells and tissues that make up the embryo. Multipotent stem cells are stem cells that produce cells of the mesoderm, endoderm, and ectoderm. Multipotent stem cells are stem cells that can differentiate into two or more cell types, while unipotent stem cells are stem cells that differentiate into only one cell type. One type of such stem cells is mesenchymal stem cells. See, for example, U.S. patent application US20190046576.

[0005] However, it is difficult to produce and store live stem cell-based therapies on a clinically relevant scale. (See, Trainor et al., Nature Biotechnology 32(1)(2014)). In addition, the therapeutic efficacy and regenerative capacity of such therapies are often variable, and cells may die before or during transplantation. (See, Newell, Seminars in Immunopathology 33(2):91(2011)). Implanted 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 "dose". Therefore, there is a need in the art for additional regenerative therapies that can overcome the current cost, storage and manufacturing quality control limitations associated with cell-based regenerative medicine therapies. Especially in the case of ocular diseases.

[0006] Blast and blunt trauma to the eye can result in a range of mechanical damage to the ocular contents, including retinal concussion, traumatic cataracts, disruption of the zonular attachment of the lens, angle recession, iridodialysis, and rupture of the pupillary sphincter and optic nerve. Treatment of these injuries is limited to mechanical repair of the iris (if possible), replacement of the lens with a plastic lens implant, and repair of retinal detachment. There is no treatment to repair the cellular structure of the retina or anterior chamber due to injury, disease, or inherited disorders such as retinitis pigmentosa. In addition, during Operation Iraqi Freedom, traumatic optic neuropathy and optic nerve avulsion were the six major types of eye injuries requiring specialized ophthalmic care (Cho and Savitsky, "Ocular Trauma Chapter 7", in Combat Casualty Care: Lessons learned from Oef and Oif, edited by Brian Eastbridge and Eric Savitsky, pp. 299-342, Ft. Detrick, Md.: Borden Institute (US) Government Printing Office, 2012), which is incorporated herein by reference in its entirety. Sixty percent of traumatic head injuries result in neuro-ophthalmological abnormalities (Van Stavern et al., J Neuro-Ophthamol 21(2): 112-117, 2001) (incorporated herein by reference in its entirety), half of which involve the optic nerve or visual pathway. Traumatic damage to neurons can result in axonal damage and irreversible neuronal loss, leading to permanent defects. Although many potential neuroprotective therapies have been identified in animals, these single agents generally fail to translate into therapies in human clinical trials (Turner et al., J Neurosurg 118(5):1072-1085, 2013, incorporated herein by reference in its entirety). Combination therapies that affect multiple cellular targets may be required to prevent neuronal damage or restore neuronal function.

[0007] The cornea, as the outermost tissue of the eye, has a protective effect, but it is extremely vulnerable to severe damage and disease. It has no blood vessels, which makes it transparent, but also limits its healing ability. Because corneal damage may lead to irreversible blindness, timely intervention and active treatment are required. The critical need for improved healing therapies for the surface of the eye is particularly evident for chemical burns and severe corneal diseases (such as acute chronic graft-versus-host disease (GvHD), Stevens-Johnson Syndrome, ocular mucous membrane pemphigoid and other diseases that cause persistent corneal epithelial defects), the total incidence of these diseases exceeds 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.)

[0008] Furthermore, the development of topical ophthalmic drugs is hampered by numerous anatomical limitations, including tear turnover and dilution, nasolacrimal drainage, and reflex blinking, with typically less than 5% of the topically applied dose reaching the deeper ocular tissues (Gaudana et al., 2009). In the case of corneal wounds, the initial injury results in a breach in the corneal epithelium, allowing topically applied MSC-S to pass through to penetrate the epithelial layer.

[0009] Therefore, there is a pressing need in the art for ocular therapies that can target the eye and deliver therapeutic payloads to hard-to-reach sensory tissues that may be degenerated due to inflammation secondary to trauma (such as, for example, burns, acute inflammation, age, and / or oxidative stress). The present invention satisfies this need by providing mesenchymal stem cell secretome compositions for such therapies and methods of preparing such compositions. Summary of the invention

[0010] The present invention provides methods and assays for the analysis of the activity of the secretome derived from mesenchymal stem cells (MSCs).

[0011] In some embodiments, the present invention provides a method for characterizing the secretome of MSCs, wherein the method comprises:

[0012] (i) subjecting the MSC secretome to one or more characterization assays, wherein the characterization assay is selected from the group consisting of: physical component characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, cellular response assays, safety analysis, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / scarring assays, inflammation / immune assays, gliosis assays, tissue explant survival and function assays, organoid development or survival / function assays, epithelial barrier integrity assays, retinal degeneration assays and / or assays for inherited retinal diseases including human and animal retinal explants, neuroprotection / neurotrophy assays, wherein the MSC secretome is optionally pre-treated; and

[0013] (ii) determining the results of the one or more determinations in (i).

[0014] In some embodiments, the present invention provides a method for determining the biological efficacy and stability of MSC secretome, comprising:

[0015] (i) subjecting the MSC secretome to one or more characterization assays, wherein the characterization assay is selected from the group consisting of: physical component characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, cellular response assays, safety analysis, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / scarring assays, inflammation assays, in vivo responses to oxidative stress (e.g., retinal ischemia reperfusion), epithelial barrier integrity assays, retinal degeneration assays and / or assays for inherited retinal diseases including human and animal retinal explants, neuroprotection / neurotrophy assays, wherein the MSC secretome is optionally pre-treated; and

[0016] (ii) determining the results of the one or more determinations in (i).

[0017] In some embodiments, the present invention provides a method for determining MSC secretome batch consistency between multiple MSC secretome batches, wherein the method comprises:

[0018] (i) subjecting the MSC secretome to one or more characterization assays, wherein the characterization assay is selected from the group consisting of: physical component characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, cellular response assays, safety analysis, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / scarring assays, inflammation assays, epithelial barrier integrity assays, retinal degeneration assays and / or assays for inherited retinal diseases including human and animal retinal explants, neuroprotection / neurotrophy assays, wherein the MSC secretome is optionally pre-treated; and

[0019] (ii) determining the results of the one or more determinations in (i).

[0020] In some embodiments, the results in (ii) from the characterization of the physical component identify the anti-angiogenic MSC secretome or a composition prepared comprising the secretome.

[0021] In some embodiments, the results in (ii) from the safety analysis provide a MSC secretome that exhibits hemocompatibility and low and / or no pyrogens and / or endotoxins.

[0022] In some embodiments, the results in (ii) from the stability assay provide a MSC secretome that exhibits stability at -20°C, 4°C, and / or 20°C (eg, room temperature) for at least 7 days or at least 14 days.

[0023] In some embodiments, the results in (ii) from the proliferation assay provide a MSC secretome that induces proliferation.

[0024] In some embodiments, the results in (ii) from the migration assay provide the MSC secretome that induces migration.

[0025] In some embodiments, the results in (ii) from the neovascularization assay provide a MSC secretome that inhibits or does not promote neovascularization.

[0026] In some embodiments, the results in (ii) from the differentiation / scarring assay provide a MSC secretome that inhibits differentiation and / or scarring.

[0027] In some embodiments, the results in (ii) from the inflammation assay provide a MSC secretome that suppresses inflammation or alters an immune response.

[0028] In some embodiments, the method further comprises:

[0029] (iii) Identify MSC secretome batches based on the results in (ii).

[0030] In some embodiments, the method comprises a pre-processing step of the secretome.

[0031] In some embodiments, the present invention provides a panel of tests and / or assays for characterizing the MSC secretome, wherein the panel comprises at least two characterization assays, wherein the characterization assays are selected from the group consisting of: physical component characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analysis, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / scarring assays, inflammation assays, epithelial barrier integrity assays, retinal degeneration assays and / or assays for inherited retinal diseases including human and animal retinal explants, neuroprotection / neurotrophy assays, wherein the secretome is optionally pre-processed.

[0032] In some embodiments, the present invention provides a panel of tests and / or assays for determining the consistency between batches of MSC secretome, wherein the panel comprises one or more characterization assays, wherein the characterization assays are selected from the group consisting of: physical component characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analysis, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / scarring assays, inflammation assays, epithelial barrier integrity assays, retinal degeneration assays and / or assays for inherited retinal diseases including human and animal retinal explants, neuroprotection / neurotrophy assays, wherein the secretome is optionally pre-processed.

[0033] In some embodiments, the physical component characterization identifies the MSC secretome characterized herein or a composition prepared from the secretome.

[0034] In some embodiments, the results in (ii) from the safety analysis provide a MSC secretome that exhibits hemocompatibility and low and / or no pyrogens and / or endotoxins.

[0035] In some embodiments, the stability assay identifies MSC secretomes that exhibit stability at -20°C, 4°C, and / or 20°C (eg, room temperature) for at least 7 days or at least 14 days.

[0036] In some embodiments, the proliferation assay identifies the MSC secretome that induces proliferation.

[0037] In some embodiments, the migration assay identifies the MSC secretome that induces migration.

[0038] In some embodiments, the neovascularization assay identifies MSC secretomes that inhibit or do not promote neovascularization.

[0039] In some embodiments, the differentiation / scarring assay identifies MSC secretomes that inhibit differentiation and / or scarring.

[0040] In some embodiments, inflammation assays identify MSC secretomes that suppress inflammation or alter immune responses.

[0041] In some embodiments, physical component characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analysis, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / scarring assays, inflammation assays and / or epithelial barrier integrity assays, retinal degeneration assays and / or assays for inherited retinal diseases including human and animal retinal explants, neuroprotection / neurotrophic assays are all performed.

[0042] In some embodiments, the test and / or assay identifies the MSC secretome.

[0043] In some embodiments, the MSC secretome is an anti-angiogenic MSC secretome and / or an anti-scarring MSC secretome.

[0044] In some embodiments, the MSC secretome is an anti-angiogenic MSC secretome and / or an anti-scarring MSC secretome.

[0045] In some embodiments, the panel of assays includes at least one migration assay.

[0046] In some embodiments, the panel of assays includes at least one adhesion assay.

[0047] In some embodiments, the migration assay is an in vitro wound closure assay.

[0048] In some embodiments, the in vitro wound closure assay is selected from the group consisting of a "scratch assay" (also referred to as a "scratch wound assay"), a circular scratch wound method, a circular scratch wound assay, and a circular wound closure assay.

[0049] In some embodiments, the panel of assays includes reactive oxygen species (ROS) measurements.

[0050] In some embodiments, the ROS measurement assay is an in vitro assay using multiple antioxidants as positive controls. In some embodiments, the ROS measurement assay is an in vitro assay using one antioxidant as a positive control. In some embodiments, N-acetylcysteine ​​(NAC) or ebselen (Ebselen) is used as a positive control. Non-limiting examples of antioxidants include vitamins and their analogs, including vitamin A, vitamin B3 (e.g., niacin [nicotinic acid] and niacinamide), 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), lutein (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 ​​(N AC), bucillamine, S-nitroso-N-acetyl-L-cysteine ​​(SNAC), S-allyl-L-cysteine ​​(SAC), S-adenosyl-L-methionine (SAM), alpha-lipoic acid and taurine; scavengers of ROS and free radicals, including carnosine, N-acetylsarcosine, 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., sodium difenton [NXY-059]), nitric oxides (e.g., XJB-5-131), polyphenols (e.g., flavonoids [e.g., apigenin, genistein, luteolin, naringenin, and quercetin]), superoxide dismutase mimetics (see below), tirilazad, vitamin C, vitamin E and its analogs (e.g., alpha-tocopherol and trolox), and xanthine derivatives (e.g., pentoxifylline); mitochondrial antioxidants / "vitamins", including ubiquinones (coenzyme Q, such as CoQ10), ubiquinols (reduced and more bioavailable forms of ubiquinone, such as quinone (e.g., ubiquinone-10), ubiquinone / ubiquinone analogs (e.g., idebenone and mitoquinone) and derivatives; antioxidants targeting mitochondria, including DMQ, DMMQ, MitoE, MitoQ, Mito-TEMPO, MitoVitE, and SkQ compounds (e.g., SkQ1, SkQ2, SkQ3, SkQB, SkQR1, SkQT, SkQT1, SkQT1(m), SkQT1(p), SkQTK1, SkQTR1, SkQBerb, and SkQPalm); inhibitors of enzymes that generate ROS,These include NADPH oxidase (NOX) inhibitors (e.g., apocynin, decursin, and decursinol angelate [all inhibit NOX-1, NOX-2, and NOX-4 activity and expression], diphenyliodonium 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, tisopurine, febuxostat, tat), topiroxostat, myo-inositol, phytic acid and flavonoids [e.g., kaempferol, myricetin and quercetin]) and myeloperoxidase inhibitors (e.g., azide, 4-aminobenzoic acid hydrazide and PF-06667272, and apoE mimetics such as AEM-28 and AEM-28-14); substances that mimic or increase the activity or production of antioxidant enzymes, including superoxide dismutase (SOD) (e.g., SOD mimetics such as manganese (III)-porphyrin complexes and zinc (III)-porphyrin complexes complexes (e.g., MnTBAP, MnTMPyP, and ZnTBAP), manganese(II) pentaazamacrocyclic complexes (e.g., M40401 and M40403), manganese(III)-Schiff base complexes (e.g., those disclosed in U.S. Pat. No. 7,122,537, which is incorporated herein by reference in its entirety), and OT-551 (a cyclopropyl ester prodrug of tempol hydroxylamine), as well as resveratrol and apoA-I mimetics such as 4F (both of which increase expression)), catalase (e.g., catalase mimetics such as manganese(III)-Schiff base complexes No. 7,122,537], and zinc [increases activity]), glutathione peroxidase (GPx) (e.g., apomorphine and zinc [both increase activity], and β-catenin, etoposide, and resveratrol [all three increase expression]), glutathione reductase (e.g., 4-tert-butylcatechol and redox cofactors such as flavin adenine dinucleotide [FAD] and NADPH [all three enhance activity]), glutathione S-transferase (GST) (e.g., phenylalkyl isothiocyanate-cysteine ​​conjugates (e.g.,S-[N-benzyl(thiocarbamoyl)]-L-cysteine), phenobarbital, rosemary extract, and carnosol [all enhanced activity]), thioredoxin (Trx) (e.g., geranylgeranylacetone, prostaglandin E1, and sulforaphane [all increased expression]), NADPH-quinone oxidoreductase 1 (NQO1) (e.g., flavonoids [e.g., β-naphthoflavone (5,6-phenylflavone)], and triterpenoids [e.g., oleanolic acid analogs such as TP-151 (CDDO), TP-155 (CDDO methyl ester), TP-190, TP-218, TP-222, TP-223 (CDDO methylamide), TP-224 (CDDO monomethylamide), TP-225, TP-226 (CDDO dimethylamide) , TP-230, TP-235 (CDDO imidazole compounds), TP-241, CDDO monoethylamide, CDDO mono(trifluoroethyl)amide, and (+)-TBE-B], all of which 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); transcription factor activators that upregulate expression of antioxidant enzymes, including activators of nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or Nrf2) (e.g., bardoxolone methyl methyl), OT-551, fumarates (e.g., dimethyl fumarate and monomethyl fumarate), dithiones (e.g., oltipraz), flavonoids (e.g., β-naphthoflavone), isoflavones (e.g., genistein), sulforaphane, trichostatin A (also upregulates glutathione synthesis), triterpenes (e.g., oleanolic acid analogs [e.g., TP-225]), and melatonin (increases Nrf2 expression)); other classes of antioxidants include anthocyanidins, benzyl alcohol diterpenes (e.g., carnosic acid), cyclopentenone prostaglandins (such as 15d-PGJ2, which also upregulates glutathione synthesis), flavonoids (e.g., Ginkgo biloba), biloba) (e.g., myricetin and quercetin [increases levels of GSH, SOD, catalase, GPx, and GST]), prenylated flavonoids (e.g., isoxanthohumol), flavonoids (e.g., apigenin), isoflavones (e.g., genistein), flavanones (e.g., naringenin), and flavanols (e.g.,catechins and epigallocatechin-3-gallate), omega-3 fatty acids and their esters (see above), phenylethanoid glycosides (e.g., tyrosol and hydroxytyrosol), retinoids (e.g., all-trans retinol [vitamin A]), stilbenes (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 monostearate]); and their analogs, derivatives, and salts.

[0051] In some embodiments, a chloromethyl-modified H 2 DCFDA(CM-H 2 DCF DA) and / or MitoSOX Red to evaluate in vitro ROS measurement assays.

[0052] In some embodiments, the panel of assays comprises a cell viability assay.

[0053] In some embodiments, the cell viability assay is a microtiter tetrazolium (MTT) assay.

[0054] In some embodiments, cells are subjected to an oxidative stress assay.

[0055] In some embodiments, the oxidative stress assay comprises:

[0056] a) inducing oxidative stress in cells; and

[0057] b) Assess the extent of oxidative stress.

[0058] In some embodiments, oxidative stress is induced by one or more inducing agents selected from the group consisting of: ketone cholesterol, FeCl 3 -Sodium nitrilotriacetate (Fe-NTA), H 2 O 2 , tert-butyl hydroperoxide (t-BHP), all-trans retinal, NaIO4, hydroquinone and oxidized cholesterol (OxLDL).

[0059] In some embodiments, oxidative stress is induced by culturing cells under low oxygen and / or hypoxic conditions.

[0060] In some embodiments, oxidative stress is induced by administering ketocholesterol at a concentration of about 0.1 mM to 1 mM, about 1 mM to 10 mM, or about 10 mM to 100 mM to the assayed cells for about 1 hour to 24 hours, about 24 hours to 48 hours, or about 48 hours to 72 hours.

[0061] In some embodiments, oxidative stress is induced by administering Fe-NTA at a concentration of about 0.1 mM to 1 mM, about 1 mM to 10 mM, or about 10 mM to 100 mM to the assayed cells for about 1 hour to 24 hours, about 24 hours to 48 hours, or about 48 hours to 72 hours.

[0062] In some embodiments, by applying to the assayed cells a concentration of about 0.01 μM to 0.1 μM, about 0.1 μM to 1 μM, about 1 μM to 10 μM, about 10 μM to 100 μM, or about 100 μM to 1000 μM H 2 O 2 Oxidative stress is induced for about 0.5 hours to 6 hours, about 6 hours to 12 hours, about 12 hours to 18 hours, or about 18 hours to 24 hours.

[0063] In some embodiments, oxidative stress is induced by administering t-BHB at a concentration of about 0.01 μM to 0.1 μM, about 0.1 μM to 1 μM, about 1 μM to 10 μM, about 10 μM to 100 μM, or about 100 μM to 1000 μM to the assayed cells for about 0.5 to 6 hours, about 6 to 12 hours, about 12 to 18 hours, or about 18 to 24 hours.

[0064] In some embodiments, oxidative stress is induced by administering all-trans retinal at a concentration of about 0.01 μM to 0.1 μM, about 0.1 μM to 1 μM, about 1 μM to 10 μM, about 10 μM to 100 μM, or about 100 μM to 1000 μM to the assayed cells for about 0.5 to 6 hours, about 6 to 12 hours, about 12 to 18 hours, or about 18 to 24 hours.

[0065] In some embodiments, by applying to the cells being assayed a concentration of about 0.01 μM to 0.1 μM, about 0.1 μM to 1 μM, about 1 μM to 10 μM, about 10 μM to 100 μM, or about 100 μM to 1000 μM NaIO 4 Oxidative stress is induced for about 0.5 hours to 6 hours, about 6 hours to 12 hours, about 12 hours to 18 hours, or about 18 hours to 24 hours.

[0066] In some embodiments, oxidative stress is induced by administering OxLDL at a concentration of about 10 μg / mL to 100 μg / mL, about 10 μg / mL to 100 μg / mL, or about 100 μg / mL to 500 μg / mL to the assayed cells for about 0.5 to 6 hours, about 6 to 12 hours, about 12 to 18 hours, or about 18 to 24 hours.

[0067] In some embodiments, assessing the extent of oxidative stress comprises one or more of the following:

[0068] i) DNA oxidation was measured using an anti-8-oxo-2'-deoxyguanosine (8-oxo-dG) assay;

[0069] ii) lipid oxidation was measured using thiobarbituric acid reactive substances (TBARS);

[0070] iii) measurement of cytoplasmic ROS using CM-H2 DCFDA;

[0071] iv) measurement of mitochondrial ROS using Mitoxed; and

[0072] v) Measuring mitochondrial potential, such as measuring JC-1.

[0073] In some embodiments, the cells assayed are assayed for one or more phenotypes selected from the group consisting of:

[0074] Alterations in epithelial polarization (e.g., Na / K ATPase polarity), tight junctions (e.g., ZO-1, transepithelial polarity, and transepithelial electrical resistance (TEER)), phagocytosis of outer segments, degeneration of outer segments, phagosome composition and function, cathepsin D distribution activity, mitochondrial potential, inflammasome activation / activity, HIf-1α 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 / susceptibility, lipid deposition, apoptosis, and necrosis.

[0075] In some embodiments, the MSC secretome is pre-treated prior to inducing oxidative stress.

[0076] In some embodiments, the MSC secretome is pre-treated during induction of oxidative stress.

[0077] In some embodiments, the MSC secretome is pre-treated following induction of oxidative stress.

[0078] In some embodiments, pre-treating the MSC secretome comprises affecting the secretion profile of the MSC, wherein the pre-treating comprises one or more of: changing the culture format (e.g., 2D planar bioreactor versus 3D bioreactor), different biomaterial scaffolds, co-culture, adding pharmacological compounds, growth factors, chemokines, adding toll-like receptor agonists, inflammatory cytokines, advanced glycation end products (AGE), oxidized phospholipids, malondialdehyde or carboxyethylpyrrole, stirring the presence of ECM, culturing under shear stress, stirring or suspending as aggregates or in a matrix, inducing a misfolded protein response, ER stress, inducing differentiation of MSCs, culturing in the presence of conditioned medium and hypoxia / hypoxia.

[0079] In some embodiments, preconditioning the MSC secretome comprises hypoxic preconditioning, which comprises culturing the MSCs in a hypoxic culture environment.

[0080] In some embodiments, the oxygen level is about 0% to 2%. In some embodiments, the hypoxic pretreatment is performed for about 4 hours to 12 hours, about 12 hours to 24 hours, about 24 hours to 36 hours, about 36 hours to 48 hours, about 48 hours to 60 hours, or about 60 hours to 72 hours.

[0081] In some embodiments, pre-treating the MSC secretome comprises treating the MSCs with one or more inflammatory cytokines.

[0082] In some embodiments, pre-treating the MSC secretome comprises treating the MSC with one or more of the following: 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.

[0083] In some embodiments, pre-treating the MSC secretome comprises culturing the MSCs in 3D (eg, by using a 3D bioreactor).

[0084] In some embodiments, the present invention provides a bone marrow-derived mesenchymal stem cell (MSC) secretome composition comprising: HGF; penetratin-3 (TSG-14); VEGF; TIMP-1; serine protease inhibitor E1; <5 ng / mL IL-8, and a tension regulator, wherein the MSC secretome is pre-treated.

[0085] In some embodiments, the MSC secretome composition further comprises:

[0086] i. at least one trophic factor / cytokine selected from the group consisting of: TIMP-2 and VEGF-A;

[0087] ii. at least one additional factor selected from the group consisting of: PEDF (serine protease inhibitor 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-β and PDGF; and

[0088] 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.

[0089] In some embodiments, the MSC secretome composition comprises 1 ng / mL-100 ng / mL of at least one factor selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentetracycline-3 (TSG-14), and serpin F1.

[0090] In some embodiments, the MSC secretome comprises 400 pg / mL-3000 pg / mL of at least one factor selected from the group consisting of angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, thrombospondin-1, angiopoietin, DPPIV, IGFBP-3 and uPA.

[0091] In some embodiments, the MSC secretome composition 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, Emmprin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15 and IFNγ.

[0092] In some embodiments, the MSC secretome composition comprises a ratio of anti-angiogenic to pro-angiogenic, wherein the ratio is >2, >3, >4, or >5.

[0093] In some embodiments, the MSC secretome composition comprises 1 pg / mL-400 pg / mL of VEGF.

[0094] In some embodiments, the level of VEGF is 5-10 times lower than the level of serpin El.

[0095] In some embodiments, the composition comprises one or more anti-angiogenic factors, and wherein the ratio of 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.

[0096] In some embodiments, the MSC secretome comprises less than 1000 pg / mL of bFGF, PLGF, and PDGF.

[0097] In some embodiments, the MSC secretome composition has a pH of about 4.7 to about 7.5.

[0098] 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 citric acid / disodium phosphate.

[0099] In some embodiments, the MSC secretome composition further comprises mono / disodium phosphate, mannitol, and trehalose, and wherein the composition has a pH of about pH 7.4.

[0100] In some embodiments, the MSC secretome composition further comprises divalent cations.

[0101] In some embodiments, the divalent cation is selected from the group consisting of Mg2+, Ca2+, and Zn2+.

[0102] In some embodiments, the MSC secretome composition further comprises disodium phosphate / citric acid, mannitol, and trehalose, wherein the composition has a pH of about pH 6.4.

[0103] In some embodiments, the MSC secretome composition further comprises an agent that increases viscosity.

[0104] In some embodiments, the binder is selected from the group consisting of: hydroxypropyl methylcellulose, poloxamer 407, poloxamer 188, poloxamer 237, poloxamer 338, hydroxypropyl methylcellulose (HPMC), polycarbophil, polyvinyl pyrrolidone (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.

[0105] In some embodiments, the MSC secretome composition does not comprise one or more components selected from the group consisting of: xenobiotic 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.

[0106] In some embodiments, the MSC secretome composition comprises:

[0107] i. 0.3-4.5 ng / mL HGF;

[0108] ii. 0.5-20 ng / mL penetrant-3 (TSG-14);

[0109] iii. 100-600 pg / mL VEGF;

[0110] iv. 10-200 ng / mL TIMP-1;

[0111] v. 20-80 ng / mL serine protease inhibitor E1; and

[0112] vi. <5ng / mL IL-8.

[0113] In some embodiments, the MSC secretome composition comprises an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.

[0114] In some embodiments, the tonicity adjuster is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerol.

[0115] In some embodiments, the present invention provides a stable bone marrow-derived mesenchymal stem cell (MSC) secretome preparation comprising:

[0116] i. 2μg-20μg MSC secretome / mL;

[0117] ii. 2mg-3mg sodium dihydrogen phosphate / mL;

[0118] iii. 11mg-12mg sodium dihydrogen phosphate / mL;

[0119] iv. 11.5mg-13mg mannitol / mL;

[0120] v.23mg-24mg trehalose dihydrate;

[0121] vi. 0.5mg-2mg hydroxypropyl methylcellulose / mL;

[0122] And wherein the pH is about 4.7 to about 7.5, wherein the MSC secretome is pre-treated.

[0123] In some embodiments, the present invention provides a stable bone marrow-derived mesenchymal stem cell (MSC) secretome preparation, comprising: i. 0.004%-0.08% w / w MSC secretome; ii. 4%-5% w / w sodium dihydrogen phosphate; iii. 21.5%-23% w / w sodium dihydrogen phosphate; iv. 23%-25% w / w mannitol; v. 46%-48% w / w trehalose dehydrate; vi. 1%-3% w / w hydroxypropyl methylcellulose; and wherein the pH is about 4.7 to about 7.5, wherein the MSC secretome is pretreated.

[0124] In some embodiments, the present invention provides a method of 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; penetratin-3 (TSG-14); VEGF; TIMP-1; serine protease inhibitor E1; and <5 ng / mL IL-8, wherein the MSC secretome is pre-treated.

[0125] In some embodiments, the MSC secretome composition further comprises:

[0126] i. at least one trophic factor / cytokine selected from the group consisting of: TIMP-2 and VEGF-A;

[0127] ii. at least one additional factor selected from the group consisting of: PEDF (serine protease inhibitor 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-β and PDGF; and

[0128] 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.

[0129] In some embodiments, the MSC secretome composition comprises 1 ng / mL-100 ng / mL of at least one factor selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentetracycline-3 (TSG-14), and serpin F1.

[0130] In some embodiments, the MSC secretome composition comprises 400 pg / mL-3000 pg / mL of at least one factor selected from the group consisting of angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, thrombospondin-1, angiopoietin, DPPIV, IGFBP-3 and uPA.

[0131] In some embodiments, the MSC secretome composition 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, Emmprin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15 and IFNγ.

[0132] In some embodiments, the MSC secretome composition comprises a ratio of anti-angiogenic to pro-angiogenic, wherein the ratio is >2, >3, >4, or >5.

[0133] In some embodiments, the MSC secretome comprises 1 pg / mL-400 pg / mL of VEGF.

[0134] In some embodiments, the level of VEGF is 5-10 times lower than the level of serpin El.

[0135] In some embodiments, the MSC secretome composition comprises one or more anti-angiogenic factors, and wherein 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.

[0136] In some embodiments, the MSC secretome comprises less than 1000 pg / mL of bFGF, PLGF, and PDGF.

[0137] In some embodiments, the MSC secretome composition has a pH of about 4.7 to about 7.5.

[0138] 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 citric acid / disodium phosphate.

[0139] In some embodiments, the MSC secretome composition further comprises a tonicity modifier.

[0140] In some embodiments, the tonicity adjuster is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerol.

[0141] In some embodiments, the MSC secretome composition further comprises mono / disodium phosphate, mannitol, and trehalose, and wherein the composition has a pH of about pH 7.4.

[0142] In some embodiments, the MSC secretome composition further comprises divalent cations.

[0143] In some embodiments, the divalent cation is selected from the group consisting of Mg2+, Ca2+, and Zn2+.

[0144] In some embodiments, the MSC secretome composition further comprises disodium phosphate / citric acid, mannitol, and trehalose, and wherein the composition has a pH of about pH 6.4.

[0145] In some embodiments, the MSC secretome composition does not comprise one or more components selected from the group consisting of: xenobiotic 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.

[0146] In some embodiments, the MSC secretome composition comprises an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.

[0147] In some embodiments, the MSC secretome composition further comprises an agent that increases viscosity.

[0148] In some embodiments, the binder is selected from the group consisting of: hydroxypropyl methylcellulose, poloxamer 407, poloxamer 188, poloxamer 237, poloxamer 338, hydroxypropyl methylcellulose (HPMC), polycarbophil, polyvinyl pyrrolidone (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.

[0149] In some embodiments, the MSC secretome composition comprises:

[0150] i. 0.3-4.5 ng / mL HGF;

[0151] ii. 0.5-20 ng / mL penetrant-3 (TSG-14);

[0152] iii. 100-600 pg / mL VEGF;

[0153] iv. 10-200 ng / mL TIMP-1;

[0154] v. 20-80 ng / mL serine protease inhibitor E1; and

[0155] vi. <5ng / mL IL-8.

[0156] In some embodiments, the present invention provides a method of 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 is a stable mesenchymal stem cell (MSC) secretome preparation comprising:

[0157] i. 2μg-20μg MSC secretome / mL;

[0158] ii. 2mg-3mg sodium dihydrogen phosphate / mL;

[0159] iii. 11mg-12mg sodium dihydrogen phosphate / mL;

[0160] iv. 11.5mg-13mg mannitol / mL;

[0161] v.23mg-24mg trehalose dihydrate;

[0162] vi. 0.5mg-2mg hydroxypropyl methylcellulose / mL;

[0163] and wherein the pH is about 4.7 to about 7.5, wherein the MSC secretome is pre-treated.

[0164] In some embodiments, the present invention provides a method of 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 is a stable mesenchymal stem cell (MSC) secretome preparation comprising:

[0165] i. 0.004%-0.08% w / w MSC secretome;

[0166] ii. 4%-5% w / w sodium dihydrogen phosphate;

[0167] iii. 21.5%-23% w / w disodium hydrogen phosphate;

[0168] iv. 23%-25% w / w mannitol;

[0169] v. 46%-48% w / w trehalose dehydrate; and

[0170] vi. 1%-3% w / w hypromellose; and

[0171] wherein the pH is about 4.7 to about 7.5, and wherein the MSC secretome is pre-treated.

[0172] In some embodiments, the present invention provides a composition for inducing ocular wound healing, comprising a mesenchymal stem cell (MSC) secretome and a tension regulator, wherein the MSC secretome is pre-treated, wherein the ability of the composition to promote ocular wound healing is indicated by a wound healing assay comprising the following steps:

[0173] a) providing a corneal cell layer;

[0174] b) introducing a wound gap into the corneal cell layer; and

[0175] c) determining whether the wound gap heals in the presence of the composition,

[0176] wherein the composition is administered to corneal cells before or after step b); wherein closure of the wound gap indicates the ability of the composition to induce ocular wound healing.

[0177] In some embodiments, the corneal cells are corneal epithelial cells.

[0178] In some embodiments, the corneal cells are corneal keratinocytes (or fibroblasts).

[0179] In some embodiments, the corneal cell layer is a confluent monolayer.

[0180] In some embodiments, the wound gap is introduced by mechanically disrupting the corneal cell layer.

[0181] In some embodiments, the wound space is introduced by chemically disrupting the corneal cell layer.

[0182] In some embodiments, the wound gap comprises a linear gap.

[0183] In some embodiments, the wound gap comprises a circular gap.

[0184] In some embodiments, determining whether the wound gap is closed in step c) comprises detecting and quantifying migration and / or proliferation of corneal cells within the wound gap on a daily basis.

[0185] In some embodiments, the migration and / or proliferation of corneal cells is characterized as the number of corneal cells that migrate and / or proliferate within the wound gap.

[0186] In some embodiments, determining whether the wound gap is healed in step c) comprises measuring the size of the wound gap daily, wherein the size of the wound gap is expressed as a percentage of an initial size of the wound gap measured immediately after introduction of the wound gap.

[0187] In some embodiments, the size of the wound gap is characterized as the surface area of ​​the wound gap.

[0188] In some embodiments, the size of the wound gap is characterized as the width of the wound gap.

[0189] In some embodiments, step c) is performed within a period of 2-4 days after step b) is completed.

[0190] In some embodiments, step c) is performed within a period of 2 days after step b) is completed.

[0191] In some embodiments, step c) is performed within a period of 3 days after step b) is completed.

[0192] In some embodiments, the wound healing assay further comprises a step of concentrating the composition, and optionally a buffer exchange step for the composition, prior to applying the composition to the corneal cells.

[0193] In some embodiments, the wound healing assay further comprises a step of diluting the composition, and optionally a buffer exchange step for the composition, prior to applying the composition to the corneal cells.

[0194] In some embodiments, the composition comprises at least 45 μg / ml of a secretome protein.

[0195] In some embodiments, the present invention provides a composition for inducing ocular wound healing, comprising a mesenchymal stem cell (MSC) secretome and a tension regulator, wherein the MSC secretome is pre-treated, wherein the ability of the composition to promote ocular wound healing is indicated by a wound healing assay comprising the following steps:

[0196] a) providing a corneal cell layer;

[0197] b) introducing a wound gap into the corneal cell layer; and

[0198] c) determining whether the wound gap heals in the presence of the composition,

[0199] wherein the composition is administered to corneal cells before or after step b); wherein closure of the wound gap indicates the ability of the composition to induce ocular wound healing.

[0200] In some embodiments, the corneal cells are corneal epithelial cells.

[0201] In some embodiments, the corneal cells are corneal keratinocytes (or fibroblasts).

[0202] In some embodiments, the corneal cell layer is a confluent monolayer.

[0203] In some embodiments, the wound gap is introduced by mechanically disrupting the corneal cell layer.

[0204] In some embodiments, the wound space is introduced by chemically disrupting the corneal cell layer.

[0205] In some embodiments, the wound gap comprises a linear gap.

[0206] In some embodiments, the wound gap comprises a circular gap.

[0207] In some embodiments, determining whether the wound gap is closed in step c) comprises detecting and quantifying migration and / or proliferation of corneal cells within the wound gap on a daily basis.

[0208] In some embodiments, the migration and / or proliferation of corneal cells is characterized as the number of corneal cells that migrate and / or proliferate within the wound gap.

[0209] In some embodiments, determining whether the wound gap is healed in step c) comprises measuring the size of the wound gap daily, wherein the size of the wound gap is expressed as a percentage of an initial size of the wound gap measured immediately after introduction of the wound gap.

[0210] In some embodiments, the size of the wound gap is characterized as the surface area of ​​the wound gap.

[0211] In some embodiments, the size of the wound gap is characterized as the width of the wound gap.

[0212] In some embodiments, step c) is performed within a period of 2-4 days after step b) is completed.

[0213] In some embodiments, step c) is performed within a period of 2 days after step b) is completed.

[0214] In some embodiments, step c) is performed within a period of 3 days after step b) is completed.

[0215] In some embodiments, the wound healing assay further comprises a step of concentrating the composition, and optionally a buffer exchange step for the composition, prior to applying the composition to the corneal cells.

[0216] In some embodiments, the wound healing assay further comprises a step of diluting the composition, and optionally a buffer exchange step for the composition, prior to applying the composition to the corneal cells.

[0217] In some embodiments, the composition comprises at least 45 μg / ml of a secretome protein.

[0218] In some embodiments, the present invention provides a method for pre-treating the secretory proteome of MSCs, comprising influencing the secretory profile of MSCs, wherein the pre-treatment comprises one or more of the following: changing the culture format (e.g., 2D planar bioreactor compared to 3D bioreactor), different biomaterial scaffolds, co-culture, adding pharmacological compounds, growth factors, chemokines, adding toll-like receptor agonists, inflammatory cytokines, advanced glycation end products (AGE), oxidized phospholipids, malondialdehyde or carboxyethylpyrrole, stirring the presence of ECM, culturing under shear stress, stirring or suspending as aggregates or in a matrix, inducing a misfolded protein response, ER stress, inducing differentiation of MSCs, culturing in the presence of conditioned medium and hypoxia / hypoxia.

[0219] In some embodiments, hypoxic preconditioning comprises culturing the MSCs in a hypoxic culture environment.

[0220] In some embodiments, the oxygen level is between about 0% and 2%.

[0221] In some embodiments, the hypoxic pretreatment is performed for about 4 hours to 12 hours, about 12 hours to 24 hours, about 24 hours to 36 hours, about 36 hours to 48 hours, about 48 hours to 60 hours, or about 60 hours to 72 hours.

[0222] In some embodiments, pre-treating the MSC secretome comprises treating the MSCs with one or more inflammatory cytokines.

[0223] In some embodiments, pre-treating the MSC secretome comprises treating the MSC with one or more of the following: 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.

[0224] In some embodiments, pre-treating the MSC secretome comprises culturing the MSCs in 3D (eg, by using a 3D bioreactor).

[0225] In some embodiments, pre-treating the MSC secretome includes inducing a misfolded protein response in the MSCs.

[0226] In some embodiments, pre-treating the MSC secretome comprises culturing the MSCs under suitable conditions to induce ER stress.

[0227] In some embodiments, pre-treating the MSC secretome includes culturing the MSCs under suitable conditions to induce differentiation of the MSCs into mature retinal cell types or precursors thereof.

[0228] In some embodiments, the present invention provides a bone marrow-derived mesenchymal stem cell (MSC) secretome composition, which includes precursors or mature retinal cell types that differentiate MSCs into mature retinal cell types. In some embodiments, the bone marrow-derived MSC secretome composition is obtained from MSC culture, wherein the MSCs are cultured under conditions suitable for inducing MSC differentiation into mature retinal cell types or precursors thereof.

[0229] In some embodiments, the present invention provides a method of treating an ocular disorder in a patient in need thereof, comprising administering to the patient the pre-treated MSC secretome disclosed herein.

[0230] In some embodiments, the present invention provides for the use of the pre-treated MSC secretome as disclosed herein for treating an ocular disorder in a patient in need thereof.

[0231] In some embodiments, the ocular disorder is selected from the group consisting of: retinal disorders, macular diseases, chronic graft-versus-host disease (GvHD), Stevens-Johnson syndrome, ocular mucous membrane pemphigoid, persistent corneal epithelial defects (PCED), limbal stem cell deficiency (LSCD), dry eyes, damage to ocular neural tissue, and concussive injury to the eye (such as concussive injury, eye contusion, or chemical burn). DETAILED DESCRIPTION

[0232] I. Introduction

[0233] The present invention provides methods, assays and protocols for the analysis of the activity of the secretome of mesenchymal stem cells, and the use of such characterized compositions. These methods and assays are described in more detail below.

[0234] A. Definition

[0235] Unless otherwise indicated, the terms used in the claims and specification are defined as follows. In the event of a direct conflict with a term used in the parent provisional patent application, the term used in this specification shall control.

[0236] As used herein, "isolated" refers to material that has been removed from its original environment and thus altered "by the hand of man" from its natural state.

[0237] As used herein, "enriched" refers to selectively concentrating or increasing the amount of one or more materials by eliminating unwanted materials from a mixture or selecting and isolating desired materials (e.g., separating cells with a particular cell marker from a heterogeneous cell population where not all cells in the population express that marker).

[0238] As used herein, the term "substantially purified" means a population of cells that are substantially homogeneous for a particular marker or marker combination. Substantially homogeneous means at least 90% and preferably 95% homogeneous for a particular marker or marker combination. As used herein, the term "pluripotent stem cells" are true stem cells, but can only differentiate into a limited number of types. For example, bone marrow contains pluripotent stem cells that can produce all cells in the blood, but may not differentiate into other cell types.

[0239] The term "animal-free" when referring to certain compositions, growth conditions, culture media, etc. described herein, means that materials derived from non-human animals, such as bovine serum, proteins, lipids, carbohydrates, nucleic acids, vitamins, etc., are not included for the preparation, growth, cultivation, amplification, storage or formulation of certain compositions or processes. "Material derived from non-human animals" means that the materials have never been in contact with or in contact with non-human animal bodies or substances, so they are not contaminated by xenobiotics. Typically, clinical-grade materials (such as recombinantly produced human proteins) are used for the preparation, growth, cultivation, amplification, storage and / or formulation of such compositions and / or processes.

[0240] With respect to cell compositions, the term "expansion" means that the cell concentration of the cell population is significantly higher than that obtained using the previous method. For example, in the amplified composition of AMP cells, the cell level per gram of amniotic tissue is at least 50 times and up to 150 times higher than the number of cells after 5 passages in the primary culture, compared to the use of the previous method to increase such cells by about 20 times. In another example, the cell level per gram of amniotic tissue in the amplified composition of AMP cells is at least 30 times and up to 100 times higher than the number of cells after 3 passages in the primary culture. Therefore, compared with the previous method, the number of cells per gram of amniotic tissue in the "expanded" population is at least 2 times higher, and up to 10 times higher. The term "expansion" is intended to cover only those situations in which people intervene to increase the number of cells.

[0241] As used herein, "conditioned medium" is a medium in which specific cells or cell groups have been cultured and then the medium is removed. When cells are cultured in a medium, they may secrete cytokines to support or influence the behavior of other cells. These factors include, but are not limited to, hormones, cytokines, extracellular matrix (ECM), proteins, vesicles, antibodies, chemokines, receptors, inhibitors, and particles. A medium containing cytokines is a conditioned medium. Examples of methods for preparing conditioned medium have been described in U.S. Patent No. 6,372,494, which is incorporated herein by reference. As used herein, conditioned medium also refers to components, such as proteins, recovered and / or purified from conditioned medium or, for example, MSC cells.

[0242] As used herein, the term "mesenchymal stem cell composition" or "MSC composition" refers to a conditioned medium derived from MSCs and, in some cases, has undergone further processing. In some embodiments, the "MSC secretome" may refer to a crude conditioned medium derived from MSCs. In some embodiments, the "MSC secretome" may refer to a composition obtained from a crude conditioned medium after further processing as described herein.

[0243] As used herein, the term "suspension" means a liquid containing dispersed components, such as cytokines. The dispersed components can be completely dissolved, partially dissolved, suspended or otherwise dispersed in a liquid. Suitable liquids include, but are not limited to, water, osmotic solutions such as salt and / or sugar solutions, cell culture media, and other aqueous or non-aqueous solutions.

[0244] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those amino acids encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure (e.g., α carbon, carboxyl, amino, and R groups bonded to hydrogen) as naturally occurring amino acids, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that are structurally different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids. Amino acids may be referred to herein by their commonly known three-letter symbols or by one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides may be referred to by their generally accepted single-letter codes.

[0245] "Amino acid substitution" refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence (the amino acid sequence of the starting polypeptide) by a second, different "replacement" amino acid residue. "Amino acid insertion" refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. Although insertions typically consist of the insertion of one or two amino acid residues, larger "peptide insertions" can be performed, such as insertions of about three to about five or even up to about ten, fifteen or twenty amino acid residues. The inserted residues can 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 predetermined amino acid sequence.

[0246] "Polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0247] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Unless otherwise limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19: 5081, 1991; Ohtsuka et al., Biol. Chem. 260: 2605-2608, 1985; and Cassol et al., 1992; Rossolini et al., Mol. Cell. Probes 8: 91-98, 1994). For arginine and leucine, the modification of the second base may also be conservative. The term nucleic acid can be used interchangeably with genes, cDNAs, and gene-encoded mRNAs. The polynucleotides used herein can be composed of any polyribonucleotides or polydeoxyribonucleotides, which can be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single-stranded and double-stranded DNA, DNA as a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNAs, and RNA as a mixture of single-stranded and double-stranded regions, and hybrid molecules comprising DNA and RNA (which can be single-stranded or more commonly double-stranded, or a mixture of single-stranded and double-stranded regions). In addition, polynucleotides can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. Polynucleotides can also include one or more modified bases or DNA or RNA backbones modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and uncommon bases, such as inosine. DNA and RNA can be modified in a variety of ways; therefore, "polynucleotides" include chemical, enzymatic, or metabolically modified forms.

[0248] As used herein, the term "secretome composition" refers to a composition comprising one or more substances secreted from a cell. In certain embodiments, the secretome composition may comprise one or more cytokines, one or more exosomes and / or one or more microvesicles. The secretome composition may be purified or unpurified. In some embodiments, the secretome composition may also comprise one or more substances that are not secreted from the cell (e.g., culture medium, additives, nutrients, etc.). In some cases, the secretome composition does not contain and or only contains trace amounts of one or more substances that are not secreted from the cell (e.g., culture medium, additives, nutrients, etc.).

[0249] As used herein, the terms "treatment", "treat" or "treating" and the like cover any treatment of humans or non-human mammals (e.g., rodents, cats, dogs, horses, cattle, sheep and primates, etc.), and include preventing the occurrence of the disease or disorder in subjects who may be susceptible to the disease or disorder but have not yet been diagnosed with the disease or disorder. It also includes inhibiting the disease, disorder and / or any related symptoms (preventing their development), alleviating or improving the disease, disorder and / or any related symptoms (causing them to disappear) or curing the disease, disorder and / or any related symptoms (permanently stopping development or progression). The terms "treatment", "treat" or "treating" as used herein encompass any treatment of a disease or disorder in a mammal, especially a human, and include: (a) preventing the occurrence of a disease or disorder in a subject who may be susceptible to the disease or disorder but has not yet been diagnosed with the disease or disorder; (b) inhibiting the disease or disorder, such as preventing its development; (c) alleviating and / or ameliorating the disease or disorder, such as causing the disease or disorder to regress; or (d) curing the disease or disorder, such as preventing its development or worsening. The subject population treated by the methods of the present invention includes subjects suffering from an undesirable disorder or disease and subjects at risk of developing the disorder or disease. In some embodiments, "treatment" (also referred to as "treat or treating") refers to the administration of any therapy that partially or completely alleviates, improves, alleviates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms, features, and / or causes of a specific disease, disorder, and / or disorder. In some embodiments, such treatments may be directed to subjects who do not show signs of the relevant disease, disorder, and / or illness, and / or subjects who only show early signs of the disease, disorder, and / or illness. Alternatively and / or additionally, such treatments may be directed to subjects who show one or more confirmed signs of the relevant disease, disorder, and / or illness. In some embodiments, treatment may be directed to subjects who have been diagnosed as suffering from the relevant disease, disorder, and / or illness. In some embodiments, treatment may be directed to subjects known to have one or more susceptibility factors that are statistically associated with an increased risk of development of the relevant disease, disorder, and / or illness.

[0250] As used herein, "wound" is any damage to normal anatomical structures (internal and / or external anatomical structures) for any reason, including but not limited to traumatic injuries, such as mechanical (e.g., contusion, penetration), thermal, chemical, electrical, radiation, concussive and incisional injuries; selective injuries, such as surgery and the resulting incisional hernias, fistulas, etc.; acute wounds, chronic wounds, infected wounds and sterile wounds, as well as wounds associated with disease states (e.g., eye contusions). Wounds are dynamic, and the healing process is a continuous process that requires a series of integrated, interrelated cellular processes that begin at the time of injury and continue after the initial wound closure until stable wound closure is achieved. These cellular processes are mediated or regulated by humoral substances, including but not limited to cytokines, lymphokines, growth factors and hormones. According to the present invention, "wound healing" refers to the improvement of natural cellular processes and humoral substances of tissue repair by some form of intervention, so that healing is faster, and / or the resulting healing area has less scars, and / or the tissue strength of the injured site is closer to that of uninjured tissue, and / or the injured tissue achieves a certain degree of functional recovery.

[0251] As used herein, the term "a" or "an" means one or more or at least one.

[0252] As used herein, a "therapeutically effective" or "effective" dose or amount of a composition is an amount sufficient to have a positive impact on a given medical condition. A therapeutically effective or effective dose or amount can provide a noticeable or measurable effect on the patient's health and well-being over a period of time, if not immediately.

[0253] As used herein, "pharmaceutical composition" refers to an effective amount of a composition described herein combined with a delivery component. A pharmaceutical composition may optionally contain other components, such as pharmaceutically suitable carriers and excipients, which can facilitate administration of the composition and / or its individual components to a subject.

[0254] 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.

[0255] The term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound.

[0256] As used herein, the terms "mixing", "mixing" and the like describe a mechanical process or mechanical treatment of the components. For example, mixing can be in the sense of performing repeated cycles of pressing and folding or similar processing steps that result in intensive compression and mixing of the provided hydrophobic matrix.

[0257] Adult stem cells can be obtained from a variety of adult tissues, including bone marrow, fat and dental pulp tissue. Although all adult stem cells are capable of (cable of) self-renewal and are considered to be multipotent, their therapeutic functions vary from source to source. Therefore, each type of adult stem cell has unique characteristics, making it suitable for certain diseases. Mesenchymal stem cells (MSC) are usually derived from mesoderm, and are multipotent, non-hematopoietic (non-blood) stem cells, which can be differentiated into a variety of tissues from a variety of tissue separations (derived from the tissue), including osteoblasts (such as osteoblasts), chondrocytes (such as chondrocytes), myocytes (such as muscle cells) and adipocytes (such as adipocytes producing marrow adipose tissue). As used herein, "isolated" refers to cells removed from their original environment. Stem cell production regulates a variety of biological processes or is very important to regulating a variety of biological processes, such as growth factors. Growth factors are agents that can stimulate cell growth and / or proliferation and / or cell differentiation, such as naturally occurring substances. Generally, growth factors are proteins or steroid hormones. Although the terms "growth factor" and "factor" and the like are used interchangeably herein, the term "biological factor" is not limited to growth factors.

[0258] 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)). In addition, suitable stem cells display CD34+ positivity upon isolation, but lose this marker during culture. Thus, a complete marker profile for one type of stem cell that can be used according to the present 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 to define cells that appear to be homologous to the above-mentioned human cells, wherein the remaining markers remain the same.

[0259] The phrase "conditioned medium" or "CM" refers to a culture medium containing biological factors secreted by MSCs. This may also be referred to herein as a "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 by, for example, filtration, purification and / or concentration procedures. "Conditioned medium" is obtained by culturing stem cells in a culture medium as described in detail herein and separating the resulting culture medium containing stem cells and their secreted stem cell products (secretome) into a conditioned medium containing biological factors and fewer stem cells than the stem cells present before separation. Conditioned medium can be used for the methods described herein and is substantially free of stem cells (may contain a small percentage of stem cells) or free of stem cells. 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., miRNA), lipids (e.g., phospholipids), polysaccharides and / or combinations thereof. Any combination of these biological factors may be bound to or on the surface of an extracellular vesicle (e.g., exosomes), or separated from an extracellular vesicle.

[0260] The phrases "cell pretreatment", "MSC pretreatment" or "secretome pretreatment" refer to any manipulation of MSCs or secretome-producing cells or secretome-producing cells prior to secretome harvesting. Such manipulations include, but are not limited to, changing the culture format (e.g., 2D planar bioreactors versus 3D bioreactors), 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, stirring in the presence of ECM, culturing under shear stress, stirring or suspending as aggregates or in a matrix, inducing a misfolded protein response, ER stress, inducing differentiation of MSCs, culturing in the presence of conditioned medium and / or hypoxia / hypoxia.

[0261] As used herein, the term "mature retinal cell" refers to a cell that can be contained in 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.

[0262] As used herein, the term "retinal precursor cell" or "retinal progenitor cell" refers to a progenitor cell that has been determined to differentiate into a mature retinal cell. Non-limiting examples of retinal precursor cells include photoreceptor progenitor cells, bipolar progenitor cells, retinal progenitor cells, horizontal progenitor cells, amacrine progenitor cells, retinal ganglion progenitor cells, Muller glial progenitor cells, and retinal pigment epithelial progenitor cells.

[0263] B. Compositions and Formulations

[0264] According to the present specification, provided herein is a composition comprising a conditioned medium comprising a mesenchymal stem cell (MSC) secretome and / or a mesenchymal stem cell (MSC) secretome (including a processed MSC secretome).

[0265] In some embodiments, the MSC secretory protein group is generally low for angiogenic factors. In some embodiments, the MSC secretory protein group does not promote angiogenesis. In some embodiments, the MSC secretory protein group exhibits anti-angiogenic properties. In some embodiments, compared with other secretory protein groups, the MSC secretory protein group provides reduced angiogenesis. In some embodiments, the MSC secretory protein group reduces angiogenesis by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%. In some embodiments, compared with another secretory protein group, the MSC secretory protein group reduces angiogenesis by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%. In some embodiments, compared with the conditioned medium before being processed into the MSC secretory protein group, the MSC secretory protein group reduces angiogenesis by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%. In some embodiments, the MSC secretory protein group has a lower angiogenesis induction. In some embodiments, the MSC secretory protein group has a reduced angiogenic response. In some embodiments, the MSC secretory protein group has a reduced angiogenic capacity. In some embodiments, the MSC secretory protein group damages and / or reduces normal blood vessel formation in the presence of a medium that supports angiogenesis. In some embodiments, when the MSC secretory protein group is compared to an untreated control, the MSC secretory protein group has a reduced angiogenic capacity. In some embodiments, the MSC secretory protein group has a reduced angiogenic capacity compared to a sample treated with a serum-containing medium. In some embodiments, the MSC secretory protein group weakens the angiogenic response. In some embodiments, the MSC secretory protein group reduces the angiogenic response induced by a serum-containing medium. In some embodiments, when the secretory protein group plus serum-containing medium (reduced or no angiogenic response) is compared with a serum-containing medium (angiogenic response), the MSC secretory protein group induces a reduction in angiogenic response. 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. In some embodiments, the angiogenic response is indicated by blood vessel formation in a CAM (Chick Chorioallantoic Membrane) assay. In some embodiments, the angiogenic response is indicated by blood vessel formation in any angiogenesis assay known in the art.

[0266] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises:

[0267] i. IDO (indoleamine-2,3-dioxygenase) enzyme activity;

[0268] ii. a "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 (serine protease inhibitor E1), VEGF-A and b-NGF;

[0269] iii. a "threshold" ppm level of at least one additional factor selected from the group consisting of: sFLT-1, PEDF (serine protease inhibitor 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-β, PDGF, SOD1, SOD2, SOD3, and HO-1; and

[0270] iv. a "threshold" ppm level of 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.

[0271] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises:

[0272] i. less than about 250 μM IDO (indoleamine-2,3-dioxygenase) enzyme activity;

[0273] ii. at least one trophic factor / cytokine selected from the group consisting of: HGF, FGF-7, TIMP-1, TIMP-2, PAI-1 (serine protease inhibitor E1), VEGF-A and / or b-NGF;

[0274] iii. at least one additional factor selected from the group consisting of: sFLT-1, PEDF (serine protease inhibitor 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-β, PDGF, SOD1, SOD2, SOD3 and / or HO-1; and

[0275] 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.

[0276] A mesenchymal stem cell (MSC) secretory proteome composition comprising:

[0277] i. at least one trophic factor / cytokine selected from the group consisting of: HGF, TIMP-1, TIMP-2, PAI-1 (serine protease inhibitor E1), VEGF-A and b-NGF;

[0278] ii. at least one additional factor selected from the group consisting of: PEDF (serine protease inhibitor 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-β and PDGF; and

[0279] 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.

[0280] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor, including but not limited to 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, Emmprin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15 and / or IFNγ.

[0281] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional 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, Emmprin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15 and IFNγ.

[0282] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor, including but not limited to members of the serpin family, including serpins: 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 serine protease inhibitor F1 (also known as PEDF). In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises serine protease inhibitor E1. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises serine protease inhibitor A1.

[0283] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor, including but not limited to proteins involved in anti-oxidation. 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, and 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.

[0284] 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.

[0285] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor, including but not limited to a protein selected from the group consisting of: soluble scavenger receptor cysteine-rich domain-containing protein SSC5D, tumor necrosis factor-induced gene 6 protein (also known as TSG-6), serum albumin, and latent transforming growth factor binding protein (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.

[0286] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises penetratin-3, TIMP-1, serpin E1, TSP-1, and HGF.

[0287] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises 2-16 ng / mL or 9.8+ / -0.5 ng / ml penetratin-3.

[0288] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises 10-200 ng / mL or 90+ / -21.5 ng / ml TIMP-1.

[0289] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises 10-100 ng / mL or 49.2+ / -9.8 ng / ml Serpin E1.

[0290] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises 0.1-10 ng / mL or 2.0+ / -0.3 ng / ml HGF.

[0291] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises 100-800 pg / mL or 304+ / -44 pg / ml VEGF.

[0292] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises 0.1-100 pg / mL or <1 ng / ml IL-8.

[0293] 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 0 μM to about 250 μM. In some embodiments, the IDO (indoleamine-2,3-dioxygenase) enzyme activity is 50 μM to about 250 μM L-kynurenine / million MSCs. In some embodiments, the IDO (indoleamine-2,3-dioxygenase) enzyme activity is 50 μM to about 200 μM L-kynurenine / million MSCs. In some embodiments, the IDO (indoleamine-2,3-dioxygenase) enzyme activity is 100 μM to about 250 μM L-kynurenine / million MSCs. In some embodiments, the IDO (indoleamine-2,3-dioxygenase) enzyme activity is 100 μM to about 200 μM L-kynurenine / 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 L-kynurenine per million MSCs.

[0294] In some embodiments, the MSC secretome further comprises a "threshold" ppm level of at least one additional factor, including but not limited to sFLT-1, PEDF (serine protease inhibitor 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-β, PDGF, SOD1, SOD2, SOD3 and / or HO-1. In some embodiments, the MSC secretome further comprises a "threshold" ppm level of at least one additional factor selected from the group consisting of: sFLT-1, PEDF (serine protease inhibitor 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-β, PDGF, SOD1, SOD2, SOD3 and HO-1. In some embodiments, the MSC secretome further comprises an additional factor in a concentration range of 200 pg / mL to 5000 pg / mL, wherein the additional factor includes but is not limited to sFLT-1, PEDF (serine protease inhibitor 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-β, PDGF, SOD1, SOD2, SOD3 and / or HO-1. In some embodiments, the MSC secretome further comprises 1000-3000 pg / mL of sFLT-1. In some embodiments, the MSC secretome further comprises 400-800 pg / mL of TSG-6.

[0295] In some embodiments, the MSC secretome further comprises 2000-8000 pg / mL of PEDF. In some embodiments, the MSC secretome further comprises 2000-7000 pg / mL of PEDF. In some embodiments, the MSC secretome further comprises 2000-6000 pg / mL of PEDF. In some embodiments, the MSC secretome further comprises 2000-5000 pg / mL of PEDF. In some embodiments, the MSC secretome further comprises 2000-4000 pg / mL of PEDF. In some embodiments, the MSC secretome further comprises 2000-3000 pg / mL of PEDF. In some embodiments, the MSC secretome further comprises 150-300 ng / mL of PEDF. In some embodiments, the MSC secretome further comprises 200-300 ng / mL of PEDF. In some embodiments, the MSC secretome further comprises 200-275 ng / mL of PEDF. In some embodiments, the MSC secretome further comprises 225-275 ng / mL of PEDF. In some embodiments, the MSC secretome further comprises 150-300 ng / mL of PEDF. In some embodiments, the MSC secretome further comprises 273±27 ng / mL of PEDF.

[0296] In some embodiments, the MSC secretome further comprises a "higher" level of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome further comprises a "higher" level of serpin E1. In some embodiments, the MSC secretome further comprises a "higher" level of serpin A1. In some embodiments, the MSC secretome further comprises a "higher" level of TIMP-1. In some embodiments, the MSC secretome further comprises a "higher" level of thrombospondin-1. In some embodiments, the MSC secretome further comprises a "higher" level of penetratin-3 (TSG-14). In some embodiments, the MSC secretome further comprises a "higher" level of platelet factor 4. In some embodiments, the MSC secretome further comprises a "higher" level of serpin F1. In some embodiments, the MSC secretome comprises 1 ng / mL to 20 ng / mL of at least one factor selected from the group consisting of: serpin E1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome comprises 1 ng / mL to 8 ng / mL of at least one factor selected from the group consisting of: serpin E1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome comprises 2 ng / mL to 8 ng / mL of at least one factor selected from the group consisting of: serpin E1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome comprises 3 ng / mL to 8 ng / mL of at least one factor selected from the group consisting of: serpin E1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome comprises 4 ng / mL to 8 ng / mL of at least one factor selected from the group consisting of: serpin E1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome comprises 5 ng / mL to 8 ng / mL of at least one factor selected from the group consisting of: serpin E1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome comprises 6 ng / mL to 8 ng / mL of at least one factor selected from the group consisting of: serpin E1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome comprises 2 ng / mL to 7 ng / mL of at least one factor selected from the group consisting of: serpin E1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1.

[0297] In some embodiments, the MSC secretome composition further comprises a "medium range" level of at least one factor including, but not limited to, angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2 and thrombospondin-1, angiopoietin, DPPIV (dipeptidyl peptidase-4), IGFBP-3 and / or uPA. In some embodiments, the MSC secretome composition further comprises a "medium range" level of at least one factor selected from the group consisting of angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, and thrombospondin-1, angiopoietin, DPPIV, IGFBP-3 and uPA. In some embodiments, the MSC secretome composition further comprises a "medium range" level of at least one factor selected from the group consisting of angiopoietin, DPPIV, IGFBP-3 and uPA. In some embodiments, the MSC secretome composition further comprises about 200 pg / mL to about 800 pg / mL of at least one factor selected from the group consisting of angiogenin, DPPIV, IGFBP-3, and uPA. In some embodiments, the MSC secretome composition further comprises 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 of at least one factor selected from the group consisting of angiogenin-1, angiogenin-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 about 200 pg / mL, 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 of 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. In some embodiments, the MSC secretome composition comprises 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 of angiogenin.In some embodiments, the MSC secretome composition further comprises 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 of DPPIV. In some embodiments, the MSC secretome composition comprises 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 of IGFBP-3. In some embodiments, the MSC secretome composition comprises 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 of uPA.

[0298] 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.

[0299] In some embodiments, the MSC secretome further comprises about 90pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 100pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 125pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 150pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 175pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 1pg / mL to about 400pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 10pg / mL to about 400pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 50pg / mL to about 350pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 50pg / mL to about 300pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 10 pg / mL to about 300 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 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 0 pg / mL to about 200 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 0 pg / mL to about 200 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 0 pg / mL to about 200 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 10 pg / mL to about 200 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 20 pg / mL to about 200 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 30 pg / mL to about 200 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 40 pg / mL to about 200 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 50 pg / mL to about 200 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 60 pg / mL to about 200 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 70 pg / mL to about 200 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 80 pg / mL to about 200 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 90 pg / mL to about 200 pg / mL of VEGF.In some embodiments, the MSC secretome further comprises 100 pg / mL to about 200 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 10 pg / mL to about 150 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 20 pg / mL to about 150 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 30 pg / mL to about 150 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 40 pg / mL to about 150 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 50 pg / mL to about 150 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 60 pg / mL to about 150 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 70 pg / mL to about 150 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 80 pg / mL to about 150 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 90 pg / mL to about 150 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 100 pg / mL to about 150 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 10 pg / mL to about 100 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 20 pg / mL to about 100 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 30 pg / mL to about 100 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 40 pg / mL to about 100 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 50 pg / mL to about 100 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 60 pg / mL to about 100 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 70 pg / mL to about 100 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 80 pg / mL to about 100 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 90 pg / mL to about 100 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 100 pg / mL to about 100 pg / mL of VEGF.

[0300] In some embodiments of the MSC secretome composition, the level of VEGF is 5-10 times lower than the level of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is 6-10 times lower than the level of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is 7-10 times lower than the level of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is 8-10 times lower than the level of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is 9-10 times lower than the level of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is 5 times lower than the level of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is 6 times lower than the level of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is 7 times lower than the level of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is 8 times lower than the level of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is 9-fold lower than the level of serpin El. In some embodiments of the MSC secretome composition, the level of VEGF is 10-fold lower than the level of serpin El.

[0301] 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 200pg / mL, less than about 150pg / mL, less than about 100pg / mL, less than about 75pg / mL, less than about 50pg / mL, or less than about 25pg / mL bFGF, PLGF, and / or PDGF. In some embodiments, the MSC secretome composition contains less than about 200pg / mL, less than about 150pg / mL, less than about 100pg / mL, less than about 75pg / mL, less than about 50pg / mL, or less than about 25pg / mL 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 comprises 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 comprise bFGF. In some embodiments, the MSC secretome composition comprises 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 comprise PLGF. In some embodiments, the MSC secretome composition comprises 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 comprise PDGF. In some embodiments, the MSC secretome composition does not comprise bFGF. In some embodiments, the MSC secretome composition does not comprise PLGF. In some embodiments, the MSC secretome composition does not comprise PDGF. In some embodiments, the MSC secretome composition comprises 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.

[0302] In some embodiments, the MSC secretome composition comprises 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, Emmprin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15 and / or IFNγ.

[0303] In some embodiments, the MSC secretome further comprises a "higher" level of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 1 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 1 ng / mL-300 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 1 ng / mL-200 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 1 ng / mL-100 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 10 ng / mL-400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 10 ng / mL-300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentetracycline-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 10 ng / mL-200 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 10 ng / mL-100 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 20 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 20 ng / mL-300 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 20 ng / mL-200 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 20 ng / mL-100 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 30 ng / mL-400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentetracycline-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 30 ng / mL-300 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 30 ng / mL-200 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 30 ng / mL-100 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 4 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 40 ng / mL-300 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 40 ng / mL-200 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 40 ng / mL-100 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentetracycline-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 50 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 50 ng / mL-300 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 50 ng / mL-200 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 50 ng / mL-100 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 60 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 60 ng / mL-300 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 60 ng / mL-200 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentetracycline-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 60 ng / mL-100 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 70 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 70 ng / mL-300 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 70 ng / mL-200 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 70 ng / mL-100 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 80 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 80 ng / mL-300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentetracycline-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 80 ng / mL-200 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 80 ng / mL-100 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 90 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 90 ng / mL-300 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 90 ng / mL-200 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 90 ng / mL-100 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 100 ng / mL-400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentetracycline-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 100 ng / mL-300 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 100 ng / mL-200 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 110 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 110 ng / mL-300 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 110 ng / mL-200 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 120 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 120 ng / mL-300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentetracycline-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 120 ng / mL-200 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 130 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 130 ng / mL-300 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 130 ng / mL-200 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 140 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 140 ng / mL-300 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 140 ng / mL-200 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentetracycline-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 150 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 150 ng / mL-300 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 150 ng / mL-200 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 160 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 160 ng / mL-300 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 160 ng / mL-200 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 170 ng / mL-400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentetracycline-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 170 ng / mL-300 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 170 ng / mL-200 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 180 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 180 ng / mL-300 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 180 ng / mL-200 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 190 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 190 ng / mL-300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentetracycline-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 190 ng / mL-200 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 200 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 200 ng / mL-300 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 210 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 210 ng / mL-300 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 220 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 220 ng / mL-300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentetracycline-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 230 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 230 ng / mL-300 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 240 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 240 ng / mL-300 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 250 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 250 ng / mL-300 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 260 ng / mL-400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentetracycline-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 260 ng / mL-300 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 270 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 270 ng / mL-300 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 280 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 280 ng / mL-300 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 290 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 290 ng / mL-300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentetracycline-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 310 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 320 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 330 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 340 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 350 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 360 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 370 ng / mL-400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentetracycline-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 380 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 390 ng / mL-400 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 10 ng / mL-90 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 10 ng / mL-80 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 20 ng / mL-100 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 30 ng / mL-100 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 40 ng / mL-100 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 50 ng / mL-100 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 10 ng / mL-70 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 10 ng / mL-60 ng / mL of at least one factor selected from the group consisting of: serpin E1, serpin A1, TIMP-1, thrombospondin-1, penetratin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 10 ng / mL-50 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentetracycline-3 (TSG-14), platelet factor 4, and serpin F1.

[0304] In some embodiments, the MSC secretome composition comprises:

[0305] i. 0.3-4.5 ng / mL HGF;

[0306] ii. 0.5-20 ng / mL penetrant-3 (TSG-14);

[0307] iii. 100-600 pg / mL VEGF;

[0308] iv. 10-200 ng / mL TIMP-1;

[0309] v. 20-80 ng / mL serine protease inhibitor E1; and

[0310] vi. <5ng / mL IL-8.

[0311] In some embodiments, the MSC secretome composition comprises:

[0312] i. 1.5-3.5 ng / mL HGF;

[0313] ii. 5-15 ng / mL penetrant-3 (TSG-14);

[0314] iii. 200-400 pg / mL VEGF;

[0315] iv. 50-120 ng / mL TIMP-1;

[0316] v. 30-70 ng / mL serine protease inhibitor E1; and

[0317] vi.<3ng / mL IL-8.

[0318] In some embodiments, the MSC secretome composition comprises:

[0319] i. 1.5-2.5 ng / mL HGF;

[0320] ii. 8-12 ng / mL penetrant-3 (TSG-14);

[0321] iii. 250-350 pg / mL VEGF;

[0322] iv. 70-110 ng / mL TIMP-1;

[0323] v. 30-70 ng / mL serine protease inhibitor E1; and

[0324] vi.<2ng / mL IL-8.

[0325] In some embodiments, the MSC secretome composition comprises:

[0326] i. 2.0+ / -0.3ng / mL HGF;

[0327] ii. 9.8+ / -0.5ng / mL penetratin-3 (TSG-14);

[0328] iii. 304+ / -44pg / mL VEGF;

[0329] iv. 90+ / -20ng / mL TIMP-1;

[0330] v.49.2+ / -10 ng / mL Serpin E1; and

[0331] vi.<1ng / mL IL-8.

[0332] 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 pH 5.0 to about pH 7.5. In some embodiments, the MSC secretome composition is formulated at a pH of about pH 5.5 to about pH 7.5. In some embodiments, the MSC secretome composition is formulated at a pH of about pH 6 to about pH 7.5.

[0333] 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.

[0334] In some embodiments, the MSC secretome composition does not include certain components. In some embodiments, the MSC secretome composition does not include certain components present in the cell culture medium. In some embodiments, the MSC secretome composition does not include one or more components selected from the group consisting of: xenobiotic components (e.g., animal serum); phenol red; peptides and biomolecules <3kDa; antibiotics; protein aggregates (e.g., protein aggregates>200nm); cells; cell debris (cell debris does not include exosomes / extracellular vesicles (EV); for example, non-exosomes, non-EV cell fragments); 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 include xenobiotic components. In some embodiments, the MSC secretome composition does not include phenol red. In some embodiments, the MSC secretome composition does not include peptides and biomolecules <3kDa. In some embodiments, the MSC secretome composition does not include antibiotics. In some embodiments, the MSC secretome composition does not include protein aggregates (e.g., protein aggregates>200nm). In some embodiments, the MSC secretome composition does not include cells. In some embodiments, the MSC secretome composition does not include cell debris (cell debris does not include exosomes / EV; for example, non-exosomes, non-EV cell debris). In some embodiments, the MSC secretome composition does not include hormones (for example, hormones include but are not limited to insulin and / or hydrocortisone). In some embodiments, the MSC secretome composition does not include L-glutamine.

[0335] 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 further comprises 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 phosphate / disodium phosphate, mannitol and trehalose, wherein the composition has a pH of about pH 7.4.

[0336] In some embodiments, the MSC secretome composition may comprise 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 may comprise 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 may comprise one or more additional agents selected from the group consisting of glycine and glycerol and dextrose. In some embodiments, the MSC secretome composition may comprise one or more additional agents selected from the group consisting of sodium chloride and potassium chloride.

[0337] 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 / monosodium 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 hydrogen phosphate buffer system.

[0338] 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 comprises disodium phosphate / citric acid, mannitol, and trehalose, wherein the composition has a pH of about pH 6.4.

[0339] In some embodiments, the MSC secretome composition further comprises a tonicity adjuster or tension modifier. In some embodiments, the tonicity adjuster or tension modifier includes but is not limited to NaCl, KCl, mannitol, dextrose, sucrose, sorbitol and / or glycerol. In some embodiments, the tonicity adjuster or tension modifier is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol and / or glycerol.

[0340] In some embodiments, the MSC secretome composition further comprises a binder. In some embodiments, the MSC secretome composition further comprises a binder, including but not limited to hydroxypropyl methylcellulose, poloxamer 407, poloxamer 188, poloxamer 237, poloxamer 338, hydroxypropyl methylcellulose (HPMC), HEC, polycarbophil, polyvinyl pyrrolidone (PVP), PVA (polyvinyl alcohol), polyimide, sodium hyaluronate, gellan gum, poly (lactic acid-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. In some embodiments, the binder is hydroxypropyl methylcellulose. In some embodiments, the binder is fibrin glue. In some embodiments, the binder is polyethylene glycol. In some embodiments, the binder is GelCORE (see Sani et al., Science Advances, Vol. 5, No. 3 (2019)).

[0341] In some embodiments, the MSC proteome composition comprises (a) a processed conditioned medium comprising a MSC secretome produced by any of the methods described herein; and (b) a polymer. In some embodiments, the MSC secretome composition comprises a conditioned medium comprising a MSC secretome produced as described herein and a polymer. In some embodiments, the MSC secretome composition comprises a processed conditioned medium comprising a MSC secretome produced as described herein and a polymer. In some embodiments, the polymer can be a biodegradable polymer from which the MSC secretome and / or processed MSC secretome components can be released. In some embodiments, the polymer is capable of sustained (slow) release of MSC secretome components.

[0342] In some embodiments, the MSC secretome composition provided herein is in the form of a therapeutic bandage (e.g., a polymer impregnated with the MSC secretome composition). The therapeutic bandage can be configured as desired, depending on the application. In some embodiments, the bandage is in the form of a patch or is configured as a mesh.

[0343] In some embodiments, the MSC secretome composition exhibits biopenetrability, such as eye 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 intrinsic biopenetrability. In some embodiments, the MSC secretome composition exhibits excipient-imparted biopenetrability. In some embodiments, the MSC secretome composition exhibits biopenetrability due to upregulation of a minor factor. In some embodiments, the MSC secretome composition exhibits biopenetrability due to the presence of a biopreservative. In some embodiments, the MSC secretome composition exhibits biopenetrability due to the presence of a biopreservative benzalkonium chloride.

[0344] In some embodiments, the MSC secretome composition exhibits a long half-life and / or has increased stability compared to other treatments. In some embodiments, the MSC secretome composition as provided herein allows for upregulation of proteins, which allows for increased stability of the MSC secretome. In some embodiments, the MSC secretome composition as provided herein allows for upregulation of chaperone proteins to improve the stability of other proteins in the MSC secretome.

[0345] In some embodiments, the MSC secretome composition exhibits super efficacy when administered to a subject in need thereof. In some embodiments, the MSC secretome composition allows for one drop or one time per day administration to have therapeutic efficacy.

[0346] C. Production / Manufacturing Methods

[0347] According to the present invention, conditioned medium (and thus mesenchymal stem cell secretory factors) can be obtained from mesenchymal stem cells obtained from a patient or individual to be treated (a patient in need) or another (donor) individual (such as a young and / or healthy donor) and / or commercially obtained mesenchymal stem cells. For example, MSCs obtained from an individual to be treated (autologous stem cells) or a donor (allogeneic stem cells) can be used to produce the conditioned medium described herein, and the conditioned medium can then be further processed into a MSC secretome composition as described herein. In some embodiments, MSCs can also be obtained from commercial suppliers. In some embodiments, commercially obtained MSCs can be used for MSC secretome production.

[0348] According to the present invention, the method for preparing an anti-angiogenic mesenchymal stem cell (MSC) secretome composition comprises:

[0349] i. culturing mesenchymal stem cells (MSCs) in a first culture medium;

[0350] ii. removing the first culture medium from step (i) from the MSCs;

[0351] iii. washing the MSCs in step (ii);

[0352] iv. adding a second culture medium and culturing for about 1-5 days;

[0353] v. harvesting the second culture medium from step (iv) as conditioned medium; and

[0354] vi. Processing the conditioned medium in step (v) into a MSC secretome composition as described herein.

[0355] In some embodiments, the culture may be carried out using a bioreactor system for culturing cells. In some embodiments, the culture may be carried out using a bioreactor system for culturing stem cells. In some embodiments, the culture may be carried out using a bioreactor system for culturing mesenchymal stem cells. In some embodiments, the culture may be carried out using a medium mixing technique. In some embodiments, the culture may be carried out using a PBS Vertical Wheel. TM Hybrid technology for cultivation.

[0356] In some embodiments, processing the conditioned medium of step (v) into a secretome composition in step (iv) comprises:

[0357] a) filtering the harvested conditioned medium from step (v) to remove cellular particles;

[0358] b) concentrating the filtered conditioned medium from step (a); and

[0359] c) Perform buffer exchange with formulation buffer.

[0360] In some embodiments, step c) comprises performing a buffer exchange with 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 citric acid / disodium phosphate.

[0361] In some embodiments, the filtering step (a) comprises using a 0.45 μm filter, a 0.22 μm filter, a 0.8 μm filter and a 0.65 micron filter, a low protein binding PVDF membrane and / or PES (polyethersulfone). In some embodiments, the filtering step (a) comprises using a 0.45 μm filter. In some embodiments, the filtering step (a) comprises using a 0.22 μm filter. In some embodiments, the filtering step (a) comprises using a 0.8 μm filter. In some embodiments, the filtering step (a) comprises using a 0.65 micron. In some embodiments, the filtering step (a) comprises using a low protein binding PVDF membrane. In some embodiments, the filtering step (a) comprises using PES (polyethersulfone).

[0362] In some embodiments, the concentration step (b) comprises using a hollow fiber filter, a tangential flow filtration system, or a centrifugal-based size exclusion technique. In some embodiments, the concentration step (b) comprises using a hollow fiber filter technique. In some embodiments, the concentration step (b) comprises using a tangential flow filtration system. In some embodiments, the concentration step (b) comprises using a centrifugal-based size exclusion technique.

[0363] In some embodiments, the centrifugation-based size exclusion technique employs a 3-10 kDa MW cutoff. In some embodiments, the centrifuge-based size exclusion technique uses a 3 kDa MW cutoff, at least a 4 kDa MW cutoff, at least a 5 kDa MW cutoff, at least a 6 kDa MW cutoff, at least a 7 kDa MW cutoff, at least a 8 kDa MW cutoff, at least a 9 kDa MW cutoff, at least a 10 kDa MW cutoff, at least a 11 kDa MW cutoff, at least a 12 kDa MW cutoff, at least a 13 kDa MW cutoff, at least a 14 kDa MW cutoff, at least a 15 kDa MW cutoff, at least a 16 kDa MW cutoff, at least a 17 kDa MW cutoff, at least a 18 kDa MW cutoff, at least a 19 kDa MW cutoff, at least a 20 kDa MW cutoff, at least a 21 kDa MW cutoff, at least a 22 kDa MW cutoff, at least a 23 kDa MW cutoff, at least a 24 kDa MW cutoff, at least a 25 kDa MW cutoff, at least a 26 kDa MW cutoff, at least a 27 kDa MW cutoff, at least a MW cutoff, at least 28 kDa MW cutoff, at least 29 kDa MW cutoff and / or at least 30 kDa MW cutoff.

[0364] In some embodiments, the method produces a MSC secretome composition and / or preparation as described herein above. In some embodiments, the first culture medium and / or the second culture medium is MSC culture medium and / or MSC-XF.

[0365] MSC or cells differentiated from MSC can be made to produce a conditioned medium containing a desired secretome, for example, it contains the desired cytokines and / or desired therapeutic properties as described herein. For example, the secretome can be produced from MSC of a super donor cell line. The secretome can also be produced from commercially available MSC. In the following embodiments, allogeneic MSC (and / or cells derived therefrom) and / or allogeneic MSC-derived secretome compositions can be prepared and stored for a large number of individuals. Allogeneic MSC (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, MSC (and / or cells derived therefrom) and / or MSC-derived secretome compositions can be processed to make a more concentrated solution or composition (e.g., a mesenchymal stem cell-derived secretome composition or a MSC secretome composition as described herein).

[0366] In some embodiments, the initial culture medium and the first culture medium are different. In some embodiments, the initial culture medium and the first culture medium are the same. Non-limiting examples of one or more cell culture media that can be used to culture MSCs to produce a conditioned medium comprising the MSC secretome according to the present invention include hMSC Media Booster XFM, hMSC High Performance Basal Medium, 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 modifications), StemPro, MSCGro, MesenCult, NutriStem, Basal Medium Eagle (BME-supplemented with Earle's salt base), Dulbecco's Modified Eagle Medium (DMEM-with or without serum), Yamane, IMEM-20, Glasgow Modified Eagle Medium (GMEM), Leibovitz L-15 medium, McCoy's 5A medium, medium M199 (M199E - with Earle's salt base), medium M199 (M199H - with Hank's salt base), minimum essential medium alpha (MEM-α), Eagle's minimum essential medium (MEM-E - with Earle's salt base), Eagle's minimum essential medium (MEM-H - with Hank's salt base), and Eagle's minimum essential medium (MEM-NAA with non-essential amino acids), as well as many other media, including medium 199, CMRL 1415, CMRL1969, CMRL 1066, NCTC 135, MB 75261, MAB 8713, DM 145, Williams'G, Neuman & Tytell, Higuchi, MCDB 301, MCDB 202, MCDB 501, MCDB 401, MCDB 411, MDBC 153. The preferred medium for use in the present invention is MEM-α. These and other useful media are available from GIBCO (Grand Island, NY, USA) and Biological Industries (Bet HaEmek, Israel), among others.Many of these media are summarized in Methods in Enzymology, Volume LVIII, "Cell Culture", pages 62-72, edited by William B. Jakoby and Ira H. Pastan, published by Academic Press, Inc.

[0367] In some embodiments, the cell culture medium for mesenchymal stem cells can 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 lysates. In some embodiments, 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 of cattle or other species. In some embodiments, the cell culture medium for mesenchymal stem cells can be supplemented with other ingredients to promote cell growth and / or promote cell health, such as mercaptoethanol and / or antibiotics. In some embodiments, the cell culture medium for mesenchymal stem cells is not supplemented with antibiotics.

[0368] In some embodiments, the oxygen percentage is changed to promote cell growth and / or promote cell health. In some embodiments, the oxygen volume is 5%, 10%, 15%, 20% or 25% to promote cell growth and / or promote cell health. In some embodiments, mesenchymal stem cells are grown under oxygen partial pressure to promote cell growth and / or promote cell health. In some embodiments, mesenchymal stem cells are grown under low oxygen partial pressure environment to promote cell growth and / or promote cell health.

[0369] On the one hand, the present invention relates to a conditioned medium (CM) comprising biological factors secreted by mesenchymal stem cells, which can be referred to as a conditioned medium comprising a MSC secretory protein group. The conditioned medium can be obtained by culturing mesenchymal stem cells in a culture medium as described herein and separating the resulting culture medium containing mesenchymal stem cells and their secreted mesenchymal stem cell products (referred to as biological factors and / or secretory protein groups) into components of a conditioned medium containing a secretory protein group and mesenchymal stem cells grown in the conditioned medium. The conditioned medium after separation comprises a mesenchymal stem cell secretory protein group and can be further processed and / or used according to the methods described herein and is substantially free of mesenchymal stem cells (may contain a small percentage of stem cells and / or a trace of stem cells) or is free of mesenchymal stem cells. The MSC secretory protein group comprises a variety of biological factors, including hormones, cytokines, extracellular matrix, proteins, vesicles, antibodies, chemokines, receptors, inhibitors and particles. As described herein, one or more conditioned mediums (CM or conditioned medium comprising a MSC secretory protein group) comprising a MSC secretory protein group can be further processed to produce a concentrated conditioned medium (pCM or concentrated MSC secretory protein group).

[0370] In some embodiments, the conditioned medium containing the MSC secretory protein group or the concentrated MSC secretory protein group is produced by culturing mesenchymal stem cells in a culture medium, replacing the culture medium in which the mesenchymal stem cells have been cultured. In some embodiments, the resulting conditioned medium containing the MSC secretory protein group is harvested (collected) and then processed to produce a concentrated MSC secretory protein group. In certain embodiments, the processing of the harvested conditioned medium containing the MSC secretory protein group includes removing some, most or substantially all of the culture medium, or removing some, most or substantially all of the selected components of the conditioned medium.

[0371] In some embodiments, the harvested conditioned medium comprising the MSC secretome is filtered to produce a concentrated MSC secretome. In some embodiments, the harvested conditioned medium comprising the MSC secretome is ultrafiltered to produce a concentrated MSC secretome.

[0372] On the one hand, provided herein is a method for producing a processed conditioned medium, comprising (a) culturing stem cells in a cell culture medium to produce a conditioned medium comprising factors secreted by mesenchymal stem cells (e.g., a conditioned medium comprising a mesenchymal stem cell secretory protein group); (b) harvesting the conditioned medium to produce a harvested conditioned medium (e.g., a harvested mesenchymal stem cell secretory protein group); and (c) filtering the harvested conditioned medium (e.g., a harvested mesenchymal stem cell secretory protein group) to produce a processed conditioned medium (mesenchymal stem cell secretory protein group). In some embodiments, the stem cells of (a) are cultured (already cultured) in a growth medium and then 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, thereby producing a conditioned medium comprising a mesenchymal stem cell secretome; (c) harvesting the conditioned medium comprising the mesenchymal stem cell secretome, thereby producing a harvested conditioned medium comprising a mesenchymal stem cell secretome; and (d) filtering the harvested conditioned medium to produce a processed conditioned medium comprising a mesenchymal stem cell secretome.

[0373] In some embodiments, the MSC secretome of the present invention is further processed. In some embodiments, the MSC secretome of the present invention is further processed using techniques known in the art, including but not limited to extraction, freeze-thaw, homogenization, permeabilization, 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.

[0374] In some embodiments, the MSC secretome of the invention is enriched by one or more of the following methods: affinity-based enrichment, size-based enrichment, cation- or anion-based enrichment, and enrichment of fractions with favorable attributes.

[0375] In some embodiments, stem cells are mesenchymal stem cells. Mesenchymal stem cells (MSCs) are multipotent (capable of differentiating into a variety of but not all cell lineages), non-hematopoietic (non-blood) stem cells that are isolated from various adult tissues (including bone marrow and adipose tissue) (derived from the adult tissue). In certain embodiments, mesenchymal stem cells are isolated from the bone marrow. "Isolated" refers to the removal of cells from their original environment. MSCs can differentiate into cells of the mesoderm lineage, such as adipocytes, osteoblasts, and chondrocytes. The cell body of MSC is small, with few cell protrusions, and is elongated. The cell body contains a large, round nucleus with prominent nucleoli, surrounded by finely dispersed chromatin particles, making the nucleus look clear. The rest of the cell body contains a small amount of Golgi bodies, rough endoplasmic reticulum, mitochondria, and polyribosomes. The elongated cells are widely dispersed, and the adjacent extracellular matrix is ​​filled with some reticular fibrils, but lacks other types of collagen fibrils [Brighton et al. 1991 The Journal of Bone and Joint Surgery 73 (6): 832-47]. The MSCs described herein may express the following molecular markers (protein molecules characteristic of the plasma membrane of cells or cell types): bone morphogenetic protein receptor ”1” (BMPR + ); CD34 + Scal + Lin ” ;CD44 + ;c-kit + ; Sca-1 + ;Thy-1 + ; NOTCH3; JAG1; ITGA11. MSCs may also express other cell type specific markers (see, stemcells.nih.gov on the World Wide Web; Kaltz et al. 2010 Exp Cell Res Oct 1; 316(16): 2609-17, incorporated herein by reference). MSCs described herein may be identified based on colony forming unit assays to detect the multipotent differentiation potential of MSCs (what cell types MSCs produce). However, slightly differentiated cells (progenitor cells) may also be used.

[0376] i. MSC secretome-pretreatment

[0377] In some embodiments, in vitro pretreatment secretory protein group can be used to produce MSC to enhance the therapeutic ability / potential of MSC. In some embodiments, this enhancement is achieved by affecting the secretion profile of MSC. In some embodiments, such pretreatment schemes may include but are not limited to changing the culture form (e.g., 2D planar bioreactors compared to 3D bioreactors), different biomaterial scaffolds, co-cultivation, adding pharmacological compounds, growth factors, chemokines, adding toll-like receptor agonists, inflammatory cytokines, advanced glycation end products (AGE), oxidized phospholipids, malondialdehyde or carboxyethyl pyrrole, stirring the presence of ECM, cultivating under shear stress, stirring or suspending as aggregates or in a matrix, inducing misfolded protein reactions, ER stress, inducing differentiation of MSC, cultivating in the presence of conditioned medium and hypoxia / hypoxia. See, Ferreira et al., 2018 Frontiers in Immunol. Vol. 9, No. 2837, incorporated herein by reference.

[0378] Physiological oxygen tension in tissues varies from 1% in cartilage and bone marrow to 12% in peripheral blood. Therefore, the 21% oxygen level routinely used in cell culture incubators is much higher than physiological conditions. In some embodiments, hypoxic pretreatment of MSCs enhances their regenerative and cytoprotective effects and / or proliferation rates, and can 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 allow the cells to adapt. In some embodiments, MSCs can adapt to very low oxygen tensions in vitro. In some embodiments, hypoxic values ​​can range from hypoxia (0% O 2 ) to 2% O 2 In some embodiments, the hypoxia value can be from hypoxia (0% O) to hypoxia (0% O) over a period of 4 hours to 72 hours, including 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, or 72 hours. 2 ) to 2% O 2 . In some embodiments, hypoxic preconditioning may cause MSCs to express factors associated with neovascularization. In some embodiments, hypoxic preconditioning may cause MSCs to express higher levels of HIF-1α, GDNF, BDNF, VEGF, Ang-1, SDF-1 and its receptor CXCR4, as well as EPO and its receptor EPOR. In some embodiments, hypoxic preconditioning may cause MSCs to express higher levels of neuroprotective factors and pro-angiogenic factors.

[0379] In some embodiments, MSC is stimulated with inflammatory cytokines. In some embodiments, MSC is stimulated with inflammatory cytokines that promote the production of secretory protein groups containing factors involved in immune response regulation. In some embodiments, MSC is stimulated with inflammatory cytokines to produce secretory protein groups containing factors that promote chemoattraction of immune cells, regulation of inflammation, and / or enhance migration / homing of other cells. Other immunomodulatory abilities of MSC secretory protein groups may include inhibition of NK cells, inhibition of complement system activation, differentiation of monocytes to M2 macrophages, inhibition of cytotoxic T cell proliferation, and increase in regulatory T cell numbers. In some embodiments, effector cytokines in these processes may include, but are not limited to, IL-6, PGE2, and IDO. In some embodiments, IFNγ pretreatment causes MSC secretory protein groups to have enhanced immunosuppressive ability. In some embodiments, IFNγ pretreatment causes MSC secretory protein groups to have enhanced immunosuppressive ability through IDO. In some embodiments, SDF-1 pretreatment may be used as part of the pretreatment process. In some embodiments, SDF-1 pretreatment provides MSC secretory protein groups that cause increased angiogenesis and reduced fibrosis. In some embodiments, TGF-α pre-treated MSCs provide a secretome with increased VEGF production. In some embodiments, melatonin pre-treated MSCs provide a MSC secretome that results in greater cell survival under oxidative stress, induced misfolded protein response, or ER stress. In some embodiments, H 2 O 2 Pre-treated MSCs resulted in an MSC secretome with increased angiogenesis, higher survival, and reduced inflammation in a rat model of ischemia / reperfusion injury. MSCs can also be pre-treated with a cocktail of growth factors, namely FGF-2, IGF-1, and BMP-2, in order to generate an MSC secretome with these activities.

[0380] In some embodiments, the pretreatment method has mechanical properties and is related to simulating MSC microenvironment by 3D culture method. In some embodiments, 3D cell culture container / bioreactor is adopted. In some embodiments, 3D culture of spheroid is adopted, so as to allow to imitate the physiological conditions in bone marrow. In some embodiments, spheroid culture produces microenvironment, wherein the inner layer is exposed to much lower oxygen and nutrient levels, so as to produce hypoxic environment. In such embodiments, MSC expresses higher levels of TSG-6, SCT-1, LIF, IL-24, TRAIL and CXCR4, so as to allow to produce MSC secretory protein group with higher concentration of these factors. In such embodiments, MSC sphere culture shows increased survival, proliferation and angiogenesis. In such embodiments, MSC produces MSC secretory protein group with enhanced immunomodulation, angiogenesis, anti-fibrosis and anti-apoptotic activity.

[0381] A variety of pharmacological agents can also be used to pre-treat MSCs. In some embodiments, MSCs can be pre-treated with atorvastatin, oxytocin, curcumin, lipopolysaccharide, nicotinamide (NIC), vasoactive intestinal peptide (VIP) and / or diazoxide. In some embodiments, MSCs can be pre-treated with nicotinamide (NIC) and / or vasoactive intestinal peptide (VIP) to protect them from oxidative stress. In some embodiments, a pharmaceutical composition based on a MSC secretome from MSC pre-treated with neuroprotective drugs VIP, NIC or both enhances the proliferation and neuroprotective effects of the secretome. See, Alonso-Alonso et al., (2020) Stem Cell Int Vol. 2020, Article ID 9463548, incorporated herein by reference.

[0382] In some embodiments, pre-treating the MSC secretome includes inducing a misfolded protein response in the MSCs.

[0383] In some embodiments, pre-treating the MSC secretome comprises culturing the MSCs under suitable conditions to induce ER stress.

[0384] In some embodiments, pre-treating the MSC secretome includes culturing the MSCs under suitable conditions to induce differentiation of the MSCs into mature retinal cell types or precursors thereof.

[0385] 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, Muller glial progenitor cells, and retinal pigment epithelial progenitor cells.

[0386] The present invention provides a pre-treated mesenchymal stem cell (MSC) secretory protein composition, comprising:

[0387] i. less than about 250 μM IDO (indoleamine-2,3-dioxygenase) enzyme activity;

[0388] ii. at least one trophic factor / cytokine selected from the group consisting of: HGF, FGF-7, TIMP-1, TIMP-2, PAI-1 (serine protease inhibitor E1), VEGF-A and / or b-NGF;

[0389] 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-β, PDGF, SOD1, SOD2, SOD3 and / or HO-1; and

[0390] 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.

[0391] In some embodiments, the pretreated MSC secretome further comprises "higher levels" of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentetracycline-3 (TSG-14), platelet factor 4 and / or serpin F1, optionally at 1 ng / mL-8 ng / mL.

[0392] In some embodiments, the pretreated MSC secretome further comprises a "mid-range" level of at least one factor selected from the group consisting of angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, thrombospondin-1, angiopoietin, DPPIV, IGFBP-3 and / or uPA, optionally 400 pg / mL-3000 pg / mL.

[0393] In some embodiments, the pretreated 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, Emmprin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15 and / or IFNγ.

[0394] In some embodiments, the pretreated MSC secretome comprises a ratio of anti-angiogenic to pro-angiogenic, wherein the ratio is >2, >3, >4, or >5.

[0395] In some embodiments, the pre-treated MSC secretome further comprises "low" levels of VEGF, optionally 0 pg / mL-200 pg / mL.

[0396] In some embodiments, the level of VEGF is 5-10 times lower than the level of serpin El.

[0397] In some embodiments, the composition comprises one or more anti-angiogenic factors, and wherein 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.

[0398] In some embodiments, the pre-treated MSC secretome contains no and / or very low levels of bFGF, PLGF, and PDGF, optionally less than 1000 pg / mL.

[0399] In some embodiments, the pre-treated MSC secretome composition has a pH of about 4.7 to about 7.5.

[0400] In some embodiments, the pre-treated MSC secretome 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 citric acid / disodium phosphate.

[0401] In some embodiments, the pre-treated MSC secretome composition further comprises a tonicity modifier.

[0402] In some embodiments, the tonicity adjuster is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerol.

[0403] In some embodiments, the pre-treated MSC secretome further comprises mono / disodium phosphate, mannitol, and trehalose, wherein the composition has a pH of about pH 7.4.

[0404] In some embodiments, the pre-treated MSC secretome further comprises divalent cations.

[0405] In some embodiments, the divalent cation is selected from the group consisting of Mg2+, Ca2+, and Zn2+.

[0406] In some embodiments, the pre-treated MSC secretome further comprises disodium phosphate / citric acid, mannitol, and trehalose, wherein the composition has a pH of about pH 6.4.

[0407] In some embodiments, the composition further comprises a binder.

[0408] In some embodiments, the binder is selected from the group consisting of: hydroxypropyl methylcellulose, poloxamer 407, poloxamer 188, poloxamer 237, poloxamer 338, hydroxypropyl methylcellulose (HPMC), polycarbophil, polyvinyl pyrrolidone (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.

[0409] The present invention also provides a method for preparing a pretreated mesenchymal stem cell (MSC) secretory protein composition, comprising:

[0410] i. culturing mesenchymal stem cells (MSCs) in a first culture medium;

[0411] ii. removing the first culture medium from step (i) from the MSCs;

[0412] iii. washing the MSCs in step (ii);

[0413] iv. adding a second culture medium and culturing for about 1-5 days;

[0414] v. harvesting the second culture medium from step (iv) as conditioned medium; and

[0415] vi. Processing the conditioned medium in step (v) into the pre-treated MSC secretome composition as described herein.

[0416] In some embodiments, the pre-treated MSC secretome composition is a secretome composition described herein.

[0417] In some embodiments, the step (vi) of processing the conditioned medium in step (v) into a secretome composition comprises:

[0418] a) filtering the harvested conditioned medium from step (v) to remove cellular particles;

[0419] b) concentrating the filtered conditioned medium from step (a); and

[0420] c) Perform buffer exchange with formulation buffer.

[0421] In some embodiments, step c) comprises performing a buffer exchange with 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 citric acid / disodium phosphate.

[0422] In some embodiments, the filtering step (a) comprises using 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).

[0423] In some embodiments, the concentration step (b) comprises the use of a hollow fiber filter, a tangential flow filtration system, or a centrifugation-based size exclusion technique.

[0424] In some embodiments, the centrifugation-based size exclusion technique employs a 3-10 kDa MW cutoff.

[0425] In some embodiments, the present invention provides a method for treating an eye disease, comprising administering to a patient in need thereof a therapeutically effective amount of a mesenchymal stem cell secretome composition as described herein or a composition prepared according to the method described herein.

[0426] In some embodiments, the composition is administered to a target area.

[0427] The present invention also provides a method for treating visual dysfunction after traumatic injury to an ocular structure in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the mesenchymal stem cell secretome composition as described herein or a composition prepared according to the method described herein.

[0428] The present invention also provides a method for inducing and / or promoting ocular wound healing in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the mesenchymal stem cell secretome composition as described herein or a composition prepared according to the method described herein.

[0429] The present invention also provides a method for reducing and / or inhibiting neovascularization, reducing and / or inhibiting scar formation, promoting and / or maintaining vision and / or increasing wound closure rate (e.g., reducing closure time) in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a mesenchymal stem cell secretome composition as described herein or a composition prepared according to the method described herein.

[0430] The present invention also provides a method for reducing and / or inhibiting neovascularization and reducing scar formation to promote vision preservation in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the mesenchymal stem cell secretome composition as described herein or a composition prepared according to the method described herein.

[0431] In some embodiments, the mesenchymal stem cell secretome composition is formulated for topical administration.

[0432] In some embodiments, the mesenchymal stem cell secretome composition is formulated for subconjunctival injection.

[0433] In some embodiments, the mesenchymal stem cell secretome composition is formulated for intravitreal injection.

[0434] The present invention also provides a method for characterizing the MSC secretory proteome, wherein the method comprises:

[0435] (i) subjecting the MSC secretome to one or more characterization assays, wherein the characterization assay is selected from the group consisting of: physical component characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analysis, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / scarring assays, inflammation assays, epithelial barrier integrity assays, retinal degeneration assays and / or assays for inherited retinal diseases including human and animal retinal explants, neuroprotection / neurotrophy assays; and

[0436] (ii) determining the results of the one or more determinations in (i).

[0437] Exemplary disclosures of characterization assays can be found 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, Artero 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.

[0438] The present invention also provides a method for determining the biological efficacy and stability of MSC secretory proteome, comprising:

[0439] (i) subjecting the MSC secretome to one or more characterization assays, wherein the characterization assay is selected from the group consisting of: physical component characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analysis, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / scarring assays, inflammation assays, epithelial barrier integrity assays, retinal degeneration assays and / or assays for inherited retinal diseases including human and animal retinal explants, neuroprotection / neurotrophy assays; and

[0440] (ii) determining the results of the one or more determinations in (i).

[0441] The present invention also provides a method for determining MSC secretome batch consistency between multiple MSC secretome batches, wherein the method comprises:

[0442] (i) subjecting the MSC secretome to one or more characterization assays, wherein the characterization assay is selected from the group consisting of: physical component characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analysis, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / scarring assays, inflammation assays, epithelial barrier integrity assays, retinal degeneration assays and / or assays for inherited retinal diseases including human and animal retinal explants, neuroprotection / neurotrophy assays; and

[0443] (ii) determining the results of the one or more determinations in (i).

[0444] In some embodiments, the results in (ii) from the characterization of the physical component identify an anti-angiogenic MSC secretome as described herein.

[0445] In some embodiments, the results in (ii) from the safety analysis provide a MSC secretome that exhibits hemocompatibility and low and / or no pyrogens and / or endotoxins.

[0446] In some embodiments, the results in (ii) from the stability assay provide a MSC secretome that exhibits stability at 4°C, 20°C, and / or 25°C (or room temperature) for at least 7 days.

[0447] In some embodiments, the results in (ii) from the proliferation assay provide a MSC secretome that induces proliferation.

[0448] In some embodiments, the results in (ii) from the migration assay provide the MSC secretome that induces migration.

[0449] In some embodiments, the results in (ii) from the adhesion assay provide a MSC secretory protein group that induces cell adhesion. Adhesion assays can be performed using techniques known in the art. Exemplary disclosures of adhesion assays are provided in U.S. Patent Publication Nos. 20170067061A1 and 20150050325A1 and Blue et al., Blood 2008, 111, 1248, each of which is incorporated herein by reference in its entirety.

[0450] In some embodiments, the results in (ii) from the neovascularization assay provide a MSC secretome that inhibits or does not promote neovascularization.

[0451] In some embodiments, the results in (ii) from the differentiation / scarring assay provide a MSC secretome that inhibits differentiation and / or scarring.

[0452] In some embodiments, the results in (ii) from the inflammation assay provide a MSC secretome that suppresses inflammation or alters an immune response.

[0453] In some embodiments, the method further comprises:

[0454] (iii) Identify MSC secretome batches based on the results in (ii).

[0455] The present invention also provides a panel of tests and / or assays for characterizing the MSC secretome, wherein the panel comprises at least two characterization assays, wherein the characterization assays are selected from the group consisting of: physical component characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analysis, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / scarring assays and / or inflammation assays.

[0456] The present invention also provides a panel of tests and / or assays for determining the consistency between batches of MSC secretome, wherein the panel comprises one or more characterization assays, wherein the characterization assays are selected from the group consisting of: physical component characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analysis, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / scarring assays and / or inflammation assays.

[0457] In some embodiments, the physical component characterization identifies the MSC secretome as described herein.

[0458] In some embodiments, the results in (ii) from the safety analysis provide a MSC secretome that exhibits hemocompatibility and low and / or no pyrogens and / or endotoxins.

[0459] In some embodiments, the stability assay identifies MSC secretomes that exhibit stability at 4°C, 20°C, and / or 25°C (or room temperature) for at least 7 days.

[0460] In some embodiments, the proliferation assay identifies the MSC secretome that induces proliferation.

[0461] In some embodiments, the migration assay identifies the MSC secretome that induces migration.

[0462] In some embodiments, the neovascularization assay identifies MSC secretomes that inhibit or do not promote neovascularization.

[0463] In some embodiments, the differentiation / scarring assay identifies MSC secretomes that inhibit differentiation and / or scarring.

[0464] In some embodiments, inflammation assays identify MSC secretomes that suppress inflammation or alter immune responses.

[0465] In some embodiments, physical component characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analysis, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / scarring assays, inflammation assays, epithelial barrier integrity assays, retinal degeneration assays and / or assays for inherited retinal diseases including human and animal retinal explants, neuroprotection / neurotrophic assays are all performed.

[0466] In some embodiments, a panel of tests and / or assays as described herein identifies the MSC secretome as described herein.

[0467] In some embodiments, a panel of tests and / or assays as described herein comprises 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 "scratch wound assay"), a circular scratch wound method, a circular scratch wound assay, and a circular wound closure assay. In some embodiments, the pretreated MSC secretome is an anti-angiogenic MSC secretome and / or an anti-scarring MSC secretome.

[0468] In some embodiments, the pre-treated MSC secretome is an anti-angiogenic MSC secretome and / or an anti-scarring MSC secretome.

[0469] In some embodiments, the pre-treated MSC secretome is an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.

[0470] The present invention also provides a stable pre-treated mesenchymal stem cell (MSC) secretory proteome preparation, comprising:

[0471] i. 1-20 μg, optionally 2 μg-8 μg MSC secretome / mL;

[0472] ii. 2mg-3mg sodium dihydrogen phosphate / mL;

[0473] iii. 11mg-12mg sodium dihydrogen phosphate / mL;

[0474] iv. 11.5mg-13mg mannitol / mL;

[0475] v.23mg-24mg trehalose dihydrate;

[0476] vi. 0.5 mg-2 mg hydroxypropyl methylcellulose / mL; and

[0477] wherein the pH is from about 4.7 to about 7.5.

[0478] The present invention also provides a stable pre-treated mesenchymal stem cell (MSC) secretory proteome preparation, comprising:

[0479] i. 0.004%-0.0375%, optionally 0.008%-0.015% w / w MSC secretome;

[0480] ii. 4%-5% w / w sodium dihydrogen phosphate;

[0481] iii. 21.5%-23% w / w disodium hydrogen phosphate;

[0482] iv. 23%-25% w / w mannitol;

[0483] v.46%-48% w / w dehydrated trehalose;

[0484] vi. 1%-3% w / w hypromellose; and

[0485] wherein the pH is from about 4.7 to about 7.5.

[0486] The present invention also provides a stable pre-treated mesenchymal stem cell (MSC) secretory proteome preparation, comprising:

[0487] i.6 μg MSC secretome / mL;

[0488] ii. 2.28 mg sodium dihydrogen phosphate / mL;

[0489] iii. 11.45 mg sodium dihydrogen phosphate / mL;

[0490] iv. 12.2 mg mannitol / mL;

[0491] v.24 mg trehalose dihydrate / mL;

[0492] vi. 1 mg of hypromellose / mL; and

[0493] The pH is about 7.4.

[0494] The present invention also provides a stable pre-treated mesenchymal stem cell (MSC) secretory proteome preparation, comprising:

[0495] i.6 μg MSC secretome / mL;

[0496] ii. 1.31 mg sodium dihydrogen phosphate / mL;

[0497] iii.5.73 mg sodium dihydrogen phosphate / mL;

[0498] iv. 12.2 mg mannitol / mL;

[0499] v.24 mg trehalose dihydrate / mL;

[0500] vi. 1 mg of Hydroxypropyl Methylcellulose / mL;

[0501] vii. 0.2 mg magnesium chloride / mL;

[0502] viii. 1.17 mg sodium chloride / mL; and

[0503] The pH is about 7.4.

[0504] The present invention also provides a stable pre-treated mesenchymal stem cell (MSC) secretory proteome preparation, comprising:

[0505] i. 0.012% w / w MSC secretome;

[0506] ii. 4.5% w / w sodium dihydrogen phosphate;

[0507] iii. 22.4% w / w disodium hydrogen phosphate;

[0508] iv. 24% w / w mannitol;

[0509] v.47.1% w / w trehalose dehydrate;

[0510] vi. 2.0% w / w hypromellose; and

[0511] The pH is about 7.4.

[0512] The present invention also provides a stable pre-treated mesenchymal stem cell (MSC) secretory proteome preparation, comprising:

[0513] i. Sodium phosphate 10 mM;

[0514] ii. Histidine HCL 10 mM;

[0515] iii. Trehalose dihydrate 10%;

[0516] iv. Polysorbate 20 0.01%;

[0517] The pH is about 5.5.

[0518] The present invention also provides a stable pre-treated mesenchymal stem cell (MSC) secretory proteome preparation, comprising:

[0519] i. Sodium phosphate 10mM

[0520] ii. Sucrose 5%

[0521] iii. Sodium chloride 10mM

[0522] The pH is about 6.2.

[0523] The present invention also provides a stable pre-treated mesenchymal stem cell (MSC) secretory proteome preparation, comprising:

[0524] i. Sodium phosphate 10mM

[0525] ii. Sucrose 5.8%

[0526] iii. Polysorbate 80 0.02%

[0527] The pH is about 6.2.

[0528] The present invention also provides a pre-treated mesenchymal stem cell (MSC) secretory protein composition, comprising:

[0529] i. at least one trophic factor / cytokine selected from the group consisting of: HGF, TIMP-1, TIMP-2, PAI-1 (serine protease inhibitor E1), VEGF-A and b-NGF;

[0530] 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-β, and PDGF; and

[0531] 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.

[0532] In some embodiments, the pretreated MSC secretome composition further comprises a high level of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentetracycline-3 (TSG-14), platelet factor 4, and serpin F1.

[0533] In some embodiments, the pretreated MSC secretome composition comprises 1 ng / mL-400 ng / mL of at least one factor selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentetracycline-3 (TSG-14), platelet factor 4, and serpin F1.

[0534] In some embodiments, the pretreated MSC secretome composition further comprises a mid-range level of at least one factor selected from the group consisting of angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, thrombospondin-1, angiopoietin, DPPIV, IGFBP-3, and uPA.

[0535] In some embodiments, the pretreated MSC secretome composition comprises 400 pg / mL-3000 pg / mL of at least one factor selected from the group consisting of angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, thrombospondin-1, angiopoietin, DPPIV, IGFBP-3 and uPA.

[0536] In some embodiments, the pretreated MSC secretome composition 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, Emmprin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15 and IFNγ.

[0537] In some embodiments, the pretreated MSC secretome composition comprises a ratio of anti-angiogenic to pro-angiogenic, wherein the ratio is >2, >3, >4, or >5.

[0538] In some embodiments of the pretreated MSC secretome composition, the anti-angiogenic factors include one or more factors selected from the group consisting of: PEDF, lower levels of VEGF and serine protease inhibitor E1, and the pro-angiogenic factors include one or more factors selected from the group consisting of: VEGF, angiopoietin, IGFBP-3, uPA, Angio-1, Angio-2, endothelin-1.

[0539] In some embodiments, the pre-treated MSC secretome composition further comprises low levels of VEGF.

[0540] In some embodiments, the pre-treated MSC secretome composition comprises 1 pg / mL-400 pg / mL of VEGF.

[0541] In some embodiments of the pre-treated MSC secretome composition, the level of VEGF is 5-10 fold lower than the level of serpin E1.

[0542] In some embodiments, the pretreated MSC secretome composition comprises one or more anti-angiogenic factors, and wherein 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.

[0543] In some embodiments, the pre-treated MSC secretome composition contains no and / or very low levels of bFGF, PLGF, and PDGF.

[0544] In some embodiments, the pre-treated MSC secretome composition comprises less than 1000 pg / mL of bFGF, PLGF, and PDGF.

[0545] In some embodiments, the pre-treated MSC secretome composition has a pH of about 4.7 to about 7.5.

[0546] In some embodiments, the pretreated 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 citric acid / disodium phosphate.

[0547] In some embodiments, the pre-treated MSC secretome composition further comprises a tonicity modifier.

[0548] In some embodiments of the pre-treated MSC secretome composition, the tonicity modifier is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerol.

[0549] In some embodiments, the pre-treated MSC secretome composition further comprises mono / disodium phosphate, mannitol, and trehalose, and wherein the composition has a pH of about pH 7.4.

[0550] In some embodiments, the pre-treated MSC secretome composition further comprises divalent cations.

[0551] In some embodiments, the divalent cations in the pretreated MSC secretome composition are selected from the group consisting of Mg2+, Ca2+, and Zn2+.

[0552] In some embodiments, the pre-treated MSC secretome composition further comprises disodium phosphate / citric acid, mannitol, and trehalose, wherein the composition has a pH of about pH 6.4.

[0553] In some embodiments, the pre-treated MSC secretome composition further comprises a binder.

[0554] In some embodiments of the pretreated MSC secretome composition, the binder is selected from the group consisting of: hydroxypropyl methylcellulose, poloxamer 407, poloxamer 188, poloxamer 237, poloxamer 338, hydroxypropyl methylcellulose (HPMC), polycarbophil, polyvinyl pyrrolidone (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.

[0555] In some embodiments, the pretreated MSC secretome composition does not comprise one or more components selected from the group consisting of: xenobiotic 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.

[0556] In some embodiments, the pre-treated MSC secretome composition comprises: HGF; penetratin-3 (TSG-14); VEGF; TIMP-1; serpin El; and <5 ng / mL IL-8.

[0557] In some embodiments, the pre-treated MSC secretome composition comprises:

[0558] i. 0.3-4.5 ng / mL HGF;

[0559] ii. 0.5-20 ng / mL penetrant-3 (TSG-14);

[0560] iii. 100-600 pg / mL VEGF;

[0561] iv. 10-200 ng / mL TIMP-1;

[0562] v. 20-80 ng / mL serine protease inhibitor E1; and

[0563] vi. <5ng / mL IL-8.

[0564] In some embodiments, the pretreated MSC secretome composition comprises an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.

[0565] The present invention also provides a stable pre-treated mesenchymal stem cell (MSC) secretory proteome preparation, comprising:

[0566] i. 2μg-20μg MSC secretome / mL;

[0567] ii. 2mg-3mg sodium dihydrogen phosphate / mL;

[0568] iii. 11mg-12mg sodium dihydrogen phosphate / mL;

[0569] iv. 11.5mg-13mg mannitol / mL;

[0570] v.23mg-24mg trehalose dihydrate;

[0571] vi. 0.5 mg-2 mg hydroxypropyl methylcellulose / mL; and

[0572] wherein the pH is from about 4.7 to about 7.5.

[0573] The present invention also provides a stable pre-treated mesenchymal stem cell (MSC) secretory proteome preparation, comprising:

[0574] i. 0.004%-0.08% w / w MSC secretome;

[0575] ii. 4%-5% w / w sodium dihydrogen phosphate;

[0576] iii. 21.5%-23% w / w disodium hydrogen phosphate;

[0577] iv. 23%-25% w / w mannitol;

[0578] v.46%-48% w / w dehydrated trehalose;

[0579] vi. 1%-3% w / w hypromellose; and

[0580] wherein the pH is from about 4.7 to about 7.5.

[0581] The present invention also provides a method for treating an eye disease in a subject in need thereof, comprising administering to the subject a pretreated mesenchymal stem cell (MSC) secretome composition, wherein the pretreated MSC secretome composition comprises:

[0582] i. at least one trophic factor / cytokine selected from the group consisting of: HGF, TIMP-1, TIMP-2, PAI-1 (serine protease inhibitor E1), VEGF-A and b-NGF;

[0583] 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-β, and PDGF; and

[0584] 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.

[0585] In some embodiments, the pretreated MSC secretome composition further comprises high levels of at least one factor selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentetracycline-3 (TSG-14), platelet factor 4, and serpin F1.

[0586] In some embodiments, the pretreated MSC secretome composition comprises 1 ng / mL-100 ng / mL of at least one factor selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentetracycline-3 (TSG-14), platelet factor 4, and serpin F1.

[0587] In some embodiments, the pretreated MSC secretome composition further comprises a mid-range level of at least one factor selected from the group consisting of angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, thrombospondin-1, angiopoietin, DPPIV, IGFBP-3, and uPA.

[0588] In some embodiments, the pretreated MSC secretome composition comprises 400 pg / mL-3000 pg / mL of at least one factor selected from the group consisting of angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, thrombospondin-1, angiopoietin, DPPIV, IGFBP-3 and uPA.

[0589] In some embodiments, the pretreated MSC secretome composition 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, Emmprin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15 and IFNγ.

[0590] In some embodiments, the pretreated MSC secretome composition comprises a ratio of anti-angiogenic to pro-angiogenic, wherein the ratio is >2, >3, >4, or >5.

[0591] In some embodiments, the anti-angiogenic factors include one or more factors selected from the group consisting of: PEDF, lower levels of VEGF, and serine protease inhibitor El and the pro-angiogenic: VEGF, angiopoietin, IGFBP-3, uPA, Angio-1, Angio-2, Endothelin-1.

[0592] In some embodiments, the pre-treated MSC secretome composition further comprises low levels of VEGF.

[0593] In some embodiments, the pre-treated MSC secretome comprises 1 pg / mL-400 pg / mL of VEGF.

[0594] In some embodiments, the level of VEGF is 5-10 times lower than the level of serpin El.

[0595] In some embodiments, the pretreated MSC secretome composition comprises one or more anti-angiogenic factors, and wherein 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.

[0596] In some embodiments, the pre-treated MSC secretome composition contains no or very low levels of bFGF, PLGF, and PDGF.

[0597] In some embodiments, the pre-treated MSC secretome composition comprises less than 1000 pg / mL of bFGF, PLGF, and PDGF.

[0598] In some embodiments, the pre-treated MSC secretome composition has a pH of about 4.7 to about 7.5.

[0599] In some embodiments, the pretreated 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 citric acid / disodium phosphate.

[0600] In some embodiments, the pre-treated MSC secretome composition further comprises a tonicity modifier.

[0601] In some embodiments, the tonicity adjuster is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerol.

[0602] In some embodiments, the pre-treated MSC secretome composition further comprises mono / disodium phosphate, mannitol, and trehalose, and wherein the composition has a pH of about pH 7.4.

[0603] In some embodiments, the pre-treated MSC secretome composition further comprises divalent cations.

[0604] In some embodiments, the divalent cation is selected from the group consisting of Mg2+, Ca2+, and Zn2+.

[0605] In some embodiments, the pre-treated MSC secretome composition further comprises disodium phosphate / citric acid, mannitol, and trehalose, and wherein the composition has a pH of about pH 6.4.

[0606] In some embodiments, the pre-treated MSC secretome composition further comprises a binder.

[0607] In some embodiments, the binder is selected from the group consisting of: hydroxypropyl methylcellulose, poloxamer 407, poloxamer 188, poloxamer 237, poloxamer 338, hydroxypropyl methylcellulose (HPMC), polycarbophil, polyvinyl pyrrolidone (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.

[0608] In some embodiments, the pretreated MSC secretome composition does not comprise one or more components selected from the group consisting of: xenobiotic 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.

[0609] In some embodiments, the pre-treated MSC secretome composition comprises: HGF; penetratin-3 (TSG-14); VEGF; TIMP-1; serpin El; and <5 ng / mL IL-8.

[0610] In some embodiments, the pre-treated MSC secretome composition comprises:

[0611] i. 0.3-4.5 ng / mL HGF;

[0612] ii. 0.5-20 ng / mL penetrant-3 (TSG-14);

[0613] iii. 100-600 pg / mL VEGF;

[0614] iv. 10-200 ng / mL TIMP-1;

[0615] v. 20-80 ng / mL serine protease inhibitor E1; and

[0616] vi. <5ng / mL IL-8.

[0617] In some embodiments, the pretreated MSC secretome composition comprises an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.

[0618] The present invention also provides a method for treating an ocular disease in a subject in need thereof, comprising administering to the subject a pretreated mesenchymal stem cell (MSC) secretome composition, wherein the pretreated MSC secretome composition is a stable pretreated mesenchymal stem cell (MSC) secretome preparation comprising:

[0619] i. 2μg-20μg MSC secretome / mL;

[0620] ii. 2mg-3mg sodium dihydrogen phosphate / mL;

[0621] iii. 11mg-12mg sodium dihydrogen phosphate / mL;

[0622] iv. 11.5mg-13mg mannitol / mL;

[0623] v.23mg-24mg trehalose dihydrate;

[0624] vi. 0.5 mg-2 mg hydroxypropyl methylcellulose / mL; and

[0625] wherein the pH is from about 4.7 to about 7.5.

[0626] The present invention also provides a method for treating an ocular disease in a subject in need thereof, comprising administering to the subject a pretreated mesenchymal stem cell (MSC) secretome composition, wherein the pretreated MSC secretome composition is a stable pretreated mesenchymal stem cell (MSC) secretome preparation comprising:

[0627] i. 0.004%-0.08% w / w MSC secretome;

[0628] ii. 4%-5% w / w sodium dihydrogen phosphate;

[0629] iii. 21.5%-23% w / w disodium hydrogen phosphate;

[0630] iv. 23%-25% w / w mannitol;

[0631] v.46%-48% w / w dehydrated trehalose;

[0632] vi. 1%-3% w / w hypromellose; and

[0633] wherein the pH is from about 4.7 to about 7.5.

[0634] The present invention also provides a stable pre-treated mesenchymal stem cell (MSC) secretory proteome preparation, comprising:

[0635] i. 1-20 μg, optionally 2 μg-8 μg MSC secretome / mL;

[0636] ii. 2mg-3mg sodium dihydrogen phosphate / mL;

[0637] iii. 11mg-12mg sodium dihydrogen phosphate / mL;

[0638] iv. 11.5mg-13mg mannitol / mL;

[0639] v.23mg-24mg trehalose dihydrate;

[0640] vi. 0.5 mg-2 mg optionally hypromellose / mL; and

[0641] wherein the pH is from about 4.7 to about 7.5.

[0642] The present invention also provides a stable pre-treated mesenchymal stem cell (MSC) secretory proteome preparation, comprising:

[0643] i. 0.004%-0.0375%, optionally 0.008%-0.015% w / w MSC secretome;

[0644] ii. 4%-5% w / w sodium dihydrogen phosphate;

[0645] iii. 21.5%-23% w / w disodium hydrogen phosphate;

[0646] iv. 23%-25% w / w mannitol;

[0647] v.46%-48% w / w dehydrated trehalose;

[0648] vi. 1%-3% w / w optionally hypromellose; and

[0649] wherein the pH is from about 4.7 to about 7.5.

[0650] The present invention also provides a stable pre-treated mesenchymal stem cell (MSC) secretory proteome preparation, comprising:

[0651] i.6 μg MSC secretome / mL;

[0652] ii. 2.28 mg sodium dihydrogen phosphate / mL;

[0653] iii. 11.45 mg sodium dihydrogen phosphate / mL;

[0654] iv. 12.2 mg mannitol / mL;

[0655] v.24mg trehalose dihydrate;

[0656] vi. 1 mg optionally hypromellose / mL; and

[0657] The pH is about 7.4.

[0658] The present invention also provides a stable pre-treated mesenchymal stem cell (MSC) secretory proteome preparation, comprising:

[0659] i.6 μg MSC secretome / mL;

[0660] ii. 1.31 mg sodium dihydrogen phosphate / mL;

[0661] iii.5.73 mg sodium dihydrogen phosphate / mL;

[0662] iv. 12.2 mg mannitol / mL;

[0663] v.24 mg trehalose dihydrate / mL;

[0664] vi. 1 mg of Hydroxypropyl Methylcellulose / mL;

[0665] vii. 0.2 mg magnesium chloride / mL;

[0666] viii. 1.17 mg sodium chloride / mL; and

[0667] The pH is about 7.4.

[0668] The present invention also provides a stable pre-treated mesenchymal stem cell (MSC) secretory proteome preparation, comprising:

[0669] i. 0.012% w / w MSC secretome;

[0670] ii. 4.5% w / w sodium dihydrogen phosphate;

[0671] iii. 22.4% w / w disodium hydrogen phosphate;

[0672] iv. 24% w / w mannitol;

[0673] v.47.1% w / w trehalose dehydrate;

[0674] vi. 2.0% w / w optionally hypromellose; and

[0675] The pH is about 7.4.

[0676] The present invention also provides a stable pre-treated mesenchymal stem cell (MSC) secretory proteome preparation, comprising:

[0677] i. 2μg-20μg MSC secretome / mL;

[0678] ii. 2mg-3mg sodium dihydrogen phosphate / mL;

[0679] iii. 11mg-12mg sodium dihydrogen phosphate / mL;

[0680] iv. 11.5mg-13mg mannitol / mL;

[0681] v.23mg-24mg trehalose dihydrate;

[0682] vi. 0.5 mg-2 mg optionally hypromellose / mL; and

[0683] wherein the pH is from about 4.7 to about 7.5.

[0684] The present invention also provides a stable pre-treated mesenchymal stem cell (MSC) secretory proteome preparation, comprising:

[0685] i. 0.004%-0.08% w / w MSC secretome;

[0686] ii. 4%-5% w / w sodium dihydrogen phosphate;

[0687] iii. 21.5%-23% w / w disodium hydrogen phosphate;

[0688] iv. 23%-25% w / w mannitol;

[0689] v.46%-48% w / w dehydrated trehalose;

[0690] vi. 1%-3% w / w optionally hypromellose; and

[0691] wherein the pH is from about 4.7 to about 7.5.

[0692] In some embodiments of the stable pre-treated mesenchymal stem cell (MSC) secretome preparation, the preparation does not comprise hypromellose.

[0693] The present invention also provides a method for treating an ocular disease in a subject in need thereof, comprising administering to the subject a pretreated mesenchymal stem cell (MSC) secretome composition, wherein the pretreated MSC secretome composition is a stable pretreated mesenchymal stem cell (MSC) secretome preparation comprising:

[0694] i. 2μg-20μg MSC secretome / mL;

[0695] ii. 2mg-3mg sodium dihydrogen phosphate / mL;

[0696] iii. 11mg-12mg sodium dihydrogen phosphate / mL;

[0697] iv. 11.5mg-13mg mannitol / mL;

[0698] v.23mg-24mg trehalose dihydrate;

[0699] vi. 0.5 mg-2 mg optionally hypromellose / mL; and

[0700] wherein the pH is from about 4.7 to about 7.5.

[0701] The present invention also provides a method for treating an ocular disease in a subject in need thereof, comprising administering to the subject a pretreated mesenchymal stem cell (MSC) secretome composition, wherein the pretreated MSC secretome composition is a stable pretreated mesenchymal stem cell (MSC) secretome preparation comprising:

[0702] i. 0.004%-0.08% w / w MSC secretome;

[0703] ii. 4%-5% w / w sodium dihydrogen phosphate;

[0704] iii. 21.5%-23% w / w disodium hydrogen phosphate;

[0705] iv. 23%-25% w / w mannitol;

[0706] v.46%-48% w / w dehydrated trehalose;

[0707] vi. 1%-3% w / w optionally hypromellose; and

[0708] wherein the pH is from about 4.7 to about 7.5.

[0709] In some embodiments of the methods of treating an ocular disorder, the pre-treated MSC secretome composition and / or formulation used in the methods of treating does not contain hypromellose.

[0710] In some embodiments of the methods described herein, the pre-treated MSC secretome composition and / or preparation does not comprise hypromellose.

[0711] In some embodiments of the pre-treated MSC secretome composition and / or preparation, the composition and / or preparation does not comprise hypromellose.

[0712] ii. MSC secretome-processing

[0713] In some embodiments, the conditioned medium containing the MSC secretory protein group described herein can be collected and filtered and / or purified to remove cell microparticles and / or other harmful components. For example, as described above, the second culture medium is harvested from step (iv) as a conditioned medium under step (v). The filter membrane used herein can be selected from any filter membrane with a suitable membrane and structure known in the art, so that they can retain the desired MSC secretory protein group components while allowing cell microparticles and / or other harmful components to pass through. Therefore, any suitable membrane can be used, which allows cells to be retained under selected fluid dynamic conditions while allowing harmful components to pass through to be removed. In some embodiments, the upper limit of the pore size is about 5 microns and the lower limit is about 0.1 microns is suitable. In some embodiments, a microporous filter can be used for filtration. In some embodiments, a 0.5 μm to 0.2 μm filter can be used for filtration. In some embodiments, 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 can be used for filtration. In some embodiments, 0.45 μm filter may be used for filtration. In some embodiments, 0.22 μm filter may be used for filtration. In some embodiments, low protein binding polyvinylidene fluoride (PVDF) membrane may be used for filtration / purification. In some embodiments, polyethersulfone (PES) may be used for filtration / purification.

[0714] In some embodiments, filtration is performed by ultrafiltration. In some embodiments, conditioned medium is filtered using a filter size of 3kD (to achieve purification, desalination and concentration of molecules greater than the filter size in the conditioned medium of processing). In some embodiments, conditioned medium is filtered using a filter size less than 3kD, and in other embodiments, a filter size greater than 3kD is used, depending on the application of the conditioned medium processed. In other embodiments, ultrafiltration of the conditioned medium harvested is performed using filters of different pore sizes (e.g., 2kD, <2kD or>2kD), and the filter is selected to determine the size of the components of the conditioned medium of the resulting processing containing the MSC secretory protein group.

[0715] In some embodiments, harmful components in the growth support medium are removed by medium exchange, preferably by "cross-flow filtration". Cross-flow filtration refers to a filtration mode in which a suspension of MSC secretory proteome 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 fluid dynamic parameters, including Re = Reynolds number, γw = wall shear rate, ΔP = pressure drop, and TMP = transmembrane pressure. Re, γw, and ΔP will depend on the geometry of the filtration system, flow conditions, and fluid properties. In some embodiments, this cross-flow process may also include a hollow fiber filtration system. See, for example, U.S. Patent No. 5,053,334, which is incorporated herein by reference in its entirety.

[0716] In some embodiments, the MSC secretome can be further concentrated without filtration and / or after filtration. In some embodiments, the MSC secretome can be concentrated using hollow fiber tangential flow technology, or

[0717] In some embodiments, the MSC secretory proteome can be concentrated using a centrifugation-based size exclusion technique, for example, an amicon and / or a centricon can be used during the centrifugation step. In some embodiments, the size cutoff is a 3-10 kDa MW cutoff. In some embodiments, the molecular weight cutoff used during the centrifugation-based size exclusion technique concentration method 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.

[0718] In some embodiments, the MSC secretome is concentrated by about 5 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, about 55 times, about 60 times, about 65 times, about 70 times, about 75 times, about 80 times, about 85 times, about 90 times, about 95 times, or about 100 times. In some embodiments, the MSC secretome is concentrated by about 5 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, about 55 times, about 60 times, about 65 times, about 70 times, about 75 times, about 80 times, about 85 times, about 90 times, about 95 times, or about 100 times compared to the conditioned medium before concentration.

[0719] In some embodiments, the MSC secretome is further buffer exchanged into a final formulation buffer after the concentration step. In some embodiments, the MSC secretome is further buffer exchanged into a final formulation buffer without a binder after the concentration step. In some embodiments, the buffer exchange comprises changing the buffer component 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.

[0720] In some embodiments, the MSC secretome is subjected to a buffer exchange after the concentration step such that all traces of culture medium components are removed. In some embodiments, the MSC secretome is subjected to a buffer exchange after the concentration step 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 culture medium components remain.

[0721] iii. MSC secretome-formulation

[0722] In some embodiments, the MSC secretome is prepared in a formulation comprising about 2 μg-20 μg / 1 mL of the MSC secretome. In some embodiments, the MSC secretome is prepared in a formulation comprising 0.004% to 0.0375% / mL of the MSC secretome.

[0723] In some embodiments, the MSC secretome is prepared in a formulation comprising about 2 μg-8 μg / 1 mL of the MSC secretome. In some embodiments, the MSC secretome is prepared in a formulation comprising 0.008% to 0.015% / mL of the MSC secretome.

[0724] In some embodiments, the MSC secretome is prepared in a formulation comprising 2 mg-3 mg / mL of sodium phosphate monobasic. In some embodiments, the MSC secretome is prepared in a formulation comprising 4% to 5% / mL of sodium phosphate monobasic.

[0725] In some embodiments, the MSC secretome is prepared in a formulation comprising 11 mg-12 mg / mL of disodium phosphate. In some embodiments, the MSC secretome is prepared in a formulation comprising 21.5% to 23% / mL of disodium phosphate.

[0726] In some embodiments, the MSC secretome is prepared in a formulation comprising 11.5 mg-13 mg / mL of mannitol. In some embodiments, the MSC secretome is prepared in a formulation comprising 23% to 25% / mL of mannitol.

[0727] In some embodiments, the MSC secretome is prepared in a formulation comprising 23 mg-25 mg / mL of trehalose dihydrate. In some embodiments, the MSC secretome is prepared in a formulation comprising 46% to 48% / mL of trehalose dihydrate.

[0728] In some embodiments, the MSC secretome is prepared in a formulation that does not include hypromellose. In some embodiments, the MSC secretome is prepared in a formulation that optionally includes hypromellose. In some embodiments, the MSC secretome is prepared in a formulation that includes 0.5 mg-2 mg / mL of hypromellose. In some embodiments, the MSC secretome is prepared in a formulation that includes 1% to 3% / mL of hypromellose.

[0729] In some embodiments, the MSC secretome is prepared in a formulation comprising hydrochloric acid and / or sodium hydroxide. In some embodiments, the MSC secretome is prepared in a formulation comprising hydrochloric acid. In some embodiments, the MSC secretome is prepared in a formulation comprising sodium hydroxide. In some embodiments, hydrochloric acid and / or sodium hydroxide are used to obtain the desired pH value.

[0730] In some embodiments, the MSC secretome is prepared in a formulation comprising the components provided in Tables 1-4 below:

[0731] Table 1a: Embodiment of MSC secretome preparation.

[0732] The ingredients that exist Amount of product per 1mL Percentage per 1mL (%) MSC secretome 0.006mg(6μg) 0.012 Sodium dihydrogen phosphate 2.28mg 4.5 Disodium hydrogen phosphate 11.45mg 22.4 Mannitol 12.2mg 24.0 Trehalose dihydrate 24mg 47.1 Hydroxypropyl methylcellulose 1mg 2.0 Hydrochloric acid and / or sodium hydroxide Adjust as needed Adjust as needed Total 50.936mg 100％

[0733] Table 1b: Embodiment of MSC secretome preparation.

[0734] The ingredients that exist Amount of product per 1mL Percentage per 1mL (%) MSC secretome 0.006mg(6μg) 0.012 Sodium dihydrogen phosphate 1.14mg 2.3 Disodium hydrogen phosphate 5.72 11.2 Mannitol 12.2mg 24.0 Trehalose dihydrate 24mg 47.1 Hydroxypropyl methylcellulose 1mg 2.0 Hydrochloric acid and / or sodium hydroxide Adjust as needed Adjust as needed

[0735] Table 1c: Embodiment of MSC secretome preparation.

[0736] The ingredients that exist Amount of product per 1mL MSC secretome 0.006mg(6μg) Sodium dihydrogen phosphate 1.31mg Disodium hydrogen phosphate 5.73mg Mannitol 12.2mg Trehalose dihydrate 24mg Hydroxypropyl methylcellulose 1mg Hydrochloric acid and / or sodium hydroxide Adjust as needed Magnesium chloride hexahydrate 0.2mg Sodium chloride 1.76mg Total 50.936mg

[0737] Table 2: Embodiment of MSC secretome preparation.

[0738] The ingredients that exist concentration MSC secretome 0.006 mg / mL (6 μg / mL) Histidine HCl 10mM α,α-Trehalose dihydrate 10％ Polysorbate 20 0.01％

[0739] In some embodiments, the MSC secretome preparations provided in Table 2 have a pH of about 5.5.

[0740] Table 3: Embodiment of MSC secretome preparation.

[0741] The ingredients that exist concentration MSC secretome 0.006 mg / mL (6 μg / mL) Sodium phosphate 10mM Sodium chloride 40mM Polysorbate 20 0.03％ sucrose 5％

[0742] In some embodiments, the MSC secretome preparations provided in Table 3 have a pH of about 6.2.

[0743] Table 4: Embodiment of MSC secretome preparation.

[0744] The ingredients that exist concentration MSC secretome 0.006 mg / mL (6 μg / mL) Sodium Citrate 10mM sucrose 5.8％ Polysorbate 20, pH 5.5 0.01％

[0745] In some embodiments, the MSC secretome preparations provided in Table 4 have a pH of about 7.2. In some embodiments, the MSC secretome is formulated with water for injection according to USP standards.

[0746] D. Assays / Therapeutic Properties

[0747] In some embodiments of the invention, the MSC secretome is processed to obtain certain component ratios / concentrations and properties of the MSC secretome.

[0748] In some embodiments, the MSC secretome composition comprises a ratio of anti-angiogenic to pro-angiogenic, wherein the ratio is >1. In some embodiments, the MSC secretome composition comprises a ratio of anti-angiogenic to pro-angiogenic, wherein the ratio is >2, >3, >4, or >5. In some embodiments, the MSC secretome composition comprises an increased concentration of pro-angiogenic factors (relative 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 more than one anti-angiogenic factor to VEGF is >2, >3, >4, or >5. In some embodiments, the MSC secretome composition comprises one or more anti-angiogenic factors, and wherein the sum of the concentrations of the one or more anti-angiogenic factors is >2, >3, >4, or >5 relative to the concentration of VEGF. In some embodiments, the pro-angiogenic factors include, but are not limited to, serine protease inhibitor El to VEGF-A. In some embodiments, the pro-angiogenic factor is serine protease inhibitor El. In some embodiments, the pro-angiogenic factor is VEGF-A.

[0749] In some embodiments of the invention, the MSC secretome is processed to achieve certain potency performance criteria. In some embodiments, a buffer exchange step facilitates obtaining a potent MSC secretome.

[0750] Extracellular vesicles are membrane-bound particles that carry the above-mentioned soluble and insoluble substances. The term "extracellular vesicle" refers to a group of secreted or shed vesicles of different species. These are generally divided into the following subtypes: 1) microvesicles or shed microvesicles, which generally show a size range of 50-1500nm; 2) exosomes, which generally show a size range of 30-120nm; and 3) vesicles, which generally show a size range of less than 500nm (i.e. <500nm). (See, for example, WO2019016799, which is incorporated herein by reference in its entirety). In some embodiments, the particle count of the MSC secretome and / or the extracellular vesicles (EVs) present in the quantitative secretome can be analyzed.

[0751] In some embodiments, EV is at about 2.5x10^5 / uL, 2.6x10^5 / uL, 2.7x10^5 / uL, 2.8x10^5 / uL, 2.9x10^5 / uL, 3.0x10^5 / uL, 3.1x10^5 / uL, 3.2x10^5 / uL, 3.3x10^5 / uL, 3.4x10^5 / uL, 3.5x10^5 / uL, 3.6x10^5 / uL, 3.7x10^5 / uL, / uL, 3.8x10^5 / uL, 3.9x10^5 / uL, 4.0x10^5 / uL, 4.1x10^5 / uL, 4.2x10^5 / uL, 4.3x10^5 / uL, 4.4x10^5 / uL, 4.5x10^5 / uL, 4.6x10^5 / uL, 4.7x10^5 / uL, 4.8x10^5 / uL, 4.9x10^5 / uL or about 5.0x10^5 / uL. In some embodiments, EVs are present at a concentration of about 3.8x10^5 / uL+ / -0.8x10^5.

[0752] In some embodiments, Ev is about 2.5x10^5 / uL, 2.6x10^5 / uL, 2.7x10^5 / uL, 2.8x10^5 / uL, 2.9x10^5 / uL, 3.0x10^5 / uL, 3.1x10^5 / uL, 3.2x10^5 / uL, 3.3x10^5 / uL, 3.4x10^5 / uL, 3.5x10^5 / uL, 3.6x10^5 / uL, 3.7x10^5 / uL, 3.8x10^5 / uL, 3.9x10^5 / uL, 4.0x10^5 / uL, 4.1x10^5 / uL, 4.2x10^5 / uL, 4.3x10^5 / uL, 4.4x10^5 / uL, 4.5x10^5 / uL, 4.6x10^5 / uL, 4.7x10^5 / uL, 4.8x10^5 / uL 10^5 / uL, 3.9x10^5 / uL, 4.0x10^5 / uL, 4.1x10^5 / uL, 4.2x10^5 / uL, 4.3x10^5 / uL, 4.4x10^5 / uL, 4.5x10^5 / uL, 4.6x10^5 / uL, 4.7x10^5 / uL, 4.8x10^5 / uL, 4.9x10^5 / uL or about 5.0x10^5 / uL and an average diameter of 110-120nm. In some embodiments, Ev is about 2.5x10^5 / uL, 2.6x10^5 / uL, 2.7x10^5 / uL, 2.8x10^5 / uL, 2.9x10^5 / uL, 3.0x10^5 / uL, 3.1x10^5 / uL, 3.2x10^5 / uL, 3.3x10^5 / uL, 3.4x10^5 / uL, 3.5x10^5 / uL, 3.6x10^5 / uL, 3.7x10^5 / uL, 3.8x10^5 / uL, 3.9x10^5 / uL, 4.0x10^5 / uL, 4.1x10^5 / uL, 4.2x10^5 / uL, 4.3x10^5 / uL, 4.4x10^5 / uL, 4.5x10^5 / uL, 4.6x10^5 / uL, 4.7x10^5 / uL, 4.8x10^5 / uL 10^5 / uL, 3.9x10^5 / uL, 4.0x10^5 / uL, 4.1x10^5 / uL, 4.2x10^5 / uL, 4.3x10^5 / uL, 4.4x10^5 / uL, 4.5x10^5 / uL, 4.6x10^5 / uL, 4.7x10^5 / uL, 4.8x10^5 / uL, 4.9x10^5 / uL or about 5.0x10^5 / uL and an average diameter of 112-116nm.In some embodiments, EV is at about 2.5x10^5 / uL, 2.6x10^5 / uL, 2.7x10^5 / uL, 2.8x10^5 / uL, 2.9x10^5 / uL, 3.0x10^5 / uL, 3.1x10^5 / uL, 3.2x10^5 / uL, 3.3x10^5 / uL, 3.4x10^5 / uL, 3.5x10^5 / uL, 3.6x10^5 / uL, 3.7x10^5 / uL, 3. 5 / uL, 4.8x10^5 / uL, 4.1x10^5 / uL, 4.2x10^5 / uL, 4.3x10^5 / uL, 4.4x10^5 / uL, 4.5x10^5 / uL, 4.6x10^5 / uL, 4.7x10^5 / uL, 4.8x10^5 / uL, 4.9x10^5 / uL or about 5.0x10^5 / uL and an average diameter of 114nm. In some embodiments, EVs are present at a concentration of about 3.8x10^5 / uL+ / -0.8x10^5 and an average diameter of 114nm.

[0753] i. MSC secretome-therapeutic properties

[0754] The MSC secretome of the present disclosure exhibits a variety of therapeutic properties, including, for example, anti-angiogenic properties (vascular and / or lymphatic), anti-fibrotic properties, anti-inflammatory properties, properties that promote cell migration and proliferation, mitogenic properties, anti-oxidative stress / damage properties,

[0755] In some embodiments, the anti-angiogenic (vascular and / or lymphatic) properties can be determined by the presence and / or level of one or more factors in the MSC secretory protein group. In some embodiments, the anti-angiogenic factors include but are not limited to one or more of PEDF, sFLT-1, lower levels of VEGF and / or serine protease inhibitor E1. In some embodiments, the anti-angiogenic factors include but are not limited to one or more of PEDF, lower levels of VEGF and / or serine protease inhibitor 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 a lower level of VEGF. In some embodiments, the anti-angiogenic factor is serine protease inhibitor E1.

[0756] In some embodiments, the angiogenic (vascular and / or lymphatic) properties can be determined by the presence and / or level of one or more factors in the MSC secretory protein group. In some embodiments, the angiogenic factors include one or more factors selected from the group consisting of: VEGF, angiogenin, IGFBP-3, uPA, Angio-1, Angio-2, endothelin-1. In some embodiments, the angiogenic factor is VEGF. In some embodiments, the angiogenic factor is angiogenin. In some embodiments, the angiogenic factor is IGFBP-3. In some embodiments, the angiogenic factor is uPA. In some embodiments, the angiogenic factor is Angio-1. In some embodiments, the angiogenic factor is Angio-2. In some embodiments, the angiogenic factor is endothelin-1.

[0757] In some embodiments, the MSC secretory protein group exhibits anti-fibrotic properties. In some embodiments, this anti-fibrotic property can be determined using standard assays. In some embodiments, the presence of various factors and / or activities about the MSC secretory protein group indicates anti-fibrotic properties. In some embodiments, factors indicating anti-fibrotic properties include but are not limited to FGF7 and / or FGF10. In some embodiments, the factor indicating anti-fibrotic properties is FGF7. In some embodiments, the factor indicating anti-fibrotic properties is FGF10. In some embodiments, the factor indicating anti-fibrotic properties is HGF. In some embodiments, the activity indicating anti-fibrotic properties includes but is not limited to the activation of SMAD, the inhibition of TGFβ pathways, the inhibition of myofibroblast differentiation and / or the inhibition of excessive ECM deposition. In some embodiments, the activity indicating anti-fibrotic properties includes the activation of SMAD. In some embodiments, the activity indicating anti-fibrotic properties includes the inhibition of TGFβ pathways. In some embodiments, the activity indicating anti-fibrotic properties includes the inhibition of myofibroblast differentiation. In some embodiments, the activity indicating anti-fibrotic properties includes the inhibition of excessive ECM deposition.

[0758] 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., inflammation is completely reduced). In some embodiments, the MSC secretome prevents mast cell degranulation.

[0759] In some embodiments, the MSC secretome promotes cell migration and proliferation, including, for example, mitogenic and prokinetic activities. In some embodiments, the MSC secretome promotes mitogenic activity. In some embodiments, the MSC secretome promotes prokinetic activity. In some embodiments, the MSC secretome comprises FGF7, which provides cell migration and proliferation activities of the MSC secretome.

[0760] In some embodiments, the MSC secretome comprises FGF7, which provides cell migration and proliferation activities of the MSC secretome.

[0761] In some embodiments, the MSC secretome comprises HGF, which provides cell migration and proliferation activities of the MSC secretome.

[0762] In some embodiments, the MSC secretome comprises an anti-apoptotic agent, which provides cell migration and proliferation activities of the MSC secretome. In some embodiments, the MSC secretome comprises an anti-apoptotic agent, which includes but is not limited to FGF-2, HGF and IGF-1, and provides cell migration and proliferation activities of the MSC secretome. In some embodiments, the MSC secretome comprises an anti-apoptotic agent selected from the group consisting of: FGF-2, HGF and IGF-1, and provides cell migration and proliferation activities of the MSC secretome.

[0763] In some embodiments, the MSC secretome comprises NGF, which provides cell migration and proliferation activities of the MSC secretome.

[0764] In some embodiments, the MSC secretome provides resistance to oxidative stress and / or cell damage reduction. In some embodiments, the MSC secretome includes resistance to oxidative stress and cell damage factor reduction. In some embodiments, resistance to oxidative stress and cell damage factor reduction include but are not limited to SOD-1, SOD-2, SOD-3, HO-1. In some embodiments, resistance to oxidative stress and cell damage factor reduction is selected from the group consisting of: SOD-1, SOD-2, SOD-3, HO-1.

[0765] ii. MSC secretome - biophysical / biochemical properties

[0766] Biochemical and biophysical characterization:

[0767] In some embodiments, the present invention provides methods for characterizing the MSC secretome. In some embodiments, the MSC secretome characterization will include: 1) comprehensive and / or quantitative positioning of molecular entities in the MSC secretome; 2) measuring the contribution of selected factors to biological activity; and 3) measuring biophysical parameters. In some embodiments, in order to determine the characteristics of the MSC secretome, various efficacy assays can be performed on the MSC secretome as described herein. In some embodiments, the MSC secretome can be subjected to comprehensive and / or quantitative positioning of molecular entities in the MSC secretome; 2) measuring the contribution of selected factors to biological activity; and 3) measuring biophysical parameters. In some embodiments, characterization assays include, but are not limited to, biophysical assays, biochemical assays, and biological assays. In some embodiments, characterization assays may include, but are not limited to, physical component characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analyses, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / scarring assays, inflammation assays, immunoassays, gliosis assays, tissue explant survival and function assays, organoid development or survival / function, epithelial barrier integrity assays, retinal degeneration assays and / or assays for inherited retinal diseases including human and animal retinal explants, neuroprotection / neurotrophy assays. In some embodiments, characterization assays are selected from the group consisting of physical component characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analyses, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / scarring assays, inflammation assays, immunoassays, gliosis assays, tissue explant survival and function assays, organoid development or survival / function, epithelial barrier integrity assays, retinal degeneration assays and / or assays for inherited retinal diseases including human and animal retinal explants, neuroprotection / neurotrophy assays.

[0768] Physical composition characterization:

[0769] In some embodiments, the characterization of the MSC secretome comprises a method using a combination of bioanalytical techniques. In some embodiments, the characterization of the MSC secretome comprises determining the physical components of the MSC secretome. In some embodiments, the characterization of the MSC secretome comprises using protein arrays, enzyme-linked immunosorbent assays (ELISA), mass spectrometry, and immunoblotting. In some embodiments, the characterization of the MSC secretome 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, quantitative techniques can be used to measure the levels of one or more factors. In some embodiments, quantitative techniques (such as ELISA) can be used to measure the level of each factor.

[0770] In some embodiments, the secretory protein group comprises protein factors and extracellular vesicles (EVs). In some embodiments, the MSC secretory protein group comprises nutritional factors. In some embodiments, the protein factors of the MSC secretory protein group include penetrant-3, TIMP-1, serine protease inhibitor E1, TSP-1, HGF. In some embodiments, the MSC secretory protein group comprises EV. In some embodiments, the simple lipid content of the MSC secretory protein group is analyzed to quantitatively measure total lipids. In some embodiments, the EV score of the MSC secretory protein group can be evaluated for EV markers. In some embodiments, the EV score of the MSC secretory protein group can be evaluated for EV markers, and the markers include but are not limited to AUX, TSG101, CD63, CD9 and CD8.

[0771] In some embodiments, the secretome comprises proteins ranging in size from 30-200 nm and 1×10 8 Up to 5x10 9 EV / mL of extracellular vesicles (EV).

[0772] In some embodiments, depletion studies can be performed to extract the individual contributions of key factors. In some embodiments, using an antibody-based pull-down method, defined factors can be removed from the MSC secretory protein group. In some embodiments, depletion can be verified by Western blot and then evaluated by one or more bioassays as described below. In some embodiments, depletion studies can be performed to evaluate the contribution of protein fractions and EV fractions. In some embodiments, TIMP1 and / or serine protease inhibitor E1 may be consumed. In some embodiments, TIMP1 and / or serine protease inhibitor E1 may be consumed.

[0773] Oxidative stress:

[0774] In some embodiments, the MSC secretome can be assayed for oxidative stress prevention. In some embodiments, the MSC secretome prevents corneal epithelial damage. 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 IL-8.

[0775] Safety Characterization:

[0776] In some embodiments, the blood compatibility of the MSC secretome can be evaluated and tests for sterility and pyrogen and endotoxin levels can be performed. In some embodiments, the blood compatibility of the MSC secretome can be evaluated. In some embodiments, assessing blood compatibility includes determinations of hemolysis and hemagglutination. In some embodiments, the MSC secretome does not exhibit harmful effects of systemic exposure. In some embodiments, the MSC secretome does not exhibit harmful effects of systemic exposure (such as severe eye burns). In some embodiments, the MSC secretome does not exhibit hemagglutination activity. In some embodiments, the MSC secretome does not induce hemolysis. In some embodiments, the MSC secretome does not induce hemolytic activity.

[0777] In some embodiments, the MSC secretome may be sterile so that it can be administered as part of a pharmaceutical formulation. In some embodiments, the MSC secretome may be free or substantially free of endotoxins. In some embodiments, the MSC secretome may be free or substantially free of microorganisms.

[0778] stability:

[0779] In some embodiments, biophysical characteristics of the MSC secretome can be evaluated and / or determined. In some embodiments, fluorescence, static light scattering, and dynamic light scattering are used to characterize protein stability metrics. In some embodiments, the following parameters can be measured to further characterize the secretome: thermal melting, thermal aggregation, ΔG, and / or viscosity. In some embodiments, a thermal melting assay is used to determine MSC secretome stability. In some embodiments, a thermal aggregation assay is used to determine MSC secretome stability. In some embodiments, ΔG is used as a measure for determining MSC secretome stability. In some embodiments, viscosity is measured as a MSC secretome characteristic. In some embodiments, viscosity is used to determine MSC secretome stability.

[0780] In some embodiments, biophysical metrics can be employed to establish stability parameters for characterizing different MSC secretome preparations.

[0781] 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 about -20°C for at least 7 days, at least 14 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, or at least 3 months. In some embodiments, the MSC secretome is stable at about 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 about 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.

[0782] In some embodiments, the MSC secretome is stable for at least 7 days at about -20°C. In some embodiments, the MSC secretome is stable for at least 7 days at about 4°C. In some embodiments, the MSC secretome is stable for at least 7 days at about 20°C. In some embodiments, the MSC secretome is stable for at least 7 days at about 25°C (room temperature).

[0783] In some embodiments, the MSC secretome is stable at about -20°C for at least 14 days. In some embodiments, the MSC secretome is stable at about 4°C for at least 14 days. In some embodiments, the MSC secretome is stable at about 20°C (or room temperature) for at least 14 days. In some embodiments, the MSC secretome is stable at about 25°C (room temperature) for at least 14 days.

[0784] Epithelial barrier integrity assay

[0785] The corneal epithelium, more precisely, the apical surface of the epithelium contributes significantly to the overall barrier properties of the cornea, and changes in the corneal barrier are used as sensitive factors for biocompatibility analysis. In some embodiments, the biophysical characteristics of the MSC secretory proteome can be evaluated and / or determined, such as by an epithelial barrier integrity assay. In some embodiments, the epithelial barrier integrity assay is transepithelial electrical resistance (TEER). In some embodiments, transepithelial electrical resistance (TEER) can be assessed to measure overall barrier performance. In some embodiments, 3D tissues can be transferred to 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 EVOMò 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:

[0786] % Barrier Integrity = 100 x [TEER (treated tissue) / TEER (placebo control)]

[0787] In some embodiments, TEER can be used to evaluate the effect on barrier integrity after topical application of MSC secretome. In some embodiments, TEER can be used to evaluate the effect on barrier integrity after topical application of MSC secretome after local exposure to corneal epithelial damage caused by nitrogen mustard (NM) using the EpiCorneal tissue model (MatTekCorp). In some embodiments, the MSC secretome can be applied topically, for example, at 6 μg / ml (diluted in placebo solution), as described in Example 6. In some embodiments, corneal epithelial tissue is cultured in 5 ml of culture medium under standard culture conditions for 24 hours.

[0788] Bioassay

[0789] In some embodiments, a bioassay can be used to characterize the MSC secretome. In some embodiments, the bioassay can be related to the following corneal wound healing: epithelial cell migration and proliferation, stromal cell differentiation (e.g., scarring); neovascularization and inflammation. In some embodiments, a bioassay can be used to evaluate the ability of the MSC secretome to mediate the following corneal wound healing: epithelial cell migration and proliferation, stromal cell differentiation (scarring); neovascularization; and inflammation.

[0790] Migration and proliferation:

[0791] 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 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 proliferation promoting ability.

[0792] In some embodiments, migration assays can be used to evaluate the ability of MSC secretory protein groups to promote migration. In some embodiments, migration assays can be used to evaluate the ability of MSC secretory protein groups to promote migration, wherein the migration assay is an in vitro wound closure assay. In some embodiments, the migration assay may include a "scratch assay" (also referred to as a "scratch wound assay"). In some embodiments, the MSC secretory protein group promotes migration and the promotion of such migration is determined and / or examined using a "scratch assay". Typically, the scratch assay method is based on when an artificial gap (also referred to as a "scratch") appears on a confluent cell monolayer. The "scratch" of cells migrating to the opening on the edge of the newly generated gap can be monitored to close / cover the "scratch". See, for example, 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).

[0793] In some embodiments, migration assays may include transwell migration assays with corneal epithelial cells (or once validated, other cell surrogates) - (e.g., wound closure) may be performed on the MSC secretome. In some embodiments, a transwell migration assay of corneal epithelium is employed as a test of the wound closure efficacy of the MSC secretome. In some embodiments, the MSC secretome promotes wound closure as determined using a transwell migration assay.

[0794] In some embodiments, in vitro wound closure assays include, but are not limited to, a "scratch assay" (also referred to as a "scratch wound assay") or a circular scratch wound method or a circular scratch wound assay or a circular wound closure assay.

[0795] In some embodiments, the MSC secretome can be subjected to a human corneal epithelial cell proliferation assay. In some embodiments, the human corneal epithelial cell proliferation assay indicates a test for 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.

[0796] In some embodiments, a circular scratch wound method or circular scratch wound assay or circular wound closure assay can be used. In some embodiments, Oris TM Cell Migration Assay Platform (see also, as described in Example 6 herein).

[0797] In some embodiments, the MSC secretory protein group can be subjected to an endothelial cell tube formation assay. In some embodiments, the endothelial cell tube formation assay can indicate that the MSC secretory protein group is not pro-angiogenic. In some embodiments, the endothelial cell tube formation assay provides a measure of the angiogenic potential of the MSC secretory protein group. In some embodiments, the MSC secretory protein group exhibits anti-angiogenic properties. In some embodiments, the MSC secretory protein group has anti-angiogenic properties. In some embodiments, the endothelial cell tube formation assay provides a ratio of anti-angiogenic signals and pro-angiogenic signals. In some embodiments, the negative results of the endothelial cell tube formation assay will confirm that the anti:pro ratio is high, and will ensure that the MSC secretory protein group will not promote new blood vessel formation. In some embodiments, the negative results of the endothelial cell tube formation assay will confirm that the anti:pro ratio is high, and will ensure that the MSC secretory protein group generally does not promote CNV (choroidal neovascularization) or new blood vessel formation. In some embodiments, the MSC secretory protein group can be subjected to the inhibition of TGFb-induced myofibroblast differentiation assay. In some embodiments, the MSC secretome can be subjected to the inhibition of TGFb-induced myofibroblast differentiation assay 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 a lower angiogenesis induction. In some embodiments, the MSC secretome has a reduced angiogenic response. In some embodiments, the MSC secretome has a reduced angiogenic capacity. In some embodiments, the MSC secretome impairs and / or reduces normal blood vessel formation in the presence of angiogenic media. In some embodiments, when the MSC secretome is compared to an untreated control, the MSC secretome has a reduced angiogenic capacity. In some embodiments, compared with a sample treated with a serum-containing medium, the MSC secretome has a reduced angiogenic capacity. In some embodiments, the MSC secretome attenuates angiogenic response. In some embodiments, the MSC secretome reduces angiogenic responses induced by serum-free medium. In some embodiments, the MSC secretome induces a reduction in angiogenic response when the secretome plus serum-containing medium (reduced or no angiogenic response) is compared to serum-containing medium (angiogenic response). 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.

[0798] Differentiation / Scarring:

[0799] In some embodiments, the ability of the MSC secretome to prevent differentiation and prevent scarring can be evaluated. In some embodiments, the MSC secretome prevents and / or weakens 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 weakens differentiation. In some embodiments, the MSC secretome prevents and / or weakens myofibroblast differentiation. 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 weakening myofibroblast differentiation.

[0800] 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 (usually upregulated during differentiation), TFGB3, MMP-2, and MMP-9 (usually downregulated during differentiation). In some embodiments, the MSC secretome regulates factors selected from the group consisting of TGFB2, collagen I, collagen III (usually upregulated during differentiation), TFGB3, MMP-2, and MMP-9 (usually 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 expression of MSC secretome inducing factors (such as SMA) is reduced. In some embodiments, the expression of MSC secretome inducing factors (such as SMA) is reduced, which indicates MSC secretome efficacy.

[0801] Neovascularization:

[0802] In some embodiments, the MSC secretome can be evaluated for the ability to prevent new blood vessel formation. In some embodiments, the MSC secretome prevents, weakens, inhibits and / or reduces new blood vessel formation. In some embodiments, the MSC secretome inhibits or does not promote new blood vessel formation. In some embodiments, the MSC secretome can be evaluated for the ability to prevent angiogenesis. In some embodiments, the MSC secretome prevents, weakens, inhibits and / or reduces angiogenesis. In some embodiments, the MSC secretome inhibits angiogenesis.

[0803] In some embodiments, the MSC secretome can be further evaluated using a depletion assay. In some embodiments, the MSC secretome can be depleted of specified factors. In some embodiments, the MSC secretome can be depleted of specified factors, including, for example, but not limited to, TIMP1 and / or serine protease inhibitor El. In some embodiments, the MSC secretome can be depleted of TIMP1 and / or serine protease inhibitor El. In some embodiments, the MSC secretome can be depleted of TIMP1. In some embodiments, the MSC secretome can be depleted of serine protease inhibitor El.

[0804] Inflammation:

[0805] In some embodiments, the MSC secretory protein group can be evaluated for the ability to prevent, weaken, inhibit and / or reduce inflammation. In some embodiments, the MSC secretory protein group prevents, weakens, inhibits and / or reduces inflammation. In some embodiments, the MSC secretory protein group inhibits inflammation. In some embodiments, the MSC secretory protein group is characterized in vitro and / or in vivo to determine the ability to prevent, weaken, inhibit and / or reduce inflammation. In some embodiments, the MSC secretory protein group prevents, weakens, inhibits and / or reduces inflammation in vitro and / or in vivo. In some embodiments, the MSC secretory protein group prevents, weakens, inhibits and / or reduces inflammation in vitro. In some embodiments, the MSC secretory protein group prevents, weakens, inhibits and / or reduces inflammation in vivo. In some embodiments, tissue models can be used to characterize the prevention, weakening, inhibition and / or reduction of inflammation in vitro. In some embodiments, 3D tissue models can be used to characterize the prevention, weakening, inhibition and / or reduction of inflammation in vitro. In some embodiments, the nitrogen mustard (NM) gas combustion model can be used to evaluate the prevention, weakening, inhibition and / or reduction of inflammation in vitro. In some embodiments, the nitrogen mustard (NM) gas combustion model can be used to evaluate the prevention, attenuation, inhibition and / or reduction of inflammation in vitro and as an alternative to in vivo conditions. In some embodiments, the cytokine profile in response to treatment and / or its administration using the MSC secretome can be determined. In some embodiments, the level of a specific cytokine can be determined. In some embodiments, the level of IL-8 can be determined. In some embodiments, the IL-8 expression level can be reduced in tissues treated with the MSC secretome. In some embodiments, the IL-8 expression level is reduced in tissues treated with the MSC secretome, and this indicates prevention, attenuation, inhibition and / or reduction of inflammation.

[0806] Oxidative stress:

[0807] In some embodiments, cell survival is assessed under conditions of oxidative stress, or an induced misfolded protein response, such as ER stress induced by tunicamycin treatment, or by thapsigargin and / or Brefeldin A treatment. In some embodiments, the oxidative stressor is selected from the group consisting of: FeCl 3 -Sodium nitrilotriacetate (Fe-NTA), sodium periodate (NaIO 4 )、7-ketocholesterol (7-KC), hydrogen peroxide (H 2 O 2 ), 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.

[0808] In some embodiments, the positive control is selected from the group consisting of: vitamin A, vitamin B3 (e.g., niacin [nicotinic acid] and niacinamide), 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 meso-zeaxanthin) and carotenoids in saffron (e.g., crocin and crocetin); sulfur-containing antioxidants, including glutathione. scavengers of ROS and free radicals, including carnosine, N-acetylsarcosine, 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., sodium difenton [NXY-059]), nitric oxides (e.g., XJB-5-131), polyphenols (e.g., flavonoids [e.g., apigenin, genistein, luteolin, naringenin, and quercetin]), superoxide dismutase mimetics (see below), tirilazad, vitamin C, vitamin E and its analogs (e.g., alpha-tocopherol and trolox), and xanthine derivatives (e.g., pentoxifylline); mitochondrial antioxidants / "vitamins", including ubiquinones (coenzyme Q, such as CoQ10), ubiquinols (reduced and more bioavailable forms, such as ubiquinone-10), ubiquinone / ubiquinone analogs (e.g., idebenone and mitoquinone) and derivatives; antioxidants targeting mitochondria, including DMQ, DMMQ, MitoE, MitoQ, Mito-TEMPO, MitoVitE, and SkQ compounds (e.g., SkQ1, SkQ2, SkQ3, SkQB, SkQR1, SkQT, SkQT1, SkQT1(m), SkQT1(p), SkQTK1, SkQTR1, SkQBerb, and SkQPalm);Inhibitors of enzymes that generate ROS, including NADPH oxidase (NOX) inhibitors (e.g., apocynin, pursedrin, and pursedrin angelate [all inhibit NOX-1, NOX-2, and NOX-4 activity and expression], diphenyliodonium, 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, mercaptopurine, febuxostat, topiroxetine, inositol, phytic acid, and flavonoids [e.g., kaempferol, myricetin, and quercetin]), and myeloperoxidase inhibitors (e.g., azide, 4-aminobenzoic acid hydrazide, and PF-06667272, and apoE mimetics such as AEM-28 and AEM-28-14);Substances that mimic or increase the activity or production of antioxidant enzymes, including superoxide dismutase (SOD) (e.g., SOD mimetics, such as manganese (III)-porphyrin complexes and zinc (III)-porphyrin complexes (e.g., MnTBAP, MnTMPyP and ZnTBAP), manganese (II) pentaazamacrocyclic complexes (e.g., M40401 and M40403), manganese (III)-Schiff base complexes (e.g., those disclosed in U.S. Pat. No. 7,122,537) and OT-551 (cyclopropyl ester prodrug of tempol hydroxylamine), as well as resveratrol and apoA-I mimetics, such as 4F (both increase expression)), catalase (e.g., catalase mimetics 7,122,537], and zinc [increases activity]), glutathione peroxidase (GPx) (e.g., apomorphine and zinc [both increase activity], and β-catenin, etoposide, and resveratrol [all three increase expression]), glutathione reductase (e.g., 4-tert-butylcatechol and redox cofactors such as flavin adenine dinucleotide [FAD] and NADPH [all three enhance activity]), glutathione S-transferase (GST) (e.g., phenylalkyl isothiocyanate-cysteine ​​conjugates (e.g., S-[N-benzyl(thiocarbamoyl)]-L-cysteine acid), phenobarbital, rosemary extract, and carnosol [all enhanced activity]), thioredoxin (Trx) (e.g., teprenone, prostaglandin E1, and sulforaphane [all increased expression]), NADPH-quinone oxidoreductase 1 (NQO1) (e.g., flavonoids [e.g., β-naphthoflavone (5,6-phenylflavone)], and triterpenes [e.g., oleanolic acid analogs such as TP-151 (CDDO), TP-155 (CDDO methyl ester), TP-190, TP-218, TP-222, TP-223 (CDDO methylamide), TP-224 (CDDO monomethylamide), TP-225, TP-226 (CDDO dimethylamide), TP-230, TP-235 (CDDO imidazole compound), TP-241, CDDO monoethylamide, CDDO mono(trifluoroethyl)amide and (+)-TBE-B], all of which 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);Transcription factor activators that upregulate the expression of antioxidant enzymes, including activators of nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or Nrf2) (e.g., bardoxolone methyl, OT-551, fumarates (e.g., dimethyl fumarate and monomethyl fumarate), dithiones (e.g., oltipraz), flavonoids (e.g., β-naphthoflavone), isoflavones (e.g., genistein), sulforaphane, trichostatin A (also upregulates glutathione synthesis), triterpenoids (e.g., oleanolic acid analogs [e.g., TP-225]), and melatonin (increases Nrf2 expression)); other classes of antioxidants, including anthocyanidins, benzyl alcohol diterpenes (e.g., carnosic acid), cyclopentenone prostaglandins (such as 15d-PGJ2, which also upregulates glutathione synthesis), flavonoids (e.g., those in Ginkgo biloba (e.g., myricetin and quercetin [increases levels of GSH, SOD, catalase, GPx, and GST]), prenylated flavonoids (e.g., isoxanthohumol), flavonoids (e.g., apigenin) , isoflavones (e.g., genistein), flavanones (e.g., naringenin), and flavanols (e.g., catechins and epigallocatechin-3-gallate)), omega-3 fatty acids and their esters (see above), phenylethanoid glycosides (e.g., tyrosol and hydroxytyrosol), retinoids (e.g., all-trans retinol [vitamin A]), stilbenes (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 monostearate]); and their analogs, derivatives, and salts. ;

[0809] In some embodiments, the positive control comprises N-acetylcysteine ​​(NAC) or ebselen.

[0810] In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 5% to 100% or more of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 10% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 15% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 20% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 25% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 30% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 35% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 40% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 45% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 50% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 55% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 60% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 65% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 70% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 75% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 80% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 85% of the response induced by the positive control. In some embodiments, the MSC secretome of the invention induces a response in an oxidative stress assay that is about 90% of the response induced by a positive control. In some embodiments, the MSC secretome of the invention induces a response in an oxidative stress assay that is about 95% of the response induced by a positive control.In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 100% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 1 to 100 times or more than the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 1 times the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 2 times the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 3 times the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 4 times the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 5 times the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 6 times the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 7 times greater than the response induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 8 times greater than the response induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 9 times greater than the response induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 10 times greater than the response induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 20 times greater than the response induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 30 times greater than the response induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 40 times greater than the response induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 50 times greater than the response induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 60 times greater than the response induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 70 times greater than the response induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in an oxidative stress assay that is about 80 times greater than the response induced by a positive control.In some embodiments, the MSC secretome of the invention induces a response in an oxidative stress assay that is about 90 times greater than that induced by a positive control. In some embodiments, the MSC secretome of the invention induces a response in an oxidative stress assay that is about 100 times greater or more than that induced by a positive control.

[0811] In some embodiments, the 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. April 1, 2019, which is incorporated herein by reference in its entirety.

[0812] In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 5% to 100% or more of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 10% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 15% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 20% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 25% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 30% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 35% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 40% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 45% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 50% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 55% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 60% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 65% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 70% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 75% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 80% of the response induced by the positive control. In some embodiments, the MSC secretome of the invention induces a response in a misfolded protein response assay that is about 85% of the response induced by a positive control. In some embodiments, the MSC secretome of the invention induces a response in a misfolded protein response assay that is about 90% of the response induced by a positive control.In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 95% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 100% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 1 to 100 times or more of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 1 times the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 2 times the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 3 times the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 4 times the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 5 times the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 6 times that induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 7 times that induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 8 times that induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 9 times that induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 10 times that induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 20 times that induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 30 times that induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 40 times that induced by a positive control. In some embodiments, the MSC secretome of the invention induces a response in a misfolded protein response assay that is about 50 times greater than the response induced by a positive control. In some embodiments, the MSC secretome of the invention induces a response in a misfolded protein response assay that is about 60 times greater than the response induced by a positive control.In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 70 times greater than the response induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 80 times greater than the response induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 90 times greater than the response induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in a misfolded protein response assay that is about 100 times greater than the response induced by a positive control or more.

[0813] 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. Oxicology Letters, Vol. 271 (2017): 26-37, which is incorporated herein by reference in its entirety.

[0814] In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 5% to 100% or more of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 10% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 15% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 20% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 25% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 30% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 35% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 40% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 45% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 50% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 55% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 60% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 65% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 70% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 75% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 80% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 85% of the response induced by the positive control. In some embodiments, the MSC secretome of the invention induces a response in an ER stress assay that is about 90% of the response induced by a positive control. In some embodiments, the MSC secretome of the invention induces a response in an ER stress assay that is about 95% of the response induced by a positive control.In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 100% of the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 1 to 100 times or more than the response induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 1 times that of the assay induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 2 times that of the assay induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 3 times that of the assay induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 4 times that of the assay induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 5 times that of the assay induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 6 times that of the assay induced by the positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 7 times greater than that induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 8 times greater than that induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 9 times greater than that induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 10 times greater than that induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 20 times greater than that induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 30 times greater than that induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 40 times greater than that induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 50 times greater than that induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 60 times greater than the response induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 70 times greater than the response induced by a positive control. In some embodiments, the MSC secretome of the present invention induces a response in an ER stress assay that is about 80 times greater than the response induced by a positive control.In some embodiments, the MSC secretome of the invention induces a response in an ER stress assay that is about 90 times greater than that induced by a positive control. In some embodiments, the MSC secretome of the invention induces a response in an ER stress assay that is about 100 times greater or more than that induced by a positive control.

[0815] In some embodiments, the cells used to assess cell survival under oxidative stress, misfolded protein response, or ER stress response are selected from the group consisting of ARPE-19, primary human RPE, 661W, and MIO-M1 human Mueller cell lines.

[0816] In some embodiments, cells were seeded at 30% confluence in 96-well plates and allowed to adhere overnight before loading the cells with the chloromethyl derivative of H2DCFDA (CM-H2DCFDA) and Mitosoxred for 30 minutes. In some embodiments, cells were plated with various concentrations of NaIO in the absence / presence of NAC. 4 , H 2 O 2 , 7-KC, ATRA or t-BHP treatment 1 hour before, washing cells. In some embodiments, after treatment 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours or longer, the relative level of reactive oxygen (ROS) production is measured in parallel with cell viability. In some embodiments, oxidative stressors increase ROS production levels by 10 times to 1000 times. In some embodiments, oxidative stressors increase ROS production levels by at least 10 times. In some embodiments, oxidative stressors increase ROS production levels by at least 100 times. In some embodiments, oxidative stressors increase ROS production levels by at least 1000 times. In some embodiments, the dose-dependent increase of ROS is evaluated using CM-H2DCFDA and MitoSOX Red, and then associated with cell survival 24 hours after injury. In some embodiments, the dose-dependent increase of ROS is compared with the increase inhibited by NAC (antioxidant positive control).

[0817] In some embodiments, the MSC secretome provides a protective benefit.

[0818] In some embodiments, the benefits of secretome therapy are evaluated based on the results and findings described herein. In some embodiments, the efficacy or activity of secretomes is evaluated based on the results and findings described herein. In some embodiments, one, two or three oxidative stress conditions are selected to test secretome activity. In most embodiments, the positive control is NAC. In some embodiments, the initial study is run in parallel to monitor the ROS levels achieved in the study. In some embodiments, the endpoint is cell survival as determined by microtiter tetrazolium (MTT). In some embodiments, cells exposed to selected oxidative damage are treated with different concentrations of secretomes, and their survival is evaluated 24 hours after damage. In other embodiments, cells are treated with various concentrations of secretomes before exposure to selective oxidative damage, and survival is evaluated 24 hours after damage. In some embodiments, the negative control consists of a basal medium containing 1% serum conditions and heat-denatured secretome conditions; the positive control is NAC. In some embodiments, the effect of the secretome on cellular ROS levels is evaluated under protective conditions.

[0819] Oxidative stress inducers

[0820] In some embodiments, one or more oxidative stress inducing agents are administered to the assayed cells to induce oxidative stress and / or cellular ROS production and the extent of oxidative stress and / or ROS production in the assayed cells is assessed.

[0821] In some embodiments, the cells assayed are RPE cells, including but not limited to ARPE-19, primary human RPE, 661W cells and MIO-M1 human Mueller cells, ocular explant tissues (including but not limited to retina, cornea, iris, trabecular meshwork and / or ciliary body).

[0822] In some embodiments, oxidative stress inducers include but are not limited to ketone cholesterol, FeCl 3 -Sodium nitrilotriacetate (Fe-NTA), H 2 O 2 , T-BHP, all-trans retinal, NaIO4, hydroquinone and oxidized cholesterol (OxLDL).

[0823] In some embodiments, oxidative stress is induced by culturing cells under low oxygen / hypoxic conditions.

[0824] In some embodiments, inducing oxidative stress in the measured cells comprises treating the cells with one or more inducers for about 0.5 hours to 96 hours. In some embodiments, inducing oxidative stress in the measured cells comprises treating the cells with one or more inducers for about 0.5 hours to 72 hours. In some embodiments, inducing oxidative stress in the measured cells comprises treating the cells with one or more inducers for about 0.5 hours to 48 hours. In some embodiments, inducing oxidative stress in the measured cells comprises treating the cells with one or more inducers for about 0.5 hours to 24 hours. In some embodiments, inducing oxidative stress in the measured cells comprises treating the cells with one or more inducers for about 0.5 hours to 12 hours. In some embodiments, inducing oxidative stress in the measured cells comprises treating the cells with one or more inducers for about 0.5 hours to 6 hours. In some embodiments, inducing oxidative stress in the measured cells comprises treating the cells with one or more inducers for about 6 hours to 96 hours. In some embodiments, inducing oxidative stress in the measured cells comprises treating the cells with one or more inducers for about 12 hours to 96 hours. In some embodiments, inducing oxidative stress in the determined cells comprises treating the cells with one or more inducers for about 24 hours to 96 hours. In some embodiments, inducing oxidative stress in the determined cells comprises treating the cells with one or more inducers for about 36 hours to 96 hours. In some embodiments, inducing oxidative stress in the determined cells comprises treating the cells with one or more inducers for about 48 hours to 96 hours. In some embodiments, inducing oxidative stress in the determined cells comprises treating the cells with one or more inducers for about 60 hours to 96 hours. In some embodiments, inducing oxidative stress in the determined cells comprises treating the cells with one or more inducers for about 72 hours to 96 hours. In some embodiments, inducing oxidative stress in the determined cells comprises treating the cells with one or more inducers for about 84 hours to 96 hours.

[0825] In some embodiments, inducing oxidative stress in the assayed cells comprises treating the cells with one or more inducing agents for about 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 11.5 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, or more.

[0826] In some embodiments, inducing oxidative stress in the assayed cells comprises culturing the cells under low oxygen / hypoxic conditions for about 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 11.5 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, or more.

[0827] In some embodiments, one or more oxidative stress inducers are applied to the measured cells at a concentration of about 0.01 μM to about 100 mM. In some embodiments, one or more oxidative stress inducers are applied to the measured cells at a concentration of about 0.01 μM to about 0.1 uM. In some embodiments, one or more oxidative stress inducers are applied to the measured cells at a concentration of about 0.1 μM to about 1 μM. In some embodiments, one or more oxidative stress inducers are applied to the measured cells at a concentration of about 1 μM to about 10 μM. In some embodiments, one or more oxidative stress inducers are applied to the measured cells at a concentration of about 10 μM to about 100 μM. In some embodiments, one or more oxidative stress inducers are applied to the measured cells at a concentration of about 100 μM to about 1 mM. In some embodiments, one or more oxidative stress inducers are applied to the measured cells at a concentration of about 1 mM to about 10 mM. In some embodiments, one or more oxidative stress inducing agents are applied to the cells being assayed at a concentration of about 10 mM to about 100 mM.

[0828] In some embodiments, the oxidative stress inducing agent is nitrilotriacetic acid ketocholesterol (Fe-NTA) applied at a concentration of 0.1 mM to 100 mM for 1-72 hours. In some embodiments, oxidative stress is induced by applying Fe-NTA at a concentration of about 0.1 mM to 1 mM, about 1 mM to 10 mM, or about 10 mM to 100 mM to the cells being assayed for about 1 hour to 24 hours, about 24 hours to 48 hours, or about 48 hours to 72 hours.

[0829] In some embodiments, the oxidative stress inducing agent is FeCl administered at a concentration of 0.1 mM to 100 mM for 1-72 hours. 3-Sodium nitrilotriacetate (Fe-NTA). In some embodiments, oxidative stress is induced by applying Fe-NTA at a concentration of about 0.1 mM to 1 mM, about 1 mM to 10 mM, or about 10 mM to 100 mM to the cells being assayed for about 1 hour to 24 hours, about 24 hours to 48 hours, or about 48 hours to 72 hours.

[0830] In some embodiments, the oxidative stress inducing agent is H administered at a concentration of 0.01 μM to 1000 μM. 2 O 2 , the exposure time is 0.5 hours to 24 hours. In some embodiments, by applying to the measured cells a concentration of about 0.01 μM to 0.1 μM, about 0.1 μM to 1 μM, about 1 μM to 10 μM, about 10 μM to 100 μM, or about 100 μM to 1000 μM H 2 O 2 Oxidative stress is induced for about 0.5 hours to 6 hours, about 6 hours to 12 hours, about 12 hours to 18 hours, or about 18 hours to 24 hours.

[0831] In some embodiments, the oxidative stress inducing agent is T-BHP at a concentration of 0.01 μM to 1000 μM, and the exposure time is 0.5 hours to 24 hours. In some embodiments, oxidative stress is induced by applying t-BHB at a concentration of about 0.01 μM to 0.1 μM, about 0.1 μM to 1 μM, about 1 μM to 10 μM, about 10 μM to 100 μM, or about 100 μM to 1000 μM to the cells being assayed for about 0.5 hours to 6 hours, about 6 hours to 12 hours, about 12 hours to 18 hours, or about 18 hours to 24 hours.

[0832] In some embodiments, the oxidative stress inducing agent is all-trans retinal applied at a concentration of 0.01 μM to 1000 μM, and the exposure time is 0.5 hours to 24 hours. In some embodiments, oxidative stress is induced by applying all-trans retinal at a concentration of about 0.01 μM to 0.1 μM, about 0.1 μM to 1 μM, about 1 μM to 10 μM, about 10 μM to 100 μM, or about 100 μM to 1000 μM to the measured cells for about 0.5 hours to 6 hours, about 6 hours to 12 hours, about 12 hours to 18 hours, or about 18 hours to 24 hours.

[0833] In some embodiments, the oxidative stress inducing agent is NaIO administered at a concentration of 0.01 μM to 1000 μM. 4, the exposure time is 0.5 hours to 24 hours. In some embodiments, by applying to the cells being measured a concentration of about 0.01 μM to 0.1 μM, about 0.1 μM to 1 μM, about 1 μM to 10 μM, about 10 μM to 100 μM, or about 100 μM to 1000 μM NaIO 4 Oxidative stress is induced for about 0.5 hours to 6 hours, about 6 hours to 12 hours, about 12 hours to 18 hours, or about 18 hours to 24 hours.

[0834] In some embodiments, the oxidative stress inducer is oxidized cholesterol / OxLDL at a concentration of 10ug / ml to 500ug / ml for 0.5 to 24 hours. In some embodiments, the oxidative stress inducer is glucose oxidase (GOx) at a concentration of 1mU / ml to 100mU / ml for about 24 hours. In some embodiments, oxidative stress is induced by administering to the measured cells a concentration of about 10μg / mL to 100μg / mL, about 10μg / mL to 100μg / mL, or about 100μg / mL to 500μg / mL of OxLDL for about 0.5 to 6 hours, about 6 hours to 12 hours, about 12 hours to 18 hours, or about 18 hours to 24 hours.

[0835] Evaluation of oxidative stress

[0836] In some embodiments, oxidative stress and / or ROS generation is assessed based on measuring DNA oxidation with anti-8-oxo-2'-deoxyguanosine (8-oxo-dG).

[0837] In some embodiments, oxidative stress and / or ROS production is assessed based on measuring lipid oxidation using thiobarbituric acid reactive substances (TBARS).

[0838] In some embodiments, oxidative stress and / or ROS production is assessed based on measuring cytoplasmic ROS using CM-H2 DCFDA.

[0839] In some embodiments, oxidative stress and / or ROS production is assessed based on measuring mitochondrial ROS using Mitoxed.

[0840] In some embodiments, oxidative stress and / or ROS production is assessed based on measurement of mitochondrial potential, including, for example, but not limited to, assessment of JC-1.

[0841] In some embodiments, 7-ketocholesterol, FeCl 3 -Sodium nitrilotriacetate (Fe-NTA), H 2 O 2, tert-butyl hydroperoxide (t-BHP), all-trans retinal, NaIO4, hydroquinone, oxidized cholesterol (OxLDL) to induce oxidative stress, and the extent 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-H2 DCFDA), mitochondria (ROS) (Mitoxed) and / or mitochondrial potential (JC-1).

[0842] 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 mitochondria (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.

[0843] 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 mitochondria (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.

[0844] In some embodiments, by H 2 O 2 Inducing oxidative stress, and evaluating the extent of induced oxidative stress based on DNA oxidation. In some embodiments, the oxidative stress is induced by H 2 O 2 Induce oxidative stress, and evaluate the extent of induced oxidative stress and / or ROS generation based on lipid oxidation. In some embodiments, the oxidative stress is induced by H 2 O2 Inducing oxidative stress, and evaluating the extent of induced oxidative stress and / or ROS production based on cytoplasmic ROS. In some embodiments, the oxidative stress is induced by H 2 O 2 Induce oxidative stress, and evaluate the extent of induced oxidative stress and / or ROS production based on mitochondria (ROS). In some embodiments, the oxidative stress is induced by H 2 O 2 Oxidative stress was induced, and the extent of induced oxidative stress and / or ROS production was assessed based on mitochondrial potential.

[0845] 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 mitochondria (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.

[0846] 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 ...

Claims

1. A method for characterizing the secretome of MSCs, wherein the method comprises: (i) subjecting the MSC secretome to one or more characterization assays, wherein the characterization assay is selected from the group consisting of: physical component characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, cellular response assays, safety analysis, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / scarring assays, inflammation assays, tissue explant survival and function assays, organoid development or survival / function assays, epithelial barrier integrity assays, retinal degeneration assays and / or assays for inherited retinal diseases including human and animal retinal explants, neuroprotection / neurotrophy assays, wherein the MSC secretome is optionally pre-treated; and (ii) determining the results of the one or more determinations in (i).

2. A method for determining the biological efficacy and stability of MSC secretory proteome, comprising: The method comprises: (i) subjecting the MSC secretome to one or more characterization assays, wherein the characterization assay is selected from the group consisting of: physical component characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, cellular response assays, safety analysis, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / scarring assays, inflammation assays, in vivo responses to oxidative stress (e.g., retinal ischemia reperfusion), epithelial barrier integrity assays, retinal degeneration assays and / or assays for inherited retinal diseases including human and animal retinal explants, neuroprotection / neurotrophy assays, wherein the MSC secretome is optionally pre-treated; and (ii) determining the results of the one or more determinations in (i).

3. A method for determining MSC secretome batch consistency between multiple MSC secretome batches, wherein the method comprises: (i) subjecting the MSC secretome to one or more characterization assays, wherein the characterization assay is selected from the group consisting of: physical component characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, cellular response assays, safety analysis, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / scarring assays, inflammation assays, epithelial barrier integrity assays, retinal degeneration assays and / or assays for inherited retinal diseases including human and animal retinal explants, neuroprotection / neurotrophy assays, wherein the MSC secretome is optionally pre-treated; and (ii) determining the results of the one or more determinations in (i).

4. The method of any one of claims 1-3, wherein the results in (ii) from the characterization of the physical component identify the anti-angiogenic MSC secretome or a composition prepared by comprising the secretome.

5. The method according to any one of claims 1 to 3, wherein the results in (ii) from the safety analysis provide a MSC secretome exhibiting hemocompatibility and low and / or no pyrogens and / or endotoxins.

6. The method of any one of claims 1-3, wherein the results in (ii) from the stability assay provide a 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.

7. The method according to any one of claims 1 to 3, wherein the results in (ii) from the proliferation assay provide a MSC secretome that induces proliferation.

8. The method according to any one of claims 1 to 3, wherein the results in (ii) from the migration assay provide the MSC secretome that induces migration.

9. The method of any one of claims 1-3, wherein the results in (ii) from the neovascularization assay provide a MSC secretome that inhibits or does not promote neovascularization.

10. The method of any one of claims 1-3, wherein the results in (ii) from the differentiation / scarring assay provide a MSC secretome that inhibits differentiation and / or scarring.

11. The method of any one of claims 1-3, wherein the results in (ii) from the inflammation assay provide a MSC secretome that inhibits inflammation or alters an immune response.

12. The method according to claim 4, further comprising: (iii) Identify MSC secretome batches based on the results in (ii).

13. The method according to any one of claims 1 to 3, further comprising a pre-processing step of the secretome.

14. A panel of tests and / or assays for characterizing the MSC secretome, wherein the panel comprises at least two characterization assays, wherein the characterization assays are selected from the group consisting of: physical component characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analysis, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / scarring assays, inflammation assays, epithelial barrier integrity assays, retinal degeneration assays and / or assays for inherited retinal diseases including human and animal retinal explants, neuroprotection / neurotrophy assays, wherein the secretome is optionally pre-treated.

15. A panel of tests and / or assays for determining the consistency between batches of MSC secretome, wherein the panel comprises one or more characterization assays, wherein the characterization assays are selected from the group consisting of: physical component characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analysis, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / scarring assays, inflammation assays, epithelial barrier integrity assays, retinal degeneration assays and / or assays for inherited retinal diseases including human and animal retinal explants, neuroprotection / neurotrophy assays, wherein the secretome is optionally pre-processed.

16. A set of tests and / or assays according to any one of claims 14-15, wherein the physical component characterizes and identifies the MSC secretome as characterized in any one of claims 1-3 or a composition prepared therefrom.

17. A panel of tests and / or assays according to any one of claims 14-15, wherein the results in (ii) from the safety analysis provide a MSC secretome exhibiting hemocompatibility and low and / or no pyrogens and / or endotoxins.

18. A panel of tests and / or assays according to any one of claims 14-15, wherein the stability assay identifies MSC secretomes that exhibit stability at -20°C, 4°C and / or 20°C (eg room temperature) for at least 7 days or at least 14 days.

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

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

21. A panel of tests and / or assays according to any one of claims 14-15, wherein the neovascularization assay identifies MSC secretomes that inhibit or do not promote neovascularization.

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

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

24. A panel of tests and / or assays according to any one of claims 14-15, wherein physical component characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analysis, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / scarring assays, inflammation assays and / or epithelial barrier integrity assays, retinal degeneration assays and / or assays for inherited retinal diseases including human and animal retinal explants, neuroprotection / neurotrophic assays are performed.

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

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

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

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

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

30. A 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 known as a "scratch wound assay"), a circular scratch wound method, a circular scratch wound assay and a circular wound closure assay.

31. A panel of tests and / or assays according to any one of claims 14-15, wherein the panel of assays comprises reactive oxygen species (ROS) measurements.

32. A 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. A panel of tests and / or assays according to claim 32, wherein the in vitro ROS measurement assay is evaluated using chloromethyl-modified H2DCFDA (CM-H2DCFDA) and / or MitoSOX Red.

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

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

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

37. A panel of tests and / or assays according to claim 36, wherein the oxidative stress assay comprises: a) inducing oxidative stress in cells; and b) assessing the extent of said oxidative stress.

38. A panel of tests and / or assays according to claim 37, wherein the oxidative stress is induced by one or more inducing agents 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 cholesterol (OxLDL).

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

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

41. A panel of tests and / or assays according to any one of claims 37 to 40, wherein the oxidative stress is induced by applying H2O2 at a concentration of about 0.01 μM to 0.1 μM, about 0.1 μM to 1 μM, about 1 μM to 10 μM, about 10 μM to 100 μM, or about 100 μM to 1000 μM to the assayed cells for about 0.5 hours to 6 hours, about 6 hours to 12 hours, about 12 hours to 18 hours, or about 18 hours to 24 hours.

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

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

44. A panel of tests and / or assays according to any one of claims 37 to 43, wherein the oxidative stress is induced by applying NaIO4 at a concentration of about 0.01 μM to 0.1 μM, about 0.1 μM to 1 μM, about 1 μM to 10 μM, about 10 μM to 100 μM, or about 100 μM to 1000 μM to the cells being assayed for about 0.5 hour to 6 hours, about 6 hours to 12 hours, about 12 hours to 18 hours, or about 18 hours to 24 hours.

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

46. ​​A panel of tests and / or assays according to any one of claims 37 to 45, wherein assessing the extent of oxidative stress comprises one or more of: i) DNA oxidation was measured using an anti-8-oxo-2'-deoxyguanosine (8-oxo-dG) assay; ii) lipid oxidation was measured using thiobarbituric acid reactive substances (TBARS); iii) measurement of cytoplasmic ROS using CM-H2 DCFDA; iv) measurement of mitochondrial ROS using Mitoxed; and v) Measuring mitochondrial potential, such as measuring JC-1.

47. A panel of tests and / or assays according to any one of claims 36 to 46, wherein the assayed cells are evaluated for one or more phenotypes selected from the group consisting of: Alterations in epithelial polarization (e.g., Na / K ATPase polarity), tight junctions (e.g., ZO-1, transepithelial polarity, and transepithelial electrical resistance (TEER)), phagocytosis of outer segments, degeneration of outer segments, phagosome composition and function, cathepsin D distribution activity, mitochondrial potential, inflammasome activation / activity, HIf-1α 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 / susceptibility, lipid deposition, apoptosis, and necrosis.

48. A panel of tests and / or assays according to any one of claims 36 to 47, wherein the MSC secretome is pre-treated prior to inducing the oxidative stress.

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

50. A panel of tests and / or assays according to any one of claims 36 to 47, wherein the MSC secretome is pre-treated after induction of the oxidative stress.

51. The method or panel of tests and / or assays of any of the preceding claims, wherein pre-treating the MSC secretome comprises affecting the secretion profile of the MSC, the pre-treating comprising one or more of: changing the culture format (e.g., 2D planar bioreactor versus 3D bioreactor), different biomaterial scaffolds, co-culturing, adding pharmacological compounds, growth factors, chemokines, adding toll-like receptor agonists, inflammatory cytokines, advanced glycation end products (AGE), oxidized phospholipids, malondialdehyde or carboxyethylpyrrole, stirring in the presence of ECM, culturing under shear stress, stirring or suspending as aggregates or in a matrix, inducing a misfolded protein response, ER stress, inducing differentiation of MSCs, culturing in the presence of conditioned medium and hypoxia / hypoxia.

52. The method or panel of tests and / or assays according to any one of the preceding claims, wherein pre-treating the MSC secretome comprises hypoxic pre-treatment comprising culturing the MSCs in a hypoxic culture environment.

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

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

55. The method or panel of tests and / or assays according to any one of the preceding claims, wherein pre-treating the MSC secretome comprises treating the MSC with one or more inflammatory cytokines.

56. The method or panel of tests and / or assays of any of the preceding claims, wherein pre-treating the MSC secretome comprises treating the MSC with one or more of 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.

57. The method or panel of tests and / or assays according to any one of the preceding claims, wherein pre-treating the MSC secretome comprises 3D culturing the MSCs (eg, by using a 3D bioreactor).

58. The method or panel of tests and / or assays according to any one of the preceding claims, wherein pre-treating the MSC secretome comprises inducing a misfolded protein response in the MSCs.

59. The method or panel of tests and / or assays according to any one of the preceding claims, wherein pre-treating the MSC secretome comprises culturing the MSCs under suitable conditions to induce ER stress.

60. The method or panel of tests and / or assays according to any one of the preceding claims, wherein pre-treating the MSC secretome comprises culturing the MSCs under suitable conditions to induce differentiation of the MSCs into mature retinal cell types or precursors thereof.

61. A bone marrow-derived mesenchymal stem cell (MSC) secretome composition comprising: HGF; penetratin-3 (TSG-14); VEGF; TIMP-1; serine protease inhibitor E1; <5 ng / mL IL-8, and a tension regulator, wherein the MSC secretome is pre-treated.

62. The MSC secretome composition of claim 61, wherein the MSC secretome composition further 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 (serine protease inhibitor 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-β and PDGF; and / or 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.

63. The MSC secretome composition according to 62, wherein the MSC secretome composition comprises 1 ng / mL-100 ng / mL of at least one factor selected from the group consisting of: serine protease inhibitor E1, TIMP-1, thrombospondin-1, pentetracycline-3 (TSG-14) and serine protease inhibitor F1.

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

65. The MSC secretome composition of claim 62, wherein the MSC secretome composition 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, Emmprin, osteopontin, vitamin DBP, MIF, RANTES, uPAR, IL-17a, GDF-15 and IFNγ.

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

67. The MSC secretome composition of claim 62, wherein the MSC secretome composition comprises 1 pg / mL-400 pg / mL of VEGF.

68. The MSC secretome composition of claim 62, wherein the level of VEGF is 5-10 times lower than the level of serpin E1.

69. The MSC secretome composition of claim 62, wherein the composition comprises one or more anti-angiogenic factors, and wherein the ratio of 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.

70. The MSC secretome composition of claim 62, wherein the MSC secretome comprises less than 1000 pg / mL of bFGF, PLGF, and PDGF.

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

5.

72. The MSC secretome composition of claim 62, wherein 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 citric acid / disodium phosphate.

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

4.

74. The MSC secretome composition of claim 62, wherein the MSC secretome composition further comprises divalent cations.

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

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

4.

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

78. The MSC secretome composition of claim 77, wherein the binder is selected from the group consisting of: hydroxypropyl methylcellulose, poloxamer 407, poloxamer 188, poloxamer 237, poloxamer 338, hydroxypropyl methylcellulose (HPMC), polycarbophil, polyvinyl pyrrolidone (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.

79. The MSC secretome composition of claim 62, wherein the MSC secretome composition does not comprise one or more components selected from the group consisting of: xenobiotic 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 of claim 62, wherein the MSC secretome composition comprises: i. 0.3-4.5 ng / mL HGF; ii. 0.5-20 ng / mL penetrant-3 (TSG-14); iii. 100-600 pg / mL VEGF; iv. 10-200 ng / mL TIMP-1; v. 20-80 ng / mL serine protease inhibitor E1; and vi.<5ng / mL IL-8.

81. The MSC secretome composition of claim 62, wherein the MSC secretome composition comprises an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.

82. The MSC secretome composition of claim 61, wherein the tonicity modifier is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerol.

83. A stable bone marrow-derived mesenchymal stem cell (MSC) secretome preparation comprising: i. 2μg-20μg MSC secretome / mL; ii. 2mg-3mg sodium dihydrogen phosphate / mL; iii. 11mg-12mg sodium dihydrogen phosphate / mL; iv. 11.5mg-13mg mannitol / mL; v.23mg-24mg trehalose dihydrate; vi. 0.5mg-2mg hydroxypropyl methylcellulose / mL; and wherein the pH is about 4.7 to about 7.5, wherein the MSC secretome is pre-treated.

84. A stable bone marrow-derived mesenchymal stem cell (MSC) secretome preparation comprising: i. 0.004%-0.08% w / w MSC secretome; ii. 4%-5% w / w sodium dihydrogen phosphate; iii. 21.5%-23% w / w disodium hydrogen phosphate; iv. 23%-25% w / w mannitol; v. 46%-48% w / w trehalose dehydrate; and vi. 1%-3% w / w hypromellose; and wherein the pH is about 4.7 to about 7.5, and wherein the MSC secretome is pre-treated.

85. A stable bone marrow-derived mesenchymal stem cell (MSC) secretome preparation comprising: i. Sodium phosphate 10 mM; ii. Histidine HCL 10 mM; iii. Trehalose dihydrate 10%; iv. Polysorbate 200.01%; and wherein the pH is about 5.5, wherein the MSC secretome is pre-treated.

86. A stable bone marrow-derived mesenchymal stem cell (MSC) secretome preparation comprising: i. Sodium phosphate 10 mM; ii. Sucrose 5%; iii. Sodium chloride 10 mM; and wherein the pH is about 6.2, wherein the MSC secretome is pre-treated.

87. A stable bone marrow-derived mesenchymal stem cell (MSC) secretome preparation comprising: i. Sodium phosphate 10 mM; ii. Sucrose 5.8%; iii. Polysorbate 80 0.02%; and wherein the pH is about 6.2, wherein the MSC secretome is pre-treated.

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; penetratin-3 (TSG-14); VEGF; TIMP-1; serine protease inhibitor E1; and <5 ng / mL IL-8, wherein the MSC secretome is pre-treated.

89. The method of claim 88, wherein the MSC secretome composition further 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 (serine protease inhibitor 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-β 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.

90. The method of claim 89, wherein the MSC secretome composition comprises 1 ng / mL-100 ng / mL of at least one factor selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentetracycline-3 (TSG-14), and serpin F1.

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

92. The method of claim 89, wherein the MSC secretome composition 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, Emmprin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15 and IFNγ.

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

94. The method of treatment of claim 89, wherein the MSC secretome comprises 1 pg / mL-400 pg / mL of VEGF.

95. The method of treatment of claim 89, wherein the level of VEGF is 5-10 times lower than the level of serpin El.

96. The method of treatment of claim 89, wherein the MSC secretome composition comprises one or more anti-angiogenic factors, and wherein 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.

97. The MSC secretome composition of claim 89, wherein the MSC secretome comprises less than 1000 pg / mL of bFGF, PLGF, and PDGF.

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

5.

99. The method of claim 89, wherein 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 citric acid / disodium phosphate.

100. The method of treatment of claim 89, wherein the MSC secretome composition further comprises a tonicity modulator.

101. The method of treatment of claim 100, wherein the tonicity modifier is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerol.

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

4.

103. The method of treatment of claim 89, wherein the MSC secretome composition further comprises divalent cations.

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

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

4.

106. The method of treatment of claim 89, wherein the MSC secretome composition does not comprise one or more components selected from the group consisting of: xenobiotic 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 method of treatment of claim 89, wherein the MSC secretome composition comprises an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.

108. The method of treatment of claim 88, wherein the MSC secretome composition further comprises an agent that increases viscosity.

109. The method of claim 108, wherein the binder is selected from the group consisting of: hydroxypropyl methylcellulose, poloxamer 407, poloxamer 188, poloxamer 237, poloxamer 338, hydroxypropyl methylcellulose (HPMC), polycarbophil, polyvinyl pyrrolidone (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 method of claim 88, wherein the MSC secretome composition comprises: i. 0.3-4.5 ng / mL HGF; ii. 0.5-20 ng / mL penetrant-3 (TSG-14); iii. 100-600 pg / mL VEGF; iv. 10-200 ng / mL TIMP-1; v. 20-80 ng / mL serine protease inhibitor E1; and vi.<5ng / mL IL-8.

111. A method of treating an ocular disorder 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 is a stable mesenchymal stem cell (MSC) secretome preparation comprising: i. 2μg-20μg MSC secretome / mL; ii. 2mg-3mg sodium dihydrogen phosphate / mL; iii. 11mg-12mg sodium dihydrogen phosphate / mL; iv. 11.5mg-13mg mannitol / mL; v.23mg-24mg trehalose dihydrate; vi. 0.5mg-2mg hydroxypropyl methylcellulose / mL; and wherein the pH is about 4.7 to about 7.5, wherein the MSC secretome is pre-treated.

112. A method of treating an ocular disorder 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 is a stable mesenchymal stem cell (MSC) secretome preparation comprising: i. 0.004%-0.08% w / w MSC secretome; ii. 4%-5% w / w sodium dihydrogen phosphate; iii. 21.5%-23% w / w disodium hydrogen phosphate; iv. 23%-25% w / w mannitol; v.46%-48% w / w trehalose dehydrate; vi. 1%-3% w / w Hydroxypropyl methylcellulose; and wherein the pH is about 4.7 to about 7.5, wherein the MSC secretome is pre-treated.

113. A method of treating an ocular disorder 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 is a stable mesenchymal stem cell (MSC) secretome preparation comprising: i. Sodium phosphate 10 mM; ii. Histidine HCL 10 mM; iii. Trehalose dihydrate 10%; iv. Polysorbate 200.01%; and wherein the pH is about 5.5, wherein the MSC secretome is pre-treated.

114. A method of treating an ocular disorder 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 is a stable mesenchymal stem cell (MSC) secretome preparation comprising: i. Sodium phosphate 10 mM; ii. Sucrose 5%; iii. Sodium chloride 10 mM; and wherein the pH is about 6.2, wherein the MSC secretome is pre-treated.

115. A method of treating an ocular disorder 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 is a stable mesenchymal stem cell (MSC) secretome preparation comprising: i. Sodium phosphate 10 mM; ii. Sucrose 5.8%; iii. Polysorbate 80 0.02%; and wherein the pH is about 6.2, wherein the MSC secretome is pre-treated.

116. A composition for inducing ocular wound healing, comprising a mesenchymal stem cell (MSC) secretome and a tension modulator, wherein the MSC secretome is pre-treated, wherein the ability of the composition to promote ocular wound healing is indicated by a wound healing assay comprising the steps of: a) providing a corneal cell layer; b) introducing a wound gap into the corneal cell layer; and c) determining whether the wound gap heals in the presence of the composition, wherein the composition is administered to the corneal cells before or after step b); wherein closure of the wound gap indicates the ability of the composition to induce ocular wound healing.

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

118. The composition of claim 116, wherein the corneal cells are corneal keratinocytes (or fibroblasts).

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

120. The composition of claim 116, wherein the wound space is introduced by mechanically disrupting the corneal cell layer.

121. The composition of claim 116, wherein the wound space is introduced by chemically disrupting the corneal cell layer.

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

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

124. The composition of claim 116, wherein determining whether the wound gap is closed in step c) comprises detecting and quantifying migration and / or proliferation of the corneal cells within the wound gap on a daily basis.

125. The composition of claim 124, wherein the migration and / or proliferation of corneal cells is characterized by the number of corneal cells that migrate and / or proliferate within the wound gap.

126. The composition of claim 116, wherein determining whether the wound gap is healed in step c) comprises measuring the size of the wound gap daily, wherein the size of the wound gap is expressed as a percentage of an initial size of the wound gap measured immediately after introduction of the wound gap.

127. The composition of claim 126, wherein the size of the wound gap is characterized by the surface area of ​​the wound gap.

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

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

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

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

132. The composition of claim 116, wherein the wound healing assay further comprises a step of concentrating the composition, and optionally a buffer exchange step for the composition, prior to applying the composition to the corneal cells.

133. The composition of claim 116, wherein the wound healing assay further comprises a step of diluting the composition, and optionally a buffer exchange step for the composition, prior to applying the composition to the corneal cells.

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

135. A composition for inducing ocular wound healing comprising a mesenchymal stem cell (MSC) secretome and a tension modulator, wherein the MSC secretome is pre-treated, wherein the ability of the composition to promote ocular wound healing is indicated by a wound healing assay comprising the steps of: a) providing a corneal cell layer; b) introducing a wound gap into the corneal cell layer; and c) determining whether the wound gap heals in the presence of the composition, wherein the composition is administered to the corneal cells before or after step b); wherein closure of the wound gap indicates the ability of the composition to induce ocular wound healing.

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

137. The composition of claim 135, wherein the corneal cells are corneal keratinocytes (or fibroblasts).

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

139. The composition of claim 135, wherein the wound space is introduced by mechanically disrupting the corneal cell layer.

140. The composition of claim 135, wherein the wound space is introduced by chemically disrupting the corneal cell layer.

141. The composition of claim 135, wherein the wound gap comprises a linear gap.

142. The composition of claim 135, wherein the wound gap comprises a circular gap.

143. The composition of claim 135, wherein determining whether the wound gap is closed in step c) comprises detecting and quantifying migration and / or proliferation of the corneal cells within the wound gap on a daily basis.

144. The composition of claim 143, wherein the migration and / or proliferation of the corneal cells is characterized by the number of corneal cells that migrate and / or proliferate within the wound gap.

145. The composition of claim 135, wherein determining whether the wound gap is healed in step c) comprises measuring the size of the wound gap daily, wherein the size of the wound gap is expressed as a percentage of an initial size of the wound gap measured immediately after introduction of the wound gap.

146. The composition of claim 145, wherein the size of the wound gap is characterized by the surface area of ​​the wound gap.

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

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

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

150. The composition of claim 135, wherein step c) is performed within a period of 3 days after step b) is completed.

151. The composition of claim 135, wherein the wound healing assay further comprises a step of concentrating the composition, and optionally a buffer exchange step for the composition, prior to applying the composition to the corneal cells.

152. The composition of claim 135, wherein the wound healing assay further comprises a step of diluting the composition, and optionally a buffer exchange step for the composition, prior to applying the composition to the corneal cells.

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

154. A method for pretreating the secretory proteome of MSCs, comprising influencing the secretory profile of the MSCs, the pretreatment comprising one or more of: changing the culture format (e.g., 2D planar bioreactor versus 3D bioreactor), different biomaterial scaffolds, co-culture, adding pharmacological compounds, growth factors, chemokines, adding toll-like receptor agonists, inflammatory cytokines, advanced glycation end products (AGE), oxidized phospholipids, malondialdehyde or carboxyethylpyrrole, stirring in the presence of ECM, culturing under shear stress, stirring or suspending as aggregates or in a matrix, inducing a misfolded protein response, ER stress, inducing differentiation of MSCs, culturing in the presence of conditioned medium and hypoxia / hypoxia.

155. The method of claim 154, comprising hypoxic pretreatment, comprising culturing the MSCs in a hypoxic culture environment.

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

157. The method of claim 155 or 156, wherein the hypoxic pretreatment is performed for about 4 hours to 12 hours, about 12 hours to 24 hours, about 24 hours to 36 hours, about 36 hours to 48 hours, about 48 hours to 60 hours, or about 60 hours to 72 hours.

158. The method of claim 154, wherein pre-treating the MSC secretome comprises treating the MSC with one or more inflammatory cytokines.

159. The method of claim 154, wherein pre-treating the MSC secretome comprises treating the MSC with one or more of 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.

160. The method of claim 154, wherein pre-treating the MSC secretome comprises culturing the MSCs in 3D (eg, by using a 3D bioreactor).

161. The method of claim 154, wherein pre-treating the MSC secretome comprises inducing a misfolded protein response in the MSC.

162. The method of claim 154, wherein pre-treating the MSC secretome comprises culturing the MSCs under suitable conditions to induce ER stress.

163. The method of claim 154, wherein pre-treating the MSC secretome comprises culturing the MSC under suitable conditions to induce the MSC to differentiate into mature retinal cell types or precursors thereof.

164. A method of treating an ocular disorder in a patient in need thereof, comprising administering to the patient the pre-treated MSC secretome according to any one of the preceding claims.

165. Use of the pre-treated MSC secretome according to any one of the preceding claims for treating an ocular disorder in a patient in need thereof.

166. The method of claim 164 or the use of claim 165, wherein the eye disorder is selected from the group consisting of: retinal disorders, macular diseases, chronic graft versus host disease (GvHD), Stevens-Johnson syndrome, ocular mucosal pemphigoid, persistent corneal epithelial defects (PCED), limbal stem cell deficiency (LSCD), dry eyes, damage to ocular neural tissue, and concussive injury to the eye (such as concussive injury, eye contusion, or chemical burn).

167. The pretreated MSC secretome of any of the preceding claims for use according to the method of any of the preceding claims.