Method for preparing and purifying secretome derived from mesenchymal stem cells

Abstract

The present application provides methods and processes for pharmaceutical formulations comprising mesenchymal stem cell secretome for use in ophthalmic treatments, as well as methods for manufacturing and packaging such formulations.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 404,455, filed September 7, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Regenerative medicine is a field of medicine concerned with replacing or regenerating 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 without limit and can differentiate into a variety of different cell types under certain conditions. Totipotent stem cells are stem cells that have the potential to give rise to 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 a mesenchymal stem cell. See, e.g., U.S. Patent Application No. 20190046576.

[0004] However, producing and storing live 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 is often variable, 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 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 drawbacks currently associated with cell-based regenerative medicine therapies. This is particularly relevant for ocular conditions.

[0005] Contusions and blunt injuries to the eye can cause a range of mechanical disruptions to the ocular contents, including retinitis concavis, traumatic cataract, disruption of the ciliary zonules attachment to the lens, recession of the iris, iris schisis, and rupture of the pupillary sphincter. Treatment of these injuries has been limited to mechanically repairing the iris (if possible), replacing the lens with a plastic lens implant, and repairing retinal detachment. No treatments have previously existed to repair the cellular structures of the retina or anterior chamber. Furthermore, traumatic optic neuropathy and optic nerve avulsion were among the six major types of ocular injuries requiring specialized ophthalmic care during the Iraq War (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). The same reference is incorporated herein by reference in its entirety. Sixty percent of traumatic head injuries result in neuro-ophthalmological abnormalities (Van Stavern, et al., J Neuro-Ophthamol 21(2):112-117, 2001) (the same reference is incorporated herein by reference in its entirety), half of which involve the optic nerve or visual pathway. Traumatic injury to neurons results in axonal damage and irreversible neuronal loss, resulting in permanent deficits. While several candidate neuroprotective therapies have been identified in animals, these single agents have generally not been successfully translated into human clinical trials (Turner, et al., J Neurosurg 118(5):1072-1085, 2013, the same reference is incorporated herein by reference in its entirety). Combination therapies affecting several cellular targets will likely be required to prevent neuronal damage.

[0006] As the outermost tissue of the eye, the cornea plays a protective role, but is highly susceptible to severe injury and disease. Its lack of blood vessels allows for permeability but also limits its ability to heal. Corneal injury can cause irreversible blindness and therefore requires prompt intervention and aggressive treatment. The urgent need for improved ocular surface healing therapies is particularly evident with respect to chemical burns and severe corneal diseases, such as acute and chronic graft-versus-host disease (GvHD), Stevens-Johnson syndrome, ocular mucous membrane pemphigoid, and ocular manifestations of other conditions that result in persistent corneal epithelial defects, which together account for an annual incidence of over 100,000 cases (Dietrich-Ntoukas et al. Cornea. 2012, 31(3):299-310; Stevenson W, et al., Clin Ophthalmol. 2013, 7:2153-2158; White KD, et al., J Allergy Clin Immunol Pract. 2018;6(1):38-69; Tauber J. (2002) Autoimmune Diseases Affecting the Ocular Surface. In: Ocular Surface Disease Medical and Surgical Management. Springer, New York, NY., and Wirostko B, et al. al., Ocul Surf. 2015 Jul;13(3):204-21, and Haring, RS., et al., JAMA Ophthalmol. 2016 Oct 1;134(10):1119-1124).

[0007] Furthermore, the development of topical ocular medications is hindered by many anatomical constraints, including tear turnover and dilution, nasolacrimal drainage, and reflex blinking, and often less than 5% of the topically administered dose reaches deep ocular tissues (Gaudana et al., 2009). In the case of corneal wounds, the initial injury creates a fissure in the corneal epithelium, allowing topically administered MSCs to penetrate the epithelial layer.

[0008] Thus, there is a significant unmet need in the art for pharmaceutically appropriate ocular therapies that can target the eye and deliver therapeutic payloads to hard-to-reach sensory tissues that may be degenerated by inflammation secondary to trauma (e.g., burns, acute inflammation, aging, and / or oxidative stress). The present invention fulfills this need by providing methods for the purification and preparation of pharmaceutical formulations comprising mesenchymal stem cell secretome for use in ocular treatments, as well as methods for manufacturing and packaging such formulations. Summary of the Invention

[0009] In one aspect, the disclosure herein provides a method for preparing and packaging a pharmaceutical formulation comprising one or more pharmaceutical agents, the method comprising: (a) preparing a formulation buffer in a container; (b) diluting a drug substance preparation comprising one or more pharmaceutical agents with a formulation buffer in a container to form a first pharmaceutical formulation; and (c) filling the first formulation into a product container using a blow-fill-seal process to produce a second pharmaceutical formulation, wherein the filled product container contains one or more pharmaceutical agents in a therapeutically effective amount, and wherein step (c) is performed in an aseptic system.

[0010] In some embodiments, the method further comprises, prior to step (a), filtering the formulation buffer and transferring the filtered formulation buffer to the container of step (a).

[0011] In some embodiments, the formulation buffer is filtered through a 0.22 μm bioburden reduction filter prior to step (a).

[0012] In some embodiments, the vessel in step (a) is a 30 L blend tank.

[0013] In some embodiments, the vessel of step (a) comprises a top-mounted mixer.

[0014] In some embodiments, the formulation buffer comprises: Sodium phosphate monobasic monohydrate, sodium phosphate dibasic anhydrous, sodium chloride, magnesium chloride hexahydrate, mannitol, trehalose dihydrate, hydroxypropyl methylcellulose, and purified water.

[0015] In some embodiments, prior to step (b), the drug substance preparation is stored frozen, and the method further comprises thawing the drug substance preparation at the start of step (b).

[0016] In some embodiments, thawing is carried out for a predetermined time period based on the state of one or more cryopreserved pharmaceutical agents.

[0017] In some embodiments, between step (b) and step (c), the first pharmaceutical formulation is subjected to filtration prior to step (c).

[0018] In some embodiments, between step (b) and step (c), the first pharmaceutical formulation is filtered using two or more sterile filters.

[0019] In some embodiments, the blow-fill-seal process of step (c) is carried out using a Weiler 624 BFS machine.

[0020] In some embodiments, the method further comprises refrigerating and storing the filled product containers after step (c).

[0021] In some embodiments, the method further includes repeating step (c) to produce a plurality of filled product containers, each of which contains one or more pharmaceutical agents in a therapeutically effective amount.

[0022] In some embodiments, step (c) is repeated to generate multiple filled product containers simultaneously.

[0023] In some embodiments, the temperature of the formulation buffer during step (a) is maintained at ambient temperature.

[0024] In some embodiments, the temperature of the formulation buffer during step (a) is maintained at about 20°C to 30°C, or about 25°C.

[0025] In some embodiments, the formulation buffer is transferred to the container in step (a) under a pressure of about 10-15 psi.

[0026] In some embodiments, the temperature of the formulation buffer and the first pharmaceutical formulation is maintained at about 2°C to 8°C during step (b).

[0027] In some embodiments, after step (b), the first pharmaceutical formulation is transferred to two or more sterilizing filters under a pressure of about 3-7 psi.

[0028] In some embodiments, between step (b) and step (c), the first pharmaceutical formulation is maintained at about 20°C to 30°C, or at about 25°C.

[0029] In some embodiments, during step (c), the first pharmaceutical formulation is exposed to about 60°C to 70°C for about 1 second to 10 seconds, about 10 seconds to 20 seconds, about 20 seconds to 30 seconds, about 30 seconds to 40 seconds, about 40 seconds to 50 seconds, or about 50 seconds to 50 seconds, and the first pharmaceutical formulation is maintained at about 20°C to 30°C for the remainder of step (c).

[0030] In some embodiments, upon completion of step (b), the first pharmaceutical formulation is collected and stored in a second container for later use prior to step (c).

[0031] In some embodiments, the second container is made of a material other than glass, hi some embodiments, the second container is made of polypropylene.

[0032] In some embodiments, the filled product container has a volume of 10 mL.

[0033] In some embodiments, the filled product container contains about 7 mL to 10 mL of the second pharmaceutical formulation.

[0034] In some embodiments, the filled product container contains between about 7.8 g and 8.65 g of the second pharmaceutical formulation.

[0035] In some embodiments, the filled product container contains about 8.28 g of the second pharmaceutical formulation.

[0036] In some embodiments, the filled product container is made from low density polyethylene (LDPE) resin.

[0037] In some embodiments, the filled product container comprises a total protein concentration of at least about 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, 190 ng / mL, 200 ng / mL, 210 ng / mL, 220 ng / mL, 230 ng / mL, 240 ng / mL, 250 ng / mL, or more.

[0038] In some embodiments, the filled product container comprises a total protein concentration of at least about 75 ng / mL.

[0039] In some embodiments, upon completion of step (c), the overall yield of each of the one or more pharmaceutical agents in the filled product container or multiple filled product containers is about 40%, 50%, 60%, 70%, 80%, 90%, or more of the pharmaceutical agents in the drug substance preparation of step (b).

[0040] In some embodiments, the one or more pharmaceutical agents comprise a bone marrow-derived mesenchymal secretome (MSC) composition.

[0041] In some embodiments, the one or more pharmaceutical agents comprise HGF, serpin A1, TIMP-1, TSG-14, IL-8, serpin E1, VEGF-A, PEDF, and fibronectin (FN).

[0042] In some embodiments, the one or more pharmaceutical agents comprise a bone marrow-derived mesenchymal stem cell (MSC) secretome composition comprising HGF, pentraxin-3 (TSG-14), VEGF, TIMP-1, serpin E1, less than 5 ng / mL IL-8, and a tonicity agent.

[0043] In some embodiments, the MSC secretome composition further comprises: i. at least one trophic factor / cytokine selected from the group consisting of TIMP-2 and VEGF-A; 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-β, and PDGF; and / or iii. At least one additional factor selected from the group consisting of DPPIV (dipeptidyl peptidase-4), uPA, angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, and thrombospondin-1.

[0044] 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), and serpin F1.

[0045] In some embodiments, the MSC secretome 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, angiogenin, DPPIV, IGFBP-3, and uPA.

[0046] In some embodiments, the MSC secretome composition further comprises at least one factor selected from the group consisting of apolipoprotein A1, complement factor D, complement factor H, complement factor I, C1 esterase inhibitor (C1-INH), C4b binding protein (C4BP), CD46, C-reactive protein, cystatin C, DKK-1, Emmprin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15, and IFNγ.

[0047] In some embodiments, the MSC secretome composition comprises a ratio of anti-angiogenic factors to pro-angiogenic factors that is greater than 2, greater than 3, greater than 4, or greater than 5.

[0048] In some embodiments, the MSC secretome composition comprises between 1 pg / mL and 400 pg / mL of VEGF.

[0049] In some embodiments, the level of VEGF is 5 to 10 times lower than the level of Serpin E1.

[0050] In some embodiments, the composition comprises one or more anti-angiogenic factors, and the ratio of the sum of the concentrations of the one or more anti-angiogenic factors to the concentration of VEGF is greater than 2, greater than 3, greater than 4, or greater than 5.

[0051] In some embodiments, the MSC secretome comprises less than 1000 pg / mL of bFGF, PLGF, and PDGF.

[0052] In some embodiments, the MSC secretome composition has a pH of about 4.7 to about 7.5.

[0053] 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 citric acid / disodium phosphate.

[0054] In some embodiments, the MSC secretome composition further comprises mono / disodium phosphate, mannitol, and trehalose, and the composition has a pH of about pH 7.4.

[0055] In some embodiments, the MSC secretome composition further comprises a divalent cation.

[0056] In some embodiments, the divalent cation is selected from the group consisting of Mg2+, Ca2+, and Zn2+.

[0057] 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.

[0058] In some embodiments, the MSC secretome composition further comprises an agent that increases viscosity.

[0059] In some embodiments, the adhesive agent is selected from the group consisting of hypromellose, Poloxamer 407, Poloxamer 188, Poloxomer 237, Poloxomer 338, hypromellose, (HPMC), polycarbophil, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyimide, sodium hyaluronate, gellan gum, poly(lactic-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethylcellulose (CMC), or hydroxypropylmethylcellulose (HPMC), hydroxymethylcellulose, hydroxyethylcellulose, sodium carboxymethylcellulose, fibrin glue, polyethylene glycol, and GelCORE.

[0060] 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 smaller than 3 kDa, antibiotics, protein aggregates larger than 200 nm, cells, non-exosomes / non-extracellular vesicle cellular debris, hormones, and L-glutamine.

[0061] In some embodiments, the MSC secretome composition comprises: i. 0.3–4.5 ng / mL HGF; ii. 0.5–20 ng / mL pentraxin-3 (TSG-14); iii. VEGF between 100 and 600 pg / mL; iv. 10–200 ng / mL TIMP-1; v. 20-80 ng / mL of serpin E1; and / or vi. IL-8 less than 5 ng / mL.

[0062] In some embodiments, the MSC secretome composition comprises an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.

[0063] In some embodiments, the tonicity adjusting agent is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin.

[0064] In some embodiments, the pharmaceutical formulation further comprises one or more ingredients selected from the group consisting of monobasic sodium phosphate, dibasic sodium phosphate, sodium hydroxide, trehalose dihydrate (such as α,α-trehalose dihydrate), hypromellose, hydrochloric acid, sodium chloride, magnesium chloride, polysorbate (such as polysorbate 20 or polysorbate 50), and hydroxypropyl methylcellulose.

[0065] In some embodiments, the pharmaceutical formulation comprises about 0.1 mg to 1 mg, 1 mg to 2 mg, 2 mg to 3 mg, 3 mg to 4 mg, or 4 mg to 5 mg, or more, of monobasic sodium phosphate per mL.

[0066] In some embodiments, the pharmaceutical formulation contains about 1.14 mg, 1.31 mg, 2.28 mg, or 2.62 mg of monobasic sodium phosphate per mL.

[0067] In some embodiments, the pharmaceutical formulation comprises about 0.5 mg to 5 mg, 5 mg to 10 mg, 10 mg to 15 mg, 15 mg to 20 mg, 20 mg to 25 mg, or 25 mg to 30 mg, or more, of sodium phosphate dibasic per mL.

[0068] In some embodiments, the pharmaceutical formulation contains about 5.7 mg or 11.4 mg of sodium phosphate dibasic per mL.

[0069] In some embodiments, the pharmaceutical formulation comprises about 0.5 mg to 30 mg, 5 mg to 25 mg, 10 mg to 20 mg, or 10 mg to 15 mg, or more, of mannitol per mL.

[0070] In some embodiments, the pharmaceutical formulation comprises about 12.2 mg of mannitol per mL.

[0071] In some embodiments, the pharmaceutical formulation contains about 0.5 mg to 50 mg, 5 mg to 45 mg, 10 mg to 40 mg, 15 mg to 35 mg, 20 mg to 30 mg, or 20 mg to 25 mg, or more, of trehalose dihydrate per mL.

[0072] In some embodiments, the pharmaceutical formulation comprises about 24 mg of trehalose dihydrate per mL.

[0073] In some embodiments, the pharmaceutical formulation contains about 0.1 mg to 5 mg, 0.2 mg to 4.5 mg, 0.3 mg to 4 mg, 0.4 mg to 3.5 mg, 0.5 mg to 3 mg, 0.6 mg to 2.5 mg, 0.7 mg to 2 mg, 0.8 mg to 2 mg, 0.9 mg to 1.5 mg, 0.9 mg to 1.4 mg, 0.9 mg to 1.3 mg, 0.9 mg to 1.2 mg, or 0.9 mg to 1.1 mg or more of hypromellose per mL.

[0074] In some embodiments, the pharmaceutical formulation comprises about 1 mg of hypromellose per mL.

[0075] In some embodiments, the pharmaceutical formulation comprises about 0.01 mg to 0.1 mg, 0.1 mg to 0.2 mg, 0.2 mg to 0.3 mg, 0.3 mg to 0.4 mg, or 0.4 mg to 0.5 mg or more of magnesium chloride per mL.

[0076] In some embodiments, the pharmaceutical formulation comprises about 0.2 mg of magnesium chloride per mL.

[0077] In some embodiments, the pharmaceutical formulation contains about 0.1 mg to 0.5 mg, 0.5 mg to 1 mg, 1 mg to 1.5 mg, 1.5 mg to 2 mg, 2 mg to 2.5 mg, 2.5 mg to 3 mg, 3 mg to 3.5 mg, 3.5 mg to 4 mg, 4 mg to 4.5 mg, or 4.5 mg to 5 mg, or more, of sodium chloride per mL.

[0078] In some embodiments, the pharmaceutical formulation comprises about 1.2 mg or 1.8 mg of sodium chloride per mL.

[0079] In some embodiments, the pharmaceutical formulation comprises about 1 mM to 5 mM, 5 mM to 10 mM, 10 mM to 15 mM, 15 mM to 20 mM, 20 mM to 25 mM, or 25 mM to 30 mM, or more, of histidine HCl.

[0080] In some embodiments, the pharmaceutical formulation comprises about 10 mM histidine HCl.

[0081] In some embodiments, the pharmaceutical formulation comprises about 0.001% to 0.005%, 0.005% to 0.01%, 0.01% to 0.015%, 0.015% to 0.02%, 0.02% to 0.025%, or 0.025% to 0.03%, or more, of polysorbate 20.

[0082] In some embodiments, the pharmaceutical formulation comprises about 0.01% polysorbate 20.

[0083] In some embodiments, the pharmaceutical formulation comprises about 0.1% to 1%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, or 9% to 10% or more sucrose.

[0084] In some embodiments, the pharmaceutical formulation comprises about 5.0%, or 5.8% sucrose.

[0085] In some embodiments, the pharmaceutical formulation comprises about 1 mM to 5 mM, 5 mM to 10 mM, 10 mM to 15 mM, 15 mM to 20 mM, 20 mM to 25 mM, or 25 mM to 30 mM, or more, sodium citrate.

[0086] In some embodiments, the pharmaceutical formulation comprises about 10 mM sodium citrate.

[0087] In some embodiments, the pharmaceutical formulation comprises about 0.1 mg to 5 mg, 0.2 mg to 4.5 mg, 0.3 mg to 4 mg, 0.4 mg to 3.5 mg, 0.5 mg to 3 mg, 0.6 mg to 2.5 mg, 0.7 mg to 2 mg, 0.8 mg to 2 mg, 0.9 mg to 1.5 mg, 0.9 mg to 1.4 mg, 0.9 mg to 1.3 mg, 0.9 mg to 1.2 mg, 0.9 mg to 1.1 mg, or more of hydroxypropyl methylcellulose per mL.

[0088] In some embodiments, the pharmaceutical formulation comprises about 1 mg of hydroxypropyl methylcellulose per mL.

[0089] In some embodiments, the pharmaceutical formulation contains about 2 μg to 10 μg, 10 μg to 50 μg, 50 μg to 100 μg, 100 μg to 150 μg, 150 μg to 200 μg, 200 μg to 250 μg, 250 μg to 300 μg, 300 μg to 350 μg, 350 μg to 400 μg, 400 μg to 450 μg, 450 μg to 500 μg per mL. g, 500 μg to 550 μg, 550 μg to 600 μg, 600 μg to 650 μg, 650 μg to 700 μg, 700 μg to 750 μg, 750 μg to 800 μg, 800 μg to 850 μg, 850 μg to 900 μg, 900 μg to 950 μg, or 950 μg to 1000 μg, or more, of MSC secretome.

[0090] In some embodiments, the pharmaceutical formulation contains about 2-20 μg, or 6 μg, of MSC secretome per mL.

[0091] In some embodiments, the pharmaceutical formulation contains about 100 to 400 μg of MSC secretome per mL.

[0092] In some embodiments, the pharmaceutical formulation comprises: i. 1 mg to 3 mg of monobasic sodium phosphate per mL; ii. 5 mg to 12 mg of dibasic sodium phosphate per mL; iii. 11.5 mg to 13 mg of mannitol per mL; and / or iv. Contains 23 mg to 25 mg of trehalose dihydrate per mL; The pH is about 4.7 to about 7.5.

[0093] In some embodiments, the pharmaceutical formulation comprises: i. 2.28 mg of monobasic sodium phosphate per mL; ii. 11.45 mg of dibasic sodium phosphate per mL; iii. 12.2g mannitol per mL; iv. 24 mg trehalose dihydrate per mL; v. 1 mg of hypromellose per mL; vi. Hydrochloric acid; and / or vii. Contains sodium hydroxide.

[0094] In some embodiments, the pharmaceutical formulation comprises: i. 1.14 mg of monobasic sodium phosphate per mL; ii. 5.72 mg of dibasic sodium phosphate per mL; iii. 12.2g mannitol per mL; iv. 24 mg trehalose dihydrate per mL; v. 1 mg of hypromellose per mL; vi. Hydrochloric acid; and / or vii. Contains sodium hydroxide.

[0095] In some embodiments, the pharmaceutical formulation comprises: i. 1.31 mg of monobasic sodium phosphate per mL; ii. 5.73 mg of dibasic sodium phosphate per mL; iii. 12.2g mannitol per mL; iv. 24 mg trehalose dihydrate per mL; v. 1 mg of hypromellose per mL; vi. Hydrochloric acid; vii. sodium hydroxide; and / or viii. Contains 1.76 mg of sodium chloride per mL.

[0096] In some embodiments, the pharmaceutical formulation comprises: i. 10 mM histidine HCl; ii. 10% α,α-trehalose dihydrate; and / or iii. containing 0.01% polysorbate 20; The pH is about 5.5.

[0097] In some embodiments, the pharmaceutical formulation comprises: i. 10 mM sodium phosphate; ii. 40mm sodium chloride; iii. 0.03% Polysorbate 20; and / or iv. containing 5% sucrose; The pH is approximately 6.2.

[0098] In some embodiments, the pharmaceutical formulation comprises: i. 10 mM sodium citrate; ii. 5.8% sucrose; and / or iii. containing 0.01% polysorbate 20, pH 5.5; The pH is approximately 7.2.

[0099] In some embodiments, the pharmaceutical formulation comprises: i. 2.62 mg of sodium phosphate monobasic monohydrate per mL; ii. 11.5 mg of dibasic sodium phosphate anhydrous per mL; iii. 1.17 mg sodium chloride per mL; iv. 0.2 mg of magnesium chloride hexahydrate per mL; v. 12.2 g of mannitol per mL; and / or vi. Contains 24 mg of trehalose dihydrate per mL.

[0100] In some embodiments, the pharmaceutical formulation comprises: i. 1.31 mg of sodium phosphate monobasic monohydrate per mL; ii. 5.73 mg of dibasic sodium phosphate anhydrous per mL; iii. 1.17 mg sodium chloride per mL; iv. 0.2 mg of magnesium chloride hexahydrate per mL; v. 12.2 mannitol per mL; vi. 24 mg of trehalose dihydrate per mL; and / or vii. Contains 1 mg of hydroxypropyl methylcellulose per mL.

[0101] In some embodiments, the pharmaceutical formulation comprises: i. 4%-5% w / w of monobasic sodium phosphate; ii. 21.5%-23% w / w of dibasic sodium phosphate; iv. 23% to 25% w / w mannitol; and / or iv. containing 46% to 48% w / w of trehalose dehydrate; The pH is about 4.7 to about 7.5.

[0102] In some embodiments, the pharmaceutical preparation is formulated for topical administration.

[0103] In some embodiments, the pharmaceutical preparation is formulated for topical administration.

[0104] In some embodiments, the pharmaceutical formulation is prepared and packaged for administration about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times per day.

[0105] In some embodiments, the pharmaceutical formulation is prepared and packaged in 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drop or more doses.

[0106] In some embodiments, the pharmaceutical formulation is prepared and packaged in a dose of 0.1-1 U / mL, 1-2 U / mL, 2-3 U / mL, 3-4 U / mL, 4-5 U / mL, 5-6 U / mL, 6-7 U / mL, 7-8 U / mL, 8-9 U / mL, or 9-10 U / mL, or more.

[0107] In some embodiments, the pharmaceutical formulation is prepared and packaged in a dosage of 1 U / mL or 3 U / mL.

[0108] In some embodiments, the pharmaceutical formulation is prepared and packaged for administration over a period of at least 1, 2, 3, 4, 5, 6, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks, or longer.

[0109] In some embodiments, the pharmaceutical formulation is prepared and packaged for administration at a dose of 3 U / mL, with one drop of the formulation administered four times per day, and optionally, the formulation is administered for at least 56 days.

[0110] In some embodiments, the pharmaceutical formulation is prepared and packaged for administration at a dose of 1 U / mL, with one drop of the formulation administered four times per day, and optionally, the formulation is administered for at least 56 days.

[0111] A kit comprising the pharmaceutical formulation provided herein.

[0112] A kit comprising a pharmaceutical formulation, wherein the pharmaceutical formulation is prepared according to the disclosure provided herein and packaged in a product container.

[0113] A pharmaceutical formulation according to the disclosure provided herein for treating an ophthalmic condition, wherein the pharmaceutical formulation is prepared according to the disclosure provided herein and packaged in a product container.

[0114] In some embodiments, the ocular condition comprises one or more of an ocular wound, ocular scarring, ocular neovascularization, elevated intraocular pressure, dry eye disease, damaged corneal surface, damaged ocular nerve tissue, a retinal condition, persistent corneal epithelial defect (PCED), graft-versus-host disease (GvHD), and Stevens-Johnson syndrome.

[0115] In some embodiments, the pharmaceutical preparation is formulated for topical administration.

[0116] In some embodiments, the pharmaceutical formulation is prepared and packaged for administration about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times per day.

[0117] In some embodiments, the pharmaceutical formulation is prepared and packaged in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more drop doses.

[0118] In some embodiments, the pharmaceutical formulation is prepared and packaged in a dose of 0.1-1 U / mL, 1-2 U / mL, 2-3 U / mL, 3-4 U / mL, 4-5 U / mL, 5-6 U / mL, 6-7 U / mL, 7-8 U / mL, 8-9 U / mL, or 9-10 U / mL, or more.

[0119] In some embodiments, the pharmaceutical formulation is prepared and packaged in a dosage of 1 U / mL or 3 U / mL.

[0120] In some embodiments, the pharmaceutical formulation is prepared and packaged for administration over a period of at least 1, 2, 3, 4, 5, 6, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks, or longer.

[0121] In some embodiments, the pharmaceutical formulation is prepared and packaged for administration at a dose of 3 U / mL, with one drop of the formulation administered four times per day, and optionally, the formulation is administered for at least 56 days.

[0122] In some embodiments, the pharmaceutical formulation is prepared and packaged for administration at a dose of 1 U / mL, with one drop of the formulation administered four times per day, and optionally, the formulation is administered for at least 56 days.

[0123] In some embodiments, the present invention provides a method of treating an ophthalmic condition comprising administering a pharmaceutical formulation according to the disclosures herein to a subject in need thereof, wherein the pharmaceutical formulation is prepared according to the disclosures provided herein and packaged in a product container.

[0124] In some embodiments, the ocular condition comprises one or more of an ocular wound, ocular scarring, ocular neovascularization, elevated intraocular pressure, dry eye disease, damaged corneal surface, damaged ocular nerve tissue, a retinal condition, persistent corneal epithelial defect (PCED), graft-versus-host disease (GvHD), and Stevens-Johnson syndrome.

[0125] In some embodiments, the pharmaceutical preparation is formulated for topical administration.

[0126] In some embodiments, the pharmaceutical formulation is prepared and packaged for administration about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times per day.

[0127] In some embodiments, the pharmaceutical formulation is prepared and packaged in 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drop or more doses.

[0128] In some embodiments, the pharmaceutical formulation is prepared and packaged in a dose of 0.1-1 U / mL, 1-2 U / mL, 2-3 U / mL, 3-4 U / mL, 4-5 U / mL, 5-6 U / mL, 6-7 U / mL, 7-8 U / mL, 8-9 U / mL, or 9-10 U / mL, or more.

[0129] In some embodiments, the pharmaceutical formulation is prepared and packaged in a dosage of 1 U / mL or 3 U / mL.

[0130] In some embodiments, the pharmaceutical formulation is prepared and packaged for administration over a period of at least 1, 2, 3, 4, 5, 6, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks, or longer.

[0131] In some embodiments, the pharmaceutical formulation is prepared and packaged for administration at a dose of 3 U / mL, with one drop of the formulation administered four times per day, and optionally, the formulation is administered for at least 56 days.

[0132] In some embodiments, the pharmaceutical formulation is prepared and packaged for administration at a dose of 1 U / mL, with one drop of the formulation administered four times per day, and optionally, the formulation is administered for at least 56 days.

[0133] In some embodiments, the pharmaceutical formulation is for use in a method of treatment.

[0134] In some embodiments, the present invention provides a unit dose formulation comprising a pharmaceutical formulation according to the disclosure provided herein.

[0135] In some embodiments, the formulation is for treating an ophthalmic condition.

[0136] In some embodiments, a formulation is prepared according to the disclosure provided herein and packaged in a product container.

[0137] In some embodiments, the unit dose formulations provided herein are for use in treatments according to the present disclosure provided herein.

[0138] In some embodiments, the ocular condition comprises one or more of an ocular wound, ocular scarring, ocular neovascularization, elevated intraocular pressure, dry eye disease, damaged corneal surface, damaged ocular nerve tissue, a retinal condition, persistent corneal epithelial defect (PCED), graft-versus-host disease (GvHD), and Stevens-Johnson syndrome.

[0139] In some embodiments, the pharmaceutical preparation is formulated for topical administration.

[0140] In some embodiments, the formulation is prepared and packaged for administration about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times per day.

[0141] In some embodiments, the formulation is prepared and packaged in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more drop doses.

[0142] In some embodiments, the formulations are prepared and packaged in doses of 0.1-1 U / mL, 1-2 U / mL, 2-3 U / mL, 3-4 U / mL, 4-5 U / mL, 5-6 U / mL, 6-7 U / mL, 7-8 U / mL, 8-9 U / mL, or 9-10 U / mL, or more.

[0143] In some embodiments, the formulation is prepared and packaged in a dosage of 1 U / mL or 3 U / mL.

[0144] In some embodiments, the formulation is sufficient to provide a dose of 0.1-1 U / mL, 1-2 U / mL, 2-3 U / mL, 3-4 U / mL, 4-5 U / mL, 5-6 U / mL, 6-7 U / mL, 7-8 U / mL, 8-9 U / mL, or 9-10 U / mL or more.

[0145] In some embodiments, the formulation is sufficient to provide a dose of 1 U / mL or 3 U / mL.

[0146] In some embodiments, the formulation is prepared and packaged for administration over a period of at least 1, 2, 3, 4, 5, 6, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks, or longer.

[0147] In some embodiments, the formulation is prepared and packaged for administration at a dose of 3 U / mL, with one drop of the formulation administered four times per day, and optionally, the formulation is administered for at least 56 days.

[0148] In some embodiments, the formulation is prepared and packaged for administration at a dose of 1 U / mL, with one drop of the formulation administered four times per day, and optionally, the formulation is administered for at least 56 days. [Brief explanation of the drawings]

[0149] [Figure 1] Compounding tank with top-mounted mixer for preparation of formulation buffers. [Figure 2] Aseptically filled using the blow-fill-seal process. [Figure 3] Sampling plan before BFS filling. [Figure 4] BFS filling sampling plan. [Figure 5] Temperature conditions during the filling process. DETAILED DESCRIPTION OF THE INVENTION

[0150] I. Introduction The present invention provides pharmaceutical formulations comprising mesenchymal stem cell secretomes for use in ophthalmic treatments, as well as methods for manufacturing and packaging such formulations.

[0151] A. Definition Terms used in the claims and specification are defined as set forth below unless otherwise specified. In the event of a direct conflict with terms used in the parent provisional patent application, the terms used herein shall control.

[0152] As used herein, "isolated" refers to material that has been removed from its original environment and has therefore been altered "by the hand of man" from the natural state.

[0153] As used herein, "enriched" means to selectively concentrate or increase the amount of one or more substances by removing unwanted substances from a mixture or by selecting and separating desired substances (e.g., separating cells having a specific cell marker from a heterogeneous cell population in which not all cells in the population express the marker).

[0154] 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 "multipotent stem cells" refers to true stem cells that can differentiate into only a limited number of types. For example, bone marrow contains multipotent stem cells that give rise to all blood cells but may not be able to differentiate into other cell types.

[0155] The term "animal-free" with respect to certain compositions, growth conditions, culture media, etc. described herein means that no non-human animal-derived materials, e.g., bovine serum, proteins, lipids, carbohydrates, nucleic acids, vitamins, etc., are used in the preparation, growth, culture, expansion, storage, or formulation of a particular composition or process. "Free of non-human animal-derived materials" means that the materials have never resided in or come into contact with the body or substances of a non-human animal and are therefore free of xenocontamination. Generally, clinical-grade materials, such as recombinantly produced human proteins, are used in the preparation, growth, culture, expansion, storage, and / or formulation of such compositions and / or processes.

[0156] The term "expanded" with respect to a cell composition means that the cell population comprises a significantly higher cell concentration than that obtained using previous methods. For example, the level of cells per gram of amniotic tissue in the expanded AMP cell composition is at least 50-fold and up to 150-fold higher than the number of cells in the primary culture after five passages, compared to an approximately 20-fold increase in such cells using previous methods. In another example, the level of cells per gram of amniotic tissue in the expanded AMP cell composition is at least 30-fold and up to 100-fold higher than the number of cells in the primary culture after three passages. Thus, the "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 encompass only situations in which human intervention has been used to increase the number of cells.

[0157] As used herein, "conditioned medium" refers to medium removed after culturing a particular cell or cell population. When cells are cultured in medium, they may secrete cellular factors that may support or influence 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. Medium containing these cellular factors is conditioned medium. An example of a method for preparing 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 components, such as proteins, that are recovered and / or purified from conditioned medium or, for example, from MSC cells.

[0158] As used herein, the term "mesenchymal stem cell composition" or "MSC composition" refers to conditioned medium derived from MSCs, and optionally, that has undergone further processing. In some embodiments, "MSC secretome" may refer to crude conditioned medium derived from MSCs. In some embodiments, "MSC secretome" may refer to a composition obtained from crude conditioned medium after it has been subjected to further processing, as described herein.

[0159] 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 solutions and / or sugar solutions, cell culture media, and other aqueous or non-aqueous solutions.

[0160] "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 amino acids that have been subsequently modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure different from the general chemical structure of an amino acid but that function in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three-letter abbreviations or the one-letter abbreviations recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter abbreviations.

[0161] 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. This insertion usually consists of the insertion of one or two amino acid residues, although larger "peptide insertions" in the present case may be made, e.g., of about 3 to about 5, or even up to about 10, 15, or 20 amino acid residues. The inserted residue(s) may 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.

[0162] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers.

[0163] "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 implicitly encompasses not only the sequence explicitly indicated, but also conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences. 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-base 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 synonymously 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, as well as hybrid molecules containing DNA and RNA, which 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 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 can be made to DNA and RNA, and thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.

[0164] As used herein, the term "secretome composition" refers to a composition comprising one or more substances secreted from 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 from cells (e.g., culture medium, additives, nutrients, etc.). In some embodiments, a secretome composition does not contain, or contains only trace amounts of, one or more substances not secreted from cells (e.g., culture medium, additives, nutrients, etc.).

[0165] As used herein, the terms "treatment," "treat," or "treating" and the like encompass 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. This also includes preventing (arresting its occurrence), alleviating or ameliorating (reversing), or curing (permanently halting the occurrence or progression) the disease, condition, and / or any associated symptoms. As used herein, the terms "treatment," "treat," or "treating" encompass any treatment of a mammalian, particularly a human, disease or condition, including (a) 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; (b) inhibiting a disease or condition, e.g., arresting its development; (c) alleviating and / or ameliorating a disease or condition, e.g., reversing the disease or condition; or (d) curing a disease or condition, e.g., halting its development or progression. The population of subjects treated by the methods of the invention includes subjects who are at risk of developing an undesirable condition or disease, as well as subjects who are suffering from the condition or disease. In some embodiments, "treatment" (also "treat" or "treating") refers to any administration of therapy that partially or completely alleviates, improves, relieves, prevents, delays the onset of, reduces the severity of, and / or reduces 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 of subjects who do not exhibit symptoms of the associated disease, disorder, and / or condition and / or who exhibit only early symptoms of the disease, disorder, and / or condition. Alternatively and / or additionally, such treatment may be of subjects who exhibit one or more established symptoms of the associated disease, disorder, and / or condition. In some embodiments, treatment may be of 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.

[0166] As used herein, a "wound" refers to any disruption of normal anatomical structure (internal and / or external anatomical structures) from any cause, including, but not limited to, traumatic injuries such as mechanical (e.g., contusion, penetration), thermal, chemical, electrical, radiation, concussive, and incisional injuries; elective surgical injuries such as surgery and resulting incisional hernias, fistulas, and the like; acute, chronic, infected, and sterile wounds; and wounds associated with disease states (e.g., eye contusion). Wounds are dynamic, and the healing process is continuous, requiring a series of integrated, interrelated cellular processes that begin at the time of wounding, continue through initial wound closure, and continue to 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" refers to any form of intervention that improves the natural cellular processes and humoral substances of tissue repair, resulting in faster healing and / or less scarring in the healed area and / or the wound area having tissue strength approaching that of uninjured tissue and / or the wound area achieving some degree of functional recovery.

[0167] As used herein, the terms "a" or "an" mean one or more or at least one.

[0168] As used herein, a "therapeutically effective" or "effective" dose or amount of a composition is an amount sufficient to favorably affect a given medical condition. A therapeutically effective or effective dose or amount may result in a detectable or measurable effect on the health and well-being of a patient over a period of time, if not immediately.

[0169] As used herein, a "pharmaceutical composition" refers to an effective amount of a composition described herein in combination with a delivery component. A pharmaceutical composition can optionally include other components, such as pharmaceutically suitable carriers and excipients, which may facilitate administration of the composition and / or its individual components to a subject.

[0170] The term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to a subject and does not abolish the biological activity and properties of the administered compound.

[0171] The term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound.

[0172] As used herein, the terms "mix," "mixing," and the like refer to a mechanical process or treatment of ingredients. For example, mixing can refer to repeated cycles of pressing and kneading or equivalent processing steps that result in strong compression and mixing of the prepared hydrophobic matrix.

[0173] Adult stem cells can be harvested from various adult tissues, including bone marrow, adipose, and dental pulp tissue. All adult stem cells are capable of self-renewal and are considered multipotent, but their therapeutic functions vary depending on their origin. As a result, each type of adult stem cell has unique characteristics that make it suitable for specific diseases. Mesenchymal stem cells (MSCs) are multipotent, non-hematopoietic (non-blood) stem cells isolated (derived) from the mesoderm, typically capable of differentiating into various tissues, including osteoblasts (e.g., bone cells), chondrocytes (e.g., cartilage cells), myocytes (e.g., muscle cells), and adipocytes (e.g., fat cells that give rise to marrow adipose tissue). As used herein, "isolated" refers to cells removed from their original environment. Stem cells produce factors, such as growth factors, that regulate or are important for the regulation of multiple biological processes. Growth factors are naturally occurring substances, such as agents, that can stimulate cell growth and / or proliferation and / or cell differentiation. Typically, growth factors are proteins or steroid hormones. Terms such as "growth factor" and "factor" are used interchangeably herein, although the term "biological factor" is not limited to growth factors.

[0174] Human mesenchymal stem cells (MSCs) can be characterized by a surface marker profile of CD45- / CD31- / CD73+ / CD90+ / CD105+ / CD44+ (or any suitable subset thereof). (See Bourin et al., Cytotherapy 15(6):641-648 (2013)). Furthermore, suitable stem cells are CD34+ positive 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 to define what is considered a homolog of the above-mentioned human cells, with the remaining markers remaining the same.

[0175] 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 from 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 the biological factors and a reduced number of stem cells than were present prior to the separation. This conditioned medium can be used in the methods described herein and is 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(s) of these biological factors may be bound to or on the surface of the extracellular vesicles (e.g., exosomes), or may be separated from the extracellular vesicles.

[0176] The phrase "drug substance" and variations thereof may refer to a composition or formulation comprising a "secretome," "MSC-CM," "MSC secretome," and / or "MSC-derived secretome," or a portion thereof, that has been further processed, including, but not limited to, by any of the methods described herein.

[0177] In some embodiments, the present disclosure provides a method for preparing and packaging a pharmaceutical formulation comprising one or more pharmaceutical agents, the method comprising: (a) preparing a formulation buffer in a container; (b) diluting a drug substance preparation comprising one or more pharmaceutical agents with a formulation buffer in a container to form a first pharmaceutical formulation; and (c) filling the first formulation into a product container using a blow-fill-seal process to produce a second pharmaceutical formulation, the product container comprising one or more pharmaceutical agents in a therapeutically effective amount, and wherein step (c) is performed in an aseptic system.

[0178] In some embodiments, the sterile system is a closed system that is closed to the external environment. Any closed system suitable for the manufacture of pharmaceutical preparations can be used in the methods of the present invention.

[0179] In some embodiments, step (c) is repeated multiple times to produce multiple filled product containers containing the final pharmaceutical formulation. In some embodiments, step (c) is repeated multiple times simultaneously to produce multiple filled product containers. In some embodiments, step (c) is repeated in continuous cycles to produce multiple filled product containers.

[0180] B. Preparation of Formulation Buffer In some embodiments, the present disclosure provides a formulation buffer for mixing and diluting one or more pharmaceutical agents. In some embodiments, the formulation buffer of step (a) is prepared outside the container before being transferred to the container. In some embodiments, the formulation buffer of step (a) is prepared inside the container before being transferred to the container. In some embodiments, the components of the formulation buffer are added to the container during step (a).

[0181] In some embodiments, the formulation buffer prior to step (a) is sterile. Methods for sterilizing aqueous solutions include, but are not limited to, filtration, heat sterilization (including autoclave sterilization, steam sterilization, and dry heat sterilization), radiation, and chemical sterilization. In some embodiments, the formulation buffer is sterilized by using sterile filtration.

[0182] In some embodiments, the vessel of step (a) is a blending tank. In some embodiments, the blending tank has a volume of 30 L. In some embodiments, the blending tank is equipped with a top-mounted mixer. In some embodiments, step (a) is performed with temperature control.

[0183] In some embodiments, the temperature of the formulation buffer during step (a) is maintained at ambient temperature.

[0184] In some embodiments, the temperature of the formulation buffer during step (a) is maintained at about 20°C to 30°C, or about 25°C.

[0185] In some embodiments, the formulation buffer is transferred to the container in step (a) under a pressure of about 10-15 psi.

[0186] In some embodiments, the formulation buffer comprises one or more buffering agents.

[0187] In some embodiments, the buffering agent includes, but is not limited to, borate, borate-polyol complex, succinate, phosphate buffer, citrate buffer, acetate buffer, carbonate buffer, organic buffer, amino acid buffer, or combinations thereof. In some embodiments, the buffering agent of the formulation buffer includes sodium phosphate monobasic monohydrate and sodium phosphate dibasic anhydrous, and any combination thereof.

[0188] In some embodiments, the formulation buffer comprises about 0.01% to 1% monobasic sodium phosphate monohydrate per mL of formulation buffer. In some embodiments, the formulation buffer comprises about 0.05% to 0.5% monobasic sodium phosphate monohydrate per mL of formulation buffer. In some embodiments, the formulation buffer comprises about 0.1% to 0.2% monobasic sodium phosphate monohydrate per mL of formulation buffer. In some embodiments, the formulation buffer comprises about 0.131% monobasic sodium phosphate monohydrate per mL of formulation buffer.

[0189] In some embodiments, the formulation buffer comprises about 0.01% to 1% dibasic sodium phosphate anhydrous per mL of formulation buffer. In some embodiments, the formulation buffer comprises about 0.1% to 0.9% dibasic sodium phosphate anhydrous per mL of formulation buffer. In some embodiments, the formulation buffer comprises about 0.2% to 0.8% dibasic sodium phosphate anhydrous per mL of formulation buffer. In some embodiments, the formulation buffer comprises about 0.3% to 0.7% dibasic sodium phosphate anhydrous per mL of formulation buffer. In some embodiments, the formulation buffer comprises about 0.4% to 0.6% dibasic sodium phosphate anhydrous per mL of formulation buffer. In some embodiments, the formulation buffer comprises about 0.573% dibasic sodium phosphate anhydrous per mL of formulation buffer.

[0190] In some embodiments, the formulation buffer comprises one or more stabilizers, including, but not limited to, magnesium chloride hexahydrate, mannitol, trehalose dihydrate, or any combination thereof.

[0191] In some embodiments, the formulation buffer comprises about 0.010% to 0.5% magnesium chloride hexahydrate per mL of formulation buffer. In some embodiments, the formulation buffer comprises about 0.015% to 0.25% magnesium chloride hexahydrate per mL of formulation buffer. In some embodiments, the formulation buffer comprises about 0.020% magnesium chloride hexahydrate per mL of formulation buffer.

[0192] In some embodiments, the formulation buffer comprises about 0.1% to 10% trehalose dihydrate per mL of formulation buffer. In some embodiments, the formulation buffer comprises about 0.5% to 8% trehalose dihydrate per mL of formulation buffer. In some embodiments, the formulation buffer comprises about 1% to 6% trehalose dihydrate per mL of formulation buffer. In some embodiments, the formulation buffer comprises about 1.5% to 4% trehalose dihydrate per mL of formulation buffer. In some embodiments, the formulation buffer comprises about 2% to 2.5% trehalose dihydrate per mL of formulation buffer. In some embodiments, the formulation buffer comprises about 2.4% trehalose dihydrate per mL of formulation buffer.

[0193] In some embodiments, the formulation buffer comprises one or more tonicity adjusters, including, but not limited to, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, mannitol, glycerol, propylene glycol, and any combination thereof.

[0194] In some embodiments, the formulation buffer comprises about 0.01% to 1% sodium chloride per mL of formulation buffer. In some embodiments, the formulation buffer comprises about 0.05% to 5% sodium chloride per mL of formulation buffer. In some embodiments, the formulation buffer comprises about 0.1% to 0.15% sodium chloride per mL of formulation buffer. In some embodiments, the formulation buffer comprises about 0.117% sodium chloride per mL of formulation buffer.

[0195] In some embodiments, the formulation buffer comprises about 0.1% to 10% mannitol per mL of formulation buffer. In some embodiments, the formulation buffer comprises about 0.5% to 5% mannitol per mL of formulation buffer. In some embodiments, the formulation buffer comprises about 1% to 2% mannitol per mL of formulation buffer. In some embodiments, the formulation buffer comprises about 1.22% mannitol per mL of formulation buffer.

[0196] In some embodiments, the formulation buffer comprises one or more viscosity agents. In some embodiments, the viscosity agents include, but are not limited to, cellulose-based polymers, polyoxyethylene-polyoxypropylene triblock copolymers, dextran-based polymers, polyvinyl alcohol, dextrin, polyvinylpyrrolidone, polyalkylene glycols, chitosan, collagen, gelatin, hyaluronic acid, or combinations thereof. In some embodiments, the viscosity agent comprises hydroxypropyl methylcellulose.

[0197] In some embodiments, the formulation buffer comprises about 0.01% to 1% hydroxypropyl methylcellulose per mL of formulation buffer. In some embodiments, the formulation buffer comprises about 0.5% to 5% hydroxypropyl methylcellulose per mL of formulation buffer. In some embodiments, the formulation buffer comprises about 0.8% to 2% hydroxypropyl methylcellulose per mL of formulation buffer. In some embodiments, the formulation buffer comprises about 1% hydroxypropyl methylcellulose per mL of formulation buffer.

[0198] In some embodiments, purified water is used as a diluent for the formulation buffer.

[0199] In some embodiments, the formulation buffer comprises the following components:

[0200] [Table 1]

[0201] C. Preparation of Pharmaceutical Formulations In some embodiments, the formulation buffer is mixed with a drug substance preparation containing one or more pharmaceutical agents prior to filling. In some embodiments, the formulation buffer is filtered prior to mixing. In some embodiments, a bioburden reduction filter is used to filter the formulation buffer to remove unwanted components from the formulation buffer. In some embodiments, the formulation is subjected to further purification before being mixed with the drug substance preparation.

[0202] In some embodiments, a drug substance preparation containing one or more pharmaceutical agents is stored frozen, thawed, and then added to a formulation buffer and diluted. In some embodiments, the drug substance preparation is thawed for a predetermined time based on its biophysical and biochemical conditions. In some embodiments, the thawed drug substance preparation is added to a compounding tank containing a formulation buffer and diluted.

[0203] In some embodiments, a quantitative assay is performed on the drug substance preparation and / or the formulation buffer prior to mixing and diluting the drug substance preparation with the formulation buffer. In some embodiments, the protein concentration of the drug substance preparation is measured prior to mixing and dilution. In some embodiments, prior to mixing and dilution, the measured protein concentration is normalized to achieve a target protein concentration in the resulting pharmaceutical formulation, and the volume of the formulation buffer is calculated based on the normalized protein concentration.

[0204] In some embodiments, a drug substance preparation comprising one or more pharmaceutical agents is diluted about 1-10,000-fold or more with a formulation buffer. In some embodiments, the drug substance preparation is diluted about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more with a formulation buffer. In some embodiments, the drug substance preparation is diluted about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more with a formulation buffer. In some embodiments, the drug substance preparation is diluted about 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold or more with a formulation buffer. In some embodiments, the drug substance preparation is diluted about 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10000-fold, or more with the formulation buffer.

[0205] In some embodiments, the pharmaceutical formulation containing the drug substance preparation is sterilized before being subjected to the filling process. In some embodiments, sterilization is achieved by using sterile filtration of the pharmaceutical formulation. In some embodiments, sterile filtration comprises passing the aqueous solution through two or more sterile filters. In some embodiments, sterile filtration comprises passing the aqueous solution through two sterile filters in series. In some embodiments, filter(s) with an average pore size of 0.1-0.45 μm are used for sterilization. In some embodiments, filter(s) with an average pore size of 0.22 μm are used for sterilization. In some embodiments, further purification and / or sterilization is performed on the pharmaceutical formulation before filling. In some embodiments, after mixing and dilution, the first pharmaceutical formulation is transferred to two or more sterile filters under a pressure of about 3-7 psi.

[0206] In some embodiments, the temperature of the formulation buffer and first pharmaceutical formulation is maintained at about 2°C to 8°C during mixing and dilution.

[0207] In some embodiments, the diluted pharmaceutical formulation is collected from the compounding tank and stored in a second container for subsequent use prior to filling.

[0208] In some embodiments, the second container is made of a material other than glass. In some embodiments, the second container is made of polypropylene. In some embodiments, the material of the second container minimizes protein loss by reducing the amount of protein that would otherwise be retained by the container wall.

[0209] In some embodiments, the second container experiences less than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% protein loss compared to the first pharmaceutical formulation upon completion of the mixing and dilution steps.

[0210] D. Blow-fill-seal process In some embodiments, following the mixing, dilution, and sterilization steps, the resulting pharmaceutical formulation comprising one or more pharmaceutical agents disclosed herein is transferred to a filling apparatus and packaged into product containers.

[0211] In some embodiments, the formulations are aseptically packaged using blow-fill-seal technology. Blow-fill-seal (BFS) describes an aseptic filling process in which a hollow container is blown, filled with a sterile product, and sealed, all in one continuous machine cycle. This technology is an alternative to traditional aseptic filling and capping operations, often resulting in cost savings due to higher output and process efficiency.

[0212] Examples of aseptic filling technologies include Weiler® (Elgin, Ill.) (see the World Wide Web at weiler-bfs.com / asep-tech-systems / applications.html) and the BottlePak® aseptic container product line by Rommelag® of Waiblingen, Germany. These technologies utilize the BFS process to form, fill, and seal packages in a single machine under aseptic conditions. The BFS method generally includes the following steps: a) Extrusion: An open blow mold receives a plastic parison extruded from a polymer and is cut from the bottom through a die at the parison head. b) Forming: The main mold closes, simultaneously sealing the bottom. A special mandrel unit lowers into the neck and uses compressed air or vacuum to form the parison into a container. c) Filling: A special mandrel unit fills the container with a precisely metered amount of product from a dosing unit. d) Sealing: After the special mandrel unit retracts, the head mold closes and a vacuum forms the necessary seal. e) Mold Opening: Opening the blow mold removes the container from the machine and the cycle repeats. BFS containers may have a threaded section which, when threaded, exposes an applicator formed integrally with the package.

[0213] Exemplary BFS products include single-dose eye drop ampoules with sharp dropper tips or small parenteral containers with Luer-Lock connectors (see the World Wide Web at rommelag.com / en / 05applications / 05otherpharmaceutical.html for details). Some BottlePak® products include an insert that is inserted into the package near the threads after filling and before sealing, so that unscrewing a breakable portion of the package exposes the insert and facilitates dispensing of the product contained within (hereafter "Blow-Fill-Insert-Seal" or "BFIS"). An example of such a product is a bellows-structured squeezable container with a stainless steel cannula insert for subcutaneous administration, approximately 3 ml. Another example of a BottlePak® BFIS product is a 1-liter infusion bottle with a rubber septum to which an infusion set can be connected via a spike. The above manufacturing method is taught by U.S. Patent Nos. 7,192,549, 7,004,213, 5,836,922, and 5,687,550, each of which is incorporated herein by reference in its entirety.

[0214] BottlePak® products and similar BFS and BFIS products are available from Weiler® (Elgin, Ill.) (see worldwide web at weiler-bfs.com / asep-techsystems / applications.html).

[0215] Additional aseptic packages similar in form and function to the BFS and BFIS unit dose containers are manufactured by the form-fill-seal process ("FFS") and are available from Sarong, Italy (www.sarong.it) and Unifil of Villafranca Di Medolla, Italy (www.unifil.it). The FFS process forms individual packages from a continuous film strip that is thermoformed, partially sealed, filled, and then sealed on a single piece of equipment. Inserts can be introduced after the forming step and before the sealing step (before or after filling), or after the package is sealed.

[0216] Similar sterile packages are produced by injection molding strips of several packages or individual packages and then filling and sealing them from the back side (i.e., not the dispensing side) in a subsequent step. Inserts can be incorporated into the package during or after the injection molding step. Such packages and equipment for making them are available from Lameplast® Rovereto di Novi, Italy (www.lameplast.it) and Sanner Plastic Technology of Bensheim, Germany (www.sanner.de).

[0217] In some embodiments, product sterility is achieved by carrying out production under aseptic and sterile conditions using pre-sterilized raw materials, components and substances, or by sterilizing the product after production or after a specific production step, or by a combination of any of the above approaches.

[0218] In some embodiments, the pre-filled pharmaceutical formulations of the present disclosure are aseptically filled into 10 mL round bottles using a Weiler® 624 BFS machine. In some embodiments, pharmaceutically acceptable packaging materials for the formulations provided herein can include, but are not limited to, polypropylene, polystyrene, low-density polyethylene (LDPE), high-density polyethylene (HDPE), polycarbonate, polyvinylidene chloride, and other materials known to those skilled in the art. In some embodiments, the packaging material of the product container is LDPE. In some embodiments, LDPE resin is used as the starting material for filling. In some embodiments, the 10 mL round bottles are made from LDPE.

[0219] In some embodiments, the BFS process produces an assembled product container comprising a 10 mL round bottle with a low viscosity tip attached to the top of the container and a removable screw-on cap, which is attached to the tip and seals the product container.

[0220] In some embodiments, the BFS machine performs multiple cycles simultaneously to produce an array of filled product containers. In some embodiments, the BFS machine performs successive cycles to produce multiple arrays of filled product containers.

[0221] In some embodiments, the target fill volume of the pharmaceutical formulation in a 10 mL bottle is about 7.1 mL, 7.2 mL, 7.3 mL, 7.4 mL, 7.5 mL, 7.6 mL, 7.7 mL, 7.8 mL, 7.9 mL, 8 mL, 8.1 mL, 8.2 mL, 8.3 mL, 8.4 mL, 8.5 mL, 8.6 mL, 8.7 mL, 8.8 mL, 8.9 mL, 9 mL, 9.1 mL, 9.2 mL, 9.3 mL, 9.4 mL, 9.5 mL, 9.6 mL, 9.7 mL, 9.8 mL, or 9.9 mL. In some embodiments, an average of 7.1 mL, 7.2 mL, 7.3 mL, 7.4 mL, 7.5 mL, 7.6 mL, 7.7 mL, 7.8 mL, 7.9 mL, 8 mL, 8.1 mL, 8.2 mL, 8.3 mL, 8.4 mL, 8.5 mL, 8.6 mL, 8.7 mL, 8.8 mL, 8.9 mL, 9 mL, 9.1 mL, 9.2 mL, 9.3 mL, 9.4 mL, 9.5 mL, 9.6 mL, 9.7 mL, 9.8 mL, or 9.9 mL of pharmaceutical formulation is filled. In some embodiments, an average of about 7.8 to 8.65 g of pharmaceutical formulation is filled. In some embodiments, an average of about 8.28 g of pharmaceutical formulation is filled.

[0222] In some embodiments, the filled product container comprises a therapeutically effective amount of one or more pharmaceutical agents for treating an ophthalmic condition.

[0223] In some embodiments, a single dose of the pharmaceutical formulation is filled into the product container. In some embodiments, multiple doses of the pharmaceutical formulation are filled into the product container.

[0224] In some embodiments, following the BFS process, the assembled product container is evaluated and the BFS process / parameters are adjusted based on the results of the evaluation. In some embodiments, the evaluation includes sampling the product container and evaluating the bottle wall thickness, tare weight, functional performance (such as the force required to remove a screw-on cap), and appearance of the product container for BFS leaks, thin / excessive plastic, attachment of the cap to the overcap, open gaps or cosmetic defects.

[0225] In some embodiments, the first pharmaceutical formulation is maintained at about 20°C to 30°C, or about 25°C, between the completion of dilution and the start of the BFS process.

[0226] In some embodiments, during the BFS process, the first pharmaceutical formulation is exposed to about 60°C to 70°C for about 1 second to 10 seconds, about 10 seconds to 20 seconds, about 20 seconds to 30 seconds, about 30 seconds to 40 seconds, about 40 seconds to 50 seconds, or about 50 seconds to 50 seconds, and the first pharmaceutical formulation is maintained at about 20°C to 30°C, and in some embodiments, 25°C, for the remainder of the BFS process.

[0227] In some embodiments, the assembled product container is stored frozen. In some embodiments, the product container is stored frozen at about -20°C immediately after the BFS process is completed.

[0228] E. Feasibility Testing In some embodiments, samples are taken from the pharmaceutical formulation during dilution and / or filling and quantitative measurements are performed for at least the following purposes: · Identifying formulation process parameters that are critical to achieving a reproducible quality finished product. Determine the critical BFS process parameters to achieve forming and filling with defined quality attributes. · Determine the initial product purge required to eliminate the dilution effect as a result of the BFS steam treatment process. · Freezer testing to determine the time required for the pellets of the finished product to freeze.

[0229] In some embodiments, a sample of the drug substance preparation is collected prior to mixing / dilution in the compounding tank and its protein concentration is measured. In some embodiments, a sample of the pharmaceutical formulation is collected at the start of mixing / dilution in the compounding tank and its protein concentration is measured.

[0230] In some embodiments, a sample of the pharmaceutical formulation is collected during mixing / dilution in the compounding tank and its protein concentration is measured. In some embodiments, the sample is collected about 15 minutes or 30 minutes after final mixing. In some embodiments, the sample is collected from the top of the compounding tank. In some embodiments, the sample is collected from the center of the compounding tank. In some embodiments, the sample is collected from the bottom of the compounding tank. In some embodiments, the sample is collected from the top of the compounding tank after about 30 minutes of final mixing. In some embodiments, the sample is collected from the center of the compounding tank after about 30 minutes of final mixing. In some embodiments, the sample is collected from the bottom of the compounding tank after about 30 minutes of final mixing. In some embodiments, a sample of the pharmaceutical formulation at the completion of mixing / dilution is collected and its protein concentration is measured.

[0231] In some embodiments, a sample of the pharmaceutical formulation after mixing / dilution in the compounding tank is collected and its protein concentration is measured. In some embodiments, a sample of the pharmaceutical formulation after mixing / dilution and before filling into the BFS is collected and its protein concentration is measured. In some embodiments, a sample of the pharmaceutical formulation from the inlet tubing before the solution passes through the first sterile filter is collected and its protein concentration is measured. In some embodiments, a sample of the pharmaceutical formulation from the tubing connecting the first and second sterile filters is collected and its protein concentration is measured. In some embodiments, a sample of the pharmaceutical formulation from the outlet tubing after passing through the second sterile filter is collected and its protein concentration is measured.

[0232] In some embodiments, a sample of the pharmaceutical formulation is collected at the start of the BFS fill and its protein concentration is measured. In some embodiments, a sample of the pharmaceutical formulation is collected during the BFS fill and its protein concentration is measured. In some embodiments, samples are collected at regular intervals during the BFS fill. In some embodiments, a sample is collected for every 0.2 kg of pharmaceutical formulation that is filled. In some embodiments, samples are collected when the BFS fill reaches 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% completion. In some embodiments, a sample of the pharmaceutical formulation is collected at the completion of the BFS fill and its protein concentration is measured.

[0233] In some embodiments, the drug substance preparations provided herein comprise mesenchymal stem cell (MSC) secretome. In some embodiments, the pharmaceutical formulations provided herein comprise HGF, serpin A1, TIMP-1, TSG-14, IL-8, serpin E1, VEGF-A, PEDF, and fibronectin (FN).

[0234] In some embodiments, the pharmaceutical formulation after filling and packaging comprises a total protein concentration of at least about 30 pg / mL, 40 pg / mL, 50 pg / mL, 60 pg / mL, 70 pg / mL, 80 pg / mL, 90 pg / mL, 100 pg / mL, 110 pg / mL, 120 pg / mL, 130 pg / mL, 140 pg / mL, 150 pg / mL, 160 pg / mL, 170 pg / mL, 180 pg / mL, 190 pg / mL, 200 pg / mL, 210 pg / mL, 220 pg / mL, 230 pg / mL, 240 pg / mL, 250 pg / mL, 260 pg / mL, 270 pg / mL, 280 pg / mL, 290 pg / mL, 300 pg / mL, or more. In some embodiments, the pharmaceutical formulation after filling and packaging comprises a total protein concentration of at least about 75 ng / mL.

[0235] In some embodiments, the pharmaceutical formulation after filling and packaging has at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46% of each of the pharmaceutical agents from the original drug substance preparation prior to the mixing and dilution steps. , 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% hold.

[0236] In some embodiments, the filled product container comprises a total protein concentration of at least about 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, 190 ng / mL, 200 ng / mL, 210 ng / mL, 220 ng / mL, 230 ng / mL, 240 ng / mL, 250 ng / mL, or more. In some embodiments, the filled product container comprises a total protein concentration of at least about 75 ng / mL. In some embodiments, upon completion of the filling step, the total yield of each of the one or more pharmaceutical agents in the filled product container or multiple filled product containers is about 40%, 50%, 60%, 70%, 80%, 90%, or more of the pharmaceutical agents in the original drug substance preparation.

[0237] In some embodiments, the pharmaceutical formulation after BFS filling and packaging contains at least about 1 pg / mL or at least about 5 pg / mL of HGF, hi some embodiments, the pharmaceutical formulation after filling and packaging contains about 0.5-30 pg / mL of HGF. In some embodiments, the pharmaceutical formulation after filling and packaging contains at least about 0.5 pg / mL, 1 pg / mL, 2 pg / mL, 3 pg / mL, 4 pg / mL, 5 pg / mL, 6 pg / mL, 7 pg / mL, 8 pg / mL, 9 pg / mL, 10 pg / mL, 11 pg / mL, 12 pg / mL, 13 pg / mL, 14 pg / mL, 15 pg / mL, 16 pg / mL, 17 pg / mL, 18 pg / mL, 19 pg / mL, 20 pg / mL, 21 pg / mL, 22 pg / mL, 23 pg / mL, 24 pg / mL, 25 pg / mL, 26 pg / mL, 27 pg / mL, 28 pg / mL, 29 pg / mL, 30 pg / mL, or more HGF.

[0238] In some embodiments, the pharmaceutical formulation after BFS filling and packaging contains at least about 50 pg / mL of serpin A1, hi some embodiments, the pharmaceutical formulation after filling and packaging contains about 50-600 pg / mL of serpin A1. In some embodiments, the pharmaceutical formulation after filling and packaging has a cytotoxicity of at least about 50 pg / mL, 60 pg / mL, 70 pg / mL, 80 pg / mL, 90 pg / mL, 100 pg / mL, 110 pg / mL, 120 pg / mL, 130 pg / mL, 140 pg / mL, 150 pg / mL, 160 pg / mL, 170 pg / mL, 180 pg / mL, 190 pg / mL, 200 pg / mL, 210 pg / mL, 220 pg / mL, 230 pg / mL, 240 pg / mL, 250 pg / mL, 260 pg / mL, 270 pg / mL, 280 pg / mL, 290 pg / mL, 300 pg / mL, 310 pg / mL, 320 pg / mL, 330 pg / mL, 340 pg / mL, 350 pg / mL, 360 pg / mL, 370 pg / mL, 380 pg / mL, 390 pg / mL, 400 pg / mL, 410 pg / mL, 420 pg / mL, 430 pg / mL, 440 pg / mL, 450 pg / mL, 460 pg / mL, 470 pg / mL, 480 pg / mL, 490 pg / mL, 500 pg / mL, 510 pg / mL, 520 pg / mL, 530 pg / mL, 540 pg / mL, 550 pg / mL, 560 pg / mL, 570 pg / mL, 580 pg / mL, 590 pg / mL pg / mL, 330 pg / mL, 340 pg / mL, 350 pg / mL, 360 pg / mL, 370 pg / mL, 380 pg / mL, 390 pg / mL, 400 pg / mL, 410 pg / mL, 420 pg / mL, 430 pg / mL, 440 pg / mL, 450 pg / mL, 460 pg / mL, 470 pg / mL, 480 pg / mL, 490 pg / mL, 500 pg / mL, 510 pg / mL, 520 pg / mL, 530 pg / mL, 540 pg / mL, 550 pg / mL, 560 pg / mL, 570 pg / mL, 580 pg / mL, 590 pg / mL, 600 pg / mL, or more of serpin A1.

[0239] In some embodiments, the pharmaceutical formulation after BFS filling and packaging contains at least about 1000 pg / mL of TIMP-1, hi some embodiments, the pharmaceutical formulation after filling and packaging contains about 1000-5000 pg / mL of TIMP-1. In some embodiments, the pharmaceutical formulation after BFS filling and packaging has a β-glucan-1-phosphate dehydrogenase (Gd) concentration of at least about 500 pg / mL, 600 pg / mL, 700 pg / mL, 800 pg / mL, 900 pg / mL, 1000 pg / mL, 1100 pg / mL, 1200 pg / mL, 1300 pg / mL, 1400 pg / mL, 1500 pg / mL, 1600 pg / mL, 1700 pg / mL, 1800 pg / mL, 1900 pg / mL, 2000 pg / mL, 2100 pg / mL, 2200 pg / mL, 2300 pg / mL, 2400 pg / mL, 2500 pg / mL, 2600 pg / mL, containing 2700pg / mL, 2800pg / mL, 2900pg / mL, 3000pg / mL, 3100pg / mL, 3200pg / mL, 3300pg / mL, 3400pg / mL, 3500pg / mL, 3600pg / mL, 3700pg / mL, 3800pg / mL, 3900pg / mL, 4000pg / mL, 4100pg / mL, 4200pg / mL, 4300pg / mL, 4400pg / mL, 4500pg / mL, 4600pg / mL, 4700pg / mL, 4800pg / mL, 4900pg / mL, 5000pg / mL, or more TIMP-1.

[0240] In some embodiments, the pharmaceutical formulation after BFS filling and packaging contains at least about 10 pg / mL of TSG-14. In some embodiments, the pharmaceutical formulation after BFS filling and packaging contains about 10-200 pg / mL of TSG-14. In some embodiments, the pharmaceutical formulation after BFS filling and packaging contains at least about 10 pg / mL, 20 pg / mL, 30 pg / mL, 40 pg / mL, 50 pg / mL, 60 pg / mL, 70 pg / mL, 80 pg / mL, 90 pg / mL, 100 pg / mL, 110 pg / mL, 120 pg / mL, 130 pg / mL, 140 pg / mL, 150 pg / mL, 160 pg / mL, 170 pg / mL, 180 pg / mL, 190 pg / mL, 200 pg / mL, or more of TSG-14.

[0241] In some embodiments, the pharmaceutical formulation after BFS filling and packaging contains at least about 0.1 pg / mL of IL-8, hi some embodiments, the pharmaceutical formulation after filling and packaging contains about 0.1-5 pg / mL of IL-8. In some embodiments, the pharmaceutical formulation after BFS filling and packaging has a cytotoxicity of at least about 0.1 pg / mL, 0.2 pg / mL, 0.3 pg / mL, 0.4 pg / mL, 0.5 pg / mL, 0.6 pg / mL, 0.7 pg / mL, 0.8 pg / mL, 0.9 pg / mL, 1.0 pg / mL, 1.1 pg / mL, 1.2 pg / mL, 1.3 pg / mL, 1.4 pg / mL, 1.5 pg / mL, 1.6 pg / mL, 1.7 pg / mL, 1.8 pg / mL, 1.9 pg / mL, 2.0 pg / mL, 2.1 pg / mL, 2.2 pg / mL, 2.3 pg / mL, 2.4 pg / mL, IL-8, 2.5pg / mL, 2.6pg / mL, 2.7pg / mL, 2.8pg / mL, 2.9pg / mL, 3.0pg / mL, 3.1pg / mL, 3.2pg / mL, 3.3pg / mL, 3.4pg / mL, 3.5pg / mL, 3.6pg / mL, 3.7pg / mL, 3.8pg / mL, 3.9pg / mL, 4.0pg / mL, 4.1pg / mL, 4.2pg / mL, 4.3pg / mL, 4.4pg / mL, 4.5pg / mL, 4.6pg / mL, 4.7pg / mL, 4.8pg / mL, 4.9pg / mL, 5.0pg / mL, or more.

[0242] In some embodiments, the pharmaceutical formulation after BFS filling and packaging contains at least about 700 pg / mL of serpin E1, hi some embodiments, the pharmaceutical formulation after filling and packaging contains about 700-5000 pg / mL of serpin E1. In some embodiments, the pharmaceutical formulation after BFS filling and packaging has a cytotoxicity of at least about 700 pg / mL, 800 pg / mL, 900 pg / mL, 1000 pg / mL, 1100 pg / mL, 1200 pg / mL, 1300 pg / mL, 1400 pg / mL, 1500 pg / mL, 1600 pg / mL, 1700 pg / mL, 1800 pg / mL, 1900 pg / mL, 2000 pg / mL, 2100 pg / mL, 2200 pg / mL, 2300 pg / mL, 2400 pg / mL, 2500 pg / mL, 2600 pg / mL, 2700 pg / mL , 2800pg / mL, 2900pg / mL, 3000pg / mL, 3100pg / mL, 3200pg / mL, 3300pg / mL, 3400pg / mL, 3500pg / mL, 3600pg / mL, 3700pg / mL, 3800pg / mL, 3900pg / mL, 4000pg / mL, 4100pg / mL, 4200pg / mL, 4300pg / mL, 4400pg / mL, 4500pg / mL, 4600pg / mL, 4700pg / mL, 4800pg / mL, 4900pg / mL, 5000pg / mL, or more of Serpin E1.

[0243] In some embodiments, the pharmaceutical formulation after BFS filling and packaging contains at least about 4 pg / mL of VEGF-A. In some embodiments, the pharmaceutical formulation after BFS filling and packaging contains about 4-40 pg / mL of VEGF-A. In some embodiments, the pharmaceutical formulation after BFS filling and packaging contains at least about 4 pg / mL, 5 pg / mL, 6 pg / mL, 7 pg / mL, 8 pg / mL, 9 pg / mL, 10 pg / mL, 11 pg / mL, 12 pg / mL, 13 pg / mL, 14 pg / mL, 15 pg / mL, 16 pg / mL, 17 pg / mL, 18 pg / mL, 19 pg / mL, 20 pg / mL, 21 pg / mL, 22 pg / mL, 23 pg / mL, 24 pg / mL, 25 pg / mL, 26 pg / mL, 27 pg / mL, 28 pg / mL, 29 pg / mL, 30 pg / mL, 31 pg / mL, 32 pg / mL, 33 pg / mL, 34 pg / mL, 35 pg / mL, 36 pg / mL, 37 pg / mL, 38 pg / mL, 39 pg / mL, 40 pg / mL, 41 pg / mL, 42 pg / mL, 43 pg / mL, 44 pg / mL, 45 pg / mL, 46 pg / mL, 47 pg / mL, 48 pg / mL, 49 pg / mL, 50 pg / mL, 51 pg / mL, 52 pg / mL, 53 pg / mL, 54 pg / mL, 55 pg / mL, 56 pg / mL, 57 pg / mL, 58 pg / mL pg / mL, 22pg / mL, 23pg / mL, 24pg / mL, 25pg / mL, 26pg / mL, 27pg / mL, 28pg / mL, 29pg / mL, 30pg / mL, 31pg / mL, 32pg / mL, 33pg / mL, 34pg / mL, 35pg / mL, 36pg / mL, 37pg / mL, 38pg / mL, 39pg / mL, 40pg / mL, and more VEGF-A.

[0244] In some embodiments, the pharmaceutical formulation after BFS filling and packaging contains at least about 100 pg / mL of PEDF. In some embodiments, the pharmaceutical formulation after BFS filling and packaging contains about 100-400 pg / mL of PEDF. In some embodiments, the pharmaceutical formulation after BFS filling and packaging contains at least about 100 pg / mL, 110 pg / mL, 120 pg / mL, 130 pg / mL, 140 pg / mL, 150 pg / mL, 160 pg / mL, 170 pg / mL, 180 pg / mL, 190 pg / mL, 200 pg / mL, 210 pg / mL, 220 pg / mL, 230 pg / mL, Contains 240pg / mL, 250pg / mL, 260pg / mL, 270pg / mL, 280pg / mL, 290pg / mL, 300pg / mL, 310pg / mL, 320pg / mL, 330pg / mL, 340pg / mL, 350pg / mL, 360pg / mL, 370pg / mL, 380pg / mL, 390pg / mL, 400pg / mL, or more of PEDF.

[0245] In some embodiments, the pharmaceutical formulation after BFS filling and packaging contains at least about 30 pg / mL of FN, hi some embodiments, the pharmaceutical formulation after filling and packaging contains about 30-300 pg / mL of FN. In some embodiments, the pharmaceutical formulation after BFS filling and packaging comprises at least about 30 pg / mL, 40 pg / mL, 50 pg / mL, 60 pg / mL, 70 pg / mL, 80 pg / mL, 90 pg / mL, 100 pg / mL, 110 pg / mL, 120 pg / mL, 130 pg / mL, 140 pg / mL, 150 pg / mL, 160 pg / mL, 170 pg / mL, 180 pg / mL, 190 pg / mL, 200 pg / mL, 210 pg / mL, 220 pg / mL, 230 pg / mL, 240 pg / mL, 250 pg / mL, 260 pg / mL, 270 pg / mL, 280 pg / mL, 290 pg / mL, 300 pg / mL, or more FN.

[0246] F. MSC Secretome Compositions and Formulations According to the present specification, there is provided herein a conditioned medium comprising a mesenchymal stem cell (MSC) secretome and / or a composition comprising a mesenchymal stem cell (MSC) secretome (including a processed MSC secretome).

[0247] The present invention provides a mesenchymal stem cell (MSC) secretome composition comprising: i. IDO (indoleamine-2,3-dioxygenase) enzyme activity 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-β, 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.

[0248] 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 at between 1 ng / mL and 8 ng / mL.

[0249] In some embodiments, the MSC secretome further comprises "mid-range" levels 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 / or uPA, optionally at between 400 pg / mL and 3000 pg / mL.

[0250] In some embodiments, the MSC secretome further comprises at least one factor selected from the group consisting of apolipoprotein A1, complement factor D, complement factor H, complement factor I, C1 esterase inhibitor (C1-INH), C4b binding protein (C4BP), CD46, C-reactive protein, cystatin C, DKK-1, Emmprin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15, and / or IFNγ.

[0251] In some embodiments, the MSC secretome comprises a ratio of anti-angiogenic factors to pro-angiogenic factors, where the ratio is greater than 2, greater than 3, greater than 4, or greater than 5.

[0252] In some embodiments, the MSC secretome further comprises "low" levels of VEGF, optionally between 0 pg / mL and 200 pg / mL.

[0253] In some embodiments, the level of VEGF is 5 to 10 times lower than the level of Serpin E1.

[0254] In some embodiments, the composition comprises one or more angiogenesis inhibitors, and the sum of the concentrations of the one or more angiogenesis inhibitors relative to the concentration of VEGF is greater than 2, greater than 3, greater than 4, or greater than 5.

[0255] In some embodiments, the MSC secretome is free of bFGF, PLGF, and PDGF and / or contains very low levels, sometimes less than 1000 pg / mL.

[0256] In some embodiments, the MSC secretome composition has a pH of about 4.7 to about 7.5.

[0257] In some embodiments, the MSC secretome 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 citric acid / disodium phosphate.

[0258] In some embodiments, the MSC secretome composition further comprises a tonicity agent.

[0259] In some embodiments, the tonicity adjusting agent is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin.

[0260] In some embodiments, the MSC secretome further comprises mono / disodium phosphate, mannitol, and trehalose, and the composition has a pH of about pH 7.4.

[0261] In some embodiments, the MSC secretome further comprises a divalent cation.

[0262] In some embodiments, the divalent cation is selected from the group consisting of Mg2+, Ca2+, and Zn2+.

[0263] 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.

[0264] In some embodiments, the composition further comprises an adhesive agent.

[0265] In some embodiments, the adhesive agent is selected from the group consisting of hypromellose, Poloxamer 407, Poloxamer 188, Poloxomer 237, Poloxomer 338, hypromellose, (HPMC), polycarbophil, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyimide, sodium hyaluronate, gellan gum, poly(lactic-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethylcellulose (CMC), or hydroxypropylmethylcellulose (HPMC), hydroxymethylcellulose, hydroxyethylcellulose, sodium carboxymethylcellulose, fibrin glue, polyethylene glycol, and GelCORE.

[0266] The present invention also provides a method of producing a mesenchymal stem cell (MSC) secretome composition, the method comprising: i. culturing mesenchymal stem cells (MSCs) in a first culture medium; ii. removing the first culture medium of step (i) from the MSCs; iii. Washing the MSCs of step (ii); iv. adding a second culture medium and culturing for about 1 to 5 days; v. collecting the second culture medium from step (iv) as conditioned medium; and vi. Processing the conditioned medium of step (v) into an MSC secretome composition as described herein.

[0267] In some embodiments, the MSC secretome composition is a secretome composition described herein.

[0268] In some embodiments, step (vi) of processing the conditioned medium of step (v) into a secretome composition comprises: a) filtering the conditioned medium collected in step (v) to remove cellular particulates; b) concentrating the filtered conditioned medium from step (a); and c) Buffer exchange with formulation buffer.

[0269] 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.

[0270] 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).

[0271] In some embodiments, the concentrating step (b) comprises using hollow fiber filters, tangential flow filtration systems, or centrifugation-based size exclusion techniques.

[0272] In some embodiments, the centrifugation-based size exclusion technique uses a MW cutoff of 3-10 kDa.

[0273] In some embodiments, the present invention provides methods for treating ocular diseases, 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.

[0274] In some embodiments, the composition is administered to a target area.

[0275] The present invention also provides a method for treating visual dysfunction following traumatic injury to an ocular structure in a patient in need thereof, 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.

[0276] The present invention also provides a method for inducing and / or promoting ocular wound healing in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a mesenchymal stem cell secretome composition described herein or a composition made according to the methods described herein.

[0277] The present invention also provides methods for reducing and / or preventing neovascularization, reducing and / or preventing scarring, improving and / or maintaining vision, and / or increasing the rate of wound closure (e.g., decreasing wound closure time) in a patient in need thereof, 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.

[0278] The present invention also provides methods for reducing and / or preventing neovascularization and reducing scarring in a patient in need thereof to promote vision preservation, 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.

[0279] In some embodiments, the mesenchymal stem cell secretome composition is formulated for topical administration.

[0280] In some embodiments, the mesenchymal stem cell secretome composition is formulated for subconjunctival injection.

[0281] In some embodiments, the mesenchymal stem cell secretome composition is formulated for intravitreal injection.

[0282] 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 (the characterization assays being selected from the group consisting of physical component characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analyses, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / citralization assays, inflammation assays, epithelial barrier integrity assays, retinal degeneration assays, and / or assays for inherited retinal diseases including human and animal retinal explants, neuroprotective / neurotrophic assays); and (ii) determining the results from one or more assays in (i);

[0283] The present invention also provides a method for determining the biopotency and stability of an MSC secretome, the method comprising: (i) subjecting the MSC secretome to one or more characterization assays (the characterization assays being selected from the group consisting of physical component characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analyses, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / citralization assays, inflammation assays, epithelial barrier integrity assays, retinal degeneration assays, and / or assays for inherited retinal diseases including human and animal retinal explants, neuroprotective / neurotrophic assays); and (ii) determining the results from one or more assays in (i);

[0284] The present invention also provides a method for determining the consistency of an MSC secretome lot between multiple MSC secretome lots, the method comprising: (i) subjecting the MSC secretome to one or more characterization assays (the characterization assays being selected from the group consisting of physical component characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analyses, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / citralization assays, inflammation assays, epithelial barrier integrity assays, retinal degeneration assays, and / or assays for inherited retinal diseases including human and animal retinal explants, neuroprotective / neurotrophic assays); and (ii) determining the results from one or more assays in (i);

[0285] In some embodiments, results from characterizing the physical components of (ii) identify the angiogenesis-suppressing MSC secretome described herein.

[0286] In some embodiments, the results from the safety analysis in (ii) demonstrate that the MSC secretome is hemocompatible and has low and / or no pyrogens and / or endotoxins.

[0287] In some embodiments, the results from the stability assay in (ii) show the MSC secretome to be stable at 4°C, 20°C, and / or 25°C (or room temperature) for at least 7 days.

[0288] In some embodiments, the results from the proliferation assay in (ii) are indicative of an MSC secretome that induces proliferation.

[0289] In some embodiments, the results from the migration assay of (ii) are indicative of an MSC secretome that induces migration.

[0290] In some embodiments, results from the adhesion assay (ii) indicate an MSC secretome that induces cell adhesion. The adhesion assay can be performed using techniques known in the art. Exemplary disclosures of adhesion assays are provided in U.S. Patent Publication Nos. 20170067061 A1 and 20150050325 A1, and Blue et al., Blood 2008, 111, 1248, each of which is incorporated herein by reference in its entirety.

[0291] In some embodiments, the results from the angiogenesis assay in (ii) indicate an MSC secretome that inhibits or does not promote angiogenesis.

[0292] In some embodiments, the results from the differentiation / scarring assay in (ii) indicate an MSC secretome that inhibits differentiation and / or scarring.

[0293] In some embodiments, the results from the inflammation assay in (ii) indicate an MSC secretome that inhibits inflammation.

[0294] In some embodiments, the method further comprises: (iii) Identifying MSC secretome lots based on the results of (ii).

[0295] 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 selected from the group consisting of physical component characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analyses, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / citralization assays, inflammation assays, epithelial barrier integrity assays, retinal degeneration assays, and / or assays for inherited retinal diseases including human and animal retinal explants, and neuroprotective / neurotrophic assays.

[0296] The present invention also provides a panel of tests and / or assays for determining lot-to-lot consistency of MSC secretomes, the panel comprising one or more characterization assays selected from the group consisting of physical characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analyses, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / citralization assays, inflammation assays, epithelial barrier integrity assays, retinal degeneration assays, and / or assays for inherited retinal diseases including human and animal retinal explants, and neuroprotective / neurotrophic assays.

[0297] In some embodiments, characterization of physical elements identifies the MSC secretome described herein.

[0298] In some embodiments, the results from the safety analysis in (ii) demonstrate that the MSC secretome is hemocompatible and has low and / or no pyrogens and / or endotoxins.

[0299] In some embodiments, a stability assay identifies an MSC secretome that exhibits stability at 4°C, 20°C, and / or 25°C (or room temperature) for at least 7 days.

[0300] In some embodiments, a proliferation assay identifies MSC secretomes that induce proliferation.

[0301] In some embodiments, a migration assay identifies the MSC secretome that induces migration.

[0302] In some embodiments, angiogenesis assays identify MSC secretomes that inhibit or do not promote angiogenesis.

[0303] In some embodiments, differentiation / scarring assays identify MSC secretomes that inhibit differentiation and / or scarring.

[0304] In some embodiments, an inflammation assay identifies MSC secretomes that inhibit inflammation.

[0305] In some embodiments, physical characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analyses, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / scarring assays, inflammation assays, epithelial barrier integrity assays, retinal degeneration assays, and assays for inherited retinal diseases including human and animal retinal explants, neuroprotective / neurotrophic assays are all performed.

[0306] In some embodiments, a panel of tests and / or assays described herein identifies the MSC secretome described herein.

[0307] In some embodiments, the panel of tests and / or assays described herein comprises at least one migration assay. In some embodiments, the migration assay is an in vitro wound closure assay. In some embodiments, the in vitro wound closure assay is selected from the group consisting of a "scratch assay" (also referred to as a "scratch wound assay"), a circular scratch wound method, a circular scratch wound assay, and a circular wound closure assay. In some embodiments, the MSC secretome is an anti-angiogenic MSC secretome and / or an anti-scarring MSC secretome.

[0308] In some embodiments, the MSC secretome is an anti-angiogenic MSC secretome and / or an anti-scarring MSC secretome.

[0309] In some embodiments, the MSC secretome is an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.

[0310] The present invention also provides a stable mesenchymal stem cell (MSC) secretome preparation comprising: i. 1-20 μg, optionally 2 μg-8 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-24mg trehalose dihydrate; vi. 0.5 mg to 2 mg of hypromellose per mL; and / or The pH is about 4.7 to about 7.5.

[0311] The present invention also provides a stable mesenchymal stem cell (MSC) secretome preparation comprising: i. 0.004%-0.0375%, optionally 0.008%-0.015% w / w of MSC secretome; ii. 4%-5% w / w of monobasic sodium phosphate; iii. 21.5%-23% w / w of dibasic sodium phosphate; iv. 23%-25% w / w mannitol; v. 46%-48% w / w trehalose dehydrate; vi. 1% to 3% w / w of hypromellose; and / or The pH is about 4.7 to about 7.5.

[0312] The present invention also provides a stable mesenchymal stem cell (MSC) secretome preparation comprising: i. 6 μg 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 trehalose dihydrate; vi. 1 mg of hypromellose per mL; and / or The pH is approximately 7.4.

[0313] The present invention also provides a stable mesenchymal stem cell (MSC) secretome preparation comprising: i. 0.012% w / w MSC secretome; ii. 4.5% w / w sodium phosphate monobasic; iii. 22.4% w / w dibasic sodium phosphate; iv. 24% w / w mannitol; v. 47.1% w / w trehalose dehydrate; vi. 2.0% w / w hypromellose; and / or The pH is approximately 7.4.

[0314] In some embodiments, the monobasic sodium phosphate is a monohydrate. In some embodiments, the dibasic sodium phosphate is anhydrous.

[0315] The present invention also provides a mesenchymal stem cell (MSC) secretome composition comprising: i. optionally, 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. optionally, at least one additional factor selected from the group consisting of PEDF (serpin F1), serpin A1, IGFBP-2, IGFBP-3, SDF-1, TSG-14, kallikrein 3, MCP-1, angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-β, and PDGF; and / or iii. Optionally, 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.

[0316] 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.

[0317] 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.

[0318] In some embodiments, the MSC secretome composition further comprises mid-range levels 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.

[0319] In some embodiments, the MSC secretome composition comprises between 400 pg / mL and 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.

[0320] In some embodiments, the MSC secretome composition further comprises at least one factor selected from the group consisting of apolipoprotein A1, complement factor D, complement factor H, complement factor I, C1 esterase inhibitor (C1-INH), C4b binding protein (C4BP), CD46, C-reactive protein, cystatin C, DKK-1, Emmprin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15, and IFNγ.

[0321] In some embodiments, the MSC secretome composition comprises a ratio of anti-angiogenic factors to pro-angiogenic factors that is greater than 2, greater than 3, greater than 4, or greater than 5.

[0322] In some embodiments of the MSC secretome composition, the anti-angiogenic factors comprise one or more factors selected from the group consisting of PEDF, lower 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.

[0323] In some embodiments, the MSC secretome composition further comprises low levels of VEGF.

[0324] In some embodiments, the MSC secretome composition comprises between 1 pg / mL and 400 pg / mL of VEGF.

[0325] In some embodiments of the MSC secretome composition, the level of VEGF is 5 to 10 times lower than the level of serpin E1.

[0326] In some embodiments, the MSC secretome composition comprises one or more anti-angiogenic factors, and the sum of the concentrations of the one or more anti-angiogenic factors relative to the concentration of VEGF is greater than 2, greater than 3, greater than 4, or greater than 5.

[0327] In some embodiments, the MSC secretome composition is free of and / or contains very low levels of bFGF, PLGF, and PDGF.

[0328] In some embodiments, the MSC secretome composition comprises less than 1000 pg / mL of bFGF, PLGF, and PDGF.

[0329] In some embodiments, the MSC secretome composition has a pH of about 4.7 to about 7.5.

[0330] 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 citric acid / disodium phosphate.

[0331] In some embodiments, the MSC secretome composition further comprises a tonicity agent.

[0332] In some embodiments of the MSC secretome composition, the tonicity agent is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin.

[0333] In some embodiments, the MSC secretome composition further comprises mono / disodium phosphate, mannitol, and trehalose, and the composition has a pH of about pH 7.4.

[0334] In some embodiments, the MSC secretome composition further comprises a divalent cation.

[0335] In some embodiments, the divalent cations of the MSC secretome composition are selected from the group consisting of Mg2+, Ca2+, and Zn2+.

[0336] 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.

[0337] In some embodiments, the MSC secretome composition further comprises an adhesive agent.

[0338] In some embodiments of the MSC secretome composition, the adhesive agent is selected from the group consisting of hypromellose, Poloxamer 407, Poloxamer 188, Poloxomer 237, Poloxomer 338, hypromellose, (HPMC), polycarbophil, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyimide, sodium hyaluronate, gellan gum, poly(lactic-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethylcellulose (CMC), or hydroxypropylmethylcellulose (HPMC), hydroxymethylcellulose, hydroxyethylcellulose, sodium carboxymethylcellulose, fibrin glue, polyethylene glycol, and GelCORE.

[0339] 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 smaller than 3 kDa, antibiotics, protein aggregates larger than 200 nm, cells, non-exosomes / non-extracellular vesicle cellular debris, hormones, and L-glutamine.

[0340] In some embodiments, the MSC secretome composition comprises HGF, pentraxin-3 (TSG-14), VEGF, TIMP-1, serpin E1, and less than 5 ng / mL of IL-8.

[0341] In some embodiments, the MSC secretome composition comprises: i. 0.3–4.5 ng / mL HGF; ii. 0.5–20 ng / mL pentraxin-3 (TSG-14); iii. VEGF between 100 and 600 pg / mL; iv. 10–200 ng / mL TIMP-1; v. 20-80 ng / mL of serpin E1; and / or vi. IL-8 less than 5 ng / mL.

[0342] In some embodiments, the MSC secretome composition comprises an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.

[0343] The present invention also provides a stable mesenchymal stem cell (MSC) secretome preparation comprising: i. 2 μg–400 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-24mg trehalose dihydrate; vi. 0.5 mg to 2 mg of hypromellose per mL; and / or The pH is about 4.7 to about 7.5.

[0344] The present invention also provides a stable mesenchymal stem cell (MSC) secretome preparation comprising: i. 0.004%~0.08% w / w MSC secretome; ii. 4%-5% w / w of monobasic sodium phosphate; iii. 21.5%-23% w / w of dibasic sodium phosphate; iv. 23%-25% w / w mannitol; v. 46%-48% w / w trehalose dehydrate; vi. 1% to 3% w / w of hypromellose; and / or The pH is about 4.7 to about 7.5.

[0345] The present invention further provides 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 comprises: i. optionally, 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. optionally, at least one additional factor selected from the group consisting of PEDF (serpin F1), serpin A1, IGFBP-2, IGFBP-3, SDF-1, TSG-14, kallikrein 3, MCP-1, angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-β, and PDGF; and / or iii. Optionally, 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.

[0346] 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.

[0347] 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.

[0348] In some embodiments, the MSC secretome composition further comprises mid-range levels 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.

[0349] 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, angiogenin, DPPIV, IGFBP-3, and uPA.

[0350] In some embodiments, the MSC secretome composition further comprises at least one factor selected from the group consisting of apolipoprotein A1, complement factor D, complement factor H, complement factor I, C1 esterase inhibitor (C1-INH), C4b binding protein (C4BP), CD46, C-reactive protein, cystatin C, DKK-1, Emmprin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15, and IFNγ.

[0351] In some embodiments, the MSC secretome composition comprises a ratio of anti-angiogenic factors to pro-angiogenic factors that is greater than 2, greater than 3, greater than 4, or greater than 5.

[0352] In some embodiments, the anti-angiogenic factor comprises one or more factors selected from the group consisting of PEDF, lower levels of VEGF, and serpin E1, and the pro-angiogenic factor comprises VEGF, angiogenin, IGFBP-3, uPA, Angio-1, Angio-2, and endothelin-1.

[0353] In some embodiments, the MSC secretome composition further comprises low levels of VEGF.

[0354] In some embodiments, the MSC secretome comprises between 1 pg / mL and 400 pg / mL of VEGF.

[0355] In some embodiments, the level of VEGF is 5 to 10 times lower than the level of Serpin E1.

[0356] In some embodiments, the MSC secretome composition comprises one or more anti-angiogenic factors, and the sum of the concentrations of the one or more anti-angiogenic factors relative to the concentration of VEGF is greater than 2, greater than 3, greater than 4, or greater than 5.

[0357] In some embodiments, the MSC secretome composition contains no or very low levels of bFGF, PLGF, and PDGF.

[0358] In some embodiments, the MSC secretome composition comprises less than 1000 pg / mL of bFGF, PLGF, and PDGF.

[0359] In some embodiments, the MSC secretome composition has a pH of about 4.7 to about 7.5.

[0360] 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 citric acid / disodium phosphate.

[0361] In some embodiments, the MSC secretome composition further comprises a tonicity agent.

[0362] In some embodiments, the tonicity adjusting agent is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin.

[0363] In some embodiments, the MSC secretome composition further comprises mono / disodium phosphate, mannitol, and trehalose, and the composition has a pH of about pH 7.4.

[0364] In some embodiments, the MSC secretome composition further comprises a divalent cation.

[0365] In some embodiments, the divalent cation is selected from the group consisting of Mg2+, Ca2+, and Zn2+.

[0366] 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.

[0367] In some embodiments, the MSC secretome composition further comprises an adhesive agent.

[0368] In some embodiments, the adhesive agent is selected from the group consisting of hypromellose, Poloxamer 407, Poloxamer 188, Poloxomer 237, Poloxomer 338, hypromellose, (HPMC), polycarbophil, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyimide, sodium hyaluronate, gellan gum, poly(lactic-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethylcellulose (CMC), or hydroxypropylmethylcellulose (HPMC), hydroxymethylcellulose, hydroxyethylcellulose, sodium carboxymethylcellulose, fibrin glue, polyethylene glycol, and GelCORE.

[0369] 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 smaller than 3 kDa, antibiotics, protein aggregates larger than 200 nm, cells, non-exosomes / non-extracellular vesicle cellular debris, hormones, and L-glutamine.

[0370] In some embodiments, the MSC secretome composition comprises HGF, pentraxin-3 (TSG-14), VEGF, TIMP-1, serpin E1, and less than 5 ng / mL of IL-8.

[0371] In some embodiments, the MSC secretome composition comprises: i. 0.3–4.5 ng / mL HGF; ii. 0.5–20 ng / mL pentraxin-3 (TSG-14); iii. VEGF between 100 and 600 pg / mL; iv. 10–200 ng / mL TIMP-1; v. 20-80 ng / mL of serpin E1; and / or vi. IL-8 less than 5 ng / mL.

[0372] In some embodiments, the MSC secretome composition comprises an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.

[0373] The present invention also provides 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 comprising: i. 2 μg–400 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-24mg trehalose dihydrate; vi. 0.5 mg to 2 mg of hypromellose per mL; and / or The pH is about 4.7 to about 7.5.

[0374] The present invention also provides 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 comprising: i. 0.004%-0.08% w / w MSC secretome ii. 4%-5% w / w of monobasic sodium phosphate; iii. 21.5%-23% w / w of dibasic sodium phosphate; iv. 23%-25% w / w mannitol; v. 46%-48% w / w trehalose dehydrate; vi. 1% to 3% w / w of hypromellose; and / or The pH is about 4.7 to about 7.5.

[0375] The present invention also provides a stable mesenchymal stem cell (MSC) secretome preparation comprising: i. 1-400 μg, optionally 2 μg-8 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-24mg trehalose dihydrate; vi. optionally, 0.5 mg to 2 mg of hypromellose per mL; and / or The pH is about 4.7 to about 7.5.

[0376] The present invention also provides a stable mesenchymal stem cell (MSC) secretome preparation comprising: i. 0.004%-0.0375%, optionally 0.008%-0.015% w / w of MSC secretome; ii. 4%-5% w / w of monobasic sodium phosphate; iii. 21.5%-23% w / w of dibasic sodium phosphate; iv. 23%-25% w / w mannitol; v. 46%-48% w / w trehalose dehydrate; vi. optionally 1% to 3% w / w of hypromellose; and / or The pH is about 4.7 to about 7.5.

[0377] The present invention also provides a stable mesenchymal stem cell (MSC) secretome preparation comprising: i. 6 μg 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 trehalose dihydrate; vi. optionally 1 mg of hypromellose per mL; and / or The pH is approximately 7.4.

[0378] The present invention also provides a stable mesenchymal stem cell (MSC) secretome preparation comprising: i. 0.012% w / w MSC secretome; ii. 4.5% w / w sodium phosphate monobasic; iii. 22.4% w / w dibasic sodium phosphate; iv. 24% w / w mannitol; v. 47.1% w / w trehalose dehydrate; vi. optionally 2.0% w / w hypromellose; and / or The pH is approximately 7.4.

[0379] The present invention also provides a stable mesenchymal stem cell (MSC) secretome preparation comprising: i. 2 μg–400 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-24mg trehalose dihydrate; vi. optionally, 0.5 mg to 2 mg of hypromellose per mL; and / or The pH is about 4.7 to about 7.5.

[0380] The present invention also provides a stable mesenchymal stem cell (MSC) secretome preparation comprising: i. 0.004%~0.08% w / w MSC secretome; ii. 4%-5% w / w of monobasic sodium phosphate; iii. 21.5%-23% w / w of dibasic sodium phosphate; iv. 23%-25% w / w mannitol; v. 46%-48% w / w trehalose dehydrate; vi. optionally 1% to 3% w / w of hypromellose; and / or The pH is about 4.7 to about 7.5.

[0381] In some embodiments of the stable mesenchymal stem cell (MSC) secretome formulation, the formulation does not include hypromellose.

[0382] The present invention also provides 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 comprising: i. 2 μg–400 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-24mg trehalose dihydrate; vi. optionally, 0.5 mg to 2 mg of hypromellose per mL; and / or The pH is about 4.7 to about 7.5.

[0383] The present invention also provides 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 comprising: i. 0.004%-0.08% w / w MSC secretome ii. 4%-5% w / w of monobasic sodium phosphate; iii. 21.5%-23% w / w of dibasic sodium phosphate; iv. 23%-25% w / w mannitol; v. 46%-48% w / w trehalose dehydrate; vi. optionally 1% to 3% w / w of hypromellose; and / or The pH is about 4.7 to about 7.5.

[0384] In some embodiments of the methods of treating an ocular condition, the MSC secretome compositions and / or formulations used in the methods of treatment do not include hypromellose.

[0385] In some embodiments of the methods described herein, the MSC secretome composition and / or formulation does not comprise hypromellose.

[0386] In some embodiments of the MSC secretome composition and / or formulation, the composition and / or formulation does not comprise hypromellose.

[0387] In some embodiments, the MSC secretome is overall 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 results in reduced angiogenesis compared to other secretomes. In some embodiments, the MSC secretome results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% reduction in angiogenesis. In some embodiments, the MSC secretome results in 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 results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% reduction in angiogenesis compared to conditioned medium prior to processing into the MSC secretome. In some embodiments, the MSC secretome has low angiogenic potential. In some embodiments, the MSC secretome exhibits 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 control. 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, when serum-containing medium plus secretome (reduced or absent angiogenic response) is compared to serum-containing medium (angiogenic response), the MSC secretome induces a reduced 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 demonstrated by tube formation in an endothelial cell tube formation assay. In some embodiments, the angiogenic response is demonstrated by blood vessel formation in a CAM (chick chorioallantoic membrane) assay. In some embodiments, the angiogenic response is demonstrated by blood vessel formation in any angiogenesis assay known in the art.

[0388] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises: i. optionally, IDO (indoleamine-2,3-dioxygenase) enzyme activity; ii. optionally, 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. optionally, a "threshold" ppm level of at least one additional factor selected from the group consisting of sFLT-1, PEDF (serpin F1), IGFBP-2, IGFBP-3, SDF-1, TSG-14, kallikrein 3, MCP-1, bFGF, angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-β, PDGF, SOD1, SOD2, SOD3, and HO-1; and / or iv. Optionally, a "threshold" ppm level of at least one additional agent selected from the group consisting of DPPIV (dipeptidyl peptidase-4), uPA, angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, and thrombospondin-1.

[0389] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises: i. optionally, an IDO (indoleamine-2,3-dioxygenase) enzyme activity of less than about 250 μM; ii. optionally, 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. optionally, 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-β, PDGF, SOD1, SOD2, SOD3, and / or HO-1; and / or iv. Optionally, 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.

[0390] 1. A mesenchymal stem cell (MSC) secretome composition, comprising: i. optionally, 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. optionally, 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-β, and PDGF; and / or iii. A mesenchymal stem cell (MSC) secretome composition, optionally comprising 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.

[0391] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor, including but not limited to apolipoprotein A1, complement factor D, complement factor H, complement factor I, C1 esterase inhibitor (C1-INH), C4b binding protein (C4BP), CD46, 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, complement factor H, complement factor I, C1 esterase inhibitor (C1-INH), C4b binding protein (C4BP), CD46, C-reactive protein, cystatin C, DKK-1, Emmprin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15, and IFNγ.

[0392] 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.

[0393] 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 protection. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor, including but not limited to, catalase, protein disulfide isomerase, protein disulfide isomerase A3, protein disulfide isomerase A4, protein disulfide isomerase A6, peroxiredoxin-6, peroxiredoxin-1, peroxiredoxin-2, and / or peroxiredoxin-4. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises catalase. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises protein disulfide isomerase, and in some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises protein disulfide isomerase A3. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises protein disulfide isomerase A4. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises protein disulfide isomerase A6, and 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.

[0394] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises at least one additional factor, including but not limited to, a 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.

[0395] 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) (including various isoforms, LTGFBP-2, LTGFBP-3, and LTGFBP-4). In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises soluble scavenger receptor cysteine-rich domain-containing protein SSC5D. In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises tumor necrosis factor-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.

[0396] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises pentraxin-3, TIMP-1, serpin E1, TSP-1, and HGF.

[0397] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises 2-16 ng / mL, or 9.8±0.5 ng / ml pentraxin-3.

[0398] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises 10-200 ng / mL, or 90±21.5 ng / ml, of TIMP-1.

[0399] 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.

[0400] 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.

[0401] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises 100-800 pg / mL, or 304±44 pg / ml, of VEGF.

[0402] In some embodiments, the mesenchymal stem cell (MSC) secretome composition comprises 0.1 to 100 pg / mL, or less than 1 ng / ml, of IL-8.

[0403] In some embodiments, the IDO (indoleamine-2,3-dioxygenase) enzyme activity is less than about 250 μM. In some embodiments, the IDO (indoleamine-2,3-dioxygenase) enzyme activity is 0 μM to about 250 μM. In some embodiments, the IDO (indoleamine-2,3-dioxygenase) enzyme activity is 50 μM to about 250 μM L-kynurenine per million MSCs. In some embodiments, the IDO (indoleamine-2,3-dioxygenase) enzyme activity is 50 μM to about 200 μM L-kynurenine per million MSCs. In some embodiments, the IDO (indoleamine-2,3-dioxygenase) enzyme activity is 100 μM to about 250 μM L-kynurenine per million MSCs. In some embodiments, the IDO (indoleamine-2,3-dioxygenase) enzyme activity is between 100 μM and about 200 μM L-kynurenine per million MSCs. In some embodiments, the IDO (indoleamine-2,3-dioxygenase) enzyme activity is about 0 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, about 100 μM, about 110 μM, about 120 μM, about 130 μM, about 140 μM, about 150 μM, about 160 μM, about 170 μM, about 180 μM, about 190 μM, about 200 μM, about 210 μM, about 220 μM, about 230 μM, about 240 μM, or about 250 μM L-kynurenine per million MSCs.

[0404] In some embodiments, the MSC secretome further comprises a "threshold" ppm level of at least one additional factor, including but not limited to, sFLT-1, PEDF (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-β, PDGF, SOD1, SOD2, SOD3, and / or HO-1. In some embodiments, the MSC secretome further comprises a "threshold" ppm level of at least one additional factor selected from the group consisting of sFLT-1, PEDF (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-β, PDGF, SOD1, SOD2, SOD3, and HO-1. In some embodiments, the MSC secretome further comprises one additional factor at a concentration ranging from 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-β, 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.

[0405] 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.

[0406] 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 4, and serpin F1. In some embodiments, the MSC secretome comprises 6 ng / mL to 8 ng / mL of at least one factor selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1. In some embodiments, the MSC secretome comprises 2 ng / mL to 7 ng / mL of at least one factor selected from the group consisting of serpin E1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), platelet factor 4, and serpin F1.

[0407] In some embodiments, the MSC secretome composition further comprises "mid-range" levels of at least one factor, including but not limited to, angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, and thrombospondin-1, angiogenin, DPPIV (dipeptidyl peptidase-4), IGFBP-3, and / or uPA. In some embodiments, the MSC secretome composition further comprises "mid-range" levels 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 "mid-range" 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, angiogenin, DPPIV, IGFBP-3, and uPA.In some embodiments, the MSC secretome composition further comprises about 200 pg / mL, about 300 pg / mL, about 400 pg / mL, about 500 pg / mL, about 600 pg / mL, about 700 pg / mL, or about 800 pg / mL of at least one factor selected from the group consisting of angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, and thrombospondin-1, angiogenin, DPPIV, IGFBP-3, and uPA. In some embodiments, the MSC secretome composition comprises about 200 pg / mL to about 800 pg / mL, about 300 pg / mL to about 800 pg / mL, about 200 pg / mL to about 500 pg / mL, or about 300 pg / mL to about 500 pg / mL of angiogenin. In some embodiments, the MSC secretome composition further comprises about 200 pg / mL to about 800 pg / mL, about 300 pg / mL to about 800 pg / mL, about 200 pg / mL to about 500 pg / mL, or about 300 pg / mL to about 500 pg / mL of DPPIV. In some embodiments, the MSC secretome composition comprises about 200 pg / mL to about 800 pg / mL, about 300 pg / mL to about 800 pg / mL, about 200 pg / mL to 500 pg / mL, or about 300 pg / mL to about 500 pg / mL IGFBP-3. In some embodiments, the MSC secretome composition comprises about 200 pg / mL to about 800 pg / mL, about 300 pg / mL to about 800 pg / mL, about 200 pg / mL to 500 pg / mL, or about 300 pg / mL to about 500 pg / mL uPA.

[0408] 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.

[0409] 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.

[0410] In some embodiments of the MSC secretome composition, the level of VEGF is 5 to 10 times lower than that of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is 6 to 10 times lower than that of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is 7 to 10 times lower than that of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is 8 to 10 times lower than that of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is 9 to 10 times lower than that of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is 5 times lower than that of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is 6 times lower than that of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is 7 times lower than that of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is eighth-fold lower than the level of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is ninth-fold lower than the level of serpin E1. In some embodiments of the MSC secretome composition, the level of VEGF is tenth-fold lower than the level of serpin E1.

[0411] 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.

[0412] In some embodiments, the MSC secretome composition comprises apolipoprotein A1, complement factor D, complement factor H, complement factor I, C1 esterase inhibitor (C1-INH), C4b binding protein (C4BP), CD46, C-reactive protein, cystatin C, DKK-1, Emmprin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15, and / or IFNγ.

[0413] 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 4 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 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 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 160 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 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 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 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 390 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.

[0414] In some embodiments, the MSC secretome composition comprises: i. 0.3–4.5 ng / mL HGF; ii. 0.5–20 ng / mL pentraxin-3 (TSG-14); iii. VEGF between 100 and 600 pg / mL; iv. 10–200 ng / mL TIMP-1; v. 20-80 ng / mL of serpin E1; and / or vi. IL-8 less than 5 ng / mL.

[0415] In some embodiments, the MSC secretome composition comprises: i. 1.5–3.5 ng / mL HGF; ii. 5–15 ng / mL pentraxin-3 (TSG-14); iii. VEGF of 200–400 pg / mL; iv. 50–120 ng / mL TIMP-1; v. 30-70 ng / mL serpin E1; and / or vi. IL-8 less than 3 ng / mL.

[0416] In some embodiments, the MSC secretome composition comprises: i. 1.5–2.5 ng / mL HGF; ii. 8–12 ng / mL pentraxin-3 (TSG-14); iii. VEGF of 250–350 pg / mL; iv. 70–110 ng / mL TIMP-1; v. 30-70 ng / mL serpin E1; and / or vi. IL-8 less than 2 ng / mL.

[0417] In some embodiments, the MSC secretome composition comprises: i. 2.0±0.3ng / mL HGF; ii. 9.8±0.5ng / mL pentraxin-3 (TSG-14); iii. VEGF of 304±44pg / mL; iv. 90±20ng / mL TIMP-1; v. 49.2±10 ng / mL serpin E1; and / or vi. IL-8 less than 1 ng / mL.

[0418] 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.

[0419] 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.

[0420] 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 less than 3 kDa, antibiotics, protein aggregates (e.g., protein aggregates greater than 200 nm), cells, cellular debris (cellular debris not including exosomes / extracellular vesicles (EVs); e.g., cellular debris other than exosomes, other than EVs), 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 include protein aggregates (e.g., protein aggregates greater than 200 nm). In some embodiments, the MSC secretome composition does not include cells. In some embodiments, the MSC secretome composition does not include cellular debris (cellular debris does not include exosomes / EVs; e.g., cellular debris other than exosomes, other than EVs). In some embodiments, the MSC secretome composition does not include hormones (e.g., hormones include, but are not limited to, insulin and / or hydrocortisone. In some embodiments, the MSC secretome composition does not include L-glutamine.

[0421] 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 mono / disodium phosphate, mannitol, and trehalose, and the composition has a pH of about pH 7.4.

[0422] In some embodiments, the MSC secretome composition may comprise one or more additional agents, including but not limited to, glycine, glycerol, sodium chloride, potassium chloride, and / or dextrose. In some embodiments, the MSC secretome composition may comprise one or more additional agents selected from the group consisting of glycine, glycerol, sodium chloride, potassium chloride, and dextrose. In some embodiments, the MSC secretome composition may comprise one or more additional agents selected from the group consisting of glycine and glycerol, and dextrose. In some embodiments, the MSC secretome composition may comprise one or more additional agents selected from the group consisting of sodium chloride and potassium chloride.

[0423] 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, boric acid / sodium citrate, boric acid / sodium tetraborate, and / or citric acid / disodium phosphate. In some embodiments, the MSC secretome composition is formulated in a buffer system selected from the group consisting of 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.

[0424] 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.

[0425] 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.

[0426] In some embodiments, the MSC secretome composition further comprises an adhesive agent. In some embodiments, the MSC secretome composition further comprises an adhesive agent, 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 agent is hypromellose. In some embodiments, the adhesive agent is fibrin glue. In some embodiments, the adhesive agent is polyethylene glycol. In some embodiments, the adhesive agent is GelCORE (Sani, et al., Science See Advances, Vol. 5, no. 3 (2019).

[0427] In some embodiments, the MSC secretome composition comprises (a) processed conditioned medium comprising an MSC secretome produced by any one of the methods described herein, and (b) a polymer. In some embodiments, the MSC secretome composition comprises conditioned medium comprising an MSC secretome produced as described herein and a polymer. In some embodiments, the MSC secretome composition comprises processed conditioned medium comprising an MSC secretome produced as described herein and a polymer. In some embodiments, the polymer can be a biodegradable polymer that can release the MSC secretome and / or processed MSC secretome components. In some embodiments, the polymer allows for sustained release of the MSC secretome components.

[0428] In some embodiments, the MSC secretome compositions provided herein are in the form of a therapeutic dressing (e.g., a polymer impregnated with the MSC secretome composition). The therapeutic dressing can be configured as needed depending on the application. In some embodiments, the dressing is in the form of a patch or configured as a mesh.

[0429] In some embodiments, the MSC secretome composition exhibits biopermeability, e.g., ocular permeability, corneal permeability, 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 biopermeability. In some embodiments, the MSC secretome composition exhibits excipient-enabled biopermeability. In some embodiments, the MSC secretome composition exhibits biopermeability due to the upregulation of smaller factors. In some embodiments, the MSC secretome composition exhibits biopermeability due to the presence of a biopreservative. In some embodiments, the MSC secretome composition exhibits biopermeability due to the presence of the biopreservative benzalkonium chloride.

[0430] 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.

[0431] In some embodiments, the MSC secretome composition exhibits ultra-high potency when administered to a subject in need thereof, hi some embodiments, the MSC secretome composition provides a therapeutic effect with one drop or one administration per day.

[0432] G. Methods for Producing / Manufacturing MSC Secretome According to the present invention, the conditioned medium (and thus the factors secreted by mesenchymal stem cells) 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, such as a young and / or healthy donor, and / or from commercially available mesenchymal stem cells. For example, MSCs obtained from the individual to be treated (autologous stem cells) or MSCs obtained from a donor (allogeneic stem cells) can be used to produce the conditioned medium described herein, which can then be further processed into an MSC secretome composition as described herein. In some embodiments, MSCs can also be obtained from commercial suppliers. In some embodiments, commercially available MSCs can be used to produce MSC secretomes.

[0433] According to the present invention, a method for producing an angiostatic mesenchymal stem cell (MSC) secretome composition comprises: i. culturing mesenchymal stem cells (MSCs) in a first culture medium; ii. removing the first culture medium of step (i) from the MSCs; iii. Washing the MSCs of step (ii); iv. adding a second culture medium and culturing for about 1 to 5 days; v. collecting the second culture medium from step (iv) as conditioned medium; and vi. Processing the conditioned medium of step (v) into an MSC secretome composition as described herein.

[0434] In some embodiments, the culturing may be carried out using a bioreactor system for culturing cells. In some embodiments, the culturing may be carried out using a bioreactor system for culturing stem cells. In some embodiments, the culturing may be carried out using a bioreactor system for culturing mesenchymal stem cells. In some embodiments, the culturing may be carried out using a media mixing technique. In some embodiments, the culturing may be carried out using a PBS Vertical Wheel™ mixing technique.

[0435] In some embodiments, in step (iv), processing the conditioned medium of step (v) into a secretome composition comprises: a) filtering the conditioned medium collected in step (v) to remove cellular particulates; b) concentrating the filtered conditioned medium from step (a); and c) Buffer exchange with formulation buffer.

[0436] 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.

[0437] 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).

[0438] 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.

[0439] In some embodiments, the centrifugation-based size exclusion technique uses a MW cutoff of 3-10 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, or A MW cutoff of at least 17 kDa, at least 18 kDa, at least 19 kDa, at least 20 kDa, at least 21 kDa, at least 22 kDa, at least 23 kDa, at least 24 kDa, at least 25 kDa, at least 26 kDa, at least 27 kDa, at least 28 kDa, at least 29 kDa, and / or at least 30 kDa is used.

[0440] In some embodiments, the method produces an MSC secretome composition and / or formulation described herein above. In some embodiments, the first and / or second culture medium is MSC medium and / or MSC-XF.

[0441] MSCs, or cells differentiated from MSCs, can be engineered to produce conditioned medium containing a desired secretome, e.g., a desired cytokine and / or a desired therapeutic property, as described herein. For example, secretomes can be produced from MSCs of superdonor cell lines. Secretomes can also be produced from commercially available MSCs. In some embodiments, allogeneic MSCs (and / or cells derived therefrom) and / or allogeneic MSC-derived secretome compositions can be prepared and stored for large populations. Allogeneic MSCs (and / or cells derived therefrom) and / or MSC-derived secretome compositions can be pre-prepared 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).

[0442] In some embodiments, the initial cell culture medium and the first cell culture medium are different. In some embodiments, the initial cell culture medium and the first cell culture medium are the same. Non-limiting examples of cell culture medium(s) useful for culturing MSCs to produce conditioned medium comprising an MSC secretome according to the present invention include hMSC Media Booster XFM, hMSC Enhanced Basal Medium, Minimum Essential Medium Eagle (MEME), ADC-1, LPM (without bovine serum albumin), F10 (HAM), F12 (HAM), DCCM1, DCCM2, RPMI 1640, BGJ Medium (with and without Fitton-Jackson modification), StemPro, MSCGro, MesenCult, NutriStem, Basal Medium Eagle (BME supplemented with Earle's salt base), Dulbecco's Modified Eagle Medium (DMEM with or without serum), Yamane, IMEM-20, Glasgow Modified Eagle Medium (GMEM), Leibovitz L-15 Medium, McCoy's, among many others. Examples of suitable medium include 5A medium, medium M199 (M199E with Earle's salts base), medium M199 (M199H with Hank's salts base), minimum essential medium alpha (MEM-α), minimum essential medium Eagle (MEM-E with Earle's salts base), minimum essential medium Eagle (MEM-H with Hank's salts base), and minimum essential medium Eagle (MEM-NAA with non-essential amino acids), including medium 199, CMRL1415, CMRL1969, CMRL1066, NCTC135, MB75261, MAB8713, DM145, Williams' G, Neuman & Tytell, Higuchi, MCDB301, MCDB202, MCDB501, MCDB401, MCDB411, and MDBC153. The preferred medium for use in the present invention is MEM-α. These and other useful media are available from, among others, GIBCO, Grand Island, NY, USA and Biological Industries, Bet HaEmek, Israel.Many of these media are summarized in Methods in Enzymology, Volume LVIII, "Cell Culture," pp. 62-72 (William B. Jakoby and Ira H. Pastan, eds., Academic Press, Inc.).

[0443] In some embodiments, the cell culture medium for mesenchymal stem cells may be serum-free. In some embodiments, the cell culture medium for mesenchymal stem cells may be supplemented with serum. In some embodiments, the cell culture medium for mesenchymal stem cells may be supplemented with human platelet lysate. In some embodiments, the serum may include fetal bovine serum (FBS). In some embodiments, the cell culture medium for mesenchymal stem cells may be supplemented with serum, such as fetal serum from bovine or other species. In some embodiments, the cell culture medium for mesenchymal stem cells may be supplemented with other components to facilitate 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.

[0444] In some embodiments, the oxygen percentage is varied to facilitate cell growth and / or promote cell health. In some embodiments, the oxygen is 5%, 10%, 15%, 20%, or 25% by volume to facilitate cell growth and / or promote cell health. In some embodiments, the mesenchymal stem cells are grown under oxygen tension to facilitate cell growth and / or promote cell health. In some embodiments, the mesenchymal stem cells are grown in a low oxygen tension environment to facilitate cell growth and / or promote cell health.

[0445] In one aspect, the present invention relates to conditioned medium (CM) containing biological factors secreted by mesenchymal stem cells, sometimes referred to as conditioned medium containing MSC secretome. 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 components: conditioned medium containing the secretome and mesenchymal stem cells grown in the conditioned medium. Once separated, the conditioned medium contains the mesenchymal stem cell secretome and can be further processed and / or used according to the methods described herein, and is substantially free of mesenchymal stem cells (which may contain a small percentage of stem cells and / or trace stem cells), or free of mesenchymal stem cells. The MSC secretome contains various biological factors, including hormones, cytokines, extracellular matrix, proteins, vesicles, antibodies, chemokines, receptors, inhibitors, and granules. As described herein, the conditioned medium(s) containing the MSC secretome (MSC secretome-containing CM or conditioned medium) can be further processed to produce concentrated conditioned medium (pCM or concentrated MSC secretome).

[0446] In some embodiments, conditioned medium or enriched MSC secretome containing MSC secretome is produced by culturing mesenchymal stem cells in a culture medium and replacing 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 specific components of the conditioned medium.

[0447] In some embodiments, the harvested conditioned medium containing the MSC secretome is filtered to produce a concentrated MSC secretome. In some embodiments, the harvested conditioned medium containing the MSC secretome is ultrafiltered to produce a concentrated MSC secretome.

[0448] In one aspect, provided herein is a method of producing processed conditioned medium, comprising: (a) culturing stem cells in a cell culture medium to generate a conditioned medium containing factors secreted by mesenchymal stem cells (e.g., a conditioned medium containing a mesenchymal stem cell secretome); (b) harvesting the conditioned medium to produce 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 (cultured) in a growth medium before being cultured in a growth factor-free medium. Thus, in some embodiments, the method comprises: (a) culturing mesenchymal stem cells in a first growth medium; (b) replacing the first growth medium with a second growth medium and culturing the stem cells in the second growth medium, thereby generating 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.

[0449] In some embodiments, the MSC secretome of the present invention is further processed using techniques known in the art, including, but not limited to, extraction, freeze-thawing, homogenization, permeabilization, centrifugation, density gradient centrifugation, CsCl gradient centrifugation, iodixanol gradient centrifugation, ultracentrifugation, fractionation, precipitation, SDS-PAGE, native PAGE, size exclusion chromatography, liquid chromatography, gas chromatography, hydrophobic interaction chromatography, ion exchange chromatography, anion exchange chromatography, cation exchange chromatography, affinity chromatography, heparin sulfate affinity chromatography, sialic acid affinity chromatography, immunoaffinity chromatography, metal binding chromatography, nickel column chromatography, epitope tag purification, or lyophilization, or any combination thereof.

[0450] In some embodiments, the MSC secretome of the present invention is enriched by one or more of the following methods: affinity-based enrichment, size-based enrichment, cation- or anion-based enrichment, and fractionation to enrich for preferred traits.

[0451] 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 (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 cells of the mesodermal lineage, such as adipocytes, osteoblasts, and chondrocytes. MSCs have small cell bodies with few elongated cell processes. The cell bodies contain large, round nuclei with prominent nucleoli, which are surrounded by finely dispersed chromatin particles, making the nuclei clearly visible. The remainder of the cell body contains a small amount of the Golgi apparatus, rough endoplasmic reticulum, mitochondria, and polyribosomes. These cells are elongated and widely dispersed, and the adjacent extracellular matrix contains a small number of reticular fibers but lacks other types of collagen fibers [Brighton, et al. 1991 The Journal of Bone and Joint Surgery 73(6):832-47]. The MSCs described herein may express the following profile of molecular markers (protein molecules characteristic of the plasma membrane of a cell or cell type): osteogenic protein receptors; ”1” (BMPR + );CD34 + Scal + Lin ” ;CD44 + ;c-kit + ;Sca-1 + ;Thy-1 + ;NOTCH3;JAG1;ITGA11. MSCs may also express other cell type specific markers (see world wide web stemcells.nih.gov; Kaltz, et al. 2010 Exp Cell Res Oct 1;316(16):2609-17, incorporated herein by reference). The MSCs described herein can be identified based on colony forming unit assays to detect the multipotent differentiation potential of MSCs (which cell types they give rise to). However, somewhat differentiated cells (progenitor cells) can also be used.

[0452] i. MSC secretome: processing In some embodiments, the conditioned medium containing the MSC secretome described herein may be collected, filtered, and / or purified to remove cellular particulates and / or other harmful components. For example, as described above in step (v), the second culture medium from step (iv) is collected as the conditioned medium. The filtration membrane used herein may be selected from any known in the art with an appropriate membrane and configuration to retain the desired MSC secretome components while allowing cellular particulates and / or other harmful components to pass through. Therefore, any suitable membrane that allows cell retention under selected hydrodynamic conditions while allowing cells to pass through to remove harmful components can be used. In some embodiments, an upper pore size limit of approximately 5 microns and a lower pore size limit of approximately 0.1 microns are suitable. In some embodiments, filtration may be performed using a micropore filter. In some embodiments, filtration may be performed using a 0.5 μm to 0.2 μm filter. In some embodiments, filtration may 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 may be performed using a 0.45 μm filter. In some embodiments, filtration may be performed using a 0.22 μm filter. In some embodiments, filtration / purification may be performed using a low protein binding polyvinylidene difluoride (PVDF) membrane. In some embodiments, filtration / purification may be performed using polyethersulfone (PES).

[0453] In some embodiments, filtration is by ultrafiltration. In some embodiments, the conditioned medium is filtered using a 3 kD filter size (to achieve purification, desalting, and concentration of molecules in the processed conditioned medium that are larger than the filter size). In some embodiments, the conditioned medium is filtered using a filter size less than 3 kD, while in other embodiments, a filter size greater 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.

[0454] 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 mode of filtration in which a suspension of MSC secretome cells flows substantially parallel to a filter that permeates non-cellular components in the suspension. Cross-flow filtration processes are characterized by a set of hydrodynamic parameters, including Re = Reynolds number, γw = wall shear rate, ΔP = pressure drop, and TMP = transmembrane pressure. Re, γw, and ΔP depend on the geometry of the filtration system, flow conditions, and fluid properties. Such cross-flow processes, in some embodiments, may also include hollow fiber filtration systems. See, e.g., U.S. Patent No. 5,053,334, incorporated herein by reference in its entirety.

[0455] In some embodiments, the MSC secretome may be further subjected to concentration without and / or after filtration. In some embodiments, the MSC secretome may be concentrated using hollow fiber tangential flow technology, or

[0456] In some embodiments, centrifugation-based size exclusion techniques can be used to enrich the MSC secretome, 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 used in the centrifugation-based size exclusion technique enrichment method is at least about 3 kDa, at least about 4 kDa, at least about 5 kDa, at least about 6 kDa, at least about 7 kDa, at least about 8 kDa, at least about 9 kDa, or at least about 10 kDa.

[0457] 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, about 95-fold, or about 100-fold compared to the conditioned medium prior to enrichment.

[0458] In some embodiments, the MSC secretome is further buffer exchanged into a final formulation buffer after the concentration step. In some embodiments, the MSC secretome is further buffer exchanged into a final formulation buffer without an adhesive agent after the concentration step. In some embodiments, the buffer exchange comprises changing the buffer composition 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 by less than 1%, less than 5%, less than 10%, less than 15%, less than 20%, or less than 25% during the buffer exchange step.

[0459] In some embodiments, the MSC secretome is buffer exchanged after the concentration step to remove all traces of culture medium components, ie, the MSC secretome is buffer exchanged after the concentration step such that less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%, or about 0% of the culture medium components remain.

[0460] ii. MSC secretome: formulation In some embodiments, the MSC secretome is prepared as a formulation containing about 2 μg to 20 μg of MSC secretome per mL, or 0.004% to 0.0375% of MSC secretome per mL.

[0461] In some embodiments, the MSC secretome is prepared as a formulation containing about 2 μg to 8 μg of MSC secretome per mL, or 0.008% to 0.015% of MSC secretome per mL.

[0462] In some embodiments, the MSC secretome is prepared as a formulation containing 2 mg to 3 mg of monobasic sodium phosphate per mL, or 4% to 5% of monobasic sodium phosphate per mL.

[0463] In some embodiments, the MSC secretome is prepared as a formulation containing 11 mg to 12 mg of dibasic sodium phosphate per mL, or 21.5% to 23% of dibasic sodium phosphate per mL.

[0464] 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.

[0465] In some embodiments, the MSC secretome is prepared in a formulation containing 23 mg to 25 mg of trehalose dihydrate per mL, or 46% to 48% trehalose dihydrate per mL.

[0466] In some embodiments, the MSC secretome is prepared as a formulation that does not contain hypromellose. In some embodiments, the MSC secretome is prepared as a formulation that optionally contains hypromellose. In some embodiments, the MSC secretome is prepared as a formulation that contains 0.5 mg to 2 mg of hypromellose per mL. In some embodiments, the MSC secretome is prepared as a formulation that contains 1% to 3% hypromellose per mL.

[0467] 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.

[0468] In some embodiments, the MSC secretome is prepared as a formulation containing the ingredients shown in Tables 1-6 below.

[0469] [Table 2]

[0470] [Table 3]

[0471] [Table 4]

[0472] [Table 5]

[0473] In some embodiments, the MSC secretome formulations shown in Table 2 have a pH of about 5.5.

[0474] [Table 6]

[0475] In some embodiments, the MSC secretome formulations shown in Table 3 have a pH of about 6.2.

[0476] [Table 7]

[0477] In some embodiments, the MSC secretome formulations shown in Table 4 have a pH of about 7.2. In some embodiments, the MSC secretome is formulated with water for injection according to USP standards.

[0478] [Table 8]

[0479] [Table 9]

[0480] H. Assay Methods / Therapeutic Properties In some embodiments of the present invention, the MSC secretome is engineered to achieve specific component ratios / concentrations and properties of the MSC secretome.

[0481] In some embodiments, the MSC secretome composition comprises a ratio of angiogenic inhibitors to pro-angiogenic factors, wherein the ratio is greater than 1. In some embodiments, the MSC secretome composition comprises a ratio of angiogenic inhibitors to pro-angiogenic factors, wherein the ratio is greater than 2, greater than 3, greater than 4, or greater than 5. In some embodiments, the MSC secretome composition comprises an elevated concentration of pro-angiogenic factors (relative to the concentration of pro-angiogenic factors in the conditioned medium from which the MSC secretome composition was produced). In some embodiments, the MSC secretome composition comprises a total of multiple angiogenic inhibitors that exceeds the level of VEGF. In some embodiments, the MSC secretome composition comprises a total of multiple angiogenic inhibitors such that the ratio of more than one angiogenic inhibitor to VEGF is greater than 2, greater than 3, greater than 4, or greater than 5. In some embodiments, the MSC secretome composition comprises one or more anti-angiogenic factors, and the sum of the concentrations of the one or more anti-angiogenic factors relative to the concentration of VEGF is greater than 2, greater than 3, greater than 4, or greater than 5. In some embodiments, the pro-angiogenic factor includes, but is not limited to, serpin E1 against VEGF-A. In some embodiments, the pro-angiogenic factor is serpin E1. In some embodiments, the pro-angiogenic factor is VEGF-A.

[0482] In some embodiments of the present invention, the MSC secretome is engineered to achieve specific efficacy performance criteria. In some embodiments, a buffer exchange step facilitates obtaining a potent MSC secretome.

[0483] Extracellular vesicles are membrane-bound particles that carry the above-mentioned soluble and insoluble cargoes. The term "extracellular vesicles" refers to a group of various types of secreted or shed vesicles. These are generally divided 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 below 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 number and / or to quantify the extracellular vesicles (EVs) present in the secretome.

[0484] In some embodiments, the EV is about 2.5x10^5 / uL, 2.6x10^5 / uL, 2.7x10^5 / uL, 2.8x10^5 / uL, 2.9x10^5 / uL, 3.0x10^5 / uL, 3.1x10^5 / uL, 3.2x10^5 / uL, 3.3x10^5 / uL, 3.4x10^5 / uL, 3.5x10^5 / uL, 3.6x10^5 / uL, 3.7x10^5 / uL uL, 3.8x10^5 / uL, 3.9x10^5 / uL, 4.0x10^5 / uL, 4.1x10^5 / uL, 4.2x10^5 / uL, 4.3x10^5 / uL, 4.4x10^5 / uL, 4.5x10^5 / uL, 4.6x10^5 / uL, 4.7x10^5 / uL, 4.8x10^5 / uL, 4.9x10^5 / uL, or about 5.0x10^5 / uL. In some embodiments, the EVs are present at a concentration of about 3.8x10^5 / uL ± 0.8x10^5.

[0485] In some embodiments, the EV is about 2.5x10^5 / uL, 2.6x10^5 / uL, 2.7x10^5 / uL, 2.8x10^5 / uL, 2.9x10^5 / uL, 3.0x10^5 / uL, 3.1x10^5 / uL, 3.2x10^5 / uL, 3.3x10^5 / uL, 3.4x10^5 / uL, 3.5x10^5 / uL, 3.6x10^5 / uL, 3.7x10^5 / uL, 3.8x10 They exist at concentrations of 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 with an average diameter of 110-120nm. In some embodiments, the EV is about 2.5x10^5 / uL, 2.6x10^5 / uL, 2.7x10^5 / uL, 2.8x10^5 / uL, 2.9x10^5 / uL, 3.0x10^5 / uL, 3.1x10^5 / uL, 3.2x10^5 / uL, 3.3x10^5 / uL, 3.4x10^5 / uL, 3.5x10^5 / uL, 3.6x10^5 / uL, 3.7x10^5 / uL, 3.8x10 They exist at concentrations of 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 with an average diameter of 112-116nm.In some embodiments, the EV is about 2.5x10^5 / uL, 2.6x10^5 / uL, 2.7x10^5 / uL, 2.8x10^5 / uL, 2.9x10^5 / uL, 3.0x10^5 / uL, 3.1x10^5 / uL, 3.2x10^5 / uL, 3.3x10^5 / uL, 3.4x10^5 / uL, 3.5x10^5 / uL, 3.6x10^5 / uL, 3.7x10^5 / uL, 3.8x10^5 / uL, 3.9x10^5 / uL, 3. ...10^5 / uL, 3.2x10^5 / uL, 3.3x10^5 / uL, 3.4x10^5 / uL, 3.5x10^5 / uL, 3. In some embodiments, the EVs are present at a concentration of about 3.8x10^5 / uL ± 0.8x10^5 and an average diameter of 114 nm.

[0486] i. MSC secretome: therapeutic properties The MSC secretome of the present disclosure exhibits a variety of therapeutic properties, including, for example, anti-angiogenic properties (blood and / or lymphatic), anti-fibrotic properties, anti-inflammatory properties, properties that promote cell migration and proliferation, mitogenic properties, and anti-oxidative stress / damage properties.

[0487] In some embodiments, angiogenesis inhibitory (blood vessel and / or lymphatic vessel) properties can be determined by the presence and / or levels of one or more factors in the MSC secretome. In some embodiments, the angiogenesis inhibitory 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 angiogenesis inhibitory factors include, but are not limited to, one or more of PEDF, lower levels of VEGF, and / or serpin E1. In some embodiments, the angiogenesis inhibitory factor is PEDF. In some embodiments, the angiogenesis inhibitory factor is sFLT-1. In some embodiments, the angiogenesis inhibitor corresponds to lower levels of VEGF. In some embodiments, the angiogenesis inhibitor is serpin E1.

[0488] In some embodiments, pro-angiogenic (vascular and / or lymphatic) properties may be determined by the presence and / or levels 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.

[0489] In some embodiments, the MSC secretome exhibits anti-fibrotic properties. In some embodiments, such anti-fibrotic properties can be assayed using standard assays. In some embodiments, the presence of various factors and / or activities associated with the MSC secretome 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, activities indicative of anti-fibrotic properties include inhibition of excessive ECM deposition.

[0490] In some embodiments, the MSC secretome exhibits anti-inflammatory properties. In some embodiments, the MSC secretome blocks inflammation. In some embodiments, the MSC secretome inhibits inflammation by 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (e.g., a complete reduction in inflammation). In some embodiments, the MSC secretome prevents mast cell degranulation.

[0491] In some embodiments, the MSC secretome promotes cell migration and proliferation, including, for example, mitogen and motility-promoting activity. In some embodiments, the MSC secretome promotes mitogen activity. In some embodiments, the MSC secretome promotes motility-promoting activity. In some embodiments, the MSC secretome comprises FGF7, which mediates cell migration and proliferation activity of the MSC secretome.

[0492] In some embodiments, the MSC secretome comprises FGF7, which mediates cell migration and proliferation activity of the MSC secretome.

[0493] In some embodiments, the MSC secretome comprises HGF, which mediates cell migration and proliferation activity of the MSC secretome.

[0494] In some embodiments, the MSC secretome comprises an anti-apoptotic agent that effects cell migration and proliferation activity of the MSC secretome. In some embodiments, the MSC secretome comprises an anti-apoptotic agent, including but not limited to, FGF-2, HGF, and IGF-1, that effects cell migration and proliferation activity of the MSC secretome. In some embodiments, the MSC secretome comprises an anti-apoptotic agent selected from the group consisting of FGF-2, HGF, and IGF-1, that effects cell migration and proliferation activity of the MSC secretome.

[0495] In some embodiments, the MSC secretome comprises NGF, which effects the cell migration and proliferation activity of the MSC secretome.

[0496] In some embodiments, the MSC secretome results in reduced anti-oxidative stress and / or cell damage. In some embodiments, the MSC secretome comprises reduced anti-oxidative stress and cell-damaging factors. In some embodiments, the reduced anti-oxidative 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 anti-oxidative stress and cell-damaging factors are selected from the group consisting of SOD-1, SOD-2, SOD-3, and HO-1.

[0497] 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 specific factors to biological activity, and 3) measuring biophysical parameters. In some embodiments, to determine the characteristics of the MSC secretome, various efficacy assays described herein can be performed on the MSC secretome. 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 specific 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 may include, but are not limited to, physical characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analyses, stability assays, proliferation assays, migration assays, adhesion assays, angiogenesis assays, differentiation / scarring assays, inflammation assays, immune assays, gliosis assays, tissue explant survival and function assays, organoid development or survival / function, epithelial barrier integrity assays, retinal degeneration assays, and / or assays of inherited retinal diseases involving human and animal retinal explants, neuroprotective / neurotrophic assays. In some embodiments, the assays are used for characterization of physical factors, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analyses, stability assays, proliferation assays, migration assays, adhesion assays, angiogenesis assays, differentiation / scarring assays, inflammation assays, immune assays, gliosis assays, tissue explant survival and function assays, organoid development or survival / function, epithelial barrier integrity assays, retinal degeneration assays, and / or assays of inherited retinal diseases involving human and animal retinal explants, neuroprotection / neurotrophy assays.

[0498] Physical element characterization: In some embodiments, characterizing the MSC secretome involves methods using a combination of bioanalytical techniques. In some embodiments, characterizing the MSC secretome involves determining physical components of the MSC secretome. In some embodiments, characterizing the MSC secretome involves using a protein array, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, and immunoblotting. In some embodiments, characterizing the MSC secretome can be used to identify molecules in the MSC secretome. In some embodiments, a protein array 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.

[0499] 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 of the MSC secretome can be evaluated for EV markers. In some embodiments, the EV fraction of the MSC secretome can be evaluated for EV markers, including, but not limited to, AUX, TSG101, CD63, CD9, and CD8.

[0500] In some embodiments, secretomes contain 1x10 extracellular vesicles (EVs) per mL in the size range of 30-200 nm. 8 ~5x10 9 Includes the number of EVs.

[0501] In some embodiments, depletion studies can be performed to identify 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, as described later, and then assessed by one or more bioassays. In some embodiments, depletion studies can be performed to assess the contributions of protein fractions 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.

[0502] Oxidative stress: In some embodiments, an oxidative stress prevention assay can be performed on the 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.

[0503] Safety characterization: In some embodiments, the MSC secretome may be evaluated for hemocompatibility by performing tests for sterility and pyrogen and endotoxin levels. In some embodiments, the MSC secretome may be evaluated for hemocompatibility. In some embodiments, evaluating hemocompatibility includes assays for hemolysis and hemagglutination. 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 in 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.

[0504] In some embodiments, the MSC secretome may be sterile so that it can be administered as part of a pharmaceutical preparation. In some embodiments, the MSC secretome may be free or substantially free of endotoxins. In some embodiments, the MSC secretome may be free or substantially free of microorganisms.

[0505] Stability: In some embodiments, biophysical characteristics of the MSC secretome may be assessed and / or determined. In some embodiments, fluorescence, static light scattering, and dynamic light scattering are used to characterize measures of protein stability. In some embodiments, the following parameters may be measured to further characterize the secretome: thermal melting, thermal aggregation, ΔG, and / or viscosity. In some embodiments, a thermal melting assay is used to determine 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, ΔG is used as a measure to determine the stability of the MSC secretome. In some embodiments, viscosity is measured as a characteristic of the MSC secretome. In some embodiments, viscosity is used to determine the stability of the MSC secretome.

[0506] In some embodiments, biophysical measures can be used to establish stability parameters to characterize various MSC secretome preparations.

[0507] 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.

[0508] 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).

[0509] 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).

[0510] Epithelial barrier integrity assay The corneal epithelium, more precisely the apical surface of the epithelium, contributes significantly to the overall barrier properties of the cornea, and changes in the corneal barrier serve as a sensitive factor for 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 × [TEER (treated tissue) / TEER (placebo control)]

[0511] In some embodiments, TEER can be used to evaluate the effect of topical application of MSC secretome on barrier integrity. In some embodiments, using an EpiCorneal tissue model (MatTek Corp), TEER can be used to evaluate the effect of topical application of MSC secretome on barrier integrity after corneal epithelial damage caused by topical exposure to nitrogen mustard (NM). 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.

[0512] 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); neovascularization, 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); neovascularization; and inflammation.

[0513] Migration and proliferation: In some embodiments, MSC secretomes may be assessed for their ability to promote proliferation and migration. In some embodiments, MSC secretomes may be assessed for their ability to promote proliferation. In some embodiments, MSC secretomes may 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 may be assessed using a transwell migration assay to determine proliferation-promoting ability.

[0514] In some embodiments, a migration assay can be used to assess the ability of an MSC secretome to promote migration. In some embodiments, a migration assay can be used to assess the ability of an MSC secretome to promote migration, and the migration assay is an in vitro wound closure assay. In some embodiments, the migration assay can include a "scratch 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," on a confluent cell monolayer. The "scratch" can be monitored for migration of cells at the edge of the newly created gap toward the opening to 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).

[0515] In some embodiments, migration assays may include transwell migration assays using corneal epithelial cells (or other validated cell surrogates), and (e.g., wound closure) may be performed on MSC secretomes. In some embodiments, transwell migration assays using corneal epithelium as a test for the wound closure efficacy of MSC secretomes. In some embodiments, MSC secretomes promote wound closure as determined using a transwell migration assay.

[0516] 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.

[0517] 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 an examination of 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.

[0518] In some embodiments, a circular scratch wound method or a circular scratch wound assay or a circular wound closure assay may be used. In some embodiments, the Oris™ cell migration assay platform may be used (see also Example 6 herein).

[0519] 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 angiogenesis inhibitory properties. In some embodiments, the MSC secretome is angiogenesis inhibitory properties. In some embodiments, the endothelial cell tube formation assay provides a ratio of angiogenesis inhibitory signals to pro-angiogenesis signals. In some embodiments, a negative result from the endothelial cell tube formation assay confirms a high inhibition:promotion ratio, ensuring that the MSC secretome does not promote neovascularization. In some embodiments, a negative result from the endothelial cell tube formation assay confirms a high inhibition:promotion ratio, ensuring that the MSC secretome does not promote CNV (choroidal neovascularization) or neovascularization in general. In some embodiments, an assay for inhibition of myofibroblast differentiation by TGFb may be performed on the MSC secretome. In some embodiments, an assay for inhibition of myofibroblast differentiation by TGFb may be performed on the MSC secretome to show that the MSC secretome prevents scarring. In some embodiments, the MSC secretome prevents scarring. In some embodiments, the MSC secretome prevents cicatricial corneal opacity. In some embodiments, the MSC secretome has low angiogenic potential. In some embodiments, the MSC secretome exhibits 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 a medium that supports angiogenesis. In some embodiments, the MSC secretome has reduced angiogenic potential when compared to an untreated control. In some embodiments, the MSC secretome has reduced angiogenic potential compared to samples treated with serum-containing medium, hi some embodiments, the MSC secretome attenuates the angiogenic response.In some embodiments, the MSC secretome reduces the angiogenic response induced by serum-free medium. In some embodiments, when serum-containing medium plus secretome (reduced or absent angiogenic response) is compared to serum-containing medium (angiogenic response), the MSC secretome induces a reduced angiogenic response. In some embodiments, the angiogenic response is demonstrated by tube formation in a cell-based assay. In some embodiments, the angiogenic response is demonstrated by tube formation in an endothelial cell tube formation assay.

[0520] Differentiation / scarring: In some embodiments, the MSC secretome may be evaluated for its ability to prevent differentiation and prevent scarring. In some embodiments, the MSC secretome prevents and / or impedes scarring. In some embodiments, the MSC secretome prevents scarring. In some embodiments, the MSC secretome reduces scarring compared to other standard treatments. In some embodiments, the MSC secretome prevents and / or impedes differentiation. In some embodiments, the MSC secretome prevents and / or impedes myofibroblast differentiation. In some embodiments, the MSC secretome reduces corneal transparency loss. In some embodiments, the MSC secretome reduces corneal transparency loss by preventing and / or impeding myofibroblast differentiation.

[0521] In some embodiments, MSC secretomes can be assessed for their ability to modulate factors involved in differentiation. In some embodiments, MSC secretomes can be assessed for their ability to modulate 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, the MSC secretome modulates a factor 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, the MSC secretome induces a decrease in a factor that is upregulated during normal differentiation. In some embodiments, the MSC secretome induces an increase in a factor that is downregulated during normal differentiation. In some embodiments, the MSC secretome induces a decrease in the expression of a factor such as SMA. In some embodiments, the MSC secretome induces a decrease in the expression of factors such as SMA, which indicates the efficacy of the MSC secretome.

[0522] Neovascularization: In some embodiments, the MSC secretome may be evaluated for its ability to prevent neovascularization. In some embodiments, the MSC secretome prevents, interferes with, inhibits, and / or reduces neovascularization. In some embodiments, the MSC secretome inhibits or does not promote neovascularization. In some embodiments, the MSC secretome may be evaluated for its ability to prevent angiogenesis. In some embodiments, the MSC secretome prevents, interferes with, inhibits, and / or reduces angiogenesis. In some embodiments, the MSC secretome inhibits angiogenesis.

[0523] 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.

[0524] inflammation: In some embodiments, the MSC secretome may be evaluated for its ability to prevent, impede, inhibit, and / or reduce inflammation. In some embodiments, the MSC secretome prevents, impedes, inhibits, and / or reduces inflammation. In some embodiments, the MSC secretome inhibits inflammation. In some embodiments, the MSC secretome is characterized in vitro and / or in vivo to determine its ability to prevent, impede, inhibit, and / or reduce inflammation. In some embodiments, the MSC secretome prevents, impedes, inhibits, and / or reduces inflammation in vitro and / or in vivo. In some embodiments, the MSC secretome prevents, impedes, inhibits, and / or reduces inflammation in vitro. In some embodiments, the MSC secretome prevents, impedes, inhibits, and / or reduces inflammation in vivo. In some embodiments, a tissue model may be used to characterize the prevention, impediment, inhibition, and / or reduction of inflammation in vitro. In some embodiments, 3D tissue models may be used to characterize the prevention, obstruction, inhibition, and / or reduction of inflammation in vitro. In some embodiments, a nitrogen mustard (NM) gas combustion model may be used to evaluate the prevention, obstruction, inhibition, and / or reduction of inflammation in vitro. In some embodiments, a nitrogen mustard (NM) gas combustion model may be used to evaluate the prevention, obstruction, inhibition, and / or reduction of inflammation in vitro and as a surrogate for in vivo conditions. In some embodiments, cytokine profiles in response to treatment with and / or administration of MSC secretome may be determined. In some embodiments, levels of specific cytokines may be determined. In some embodiments, levels of IL-8 may be determined. In some embodiments, IL-8 expression levels may be reduced in tissues treated with MSC secretome.In some embodiments, the expression levels of IL-8 are reduced in tissues treated with MSC secretome, indicating prevention, disruption, inhibition, and / or reduction of inflammation.

[0525] I. Treatment Method The present disclosure also provides methods of treatment 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 is used in the treatment of ocular conditions, including accelerating wound healing. In some embodiments, the MSC secretome is used in the treatment of ocular conditions, including reducing scarring. In some embodiments, the MSC secretome is used in the treatment of ocular conditions, including reducing inflammation. In some embodiments, the MSC secretome is used in the treatment of ocular conditions, including reducing inflammation and therefore promoting growth. In some embodiments, the MSC secretome is used in the treatment of ocular conditions, such as reducing inflammation at the ocular surface. In some embodiments, the MSC secretome is used in the treatment of ocular conditions, including reducing neovascularization. In some embodiments, the MSC secretome is used in the treatment of ocular conditions, including reducing neovascularization in the cornea. In some embodiments, the MSC secretome is used in the treatment of ocular conditions, including the treatment of dry eye (e.g., including the treatment of severe dry eye, including epithelial cell damage). In some embodiments, the MSC secretome is used in the treatment of ocular conditions, such as restoring the integrity of damaged ocular tissue. In some embodiments, the MSC secretome is used in the treatment of ocular conditions, such as accelerating the healing of damaged ocular tissue. In some embodiments, the MSC secretome is used in the treatment of ocular conditions, such as treating retinal conditions. In some embodiments, the MSC secretome is used in the treatment of ocular conditions, such as treating macular disease. In some embodiments, the MSC secretome is used in the treatment of ocular conditions, such as regenerating damaged ocular nerve tissue. In some embodiments, the MSC secretome is used in the treatment of ocular conditions, such as regenerating damaged ocular nerve tissue associated with PCED.In some embodiments, the MSC secretome is used in the treatment of an ocular condition, such as PCED. In some embodiments, the MSC secretome is used in the treatment of an ocular condition, such as inflammatory damage to the ocular surface. In some embodiments, the MSC secretome is used in the treatment of an ocular condition, such as GvHD and / or Sjogren's syndrome. In some embodiments, the MSC secretome is used in the treatment of an ocular condition, such as corneal stem cell deficiency (LSCD). In some embodiments, the MSC secretome is used to accelerate wound healing. In some embodiments, the MSC secretome is used to reduce scarring. In some embodiments, the MSC secretome is used to reduce inflammation. In some embodiments, the MSC secretome is used to reduce inflammation and thus promote growth. In some embodiments, the MSC secretome is used to reduce inflammation at the ocular surface. In some embodiments, the MSC secretome is used to reduce neovascularization. In some embodiments, the MSC secretome is used to reduce neovascularization in the cornea. In some embodiments, the MSC secretome is used to protect and repair retinal epithelial cells and retinal ganglion cells. In some embodiments, the MSC secretome is used to induce regeneration of the trabecular meshwork and reduce intraocular pressure.

[0526] In some embodiments, the mesenchymal stem cell secretome is administered to treat an ocular disease. In some embodiments, the treatment comprises administering a therapeutically effective amount of a mesenchymal stem cell secretome composition 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 prevent neovascularization, reduce and / or prevent scarring, improve and / or maintain vision, and / or increase the rate of wound closure (e.g., decrease wound closure time). In some embodiments, the mesenchymal stem cell secretome is administered to a patient in need thereof to prevent, reduce, and / or prevent neovascularization. In some embodiments, the mesenchymal stem cell secretome is administered to a patient in need thereof to prevent, reduce, and / or prevent 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 accelerated wound closure of a closing wound (e.g., shortening the time required for wound closure). In some embodiments, the mesenchymal stem cell secretome prevents, reduces, and / or inhibits or does not promote neovascularization and reduced scarring to promote the maintenance of vision. In some embodiments, the mesenchymal stem cell secretome is administered to a patient in need thereof to prevent, reduce, and / or inhibit neovascularization and reduced scarring to promote the maintenance of vision. 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.

[0527] In some embodiments, the mesenchymal stem cell secretome is administered to treat 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 described herein.

[0528] In some embodiments, the mesenchymal stem cell secretome is administered for the treatment of traumatic injury of optic nerve degeneration following concussive injury. In some embodiments, the ocular concussive injury is selected from the group consisting of ocular contusion and blunt ocular injury. In some embodiments, the mesenchymal stem cell secretome is administered for the treatment of 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 described herein.

[0529] In some embodiments, a mesenchymal stem cell secretome is administered to ameliorate optic nerve degeneration following ocular concussive injury. 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 described herein. In some embodiments, the ocular concussive injury is selected from the group consisting of an ocular contusion and a blunt ocular injury. In some embodiments, the ocular concussive injury comprises an ocular contusion. In some embodiments, the ocular concussive injury is a blunt ocular injury.

[0530] Efficacy readouts can include, for example, a reduction in symptoms and / or a decrease in disease state, including an improvement in quality of life. In some embodiments, a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% reduction in symptoms and / or a decrease in 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 neovascularization indicates therapeutic efficacy.

[0531] In some embodiments, the disease or condition is an ocular disease or condition. In some embodiments, the disease or condition is visual dysfunction following traumatic injury to an ocular structure. In some embodiments, the disease or condition is a concussive injury (e.g., blunt or non-blunt injury) of the eye. In some embodiments, the disease or condition is a burn, including a chemical burn, of the eye.

[0532] 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.

[0533] 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.

[0534] 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.

[0535] 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.

[0536] 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 mesenchymal stem cell secretome is administered by intravitreal injection. In some embodiments, the MSC secretome composition exhibits ultra-high potency when administered to a subject in need thereof. In some embodiments, the mesenchymal stem cell secretome is administered locally once, twice, three times, four times, five times, and / or up to six times daily. In some embodiments, the MSC secretome composition provides a therapeutic effect with one drop or one administration per day. In some embodiments, one drop is administered 1, 2, 3, 4, 5, or 6 times per day. In some embodiments, one drop is administered at intervals of 1 hour, 2 hours, 3 hours, or 4 hours. In some embodiments, one drop is administered at least once per 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 twice per 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 per 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 per 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 per 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 per day for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks.

[0537] In some embodiments, the mesenchymal stem cell secretome is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times per day. In some embodiments, the mesenchymal stem cell secretome is administered about 1-10 times per day. In some embodiments, the mesenchymal stem cell secretome is administered once per day. In some embodiments, the mesenchymal stem cell secretome is administered twice per day. In some embodiments, the mesenchymal stem cell secretome is administered three times per day. In some embodiments, the mesenchymal stem cell secretome is administered four times per day. In some embodiments, the mesenchymal stem cell secretome is administered five times per day. In some embodiments, the mesenchymal stem cell secretome is administered six times per day. In some embodiments, the mesenchymal stem cell secretome is administered seven times per day. In some embodiments, the mesenchymal stem cell secretome is administered eight times per day. In some embodiments, the mesenchymal stem cell secretome is administered 9 times per day. In some embodiments, the mesenchymal stem cell secretome is administered 10 times per day.

[0538] In some embodiments, about 1-10 drops of mesenchymal stem cell secretome are administered per day. In some embodiments, about 1 drop of mesenchymal stem cell secretome is administered per day. In some embodiments, about 2 drops of mesenchymal stem cell secretome is administered per day. In some embodiments, about 3 drops of mesenchymal stem cell secretome is administered per day. In some embodiments, about 4 drops of mesenchymal stem cell secretome is administered per day. In some embodiments, about 5 drops of mesenchymal stem cell secretome is administered per day. In some embodiments, about 6 drops of mesenchymal stem cell secretome is administered per day. In some embodiments, about 7 drops of mesenchymal stem cell secretome is administered per day. In some embodiments, about 8 drops of mesenchymal stem cell secretome is administered per day. In some embodiments, about 9 drops of mesenchymal stem cell secretome is administered per day. In some embodiments, about 10 drops of mesenchymal stem cell secretome is administered per day.

[0539] In some embodiments, a high dose of mesenchymal stem cell secretome is administered. In some embodiments, a low dose of mesenchymal stem cell secretome is administered.

[0540] In some embodiments, about 0.5-10 U / mL of mesenchymal stem cell secretome is administered. In some embodiments, at least 1 U / mL of mesenchymal stem cell secretome is administered. In some embodiments, at least 2 U / mL of mesenchymal stem cell secretome is administered. In some embodiments, at least 3 U / mL of mesenchymal stem cell secretome is administered. In some embodiments, at least 4 U / mL of mesenchymal stem cell secretome is administered. In some embodiments, at least 5 U / mL of mesenchymal stem cell secretome is administered. In some embodiments, at least 6 U / mL of mesenchymal stem cell secretome is administered. In some embodiments, at least 7 U / mL of mesenchymal stem cell secretome is administered. In some embodiments, at least 8 U / mL of mesenchymal stem cell secretome is administered. In some embodiments, at least 9 U / mL of mesenchymal stem cell secretome is administered. In some embodiments, at least 10 U / mL of mesenchymal stem cell secretome is administered.

[0541] In some embodiments, about 1 U / mL of mesenchymal stem cell secretome is administered. In some embodiments, about 2 U / mL of mesenchymal stem cell secretome is administered. In some embodiments, about 3 U / mL of mesenchymal stem cell secretome is administered. In some embodiments, about 4 U / mL of mesenchymal stem cell secretome is administered. In some embodiments, about 5 U / mL of mesenchymal stem cell secretome is administered. In some embodiments, about 6 U / mL of mesenchymal stem cell secretome is administered. In some embodiments, about 7 U / mL of mesenchymal stem cell secretome is administered. In some embodiments, about 8 U / mL of mesenchymal stem cell secretome is administered. In some embodiments, about 9 U / mL of mesenchymal stem cell secretome is administered. In some embodiments, about 10 U / mL of mesenchymal stem cell secretome is administered.

[0542] In some embodiments, one drop of mesenchymal stem cell secretome at a concentration of 1 U / mL is administered once per day. In some embodiments, one drop of mesenchymal stem cell secretome at a concentration of 1 U / mL is administered twice per day. In some embodiments, one drop of mesenchymal stem cell secretome at a concentration of 1 U / mL is administered three times per day. In some embodiments, one drop of mesenchymal stem cell secretome at a concentration of 1 U / mL is administered four times per day. In some embodiments, one drop of mesenchymal stem cell secretome at a concentration of 1 U / mL is administered once per day for at least 56 days. In some embodiments, one drop of mesenchymal stem cell secretome at a concentration of 1 U / mL is administered two times per day for at least 56 days. In some embodiments, one drop of mesenchymal stem cell secretome at a concentration of 1 U / mL is administered three times per day for at least 56 days. In some embodiments, one drop of mesenchymal stem cell secretome at a concentration of 1 U / mL is administered four times per day for at least 56 days.

[0543] In some embodiments, one drop of mesenchymal stem cell secretome at a concentration of 3 U / mL is administered once per day. In some embodiments, one drop of mesenchymal stem cell secretome at a concentration of 3 U / mL is administered twice per day. In some embodiments, one drop of mesenchymal stem cell secretome at a concentration of 3 U / mL is administered three times per day. In some embodiments, one drop of mesenchymal stem cell secretome at a concentration of 3 U / mL is administered four times per day. In some embodiments, one drop of mesenchymal stem cell secretome at a concentration of 3 U / mL is administered once per day for at least 56 days. In some embodiments, one drop of mesenchymal stem cell secretome at a concentration of 3 U / mL is administered two times per day for at least 56 days. In some embodiments, one drop of mesenchymal stem cell secretome at a concentration of 3 U / mL is administered three times per day for at least 56 days. In some embodiments, one drop of mesenchymal stem cell secretome at a concentration of 3 U / mL is administered four times per day for at least 56 days.

[0544] In some embodiments of 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, the MSC secretome composition comprises: i. optionally, 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. optionally, 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-β, and PDGF; and / or iii. Optionally, 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.

[0545] In some embodiments, the MSC secretome composition for use in the treatment method 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.

[0546] In some embodiments, an MSC secretome composition for use in a treatment method 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 treatment method 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 treatment method 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.

[0547] In some embodiments, the MSC secretome composition for use in the treatment method further comprises mid-range levels 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.

[0548] In some embodiments, the MSC secretome composition for use in the treatment method 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, angiogenin, DPPIV, IGFBP-3, and uPA.

[0549] In some embodiments, the MSC secretome composition for use in the treatment method further comprises at least one factor selected from the group consisting of apolipoprotein A1, complement factor D, complement factor H, complement factor I, C1 esterase inhibitor (C1-INH), C4b binding protein (C4BP), CD46, C-reactive protein, cystatin C, DKK-1, Emmprin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15, and IFNγ.

[0550] In some embodiments, the MSC secretome composition for use in the treatment method comprises a ratio of anti-angiogenic factors to pro-angiogenic factors that is greater than 2, greater than 3, greater than 4, or greater than 5. In some embodiments, the anti-angiogenic factors comprise one or more factors selected from the group consisting of PEDF, lower levels of VEGF, and serpin E1, and the pro-angiogenic factors comprise VEGF, angiogenin, IGFBP-3, uPA, Angio-1, Angio-2, endothelin-1.

[0551] In some embodiments, the MSC secretome composition for use in the treatment method further comprises a low level of VEGF. In some embodiments, the MSC secretome for use in the treatment method 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 treatment method comprises one or more angiogenesis inhibitors, and the sum of the concentrations of the one or more angiogenesis inhibitors relative to the concentration of VEGF is greater than 2, greater than 3, greater than 4, or greater than 5.

[0552] In some embodiments, the MSC secretome composition for use in the treatment methods is free of or contains very low levels of bFGF, PLGF, and PDGF.

[0553] In some embodiments, the MSC secretome composition for use in the treatment methods comprises less than 1000 pg / mL of bFGF, PLGF, and PDGF.

[0554] In some embodiments, the MSC secretome composition for use in the treatment methods has a pH of about 4.7 to about 7.5.

[0555] In some embodiments, the MSC secretome composition for use in the treatment methods 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 citric acid / disodium phosphate.

[0556] In some embodiments, the MSC secretome composition for use in the treatment methods further comprises a tonicity adjusting agent, hi some embodiments, the tonicity adjusting agent is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin.

[0557] In some embodiments, the MSC secretome composition for use in the treatment method further comprises mono / disodium phosphate, mannitol, and trehalose, and the composition has a pH of about pH 7.4.

[0558] In some embodiments, the MSC secretome composition for use in the treatment method further comprises a divalent cation, hi some embodiments, the divalent cation is selected from the group consisting of Mg2+, Ca2+, and Zn2+.

[0559] In some embodiments, the MSC secretome composition for use in the treatment method further comprises disodium phosphate / citric acid, mannitol, and trehalose, and the composition has a pH of about pH 6.4.

[0560] In some embodiments, the MSC secretome composition for use in the treatment method further comprises an adhesive agent, hi some embodiments, the adhesive agent is selected from the group consisting of hypromellose, Poloxamer 407, Poloxamer 188, Poloxomer 237, Poloxomer 338, hypromellose, (HPMC), polycarbophil, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyimide, sodium hyaluronate, gellan gum, poly(lactic-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethylcellulose (CMC), or hydroxypropylmethylcellulose (HPMC), hydroxymethylcellulose, hydroxyethylcellulose, sodium carboxymethylcellulose, fibrin glue, polyethylene glycol, and GelCORE.

[0561] In some embodiments, the MSC secretome composition for use in the treatment method does not contain one or more components selected from the group consisting of xenobiotic components, phenol red, peptides and biomolecules smaller than 3 kDa, antibiotics, protein aggregates larger than 200 nm, cells, non-exosomes / non-extracellular vesicle cellular debris, hormones, and L-glutamine.

[0562] In some embodiments, the MSC secretome composition for use in the treatment method comprises HGF, pentraxin-3 (TSG-14), VEGF, TIMP-1, serpin E1, and less than 5 ng / mL of IL-8.

[0563] In some embodiments, the MSC secretome for use in the method of treatment composition comprises: i. 0.3–4.5 ng / mL HGF; ii. 0.5–20 ng / mL pentraxin-3 (TSG-14); iii. VEGF between 100 and 600 pg / mL; iv. 10–200 ng / mL TIMP-1; v. 20-80 ng / mL of serpin E1; and / or vi. IL-8 less than 5 ng / mL.

[0564] In some embodiments, the MSC secretome composition for use in the treatment methods comprises an anti-angiogenic MSC secretome or an anti-scarring MSC secretome.

[0565] In some embodiments, the present disclosure provides 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 comprising: i. 2 μg–400 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-24mg trehalose dihydrate; vi. 0.5 mg to 2 mg of hypromellose per mL; and / or The pH is about 4.7 to about 7.5.

[0566] In some embodiments, the present disclosure provides 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 comprising: i. 0.004%-0.08% w / w MSC secretome ii. 4%-5% w / w of monobasic sodium phosphate; iii. 21.5%-23% w / w of dibasic sodium phosphate; iv. 23%-25% w / w mannitol; v. 46%-48% w / w trehalose dehydrate; vi. 1% to 3% w / w of hypromellose; and / or The pH is about 4.7 to about 7.5.

[0567] In some embodiments, the present disclosure provides 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 comprising: i. 2 μg–400 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-24mg trehalose dihydrate; vi. optionally, 0.5 mg to 2 mg of hypromellose per mL; and / or The pH is about 4.7 to about 7.5.

[0568] In some embodiments, the present disclosure provides 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 comprising: i. 0.004%-0.08% w / w MSC secretome ii. 4%-5% w / w of monobasic sodium phosphate; iii. 21.5%-23% w / w of dibasic sodium phosphate; iv. 23%-25% w / w mannitol; v. 46%-48% w / w trehalose dehydrate; vi. optionally 1% to 3% w / w of hypromellose; and / or The pH is about 4.7 to about 7.5.

[0569] In some embodiments, the present disclosure provides 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 comprising: i. 2 μg–400 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; and / or The pH is about 4.7 to about 7.5.

[0570] In some embodiments, the present disclosure provides 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 comprising: i. 0.004%-0.08% w / w MSC secretome ii. 4%-5% w / w of monobasic sodium phosphate; iii. 21.5%-23% w / w of dibasic sodium phosphate; iv. 23%-25% w / w mannitol; v. 46% to 48% w / w trehalose dehydrate; and / or The pH is about 4.7 to about 7.5.

[0571] J. Kit The kit may include an MSC secretome in a container, or a conditioned medium for use in preparing an MSC secretome, also in a container, as disclosed herein, and instructions for use. The kit may also include components for mixing to prepare a solution for use in treating the eye, and instructions for mixing and use.

[0572] The container can include at least one vial, well, test tube, flask, bottle, syringe, or other container means, into which the MSC secretome in the container or conditioned medium for use in preparing the MSC secretome is placed, optionally suitably aliquoted. If additional components are provided, the kit can include additional containers into which the components can be placed. Such containers can include injection-molded or blow-molded plastic containers that hold the desired vials. The container and / or kit can include instructions for use and / or warning labels.

[0573] 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 hereby expressly incorporated by reference.

[0574] The invention may 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 characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analyses, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / citralization assays, inflammation assays, epithelial barrier integrity assays, retinal degeneration assays, and / or assays for inherited retinal diseases involving human and animal retinal explants, and neuroprotective / neurotrophic assays. In some embodiments, the panel of tests and / or assays identifies the MSC secretome described herein.

[0575] The invention may 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 characterization, oxidative stress assays, misfolded protein response assays, ER stress assays, safety analyses, stability assays, proliferation assays, migration assays, adhesion assays, neovascularization assays, differentiation / citralization assays, inflammation assays, epithelial barrier integrity assays, retinal degeneration assays, and / or assays for inherited retinal diseases involving human and animal retinal explants, and neuroprotective / neurotrophic assays. In some embodiments, the panel of tests and / or assays identifies the MSC secretome described herein. [Example]

[0576] Example 1: MSC secretome: manufacturing process The manufacturing process consists of the following steps: Production of formulation buffers in compounding tanks, Dilution of the drug substance with formulation buffer in the holding tank; and · Aseptic filling process using blow-fill-seal process.

[0577] MSC secretome: preparation of formulation buffer Add the following ingredients to a 30 L blending tank equipped with a top-mounted mixer (Figure 1):

[0578] [Table 10]

[0579] MSC secretome: Dilution of drug substance to obtain final bulk for filling Calculations are performed at the start of compounding based on available protein assays and normalized to the target concentration in the filled product. A known amount of formulation buffer is filtered through a 0.22μ bioburden reduction filter and transferred to a holding tank.

[0580] The frozen drug substance is thawed (for a predetermined time based on conditions) and then added to a holding tank.

[0581] The drug substance is added to a holding tank containing the formulation buffer.

[0582] A summary flow chart for aseptic filling using the blow-fill-seal process is shown in Figure 2.

[0583] MSC secretome: a feasibility study Three feasibility batches were run with the following objectives: Identifying formulation process parameters that are critical to achieving a reproducible quality finished product Determine the critical BFS process parameters to achieve forming and filling with defined quality attributes · Determine the initial product purge required to eliminate the dilution effect as a result of the BFS steam treatment process. · Freezer testing to determine the time required for the pellets of the finished product to freeze.

[0584] Sampling Plan

[0585] [Table 11]

[0586] Example 2: Evaluation of the MSC secretome in subjects with persistent corneal epithelial defects (PCED) Summary:

[0587] The primary objective of this study is to investigate the safety and efficacy of a pharmaceutical composition comprising bone marrow-derived MSC secretome as disclosed herein compared to vehicle in participants with a clinically documented diagnosis of persistent corneal epithelial defect (PCED). The pharmaceutical composition comprising bone marrow-derived MSC secretome is prepared according to the manufacturing process described herein and in Example 1.

[0588] Detailed Description:

[0589] Approximately 90 participants diagnosed with persistent corneal epithelial defects (PCEDs) will be enrolled at multiple sites in a study to evaluate the safety and efficacy of a topical mesenchymal stem cell secretome therapy, a pharmaceutical composition containing bone marrow-derived MSC secretome. After a first cohort of at least two participants to evaluate the safety of the high-potency pharmaceutical composition containing bone marrow-derived MSC secretome, participants in a second cohort will be randomized to 8 weeks of treatment with either the active drug or vehicle (placebo). Cure rates will be compared between the groups treated with the pharmaceutical composition containing bone marrow-derived MSC secretome and the vehicle-treated group. Total study participation will last approximately 34 weeks.

[0590] Test Arm:

[0591] Experiment: Cohort 1, Arm 1 High-dose subjects will receive one drop of bone marrow-derived MSC Secretome eye drop at 3U / mL four times daily for 56 days.

[0592] Experiment: Cohort 2, Arm 1 Low-dose subjects will receive one drop of bone marrow-derived MSC secretome eye drops at 1U / mL four times a day for 56 days.

[0593] Experiment: Cohort 2, Arm 2 High-dose subjects will receive one drop of bone marrow-derived MSC Secretome eye drop at 3U / mL four times daily for 56 days.

[0594] Placebo comparison: Cohort 2, Arm 3 Vehicle subjects received bone marrow-derived MSC secretome eye drops (no active drug) at 0 U / mL, one drop four times daily for 56 days.

[0595] Primary endpoint: Response status [duration: 56 days]: Complete resolution of PCED and no corneal fluorescein staining in the test lesions.

[0596] Eligibility Criteria: Eligible age for the test: 18 years or older (adults, elderly) Eligible genders: All Accepting healthy volunteers: No

[0597] Selection Criteria - Have had PCED for at least the past 7 days due to underlying conditions such as diabetic keratopathy, herpetic eye disease, severe dry eye disease, corneal epithelial stem cell deficiency, infectious keratitis, neurotrophic keratitis, post-ocular surgery, medical trauma, or chemical burns. · PCED measurements meet test criteria.

[0598] Exclusion criteria · Have an active eye infection or active infectious disease that may affect PCED. · Severe corneal burns in the test eye. Severe corneal epithelial stem cell deficiency in either eye. The circumference of the area affected by limbal vascular ischemia exceeds 75% of the circumference in the study eye. · You have severe blepharitis or severe meibomian gland disease. The study eye has severe eyelid abnormalities that contribute to the persistence of PCED. · Evidence of corneal ulceration. · The need for punctal occlusion is anticipated. - Use of Oxervate in the study eye within the past 30 days. · History of surgical procedures to treat the PCED being studied. - History of other ophthalmic surgery in the study eye within 90 days prior to screening. · Unwillingness to temporarily discontinue contact lens use in the test eye. · Use of Botox injections to induce pharmacological ptosis within the last 90 days. · Systemic doxycycline use is anticipated. - Chemotherapeutic agents have been used within 7 days prior to the study or are expected to be used during the study. · History of abuse of or dependence on study drugs or alcohol. - Taking another investigational drug within the last 30 days. Patients who are pregnant, nursing, or intend to become pregnant during the study.

[0599] The foregoing examples are provided so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use embodiments of the compositions, systems, and methods of this invention, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes for carrying out the invention that are obvious to those of ordinary skill in the art are intended to be within the scope of the following claims. All patents and publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference was individually incorporated by reference in its entirety.

[0600] All heading and section designations are used for clarity and reference purposes only and should not be construed as limiting in any way. For example, those skilled in the art will understand the utility of combining various aspects from different headings and sections as appropriate in accordance with the spirit and scope of the invention described herein.

[0601] All references cited in this specification are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0602] It will be apparent to those skilled in the art that many modifications and variations of this application can be made without departing from its spirit and scope. The specific embodiments and examples described herein are offered by way of example only, and this application is limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

1. A method for preparing and packaging a pharmaceutical preparation containing one or more pharmaceutically active substances, (a) Prepare the formulation buffer in a container. (b) Adding a drug substance preparation containing one or more pharmaceutically active substances to the formulation buffer in the container to dilute it and produce a first pharmaceutical formulation, and (c) The method comprising filling the first pharmaceutical formulation into a product container using a blow-fill-seal process to produce a second pharmaceutical formulation, wherein the filled product container contains one or more of the pharmaceutically active substances in a therapeutically effective amount, and step (c) is performed in a sterile system.

2. (i) The method further includes filtering the formulation buffer before step (a) and transferring the filtered formulation buffer to the container of step (a), (ii) The formulation buffer is filtered through a 0.22 μm bioburden reduction filter before step (a). (iii) The container in step (a) is a 30L mixing tank. (iv) The container of step (a) is equipped with a top-mounted mixer, (v) The formulation buffer comprises monobasic sodium phosphate monohydrate, dibasic sodium phosphate anhydrous, sodium chloride, magnesium chloride hexahydrate, mannitol, trehalose dihydrate, hydroxypropyl methylcellulose, and purified water. (vi) Prior to step (b), the active pharmaceutical ingredient preparation is cryopreserved, and the method further comprises thawing the active pharmaceutical ingredient preparation at the start of step (b), optionally, the thawing is performed over a predetermined period of time based on the state of one or more cryopreserved pharmaceutically active substances. (vii) Between step (b) and step (c), the first pharmaceutical preparation is subjected to filtration before step (c). (viiii) Between step (b) and step (c), the first pharmaceutical preparation is filtered using two or more sterile filters. (ix) The blow-fill seal process of step (c) is performed using a Weiler 624 BFS machine. (x) After step (c), further comprising freezing the filled product container, (xi) Further comprising repeating step (c) to produce a plurality of filled product containers, each of which filled product containers contains one or more of the pharmaceutically active substances in a therapeutically effective amount. (xi) Step (c) is repeated simultaneously to produce a plurality of filled product containers, (xiiii) The temperature of the formulation buffer during step (a) is maintained at ambient temperature. (xiv) The temperature of the formulation buffer during step (a) is maintained at approximately 20°C to 30°C, or approximately 25°C. (xv) The formulation buffer is transferred to the container in step (a) under a pressure of about 10 to 15 psi. (xvi) The temperature of the formulation buffer and the first pharmaceutical formulation is maintained at approximately 2°C to 8°C during step (b). (xvii) After step (b), the first pharmaceutical preparation is transferred to two or more sterile filters under a pressure of approximately 3 to 7 psi. (xviiii) Between step (b) and step (c), the first pharmaceutical preparation is maintained at approximately 20°C to 30°C, or approximately 25°C. (xix) During step (c), the first pharmaceutical formulation is exposed to a temperature of approximately 60°C to 70°C for approximately 1 to 10 seconds, approximately 10 to 20 seconds, approximately 20 to 30 seconds, approximately 30 to 40 seconds, approximately 40 to 50 seconds, or approximately 50 to 50 seconds, and the first pharmaceutical formulation is maintained at approximately 20°C to 30°C for the remainder of step (c). (xx) Upon completion of step (b), the first pharmaceutical preparation is collected and stored in a second container for further use prior to step (c), and optionally (i) the second container is made of a material other than glass and / or (ii) the second container is made of polypropylene, (xxi) The filled product container has a volume of 10 mL, (xxii) The filled product container contains approximately 7 mL to 10 mL of the second pharmaceutical preparation. (xxiii) The filled product container contains approximately 7.8 g to 8.65 g of the second pharmaceutical preparation. (xxiv) The filled product container contains approximately 8.28 g of the second pharmaceutical preparation. (xxv) The filled product container is made of low-density polyethylene (LDPE) resin. (xxvi) The filled product container contains a total protein concentration of at least about 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL, 170 ng / mL, 180 ng / mL, 190 ng / mL, 200 ng / mL, 210 ng / mL, 220 ng / mL, 230 ng / mL, 240 ng / mL, 250 ng / mL, or more. (xxvii) The filled product container contains a total protein concentration of at least about 75 ng / mL, and / or (xxviiii) Upon completion of step (c), the total yield of one or more of the pharmaceutically active substances in the filled product container or a plurality of filled product containers is about 40%, 50%, 60%, 70%, 80%, 90%, or more of the pharmaceutically active substances in the active pharmaceutical ingredient preparation of step (b). The method according to claim 1.

3. (i) One or more of the pharmaceutically active substances include a bone marrow-derived mesenchymal secretome (MSC) composition, and / or (ii) One or more of the pharmaceutically active substances include HGF, serpine A1, TIMP-1, TSG-14, IL-8, serpine E1, VEGF-A, PEDF, and fibronectin (FN), The method according to claim 1.

4. (a) A bone marrow-derived mesenchymal stem cell (MSC) secretome composition comprising one or more of the pharmaceutically active substances, including HGF, pentraxin-3 (TSG-14), VEGF, TIMP-1, serpine E1, IL-8 less than 5 ng / mL, and a tonicity modifier. (b) The MSC secretome composition is i. At least one trophic factor / cytokine selected from the group consisting of TIMP-2 and VEGF-A; ii. At least one additional factor selected from the group consisting of PEDF (serpine F1), IGFBP-2, IGFBP-3, SDF-1, kallikrein 3, MCP-1, angiogenin, MCP-2, Angio-2, IL-6, IL-17, G-CSF, M-CSF, GM-CSF, IL-8, TNF-β, and PDGF; and / or iii. Further comprising 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, (c) The MSC secretome composition contains at least one factor selected from the group consisting of serpine E1, TIMP-1, thrombospondin-1, pentraxin-3 (TSG-14), and serpine F1 at a concentration of 1 ng / mL to 100 ng / mL. (d) The MSC secretome contains at least one factor selected from the group consisting of angiopoietin-1, angiopoietin-2, amphiregulin, endostatin, endothelin-1, thrombospondin-2, thrombospondin-1, angiogenin, DPPIV, IGFBP-3, and uPA in a concentration of 400 pg / mL to 3000 pg / mL. (e) The MSC secretome composition further comprises at least one factor selected from the group consisting of apolipoprotein A1, complement factor D, complement factor H, complement factor I, C1 esterase inhibitor (C1-INH), C4b binding protein (C4BP), CD46, C-reactive protein, cystatin C, DKK-1, Emprin, osteopontin, vitamin D BP, MIF, RANTES, uPAR, IL-17a, GDF-15, and IFNγ. (f) The MSC secretome composition includes a ratio of angiogenesis inhibitors to angiogenesis promoters, wherein the ratio is greater than 2, greater than 3, greater than 4, or greater than 5. (g) The MSC secretome composition contains VEGF in an amount of 1 pg / mL to 400 pg / mL, and optionally the level of VEGF is one-fifth to one-tenth the level of serpine E1. (h) The composition comprises one or more angiogenesis inhibitors, and the ratio of the total concentration of one or more angiogenesis inhibitors to the concentration of VEGF is greater than 2, greater than 3, greater than 4, or greater than 5. (i) The MSC secretome contains bFGF, PLGF, and PDGF at a concentration of less than 1000 pg / mL (j) The MSC secretome composition has a pH of about 4.7 to about 7.

5. (k) The MSC secretome composition is formulated in a buffer system selected from the group consisting of disodium phosphate, sodium citrate, boric acid, sodium citrate, boric acid, sodium tetraborate, and citrate / disodium phosphate. (l) The MSC secretome composition further comprises monosodium phosphate, disodium mannitol, and trehalose, and the composition has a pH of approximately 7.

4. (m) The MSC secretome composition further comprises a divalent cation, optionally selected from the group consisting of Mg2+, Ca2+, and Zn2+. (n) The MSC secretome composition further comprises disodium phosphate / citric acid, mannitol, and trehalose, and the composition has a pH of approximately 6.

4. (o) The MSC secretome composition further comprises a viscosity-increasing agent, optionally the tackifying agent being selected from the group consisting of hypromellose, Poloxamere 407, Poloxamere 188, Poloxamere 237, Poloxamere 338, hypromellose (HPMC), polycarbophil, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyimide, sodium hyaluronate, gellan gum, poly(lactic acid-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethylcellulose (CMC), or hydroxypropylmethylcellulose (HPMC), hydroxymethylcellulose, hydroxyethylcellulose, sodium carboxymethylcellulose, fibrin glue, polyethylene glycol, and GelCORE. (p) The MSC secretome composition does not contain one or more components selected from the group consisting of xenobiotic components, phenol red, peptides and biomolecules less than 3 kDa, antibiotics, protein aggregates greater than 200 nm, cells, cellular debris other than exosomes / extracellular vesicles, hormones, and L-glutamine. (q) The MSC secretome composition is i. HGF at 0.3–4.5 ng / mL; ii. Pentraxin-3 (TSG-14) at 0.5–20 ng / mL; iii. VEGF at 100-600 pg / mL; iv. TIMP-1 at 10–200 ng / mL; v. Serpine E1 in a dose of 20-80 ng / mL; and / or vi. Containing IL-8 less than 5 ng / mL, (r) The MSC secretome composition comprises an angiogenesis-inhibiting MSC secretome or an anti-scarring MSC secretome, and / or (s) The tension adjusting agent is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin. The method according to claim 3.

5. (a) The pharmaceutical preparation further comprises one or more components selected from the group consisting of monobasic sodium phosphate, dibasic sodium phosphate, sodium hydroxide, trehalose dihydrate (such as α,α-trehalose dihydrate), hypromellose, hydrochloric acid, sodium chloride, magnesium chloride, polysorbate (such as polysorbate 20 or polysorbate 50), and hydroxypropyl methylcellulose. (b) The pharmaceutical preparation contains approximately 0.1 mg to 1 mg, 1 mg to 2 mg, 2 mg to 3 mg, 3 mg to 4 mg, or 4 mg to 5 mg or more of monobasic sodium phosphate per 1 mL. (c) The pharmaceutical preparation contains approximately 1.14 mg, 1.31 mg, 2.28 mg, or 2.62 mg of monobasic sodium phosphate per 1 mL. (d) The pharmaceutical preparation contains approximately 0.5 mg to 5 mg, 5 mg to 10 mg, 10 mg to 15 mg, 15 mg to 20 mg, 20 mg to 25 mg, or 25 mg to 30 mg or more of dibasic sodium phosphate per 1 mL. (e) The pharmaceutical preparation contains approximately 5.7 mg or 11.4 mg of dibasic sodium phosphate per 1 mL. (f) The pharmaceutical preparation contains approximately 0.5 mg to 30 mg, 5 mg to 25 mg, 10 mg to 20 mg, or 10 mg to 15 mg, or more of mannitol per 1 mL. (g) The pharmaceutical preparation contains approximately 12.2 mg of mannitol per 1 mL. (h) The pharmaceutical preparation contains approximately 0.5 mg to 50 mg, 5 mg to 45 mg, 10 mg to 40 mg, 15 mg to 35 mg, 20 mg to 30 mg, or 20 mg to 25 mg or more of trehalose dihydrate per 1 mL. (i) The pharmaceutical preparation contains approximately 24 mg of trehalose dihydrate per 1 mL, (j) The pharmaceutical preparation contains approximately 0.1 mg to 5 mg, 0.2 mg to 4.5 mg, 0.3 mg to 4 mg, 0.4 mg to 3.5 mg, 0.5 mg to 3 mg, 0.6 mg to 2.5 mg, 0.7 mg to 2 mg, 0.8 mg to 2 mg, 0.9 mg to 1.5 mg, 0.9 mg to 1.4 mg, 0.9 mg to 1.3 mg, 0.9 mg to 1.2 mg, or 0.9 mg to 1.1 mg or more of hypromellose per 1 mL. (k) The pharmaceutical preparation contains approximately 1 mg of hypromellose per 1 mL. (l) The pharmaceutical preparation contains approximately 0.01 mg to 0.1 mg, 0.1 mg to 0.2 mg, 0.2 mg to 0.3 mg, 0.3 mg to 0.4 mg, or 0.4 mg to 0.5 mg or more of magnesium chloride per 1 mL. (m) The pharmaceutical preparation contains approximately 0.2 mg of magnesium chloride per 1 mL. (n) The pharmaceutical preparation contains approximately 0.1 mg to 0.5 mg, 0.5 mg to 1 mg, 1 mg to 1.5 mg, 1.5 mg to 2 mg, 2 mg to 2.5 mg, 2.5 mg to 3 mg, 3 mg to 3.5 mg, 3.5 mg to 4 mg, 4 mg to 4.5 mg, or 4.5 mg to 5 mg or more of sodium chloride per 1 mL. (o) The pharmaceutical preparation contains approximately 1.2 mg or 1.8 mg of sodium chloride per 1 mL. (p) The pharmaceutical preparation contains approximately 1 mM to 5 mM, 5 mM to 10 mM, 10 mM to 15 mM, 15 mM to 20 mM, 20 mM to 25 mM, or 25 mM to 30 mM, or more histidine HCl. (q) The pharmaceutical preparation contains approximately 10 mM histidine HCl, (r) The pharmaceutical preparation contains approximately 0.001% to 0.005%, 0.005% to 0.01%, 0.01% to 0.015%, 0.015% to 0.02%, 0.02% to 0.025%, or 0.025% to 0.03%, or more of polysorbate 20. (s) The pharmaceutical preparation contains approximately 0.01% polysorbate 20, (t) The pharmaceutical preparation contains approximately 0.1% to 1%, 1% to 2%, 2% to 3%, 3% to 4%, 4% to 5%, 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, or 9% to 10%, or more of sucrose. (u) The pharmaceutical preparation contains approximately 5.0% or 5.8% sucrose, (v) The pharmaceutical preparation contains approximately 1 mM to 5 mM, 5 mM to 10 mM, 10 mM to 15 mM, 15 mM to 20 mM, 20 mM to 25 mM, or 25 mM to 30 mM, or more sodium citrate. (w) The pharmaceutical preparation contains approximately 10 mM sodium citrate, (x) The pharmaceutical preparation contains approximately 0.1 mg to 5 mg, 0.2 mg to 4.5 mg, 0.3 mg to 4 mg, 0.4 mg to 3.5 mg, 0.5 mg to 3 mg, 0.6 mg to 2.5 mg, 0.7 mg to 2 mg, 0.8 mg to 2 mg, 0.9 mg to 1.5 mg, 0.9 mg to 1.4 mg, 0.9 mg to 1.3 mg, 0.9 mg to 1.2 mg, 0.9 mg to 1.1 mg, or more of hydroxypropyl methylcellulose per 1 mL. (y) The pharmaceutical preparation contains approximately 1 mg of hydroxypropyl methylcellulose per 1 mL. (z) The pharmaceutical preparation contains approximately 2 μg to 10 μg, 10 μg to 50 μg, 50 μg to 100 μg, 100 μg to 150 μg, 150 μg to 200 μg, 200 μg to 250 μg, 250 μg to 300 μg, 300 μg to 350 μg, 350 μg to 400 μg, 400 μg to 450 μg, 450 μg to 500 μg, 500 μg to Containing 550 μg, 550 μg to 600 μg, 600 μg to 650 μg, 650 μg to 700 μg, 700 μg to 750 μg, 750 μg to 800 μg, 800 μg to 850 μg, 850 μg to 900 μg, 900 μg to 950 μg, or 950 μg to 1000 μg, or more of the MSC secretome, and / or (aa) The pharmaceutical preparation contains approximately 2 to 20 μg or 6 μg of MSC secretome per 1 mL, or the pharmaceutical preparation contains approximately 100 to 400 μg of MSC secretome per 1 mL. The method according to claim 1.

6. (a) The pharmaceutical preparation is i. 1 mg to 3 mg of monobasic sodium phosphate per 1 mL; ii. 5 mg to 12 mg of dibasic sodium phosphate per 1 mL; iii. 11.5 mg to 13 mg of mannitol per 1 mL; and / or iv. Contains 23 mg to 25 mg of trehalose dihydrate per 1 mL. The aforementioned pH is approximately 4.7 to approximately 7.

5. (b) The pharmaceutical preparation is i. 2.28 mg of monobasic sodium phosphate per 1 mL; ii. 11.45 mg of dibasic sodium phosphate per 1 mL; iii. 12.2 mg of mannitol per 1 mL; iv. 24 mg of trehalose dihydrate per 1 mL; v. 1 mg of hypromellose per 1 mL; vi. Hydrochloric acid; and / or vii. Containing sodium hydroxide, (c) The pharmaceutical preparation is i. 1.14 mg of monobasic sodium phosphate per 1 mL; ii. 5.72 mg of dibasic sodium phosphate per 1 mL; iii. 12.2 mg of mannitol per 1 mL; iv. 24 mg of trehalose dihydrate per 1 mL; v. 1 mg of hypromellose per 1 mL; vi. Hydrochloric acid; and / or vii. Containing sodium hydroxide, (d) The pharmaceutical preparation is i. 1.31 mg of monobasic sodium phosphate per 1 mL; ii. 5.73 mg of dibasic sodium phosphate per 1 mL; iii. 12.2 mg of mannitol per 1 mL; iv. 24 mg of trehalose dihydrate per 1 mL; v. 1 mg of hypromellose per 1 mL; vi. Hydrochloric acid; vii. Sodium hydroxide; and / or viiii. Contains 1.76 mg of sodium chloride per 1 mL. (e) The pharmaceutical preparation is i. 10 mM histidine HCl; ii. 10% α,α-trehalose dihydrate; and / or iii. Contains 0.01% polysorbate 20, The aforementioned pH is approximately 5.

5. (f) The pharmaceutical preparation is i. 10 mM sodium phosphate; ii. 40 mm of sodium chloride; iii. 0.03% polysorbate 20; and / or iv. Contains 5% sucrose, The aforementioned pH is approximately 6.

2. (g) The pharmaceutical preparation is i. 10 mM sodium citrate; ii. 5.8% sucrose; and / or iii. Contains 0.01% polysorbate 20, pH 5.

5. The aforementioned pH is approximately 7.

2. (h) The pharmaceutical preparation is i. 2.62 mg of monobasic sodium phosphate monohydrate per 1 mL; ii. 11.5 mg of dibasic sodium phosphate anhydrous per 1 mL; iii. 1.17 mg of sodium chloride per 1 mL; iv. 0.2 mg of magnesium chloride hexahydrate per 1 mL; v. 12.2 mannitol per 1 mL; and / or vi. Contains 24 mg of trehalose dihydrate per 1 mL. (i) The pharmaceutical preparation is i. 1.31 mg of monobasic sodium phosphate monohydrate per 1 mL; ii. 5.73 mg of dibasic sodium phosphate anhydrous per 1 mL; iii. 1.17 mg of sodium chloride per 1 mL; iv. 0.2 mg of magnesium chloride hexahydrate per 1 mL; v. 12.2g of mannitol per 1 mL; vi. 24 mg of trehalose dihydrate per 1 mL; and / or vii. Containing 1 mg of hydroxypropyl methylcellulose per 1 mL, or (j) The pharmaceutical preparation is i. 4% to 5% w / w monobasic sodium phosphate; ii. 21.5% to 23% w / w dibasic sodium phosphate; iv. 23%–25% w / w mannitol; and / or iv. Contains 46% to 48% w / w trehalose dehydrated product. The aforementioned pH is approximately 4.7 to approximately 7.

5. The method according to claim 1.

7. (a) The pharmaceutical preparation is prepared for local administration. (b) The pharmaceutical preparation is prepared and packaged for approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses per day, or more. (c) The pharmaceutical preparation is prepared and packaged in doses of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drops, or more. (d) The pharmaceutical preparation is prepared and packaged in doses of 0.1 to 1 U / mL, 1 to 2 U / mL, 2 to 3 U / mL, 3 to 4 U / mL, 4 to 5 U / mL, 5 to 6 U / mL, 6 to 7 U / mL, 7 to 8 U / mL, 8 to 9 U / mL, or 9 to 10 U / mL, or more. (e) The pharmaceutical preparation is prepared and packaged in a dose of 1 U / mL or 3 U / mL. (f) The pharmaceutical preparation is prepared and packaged for administration over a period of at least 1, 2, 3, 4, 5, 6, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks, and / or (g) (i) The pharmaceutical preparation is prepared and packaged for administration at a dose of 3 U / mL, one drop of the preparation is administered four times a day, and optionally the preparation is administered for at least 56 days, (ii) The pharmaceutical preparation is prepared and packaged for administration at a dose of 1 U / mL, and one drop of the preparation is administered four times a day, and optionally the preparation is administered for at least 56 days. The method according to claim 1.

8. A kit comprising a pharmaceutical preparation, wherein the pharmaceutical preparation is prepared according to any one of claims 1 to 7 and packaged in a product container.

9. A pharmaceutical preparation for treating an eye condition, which is prepared according to any one of claims 1 to 7 and packaged in a product container.

10. (a) The eye condition includes one or more of the following: eye wound, eye scarring, neovascularization of the eye, increased intraocular pressure, dry eye disease, damaged corneal surface, damaged ophthalmic nerve tissue, retinal condition, persistent corneal epithelial defect (PCED), graft-versus-host disease (GvHD), and Stevens-Johnson syndrome. (b) The pharmaceutical preparation is formulated for topical administration. (c) The pharmaceutical preparation is prepared and packaged for administration approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per day, or more. (d) The pharmaceutical preparation is prepared and packaged in doses of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 drops or more. (e) The pharmaceutical preparation is prepared and packaged in doses of 0.1 to 1 U / mL, 1 to 2 U / mL, 2 to 3 U / mL, 3 to 4 U / mL, 4 to 5 U / mL, 5 to 6 U / mL, 6 to 7 U / mL, 7 to 8 U / mL, 8 to 9 U / mL, or 9 to 10 U / mL, or more. (f) The pharmaceutical preparation is prepared and packaged in doses of 1 U / mL or 3 U / mL. (g) The pharmaceutical preparation is prepared and packaged for administration over a period of at least 1, 2, 3, 4, 5, 6, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks, or longer, and / or (h)(i) The pharmaceutical preparation is prepared and packaged for administration at a dose of 3 U / mL, one drop of the preparation is administered four times a day, and optionally the preparation is administered for at least 56 days, (ii) The pharmaceutical preparation is prepared and packaged for administration at a dose of 1 U / mL, and one drop of the preparation is administered four times a day, and optionally the preparation is administered for at least 56 days. The pharmaceutical preparation according to claim 9.

11. A pharmaceutical preparation according to claim 9, which is administered to a subject in need thereof.

12. (a) The eye condition includes one or more of the following: eye wound, eye scarring, neovascularization of the eye, increased intraocular pressure, dry eye disease, damaged corneal surface, damaged ophthalmic nerve tissue, retinal condition, persistent corneal epithelial defect (PCED), graft-versus-host disease (GvHD), and Stevens-Johnson syndrome. (b) The pharmaceutical preparation is prepared for topical administration. (c) The pharmaceutical preparation is prepared and packaged for approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses per day, or more. (d) The pharmaceutical preparation is prepared and packaged in doses of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drops, or more. (e) The pharmaceutical preparation is prepared and packaged in doses of 0.1 to 1 U / mL, 1 to 2 U / mL, 2 to 3 U / mL, 3 to 4 U / mL, 4 to 5 U / mL, 5 to 6 U / mL, 6 to 7 U / mL, 7 to 8 U / mL, 8 to 9 U / mL, or 9 to 10 U / mL, or more. (f) The pharmaceutical preparation is prepared and packaged in a dose of 1 U / mL or 3 U / mL. (g) The pharmaceutical preparation is prepared and packaged for administration over a period of at least 1, 2, 3, 4, 5, 6, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks, or longer. (h)(i) The pharmaceutical preparation is prepared and packaged for administration at a dose of 3 U / mL, with one drop of the preparation administered four times a day, optionally for at least 56 days, or (ii) The pharmaceutical preparation is prepared and packaged for administration at a dose of 1 U / mL, with one drop of the preparation administered four times a day, optionally for at least 56 days, and / or (i) The pharmaceutical preparation is for use in the treatment method, The pharmaceutical preparation according to claim 9.

13. A unit dose formulation comprising the pharmaceutical formulation described in claim 9.

14. (a) The preparation is for treating the condition of the eye. (b) The formulation is prepared according to any one of claims 1 to 7 and packaged in a product container. (c) For use in treating eye conditions, (d) The eye condition includes one or more of the following: eye wound, eye scarring, neovascularization of the eye, increased intraocular pressure, dry eye disease, damaged corneal surface, damaged ophthalmic nerve tissue, retinal condition, persistent corneal epithelial defect (PCED), graft-versus-host disease (GvHD), and Stevens-Johnson syndrome. (e) The pharmaceutical preparation is formulated for topical administration. (f) The formulation is prepared and packaged for approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses per day, or more. (g) The formulation is prepared and packaged in doses of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 drops, or more. (h) The formulation is prepared and packaged in doses of 0.1-1 U / mL, 1-2 U / mL, 2-3 U / mL, 3-4 U / mL, 4-5 U / mL, 5-6 U / mL, 6-7 U / mL, 7-8 U / mL, 8-9 U / mL, or 9-10 U / mL, or more. (i) The formulation is prepared and packaged in doses of 1 U / mL or 3 U / mL, (j) The formulation is sufficient to provide doses of 0.1 to 1 U / mL, 1 to 2 U / mL, 2 to 3 U / mL, 3 to 4 U / mL, 4 to 5 U / mL, 5 to 6 U / mL, 6 to 7 U / mL, 7 to 8 U / mL, 8 to 9 U / mL, or 9 to 10 U / mL, or more. (k) The formulation is sufficient to provide a dose of 1 U / mL or 3 U / mL. (l) The formulation is prepared and packaged for administration over a period of at least 1, 2, 3, 4, 5, 6, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks, and / or (m)(i) The formulation is prepared and packaged for administration at a dose of 3 U / mL, one drop of the formulation is administered four times a day, and optionally the formulation is administered for at least 56 days, (ii) The formulation is prepared and packaged for administration at a dose of 1 U / mL, one drop of the formulation is administered four times a day, and optionally the formulation is administered for at least 56 days. The unit dose formulation according to claim 13.

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