Process for producing and using secretomes derived from mesenchymal stem cells - Patent Application 20070122999

Abstract

The present application provides methods and processes for making and using mesenchymal stem cell secretomes, as well as methods for treating ocular conditions and / or disorders using the mesenchymal stem cell secretomes described herein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Application No. 16 / 785,463, filed February 7, 2020, U.S. Application No. 16 / 785,470, filed February 7, 2020, U.S. Provisional Application No. 62 / 831,371, filed April 9, 2019, and U.S. Provisional Application No. 62 / 929,035, filed October 31, 2019, the disclosures of which are incorporated herein by reference in their entireties for all purposes. [Background technology]

[0002] Regenerative medicine is a branch 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 disorders.

[0003] Stem cells are cells that have the ability to divide indefinitely and, under certain conditions, can differentiate into a variety of different cell types. Totipotent stem cells are stem cells that have the potential to generate all of the cells and tissues that make up an embryo. Pluripotent stem cells are stem cells that give rise to cells of the mesoderm, endoderm, and ectoderm. Multipotent stem cells are stem cells that have the ability to differentiate into two or more cell types, and unipotent stem cells are stem cells that differentiate into only one cell type. One type of such stem cell is the mesenchymal stem cell. See, e.g., U.S. Patent Application No. 20190046576.

[0004] However, generating and preserving viable stem cell-based therapies on a clinically relevant scale is challenging (see Trainor et al., Nature Biotechnology 32(1) (2014)). Furthermore, the therapeutic efficacy and regenerative capacity of such therapies often vary, and cells may die before or during transplantation (see Newell, Seminars in Immunopathology 33(2):91 (2011)). Transplanted stem cells are also susceptible to attack and / or rejection by the host's immune system, and assessing efficacy and / or controlling "dosing" is often difficult. Thus, there is a need in the art for additional regenerative therapies that can overcome the cost, storage, and manufacturing quality control limitations currently associated with cell-based regenerative medicine therapies, particularly for ocular conditions.

[0005] Blast and blunt trauma to the eye can cause a range of mechanical disruptions to the ocular contents, including retinal coma, traumatic cataract, disruption of the zonular attachment to the lens, angle recession, iris dialysis, and pupillary sphincter rupture. Treatment for these injuries is limited to mechanical repair of the iris (when possible), replacement of the lens with a plastic lens implant, and repair of retinal detachment. There are no treatments to repair the cellular structures of the retina or anterior chamber. Furthermore, traumatic optic neuropathy and optic nerve detachment were among the six major types of ocular injuries requiring specialized eye care during Operation Iraqi Freedom (Cho and Savitsky, “Ocular Trauma Chapter 7,” in Combat Casualty Care: Lessons learned from Oef and Oef, 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 (VanStavern, et al., JNeuro-Ophthamol 21(2):112-117, 2001) (incorporated herein by reference in its entirety), half of which involve nerves or visual pathways related to the optic nerve. Traumatic injury to neurons results in axonal damage and irreversible neuronal loss, resulting in permanent deficits. While many potential neuroprotective therapies have been identified in animals, these single agents have generally failed to translate into human clinical trials (Turner, et al., JNeurosurg 118(5):1072-1085, 2013, incorporated herein by reference in its entirety). Combination therapies affecting several cellular targets may be necessary to prevent neuronal damage.

[0006] As the outermost tissue of the eye, the cornea plays a protective role, but is highly vulnerable to severe injury and disease. Its lack of blood vessels gives it transparency, but it also limits its healing ability. Corneal injury can cause irreversible blindness, requiring prompt intervention and aggressive treatment. The critical need for improved ocular surface healing therapies is particularly evident in severe corneal diseases such as chemical burns and ocular manifestations of acute and chronic graft-versus-host disease (GvHD), Stevens-Johnson syndrome, ocular mucous membrane pemphigoid, and other conditions that cause persistent corneal epithelial defects, which together account for over 100,000 cases annually (Dietrich-Ntoukas et al. Cornea. 2012, 31(3):299-310; Stevenson W, et al., Clin Ophthalmol. 2013, 7:2153-2158; White K D, 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.; Wirostko B, et al. al., OculSurf. 2015Jul;13(3):204-21; Haring, R.S., et al., JAMA Ophthalmol. 2016Oct1;134(10):1119-1124).

[0007] The development of topical eye drops is hindered by many anatomical constraints, including tear turnover and dilution, nasolacrimal drainage, and reflex blinking, often resulting in less than 5% of the topical dose reaching deep ocular tissues (Gaudana et al., 2009). In the case of corneal wounds, the initial trauma causes a breach in the corneal epithelium, thereby allowing the passage of topically applied MSC-S to penetrate the epithelial layer.

[0008] Thus, there is a significant unmet need in the art for ocular therapies that can target the eye and deliver therapeutic payloads to hard-to-reach sensory cells that may be exacerbated by inflammation secondary to trauma (e.g., burns, acute inflammation, age, and / or oxidative stress). The present invention fulfills this need by providing mesenchymal stem cell secretome compositions for use in such therapies, as well as methods for making such compositions. Summary of the Invention

[0009] The present invention provides a mesenchymal stem cell (MSC) secretome composition, the composition comprising: i. an IDO (indoleamine-2,3-dioxygenase) enzyme activity of less than about 250 μM; ii. at least one trophic factor / cytokine selected from the group consisting of HGF, FGF-7, TIMP-1, TIMP-2, PAI-1 (serpin E1), VEGF-A, and / or b-NGF; iii. at least one additional factor selected from the group consisting of sFLT-1, PEDF (serpin F1), serpin A1, IGFBP-2, IGFBP-3, SDF-1, TSG-14, Kallikrein 3, MCP-1, bFGF, Angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, PDGF, SOD1, SOD2, SOD3, and / or HO-1; 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.

[0010] In some embodiments, the 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, pentraxin-3 (TSG-14), platelet factor 4, and / or serpin F1, optionally between 1 ng / mL and 8 ng / mL.

[0011] In some embodiments, the MSC secretome further comprises "moderate" levels 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 between 400 pg / mL and 3000 pg / mL.

[0012] In some embodiments, the MSC secretome further comprises at least one factor selected from the group consisting of apolipoprotein A1, complement factor D, C-reactive protein, cystatin C, DKK-1, EMMPRIN, osteopontin, vitamin DBP, MIF, RANTES, uPAR, IL-17a, GDF-15, and / or IFNγ.

[0013] In some embodiments, the MSC secretome comprises an anti-angiogenic to pro-angiogenic ratio, wherein the ratio is >2, >3, >4, or >5.

[0014] In some embodiments, the MSC secretome further comprises "low" levels of VEGF, optionally between 0 pg / mL and 200 pg / mL.

[0015] In some embodiments, the level of VEGF is 5-10 times lower than the level of serpin E1.

[0016] In some embodiments, the composition comprises one or more anti-angiogenic factors, 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.

[0017] In some embodiments, the MSC secretome is free of and / or contains very low levels of bFGF, PLGF, and PDGF, optionally less than 1000 pg / mL.

[0018] In some embodiments, the MSC secretome composition has a pH of about 4.7 to about 7.5.

[0019] In some embodiments, the MSC secretome is formulated with a buffer system selected from the group consisting of di / monosodium phosphate, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and citric acid / disodium phosphate.

[0020] In some embodiments, the MSC secretome composition further comprises a tonicity modifying agent.

[0021] In some embodiments, the tonicity modifying agent is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin.

[0022] In some embodiments, the MSC secretome further comprises monosodium phosphate / disodium phosphate, mannitol, and trehalose, and the composition has a pH of about pH 7.4.

[0023] In some embodiments, the MSC secretome further comprises a divalent cation.

[0024] In some embodiments, the divalent cation is selected from the group consisting of Mg2+, Ca2+, and Zn2+.

[0025] In some embodiments, the MSC secretome further comprises disodium phosphate / citric acid, mannitol, and trehalose, and the composition has a pH of about pH 6.4.

[0026] In some embodiments, the composition further comprises an adhesive.

[0027] In some embodiments, the adhesive is selected from the group consisting of hypromellose, poloxamer 407, poloxamer 188, poloxomer 237, poloxomer 338, hypromellose, (HPMC), polycarbophil, polyvinylpyrrolidone (PVP), PVA (polyvinyl alcohol), polyimide, sodium hyaluronate, gellan gum, poly(lactic-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethylcellulose (CMC), or hydroxypropylmethylcellulose (HPMC), hydroxymethylcellulose, hydroxyethylcellulose, sodium carboxymethylcellulose, fibrin glue, polyethylene glycol, and GelCORE.

[0028] The present invention also provides a method for producing a mesenchymal stem cell (MSC) secretome composition, the method comprising: i. culturing mesenchymal stem cells (MSCs) in a first medium; ii. removing the initial medium from step (i) from the MSCs; iii. Washing the MSCs of step (ii); iv. Add the second medium and incubate for approximately 1 to 5 days; v. harvesting the second medium from step (iv) as conditioned medium; vi. Processing the conditioned medium of step (v) into an MSC secretome composition as described herein.

[0029] In some embodiments, the MSC secretome composition is a secretome composition as described herein.

[0030] In some embodiments, step (vi) of processing the conditioned medium of step (v) into a secretome composition comprises: a) filtering the conditioned medium collected from step (v) to remove cell particles; b) concentrating the filtered conditioned medium from step (a); and c) buffer exchanging with the formulation buffer.

[0031] In some embodiments, step c) comprises buffer exchanging with a buffer system selected from the group consisting of di / monosodium phosphate, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and citric acid / disodium phosphate.

[0032] In some embodiments, the filtering step (a) comprises the use of 0.45 μm filters, 0.22 μm filters, 0.8 μm filters, and 0.65 μm filters, low protein binding PVDF membranes, and / or PES (polyethersulfone).

[0033] In some embodiments, the concentration step (b) comprises using hollow fiber filters, tangential flow filtration systems, or centrifugation-based size exclusion techniques.

[0034] In some embodiments, the centrifugation-based size exclusion technique uses a MW cutoff of 3-10 kDa.

[0035] In some embodiments, the present invention provides methods for treating ocular diseases, the methods comprising administering to a patient in need thereof a therapeutically effective amount of a mesenchymal stem cell secretome composition described herein or a composition made according to the methods described herein.

[0036] In some embodiments, the composition is administered to a target area.

[0037] The present invention also provides a method for treating visual dysfunction following trauma to an ocular structure in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a mesenchymal stem cell secretome composition described herein or a composition made according to the methods described herein.

[0038] The present invention also provides a method for inducing and / or promoting ocular wound healing in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a mesenchymal stem cell secretome composition described herein or a composition made according to the methods described herein.

[0039] The present invention also provides methods for reducing and / or inhibiting angiogenesis, reducing and / or inhibiting scarring, promoting and / or maintaining vision, and / or increasing wound closure rates (e.g., decreasing wound closure time) in a patient in need thereof, the methods comprising administering to the patient a therapeutically effective amount of a mesenchymal stem cell secretome composition described herein or a composition made according to the methods described herein.

[0040] The present invention also provides methods for reducing and / or inhibiting angiogenesis, reducing scarring and promoting vision preservation in a patient in need thereof, the methods comprising administering to the patient a therapeutically effective amount of a mesenchymal stem cell secretome composition described herein or a composition made according to the methods described herein.

[0041] In some embodiments, the mesenchymal stem cell secretome composition is formulated for topical administration.

[0042] In some embodiments, the mesenchymal stem cell secretome composition is formulated for subconjunctival injection.

[0043] The present invention also provides a method for characterizing an MSC secretome, the method comprising: (i) subjecting the MSC secretome to one or more characterization assays, wherein the characterization assays are selected from the group consisting of physical component characterization, oxidative stress assays, safety analyses, stability assays, proliferation assays, migration assays, angiogenesis assays, differentiation / scarring assays, inflammation assays, and / or epithelial barrier integrity assays; (ii) determining a result from one or more assays of (i).

[0044] The present invention also provides a method for determining the biological potency and stability of an MSC secretome, the method comprising: (i) subjecting the MSC secretome to one or more characterization assays, wherein the characterization assays are selected from the group consisting of physical component characterization, oxidative stress assays, safety analyses, stability assays, proliferation assays, migration assays, angiogenesis assays, differentiation / scarring assays, inflammation assays, and / or epithelial barrier integrity assays; (ii) determining a result from one or more assays of (i).

[0045] The present invention also provides a method for determining the consistency of MSC secretome lots among multiple MSC secretome lots, the method comprising: (i) subjecting the MSC secretome to one or more characterization assays, wherein the characterization assays are selected from the group consisting of physical component characterization, oxidative stress assays, safety analyses, stability assays, proliferation assays, migration assays, angiogenesis assays, differentiation / scarring assays, inflammation assays, and / or epithelial barrier integrity assays; (ii) determining a result from one or more assays of (i).

[0046] In some embodiments, the results of (ii) from the characterization of the physical components identify the anti-angiogenic MSC secretome described herein.

[0047] In some embodiments, the results of (ii) from the safety analysis indicate hemocompatibility and provide an MSC secretome that is low in and / or free of pyrogens and / or endotoxins.

[0048] In some embodiments, the results of (ii) from the stability assay provide an MSC secretome that exhibits stability for at least 7 days at 4°C, 20°C, and / or 25°C (or room temperature).

[0049] In some embodiments, the results of (ii) from the proliferation assay provide a proliferation-inducing MSC secretome.

[0050] In some embodiments, the results of (ii) from the migration assay provide an MSC secretome that induces migration.

[0051] In some embodiments, the results of (ii) from the angiogenesis assay provide an MSC secretome that inhibits or does not promote angiogenesis.

[0052] In some embodiments, the results of (ii) from the differentiation / scarring assay provide an MSC secretome that inhibits differentiation and / or scarring.

[0053] In some embodiments, the results of (ii) from the inflammation assay provide an MSC secretome that suppresses inflammation.

[0054] In some embodiments, the method further comprises: (iii) identifying an MSC secretome lot based on the results of (ii);

[0055] The present invention also provides a panel of tests and / or assays for characterizing the MSC secretome, the panel comprising at least two characterization assays, wherein the characterization assays are selected from the group consisting of physical component characterization, oxidative stress assays, safety analysis, stability assays, proliferation assays, migration assays, angiogenesis assays, differentiation / scarring assays, and / or inflammation assays.

[0056] The present invention also provides a panel of tests and / or assays for determining MSC secretome lot-to-lot consistency, the panel comprising one or more characterization assays, wherein the characterization assays are selected from the group consisting of physical component characterization, oxidative stress assays, safety analyses, stability assays, proliferation assays, migration assays, angiogenesis assays, differentiation / scarring assays, and / or inflammation assays.

[0057] In some embodiments, characterization of the physical components identifies the MSC secretome as described herein.

[0058] In some embodiments, the results of (ii) from the safety analysis indicate hemocompatibility and provide an MSC secretome that is low in and / or free of pyrogens and / or endotoxins.

[0059] 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.

[0060] In some embodiments, the proliferation assay identifies MSC secretomes that induce proliferation.

[0061] In some embodiments, the migration assay identifies the MSC secretome that induces migration.

[0062] In some embodiments, the angiogenesis assay identifies MSC secretomes that inhibit or do not promote angiogenesis.

[0063] In some embodiments, the differentiation / scarring assay identifies MSC secretomes that inhibit differentiation and / or scarring.

[0064] In some embodiments, the inflammation assay identifies MSC secretomes that suppress inflammation.

[0065] In some embodiments, characterization of physical components, oxidative stress assays, safety analyses, stability assays, proliferation assays, migration assays, angiogenesis assays, differentiation / scarring assays, inflammation assays, and / or epithelial barrier integrity assays are all performed.

[0066] In some embodiments, a panel of tests and / or assays described herein identifies an MSC secretome as described herein.

[0067] In some embodiments, the panel of tests and / or assays described herein includes at least one migration assay. In some embodiments, the migration assay is an in vitro wound closure assay. In some embodiments, the in vitro wound closure assay is selected from the group consisting of a "scratch assay" (also referred to as a "scratch wound assay"), a circular scratch wound method, a circular scratch wound assay, and a circular wound closure assay. In some embodiments, the MSC secretome is an anti-angiogenic MSC secretome and / or an anti-scarring MSC secretome.

[0068] In some embodiments, the MSC secretome is an anti-angiogenic MSC secretome and / or an anti-scarring MSC secretome.

[0069] In some embodiments, the MSC secretome is an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.

[0070] The present invention also provides a stable mesenchymal stem cell (MSC) secretome preparation, the preparation comprising: i. 1-20μg, optionally 2μg-8μg, of MSC secretome per mL; ii. 2 mg to 3 mg of monobasic sodium phosphate per mL; iii. 11 mg to 12 mg of dibasic sodium phosphate per mL; iv. 11.5 mg to 13 mg of mannitol per mL; v. 23mg to 24mg trehalose dihydrate, vi. 0.5 mg to 2 mg of hypromellose per 1 mL; Here, the pH is about 4.7 to about 7.5.

[0071] The present invention also provides a stable mesenchymal stem cell (MSC) secretome preparation, the preparation comprising: i. 0.004% to 0.0375%, optionally 0.008% to 0.015% w / w of MSC secretome; ii. 4% to 5% w / w of monobasic sodium phosphate; iii. 21.5% to 23% w / w of dibasic sodium phosphate; iv. 23%-25% w / w mannitol; v. 46% to 48% w / w dehydrated trehalose, vi. 1% to 3% w / w of hypromellose; Here, the pH is about 4.7 to about 7.5.

[0072] The present invention also provides a stable mesenchymal stem cell (MSC) secretome preparation, the preparation comprising: i. 6 μg of MSC secretome per mL; ii. 2.28 mg of monobasic sodium phosphate per mL; iii. 11.45 mg of dibasic sodium phosphate per mL; iv. 12.2 mg of mannitol per mL; v.24mg trehalose dihydrate, vi. 1 mg of hypromellose per mL; Here, the pH is about 7.4.

[0073] The present invention also provides a stable mesenchymal stem cell (MSC) secretome preparation, the preparation comprising: i. 0.012% w / w MSC secretome; ii. 4.5% w / w of monobasic sodium phosphate; iii. 22.4% w / w of dibasic sodium phosphate; iv. 24% w / w mannitol; v. 47.1% w / w dehydrated trehalose and vi. 2.0% w / w hypromellose, Here, the pH is about 7.4.

[0074] The present invention also provides a mesenchymal stem cell (MSC) secretome composition, the composition comprising: i. at least one trophic factor / cytokine selected from the group consisting of HGF, TIMP-1, TIMP-2, PAI-1 (serpin E1), VEGF-A, and b-NGF; ii. at least one additional factor selected from the group consisting of PEDF (serpin F1), serpin A1, IGFBP-2, IGFBP-3, SDF-1, TSG-14, Kallikrein 3, MCP-1, Angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, and PDGF; 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.

[0075] In some embodiments, the MSC secretome composition further comprises elevated levels of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1.

[0076] In some embodiments, the MSC secretome composition comprises 1 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1.

[0077] In some embodiments, the MSC secretome composition further comprises a moderate level of at least one factor selected from the group consisting of angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, thrombospondin-1, angiogenin, DPPIV, IGFBP-3, and uPA.

[0078] In some embodiments, the MSC secretome composition comprises 400 pg / mL to 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.

[0079] In some embodiments, the MSC secretome composition further comprises at least one factor selected from the group consisting of apolipoprotein A1, complement factor D, C-reactive protein, cystatin C, DKK-1, EMMPRIN, osteopontin, vitamin DBP, MIF, RANTES, uPAR, IL-17a, GDF-15, and IFNγ.

[0080] In some embodiments, the MSC secretome composition comprises an anti-angiogenic to pro-angiogenic ratio, where the ratio is >2, >3, >4, or >5.

[0081] In some embodiments of the MSC secretome composition, the anti-angiogenic factors comprise one or more factors selected from the group consisting of PEDF, low levels of VEGF, and serpin E1, and the pro-angiogenic factors comprise one or more factors selected from the group consisting of VEGF, angiogenin, IGFBP-3, uPA, angio-1, angio-2, and endothelin-1.

[0082] In some embodiments, the MSC secretome composition further comprises low levels of VEGF.

[0083] In some embodiments, the MSC secretome composition comprises between 1 pg / mL and 400 pg / mL of VEGF.

[0084] In some embodiments of the MSC secretome composition, the level of VEGF is 5-10 times lower than the level of serpin E1.

[0085] In some embodiments, the MSC secretome composition comprises one or more anti-angiogenic factors, 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.

[0086] In some embodiments, the MSC secretome composition is free of and / or contains very low levels of bFGF, PLGF, and PDGF.

[0087] In some embodiments, the MSC secretome composition comprises less than 1000 pg / mL of bFGF, PLGF, and PDGF.

[0088] In some embodiments, the MSC secretome composition has a pH of about 4.7 to about 7.5.

[0089] In some embodiments, the MSC secretome composition is formulated with a buffer system selected from the group consisting of disodium phosphate / monosodium, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and citric acid / disodium phosphate.

[0090] In some embodiments, the MSC secretome composition further comprises a tonicity modifying agent.

[0091] In some embodiments of the MSC secretome composition, the tonicity modifying agent is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin.

[0092] In some embodiments, the MSC secretome composition further comprises monosodium phosphate / disodium phosphate, mannitol, and trehalose, and the composition has a pH of about pH 7.4.

[0093] In some embodiments, the MSC secretome composition further comprises a divalent cation.

[0094] In some embodiments, the MSC secretome composition has divalent cations selected from the group consisting of Mg2+, Ca2+, and Zn2+.

[0095] In some embodiments, the MSC secretome composition further comprises disodium phosphate / citric acid, mannitol, and trehalose, and the composition has a pH of about pH 6.4.

[0096] In some embodiments, the MSC secretome composition further comprises an adhesive.

[0097] In some embodiments of the MSC secretome composition, the adhesive is selected from the group consisting of hypromellose, poloxamer 407, poloxamer 188, poloxomer 237, poloxomer 338, hypromellose (HPMC), polycarbophil, polyvinylpyrrolidone (PVP), PVA (polyvinyl alcohol), polyimide, sodium hyaluronate, gellan gum, poly(lactic-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethylcellulose (CMC), or hydroxypropylmethylcellulose (HPMC), hydroxymethylcellulose, hydroxyethylcellulose, sodium carboxymethylcellulose, fibrin glue, polyethylene glycol, GelCORE.

[0098] In some embodiments, the MSC secretome composition does not contain 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-exosomes / non-extracellular vesicle cellular debris, hormones, and L-glutamine.

[0099] In some embodiments, the MSC secretome composition comprises HGF, pentraxin-3 (TSG-14), VEGF, TIMP-1, serpin E1, and <5 ng / mL IL-8.

[0100] In some embodiments, the MSC secretome composition comprises: i. 0.3 to 4.5 ng / mL HGF and ii. 0.5 to 20 ng / mL of pentraxin-3 (TSG-14); iii. 100-600pg / mL VEGF; iv. 10 to 200 ng / mL TIMP-1 and v. 20-80ng / mL serpin E1 and vi. <5ng / mL IL-8;

[0101] In some embodiments, the MSC secretome composition comprises an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.

[0102] The present invention also provides a stable mesenchymal stem cell (MSC) secretome preparation, the preparation comprising: i. 2 μg-20 μg of MSC secretome per mL; ii. 2 mg to 3 mg of monobasic sodium phosphate per mL; iii. 11 mg to 12 mg of dibasic sodium phosphate per mL; iv. 11.5 mg to 13 mg of mannitol per mL; v. 23mg to 24mg trehalose dihydrate, vi. 0.5 mg to 2 mg of hypromellose per 1 mL; Here, the pH is about 4.7 to about 7.5.

[0103] The present invention also provides a stable mesenchymal stem cell (MSC) secretome preparation, the preparation comprising: i. 0.004%-0.08% w / w MSC secretome; ii. 4% to 5% w / w of monobasic sodium phosphate; iii. 21.5% to 23% w / w of dibasic sodium phosphate; iv. 23%-25% w / w mannitol; v. 46% to 48% w / w dehydrated trehalose, vi. 1% to 3% w / w of hypromellose; Here, the pH is about 4.7 to about 7.5.

[0104] The present invention further provides a method for treating an ocular condition in a subject in need thereof, the method comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, the MSC secretome composition comprising: i. at least one trophic factor / cytokine selected from the group consisting of HGF, TIMP-1, TIMP-2, PAI-1 (serpin E1), VEGF-A, and b-NGF; ii. at least one additional factor selected from the group consisting of PEDF (serpin F1), serpin A1, IGFBP-2, IGFBP-3, SDF-1, TSG-14, Kallikrein 3, MCP-1, Angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, and PDGF; 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.

[0105] In some embodiments, the MSC secretome composition further comprises elevated levels of at least one factor selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1.

[0106] In some embodiments, the MSC secretome composition comprises 1 ng / mL to 100 ng / mL of at least one factor selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1.

[0107] In some embodiments, the MSC secretome composition further comprises a moderate level of at least one factor selected from the group consisting of angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, thrombospondin-1, angiogenin, DPPIV, IGFBP-3, and uPA.

[0108] In some embodiments, the MSC secretome composition comprises 400 pg / mL to 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.

[0109] In some embodiments, the MSC secretome composition further comprises at least one factor selected from the group consisting of apolipoprotein A1, complement factor D, C-reactive protein, cystatin C, DKK-1, EMMPRIN, osteopontin, vitamin DBP, MIF, RANTES, uPAR, IL-17a, GDF-15, and IFNγ.

[0110] In some embodiments, the MSC secretome composition comprises an anti-angiogenic to pro-angiogenic ratio, where the ratio is >2, >3, >4, or >5.

[0111] In some embodiments, the anti-angiogenic factors include PEDF, low levels of VEGF, and one or more factors selected from the group consisting of serpin E1 and the pro-angiogenic agents VEGF, angiogenin, IGFBP-3, uPA, angio-1, angio-2, and endothelin-1.

[0112] In some embodiments, the MSC secretome composition further comprises low levels of VEGF.

[0113] In some embodiments, the MSC secretome comprises between 1 pg / mL and 400 pg / mL of VEGF.

[0114] In some embodiments, the level of VEGF is 5-10 times lower than the level of serpin E1.

[0115] In some embodiments, the MSC secretome composition comprises one or more anti-angiogenic factors, 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.

[0116] In some embodiments, the MSC secretome composition is free of and / or contains very low levels of bFGF, PLGF, and PDGF.

[0117] In some embodiments, the MSC secretome composition comprises less than 1000 pg / mL of bFGF, PLGF, and PDGF.

[0118] In some embodiments, the MSC secretome composition has a pH of about 4.7 to about 7.5.

[0119] In some embodiments, the MSC secretome composition is formulated with a buffer system selected from the group consisting of disodium phosphate / monosodium, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and citric acid / disodium phosphate.

[0120] In some embodiments, the MSC secretome composition further comprises a tonicity modifying agent.

[0121] In some embodiments, the tonicity modifying agent is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin.

[0122] In some embodiments, the MSC secretome composition further comprises monosodium phosphate / disodium phosphate, mannitol, and trehalose, and the composition has a pH of about pH 7.4.

[0123] In some embodiments, the MSC secretome composition further comprises a divalent cation.

[0124] In some embodiments, the divalent cation is selected from the group consisting of Mg2+, Ca2+, and Zn2+.

[0125] In some embodiments, the MSC secretome composition further comprises disodium phosphate / citric acid, mannitol, and trehalose, and the composition has a pH of about pH 6.4.

[0126] In some embodiments, the MSC secretome composition further comprises an adhesive.

[0127] In some embodiments, the adhesive is selected from the group consisting of hypromellose, poloxamer 407, poloxamer 188, poloxomer 237, poloxomer 338, hypromellose, (HPMC), polycarbophil, polyvinylpyrrolidone (PVP), PVA (polyvinyl alcohol), polyimide, sodium hyaluronate, gellan gum, poly(lactic-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethylcellulose (CMC), or hydroxypropylmethylcellulose (HPMC), hydroxymethylcellulose, hydroxyethylcellulose, sodium carboxymethylcellulose, fibrin glue, polyethylene glycol, and GelCORE.

[0128] In some embodiments, the MSC secretome composition does not contain 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-exosomes / non-extracellular vesicle cellular debris, hormones, and L-glutamine.

[0129] In some embodiments, the MSC secretome composition comprises HGF, pentraxin-3 (TSG-14), VEGF, TIMP-1, serpin E1, and <5 ng / mL IL-8.

[0130] In some embodiments, the MSC secretome composition comprises: i. 0.3 to 4.5 ng / mL HGF and ii. 0.5 to 20 ng / mL of pentraxin-3 (TSG-14); iii. 100-600pg / mL VEGF; iv. 10 to 200 ng / mL TIMP-1 and v. 20-80ng / mL serpin E1 and vi. <5ng / mL IL-8;

[0131] In some embodiments, the MSC secretome composition comprises an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.

[0132] The present invention also provides a method for treating an ocular condition in a subject in need thereof, the method comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, the MSC secretome composition comprising: i. 2 μg-20 μg of MSC secretome per mL; ii. 2 mg to 3 mg of monobasic sodium phosphate per mL; iii. 11 mg to 12 mg of dibasic sodium phosphate per mL; iv. 11.5 mg to 13 mg of mannitol per mL; v. 23 mg to 24 mg of trehalose dihydrate; vi. 0.5 mg to 2 mg of hypromellose per 1 mL; Here, the pH is about 4.7 to about 7.5. It is a stable mesenchymal stem cell (MSC) secretome preparation.

[0133] The present invention also provides a method for treating an ocular condition in a subject in need thereof, the method comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, the MSC secretome composition comprising: i. 0.004%-0.08% w / w MSC secretome; ii. 4% to 5% w / w of monobasic sodium phosphate; iii. 21.5% to 23% w / w of dibasic sodium phosphate; iv. 23%-25% w / w mannitol; v. 46% to 48% w / w dehydrated trehalose, vi. 1% to 3% w / w of hypromellose; Here, the pH is about 4.7 to about 7.5. It is a stable mesenchymal stem cell (MSC) secretome preparation.

[0134] The present invention also provides a stable mesenchymal stem cell (MSC) secretome preparation, the preparation comprising: i. 1-20μg, optionally 2μg-8μg, of MSC secretome per mL; ii. 2 mg to 3 mg of monobasic sodium phosphate per mL; iii. 11 mg to 12 mg of dibasic sodium phosphate per mL; iv. 11.5 mg to 13 mg of mannitol per mL; v. 23 mg to 24 mg of trehalose dihydrate; vi. 0.5 mg to 2 mg of any hypromellose per 1 mL; Here, the pH is about 4.7 to about 7.5.

[0135] The present invention also provides a stable mesenchymal stem cell (MSC) secretome preparation, the preparation comprising: i. 0.004% to 0.0375%, optionally 0.008% to 0.015% w / w of MSC secretome; ii. 4% to 5% w / w of monobasic sodium phosphate; iii. 21.5% to 23% w / w of dibasic sodium phosphate; iv. 23%-25% w / w mannitol; v. 46% to 48% w / w dehydrated trehalose, vi. 1%-3% w / w of optional hypromellose, Here, the pH is about 4.7 to about 7.5.

[0136] The present invention also provides a stable mesenchymal stem cell (MSC) secretome preparation, the preparation comprising: i. 6 μg of MSC secretome per mL; ii. 2.28 mg of monobasic sodium phosphate per mL; iii. 11.45 mg of dibasic sodium phosphate per mL; iv. 12.2 mg of mannitol per mL; v.24mg trehalose dihydrate, and 1 mg of optional hypromellose per 1 mL of Here, the pH is about 7.4.

[0137] The present invention also provides a stable mesenchymal stem cell (MSC) secretome preparation, the preparation comprising: i. 0.012% w / w MSC secretome; ii. 4.5% w / w of monobasic sodium phosphate; iii. 22.4% w / w of dibasic sodium phosphate; iv. 24% w / w mannitol; v. 47.1% w / w dehydrated trehalose and vi. 2.0% w / w of optional hypromellose, Here, the pH is about 7.4.

[0138] The present invention also provides a stable mesenchymal stem cell (MSC) secretome preparation, the preparation comprising: i. 2 μg-20 μg of MSC secretome per mL; ii. 2 mg to 3 mg of monobasic sodium phosphate per mL; iii. 11 mg to 12 mg of dibasic sodium phosphate per mL; iv. 11.5 mg to 13 mg of mannitol per mL; v. 23 mg to 24 mg of trehalose dihydrate; vi. 0.5 mg to 2 mg of any hypromellose per 1 mL; Here, the pH is about 4.7 to about 7.5.

[0139] The present invention also provides a stable mesenchymal stem cell (MSC) secretome preparation, the preparation comprising: i. 0.004%-0.08% w / w MSC secretome; ii. 4% to 5% w / w of monobasic sodium phosphate; iii. 21.5% to 23% w / w of dibasic sodium phosphate; iv. 23%-25% w / w mannitol; v. 46% to 48% w / w dehydrated trehalose, vi. 1%-3% w / w of optional hypromellose, Here, the pH is about 4.7 to about 7.5.

[0140] In some embodiments of the stable mesenchymal stem cell (MSC) secretome formulation, the formulation does not include hypromellose.

[0141] The present invention also provides a method for treating an ocular condition in a subject in need thereof, the method comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, the MSC secretome composition comprising: i. 2 μg to 20 μg of MSC secretome per mL; ii. 2 mg to 3 mg of monobasic sodium phosphate per mL; iii. 11 mg to 12 mg of dibasic sodium phosphate per mL; iv. 11.5 mg to 13 mg of mannitol per mL; v. 23mg to 24mg trehalose dihydrate, vi. 0.5 mg to 2 mg of any hypromellose per 1 mL; Here, the pH is about 4.7 to about 7.5.

[0142] The present invention also provides a method for treating an ocular condition in a subject in need thereof, the method comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, the MSC secretome composition comprising: i. 0.004%-0.08% w / w MSC secretome; ii. 4% to 5% w / w of monobasic sodium phosphate; iii. 21.5% to 23% w / w of dibasic sodium phosphate; iv. 23%-25% w / w mannitol; v. 46% to 48% w / w trehalose dehydrate; vi. 1% to 3% w / w of any hypromellose; Here, the pH is about 4.7 to about 7.5.

[0143] In some embodiments of the methods for treating an ocular condition, the MSC secretome compositions and / or formulations used in the treatment methods do not include hypromellose.

[0144] In some embodiments of the methods described herein, the MSC secretome compositions and / or formulations do not comprise hypromellose.

[0145] In some embodiments of the MSC secretome composition and / or formulation, the composition and / or formulation does not comprise hypromellose. Further aspects of the present invention are described below: [Section 1] 1. A mesenchymal stem cell (MSC) secretome composition, the composition comprising: i. IDO (indoleamine-2,3-dioxygenase) enzyme activity less than about 250 μM; ii. at least one trophic factor or cytokine selected from the group consisting of HGF, FGF-7, TIMP-1, TIMP-2, PAI-1 (serpin E1), VEGF-A, and / or b-NGF; iii. at least one additional factor selected from the group consisting of sFLT-1, PEDF (serpin F1), serpin A1, IGFBP-2, IGFBP-3, SDF-1, TSG-14, Kallikrein 3, MCP-1, bFGF, Angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, PDGF, SOD1, SOD2, SOD3, and / or HO-1; and iv. at least one additional factor selected from the group consisting of DPPIV (dipeptidyl peptidase-4), uPA, angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, and / or thrombospondin-1; A mesenchymal stem cell (MSC) secretome composition comprising: [Section 2] Item 2. The MSC secretome composition of Item 1, wherein 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, pentraxin-3 (TSG-14), platelet factor 4, and / or serpin F1, optionally at 1 ng / mL to 400 ng / mL, optionally 1 ng / mL to 300 ng / mL, optionally 1 ng / mL to 200 ng / mL, optionally 1 ng / mL to 100 ng / mL, optionally 1 ng / mL to 50 ng / mL, optionally 1 ng / mL to 10 ng / mL, or optionally 1 ng / mL to 8 ng / mL. [Section 3] Item 3. The MSC secretome composition according to Item 1 or 2, wherein the MSC secretome further comprises a moderate level, optionally 400 pg / mL to 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 / or uPA. [Section 4] The MSC secretome composition according to any one of items 1 to 3, wherein the MSC secretome further comprises at least one factor selected from the group consisting of apolipoprotein A1, complement factor D, C-reactive protein, cystatin C, DKK-1, EMMPRIN, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15, and / or IFNγ. [Section 5] Item 5. The MSC secretome composition of any one of items 1 to 4, wherein the MSC secretome comprises an anti-angiogenic to pro-angiogenic ratio, and the ratio is >2, >3, >4, or >5. [Section 6] Item 5. The MSC secretome composition according to any one of items 1 to 4, wherein the MSC secretome further comprises a low level of VEGF, optionally 0 pg / mL to 200 pg / mL, or optionally 1 pg / mL to 400 pg / mL. [Section 7] Item 7. The MSC secretome composition according to any one of Items 1 to 6, wherein the level of VEGF is 5 to 10 times lower than the level of serpin E1. [Section 8] 8. The MSC secretome composition according to any one of items 1 to 7, wherein the composition comprises one or more anti-angiogenic factors, and the sum of the concentrations of the one or more anti-angiogenic factors relative to the concentration of VEGF is >2, >3, >4, or >5. [Section 9] Item 9. The MSC secretome composition according to any one of items 1 to 8, wherein the MSC secretome does not contain bFGF, PLGF, and PDGF and / or contains them at very low levels, optionally less than 1000 pg / mL. [Section 10] Item 10. The MSC secretome composition according to any one of Items 1 to 9, wherein the MSC secretome composition has a pH of about 4.7 to about 7.5. [Section 11] The MSC secretome composition according to any one of items 1 to 10, wherein the MSC secretome is formulated in a buffer system selected from the group consisting of disodium phosphate / monosodium, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and citric acid / disodium phosphate. [Section 12] Item 12. The MSC secretome composition according to any one of items 1 to 11, further comprising a tonicity modifying agent. [Section 13] Item 13. The MSC secretome composition according to Item 12, wherein the tonicity modifying agent is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin. [Section 14] Item 14. The MSC secretome composition according to any one of items 1 to 13, wherein the MSC secretome further comprises monosodium / disodium phosphate, mannitol, and trehalose, and the composition has a pH of about pH 7.4. [Section 15] Item 15. The MSC secretome composition according to any one of Items 1 to 14, wherein the MSC secretome further comprises a divalent cation. [Section 16] Item 16. The MSC secretome composition of item 15, wherein the divalent cation is selected from the group consisting of Mg2+, Ca2+, and Zn2+. [Section 17] Item 17. The MSC secretome composition according to any one of items 1 to 16, wherein the MSC secretome further comprises disodium phosphate / citric acid, mannitol, and trehalose, and the composition has a pH of about pH 6.4. [Section 18] Item 18. The MSC secretome composition according to any one of items 1 to 17, wherein the composition further comprises an adhesive. [Section 19] Item 19. The MSC secretome composition of item 18, wherein the adhesive is selected from the group consisting of hypromellose, poloxamer 407, poloxamer 188, poloxomer 237, poloxomer 338, hypromellose (HPMC), polycarbophil, polyvinylpyrrolidone (PVP), PVA (polyvinyl alcohol), polyimide, sodium hyaluronate, gellan gum, poly(lactic-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethylcellulose (CMC), or hydroxypropyl methylcellulose (HPMC), hydroxymethylcellulose, hydroxyethylcellulose, sodium carboxymethylcellulose, fibrin glue, polyethylene glycol, and GelCORE. [Section 20] 1. A method of producing a mesenchymal stem cell (MSC) secretome composition, comprising: i. culturing mesenchymal stem cells (MSCs) in a first medium; ii. removing the initial medium from step (i) from the MSCs; iii. washing the MSCs of step (ii); iv. Add the second medium and incubate for approximately 1 to 5 days; v. harvesting the second medium from step (iv) as conditioned medium; vi. Processing the conditioned medium of step (v) into an MSC secretome composition according to any one of items 1 to 19; A method comprising: [Section 21] Item 20. The method according to Item 19, wherein the MSC secretome composition is the secretome composition according to any one of Items 1 to 19. [Section 22] Step (vi) of processing the conditioned medium of step (v) into the secretome composition comprises: a) filtering the conditioned medium harvested from step (v) to remove cell particles; b) concentrating the filtered conditioned medium from step (a); and c) buffer exchanging with the formulation buffer; 22. The method according to item 20 or 21, comprising: [Section 23] 23. The method of claim 22, wherein step c) comprises buffer exchanging with a buffer system selected from the group consisting of di / monosodium phosphate, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and citric acid / disodium phosphate. [Section 24] 24. The method of claim 22 or 23, wherein the filtration step (a) comprises the use of a 0.45 μm filter, a 0.22 μm filter, a 0.8 μm filter, and a 0.65 μm filter, a low protein binding PVDF membrane, and / or a PES (polyethersulfone) membrane. [Section 25] 25. The method of any one of paragraphs 22 to 24, wherein the concentration step (b) comprises using a hollow fiber filter, a tangential flow filtration system, or a centrifugation-based size exclusion technique. [Section 26] 26. The method of paragraph 25, wherein the centrifugation-based size exclusion technique uses a MW cutoff of 3-10 kDa. [Section 27] A method for treating an eye disease, comprising administering a therapeutically effective amount of the mesenchymal stem cell secretome composition according to any one of items 1 to 19 to a patient in need thereof, or a composition prepared according to any one of items 20 to 26 to a patient in need thereof. [Section 28] 28. The method of treatment according to paragraph 27, wherein the composition is administered to a target area. [Section 29] A method for treating visual dysfunction following trauma 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 described in any one of items 1 to 19, or a composition produced according to any one of items 20 to 26. [Section 30] 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 described in any one of items 1 to 19, or a composition produced according to any one of items 20 to 26. [Section 31] A method for reducing and / or inhibiting angiogenesis, reducing and / or inhibiting scarring, promoting and / or maintaining vision, and / or increasing wound closure rate (e.g., decreasing wound closure time) in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of the mesenchymal stem cell secretome composition described in any one of paragraphs 1 to 19, or a composition made according to any one of paragraphs 20 to 26. [Section 32] A method for reducing and / or inhibiting angiogenesis and reducing scarring to promote vision preservation in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of the mesenchymal stem cell secretome composition described in any one of items 1 to 19, or a composition produced according to any one of items 20 to 26. [Section 33] Item 33. The method according to any one of Items 27 to 32, wherein the mesenchymal stem cell secretome composition is formulated for topical administration. [Section 34] Item 33. The method according to any one of Items 27 to 32, wherein the mesenchymal stem cell secretome composition is formulated for subconjunctival injection. [Section 35] 1. A method for characterizing an MSC secretome, comprising: (i) subjecting the MSC secretome to one or more characterization assays, wherein the characterization assays are selected from the group consisting of physical component characterization, oxidative stress assays, safety analyses, stability assays, proliferation assays, migration assays, angiogenesis assays, differentiation / scarring assays, inflammation assays, and / or epithelial barrier integrity assays; (ii) determining results from one or more assays of (i); and A method comprising: [Section 36] 1. A method for determining the biopotency and stability of an MSC secretome, comprising: (i) subjecting the MSC secretome to one or more characterization assays, wherein the characterization assays are selected from the group consisting of physical component characterization, oxidative stress assays, safety analyses, stability assays, proliferation assays, migration assays, angiogenesis assays, differentiation / scarring assays, inflammation assays, and / or epithelial barrier integrity assays; (ii) determining results from one or more assays of (i); and A method comprising: [Section 37] 1. A method for determining MSC secretome lot consistency between multiple MSC secretome lots, comprising: (i) subjecting the MSC secretome to one or more characterization assays, wherein the characterization assays are selected from the group consisting of physical component characterization, oxidative stress assays, safety analyses, stability assays, proliferation assays, migration assays, angiogenesis assays, differentiation / scarring assays, inflammation assays, and / or epithelial barrier integrity assays; (ii) determining results from one or more assays of (i); and A method comprising: [Section 38] The method of any one of paragraphs 35 to 37, wherein the results of (ii) from the characterization of the physical components identify the antiangiogenic MSC secretome of any one of paragraphs 1 to 19 or the composition produced according to any one of paragraphs 20 to 26. [Section 39] 38. The method according to any one of paragraphs 35 to 37, wherein the results of (ii) from the safety analysis indicate hemocompatibility and provide an MSC secretome with low and / or no pyrogens and / or endotoxins. [Section 40] 38. The method of any one of paragraphs 35 to 37, wherein the result of (ii) from the stability assay provides an MSC secretome that is stable at -20°C, 4°C, and / or 20°C (e.g., room temperature) for at least 7 days or at least 14 days. [Section 41] 38. The method of any one of paragraphs 35 to 37, wherein the results of (ii) from the proliferation assay provide a proliferation-inducing MSC secretome. [Section 42] 38. The method of any one of paragraphs 35 to 37, wherein the results of (ii) from the migration assay provide an MSC secretome that induces migration. [Section 43] 38. The method according to any one of items 35 to 37, wherein the result (ii) from the angiogenesis assay provides an MSC secretome that inhibits or does not promote angiogenesis. [Section 44] 38. The method of any one of paragraphs 35 to 37, wherein the results of (ii) from the differentiation / scarring assay provide an MSC secretome that inhibits differentiation and / or scarring. [Section 45] 38. The method of any one of paragraphs 35 to 37, wherein the results of (ii) from the inflammation assay provide an MSC secretome that suppresses inflammation. [Section 46] (iii) The method of claim 37, further comprising identifying an MSC secretome lot based on the results of (ii). [Section 47] 1. A panel of tests and / or assays for characterizing the MSC secretome, said panel comprising at least two characterization assays selected from the group consisting of: physical component characterization, oxidative stress assay, safety analysis, stability assay, proliferation assay, migration assay, angiogenesis assay, differentiation / scarring assay, inflammation assay, and / or epithelial barrier integrity assay. [Section 48] 1. A panel of tests and / or assays for determining the consistency between MSC secretome lots, said panel comprising one or more characterization assays, wherein the characterization assays are selected from the group consisting of physical component characterization, oxidative stress assays, safety analyses, stability assays, proliferation assays, migration assays, angiogenesis assays, differentiation / scarring assays, inflammation assays, and / or epithelial barrier integrity assays. [Section 49] 49. The panel of tests and / or assays of paragraph 47 or 48, wherein characterization of the physical components identifies the MSC secretome of any one of paragraphs 1 to 19 or a composition produced according to the method of any one of paragraphs 20 to 26. [Section 50] 49. The panel of tests and / or assays of clause 47 or 48, wherein (ii) the results from the safety analysis indicate hemocompatibility and provide an MSC secretome with low and / or no pyrogens and / or endotoxins. [Section 51] 49. The panel of tests and / or assays of paragraph 47 or 48, wherein the stability assay identifies an MSC secretome that exhibits stability at -20°C, 4°C, and / or 20°C (e.g., room temperature) for at least 7 days or at least 14 days. [Section 52] 49. The test and / or assay panel of paragraph 47 or 48, wherein said proliferation assay identifies an MSC secretome that induces proliferation. [Section 53] 49. The panel of tests and / or assays of paragraph 47 or 48, wherein said migration assay identifies an MSC secretome that induces migration. [Section 54] 49. The panel of tests and / or assays of paragraph 47 or 48, wherein said angiogenesis assay identifies an MSC secretome that inhibits or does not promote angiogenesis. [Section 55] 49. The panel of tests and / or assays of paragraph 47 or 48, wherein said differentiation / scarring assay identifies an MSC secretome that inhibits differentiation and / or scarring. [Section 56] 49. The panel of tests and / or assays of paragraph 47 or 48, wherein said inflammation assay identifies an MSC secretome that suppresses inflammation. [Section 57] 49. The panel of tests and / or assays of paragraph 47 or 48, wherein physical component characterization, oxidative stress assay, safety analysis, stability assay, proliferation assay, migration assay, angiogenesis assay, differentiation / scarring assay, inflammation assay, and / or epithelial barrier integrity assay are all performed. [Section 58] 58. The panel of tests and / or assays of paragraph 57, wherein said tests and / or assays identify the MSC secretome of any one of paragraphs 49 to 56. [Section 59] 59. The panel of tests and / or assays of any one of paragraphs 47 to 58, wherein the MSC secretome is an anti-angiogenic MSC secretome and / or an anti-scarring MSC secretome. [Section 60] Item 47. The method according to any one of items 20 to 46, wherein the MSC secretome is an anti-angiogenic MSC secretome and / or an anti-scarring MSC secretome. [Section 61] 27. The composition according to any one of items 1 to 19 or the composition produced according to the method according to any one of items 20 to 26, wherein the MSC secretome is an anti-angiogenic MSC secretome or an anti-scarring MSC secretome. [Section 62] A stable mesenchymal stem cell (MSC) secretome preparation, comprising: i. 1-20μg, optionally 2μg-8μg, of MSC secretome per mL; ii. 2 mg to 3 mg of monobasic sodium phosphate per mL; iii. 11 mg to 12 mg of dibasic sodium phosphate per mL; iv. 11.5 mg to 13 mg of mannitol per mL; v. 23 mg to 24 mg of trehalose dihydrate; vi. 0.5 mg to 2 mg of hypromellose per 1 mL; wherein the pH is about 4.7 to about 7.5. A stable mesenchymal stem cell (MSC) secretome preparation. [Section 63] A stable mesenchymal stem cell (MSC) secretome preparation, comprising: i. 0.004% to 0.0375%, optionally 0.008% to 0.015% w / w of MSC secretome; ii. 4% to 5% w / w of monobasic sodium phosphate; iii. 21.5% to 23% w / w of dibasic sodium phosphate; iv. 23%-25% w / w mannitol; v. 46% to 48% w / w dehydrated trehalose, vi. 1% to 3% w / w of hypromellose; wherein the pH is about 4.7 to about 7.5. A stable mesenchymal stem cell (MSC) secretome preparation. [Section 64] A stable mesenchymal stem cell (MSC) secretome preparation, comprising: i. 6 μg of MSC secretome per mL; ii. 2.28 mg of monobasic sodium phosphate per mL; iii. 11.45 mg of dibasic sodium phosphate per mL; iv. 12.2 mg of mannitol per mL; v. 24 mg of trehalose dihydrate, vi. 1 mg of hypromellose per mL; where the pH is about 7.4, A stable mesenchymal stem cell (MSC) secretome preparation. [Section 65] A stable mesenchymal stem cell (MSC) secretome preparation, comprising: i. 0.012% w / w MSC secretome; ii. 4.5% w / w of monobasic sodium phosphate; iii. 22.4% w / w of dibasic sodium phosphate; iv. 24% w / w mannitol; v. 47.1% w / w dehydrated trehalose and vi. 2.0% w / w hypromellose, where the pH is about 7.4, A stable mesenchymal stem cell (MSC) secretome preparation. [Section 66] 1. A mesenchymal stem cell (MSC) secretome composition, comprising: at least one trophic factor or cytokine selected from the group consisting of HGF, TIMP-1, TIMP-2, PAI-1 (serpin E1), VEGF-A, and b-NGF; b. at least one additional factor selected from the group consisting of PEDF (serpin F1), IGFBP-2, IGFBP-3, SDF-1, TSG-14, Kallikrein 3, MCP-1, Angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, and PDGF; c. 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; 1. An MSC secretome composition comprising: [Section 67] 67. The MSC secretome composition of paragraph 66, further comprising elevated levels of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. [Section 68] Item 66 or 67, the MSC secretome composition comprising 1 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. [Section 69] Item 69. The MSC secretome composition of any one of items 66 to 68, further comprising a "moderate" 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. [Section 70] Item 66 to 69, wherein the MSC secretome composition comprises 400 pg / mL to 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. The MSC secretome composition of any one of items 66 to 69. [Section 71] Item 71. The MSC secretome composition of any one of Items 66 to 70, further comprising at least one factor selected from the group consisting of apolipoprotein A1, complement factor D, C-reactive protein, cystatin C, DKK-1, EMMPRIN, osteopontin, vitamin DBP, MIF, RANTES, uPAR, IL-17a, GDF-15, and IFNγ. [Section 72] 72. The MSC secretome composition of any one of items 66 to 71, wherein the MSC secretome composition comprises an anti-angiogenic to pro-angiogenic ratio, wherein the ratio is >2, >3, >4, or >5. [Section 73] Item 73. The MSC secretome composition of any one of Items 66 to 72, wherein the anti-angiogenic factor comprises one or more factors selected from the group consisting of PEDF, low levels of VEGF, and serpin E1, and the pro-angiogenic factor comprises one or more factors selected from the group consisting of VEGF, angiogenin, IGFBP-3, uPA, angio-1, angio-2, and endothelin-1. [Section 74] Item 74. The MSC secretome composition according to any one of Items 66 to 73, wherein the MSC secretome composition further comprises a low level of VEGF. [Section 75] Item 75. The MSC secretome composition according to any one of Items 66 to 74, wherein the MSC secretome composition contains 1 pg / mL to 400 pg / mL of VEGF. [Section 76] Item 76. The MSC secretome composition according to any one of Items 66 to 75, wherein the level of VEGF is 5 to 10 times lower than the level of serpin E1. [Section 77] Item 77. The MSC secretome composition of any one of items 66 to 76, wherein the composition comprises one or more antiangiogenic factors, and the sum of the concentrations of the one or more antiangiogenic factors relative to the concentration of VEGF is >2, >3, >4, or >5. [Section 78] Item 78. The MSC secretome composition of any one of items 66 to 77, wherein the MSC secretome composition does not contain and / or contains very low levels of bFGF, PLGF, and PDGF. [Section 79] Item 79. The MSC secretome composition of any one of items 66 to 78, wherein the MSC secretome contains less than 1000 pg / mL of bFGF, PLGF, and PDGF. [Section 80] Item 80. The MSC secretome composition according to any one of Items 66 to 79, wherein the MSC secretome composition has a pH of about 4.7 to about 7.5. [Section 81] Item 66 to 80, wherein the MSC secretome composition is formulated with a buffer system selected from the group consisting of disodium phosphate / monosodium, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and citric acid / disodium phosphate. The MSC secretome composition of any one of items 66 to 80. [Section 82] Item 82. The MSC secretome composition according to any one of items 66 to 81, wherein the MSC secretome composition further comprises a tonicity modifying agent. [Section 83] Item 83. The MSC secretome composition of Item 82, wherein the tonicity modifying agent is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin. [Section 84] Item 84. The MSC secretome composition of any one of items 66 to 83, wherein the MSC secretome composition further comprises monosodium / disodium phosphate, mannitol, and trehalose, and the composition has a pH of about pH 7.4. [Section 85] Item 66 to 84, the MSC secretome composition further comprising a divalent cation. [Section 86] Item 86. The MSC secretome composition of Item 85, wherein the divalent cations are selected from the group consisting of Mg2+, Ca2+, and Zn2+. [Section 87] Item 87. The MSC secretome composition of any one of items 66 to 86, wherein the MSC secretome composition further comprises disodium phosphate / citric acid, mannitol, and trehalose, and the composition has a pH of about pH 6.4. [Section 88] Item 66 to 87, the MSC secretome composition further comprising an adhesive. [Section 89] Item 89. The MSC secretome composition of Item 88, wherein the adhesive is selected from the group consisting of hypromellose, poloxamer 407, poloxamer 188, poloxomer 237, poloxomer 338, hypromellose (HPMC), polycarbophil, polyvinylpyrrolidone (PVP), PVA (polyvinyl alcohol), polyimide, sodium hyaluronate, gellan gum, poly(lactic-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethylcellulose (CMC), or hydroxypropyl methylcellulose (HPMC), hydroxymethylcellulose, hydroxyethylcellulose, sodium carboxymethylcellulose, fibrin glue, polyethylene glycol, and GelCORE. [Section 90] Item 89. The MSC secretome composition according to any one of Items 66 to 89, wherein the MSC secretome composition does not contain 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-exosomes / non-extracellular vesicle cellular debris, hormones, and L-glutamine. [Section 91] Item 91. The MSC secretome composition according to any one of Items 66 to 90, wherein the MSC secretome composition comprises HGF, pentraxin-3 (TSG-14), VEGF, TIMP-1, serpin E1, and <5 ng / mL of IL-8. [Section 92] The MSC secretome composition, i. 0.3 to 4.5 ng / mL HGF and ii. 0.5 to 20 ng / mL of pentraxin-3 (TSG-14); iii. VEGF levels of 100–600 pg / mL; iv. 10 to 200 ng / mL TIMP-1 and v. 20-80ng / mL serpin E1 and vi. IL-8 <5ng / mL; Item 66 to 91, the MSC secretome composition according to any one of items 66 to 91. [Section 93] The MSC secretome composition, i. 0.3 to 4.5 ng / mL HGF and ii. 0.5 to 20 ng / mL of pentraxin-3 (TSG-14); iii. VEGF levels of 100–600 pg / mL; iv. 10 to 200 ng / mL TIMP-1 and v. 20-80ng / mL serpin E1 and vi. 100-400ng / mL of serpin F1; vii. IL-8 <5ng / mL; Item 66 to 92, the MSC secretome composition according to any one of items 66 to 92. [Section 94] Item 66. The MSC secretome composition according to any one of items 66 to 93, wherein the MSC secretome composition comprises an anti-angiogenic MSC secretome or an anti-scarring MSC secretome. [Section 95] A stable mesenchymal stem cell (MSC) secretome preparation, comprising: i. 2 μg to 20 μg of MSC secretome per mL; ii. 2 mg to 3 mg of monobasic sodium phosphate per mL; iii. 11 mg to 12 mg of dibasic sodium phosphate per mL; iv. 11.5 mg to 13 mg of mannitol per mL; v. 23 mg to 24 mg of trehalose dihydrate; vi. 0.5 mg to 2 mg of hypromellose per 1 mL; wherein the pH is about 4.7 to about 7.5. A stable mesenchymal stem cell (MSC) secretome preparation. [Section 96] A stable mesenchymal stem cell (MSC) secretome preparation, comprising: i. 0.004%-0.08% w / w MSC secretome; ii. 4% to 5% w / w of monobasic sodium phosphate; iii. 21.5% to 23% w / w of dibasic sodium phosphate; iv. 23%-25% w / w mannitol; v. 46% to 48% w / w dehydrated trehalose, vi. 1% to 3% w / w of hypromellose; wherein the pH is about 4.7 to about 7.5. A stable mesenchymal stem cell (MSC) secretome preparation. [Section 97] 1. A method for treating an ocular condition in a subject in need thereof, comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, wherein the MSC secretome composition comprises: i. at least one trophic factor or cytokine selected from the group consisting of HGF, TIMP-1, TIMP-2, PAI-1 (serpin E1), VEGF-A, and b-NGF; ii. at least one additional factor selected from the group consisting of PEDF (serpin F1), serpin A1, IGFBP-2, IGFBP-3, SDF-1, TSG-14, Kallikrein 3, MCP-1, Angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, and PDGF; 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; and A method comprising: [Section 98] 98. The method of treatment of paragraph 97, wherein the MSC secretome composition further comprises elevated levels of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. [Section 99] Item 99. The method of treatment according to Item 97 or 98, wherein the MSC secretome composition comprises 1 ng / mL to 100 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. [Section 100] 99. The method of any one of items 97 to 99, wherein the MSC secretome composition further comprises a moderate 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. [Section 101] The method of any one of items 97 to 100, wherein the MSC secretome composition comprises 400 pg / mL to 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. [Section 102] Item 97. The method of any one of Items 97 to 101, wherein the MSC secretome composition further comprises at least one factor selected from the group consisting of apolipoprotein A1, complement factor D, C-reactive protein, cystatin C, DKK-1, EMMPRIN, osteopontin, vitamin DBP, MIF, RANTES, uPAR, IL-17a, GDF-15, and IFNγ. [Section 103] 103. The method of any one of paragraphs 97 to 102, wherein the MSC secretome composition comprises an anti-angiogenic to pro-angiogenic ratio, wherein the ratio is >2, >3, >4, or >5. [Section 104] 104. The method of any one of items 97 to 103, wherein the anti-angiogenic factors include PEDF, low levels of VEGF, and one or more factors selected from the group consisting of serpin E1 and the angiogenic agents VEGF, angiogenin, IGFBP-3, uPA, angio-1, angio-2, and endothelin-1. [Section 105] Item 105. The method of treatment according to any one of Items 97 to 104, wherein the MSC secretome composition further comprises a low level of VEGF. [Section 106] Item 105. The method of treatment according to any one of Items 97 to 104, wherein the MSC secretome contains 1 pg / mL to 400 pg / mL of VEGF. [Section 107] Item 107. The method of treatment according to any one of Items 97 to 106, wherein the level of VEGF is 5 to 10 times lower than the level of Serpin E1. [Section 108] Item 97. The method of any one of items 97 to 107, 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. [Section 109] Item 109. The method of treatment according to any one of items 97 to 108, wherein the MSC secretome composition does not contain bFGF, PLGF, and PDGF or contains them at very low levels. [Section 110] Item 109. The method of any one of Items 97 to 109, wherein the MSC secretome composition contains less than 1000 pg / mL of bFGF, PLGF, and PDGF. [Section 111] Item 111. The method of treatment according to any one of Items 97 to 110, wherein the MSC secretome composition has a pH of about 4.7 to about 7.5. [Section 112] Item 97. The method of any one of items 97 to 111, wherein the MSC secretome composition is formulated in a buffer system selected from the group consisting of disodium phosphate / monosodium, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and citric acid / disodium phosphate. [Section 113] Item 113. The method of treatment according to any one of Items 97 to 112, wherein the MSC secretome composition further comprises a tonicity modifying agent. [Section 114] 114. The method of treatment of claim 113, wherein the tonicity modifying agent is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin. [Section 115] Item 115. The method of any one of items 97 to 114, wherein the MSC secretome composition further comprises monosodium / disodium phosphate, mannitol, and trehalose, and the composition has a pH of about pH 7.4. [Section 116] Item 116. The method of treatment according to any one of Items 97 to 115, wherein the MSC secretome composition further comprises a divalent cation. [Section 117] 117. The method of treatment of claim 116, wherein the divalent cation is selected from the group consisting of Mg2+, Ca2+, and Zn2+. [Section 118] Item 118. The method of any one of items 97 to 117, wherein the MSC secretome composition further comprises disodium phosphate / citric acid, mannitol, and trehalose, and the composition has a pH of about pH 6.4. [Section 119] Item 119. The method of treatment according to any one of Items 97 to 118, wherein the MSC secretome composition further comprises an adhesive. [Section 120] Item 119. The method of treatment of paragraph 119, wherein the adhesive is selected from the group consisting of hypromellose, poloxamer 407, poloxamer 188, poloxomer 237, poloxomer 338, hypromellose (HPMC), polycarbophil, polyvinylpyrrolidone (PVP), PVA (polyvinyl alcohol), polyimide, sodium hyaluronate, gellan gum, poly(lactic-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethylcellulose (CMC), or hydroxypropyl methylcellulose (HPMC), hydroxymethylcellulose, hydroxyethylcellulose, sodium carboxymethylcellulose, fibrin glue, polyethylene glycol, and GelCORE. [Section 121] Item 121. The method of any one of Items 97 to 120, wherein the MSC secretome composition does not contain 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-exosomes / non-extracellular vesicle cellular debris, hormones, and L-glutamine. [Section 122] Item 122. The method of treatment of any one of items 97 to 121, wherein the MSC secretome composition comprises HGF, pentraxin-3 (TSG-14), VEGF, TIMP-1, serpin E1, and less than 5 ng / mL of IL-8. [Section 123] The MSC secretome composition, i. 0.3 to 4.5 ng / mL HGF and ii. 0.5 to 20 ng / mL of pentraxin-3 (TSG-14); iii. VEGF levels of 100–600 pg / mL; iv. 10 to 200 ng / mL TIMP-1 and v. 20-80ng / mL serpin E1 and vi. IL-8 <5ng / mL; Item 97 to 122. The method of treatment according to any one of Items 97 to 122, comprising: [Section 124] Item 97. The method of treatment according to any one of items 97 to 123, wherein the MSC secretome composition comprises an anti-angiogenic MSC secretome or an anti-scarring MSC secretome. [Section 125] 1. A method of treating an ocular condition in a subject in need thereof, comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, wherein the MSC secretome composition is a stable mesenchymal stem cell (MSC) secretome preparation, the stable mesenchymal stem cell (MSC) secretome preparation comprising: i. 2 μg to 20 μg of MSC secretome per mL; ii. 2 mg to 3 mg of monobasic sodium phosphate per mL; iii. 11 mg to 12 mg of dibasic sodium phosphate per mL; iv. 11.5 mg to 13 mg of mannitol per mL; v. 23 mg to 24 mg of trehalose dihydrate; vi. 0.5 mg to 2 mg of hypromellose per 1 mL; wherein the pH is about 4.7 to about 7.5. Treatment method. [Section 126] A method for treating an ocular condition in a subject in need thereof comprises administering to the subject a mesenchymal stem cell (MSC) secretome composition, wherein the MSC secretome composition is a stable mesenchymal stem cell (MSC) secretome preparation, the stable mesenchymal stem cell (MSC) secretome preparation comprising: i. 0.004%-0.08% w / w MSC secretome; ii. 4% to 5% w / w of monobasic sodium phosphate; iii. 21.5% to 23% w / w of dibasic sodium phosphate; iv. 23%-25% w / w mannitol; v. 46% to 48% w / w dehydrated trehalose, vi. 1% to 3% w / w of hypromellose; wherein the pH is about 4.7 to about 7.5. Treatment method. [Section 127] 49. The test and / or assay panel of any one of paragraphs 47 to 48, wherein said panel of assays comprises at least one migration assay. [Section 128] 128. The test and / or assay panel of any one of paragraphs 47-48 or 127, wherein the migration assay is an in vitro wound closure assay. [Section 129] 129. The test and / or assay panel of claim 128, wherein said 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. [Section 130] A stable mesenchymal stem cell (MSC) secretome preparation, comprising: i. 1-20μg, optionally 2μg-8μg, of MSC secretome per mL; ii. 2 mg to 3 mg of monobasic sodium phosphate per mL; iii. 11 mg to 12 mg of dibasic sodium phosphate per mL; iv. 11.5 mg to 13 mg of mannitol per mL; v. 23 mg to 24 mg of trehalose dihydrate; vi. 0.5 mg to 2 mg of any hypromellose per 1 mL; wherein the pH is about 4.7 to about 7.5. A stable mesenchymal stem cell (MSC) secretome preparation. [Section 131] A stable mesenchymal stem cell (MSC) secretome preparation, comprising: i. 0.004% to 0.0375%, optionally 0.008% to 0.015% w / w of MSC secretome; ii. 4% to 5% w / w of monobasic sodium phosphate; iii. 21.5% to 23% w / w of dibasic sodium phosphate; iv. 23%-25% w / w mannitol; v. 46% to 48% w / w dehydrated trehalose, vi. 1% to 3% w / w of any hypromellose; wherein the pH is about 4.7 to about 7.5. A stable mesenchymal stem cell (MSC) secretome preparation. [Section 132] A stable mesenchymal stem cell (MSC) secretome preparation, comprising: i. 6 μg of MSC secretome per mL; ii. 2.28 mg of monobasic sodium phosphate per mL; iii. 11.45 mg of dibasic sodium phosphate per mL; iv. 12.2 mg of mannitol per mL; v. 24 mg of trehalose dihydrate, vi. 1 mg of optional hypromellose per mL; where the pH is about 7.4, A stable mesenchymal stem cell (MSC) secretome preparation. [Section 133] A stable mesenchymal stem cell (MSC) secretome preparation, comprising: i. 0.012% w / w MSC secretome; ii. 4.5% w / w of monobasic sodium phosphate; iii. 22.4% w / w of dibasic sodium phosphate; iv. 24% w / w mannitol; v. 47.1% w / w dehydrated trehalose and vi. 2.0% w / w of optional hypromellose, where the pH is about 7.4, A stable mesenchymal stem cell (MSC) secretome preparation. [Section 134] A stable mesenchymal stem cell (MSC) secretome preparation, comprising: i. 2 μg to 20 μg of MSC secretome per mL; ii. 2 mg to 3 mg of monobasic sodium phosphate per mL; iii. 11 mg to 12 mg of dibasic sodium phosphate per mL; iv. 11.5 mg to 13 mg of mannitol per mL; v. 23 mg to 24 mg of trehalose dihydrate; vi. 0.5 mg to 2 mg of any hypromellose per 1 mL; wherein the pH is about 4.7 to about 7.5. A stable mesenchymal stem cell (MSC) secretome preparation. [Section 135] A stable mesenchymal stem cell (MSC) secretome preparation, comprising: i. 0.004%-0.08% w / w MSC secretome; ii. 4% to 5% w / w of monobasic sodium phosphate; iii. 21.5% to 23% w / w of dibasic sodium phosphate; iv. 23%-25% w / w mannitol; v. 46% to 48% w / w dehydrated trehalose, vi. 1%-3% w / w of optional hypromellose, Here, the pH is about 4.7 to about 7.5. A stable mesenchymal stem cell (MSC) secretome preparation. [Section 136] Item 136. The stable mesenchymal stem cell (MSC) secretome preparation of any one of items 130 to 135, wherein the preparation does not contain hypromellose. [Section 137] 1. A method of treating an ocular condition in a subject in need thereof, comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, wherein the MSC secretome composition is a stable mesenchymal stem cell (MSC) secretome preparation, the stable mesenchymal stem cell (MSC) secretome preparation comprising: i. 2 μg to 20 μg of MSC secretome per mL; ii. 2 mg to 3 mg of monobasic sodium phosphate per mL; iii. 11 mg to 12 mg of dibasic sodium phosphate per mL; iv. 11.5 mg to 13 mg of mannitol per mL; v. 23 mg to 24 mg of trehalose dihydrate; vi. 0.5 mg to 2 mg of any hypromellose per 1 mL; wherein the pH is about 4.7 to about 7.5. Treatment method. [Section 138] 1. A method of treating an ocular condition in a subject in need thereof, comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, wherein the MSC secretome composition is a stable mesenchymal stem cell (MSC) secretome preparation, the stable mesenchymal stem cell (MSC) secretome preparation comprising: i. 0.004%-0.08% w / w MSC secretome; ii. 4% to 5% w / w of monobasic sodium phosphate; iii. 21.5% to 23% w / w of dibasic sodium phosphate; iv. 23%-25% w / w mannitol; v. 46% to 48% w / w trehalose dehydrate; vi. 1% to 3% w / w of any hypromellose; wherein the pH is about 4.7 to about 7.5. Treatment method. [Section 139] 139. A method of treating an eye condition according to paragraph 137 or 138, wherein the formulation does not contain hypromellose. [Section 140] Item 139. The method according to any one of items 1 to 139, wherein the MSC secretome composition and / or formulation does not contain hypromellose. [Section 141] Item 139. The MSC secretome composition and / or formulation according to any one of items 1 to 139, wherein the composition and / or formulation does not contain hypromellose. [Brief explanation of the drawings]

[0146] [Figure 1] Schematic of an embodiment of the purification, processing, and use of the MSC secretome. [Figure 2] MSC-S contain wound healing factors. Using the Human XL Cytokine Array (R&D Systems), several protein factors present in MSC-S were identified. Dashed box: reference spot. [Figure 3A-3C]MSC-S demonstrates a lower tendency for aggregation. A) MSC-S formulated in pH 7.4 phosphate buffer was analyzed by SEC. After initial thawing, MSC-S were maintained at 4°C, 20°C, or 37°C for 7 days and analyzed by SEC. Only a slight increase in higher-order aggregates was observed, correlating with the higher storage temperature. Samples were separated on an Agilent 1100 HPLC system equipped with a diode array detector and a TSKgel SuperSW2000, 4.6 x 300 mm column using a flow rate of 0.25 mg / mL and a mobile phase of 1x PBS. The observed wavelength was 214 nm. 28 μg of MSC-S was loaded. B) MSC-S formulated at pH 7.4 is more stable than C) pH 6.4 due to its tendency for thermal aggregation. Samples were evaluated, and signals were observed at 266 nm and 473 nm (for large aggregates). 8.8 μL of 1 mg / mL MSC-S was loaded into a Uni-cuvette in quadruplicate. Temperature scans were performed from 20°C to 90°C at a scan rate of 0.6°C per minute. [Figure 4A-4B] MSC-S promotes migration of primary human corneal epithelial cells. A) Photograph shows the underside of a transwell membrane after 24 incubations with MSC-S or 0.1% growth supplement. B) MSC-S significantly promotes migration. Migrated cells were counted in three fields per replicate, with three replicates per treatment. A two-tailed unpaired t-test was performed using GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, California USA (see WorldWideWeb at graphpad.com). Data presented are mean ± SE; ****p<0.0001. [Figure 5A-5B]MSC-S promotes migration of primary human corneal epithelial cells. A) Photograph shows the underside of a transwell membrane after 24 incubations with MSC-S or 0.1% growth supplement. B) MSC-S significantly promotes migration. Migrated cells were counted in three fields per replicate, with three replicates per treatment. A two-tailed unpaired t-test was performed using GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, California USA (see WorldWideWeb at graphpad.com). Data presented are mean ± SE; ****p<0.0001. [Figures 6A-6B] MSC-S have an anti-angiogenic profile based on protein expression. A) Pro- and anti-angiogenic factors in MSC-S were assessed using a human angiogenesis array. B) Anti-angiogenic factors (black) are present at high levels, and pro-angiogenic factors (white) are present at low levels. Average pixel intensity was determined using ImageJ (NIH, Bethesda, MD). [Figures 7A-7B]MSC-S exhibit antiangiogenic properties in a tube formation assay. HUVEC cells (10K in 100 μL) were seeded on Matrigel with 10K cells in 100 μL of endothelial cell basal medium and low-serum growth supplement (Life Technologies) in the absence or presence of MSC-S (300 μg / mL). Cells were incubated for 6 hours to form endothelial tubes, after which images were acquired and processed. Five replicates were performed per condition. A) Image panels show that MSC-S attenuates tube formation. B) MSC-S reduces angiogenesis metrics (total loops, total tube length, total branch points). Images were processed using WimTube: Tube Formation Assay Image Analysis Solution, Release 4.0. Two-tailed unpaired t-tests were performed using GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, California, USA, www.graphpad.com. Data presented are mean ± SE: **p<0.001, and ***p<0.0005. [Figure 8] MSC-S demonstrate anti-scar properties. Human dermal fibroblasts were differentiated into myofibroblasts using TGFβ-1 in the presence of MSC-S. After 24 hours, qPCR was performed on total isolated RNA. MSC-S reduces the expression of scarring biomarkers TGFβ2 and SMA. Data represent the mean ± SD of nine replicates. Comparisons were performed using one-way analysis of variance (ANOVA) followed by post-hoc Dunnett's test. *p<0.05, ***p<0.0001. [Figure 9]MSC-S treatment significantly improved wound closure following corneal alkali burn. Alkali burn animal model: Norway rats (150 g, 10 weeks old) were deeply anesthetized with subcutaneous ketoprofen and then injected with ketamine / xylazine subcutaneously. One drop of topical proparacaine was then applied to the right eye to treat the burn. Next, alkali burn was induced in the right eye of each deeply anesthetized rat by placing a 5 mm diameter circular piece of filter paper soaked in 1 N NaOH on the central cornea for 30 seconds and irrigating with 100 mL of balanced salt solution (BSS). Postoperative pain management was achieved by administering ketoprofen via subcutaneous injection three times every 24 hours. Animals were then randomly assigned to two treatment groups: vehicle (n = 4) and MSC-S (n = 4-5). Eyes were stained with fluorescein (20 μL of 1.0% fluorescein phosphate diluted in BSS), and photographs were taken at the time of initial wounding and daily after induction of alkali burn. The area of ​​the epithelial defect was measured. Immediately after the initial fluorescein staining, topical treatment was initiated: vehicle (BSS, 5 μL) and MSC-S (5 mg / mL, 5 μL) three times daily (TID) for 7 days. The area of ​​the corneal abrasion was quantified from the photographs using a computer-assisted image analyzer (ImageJ 1.38x; National Institutes of Health, Bethesda, MD). The degree of healing was determined by the ratio of the difference between the immediate insult and the remaining wound area every 24 hours. Two-tailed unpaired t-tests were performed using GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, California, USA, www.graphpad.com. Data are expressed as mean ± SE: *p<0.05, **p<0.01, and ***p<0.001. [Figure 10]MSC-S maintain migration-promoting bioactivity for 7 days when stored at 5°C and 25°C (ambient temperature) [Study CM19_TWS01]. The cell-based migration stability assay utilizes the transwell migration principle and primary corneal fibroblasts. Briefly, cells are seeded in basal (nutrient-depleted) medium in an upper chamber with a porous membrane. The chamber is then placed in a well of basal medium containing MSC-S. After 24 hours, cells that have migrated to the bottom surface of the membrane are stained, imaged, and quantified. [Figures 11A-11B] MSC-S promotes migration of primary human corneal epithelial cells. Photographs show the underside of a transwell membrane after 24 hours of incubation with 0.1% growth supplement (GS, A) or MSC-S (300 μg / mL, B). Migrated cells were counted in three fields per replicate, with three replicates per treatment. Denat: heat denatured at 90°C for 10 minutes. Data presented are mean ± SE, ****p<0.0001 using a two-tailed unpaired t-test. [Figures 12A-12B] MSC-S improves wound gap closure in confluent monolayers of human primary corneal epithelial cells. A) Photographs are representative images of wound gaps in primary human corneal epithelial cell monolayers treated with vehicle control (top panel) or MSC-S (45 μg / ml; bottom panel), imaged at T = 0, 24, and 48 h. The cell monolayer is depicted in green, and the dashed line (red) corresponds to the wound gap. B) Treatment of cells with 45 μg / ml MSC-S for 48 h significantly improves wound gap closure. Two-tailed unpaired t-tests were performed using GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, California, USA. Data presented are mean ± SE; *p<0.001. [Figure 13] MSC-S promotes migration of human primary corneal fibroblasts. Pictured is the underside of a transwell membrane after 20 hours of incubation with MSC-S or vehicle control. [Figures 14A-14B]MSC-S exhibit an anti-angiogenic protein profile. A) A human angiogenesis array was used to identify pro- and anti-angiogenic protein factors in MSC-S. Numbers (1-14) in panel A correspond to the labeled density bar graph in panel B. B) Anti-angiogenic factors (black) are present at high levels, while pro-angiogenic factors (white) are present at low levels. Average pixel intensity was determined using image processing software, ImageJ (NIH, Bethesda, MD). [Figures 15A-15B] MSC-S exhibit antiangiogenic properties in a tube formation assay. 12,000 cells were cultured in 100 μL of endothelial cell basal medium with low serum growth supplement (Life Technologies) in the presence or absence of MSC-S (40 μg / mL) in Matrigel. Cells were incubated for 6 hours to form endothelial tubes, after which images were acquired and processed. Five replicates were performed per condition. A) Image panels show that MSC-S attenuate tube formation compared to a VEGF-positive control. B) MSC-S reduce angiogenesis metrics (total tube length, total loops, and total branch points). Images were processed using WimTube: Tube Formation Assay Image Analysis Solution, Release 4.0. Two-tailed unpaired t-tests were performed using GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, California, USA. Data presented are mean ± SE; **p<0.05. [Figure 16] MSC-S reduces corneal myofibroblast differentiation induced by TGFβ-1. Shown are human primary corneas induced to differentiate into myofibroblasts by treatment with TGFβ-1 in the presence of vehicle buffer or MSC-S (10 μg / mL). After 24 hours, cells were fixed and stained with nuclear dye (DAPI; blue channel) and smooth muscle actin (green channel). The same imaging and camera settings were applied to all treatment groups. [Figures 17A-17B]MSC-S treatment accelerates wound closure. Corneal alkali burns were performed on Norway rats (approximately 150 g, 12 weeks old) and 5 µL of vehicle (HPMC, n = 7) or MSC-S (0.6 µg / mL, n = 7; 6 µg / mL, n = 7; 60 µg / mL, n = 8) was placed on the right eye of animals treated with either 0.6 µg / mL MSC-S three times daily or 6 µg / mL MSC-S six times daily (n = 8). Data from day 4 are shown. Panel (a) shows the wound closure rate, and panel (b) shows representative fluorescein staining. Animal identification numbers are displayed in white in the upper left corner. The extent of wound closure was assessed via fluorescein staining by the ratio of the difference between the immediate wound area and the remaining wound area. [Figures 18A-18B] MSC-S treatment significantly improves corneal opacity and neovascularization. Corneal opacity (A) and neovascularization (B) data are shown for corneal burns treated with 5 μL of vehicle (HPMC, n = 6) or MSC-S (0.6 μg / mL, n = 6; 6 μg / mL, n = 6; 60 μg / mL, n = 6) three times a day or 6 μg / mL MSC-S (n = 7) six times a day for 10 days. Corneal opacity on a scale of 0 to 4: 0 = completely clear; 1 = slightly hazy, iris and pupil easily visible; 2 = slightly opaque, iris and pupil still detectable; 3 = opaque, pupil barely detectable; 4 = completely opaque, pupil not visible. Neovascularization on a scale of 0 to 4. 0 = no neovascularization; 1 = limbal neovascularization; 2 = limbal neovascularization; 3 = neovascularization reaching the center of the cornea, leaving a clear zone elsewhere in the cornea; 4 = neovascularization in all four quadrants of the cornea, including the center. Data are expressed as means with standard errors. Comparisons of parameters were performed using one-way analysis of variance followed by a post-hoc Bonferroni test. *P values ​​of <0.05 were considered statistically significant (Graph Pad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, CA USA). [Figures 19A-19B]MSC-S at 6 μg / mL is significantly superior to vehicle in ameliorating the effects of corneal injury. Changes in corneal injury following topical application of (5 μL) vehicle (a) and 6 μg / mL MSC-S (b) three times daily for 10 days to rats with corneal alkali injury. Digital images of the eyes were taken on days 7 and 14 to document the clinical appearance of the eyes. Animal identification numbers are displayed in the upper right corner of each rat complex. [Figure 20] Size distribution of EVs. The shaded area represents particles remaining after detergent treatment, indicating that more than 85% of the particles are detergent-labile lipid-based vesicles. [Figure 21] Proliferation assays were performed using primary keratocytes and MSC secretome. MSC secretome promotes keratocyte proliferation. Primary keratocytes were seeded at 5,000 cells / cm² in overnight culture. Cells were then serum-starved for 1 hour and treated with various concentrations of MSC secretome. Proliferation was monitored daily for up to 3 days after treatment using a CCK-8 assay (cell counting kit; Dojindo Molecular Technologies). [Figure 22] MSC secretome stability - composition. New batches of MSC secretome were manufactured and the stability of the drug substance (in formulation buffer) was evaluated. Aliquots were stored at -20°C, 4°C, or room temperature (RT) as part of a 7-day or 14-day program. At the end of the study, samples were analyzed by ELISA to measure serpin E1, serpin F1, and TIMP-1. MSC secretome factors do not degrade in the liquid formulation for at least 14 days. The stability of multiple factors suggests that the formulation supports broad protein stabilization. [Figure 23]In vitro wound closure assay. Top panel: MSC secretome stimulates migration of primary keratocytes at the site of a circular wound. Circular wounds were created in a confluent monolayer of keratocytes, and wound gap closure was observed daily. MSC secretome at 100 and 50 μg / mL in serum-free medium closes the wound within 48 hours. Shown is the image of the wound area stained with gentian violet, imaged 2 days after wounding. Bottom panel: MSC secretome stimulates migration of corneal epithelial cells at the site of a circular wound. Circular wounds were created in a confluent monolayer of corneal epithelial cells, and wound gap closure was observed daily. At doses of 100 and 50 μg / mL in serum-free medium, MSC secretome closes the wound within 72 hours. Shown is the image of the wound area stained with gentian violet, imaged 2 days after wounding. [Figure 24] Stability and Bioactivity of MSC Secretome. New batches of MSC secretome were manufactured and the stability of the drug substance (in formulation buffer) was evaluated. Aliquots were stored at -20°C, 4°C, or room temperature as part of a 7-day or 14-day program. At the end of the study, samples were evaluated in a circular wound assay using corneal epithelial cells. Wound closure was observed daily for 3 days. Snap-frozen drug substance at the time of manufacture served as the reference standard. Designated wound closure rate (%WC). MSC secretome maintains wound-healing bioactivity in vitro for at least 14 days when stored refrigerated at -20°C, 4°C, and at room temperature (RT). Stability was observed in both corneal epithelial cells and fibroblasts. [Figure 25]3D tissue model. 24 hours after injury, injured tissue exhibits 10% of control TEER (compared to untreated tissue), while tissue treated with MSC secretome after injury exhibits 55% of control TEER (compared to untreated tissue). The purpose of this study was to utilize the EpiCorneal tissue model (MatTekCorp) to evaluate the effect of topical application of a test product (MSC secretome) on barrier integrity following corneal epithelial injury caused by topical exposure to nitrogen mustard (NM). MSC secretome was applied topically at 6 μg / ml (diluted in placebo solution). EpiCorneal tissue was cultured in 5 ml of medium under standard culture conditions for 24 hours. DETAILED DESCRIPTION OF THE INVENTION

[0147] I. Introduction The present invention provides mesenchymal stem cell secretome compositions for use in such treatments, as well as methods for making such compositions, which compositions, uses, and related methods are described in further detail below.

[0148] A.Definition

[0149] Terms used in the claims and specification are defined as set forth below unless otherwise stated. In the event of a direct conflict with a term used in the parent provisional patent application, the term used herein shall control.

[0150] As used herein, "isolated" means material that has been removed from its original environment and thus altered from its natural state "by the hand of man."

[0151] As used herein, "enrichment" means selectively concentrating or increasing the amount of one or more materials by eliminating unwanted materials or by selecting and separating desirable materials from a mixture (e.g., separating cells having a particular cell marker from a heterogeneous cell population where not all cells in the population express the marker).

[0152] As used herein, the term "substantially purified" refers to a population of cells that is substantially homogeneous for a particular marker or combination of markers. Substantially homogeneous means at least 90%, preferably 95%, homogeneous for a particular marker or combination of markers. As used herein, the term "pluripotent stem cells" refers to true stem cells that can differentiate into only a limited number of types. For example, bone marrow contains pluripotent stem cells that generate all the cells of the blood but may not be able to differentiate into other types of cells.

[0153] The term "animal-free" when referring to certain compositions, growth conditions, media, etc. described herein means that no non-human animal-derived materials, such as bovine serum, proteins, lipids, carbohydrates, nucleic acids, vitamins, etc., are used in preparing, growing, culturing, expanding, storing, or formulating a particular composition or process. "Free of non-human animal-derived materials" means that the material has not come into contact with or been in contact with the body or materials of a non-human animal and is free of xenocontamination. Generally, clinical-grade materials, such as recombinantly produced human proteins, are used in preparing, growing, culturing, expanding, storing, and / or formulating such compositions and / or processes.

[0154] The term "expanded," with respect to a cell composition, means that the cell population comprises a significantly higher concentration of cells than that obtained using previous methods. For example, the cell level per gram of amniotic tissue in an AMP cell expansion composition is at least 50-fold and up to 150-fold higher than the number of cells in a primary culture after five passages, compared to an approximately 20-fold doubling of such cells using previous methods. In another example, the cell level per gram of amniotic tissue in an AMP cell expansion composition is at least 30-fold and up to 100-fold higher than the number of cells in a primary culture after three passages. Thus, an "expanded" population has at least a two-fold and up to a ten-fold improvement in the number of cells per gram of amniotic tissue compared to previous methods. The term "expanded" is intended to include only situations in which human intervention has been used to increase the number of cells.

[0155] As used herein, "conditioned medium" refers to a medium in which a specific cell or cell population has been cultured and then removed. When cells are cultured in the medium, they may secrete cellular factors that may support or affect the behavior of other cells. Such factors include, but are not limited to, hormones, cytokines, extracellular matrix (ECM), proteins, vesicles, antibodies, chemokines, receptors, inhibitors, and granules. A medium containing cellular factors is a conditioned medium. An example of a method for preparing a conditioned medium is described in U.S. Patent No. 6,372,494, which is incorporated herein by reference in its entirety. As used herein, conditioned medium also refers to conditioned medium or components thereof, such as proteins, recovered and / or purified from MSC cells.

[0156] As used herein, the term "mesenchymal stem cell composition" or "MSC composition" refers to conditioned medium derived from MSCs, optionally subjected to further processing. In some embodiments, "MSC secretome" can refer to crude conditioned medium derived from MSCs. In some embodiments, "MSC secretome" can refer to a composition obtained from crude conditioned medium after it has been subjected to further processing as described herein.

[0157] As used herein, the term "suspension" refers to a liquid containing dispersed components, e.g., cytokines. The dispersed components may be fully solubilized, partially solubilized, suspended, or otherwise dispersed in the 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.

[0158] "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 encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. An amino acid analog refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon atom attached to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, or methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. An amino acid mimetic refers to a chemical compound that has a structure different from the general chemical structure of an amino acid but functions similarly to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted one-letter symbols.

[0159] An "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) with a second, different, "replacement" amino acid residue. An "amino acid insertion" refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. Insertions typically consist of the insertion of one or two amino acid residues, although larger "peptide insertions" can now be made, e.g., insertions of about 3 to about 5 or up to about 10, 15, or 20 amino acid residues. The inserted residues can be naturally occurring or non-naturally occurring, as disclosed above. An "amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

[0160] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. This term includes amino acid polymers in which one or more amino acid residues are artificial chemical analogues of a corresponding naturally occurring amino acid, as well as naturally occurring and non-naturally occurring amino acid polymers.

[0161] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence 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 substituted with 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). In the case of arginine and leucine, modifications at the second base can also be conservative. The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene. As used herein, a polynucleotide can be composed of any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. For example, a polynucleotide can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules containing DNA and RNA that can be single-stranded, or more typically double-stranded, or a mixture of single- and double-stranded regions. Furthermore, a polynucleotide can be composed of triple-stranded regions containing either RNA or DNA, or both RNA and DNA. A polynucleotide can also contain one or more modified bases or DNA or RNA backbones modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine.A variety of modifications are possible to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.

[0162] As used herein, the term "secretome composition" refers to a composition comprising one or more substances secreted by cells. In certain embodiments, a secretome composition may comprise one or more cytokines, one or more exosomes, and / or one or more microvesicles. A secretome composition may be purified or unpurified. In some embodiments, a secretome composition may further comprise one or more substances not secreted by cells (e.g., culture medium, additives, nutrients, etc.). Some secretome compositions contain only trace amounts of one or more substances not secreted by cells (e.g., culture medium, additives, nutrients, etc.), or none at all.

[0163] As used herein, the terms "treatment," "treat," or "treating" and the like include any treatment of a human or non-human mammal (e.g., rodents, cats, dogs, horses, cattle, sheep, primates, etc.), including preventing a disease or condition from occurring in a subject who may be predisposed to, but has not yet been diagnosed with, the disease or condition. It also includes suppressing (halting the onset), alleviating, or ameliorating (causing regression) the disease, condition, and / or associated symptoms, or curing (permanently halting the onset or progression) the disease, condition, and / or any associated symptoms. As used herein, the terms "treatment," "treating," or "treating" include any treatment of a mammal, particularly a human, disease or condition, including (a) preventing a disease or condition from occurring in a subject who may be predisposed to the disease or condition but has not yet been diagnosed as having it; (b) inhibiting a disease or condition, e.g., arresting its onset; (c) alleviating and / or ameliorating a disease or condition, e.g., causing regression of the disease or condition; or (d) curing a disease or condition, e.g., halting its onset or progression. The population of subjects treated by the methods of the invention includes subjects afflicted with an undesirable condition or disease as well as subjects at risk of developing a condition or disease. In some embodiments, "treatment" (also "treat" or "treating") refers to any administration of a therapy that results in partial or complete relief, improvement, reversal, suppression, delay in onset, reduction in severity, and / or reduction in the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be for subjects who do not exhibit symptoms of the associated disease, disorder, and / or condition, and / or who exhibit only early signs of the disease, disorder, and / or condition. Alternatively and / or in addition, such treatment may be for subjects who exhibit one or more established signs of the associated disease, disorder, and / or condition. In some embodiments, treatment may be for subjects who have been diagnosed with the associated disease, disorder, and / or condition.In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that statistically correlate with an increased risk of developing the relevant disease, disorder, and / or condition.

[0164] As used herein, a "wound" refers to any wound that disrupts normal anatomy in any way, including, but not limited to, traumatic injuries such as mechanical (e.g., contusion, penetration), thermal, chemical, electrical, radiation, percussion, and incisional injuries; elective wounds, such as surgical procedures and resulting incisional hernias, fistulas, etc.; acute, chronic, infected, and sterile wounds; and wounds associated with pathologies (e.g., eye contusions). Wounds are dynamic, and the healing process is continuous, requiring a series of integrated and interrelated cellular processes that begin at the time of wounding and progress beyond initial wound closure to achieve stable wound closure. 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" means any form of intervention that improves the natural cellular processes and humoral substances of tissue repair such that healing is more rapid and / or the resulting healed area is less scarred and / or the healed area has tissue strength approaching that of uninjured tissue and / or the wounded tissue achieves some degree of functional recovery.

[0165] As used herein, the terms "a" or "an" mean one or more or at least one.

[0166] As used herein, a "therapeutically effective" or "effective" dose or amount of a composition is an amount sufficient to produce a positive effect on a given condition. If not immediately, a therapeutically effective or effective dose or amount produces a noticeable or measurable effect on the health and well-being of a patient over a period of time.

[0167] As used herein, "pharmaceutical composition" refers to an effective amount of the composition described herein in combination with a delivery component. The pharmaceutical composition may optionally contain other components, such as pharmaceutically suitable carriers and excipients, that can facilitate administration of the composition and / or its individual components to a subject.

[0168] The term "pharmaceutically acceptable carrier" means a carrier or diluent that does not cause significant irritation to a subject and does not abolish the biological activity and properties of the compound with which it is administered.

[0169] The term "excipient" means an inert substance added to a pharmaceutical composition to further facilitate administration of a compound.

[0170] As used herein, the terms "mixing," "mixing," and the like describe a mechanical process or treatment of the ingredients. For example, mixing can mean performing repeated cycles of pressing and folding or equivalent processing steps that result in strong compression and mixing of the provided hydrophobic matrix.

[0171] Adult stem cells can be harvested from various adult tissues, such as bone marrow, adipose tissue, and dental pulp tissue. While all adult stem cells are considered self-renewing and multipotent, their therapeutic functions vary depending on their origin. As a result, each type of adult stem cell has unique characteristics suited to specific diseases. Mesenchymal stem cells (MSCs) are non-hematopoietic (non-blood) stem cells isolated (derived) from the mesoderm, are multipotent, and can differentiate into various tissues, including osteoblasts (e.g., bone cells), chondrocytes (e.g., cartilage cells), myocytes (e.g., muscle cells), and adipocytes (e.g., adipocytes that give rise to bone marrow and adipose tissue). As used herein, "isolated" refers to cells that have been removed from their original environment. Stem cells produce factors, such as growth factors, that regulate or are important for regulating multiple biological processes. Growth factors are naturally occurring substances or agents that can stimulate cell growth and / or proliferation and / or cell differentiation. Growth factors are typically proteins or steroid hormones. As used herein, terms such as "growth factor" and "factor" are used interchangeably, however the term "biological factor" is not limited to growth factors.

[0172] Human mesenchymal stem cells (MSCs) can be characterized by a surface marker profile of CD45- / CD31- / CD73+ / CD90+ / CD105+ / CD44+ (or a suitable subset thereof) (see Bourin et al., Cytotherapy 15(6):641-648 (2013)). Furthermore, suitable stem cells exhibit CD34+ positivity upon isolation but lose this marker during culture. Thus, the complete marker profile of one stem cell type that can be used in accordance with 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, with the remaining markers remaining the same to define what appears to be homologous to the human cells described above.

[0173] The phrase "conditioned medium" or "CM" refers to a medium containing biological factors secreted by MSCs. This may also be referred to herein as "secretome," "MSC-CM," "MSC secretome," and / or "MSC-derived secretome." Also provided is a processed "conditioned medium" that contains biological factors secreted by MSCs and has been further processed, for example, by filtration, purification, and / or concentration procedures. Conditioned medium is obtained by culturing stem cells in a medium, as described in detail herein, and separating the resulting medium containing stem cells and their secreted stem cell products (secretome) into a conditioned medium containing biological factors and fewer stem cells than were present prior to separation. Conditioned medium can be used in the methods described herein and may be substantially free of stem cells (although it may contain a small percentage of stem cells) or free of stem cells. Biological factors that may be present in 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 can be bound to the interior or surface of extracellular vesicles (e.g., exosomes) or separated from the extracellular vesicles. Compositions and Formulations

[0174] According to the present specification, provided herein are compositions comprising mesenchymal stem cell (MSC) secretome and / or conditioned medium comprising mesenchymal stem cell (MSC) secretome (including processed MSC secretome).

[0175] In some embodiments, the MSC secretome is generally low in angiogenic factors. In some embodiments, the MSC secretome does not promote angiogenesis. In some embodiments, the MSC secretome exhibits anti-angiogenic properties. In some embodiments, the MSC secretome provides reduced angiogenesis compared to other secretomes. In some embodiments, the MSC secretome provides a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% reduction in angiogenesis. In some embodiments, the MSC secretome provides a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% reduction in angiogenesis compared to another secretome. In some embodiments, the MSC secretome provides a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% reduction in angiogenesis compared to conditioned medium prior to treatment with the MSC secretome. In some embodiments, the MSC secretome has low angiogenic induction. In some embodiments, the MSC secretome has a reduced angiogenic response. In some embodiments, the MSC secretome has reduced angiogenic potential. In some embodiments, the MSC secretome impairs and / or reduces the normal formation of blood vessels in the presence of an angiogenesis-supportive medium. In some embodiments, the MSC secretome has reduced angiogenic potential when compared to an untreated control MSC secretome. In some embodiments, the MSC secretome has reduced angiogenic potential compared to a sample treated with serum-containing medium. In some embodiments, the MSC secretome attenuates the angiogenic response. In some embodiments, the MSC secretome reduces the angiogenic response induced by serum-containing medium. In some embodiments, the reduced angiogenic response is induced by the MSC secretome when secretome plus serum-containing medium (reduced or no angiogenic response) is compared to serum-containing medium (an 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.

[0176] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises: i. IDO (indoleamine-2,3-dioxygenase) enzyme activity, 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 (serpin E1), VEGF-A, and b-NGF; iii. a "threshold" ppm level of at least one additional factor selected from the group consisting of sFLT-1, PEDF (serpin F1), IGFBP-2, IGFBP-3, SDF-1, TSG-14, Kallikrein3, MCP-1, bFGF, angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, PDGF, SOD1, SOD2, SOD3, and HO-1; iv. 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.

[0177] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises: i. an IDO (indoleamine-2,3-dioxygenase) enzyme activity of less than about 250 μM; ii. at least one trophic factor / cytokine selected from the group consisting of HGF, FGF-7, TIMP-1, TIMP-2, PAI-1 (serpin E1), VEGF-A, and / or b-NGF; iii. at least one additional factor selected from the group consisting of sFLT-1, PEDF (serpin F1), IGFBP-2, IGFBP-3, SDF-1, TSG-14, Kallikrein3, MCP-1, bFGF, Angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, PDGF, SOD1, SOD2, SOD3, and / or HO-1; and iv. At least one additional factor selected from the group consisting of DPPIV (dipeptidyl peptidase-4), uPA, angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, and / or thrombospondin-1.

[0178] A mesenchymal stem cell (MSC) secretome composition, the composition comprising: i. at least one trophic factor / cytokine selected from the group consisting of HGF, TIMP-1, TIMP-2, PAI-1 (serpin E1), VEGF-A, and b-NGF; ii. PEDF (serpin F1), IGFBP-2, IGFBP-3, SDF-1, TSG-14, Kallikrein3, MCP-1, Angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, and PDGF; iii. At least one 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.

[0179] 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, C-reactive protein, cystatin C, DKK-1, EMMPRIN, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15, and / or IFNγ. 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, C-reactive protein, cystatin C, DKK-1, EMMPRIN, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15, and IFNγ.

[0180] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor including, but not limited to, a serpin family member, including a serine protease inhibitor: serpin F1, serpin E1, serpin A1, serpin G1, serpin H1, serpin B6, serpin E2, serpin A3, serpin C1, serpin F2, serpin I1, serpin B1, serpin B7, serpin D1, serpin B3, serpin B8, serpin B2, serpin B12, serpin A7, serpin A4, and / or serpin A6. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor including, but not limited to, serpin F1 (also known as PEDF), serpin E1, and serpin A1. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises serpin F1 (also known as PEDF). In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises serpin E1. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises serpin A1.

[0181] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor, including but not limited to, a protein involved in antioxidant activity. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor, including but not limited to, catalase, protein disulfide isomerase, protein disulfide isomerase A3, protein disulfide isomerase A4, protein disulfide isomerase A6, peroxiredoxin-6, peroxiredoxin-1, peroxiredoxin-2, and / or peroxiredoxin-4. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises catalase. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises protein disulfide isomerase. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises protein disulfide isomerase A3. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises protein disulfide isomerase A4. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises protein disulfide isomerase A6. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises peroxiredoxin-6. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises peroxiredoxin-1. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises peroxiredoxin-2. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises peroxiredoxin-4.

[0182] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor, including but not limited to, matrix metalloproteinase. 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.

[0183] 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-inducible gene 6 protein (also known as TSG-6), serum albumin, and latent transforming growth factor binding protein (LTGFBP-1). Various isoforms include 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-inducible 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.

[0184] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises pentraxin-3, TIMP-1, serpin E1, TSP-1, and HGF.

[0185] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises 2-16 ng / mL, or 9.8±0.5 ng / ml pentraxin-3.

[0186] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises 10-200 ng / mL, or 90±21.5 ng / ml, of TIMP-1.

[0187] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises 10-100 ng / mL, or 49.2±9.8 ng / ml of Serpin E1.

[0188] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises 0.1-10 ng / mL, or 2.0±0.3 ng / mL, of HGF.

[0189] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises 100-800 pg / mL, or 304±44 pg / ml, of VEGF.

[0190] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises 0.1-100 pg / mL, or <1 ng / ml, of IL-8.

[0191] 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 between 0 μM and about 250 μM. In some embodiments, the IDO (indoleamine-2,3-dioxygenase) enzyme activity is between 50 μM and about 250 μM L-kynurenine / million MSCs. In some embodiments, the IDO (indoleamine-2,3-dioxygenase) enzyme activity is between 50 μM and about 200 μM L-kynurenine / million MSCs. In some embodiments, the IDO (indoleamine-2,3-dioxygenase) enzyme activity is between 100 μM and about 250 μM L-kynurenine / million MSCs. In some embodiments, the IDO (indoleamine-2,3-dioxygenase) enzyme activity is between 100 μM and 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 / million MSCs.

[0192] In some embodiments, the MSC secretome further comprises a "threshold" ppm level for at least one additional factor, including but not limited to, sFLT-1, PEDF (serpin F1), IGFBP-2, IGFBP-3, SDF-1, TSG-14, kallikrein 3, MCP-1, bFGF, angiogenin, MCP-2, angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, PDGF, SOD1, SOD2, SOD3, and / or HO-1. In some embodiments, the MSC secretome further comprises a "threshold" ppm level for at least one additional factor selected from the group consisting of sFLT-1, PEDF (serpin F1), IGFBP-2, IGFBP-3, SDF-1, TSG-14, kallikrein 3, MCP-1, bFGF, angiogenin, MCP-2, angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, PDGF, SOD1, SOD2, SOD3, and HO-1. In some embodiments, the MSC secretome further comprises one additional factor at a concentration range of 200 pg / mL to 5000 pg / mL, including, but not limited to, sFLT-1, PEDF (serpin F1), IGFBP-2, IGFBP-3, SDF-1, TSG-14, kallikrein 3, MCP-1, bFGF, angiogenin, MCP-2, angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, PDGF, SOD1, SOD2, SOD3, and / or HO-1. In some embodiments, the MSC secretome further comprises 1000 to 3000 pg / mL of sFLT-1. In some embodiments, the MSC secretome further comprises 400 to 800 pg / mL of TSG-6.

[0193] 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 to 275 ng / mL of PEDF. In some embodiments, the MSC secretome further comprises 225 to 275 ng / mL of PEDF. In some embodiments, the MSC secretome further comprises 150 to 300 ng / mL of PEDF. In some embodiments, the MSC secretome further comprises 273±27 ng / mL of PEDF.

[0194] In some embodiments, the 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, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome further comprises "higher" levels of serpin E1. In some embodiments, the MSC secretome further comprises "higher" levels of serpin A1. In some embodiments, the MSC secretome further comprises "higher" levels of TIMP-1. In some embodiments, the MSC secretome further comprises "higher" levels of thrombospondin-1. In some embodiments, the MSC secretome further comprises "higher" levels of pentraxin-3 (TSG-14). In some embodiments, the MSC secretome further comprises "higher" levels of platelet factor 4. In some embodiments, the MSC secretome further comprises "higher" levels 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, pentraxin-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, pentraxin-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, pentraxin-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, pentraxin-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, pentraxin-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, pentraxin-3 (TSG-14), platelet factor 5, 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, pentraxin-3 (TSG-14), platelet factor 6, 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, pentraxin-3 (TSG-14), platelet factor 2, and serpin F1.

[0195] In some embodiments, the MSC secretome composition further comprises a "moderate" 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 "moderate" 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, angiogenin, DPPIV, IGFBP-3, and uPA. In some embodiments, the MSC secretome composition further comprises a "moderate" level 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 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 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 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, angiopoietin, 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 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 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.

[0196] In some embodiments, the MSC secretome further comprises "low" levels 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.

[0197] In some embodiments, the MSC secretome further comprises about 90 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 100 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 125 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 150 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises about 175 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises between 1 pg / mL and about 400 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises between 10 pg / mL and about 400 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises between 50 pg / mL and about 350 pg / mL of VEGF. In some embodiments, the MSC secretome further comprises 50 pg / mL to about 300 pg / 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 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.

[0198] 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-fold 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 E1. In some embodiments of the MSC secretome composition, the level of VEGF is 10-fold lower than the level of serpin E1.

[0199] In some embodiments, the MSC secretome composition is free of and / or contains very low levels of bFGF, PLGF, and PDGF. In some embodiments, the MSC secretome composition contains less than about 200 pg / mL, less than about 150 pg / mL, less than about 100 pg / mL, less than about 75 pg / mL, less than about 50 pg / mL, or less than about 25 pg / mL of bFGF, PLGF, and / or PDGF. In some embodiments, the MSC secretome composition contains less than about 200 pg / mL, less than about 150 pg / mL, less than about 100 pg / mL, less than about 75 pg / mL, less than about 50 pg / mL, or less than about 25 pg / mL of bFGF, PLGF, and PDGF. In some embodiments, the MSC secretome composition is free of bFGF, PLGF, and / or PDGF. In some embodiments, the MSC secretome composition is free of bFGF, PLGF, and / or 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.

[0200] In some embodiments, the MSC secretome composition comprises apolipoprotein A1, complement factor D, C-reactive protein, cystatin C, DKK-1, EMMPRIN, osteopontin, vitamin DBP, MIF, RANTES, uPAR, IL-17a, GDF-15, and / or IFNγ.

[0201] In some embodiments, the 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, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 1 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 1 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 1 ng / mL to 200 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 1 ng / mL to 100 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 10 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 10 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 10 ng / mL to 200 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 10 ng / mL to 100 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 20 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 20 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 20 ng / mL to 200 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 20 ng / mL to 100 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 30 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 30 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 30 ng / mL to 200 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 30 ng / mL to 100 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 30 ng / mL to 100 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 40 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 40 ng / mL to 200 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 40 ng / mL to 100 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 50 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 50 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 50 ng / mL to 200 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 50 ng / mL to 100 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 60 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 60 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 60 ng / mL to 200 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 60 ng / mL to 100 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 70 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 70 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 70 ng / mL to 200 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 70 ng / mL to 100 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 80 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 80 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 80 ng / mL to 200 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 80 ng / mL to 100 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 90 ng / mL to 400 ng / mL of serpin E1, serpin A1, TIMP-1. In some embodiments, the MSC secretome composition comprises at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 90 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 90 ng / mL to 200 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 90 ng / mL to 100 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 100 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 100 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 100 ng / mL to 200 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 110 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 110 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 110 ng / mL to 200 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 120 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 120 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 120 ng / mL to 200 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 130 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 130 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 130 ng / mL to 200 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 140 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 140 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 140 ng / mL to 200 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 150 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 150 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 150 ng / mL to 200 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 160 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 160 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 1560 ng / mL to 200 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 170 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 170 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 170 ng / mL to 200 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 180 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 180 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 180 ng / mL to 200 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 190 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 190 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 190 ng / mL to 200 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 200 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 200 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 210 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 210 ng / mL to 300 ng / mL of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 220 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 220 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 230 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 230 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 240 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 240 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 250 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 250 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 260 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 260 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 270 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 270 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 280 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 280 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 290 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 290 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 310 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 320 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 330 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 340 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 350 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 360 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 370 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 380 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 1390 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 10 ng / mL to 90 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 10 ng / mL to 80 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1.In some embodiments, the MSC secretome composition comprises 20 ng / mL to 100 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 30 ng / mL to 100 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 40 ng / mL to 100 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 50 ng / mL to 100 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 10 ng / mL to 70 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 10 ng / mL to 60 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition comprises 10 ng / mL to 50 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1.

[0202] In some embodiments, the MSC secretome composition comprises: i. 0.3 to 4.5 ng / mL HGF and ii. 0.5 to 20 ng / mL of pentraxin-3 (TSG-14); iii. 100-600pg / mL VEGF; iv. 10 to 200 ng / mL TIMP-1 and v. 20-80ng / mL serpin E1 and vi. <5ng / mL IL-8;

[0203] In some embodiments, the MSC secretome composition comprises: i. 1.5 to 3.5 ng / mL HGF; ii. 5 to 15 ng / mL of pentraxin-3 (TSG-14); iii. 200-400pg / mL VEGF; iv. 50-200ng / mL TIMP-1 and v. 30-70ng / mL serpin E1 and vi. <3ng / mL IL-8;

[0204] In some embodiments, the MSC secretome composition comprises: i. 1.5 to 2.5 ng / mL HGF; ii. 8-12 ng / mL of pentraxin-3 (TSG-14); iii. 250-350pg / mL VEGF; iv. 70-110ng / mL TIMP-1 and v. 30-70ng / mL serpin E1 and vi. <2ng / mL IL-8,

[0205] In some embodiments, the MSC secretome composition comprises: i. 2.0 ± 0.3 ng / mL HGF; ii. 9.8 ± 0.5 ng / mL pentraxin-3 (TSG-14); iii. VEGF of 304±44pg / mL; iv. 90±20ng / mL TIMP-1; v. 49.2 ± 10 ng / mL of serpin E1 and vi. <1 ng / mL IL-8,

[0206] 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.

[0207] 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, or 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.

[0208] In some embodiments, the MSC secretome composition does not contain certain components. In some embodiments, the MSC secretome composition does not contain certain components found in cell culture media. In some embodiments, the MSC secretome composition does not contain one or more components selected from the group consisting of xenobiotic components (e.g., animal serum); phenol red; peptides and biomolecules <3 kDa; antibiotics; protein aggregates (e.g., protein aggregates >200 nm); cells; cellular debris (cellular debris does not include exosomes / extracellular vesicles (EVs); e.g., non-exosome, non-EV cellular debris); hormones (e.g., hormones include, but are not limited to, insulin and / or hydrocortisone); and / or L-glutamine. In some embodiments, the MSC secretome composition does not contain xenobiotic components. In some embodiments, the MSC secretome composition does not contain phenol red. In some embodiments, the MSC secretome composition does not contain peptides and biomolecules less than 3 kDa. In some embodiments, the MSC secretome composition does not contain antibiotics. In some embodiments, the MSC secretome composition does not contain protein aggregates (e.g., protein aggregates greater than 200 nm). In some embodiments, the MSC secretome composition does not contain cells. In some embodiments, the MSC secretome composition does not contain cellular debris (cellular debris does not include exosomes / EVs; e.g., non-exosome, non-EV cellular debris). In some embodiments, the MSC secretome composition does not contain hormones (e.g., hormones include, but are not limited to, insulin and / or hydrocortisone). In some embodiments, the MSC secretome composition does not contain L-glutamine.

[0209] 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 sodium monophosphate / diphosphate, mannitol, and trehalose, and the composition has a pH of about pH 7.4.

[0210] In some embodiments, the MSC secretome composition can 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 can 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 can comprise one or more additional agents selected from the group consisting of glycine and glycerol, and dextrose. In some embodiments, the MSC secretome composition can comprise one or more additional agents selected from the group consisting of sodium chloride and potassium chloride.

[0211] 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, di / monosodium phosphate, sodium citrate / citric acid, citric 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 di / 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 di / 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 citrate / disodium phosphate buffer system.

[0212] 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, and the composition has a pH of about pH 6.4.

[0213] In some embodiments, the MSC secretome composition further comprises a tonicity adjuster or tonicity modifier. In some embodiments, the tonicity adjuster or tonicity modifier includes, but is not limited to, NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and / or glycerin. In some embodiments, the tonicity adjuster or tonicity modifier is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and / or glycerin.

[0214] In some embodiments, the MSC secretome composition further comprises an adhesive, including, but not limited to, hypromellose, poloxamer 407, poloxamer 188, poloxomer 237, poloxomer 338, hypromellose (HPMC), HEC, polycarbophil, polyvinylpyrrolidone (PVP), PVA (polyvinyl alcohol), polyimide, sodium hyaluronate, gellan gum, poly(lactic-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethylcellulose (CMC), or hydroxypropylmethylcellulose (HPMC), hydroxymethylcellulose, hydroxyethylcellulose, sodium carboxymethylcellulose, fibrin glue, polyethylene glycol, and GelCORE. In some embodiments, the adhesive is hypromellose. In some embodiments, the adhesive is fibrin glue. In some embodiments, the adhesive is polyethylene glycol. In some embodiments, the adhesive is GelCORE (see Sani, et al., Science Advances, Vol. 5, no. 3 (2019)).

[0215] In some embodiments, the MSC secretome composition comprises (a) a processed conditioned medium containing an MSC secretome generated by any one of the methods described herein, and (b) a polymer. In some embodiments, the MSC secretome composition comprises a conditioned medium containing an MSC secretome generated as described herein and a polymer. In some embodiments, the MSC secretome composition comprises a processed conditioned medium containing an MSC secretome generated 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 allows for sustained (slow) release of the MSC secretome components.

[0216] In some embodiments, the MSC secretome compositions provided herein are in the form of a therapeutic bandage (e.g., a polymer impregnated with the MSC secretome composition). The therapeutic bandage can be configured as needed depending on the application. In some embodiments, the bandage is a form or patch, or configured as a mesh.

[0217] In some embodiments, the MSC secretome composition exhibits biopenetrance, e.g., ocular penetration, corneal penetration, and / or corneal penetration. In some embodiments, the MSC secretome composition exhibits the ability to be absorbed by the eye. In some embodiments, the MSC secretome composition exhibits inherent biopenetrance. In some embodiments, the MSC secretome composition exhibits excipient-compatible biopenetrance. In some embodiments, the MSC secretome composition exhibits biopenetrance due to upregulation of smaller factors. In some embodiments, the MSC secretome composition exhibits biopenetrance due to the presence of a biopreservative. In some embodiments, the MSC secretome composition exhibits biopenetrance due to the presence of the biopreservative benzalkonium chloride.

[0218] In some embodiments, the MSC secretome compositions exhibit a longer half-life and / or have increased stability compared to other treatments. In some embodiments, the MSC secretome compositions provided herein allow for the upregulation of proteins that allow for increased stability of the MSC secretome. In some embodiments, the MSC secretome compositions provided herein allow for the upregulation of chaperone proteins to improve the stability of other proteins in the MSC secretome.

[0219] In some embodiments, the MSC secretome composition exhibits ultra-potency when administered to a subject in need thereof, hi some embodiments, the MSC secretome composition enables a therapeutic effect with a single drop or single administration per day.

[0220] C. Production / Manufacturing Method According to the present invention, the conditioned medium (and thus the mesenchymal stem cell-secreted factors) can be obtained from mesenchymal stem cells obtained from the patient or individual to be treated (the patient in need thereof) or from another (donor) individual, i.e., from a young and / or healthy donor and / or commercially obtained mesenchymal stem cells. For example, MSCs obtained from the individual to be treated (autologous stem cells) or from a donor (allogeneic stem cells) can be used to generate the conditioned medium described herein, which can then be further processed into an MSC secretome composition as described. In some embodiments, MSCs can also be obtained from commercial suppliers. In some embodiments, commercially obtained MSCs can be used in MSC secretome production.

[0221] According to the present invention, a method for producing an anti-angiogenic mesenchymal stem cell (MSC) secretome composition comprises: i. culturing mesenchymal stem cells (MSCs) in a first medium; ii. removing the initial medium from step (i) from the MSCs; iii. Washing the MSCs of step (ii); iv. Add the second medium and incubate for approximately 1 to 5 days; v. harvesting the second medium from step (iv) as conditioned medium; vi. Processing the conditioned medium of step (v) into an MSC secretome composition as described herein.

[0222] In some embodiments, the culturing can be performed using a bioreactor system for culturing cells. In some embodiments, the culturing can be performed using a bioreactor system for culturing stem cells. In some embodiments, the culturing can be performed using a bioreactor system for culturing mesenchymal stem cells. In some embodiments, the culturing can be performed using a media mixing technique. In some embodiments, the culturing can be performed using a PBSVerticalWheel™ mixing technique.

[0223] In some embodiments, in step (iv), processing the conditioned medium of step (v) into a secretome composition comprises: a) filtering the conditioned medium collected from step (v) to remove cell particles; b) concentrating the filtered conditioned medium from step (a); and c) buffer exchanging with the formulation buffer.

[0224] In some embodiments, step c) comprises buffer exchanging with a buffer system selected from the group consisting of di / monosodium phosphate, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and citric acid / disodium phosphate.

[0225] In some embodiments, the filtering step (a) comprises the use of a 0.45 μm filter, a 0.22 μm filter, a 0.8 μm filter, and a 0.65 micron, low protein binding PVDF membrane, and / or PES (polyethersulfone). In some embodiments, the filtering step (a) comprises the use of a 0.45 μm filter. In some embodiments, the filtering step (a) comprises the use of a 0.22 μm filter. In some embodiments, the filtering step (a) comprises the use of a 0.8 μm filter. In some embodiments, the filtering step (a) comprises the use of a 0.65 micron. In some embodiments, the filtering step (a) comprises the use of a low protein binding PVDF membrane. In some embodiments, the filtering step (a) comprises the use of PES (polyethersulfone).

[0226] In some embodiments, the concentrating step (b) comprises using a hollow fiber filter, a tangential flow filtration system, or a centrifugation-based size exclusion technique. In some embodiments, the concentrating step (b) comprises using a hollow fiber filter technique. In some embodiments, the concentrating step (b) comprises using a tangential flow filtration system. In some embodiments, the concentrating step (b) comprises using a centrifugation-based size exclusion technique.

[0227] In some embodiments, the centrifugation-based size exclusion technique uses a MW cutoff of 3 to 10 kDa. In some embodiments, the centrifugation-based size exclusion technique uses a MW cutoff of at least 3 kDa, at least 4 kDa, at least 5 kDa, at least 6 kDa, at least 7 kDa, at least 8 kDa, at least 9 kDa, at least 10 kDa, at least 11 kDa, at least 12 kDa, at least 13 kDa, at least 14 kDa, at least 15 kDa, at least 16 kDa. At least a 17 kDa MW cutoff, at least an 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 28 kDa MW cutoff, at least a 29 kDa MW cutoff, and / or at least a 30 kDa MW cutoff may be used.

[0228] In some embodiments, the method produces an MSC secretome composition and / or formulation as described hereinabove. In some embodiments, the first and / or second culture medium is MSC medium and / or MSC-XF.

[0229] MSCs, or cells differentiated from MSCs, can be engineered to produce conditioned medium containing a desired secretome, e.g., containing desired cytokines and / or desired therapeutic properties as described herein. For example, secretomes can be produced from MSCs of superdonor cell lines. Secretomes can also be produced from commercially obtained MSCs. In upcoming embodiments, allogeneic MSCs (and / or cells derived therefrom) and / or allogeneic MSC-derived secretome compositions can be prepared and stored for large groups of individuals. Allogeneic MSCs (and / or cells derived therefrom) and / or MSC-derived secretome compositions can be prepared in advance so that they are ready when people need them. In certain embodiments, MSCs (and / or cells derived therefrom) and / or MSC-derived secretome compositions can be processed to produce more concentrated solutions or compositions (e.g., mesenchymal stem cell-derived secretome compositions or MSC secretome compositions described herein).

[0230] In some embodiments, the initial culture medium and the first culture medium are different. In some embodiments, the initial medium and the first medium are the same. Non-limiting examples of cell culture media or media useful for culturing MSCs to produce conditioned medium comprising an MSC secretome according to the present invention include hMSC Media Booster XFM, hMSC High Performance Basal Media, Minimum Essential Medium Eagle (MEME), ADC-1, LPM (Bovine Serum Albumin-free), F10 (HAM), F12 (HAM), DCCM1, DCCM2, RPMI 1640, BGJ Medium (with and without Fitton-Jackson Modification), StemPro, MSCGro, MesenCult, NutriStem, Basal Medium Eagle (BME - with the addition of 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, and Medium. Examples include M199 (M199E-with Earle's salt base), Medium M199 (M199H-with Hank's salt base), Minimum Essential Medium Alpha (MEM-alpha), Minimum Essential Medium Eagle (MEM-E-with Earle's salt base), Minimum Essential Medium Eagle (MEM-H-with Hank's salt base), and Minimum Essential Medium Eagle (MEM-with NAA-lacking amino acids), but also include Medium 199, CMRL1415, CMRL1969, CMRL1066, NCTC135, MB75261, MAB8713, DM145, Williams'G, Neuman & Tytell, Higuchi, MCDB301, MCDB202, MCDB501, MCDB401, MCDB411, MDBC153, among others.A preferred medium for use in the present invention is MEM-alpha. These and other useful media are available from, among others, GIBCO (Grand Island, NY, USA) and Biological Industries (BetHaEmek, Israel). Many of these media are summarized in Enzymology, Volume LVIII, "Cell Culture," pp. 6272, edited by William B. Jakoby and Ira H. Pastan, published by Academic Press, Inc.

[0231] In some embodiments, the cell culture medium for mesenchymal stem cells can be serum-free. In some embodiments, the cell culture medium for mesenchymal stem cells can be supplemented with serum. In some embodiments, the cell culture medium for mesenchymal stem cells can be supplemented with human platelet lysate. In some embodiments, the serum can include fetal bovine serum (FBS). In some embodiments, the cell culture medium for mesenchymal stem cells can be supplemented with serum, such as fetal serum from bovine or other species. In some embodiments, the cell culture medium for mesenchymal stem cells can be supplemented with other components 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.

[0232] In some embodiments, the percentage of oxygen is varied to promote cell growth and / or cell health. In some embodiments, the oxygen is in an amount of 5%, 10%, 15%, 20%, or 25% to promote cell growth and / or cell health. In some embodiments, the mesenchymal stem cells are grown under partial oxygen tension to promote cell growth and / or cell health. In some embodiments, the mesenchymal stem cells are grown under a low oxygen tension environment to promote cell growth and / or cell health.

[0233] In one aspect, the present invention is directed to conditioned medium (CM) containing biological factors secreted by mesenchymal stem cells, which may be referred to as conditioned medium containing MSC secretome. The conditioned medium can be obtained by culturing mesenchymal stem cells in a medium as described herein and separating the resulting medium containing mesenchymal stem cells and their secreted mesenchymal stem cell products (referred to as biological factors and / or secretome) into component portions: the secretome grown in the conditioned medium and the conditioned medium containing mesenchymal stem cells. The separated conditioned medium contains the mesenchymal stem cell secretome and can be further processed and / or used according to the methods described herein, and is substantially free of mesenchymal stem cells (which may contain small amounts of stem cells and / or trace stem cells) or free of mesenchymal stem cells. The MSC secretome is composed of various biological factors, such as hormones, cytokines, extracellular matrix, proteins, vesicles, antibodies, chemokines, receptors, inhibitors, and granules. As described herein, the conditioned medium or media containing the MSC secretome (CM or MSC secretome-containing conditioned medium) can be further processed to generate concentrated conditioned medium (pCM or concentrated MSC secretome).

[0234] In some embodiments, conditioned medium containing MSC secretome or enriched MSC secretome is produced by culturing mesenchymal stem cells in a culture medium that replaces the culture medium in which the mesenchymal stem cells were cultured. In some embodiments, the resulting conditioned medium containing MSC secretome is harvested (collected) and then processed to produce enriched MSC secretome. In certain embodiments, processing the harvested conditioned medium containing MSC secretome involves removing some, most, or essentially all of the medium, or removing some, most, or essentially all of selected components of the conditioned medium.

[0235] In some embodiments, the harvested conditioned medium containing the MSC secretome is filtered to produce an enriched MSC secretome. In some embodiments, the harvested conditioned medium containing the MSC secretome is ultrafiltered to produce an enriched MSC secretome.

[0236] In one aspect, provided herein is a method of producing a processed conditioned medium, the method comprising: (a) culturing stem cells in a cell culture medium, thereby producing a conditioned medium containing factors secreted by mesenchymal stem cells (e.g., a conditioned medium containing mesenchymal stem cells); (b) harvesting the conditioned medium, thereby producing a harvested conditioned medium (e.g., a harvested mesenchymal stem cell secretome); and (c) filtering the harvested conditioned medium (e.g., a harvested mesenchymal stem cell secretome) to produce a processed conditioned medium (mesenchymal stem cell secretome). In some embodiments, the stem cells of (a) are cultured in a proliferation medium before being cultured in a medium that does not contain growth factors. 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 the mesenchymal stem cell secretome; and (d) filtering the harvested conditioned medium to produce a processed conditioned medium comprising the mesenchymal stem cell secretome.

[0237] In some embodiments, the stem cells are mesenchymal stem cells. Mesenchymal stem cells (MSCs) are multipotent (capable of differentiating into multiple, but not all, cell lineages) non-hematopoietic (non-blood) stem cells isolated from (or derived from) various adult tissues, including bone marrow and adipose tissue. In certain embodiments, mesenchymal stem cells are isolated from bone marrow. "Isolated" refers to cells removed from their original environment. MSCs can differentiate into mesodermal cells, such as adipocytes, osteoblasts, and chondrocytes. MSCs have long, thin, small cell bodies with few cellular processes. The cell bodies contain large, round nuclei with prominent nucleoli, surrounded by finely dispersed chromatin particles, giving the nucleus a distinct appearance. The remainder of the cell body contains a small amount of Golgi apparatus, rough endoplasmic reticulum, mitochondria, and polysomes. The long, thin cells are widely dispersed, and the adjacent extracellular matrix contains 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 marker (protein molecules characteristic of the plasma membrane of a cell or cell type) profile: bone morphogenetic protein receptor "1" (BMPR+), CD34 + Scal + ;Lin”;CD44 + ;c-kit + ;Sca-1 + ;Thy-1 + ;NOTCH3;JAG1;ITGA11. MSCs express other cell type-specific markers (stemcells.nih.gov; Kaltz, et al. 2010 Exp Cell Res Oct1;316(16):2609-17, incorporated herein by reference). The MSCs described herein can be identified based on colony-forming unit assays to detect the pluripotent differentiation potential of MSCs (which cell types MSCs can give rise to). However, some differentiated cells (progenitor cells) can also be used.

[0238] i.MSC secretome - processing In some embodiments, the conditioned medium containing the MSC secretome described herein can be collected and filtered and / or purified to remove cell particles and / or other harmful components. For example, as described in step (v) above, the second culture medium from step (iv) is harvested as the conditioned medium. The filtration membrane used herein can be selected from any known in the art with an appropriate membrane and configuration to retain the desired MSC secretome components while allowing cell particles and / or other harmful components to pass through. Therefore, any suitable membrane can be used that allows cell retention under the selected hydrodynamic conditions while allowing harmful components to pass through for removal. In some embodiments, an upper pore size limit of approximately 5 microns and a lower pore size limit of approximately 0.1 microns would be appropriate. In some embodiments, filtration can be performed using a micropore filter. In some embodiments, filtration can be performed using a 0.5 μm to 0.2 μm filter. In some embodiments, filtration can be performed using 0.5 μm, 0.45 μm, 0.4 μm, 0.35 μm, 0.3 μm, 0.25 μm, 0.22 μm, and / or 0.2 μm filters. In some embodiments, filtration can be performed using a 0.45 μm filter. In some embodiments, filtration can be performed using a 0.22 μm filter. In some embodiments, filtration / purification can be performed using a low protein binding polyvinylidene fluoride (PVDF) membrane. In some embodiments, filtration / purification can be performed using polyethersulfone (PES).

[0239] In some embodiments, filtration is by ultrafiltration. In some embodiments, the conditioned medium is filtered using a filter size of 3 kD (to achieve purification, desalting, and concentration in the processed conditioned medium of molecules larger than the filter size). In some embodiments, a filter size of less than 3 kD is used to filter the conditioned medium, while in other embodiments, a filter size of more than 3 kD is used depending on the application for which the processed conditioned medium will be used. In other embodiments, ultrafiltration of the harvested conditioned medium is performed using filters of different pore sizes (e.g., 2 kD, less than 2 kD, or more than 2 kD), selected to determine the size of the components of the resulting processed conditioned medium, including the MSC secretome.

[0240] In some embodiments, harmful components in the growth support medium are removed by medium exchange, preferably via "cross-flow filtration." Cross-flow filtration refers to a filtration mode in which a suspension of MSC secretome cells flows substantially parallel to a filter that permeates components of the suspension other than the cells. Cross-flow filtration processes are 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 depend on the structure of the filtration system, flow conditions, and fluid properties. Such cross-flow processes, in some embodiments, can also include hollow fiber filtration systems. See, e.g., U.S. Patent No. 5,053,334, incorporated herein by reference in its entirety.

[0241] In some embodiments, the MSC secretome may be further concentrated in the absence and / or after filtration. In some embodiments, the MSC secretome may be concentrated using hollow fiber tangential flow technology, or

[0242] In some embodiments, MSC secretomes can be enriched using centrifugation-based size exclusion techniques, e.g., Amicon and / or Centricon, can be used during the enrichment step. In some embodiments, the size cutoff is a MW cutoff of 3-10 kDa. In some embodiments, the molecular weight cutoff for use during centrifugation-based size exclusion techniques enrichment methods is at least about 3 kDa, at least about 4 kDa, at least about 5 kDa, at least about 6 kDa, at least about 7 kDa, at least about 8 kDa, at least about 9 kDa, or at least about 10 kDa.

[0243] In some embodiments, the MSC secretome is enriched about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, or about 100-fold. In some embodiments, the MSC secretome is enriched about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, 95-fold, or about 100-fold compared to the conditioned medium prior to enrichment.

[0244] In some embodiments, the MSC secretome is further buffer exchanged after the concentration step into the final formulation buffer. In some embodiments, the MSC secretome is further buffer exchanged after the concentration step into the final formulation buffer without the adhesive. In some embodiments, the buffer exchange comprises changing the buffer components of the MSC secretome. In some embodiments, the MSC secretome is not diluted during the buffer exchange step. In some embodiments, the MSC secretome is diluted to less than 1%, less than 5%, less than 10%, less than 10%, less than 15%, less than 20%, or less than 25% during the buffer exchange step.

[0245] In some embodiments, the MSC secretome is buffer exchanged after the concentration step so that all traces of culture medium components are removed, ie, the MSC secretome is buffer exchanged after the concentration step so 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 less than about 0% of the culture medium components remain.

[0246] ii. MSC secretome-Formulating In some embodiments, the MSC secretome is prepared in a formulation containing about 2 μg to 20 μg per mL of MSC secretome, hi some embodiments, the MSC secretome is prepared in a formulation containing 0.004% to 0.0375% per mL of MSC secretome.

[0247] In some embodiments, the MSC secretome is prepared at a concentration of about 2 μg to 8 μg of MSC per mL of MSC secretome, or at a concentration of 0.008% to 0.015% of MSC per mL of MSC secretome.

[0248] In some embodiments, the MSC secretome is prepared in a formulation containing 2 mg to 3 mg / mL of monobasic sodium phosphate. In some embodiments, the MSC secretome is prepared in a formulation containing 4% to 5% monobasic sodium phosphate per mL.

[0249] In some embodiments, the MSC secretome is prepared in a formulation comprising 11 mg to 12 mg of dibasic sodium phosphate per mL, hi some embodiments, the MSC secretome is prepared in a formulation comprising 21.5% to 23% dibasic sodium phosphate per mL.

[0250] In some embodiments, the MSC secretome is prepared in a formulation containing 11.5 mg to 13 mg of mannitol per mL, hi some embodiments, the MSC secretome is prepared in a formulation containing 23% to 25% mannitol per mL.

[0251] In some embodiments, the MSC secretome is prepared in a formulation comprising 23 mg to 25 mg of trehalose dihydrate per mL, hi some embodiments, the MSC secretome is prepared in a formulation comprising 46% to 48% of trehalose dihydrate per mL.

[0252] In some embodiments, the MSC secretome is prepared without 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 to 2 mg / mL of hypromellose. In some embodiments, the MSC secretome is prepared in a formulation that includes 1% to 3% hypromellose per mL.

[0253] 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 achieve a desired pH.

[0254] In some embodiments, the MSC secretome is prepared in a formulation including ingredients as provided in Table 1 below.

[0255] [Table 1]

[0256] D. Assay Methods / Therapeutic Characteristics In some embodiments of the present invention, the MSC secretome is processed to achieve specific component ratios / concentrations as well as properties of the MSC secretome.

[0257] In some embodiments, the MSC secretome composition comprises an anti-angiogenic to pro-angiogenic ratio, where the ratio is >1. In some embodiments, the MSC secretome composition comprises an anti-angiogenic to pro-angiogenic ratio, where the ratio is >2, >3, >4, or >5. In some embodiments, the MSC secretome composition comprises an increased concentration of a pro-angiogenic factor (relative to the concentration of the pro-angiogenic factor in the conditioned medium from which the MSC secretome composition is generated). In some embodiments, the MSC secretome composition comprises a sum of several anti-angiogenic factors that exceeds the level of VEGF. In some embodiments, the MSC secretome composition comprises a sum of several anti-angiogenic factors such that the ratio of the two or more anti-angiogenic factors to VEGF is >2, >3, >4, or >5. In some embodiments, the MSC secretome composition comprises one or more anti-angiogenic factors, where the sum of the concentrations of the one or more anti-angiogenic factors relative to the concentration of VEGF is >2, >3, >4, or >5. In some embodiments, pro-angiogenic factors include, but are not limited to, serpin E1 to VEGF-A. In some embodiments, the pro-angiogenic factor is serpin E1. In some embodiments, the pro-angiogenic factor is VEGF-A.

[0258] In some embodiments of the present invention, the MSC secretome is processed to achieve specific potency performance criteria. In some embodiments, a buffer exchange step facilitates obtaining a potent MSC secretome.

[0259] Extracellular vesicles are membrane-bound particles that carry the above-mentioned cargo of soluble and insoluble substances. The term "extracellular vesicles" refers to a group of various species of secreted or shed vesicles. These are generally classified into the following subtypes: 1) microvesicles or shed microvesicles, which typically range in size from 50 to 1500 nm; 2) exosomes, which typically range in size from 30 to 120 nm; and 3) vesicles, which typically range in size from less than 500 nm (i.e., <500 nm). (See, e.g., WO2019016799, incorporated herein by reference in its entirety.) In some embodiments, MSC secretomes can be analyzed for particle counting and / or to quantify the extracellular vesicles (EVs) present in the secretome.

[0260] In some embodiments, EVs are about 2.5 x 10 5 / uL, 2.6×10 5 / uL, 2.7 x 10 5 / uL, 2.8 × 10 5 / uL, 2.9 × 10 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, 4.9x10 5 / uL, or approximately 5.0x10 5 / uL. In some embodiments, the EVs are present at a concentration of about 3.8x10 5 / uL±0.8x10 5 It is present in a concentration of

[0261] In some embodiments, EVs are about 2.5 x 10 5 / uL, 2.6×10 5 / uL, 2.7 x 10 5 / uL, 2.8 × 10 5 / uL, 2.9 × 10 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, 4.9x10 5 / uL, or approximately 5.0x10 5 EVs are present at a concentration of about 2.5 x 10 / uL and have an average diameter of 110-120 nm. 5 / uL, 2.6×10 5 / uL, 2.7 x 10 5 / uL, 2.8 × 10 5 / uL, 2.9 × 10 5 / uL, 3.0x10 5 / uL, 3.1x10 5 / uL, 3.2x10 5 / uL, 3.3x10 5 / uL, 3.4x105 / 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, 4.9x10 5 / uL, or approximately 5.0x10 5 EVs are present at a concentration of about 2.5 x 10 / uL and have an average diameter of 112-116 nm. 5 / uL, 2.6×10 5 / uL, 2.7 x 10 5 / uL, 2.8 × 10 5 / uL, 2.9 × 10 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, 4.9x10 5 / uL, or approximately 5.0x10 5 / uL and have an average diameter of 114 nm. In some embodiments, the EVs are present at a concentration of about 3.8 x 10 5 / uL±0.8×10 5 They are present in concentrations of 114 nm in diameter on average.

[0262] i.MSC secretome - therapeutic properties The MSC secretome of the present disclosure exhibits various therapeutic properties, including, for example, anti-angiogenic properties (blood and / or lymphatic vessels), anti-fibrotic properties, anti-inflammatory properties, properties that promote cell migration and proliferation, mitogenic properties, and anti-oxidative stress / damage properties.

[0263] In some embodiments, anti-angiogenic (blood vessel and / or lymphatic vessel) properties can be determined by the presence and / or level of one or more factors in the MSC secretome. In some embodiments, the anti-angiogenic factors include, but are not limited to, one or more of PEDF, sFLT-1, lower levels of VEGF, and / or serpin E1. In some embodiments, the anti-angiogenic factors include, but are not limited to, one or more of PEDF, lower levels of VEGF, and / or serpin E1. In some embodiments, the anti-angiogenic factor is PEDF. In some embodiments, the anti-angiogenic factor is sFLT-1. In some embodiments, the anti-angiogenic factor corresponds to lower levels of VEGF. In some embodiments, the anti-angiogenic factor is serpin E1.

[0264] In some embodiments, pro-angiogenic (vascular and / or lymphatic) properties can be determined by the presence and / or level of one or more factors in the MSC secretome. In some embodiments, the pro-angiogenic factors include one or more factors selected from the group consisting of VEGF, angiogenin, IGFBP-3, uPA, angio-1, angio-2, and endothelin-1. In some embodiments, the pro-angiogenic factor is VEGF. In some embodiments, the pro-angiogenic factor is angiogenin. In some embodiments, the pro-angiogenic factor is IGFBP-3. In some embodiments, the pro-angiogenic factor is uPA. In some embodiments, the pro-angiogenic factor is angio-1. In some embodiments, the pro-angiogenic factor is angio-2. In some embodiments, the pro-angiogenic factor is endothelin-1.

[0265] In some embodiments, the MSC secretome exhibits anti-fibrotic properties. In some embodiments, such anti-fibrotic properties can be assayed using standard assays. In some embodiments, the presence of various factors and / or activities on the MSC secretome is indicative of anti-fibrotic properties. In some embodiments, factors exhibiting anti-fibrotic properties include, but are not limited to, FGF7 and / or FGF10. In some embodiments, a factor exhibiting anti-fibrotic properties is FGF7. In some embodiments, a factor exhibiting anti-fibrotic properties is FGF10. In some embodiments, a factor exhibiting anti-fibrotic properties is HGF. In some embodiments, activities exhibiting anti-fibrotic properties include, but are not limited to, activation of SMADs, inhibition of the TGFβ pathway, inhibition of myofibroblast differentiation, and / or inhibition of excessive ECM deposition. In some embodiments, activities exhibiting anti-fibrotic properties include activation of SMADs. In some embodiments, activities exhibiting anti-fibrotic properties include inhibition of the TGFβ pathway. In some embodiments, activities exhibiting anti-fibrotic properties include inhibition of myofibroblast differentiation. In some embodiments, the activity exhibiting anti-fibrotic properties includes the inhibition of excessive ECM deposition.

[0266] In some embodiments, the MSC secretome exhibits anti-inflammatory properties. In some embodiments, the MSC secretome suppresses inflammation. In some embodiments, the MSC secretome suppresses inflammation by 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (e.g., a complete reduction in inflammation). In some embodiments, the MSC secretome prevents mast cell degranulation.

[0267] In some embodiments, the MSC secretome promotes cell migration and proliferation, including, for example, mitogenic and motility-promoting activity. In some embodiments, the MSC secretome promotes mitogenic activity. In some embodiments, the MSC secretome promotes motility activity. In some embodiments, the MSC secretome comprises FGF7, which provides the cell migration and proliferation activity of the MSC secretome.

[0268] In some embodiments, the MSC secretome comprises FGF7, which provides the cell migration and proliferation activity of the MSC secretome.

[0269] In some embodiments, the MSC secretome comprises HGF, which provides the cell migration and proliferation activity of the MSC secretome.

[0270] In some embodiments, the MSC secretome comprises an anti-apoptotic agent that provides cell migration and proliferation activity of the MSC secretome. In some embodiments, the MSC secretome comprises an anti-apoptotic agent that provides cell migration and proliferation activity of the MSC secretome, including but not limited to FGF-2, HGF, and IGF-1. In some embodiments, the MSC secretome comprises an anti-apoptotic agent that provides cell migration and proliferation activity of the MSC secretome, selected from the group consisting of FGF-2, HGF, and IGF-1.

[0271] In some embodiments, the MSC secretome comprises NGF, which provides cell migration and proliferation activity of the MSC secretome.

[0272] In some embodiments, the MSC secretome provides reduced antioxidant stress and / or cell damage. In some embodiments, the MSC secretome comprises reduced antioxidant stress and cell-damaging factors. In some embodiments, the reduced antioxidant stress and cell-damaging factors include, but are not limited to, SOD-1, SOD-2, SOD-3, and HO-1. In some embodiments, the reduced antioxidant stress and cell-damaging factors are selected from the group consisting of SOD-1, SOD-2, SOD-3, and HO-1.

[0273] ii. MSC secretome - biophysical / biochemical characterization Biochemical and biophysical characterization: In some embodiments, the present invention provides methods for characterizing the MSC secretome. In some embodiments, characterizing the MSC secretome includes 1) comprehensive and / or quantitative mapping 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, various efficacy assays can be performed on the MSC secretome as described herein to determine its properties. In some embodiments, the MSC secretome can be subjected to comprehensive and / or quantitative mapping 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 bioassays. In some embodiments, characterization assays can include, but are not limited to, characterization of physical components, oxidative stress assays, safety analyses, stability assays, proliferation assays, migration assays, angiogenesis assays, differentiation / scarring assays, inflammation assays, and / or epithelial barrier integrity assays. In some embodiments, the characterization assay is selected from the group consisting of physical component characterization, oxidative stress assay, safety analysis, stability assay, proliferation assay, migration assay, angiogenesis assay, differentiation / scarring assay, inflammation assay, and / or epithelial barrier integrity assay.

[0274] Physical component characterization: In some embodiments, characterizing the MSC secretome involves a method using a combination of bioanalytical techniques. In some embodiments, characterizing the MSC secretome involves determining the physical components of the MSC secretome. In some embodiments, characterizing the MSC secretome involves the use of protein arrays, enzyme-linked immunosorbent assays (ELISAs), mass spectrometry, and immunoblotting. In some embodiments, characterizing the MSC secretome can be used to identify molecules within 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 levels of each factor.

[0275] In some embodiments, the secretome comprises protein factors and extracellular vesicles (EVs). In some embodiments, the MSC secretome comprises trophic factors. In some embodiments, the protein factors of the MSC secretome include pentraxin-3, TIMP-1, serpin E1, TSP-1, and HGF. In some embodiments, the MSC secretome comprises EVs. In some embodiments, the MSC secretome is analyzed for simple lipid content to quantitatively measure total lipids. In some embodiments, the EV fraction, where the MSC secretome can be evaluated for EV markers, can be evaluated for EV markers, including, but not limited to, AUX, TSG101, CD63, CD9, and CD8.

[0276] In some embodiments, secretomes are extracellular vesicles (EVs) ranging in size from 30 to 200 nm and 1x10 per mL. 8 ~5x10 9 This includes EVs.

[0277] In some embodiments, depletion studies can be performed to tease out the individual contributions of key factors. In some embodiments, antibody-based pull-down methods can be used to remove defined factors from the MSC secretome. 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 assess the contributions of protein and EV fractions. In some embodiments, TIMP1 and / or serpin E1 can be depleted. In some embodiments, TIMP1 and / or serpin E1 can be depleted.

[0278] Oxidative stress: In some embodiments, an oxidative stress prevention assay can be performed on MSC secretome. In some embodiments, the MSC secretome prevents damage to the corneal epithelium. In some embodiments, the MSC secretome reduces the presence of inflammation. In some embodiments, the MSC secretome reduces the presence of inflammation, as determined by an increased presence of anti-inflammatory markers. In some embodiments, the MSC secretome reduces the presence of inflammation, as determined by an increased presence of anti-inflammatory markers, such as IL-8.

[0279] Safety Characterization: In some embodiments, the MSC secretome can be evaluated for hemocompatibility, and tests for sterility and pyrogen and endotoxin levels can be performed. In some embodiments, the MSC secretome can be evaluated for hemocompatibility. In some embodiments, the evaluation of hemocompatibility includes hemolysis and hemagglutination assays. In some embodiments, the MSC secretome does not exhibit adverse effects upon systemic exposure. In some embodiments, the MSC secretome does not exhibit adverse effects upon 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.

[0280] In some embodiments, the MSC secretome can be sterile so that it can be administered as part of a formulation. In some embodiments, the MSC secretome can be free or substantially free of endotoxins. In some embodiments, the MSC secretome can be free or substantially free of microorganisms.

[0281] Stability: In some embodiments, biophysical properties of the MSC secretome can be assessed and / or determined. In some embodiments, fluorescence, static light scattering, and dynamic light scattering characterize protein stability metrics. In some embodiments, the following parameters can be measured to further characterize the secretome: thermal melting, thermal aggregation, delta G, and / or viscosity. In some embodiments, a thermal melting assay is used to determine the stability of the MSC secretome. In some embodiments, a thermal aggregation assay is used to determine the stability of the MSC secretome. In some embodiments, delta G is used as a measure to determine the stability of the MSC secretome. In some embodiments, viscosity is measured as an MSC secretome property. In some embodiments, viscosity determines the stability of the MSC secretome.

[0282] In some embodiments, biophysical metrics can be used to establish stability parameters to characterize different MSC secretome preparations.

[0283] 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.

[0284] 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).

[0285] In some embodiments, the MSC secretome is stable for at least 14 days at about -20°C. In some embodiments, the MSC secretome is stable for at least 14 days at about 4°C. In some embodiments, the MSC secretome is stable for at least 14 days at about 20°C (or room temperature). In some embodiments, the MSC secretome is stable for at least 14 days at about 25°C (room temperature).

[0286] Epithelial barrier integrity assay The corneal epithelium, or more precisely, the apical surface of the epithelium, contributes significantly to the overall barrier properties of the cornea, making changes to the corneal barrier a sensitive factor in biocompatibility analysis. In some embodiments, biophysical characteristics of the MSC secretome can be assessed 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 properties. In some embodiments, the 3D tissue 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 EVOMO epithelial volt-ohm meter and an EndOhm-12 chamber (World Precision, Sarasota, FL). In some embodiments, at the end of the procedure, the tissue can be used for tissue viability assessment using the following formula: % Barrier Integrity = 100 x [TEER (treated tissue) / TEER (placebo control)]

[0287] In some embodiments, TEER can be used to evaluate the effect of topical application of MSC secretome on barrier integrity. In some embodiments, TEER can be used to evaluate the effect of topical application of MSC secretome on barrier integrity after corneal epithelial injury caused by topical exposure to nitrogen mustard (NM) using an EpiCorneal tissue model (MatTekCorp). In some embodiments, MSC secretome can be applied topically at, for example, 6 μg / ml (diluted with placebo solution), as described in Example 6. In some embodiments, EpiCorneal tissue was cultured in 5 ml of medium under standard culture conditions for 24 hours.

[0288] Bioassay In some embodiments, bioassays can be used to characterize the MSC secretome. In some embodiments, bioassays can be related to corneal wound healing: epithelial cell migration and proliferation, stromal cell differentiation (e.g., scarring), angiogenesis, and inflammation. In some embodiments, bioassays can be used to evaluate the ability of the MSC secretome to mediate corneal wound healing: epithelial cell migration and proliferation, stromal cell differentiation (e.g., scarring), angiogenesis, and inflammation.

[0289] Migration and proliferation: In some embodiments, MSC secretomes can be assessed for their ability to promote proliferation and migration. In some embodiments, MSC secretomes can be assessed for their ability to promote proliferation. In some embodiments, MSC secretomes can be assessed for their ability to promote migration. In some embodiments, MSC secretomes promote proliferation and / or migration. In some embodiments, MSC secretomes promote proliferation. In some embodiments, MSC secretomes promote migration. In some embodiments, MSC secretomes can be assessed using a transwell migration assay to determine proliferation-promoting ability.

[0290] In some embodiments, a migration assay can be used to evaluate the ability of an MSC secretome to promote migration. In some embodiments, a migration assay can be used to evaluate the ability of an MSC secretome to promote migration, and the migration assay is an in vitro wound closure assay (also referred to as a "scratch wound assay"). In some embodiments, an MSC secretome promotes migration, and this promotion of migration is determined and / or examined using a "scratch assay." Generally, scratch assay methods are based on the creation of an artificial gap, also referred to as a "scratch," in a confluent cell monolayer. The "scratch" can be monitored to see whether cells at the edge of the newly created gap migrate toward the opening and close / cover the "scratch." See, e.g., Liang, C., Park, A. & Guan, J. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2, 329-333 (2007)).

[0291] In some embodiments, migration assays can include transwell migration assays using corneal epithelial cells (or other cell surrogates after validation)—e.g., wound closure—can be performed on MSC secretomes. In some embodiments, the transwell migration assay uses corneal epithelium as a test of the wound closure efficacy of MSC secretomes. In some embodiments, MSC secretomes promote wound closure as determined using the transwell migration assay.

[0292] 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.

[0293] In some embodiments, a human corneal epithelial cell proliferation assay can be performed on the MSC secretome. In some embodiments, the human corneal epithelial cell proliferation assay demonstrates a test for the wound closure properties of the MSC secretome. In some embodiments, the MSC secretome promotes wound closure as determined using the human corneal epithelial cell proliferation assay.

[0294] In some embodiments, a circular scratch method or circular scratch assay or circular wound closure assay can be used. In some embodiments, the Oris™ cell migration assay platform can be used (see also Example 6 herein).

[0295] In some embodiments, an endothelial cell tube formation assay may be performed on the MSC secretome. In some embodiments, the endothelial cell tube formation assay may indicate that the MSC secretome is not pro-angiogenic. In some embodiments, the endothelial cell tube formation assay provides a measure of the angiogenic potential of the MSC secretome. In some embodiments, the MSC secretome exhibits anti-angiogenic properties. In some embodiments, the MSC secretome is anti-angiogenic. In some embodiments, the endothelial cell tube formation assay provides a ratio of anti-angiogenic and pro-angiogenic signals. In some embodiments, a negative result from the endothelial cell tube formation assay will confirm a high anti:promotion ratio, ensuring that the MSC secretome does not promote angiogenesis. In some embodiments, a negative result from the endothelial cell tube formation assay will confirm a high anti:promotion ratio, ensuring that the MSC secretome does not promote CNV (choroidal neovascularization) or general angiogenesis. In some embodiments, an inhibition of TGFb-induced myofibroblast differentiation assay can be performed on MSC secretome. In some embodiments, an inhibition of TGFb-induced myofibroblast differentiation assay can be performed on MSC secretome to demonstrate that the MSC secretome inhibits scarring. In some embodiments, the MSC secretome prevents scarring. In some embodiments, the MSC secretome prevents corneal opacity scarring. In some embodiments, the MSC secretome has low angiogenic induction. In some embodiments, the MSC secretome has a reduced angiogenic response. In some embodiments, the MSC secretome has reduced angiogenic potential. In some embodiments, the MSC secretome impairs and / or reduces the normal formation of blood vessels in the presence of angiogenesis-supportive medium. In some embodiments, the MSC secretome has reduced angiogenic potential when compared to an untreated MSC secretome control. In some embodiments, the MSC secretome has reduced angiogenic potential compared to samples treated with serum-containing medium. In some embodiments, the MSC secretome attenuates the angiogenic response.In some embodiments, the MSC secretome reduces the angiogenic response induced by serum-free medium. In some embodiments, a reduced angiogenic response is induced by the MSC secretome when comparing secretome and serum-containing medium (reduced or no angiogenic response) with 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.

[0296] Differentiation / scarring: In some embodiments, the MSC secretome can be assessed for its ability to prevent differentiation and prevent scarring. In some embodiments, the MSC secretome prevents and / or impairs scarring. In some embodiments, the MSC secretome prevents scarring. In some embodiments, the MSC secretome reduces scarring compared to other standard of care. In some embodiments, the MSC secretome prevents and / or impairs differentiation. In some embodiments, the MSC secretome prevents and / or impairs myofibroblast differentiation. In some embodiments, the MSC secretome reduces loss of corneal transparency. In some embodiments, the MSC secretome reduces loss of corneal transparency by preventing and / or impairing myofibroblast differentiation.

[0297] In some embodiments, MSC secretomes can be assessed for their ability to regulate factors involved in differentiation. In some embodiments, MSC secretomes can be assessed for their ability to regulate factors involved in differentiation, including, but not limited to, TGFB2, collagen I, collagen III (normally upregulated during differentiation), TFGB3, MMP-2, and MMP-9 (normally downregulated during differentiation). In some embodiments, MSC secretomes regulate factors selected from the group consisting of TGFB2, collagen I, collagen III (normally upregulated during differentiation), TFGB3, MMP-2, and MMP-9 (normally downregulated during differentiation). In some embodiments, MSC secretomes induce a decrease in factors that are upregulated during normal differentiation. In some embodiments, MSC secretomes induce an increase in factors that are downregulated during normal differentiation. In some embodiments, MSC secretomes induce a decrease in the expression of factors such as SMA. In some embodiments, the MSC secretome induces a decrease in the expression of factors such as SMA, which indicates MSC secretome efficacy.

[0298] Angiogenesis: In some embodiments, the MSC secretome can be evaluated for its ability to prevent angiogenesis. In some embodiments, the MSC secretome prevents, impairs, suppresses, and / or reduces angiogenesis. In some embodiments, the MSC secretome suppresses or does not promote angiogenesis. In some embodiments, the MSC secretome can be evaluated for its ability to prevent angiogenesis. In some embodiments, the MSC secretome prevents, impairs, suppresses, and / or reduces angiogenesis. In some embodiments, the MSC secretome suppresses angiogenesis.

[0299] In some embodiments, the MSC secretome can be further evaluated using a depletion assay. In some embodiments, the MSC secretome can be depleted of specific factors. In some embodiments, the MSC secretome can be depleted of specific factors, including, but not limited to, TIMP1 and / or serpin E1. In some embodiments, the MSC secretome can be depleted of TIMP1 and / or serpin E1. In some embodiments, the MSC secretome can be depleted of TIMP1. In some embodiments, the MSC secretome can be depleted of serpin E1.

[0300] inflammation: In some embodiments, MSC secretomes can be evaluated for their ability to prevent, impair, suppress, and / or reduce inflammation. In some embodiments, MSC secretomes prevent, impair, suppress, and / or reduce inflammation. In some embodiments, MSC secretomes suppress inflammation. In some embodiments, MSC secretomes are characterized in vitro and / or in vivo to determine their ability to prevent, impair, suppress, and / or reduce inflammation. In some embodiments, MSC secretomes prevent, impair, suppress, and / or reduce inflammation in vitro and / or in vivo. In some embodiments, MSC secretomes prevent, impair, suppress, and / or reduce inflammation in vitro. In some embodiments, MSC secretomes prevent, impair, suppress, and / or reduce inflammation in vivo. In some embodiments, tissue models can be used to characterize the prevention, impair, suppress, and / or reduction of inflammation in vitro. In some embodiments, 3D tissue models can be used to characterize the prevention, impairment, suppression, and / or reduction of inflammation in vitro. In some embodiments, a nitrogen mustard (NM) gas combustion model can be used to evaluate the prevention, impairment, suppression, and / or reduction of inflammation in vitro. In some embodiments, a nitrogen mustard (NM) gas combustion model can be used to evaluate the prevention, impairment, suppression, and / or reduction of inflammation in vitro and can be used as a surrogate for in vivo conditions. In some embodiments, cytokine profiles can be determined in response to treatment and / or administration with MSC secretome. In some embodiments, levels of specific cytokines can be determined. In some embodiments, levels of IL-8 can be determined. In some embodiments, levels of IL-8 expression can be reduced in tissues treated with MSC secretome. In some embodiments, levels of IL-8 expression are reduced in tissues treated with MSC secretome, indicating the prevention, impairment, suppression, and / or reduction of inflammation.

[0301] E. Treatment methods The present disclosure also provides therapeutic methods using the MSC secretome of the present disclosure. In particular, the MSC secretome is used in the treatment of ocular conditions. In particular, the MSC secretome is used in the treatment of ocular conditions, including, but not limited to, ocular diseases. In some embodiments, the ocular disease is associated with the ocular surface. In some embodiments, the ocular disease is associated with damaged ocular tissue and / or symptoms of damaged ocular tissue. In some embodiments, the MSC secretome finds use in the treatment of ocular conditions, including accelerating wound healing. In some embodiments, the MSC secretome finds use in the treatment of ocular conditions, including reducing scarring. In some embodiments, the MSC secretome finds use in the treatment of ocular conditions, including reducing inflammation. In some embodiments, the MSC secretome finds use in the treatment of ocular conditions, including reducing inflammation and therefore promoting growth. In some embodiments, the MSC secretome finds use in the treatment of ocular conditions, such as reducing inflammation at the ocular surface. In some embodiments, the MSC secretome finds use in the treatment of ocular conditions, including reducing angiogenesis. In some embodiments, the MSC secretome finds use in treating ocular conditions, including reducing neovascularization in the cornea. In some embodiments, the MSC secretome finds use in treating ocular conditions, including dry eye treatment (e.g., treating severe dry eye, including where epithelial cells are damaged). In some embodiments, the MSC secretome finds use in treating ocular conditions, such as restoring integrity to damaged ocular tissue. In some embodiments, the MSC secretome finds use in treating ocular conditions, such as accelerating the healing of damaged ocular tissue. In some embodiments, the MSC secretome finds use in treating ocular conditions, such as regenerating damaged ocular nerve tissue. In some embodiments, the MSC secretome finds use in treating ocular conditions, such as regenerating damaged ocular nerve tissue associated with PCED. In some embodiments, the MSC secretome finds use in treating ocular conditions, such as PCED.In some embodiments, the MSC secretome finds use in treating ocular conditions, such as inflammatory damage to the ocular surface, hi some embodiments, the MSC secretome finds use in treating ocular conditions, such as, for example, GvHD and / or Sjogren's syndrome.

[0302] In some embodiments, the MSC secretome finds use in accelerating wound healing. In some embodiments, the MSC secretome finds use in reducing scarring. In some embodiments, the MSC secretome finds use in reducing inflammation. In some embodiments, the MSC secretome finds use in reducing inflammation and therefore promoting growth. In some embodiments, the MSC secretome finds use in reducing inflammation at the ocular surface. In some embodiments, the MSC secretome finds use in reducing angiogenesis. In some embodiments, the MSC secretome finds use in reducing angiogenesis in the cornea. In some embodiments, the MSC secretome finds use in protecting and repairing retinal epithelial cells and retinal ganglion cells. In some embodiments, the MSC secretome finds use in inducing trabecular meshwork regeneration and reducing intraocular pressure.

[0303] In some embodiments, the mesenchymal stem cell secretome is administered for the treatment of an ocular disease. In some embodiments, this comprises administering a therapeutically effective amount of a mesenchymal stem cell secretome composition as described herein to a patient in need thereof. In some embodiments, the mesenchymal stem cell secretome is administered to a patient in need thereof to promote or induce ocular wound healing. In some embodiments, the mesenchymal stem cell secretome is administered to a patient in need thereof to reduce and / or inhibit angiogenesis, reduce and / or inhibit scarring, promote and / or maintain vision, and / or increase wound closure rate (e.g., shorten wound closure time). In some embodiments, the mesenchymal stem cell secretome is administered to a patient in need thereof to prevent, reduce, and / or inhibit angiogenesis. In some embodiments, the mesenchymal stem cell secretome is administered to a patient in need thereof to prevent, reduce, and / or inhibit scarring. In some embodiments, the mesenchymal stem cell secretome is administered to a patient in need thereof to promote and / or maintain vision. In some embodiments, the mesenchymal stem cell secretome is administered to promote and / or induce faster wound closure (e.g., shorten the time required for wound closure). In some embodiments, the mesenchymal stem cell secretome prevents, reduces, and / or inhibits or does not promote angiogenesis and reduces scarring to promote vision preservation. In some embodiments, the mesenchymal stem cell secretome is administered to a patient in need thereof to prevent, reduce, and / or inhibit angiogenesis and reduce scarring to promote vision preservation. In some embodiments, the mesenchymal stem cell secretome prevents, reduces, and / or inhibits inflammation. In some embodiments, the mesenchymal stem cell secretome is administered to a patient in need thereof to prevent, reduce, and / or inhibit inflammation.

[0304] In some embodiments, the mesenchymal stem cell secretome is administered for the treatment of visual dysfunction following traumatic injury to an ocular structure, hi some embodiments, the treatment comprises administering to a patient in need thereof a therapeutically effective amount of a mesenchymal stem cell secretome composition as described herein.

[0305] In some embodiments, the mesenchymal stem cell secretome is administered to treat traumatic injury of optic nerve degeneration after percussive injury. In some embodiments, the percussive injury to the eye is selected from the group consisting of an ocular contusion and a blunt injury to the eye. In some embodiments, the mesenchymal stem cell secretome is administered to treat traumatic injury of the optic nerve. In some embodiments, the treatment comprises administering to a patient in need thereof a therapeutically effective amount of a mesenchymal stem cell secretome composition as described herein.

[0306] In some embodiments, a mesenchymal stem cell secretome is administered to ameliorate optic nerve degeneration following a percussive injury to the eye. In some embodiments, a method for ameliorating optic nerve degeneration comprises administering to a patient a therapeutically effective amount of a mesenchymal stem cell secretome composition as described herein. In some embodiments, the percussive injury to the eye is selected from the group consisting of an ocular contusion and a blunt ocular injury. In some embodiments, the percussive injury to the eye is an ocular contusion. In some embodiments, the percussive injury to the eye is a blunt ocular injury.

[0307] Efficacy readouts can include, for example, symptom relief and / or reduction in disease state, including improved quality of life. In some embodiments, a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% reduction in symptoms and / or disease state indicates therapeutic efficacy. In some embodiments, a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% reduction in inflammation indicates therapeutic efficacy. In some embodiments, a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% reduction in scarring indicates therapeutic efficacy. In some embodiments, a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% reduction in angiogenesis indicates a therapeutic effect.

[0308] In some embodiments, the disease or condition is an ocular disease or condition. In some embodiments, the disease or condition is visual dysfunction following trauma to an ocular structure. In some embodiments, the disease or condition is a percussive (e.g., blunt or non-blunt) injury to the eye. In some embodiments, the disease or condition is a burn, including a chemical burn, to the eye.

[0309] In some embodiments, the mesenchymal stem cell secretome is administered to a specific target area. In some embodiments, the specific target area is the eye. In some embodiments, the mesenchymal stem cell secretome is administered to the specific target area and is formulated to prevent spread to other surrounding areas.

[0310] In some embodiments, the mesenchymal stem cell secretome is administered to a specific target area and is formulated to prevent spread to other surrounding areas.

[0311] In some embodiments, the mesenchymal stem cell secretome is administered to a specific target area and is formulated to remain in the target area for at least 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about 60 minutes, at least about 70 minutes, at least about 80 minutes, at least about 90 minutes, or at least about 2 hours.

[0312] In some embodiments, the mesenchymal stem cell secretome is administered to the affected area immediately after the wound or injury. In some embodiments, the mesenchymal stem cell secretome is administered to the affected area within 15 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, or 96 hours.

[0313] In some embodiments, the mesenchymal stem cell secretome is administered locally. In some embodiments, the mesenchymal stem cell secretome is administered by subconjunctival injection. In some embodiments, the MSC secretome composition exhibits ultra-potency when administered to a subject in need thereof. In some embodiments, the mesenchymal stem cell secretome is administered locally once, two, three, four, five, and / or up to six times daily. In some embodiments, the MSC secretome composition enables therapeutic effects with one drop or one administration per day. In some embodiments, one drop is administered one, two, three, four, five, or six times daily. In some embodiments, one drop is administered at intervals of one hour, two hours, three hours, or four hours. In some embodiments, one drop is administered at least once daily for one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, or ten weeks. In some embodiments, one drop is administered at least twice a day for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks. In some embodiments, one drop is administered at least three times a day for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks. In some embodiments, one drop is administered at least four times a day for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks. In some embodiments, one drop is administered at least five times a day for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks. In some embodiments, one drop is administered at least six times a day for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks.

[0314]

[0010] In some embodiments, a method for treating an ocular condition in a subject in need thereof comprises administering to the subject a mesenchymal stem cell (MSC) secretome composition, wherein the MSC secretome composition comprises: i. at least one trophic factor / cytokine selected from the group consisting of HGF, TIMP-1, TIMP-2, PAI-1 (serpin E1), VEGF-A, and b-NGF; ii. PEDF (serpin F1), IGFBP-2, IGFBP-3, SDF-1, TSG-14, Kallikrein3, MCP-1, Angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, and PDGF; iii. At least one 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.

[0315] In some embodiments, the MSC secretome composition for use in the method of treatment further comprises elevated levels of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1.

[0316] In some embodiments, an MSC secretome composition for use in a method of treatment comprises 1 ng / mL to 100 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, an MSC secretome composition for use in a method of treatment comprises 1 ng / mL to 200 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, an MSC secretome composition for use in a method of treatment comprises 1 ng / mL to 300 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome composition for use in the treatment method comprises 1 ng / mL to 400 ng / mL of at least one factor selected from the group consisting of serpin E1, serpin A1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1.

[0317] In some embodiments, the MSC secretome composition for use in the method of treatment further comprises a moderate level of at least one factor selected from the group consisting of angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, thrombospondin-1, angiogenin, DPPIV, IGFBP-3, and uPA.

[0318] In some embodiments, the MSC secretome composition for use in the method of treatment comprises 400 pg / mL to 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.

[0319] In some embodiments, the MSC secretome composition for use in the method of treatment further comprises at least one factor selected from the group consisting of apolipoprotein A1, complement factor D, C-reactive protein, cystatin C, DKK-1, EMMPRIN, osteopontin, vitamin DBP, MIF, RANTES, uPAR, IL-17a, GDF-15, and IFNγ.

[0320] In some embodiments, the MSC secretome composition for use in the method of treatment comprises an anti-angiogenic to pro-angiogenic ratio, wherein the ratio is >2, >3, >4, or >5. In some embodiments, the anti-angiogenic factors comprise PEDF, low levels of VEGF, and one or more factors selected from the group consisting of serpin E1 and the pro-angiogenic agents VEGF, angiogenin, IGFBP-3, uPA, angio-1, angio-2, endothelin-1.

[0321] In some embodiments, the MSC secretome composition for use in the method of treatment further comprises a low level of VEGF. In some embodiments, the MSC secretome for use in the method of treatment comprises 1 pg / mL to 400 pg / mL of VEGF. In some embodiments, the level of VEGF is 5 to 10 times lower than the level of serpin E1. In some embodiments, the MSC secretome composition for use in the method of treatment comprises one or more anti-angiogenic factors, 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.

[0322] In some embodiments, the MSC secretome compositions for use in the methods of treatment are free of and / or contain very low levels of bFGF, PLGF, and PDGF.

[0323] In some embodiments, the MSC secretome composition for use in the method of treatment comprises less than 1000 pg / mL of bFGF, PLGF, and PDGF.

[0324] In some embodiments, the MSC secretome composition for use in the treatment methods has a pH of about 4.7 to about 7.5.

[0325] In some embodiments, the MSC secretome composition for use in the treatment methods is formulated with a buffer system selected from the group consisting of disodium phosphate / monosodium, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and citric acid / disodium phosphate.

[0326] In some embodiments, the MSC secretome composition for use in the method of treatment further comprises a tonicity modifying agent, hi some embodiments, the tonicity modifying agent is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin.

[0327] In some embodiments, the MSC secretome composition for use in the method of treatment further comprises monosodium phosphate / di-sodium phosphate, mannitol, and trehalose, and the composition has a pH of about pH 7.4.

[0328] In some embodiments, the MSC secretome composition for use in the method of treatment further comprises a divalent cation, hi some embodiments, the divalent cation is selected from the group consisting of Mg2+, Ca2+, and Zn2+.

[0329] In some embodiments, the MSC secretome composition for use in the method of treatment further comprises disodium phosphate / citric acid, mannitol, and trehalose, and the composition has a pH of about pH 6.4.

[0330] In some embodiments, the MSC secretome composition for use in the method of treatment further comprises an adhesive, in some embodiments, the adhesive is selected from the group consisting of hypromellose, poloxamer 407, poloxamer 188, poloxomer 237, poloxomer 338, hypromellose, (HPMC), polycarbophil, polyvinylpyrrolidone (PVP), PVA (polyvinyl alcohol), polyimide, sodium hyaluronate, gellan gum, poly(lactic-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethylcellulose (CMC), or hydroxypropylmethylcellulose (HPMC), hydroxymethylcellulose, hydroxyethylcellulose, sodium carboxymethylcellulose, fibrin glue, polyethylene glycol, and GelCORE.

[0331] In some embodiments, the MSC secretome composition for use in the method of treatment does not contain 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-exosomes / non-extracellular vesicle cellular debris, hormones, and L-glutamine.

[0332] In some embodiments, the MSC secretome composition for use in the method of treatment comprises HGF, pentraxin-3 (TSG-14), VEGF, TIMP-1, serpin E1, and <5 ng / mL IL-8.

[0333] In some embodiments, the MSC secretome for use in the methods of therapeutic composition comprises: i. 0.3 to 4.5 ng / mL HGF and ii. 0.5 to 20 ng / mL of pentraxin-3 (TSG-14); iii. 100-600pg / mL VEGF; iv. 10 to 200 ng / mL TIMP-1 and v. 20-80ng / mL serpin E1 and vi. <5ng / mL IL-8;

[0334] In some embodiments, the MSC secretome composition for use in the treatment method comprises an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.

[0335] In some embodiments, the present disclosure provides a method for treating an ocular condition in a subject in need thereof, the method comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, the MSC secretome composition comprising: i. 2 μg-20 μg of MSC secretome per mL; ii. 2 mg to 3 mg of monobasic sodium phosphate per mL; iii. 11 mg to 12 mg of dibasic sodium phosphate per mL; iv. 11.5 mg to 13 mg of mannitol per mL; v. 23mg to 24mg trehalose dihydrate, vi. 0.5 mg to 2 mg of hypromellose per 1 mL; A stable mesenchymal stem cell (MSC) secretome preparation with a pH of approximately 4.7 to approximately 7.5.

[0336] In some embodiments, the present disclosure provides a method for treating an ocular condition in a subject in need thereof, the method comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, the MSC secretome composition comprising: i. 0.004%-0.08% w / w MSC secretome; ii. 4% to 5% w / w of monobasic sodium phosphate; iii. 21.5% to 23% w / w of dibasic sodium phosphate; iv. 23%-25% w / w mannitol; v. 46% to 48% w / w dehydrated trehalose, vi. 1% to 3% w / w of hypromellose; A stable mesenchymal stem cell (MSC) secretome preparation with a pH of approximately 4.7 to approximately 7.5.

[0337] In some embodiments, the present disclosure provides a method for treating an ocular condition in a subject in need thereof, the method comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, the MSC secretome composition comprising: i. 2 μg-20 μg of MSC secretome per mL; ii. 2 mg to 3 mg of monobasic sodium phosphate per mL; iii. 11 mg to 12 mg of dibasic sodium phosphate per mL; iv. 11.5 mg to 13 mg of mannitol per mL; v. 23mg to 24mg trehalose dihydrate, vi. 0.5 mg to 2 mg of any hypromellose per 1 mL; A stable mesenchymal stem cell (MSC) secretome preparation with a pH of approximately 4.7 to approximately 7.5.

[0338] In some embodiments, the present disclosure provides a method for treating an ocular condition in a subject in need thereof, the method comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, the MSC secretome composition comprising: i. 0.004%-0.08% w / w MSC secretome; ii. 4% to 5% w / w of monobasic sodium phosphate; iii. 21.5% to 23% w / w of dibasic sodium phosphate; iv. 23%-25% w / w mannitol; v. 46% to 48% w / w dehydrated trehalose, vi. 1%-3% w / w of optional hypromellose, A stable mesenchymal stem cell (MSC) secretome preparation with a pH of approximately 4.7 to approximately 7.5.

[0339] In some embodiments, the present disclosure provides a method for treating an ocular condition in a subject in need thereof, the method comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, the MSC secretome composition comprising: i. 2 μg-20 μg of MSC secretome per mL; ii. 2 mg to 3 mg of monobasic sodium phosphate per mL; iii. 11 mg to 12 mg of dibasic sodium phosphate per mL; iv. 11.5 mg to 13 mg of mannitol per mL; v. 23 mg to 24 mg of trehalose dihydrate; vi. A stable mesenchymal stem cell (MSC) secretome preparation having a pH of about 4.7 to about 7.5.

[0340] In some embodiments, the present disclosure provides a method for treating an ocular condition in a subject in need thereof, the method comprising administering to the subject a mesenchymal stem cell (MSC) secretome composition, the MSC secretome composition comprising: i. 0.004%-0.08% w / w MSC secretome; ii. 4% to 5% w / w of monobasic sodium phosphate; iii. 21.5% to 23% w / w of dibasic sodium phosphate; iv. 23%-25% w / w mannitol; v. 46%-48% w / w trehalose dehydrate; vi. A stable mesenchymal stem cell (MSC) secretome preparation having a pH of about 4.7 to about 7.5.

[0341] F. Kit The kit can include a containerized MSC secretome or a conditioned medium for use in preparing a containerized MSC secretome, as disclosed herein, and instructions for use. Additionally, the kit can include components for mixing to prepare a solution for use in treating the eye, and instructions for mixing and use.

[0342] The container can include at least one vial, well, test tube, flask, bottle, syringe, or other container means containing the containerized MSC secretome or conditioned medium for use in preparing the MSC secretome, optionally aliquoted appropriately. If additional components are provided, the kit can include additional containers into which the components can be placed. Such containers can include injection or blow-molded plastic containers into which the desired vials are held. The container and / or kit can include a label with instructions and / or warnings for use.

[0343] The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, Genbank sequences, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0344] The present invention can provide kits comprising a panel of tests and / or assays for characterizing the MSC secretome, the panel comprising at least two characterization assays selected from the group consisting of physical component characterization, oxidative stress assays, safety analysis, stability assays, proliferation assays, migration assays, angiogenesis assays, differentiation / scarring assays, inflammation assays, and / or epithelial barrier integrity assays. In some embodiments, the panel of tests and / or assays identifies the MSC secretome as described herein.

[0345] The present invention can provide kits comprising a panel of tests and / or assays for determining lot-to-lot consistency of MSC secretome, the panel comprising one or more characterization assays selected from the group consisting of physical component characterization, oxidative stress assays, safety analysis, stability assays, proliferation assays, migration assays, angiogenesis assays, differentiation / scarring assays, inflammation assays, and / or epithelial barrier integrity assays. In some embodiments, the panel of tests and / or assays identifies the MSC secretome as described herein. [Example]

[0346] Example 1: Secretome characterization biochemical composition The biochemical composition of the MSC secretome can be analyzed for the presence of various factors and activities, including: IDO (indoleamine-2,3-dioxygenase) enzyme activity assay, Threshold ppm levels (assessed by ELISA or luminex) of several trophic factors / cytokines, including but not limited to HGF, FGF-7, TIMP-1, TIMP-2 thrombospondin, PAI-1 (serpin E1), VEGF-A, b-NGF, ·Additional factors: sFLT-1, PEDF (serpin F1), IGFBP-2, IGFBP-3, SDF-1, TSG-14, Kallikrein3, MCP-1, bFGF (FGF2), Angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, PDGF.

[0347] The biochemical composition of the MSC secretome is approximately 10–150,000 pg / 10 5 Cells can be analyzed based on a range of factors.

[0348] Particle counting to quantify EVs (extracellular vesicles) within the secretome is also performed.

[0349] Lipid content is assessed as needed.

[0350] Biophysical characterization / release can be performed, including 1) FTIR spectroscopy, and 2) buffer exchange / enrichment secretome process (a buffer exchange process is used to enrich the secretome and, in some cases, a size exclusion process that can remove molecules below a predefined molecular weight cutoff). For example, VEGF is a very small protein (42 kDa), so based on the filtration cutoff employed, it is possible to remove nearly all (or virtually all) of the VEGF.

[0351] Once purified and concentrated, the MSC secretome can be further processed downstream. This includes a buffer exchange process to prepare the MSC secretome for administration. Buffer exchange can employ hollow fiber tangential flow techniques to remove media components and replace them with desired formulation components. The buffer exchange process can also result in the concentration of the secretome.

[0352] The secretome can then be formulated for administration.

[0353] Example 2: Biomechanical and biophysical properties of the secretome This example describes methods for determining secretome identity, compositional analysis, biochemical and biophysical characterization, and cell-based assays for use in secretome characterization.

[0354] The analytical techniques described in this example can be used to evaluate the performance and stability of various formulations. Formulations with the best stability and tolerance profiles undergo dose-response and dosing regimen evaluation using an alkaline rodent burn model. Time to wound healing and histopathology at the wound site, as well as local toxicity, can be assessed.

[0355] Efficacy Assay: To determine the properties of the conditioned medium, various efficacy assays can be performed, including the following:

[0356] A transwell migration assay of corneal epithelial cells (or other cell surrogates after validation) (e.g., wound closure) can be performed. This assay provides a measure of trophic growth factors (e.g., HGF, FGF) present in the secretome.

[0357] Human corneal epithelial cell proliferation assay (e.g., wound closure). This assay provides a measure of trophic growth factors (e.g., HGF, FGF) present in the secretome.

[0358] An endothelial cell tube formation assay can be performed, which can be a characterization assay to demonstrate that a lot is not pro-angiogenic. This assay provides a measure of the angiogenic potential of the secretome product—it must be inhibitory (e.g., exhibit anti-angiogenic properties). This assay can test for a reduction in angiogenic potential. In the assay, the angiogenic response or angiogenic potential is reduced compared to untreated. For example, an experiment could include two sample groups: 1) not treated with secretome but treated with serum-containing medium (which induces tube formation, a normal angiogenic response), and 2) treated with secretome and serum-containing medium. Because secretome attenuates this normal angiogenic response, the angiogenic potential is reduced. See Figures 5 and 6 for examples. This assay reflects the ratio of anti-angiogenic to pro-angiogenic signals. A negative result confirms a high anti-:pro-ratio and ensures that the secretome product does not promote CNV (choroidal neovascularization) or general angiogenesis. For example, to assess the angiogenesis-regulating ability of MSC secretomes, other assays to evaluate the expression levels and activity of several factors (as revealed through the depletion experiments described herein) can also be used. An inhibition (e.g., anti-scarring) assay of TGFβ-induced myofibroblast differentiation can be performed. This assay evaluates the ability of secretomes to inhibit scarring (or corneal opacification), and factors characteristic of stromal fibroblast differentiation into myofibroblasts (e.g., smooth muscle actin) and matrix deposition are assessed by qPCR, fluorescence microscopy, and Western blot.

[0359] Oxidative stress prevention assays can be performed. 3D model assays can also be used to assess efficacy and prevent / repair nitrogen mustard gas damage to the corneal epithelium as a readout. Such assays include macroscopic observation to confirm tissue health as well as assays for various anti-inflammatory markers (e.g., IL-8).

[0360] FDA guidance provides industry guidance for cellular and genetic products (see fda.gov / downloads / biologicsbloodvaccines / guidancecomplianceregulatoryinformation / guidances / cellularandgenetherapy / ucm243392.pdf, incorporated herein by reference). This guidance specifies the conduct of efficacy assays and tests, and how such efficacy assays correlate with the therapeutic effect of MSC secretomes so that they can be evaluated for efficacy under standard FDA guidelines.

[0361] Safety Characterization Secretomes can assess blood compatibility and perform tests for sterility, pyrogen, and endotoxin levels. Current, up-to-date FDA guidance is generally available for such analyses (e.g., 2012 FDA Guidance: Pyrogen and Endotoxin Testing). (See fda.gov / downloads / Drugs / GuidanceComplianceRegulatoryInformation / Guidances / UCM310098.pdf, incorporated herein by reference).

[0362] Methods to assess hemocompatibility, including hemolysis and hemagglutination assays, are also used to confirm that there are no systemic exposure issues with the prepared MSC secretome, for example, in cases where systemic exposure occurs in severe eye burns. These methods are based on existing techniques (Maji et al.; Saswati et al.; Nayak et al.) and use blood from healthy consenting donors. Briefly, for hemolysis, red blood cells (RBCs) can be co-incubated with MSC secretomes at 37°C for 1 hour. RBCs treated with osmotic PBS serve as a negative control, and distilled water serves as a positive control. After incubation and centrifugation, the RBCs are analyzed by ion exchange at 540 nm (A). max Absorbance of hemoglobin can be measured. The percentage of hemolysis is: 100% x (A MSC-S -Aブランク ) / A 蒸留水 This can be calculated as: This study allows us to establish the dose that begins to induce hemolysis and to assess whether MSC secretomes are hemocompatible according to ISO / TR7406 (hemolysis rate <5%). For hemagglutination, RBC suspensions can be co-incubated with MSC secretomes in 96-well U-bottom dishes for 2 hours at 37°C. A positive result for hemagglutination is an RBC suspension (because the agglutinated RBC lattice does not fall out of solution), whereas a negative result is RBCs falling out of solution, resulting in an RBC "button" at the bottom of the well. MSC secretomes do not exhibit hemagglutination activity.

[0363] Sterility and endotoxin assays will also be developed. These methods will aid in performing sterility and endotoxin testing on each MSC secretome generated. These assays can be used to demonstrate that the test can reliably detect the presence of viable contaminating microorganisms or endotoxins. All test components can also be validated to demonstrate that the test method can consistently detect the presence of viable microorganisms or endotoxins.

[0364] Biochemical and biophysical characterization Due to the complex composition of the MSC secretome, it is beneficial to characterize secretome components to develop the most relevant and meaningful efficacy assays. Different combinations of characterization methods, including molecular, biochemical, and biophysical analyses, can be used to measure efficacy.

[0365] Such characterization studies will focus on: 1) comprehensive and / or quantitative mapping of molecular entities in the MSC secretome, 2) measuring the contribution of selected factors to biological activity, and 3) measuring biophysical parameters. Importantly, each focus area can be applied to each MSC secretome preparation to assess the consistency of the preparation.

[0366] This example describes the characterization of the molecular composition of the MSC secretome. Because the MSC secretome is composed of protein factors and extracellular vesicles (EVs), both fractions must be characterized. A combination of bioanalytical techniques (including protein arrays, enzyme-linked immunosorbent assays (ELISAs), mass spectrometry, and immunoblotting) can be used to characterize these molecules within the MSC secretome. Protein arrays and mass spectrometry have been employed to determine the presence and identify factors. More quantitative techniques, such as ELISA, can be used to measure the levels of each factor present and establish ranges for some analytes.

[0367] Several factors have been identified in the MSC secretome, including its trophic and overall low angiogenic properties. Factors identified in the MSC secretome to date include pentraxin-3, TIMP-1, serpin E1, TSP-1, and HGF.

[0368] In addition to nutrients and other factors, EVs are a major component of the secretome, making them the focus of therapeutic campaigns. A series of analyses can be performed to assess the EV component of APIs. First, a simple lipid content analysis can be performed using a lipid-binding dye (e.g., Nile Red) to generate a quantitative measurement of total lipids in a product lot. Additionally, particle count and size distribution assessment can be performed using the NanoSight NS300. This analysis can be performed on each lot to establish particle concentration and a consistent particle distribution size range. Biochemical characterization of the EV fraction using immunoblotting for standard markers (including AUX, TSG101, CD63, CD9, and CD81) can be performed.

[0369] To complement our biochemical analysis, depletion studies can be performed to extract the individual contributions of key factors. Briefly, an antibody-based pull-down method can be used to remove defined factors from the MSC secretome. Depletion can be confirmed by Western blot and assessed with a series of bioassays (described below). Similar studies can be performed to assess the contributions of protein and EV fractions.

[0370] The biophysical properties of the MSC secretome can also be assessed using platforms that characterize protein stability indicators using fluorescence, static light scattering, and dynamic light scattering. The following parameters can be measured: Thermal Melting: Measures the intrinsic fluorescence of exposed tryptophan or tyrosine residues as proteins undergo conformational changes. This assay measures the change in fluorescence intensity, or peak shift, as the protein begins to unfold. Allows for the ranking and comparison of different formulations. Thermal melting with SYPRO: Differential scanning calorimetry (DSF) is used to measure the spectral shift of a dye (SYPROO range, Molecular Probes, Inc.) as it binds to exposed hydrophobic residues as the protein unfolds. The sensitivity of fluorescence allows measurements at very low concentrations of the MSC secretome. Thermal Aggregation: Monitor the aggregation behavior and temperature at which API aggregates during a thermal ramp. Measure at two wavelengths (266nm and 463nm) using SLS to distinguish between small and large particles. Allows ranking and comparison of different formulations and provides information on the dynamics of protein unfolding and aggregation. Delta G: Measures the change in fluorescence of an API after the addition of a chemical denaturant, allowing for the calculation of a quantitative value of protein stability or the amount of energy required to unfold the MSC secretome, Delta G. Viscosity: Measure the viscosity of MSC secretome preparations using small amounts of material, allowing for evaluation and comparison of different formulations.

[0371] The biophysical metrics described above establish key stability parameters for evaluating various MSC secretome formulations and performing MSC secretome proliferation and / or accelerated stability studies. Stability analysis of two different MSC secretomes formulated at pH 6.4 and 7.4 was performed.

[0372] Biological potency Rationale: Development of a cell-based assay program to assess the efficacy of the MSC secretome was initiated. The cell-based assay program aims to achieve the following: · Demonstrate product activity, quality and consistency throughout product development; · Generating a collection of data supporting lot release specifications; · Providing a basis for evaluating manufacturing changes; To assess the stability of MSC secretome.

[0373] To properly evaluate the MSC secretome, a series of bioassays can be developed to assess the biopotency of the MSC secretome, as described in the Examples provided herein.

[0374] Bioassay To develop a series of bioassays for the MSC secretome, we can focus on processes including key events that orchestrate corneal wound healing, epithelial cell migration and proliferation, stromal cell differentiation (scarring), angiogenesis, and inflammation. Bioassays can be employed to assess the ability of the MSC secretome to mediate each of these processes.

[0375] Migration and proliferation: Following trauma, corneal epithelial cells must divide and migrate at the leading edge of the wound to mediate closure (Ljubimov AV., Prog Retin Eye Res. 2015 Nov;49:17-45). We evaluate the ability of the MSC secretome to promote proliferation and migration in primary corneal epithelial cells (or immortalized corneal epithelial cells) and in vitro cell assays.

[0376] For proliferation, corneal epithelial cells can be cultured in the presence of MSC-S at a defined dose. Live cell stains (e.g., MTT or WST-8) can be used to quantify viable cells after defined incubation periods (e.g., 24 and 48 hours). For migration, a transwell migration assay can be used in which corneal epithelial cells can be seeded into a chamber with an 8-micron polycarbonate membrane in basal medium minus growth factors. The chamber can be placed in a culture dish well (24-well dish) containing basal medium supplemented with API at a defined dose. After 24 hours of incubation, cells that have migrated through the membrane can be quantified using either fluorescent (e.g., calcein AM) or colorimetric (e.g., crystal violet) staining.

[0377] Differentiation / scarring: Following ocular trauma that breaches the corneal stromal layer (the layer beneath the epithelium), corneal fibroblasts are induced to differentiate into myofibroblasts that express smooth muscle actin (SMA) in a process mediated by transforming growth factor-β (TGFβ). This process accompanies the normal wound healing process but leads to undesirable consequences for the eye, including scarring and vision loss. This vision loss occurs when myofibroblasts deposit large amounts of disorganized extracellular matrix, resulting in a loss of corneal transparency. Assays have been developed to assess the ability of the MSC secretome to prevent or impair myofibroblast differentiation and, therefore, are a measure of MSC secretome efficacy.

[0378] Human corneal fibroblasts can be used as a model cell system. Cells are treated with TGFB1 to induce differentiation in the presence (or absence) of increasing concentrations of API and then incubated for an additional 24 hours. TGFB1-treated fibroblasts rapidly differentiate into myofibroblasts, characterized by a significant increase in smooth muscle actin expression levels and modulation of several other factors, such as TGFB2, collagen I, and collagen III (normally upregulated during differentiation), and TFGB3, MMP-2, and MMP-9 (normally downregulated during differentiation). qPCR and immunoblotting (mRNA and protein, respectively) can be used to measure the effect of MSC secretome on the expression levels of these molecular hallmarks of differentiation. MSC secretome treatment reduces the expression of factors such as SMA, which may be a reflective indicator of its effectiveness.

[0379] Angiogenesis: A healthy cornea is avascular and lacks blood vessels. However, after trauma, corneal neovascularization often occurs, which contributes to visual impairment and even vision loss. More specifically, neovascularization can induce tissue scarring, lipid deposition, interstitial hemorrhage, and corneal edema, significantly altering vision. A key feature of the MSC secretome is its ability to impair angiogenesis during the healing process. Cell-based assays can be performed to assess the ability of the MSC secretome to impair angiogenesis.

[0380] The endothelial tube formation assay can be performed using human umbilical vein endothelial cells (HUVECs). This assay is a standard assay for assessing vascular development by measuring endothelial cell reorganization to form capillary-like vasculature. Briefly, HUVEC cells are seeded on a thin layer of basement membrane extract (collagen or Matrigel®; BD Biosciences) and incubated in the presence or absence of an API. Within 2–6 h, when supported by pro-angiogenic molecules, the endothelial cells begin to divide and form a vascular network. It is possible to assess whether MSC secretome can impair the formation of this network. Images can be acquired and image analysis software can be used to measure branching points, tube length and number, loop number and area, and cell-covered area. The MSC secretome can inhibit angiogenesis.

[0381] To complement the tube formation assay, depletion studies can be performed in which specific factors (e.g., TIMP1, serpin E1) are deleted or depleted from the API to help assess their impact in the assay. The goal in performing these assays is to identify key anti-angiogenic molecules in MSC-S (numerous), which will aid in the development of release criteria.

[0382] inflammation: Several animal models (Yamagami S., et al., Invest Ophthalmol Vis Sci. 2005 Apr;46(4):1201-7; Gao N., et al., Am J Pathol. 2011 Nov;179(5):2243-53; and Jin Y., et al., MolVis. 2007 Apr;13:626-34) have shown that corneal epithelial injury induces an acute inflammatory response in the blood vessels of the limbus, leading to the accumulation of leukocytes and neutrophils and the migration of dendritic cells, macrophages, and lymphocytes into the stroma and injured epithelium. This increased immune infiltration of the cornea leads to neovascularization, which can result in vision loss. Indeed, clinicians on our scientific advisory board emphasize the importance of attenuating the inflammatory response during the acute phase of corneal injury. In vitro cell and in vitro tissue assays can be developed and used to characterize this important capability of the MSC secretome and develop bioassays for the process.

[0383] The ability of lipopolysaccharide (LPS) to induce an inflammatory response can be exploited to develop an in vitro cell assay. LPS is a component of the cell membrane of Gram-negative bacteria and a potent trigger for the secretion of various cytokines produced by inflammatory cells. When LPS is injected into the corneal stroma, inflammatory cells such as neutrophils and monocytes rapidly infiltrate the stroma, leading to corneal ulcers in rabbits. Specifically, stromal fibroblasts recognize the presence of LPS and trigger inflammatory cell infiltration through the expression of chemokines and adhesion molecules (e.g., IL-8 and MCP-1) (Fukuda K., Int J Mol Sci. 2017 Aug 23;18(9)). Primary human corneal fibroblasts can be treated with LPS in the presence or absence of MSC secretome. Cells can be incubated for 24 hours before evaluation, during which time the levels of IL-8 and MCP-1 (immunocytochemical chemoattractants) can be measured in the culture medium. ELISA can be used as a quantitative method to measure cytokine levels. Treatment with MSC secretome reduces the levels of IL-8 and MCP-1 in the culture medium.

[0384] Second, a 3D tissue model can be used: an in vitro reconstructed human corneal tissue model, which structurally and functionally recapitulates key features of in vivo corneal tissue. Importantly, this model behaves like tri-denatured corneal tissue and replicates the inflammatory response to chemical burns (e.g., nitrogen mustard). A model of nitrogen mustard (NM) gas burn can be implemented and used to evaluate the ability of MSC secretome to attenuate the immune response. This model is commonly used to mimic in vivo conditions to evaluate inflammatory responses. Similar to in vitro models, cytokine profiles (e.g., IL-8 levels) can be evaluated in response to treatment with MSC secretome after NM burn. IL-8 levels can be reduced in tissue treated with MSC secretome.

[0385] Example 3: Characteristics and properties of the MSC secretome MSC-S are composed of key elements related to wound healing. Significant factors related to wound healing and repair constitute MSC-S. Biochemical characterization of MSC-S was performed using a human growth factor / cytokine array containing 105 proteins (Figure 2), and most of the highly expressed proteins are involved in promoting wound healing or regulating important processes in tissue repair (e.g., reducing oxidative stress).

[0386] MSC-S demonstrates a low tendency to aggregate. MSC-S was analyzed to assess its stability and tendency to aggregate. In this study, MSC-S was prepared in phosphate buffer at pH 6.4 and 7.4. MSC-S was first characterized by size-exclusion chromatography (SEC) by storing MSC-S at 4°C, room temperature (20°C), and 37°C for 7 days, and then re-evaluating the samples by SEC. Only a slight increase in one SEC peak (RT 8.5 min) was observed, which correlated with the increase in temperature and reflected a slight increase in aggregated material over time.

[0387] To further analyze aggregation propensity, static light scattering (SLS) thermal aggregation analysis was performed (Figure 3). This assay measures SLS at 266 nm and 473 nm; SLS266 measurements are more sensitive to changes in sample intensity and signal the onset of small particle formation, while SLS473 measurements are useful for detecting larger aggregates. Small aggregates (T agg-266nm It was observed that the onset temperature of aggregation was higher for the samples at pH 7.4 (68-70°C, Fig. 3A) compared to pH 6.4 (54°C, Fig. 3B). This indicates that MSC-S is more stable at pH 7.4 in terms of thermal aggregation tendency. Importantly, the formation of large aggregates (SLS 473nm ) was not observed throughout the temperature ramp at both pH values ​​tested (FIGS. 3A, 3B), indicating that the extent of formation of large aggregates was very low.

[0388] MSC-S promotes migration of primary human corneal epithelial cells. The ability of MSC-S (lot 2) to promote corneal epithelial cell migration, a critical process for rapid wound closure, was evaluated. A transwell migration assay was performed to measure migration using primary human corneal epithelial cells. Cells were seeded in basal medium in an upper chamber with a porous membrane bottom. The chamber was then placed in medium containing MSC-S. After 24 hours, the migrated cells were quantified. The data demonstrated that MSC-S indeed promotes epithelial cell migration (Figure 4A, B). Further data showed that heat denaturation (90°C / 10 min) abolished the migration-promoting activity of MSC-S (Figure 4B), establishing the assay as a stable assay.

[0389] MSC-S exhibit antiangiogenic properties. Characterization of MSC-S was performed to identify key proteins associated with angiogenesis and assess their impact on endothelial tube formation in vitro. The content of MSC-S was analyzed using a human angiogenesis protein array (R&D Systems) (Figure 5A). Various production lots were analyzed, and the protein composition, both identity and relative abundance, was found to be consistent. Array analysis showed that the most prominent proteins classified as antiangiogenic were present in 2- to 7-fold excess over proangiogenic molecules (Figure 5B). In particular, TIMP-1, a matrix metalloproteinase inhibitor, was present at high levels, consistent with a report identifying TIMP-1 as a major antiangiogenic modulator produced by MSCs (Zanotti, et al., Leukemia. 2016 May;30(5):1143-54). The antiangiogenic biochemical fingerprint was translated into a cell-based assay in which inhibition of angiogenic function was observed. Using a HUVEC tube formation assay, we demonstrated that incubation with MSC-S attenuated tube formation parameters, such as total tube length, number of branch points, and number of loops (Figure 6). Collectively, the suppression of these properties highlights that MSC-S impairs angiogenesis. These anti-angiogenic properties of MSC-S may impair corneal angiogenesis during wound healing, thereby reducing the likelihood of vision impairment or blindness, making them highly suitable as an ophthalmic treatment.

[0390] MSC-S exhibit anti-scar properties. The anti-scarring ability of MSC-S was evaluated. Following corneal trauma, stromal fibroblasts differentiate into myofibroblasts, a hallmark of scarring, in a process characterized by several biomarkers. Myofibroblast differentiation can be replicated in vitro using dermal fibroblasts and treating them with TGFβ-1, a potent inducer of differentiation (Li, M. et al., Int Wound J. 2017 Feb;14(1):64-73). This assay was used to assess the ability of MSC-S to impair differentiation, and qPCR was used to evaluate the expression of TGFβ-2 and smooth muscle actin (SMA), key biomarkers upregulated during myofibroblast differentiation (Figure 7). The data showed that MSC-S had a potent effect in attenuating the expression of both TGFβ-2 and SMA, indicating the inhibition of myofibroblast differentiation. Thus, MSC-Ss demonstrated anti-scarring properties and the potential to mediate scar-free corneal wound healing.

[0391] MSC-S increases in vitro corneal cell proliferation. To evaluate the ability of MSC-S to promote cell proliferation, an in vitro assay was performed using primary rabbit corneal epithelial cells. When incubated with MSC-S, cell proliferation increased dose-dependently after incubation in primary rabbit corneal cells (see, e.g., Fernandes-Cunha, GM, et al., Stem Cells Transl. Med. 2019).

[0392] MSC-S improve in vivo wound closure in various corneal injury models. After demonstrating increased cell proliferation in an in vitro setting, wound healing was assessed following mechanical corneal injury in a mouse model. MSC-S were administered as a single drop daily for 4 days and assessed 24 hours after treatment cessation. Significant wound closure was observed in eyes receiving MSC-S compared to saline (see, e.g., Fernandes-Cunha, GM, et al., Stem Cells Transl. Med. 2019) (*, p<.05).

[0393] The second lot of MSC-S (formulated with BSS) was evaluated using a rat corneal alkali burn model. Topical application of MSC-S to rats with corneal alkali burn-induced injury significantly reduced corneal damage (Figure 8). Alkali burn induced highly evident severe corneal damage, including severe epithelial wounding, neovascularization, and opacification, which persisted until day 8 (Figure 8). Figure 8 shows that the substantial epithelial damage induced by alkali burn was only 16.7 ± 14.3%, 48.0 ± 6.04%, and 69.4 ± 6.74% wound closure in the vehicle control group on days 2, 3, and 4 after injury, with the MSC-S treatment group achieving a substantial improvement of 47.0 ± 37.2%, 75.7 ± 23.6%, and 92.1 ± 5.73%, respectively.

[0394] MSC-S reduces opacity and neovascular size in corneal chemical burns. MSC-S significantly reduced opacity and neovascular size in the corneal alkali burn model (Figure 10). The opacity scores were 2.25.0±0.5, 2.75±0.5, and 2.0±0.25±0.6 in the MSC-S-treated group compared with 3.0±0, 4.0±0, 3.75±0.5, and 3.75±0.5 in the vehicle-treated group on days 5, 6, 7, and 8 (*p<0.05, **p<0.01, ***p<0.001, and *p<0.05, respectively). Furthermore, the neovessel size scores were 1.5.0 ± 0.6, 1.75 ± 0.5, and 1.75 ± 0.5 in the MSC-S-treated group compared with 2.5.0 ± 0.6, 3.0 ± 0, and 3.0 ± 0 in the vehicle control-treated group on days 5, 6, and 7 (*p<0.05, **p<0.01, **p<0.01, respectively). This data is consistent with in vitro data demonstrating the antiangiogenic properties of MSC-S (Figures 5 and 6) and the anti-scarring properties of MSC-S (Figure 7).

[0395] Example 4: Pharmaceutical ophthalmic preparations containing MSC secretome MSC-S that are safe and effective for the treatment of corneal wounds Agent The development of topical eye drops is hindered by many anatomical constraints, including tear turnover and dilution, nasolacrimal drainage, and reflex blinking, often resulting in less than 5% of the topical dose reaching deep ocular tissues (Gaudana et al., 2009). In the case of corneal wounds, the initial trauma causes a breach in the corneal epithelium, thereby allowing the passage of topically applied MSC-S to penetrate the epithelial layer. Because the residence time of formulated therapeutics on the cornea may be too limited, formulation strategies include: 1) evaluating pH, including mimicking the pH of tears (pH 7.4) to reduce stinging and tearing; 2) maximizing MSC secretome concentration to overcome tear dilution and optimize the delivered effective dose without causing adverse events; 3) utilizing the FDA-approved mucoadhesive agent, hydroxyl propyl methylcellulose (HPMC hypromellose), to increase residence time, enhance ocular penetration, and sustain therapeutic delivery; and 4) increasing the lipophilicity of the formulation to optimize corneal penetration without causing nonselective eukaryotic toxicity. All of these formulation changes are necessary to ensure that MSC-S remain stable and potent.

[0396] The formulation can be the most stable and tolerable while maintaining therapeutic efficacy. The FDA-approved pharmaceutical excipient, HPMC, is available for initial evaluation and can be modified as needed to achieve an effective, suitable, and stable formulation. HPMC is a semisynthetic, inert, viscoelastic polymer widely used as an ophthalmic lubricant and controlled delivery agent. Its bioadhesive properties result from the formation of hydr...

Claims

1. 1. A composition for preventing, reducing and / or inhibiting angiogenesis and / or reducing scarring, comprising a treated conditioned medium obtained by culturing bone marrow-derived mesenchymal stem cells (MSCs) in a culture medium and treating the resulting conditioned medium, wherein the treated conditioned medium is subjected to the following steps (i) to (vi): i. culturing MSCs in a first medium, wherein said MSCs are CD166 ＋ CD90 ＋ CD34 － CD45 － including MSCs that are ii. removing the first medium from step (i) from the MSCs; iii. Washing the MSCs of step (ii); iv. adding the second medium to step (iii) and culturing for about 1 to 5 days; v. harvesting the second medium from step (iv) as conditioned medium; and vi. treating the conditioned medium of step (v) by the following steps (a) to (c): a) filtering the harvested conditioned medium from step (v) to remove cell particles; b) concentrating the filtered conditioned medium from step (a); and c) buffer exchange with formulation buffer is obtained by the treated conditioned medium contains HGF, pentraxin-3 (TSG-14), VEGF, TIMP-1, serpin E1, and IL-8, wherein IL-8 is at a concentration of <5 ng / mL; and The composition is in liquid form.

2. 10. The composition of claim 1 for use in treating an ophthalmic condition in a subject.

3. 3. The composition of claim 1 or 2, further comprising a tonicity modifying agent.

4. 4. The composition of claim 3, wherein the tonicity modifying agent is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin.

5. 5. The composition of any one of claims 2 to 4, wherein the ocular condition is selected from the group consisting of ocular damage, scarring of ocular tissue, neovascularization of ocular tissue, increased intraocular pressure, dry eye disease, corneal stem cell deficiency (LSCD), damaged corneal surface, damaged ocular nerve tissue, retinal conditions, persistent corneal epithelial defect (PCED), graft-versus-host disease (GvHD), and Stevens-Johnson syndrome.

6. i. at least one trophic factor or cytokine selected from the group consisting of TIMP-2 and VEGF-A; ii. at least one additional factor selected from the group consisting of PEDF (serpin F1), IGFBP-2, IGFBP-3, SDF-1, Kallikrein 3, MCP-1, angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-beta, and PDGF; 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; 6. The composition of claim 1, further comprising:

7. 7. The composition of any one of claims 1 to 6, comprising 1 ng / mL to 100 ng / mL of at least one factor selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), and serpin F1.

8. 7. The composition of any one of claims 1 to 6, comprising 400 pg / mL to 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, angiogenin, DPPIV, IGFBP-3, and uPA.

9. 9. The composition of any one of claims 1 to 8, further comprising at least one factor selected from the group consisting of apolipoprotein A1, complement factor D, C-reactive protein, cystatin C, DKK-1, EMMPRIN, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15, and IFNγ.

10. 10. The composition of any one of claims 1 to 9, comprising 1 pg / mL to 400 pg / mL of VEGF.

11. The composition of any one of claims 1 to 10, wherein the level of VEGF is 5 to 10 times lower than the level of serpin E1.

12. 12. The composition of claim 1, comprising less than 1000 pg / mL of bFGF, PLGF, and PDGF.

13. 13. The composition of any one of claims 1 to 12, having a pH of from about 4.7 to about 7.

5.

14. 14. The composition of any one of claims 1 to 13, formulated with a buffer system selected from the group consisting of disodium phosphate / monosodium, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and citric acid / disodium phosphate.

15. 15. The composition of any one of claims 1 to 14, further comprising monosodium / disodium phosphate, mannitol, and trehalose, and having a pH of about pH 7.

4.

16. 16. The composition of claim 1, further comprising a divalent cation.

17. The divalent cation is Mg2 ＋ , Ca2 ＋ , and Zn2 ＋ 17. The composition of claim 16, selected from the group consisting of:

18. 18. The composition of any one of claims 1 to 17, further comprising disodium phosphate / citric acid, mannitol, and trehalose, and having a pH of about pH 6.

4.

19. 19. The composition of any one of claims 1 to 18, which is free of 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-exosomes / non-extracellular vesicle cellular debris, hormones, and L-glutamine.

20. i. 0.3-4.5 ng / mL HGF; ii. 0.5-20 ng / mL of pentraxin-3 (TSG-14); iii. 100-600 pg / mL VEGF; iv. 10-200 ng / mL TIMP-1; v. 20-80 ng / mL of serpin E1; vi. <5 ng / mL of IL-8; 20. The composition of any one of claims 1 to 19, comprising:

Images