Processes for making and using cellular fibronectin compositions

Abstract

The present application provides methods and processes for making and using the fibronectin compositions, as well as methods for treating ocular conditions and / or disorders using the cellular fibronectin compositions described herein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 217,952, filed July 2, 2021, and U.S. Provisional Application No. 63 / 235,605, filed August 20, 2021, all of which are incorporated by reference in their entireties herein. [Background technology]

[0002] Blast and blunt trauma to the eye can cause a series of mechanical disruptions to the ocular contents, including retinal comorbidity, traumatic cataracts, disruption of the zonular attachment to the lens, angle recession, iris dialysis, and rupture of the pupillary sphincter. Treatment of these injuries is limited to mechanical repair of the iris (when possible), replacement of the lens with a plastic lens implant, and repair of retinal detachment. There are no therapies to repair the cellular structures of the retina or anterior chamber. Furthermore, traumatic optic neuropathy and optic nerve detachment were one of the six major types of ocular injuries that required specialized eye care during Operation Iraqi Freedom (Choand Savitsky, “Ocular Trauma Chapter 7”, in Combat Casualty Care: Lessons learned from Oef and Oif, by Brian East bridgeand Eric Savitsky, pp. 299-342, Ft. Detrick, Md.: Borden Institute (US) Government Printing Office, 2012), which is incorporated herein by reference in its entirety. Sixty percent of traumatic head injuries result in neuro-ophthalmological abnormalities (VanStavern, et al., JNeuro-Ophthamol 21(2):112-117, 2001), incorporated herein by reference in its entirety, half of which involve nerves or visual pathways related to the optic nerve. Traumatic injury to neurons leads to axonal damage and irreversible neuronal loss, resulting in permanent deficits. Although many potential neuroprotective therapies have been identified in animals, these single agents have generally failed to translate into therapies in human clinical trials (Turner, et al., JNeurosurg 118(5):1072-1085, 2013, incorporated herein by reference in its entirety). Combination therapies affecting several cellular targets may be required to prevent neuronal damage.

[0003] As the outermost tissue of the eye, the cornea plays a protective role but is highly vulnerable to severe injury and disease. Its lack of blood vessels gives it transparency but also limits its ability to heal. Corneal damage can cause irreversible blindness, so prompt intervention and aggressive treatment are necessary. The critical need for improved ocular surface healing therapies is particularly evident in severe corneal diseases such as chemical burns and ocular manifestations of acute-chronic graft-versus-host disease (GvHD), Stevens-Johnson syndrome, ocular mucous membrane pemphigoid, and other conditions that cause persistent corneal epithelial defects, which together account for more than 100,000 cases per year. (Dietrich-Ntoukas et al.Cornea.2012,31(3):299-310; StevensonW,et al.,ClinOphthalmol.2013,7:2153-2158. WhiteKD,et al.,JAllergyClinImmunolPract.2018;6(1):38-69;TauberJ.(2002)Autoimmune Diseases Affecting the Ocular Surface.In:Ocular Surface Disease Medicaland Surgical Management.Springer,NewYork,NY.;WirostkoB,et al.,OculSurf.2015Jul;13(3):204-21;Haring,RS.,et al.,JAMAOphthalmol.2016Oct1;134(10):1119-1124).

[0004] The development of topical eye drops is hampered by many anatomical constraints, including tear turnover and dilution, nasolacrimal drainage, and reflex blinking, often resulting in less than 5% of the topical dose reaching deep ocular tissues (Gaudana et al., 2009). In the case of corneal wounds, the initial trauma induces a breach in the corneal epithelium, thereby allowing the passage of topically applied MSC-S to penetrate the epithelial layer.

[0005] Thus, there is a significant unmet need in the art for ocular therapies that can target the eye and deliver therapeutic payloads to hard-to-reach sensory cells that can be exacerbated by inflammation secondary to trauma (e.g., burns, acute inflammation, age, and / or oxidative stress). Summary of the Invention

[0006] The present invention meets this need by providing compositions comprising fibronectin, optionally one or more growth factors non-covalently attached to the fibronectin (FN), for use in such treatments, and methods of making such compositions.

[0007] In some embodiments, the FN is FN derived from MSCs.

[0008] In some embodiments, the FN is MSC-secreted FN.

[0009] In some embodiments, the FN is cellular FN.

[0010] In some embodiments, the cellular FN is cell-derived FN, where the FN is non-covalently attached to one or more growth factors.

[0011] In some embodiments, the cellular FN is EDA+ and / or EDB+.

[0012] In some embodiments, the cellular fibronectin is obtained from conditioned medium.

[0013] In some embodiments, cellular fibronectin is secreted by mesenchymal stem cells (MSCs).

[0014] In some embodiments, the conditioned medium is obtained from a culture of MSCs.

[0015] In some embodiments, the composition comprises an MSC secretome.

[0016] In some embodiments, the MSCs are derived from bone marrow. In some embodiments, the MSCs are derived from bone marrow from healthy human donors.

[0017] In some embodiments, the composition further comprises one or more growth factors selected from the group consisting of FGF (such as FGF-2), PDGF, HGF, VEGF, TGFβ1, TGFβ2, IGF-1, IGF-2, NGF, neurotrophins, and EGF.

[0018] In some embodiments, the cellular fibronectin is bound to one or more growth factors selected from the group consisting of FGF (such as FGF-2), PDGF, HGF, VEGF, TGFβ1, TGFβ2, IGF-1, IGF-2, NGF, neurotrophins, and EGF.

[0019] In some embodiments, the cellular fibronectin in the composition is at a concentration of about 0.5-50 ng / mL.

[0020] In some embodiments, the cellular fibronectin in the composition is at a concentration of about 25 ng / mL.

[0021] In some embodiments, the composition further comprises at least about 0.1 ng / mL of PDGF.

[0022] In some embodiments, the composition further comprises about 0.3-4.5 ng / mL of HGF.

[0023] In some embodiments, the composition further comprises about 1 pg / mL to 400 pg / mL of VEGF.

[0024] In some embodiments, the composition further comprises a tonicity modifying agent, hi some embodiments, the tonicity modifying agent is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin.

[0025] In some embodiments, the composition comprises 0.5-50 ng / ml FN, 2.28 mg / ml sodium phosphate monobasic, 10-12 mg / ml sodium phosphate dibasic, 11-13 mg / ml mannitol, 2-25 mg / ml trehalose dihydrate, and 0.5-2 mg / ml hypromellose.

[0026] In some embodiments, the composition comprises 0.5-50 ng / ml FN, 2.28 mg / ml sodium phosphate monobasic, 11.45 mg / ml sodium phosphate dibasic, 12.2 mg / ml mannitol, 24 mg / ml trehalose dihydrate, and 1 mg / ml hypromellose.

[0027] In some embodiments, the composition comprises 0.5-50 ng / ml FN, 1.31 mg / ml monobasic sodium phosphate, 4.5-7 mg / ml dibasic sodium phosphate, 5.5-7.5 mg / ml mannitol, 11-13 mg / ml trehalose dihydrate, and 0.1-1.5 mg / ml hypromellose. In some embodiments, the FN composition does not include NaCl and / or MgCl2.

[0028] In some embodiments, the composition comprises 0.5-50 ng / ml FN, 1.31 mg / ml sodium phosphate monobasic, 5.73 mg / ml sodium phosphate dibasic, 6.1 mg / ml mannitol, 12 mg / ml trehalose dihydrate, and 0.5 mg / ml hypromellose.

[0029] In some embodiments, the composition does not include NaCl and / or MgCl2.

[0030] In some embodiments, the disclosure herein provides a method of treating an ocular condition in a subject in need thereof, the method comprising administering to the subject a composition provided herein.

[0031] In some embodiments, the ocular condition is selected from the group consisting of retinal pathology, chronic graft-versus-host disease (GvHD), Stevens-Johnson syndrome, ocular mucosa pemphigoid, persistent corneal epithelial defect (PCED), dry eye, ocular nerve tissue injury, and traumatic injury to the eye (such as a percussive injury, ocular contusion, or chemical burn).

[0032] In some embodiments, the disclosure herein provides for the use of a composition according to the methods disclosed herein for treating an ophthalmic condition in a subject in need thereof.

[0033] In some embodiments, the disclosure herein provides a method of making a FN composition, the method comprising: (a) culturing stem cells in a cell culture medium, thereby producing a conditioned medium comprising factors secreted by the stem cells; (b) harvesting the conditioned medium, thereby obtaining a harvested conditioned medium; (c) filtering the harvested conditioned medium to obtain a processed conditioned medium; Includes.

[0034] In some embodiments, the method further comprises concentrating the treated conditioned medium.

[0035] In some embodiments, the processed conditioned medium is further subjected to buffer exchange with a formulation buffer.

[0036] In some embodiments, the formulation buffer comprises one or more of di / monosodium phosphate, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and citric acid / disodium phosphate.

[0037] In some embodiments, the stem cells are mesenchymal stem cells (MSCs).

[0038] In some embodiments, the cell culture medium is serum-free.

[0039] In some embodiments, the method further comprises, prior to step (a), (i) culturing stem cells in a growth medium; (ii) replacing the growth medium with the cell culture medium of step (a); Further includes: [Brief description of the drawings]

[0040] [Figure 1] Schematic diagram of an embodiment of fibronectin preparation, processing, and use. [Diagram 2] Fibronectin stimulates the migration of human corneal epithelial cells. [Diagram 3] Depletion of fibronectin impairs human corneal epithelial cell migration. [Figure 4] Depletion of fibronectin impairs in vitro wound closure of human corneal epithelial cells. [Diagram 5] We provide data showing that immunoprecipitated fibronectin contains bound HGF. [Figure 6] Characterization of fibronectin in secretomes. Secretomes isolated from MSCs were assessed by immunoblotting using antibodies specific for the EDA sequence, EDB+fibronectin, and general fibronectin (FN). Both anti-EDA and anti-EDB antibodies cross-reacted with fibronectin, indicating that the species present in the secretome is cellular fibronectin. [Figure 7] Detection of cellular EDA+ fibronectin using sandwich ELISA. An ELISA standard curve for EDA+ fibronectin (open circles) was established using recombinant cellular fibronectin and an EDA sequence-specific capture antibody. The recombinant fibronectin used to generate the standard curve contained the EDA sequence used as an immunogen to generate an anti-EDA antibody. MSC secretome was assayed in an EDA sandwich ELISA and cellular fibronectin was readily detected (blue dots). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] I. Introduction Fibronectin (FN) is a large glycoprotein with a carbohydrate content of about 5%. The characteristic form of plasma fibronectin is a disulfide-linked dimer of 440,000 daltons, with each subunit having a molecular weight of about 220,000 daltons. Fibronectin is usually found in plasma at a concentration of about 300 μg / mL and is extracted and purified using the method reported by Hynes, RO, Methods for identification of fibronectin (chapter 2, page 12), IN: Fibronectins New-York: Springer-Verlag, 1990. Plasma fibronectin is also known by a variety of other names, including cold-insoluble globulin, anti-gelatin factor, cell attachment protein, cell spreading factor, and opsonin α2 surface-associated glycoprotein. These names reflect the biological activities of fibronectin, such as cell recruitment, opsonization of particulate debris, and promotion of wound contraction. A description of the structure and activity of fibronectin can be found in Hynes, RO, Methods for identification of fibronectin (Chapter 2, page 12), IN: Fibronectins New-York: Springer-Verlag, 1990, as well as Hynes, RO, Methods for identification of fibronectin (Chapter 2, pages 7-23) and Wound healing, inflammation, and fibrosis (Chapter 14, pages 349-64), IN: Fibronectins New-York: Springer-Verlag, March 1990, as well as Brotchie, H., Wakefield, D. Australas J Dermatol 1990;31:47-56, all of which are incorporated herein by reference in their entireties.

[0042] Wound healing is usually divided into three phases: inflammatory, proliferative, and remodeling. Fibronectin has been reported to be involved in each stage of the wound healing process, particularly by creating a scaffold to allow the adhesion of infiltrating cells. Initially, many mediators (such as fibronectin and fibrinogen) are released at the wound site. Fibronectin promotes the migration of inflammatory cells into the wound and the phagocytosis of debris by monocytes. Angiogenesis and re-epithelialization then occur. In this stage, fibronectin exerts migratory activity on endothelial cells and promotes the migration of epithelial cells and fibroblasts onto the basement membrane.

[0043] Fibronectin also appears to be an essential component of the remodeling phase, where it plays a major role in structuring collagen fibrils. Fibrillar collagen eventually forms fiber bundles that greatly increase the tensile strength of tissues, resulting in wound closure. Plasma fibronectin has been reported to be useful in increasing the rate of wound healing, such as in corneal wounds and leg ulcers.

[0044] Fibronectin exists in two forms: plasma fibronectin, which is synthesized by hepatocytes and secreted into plasma, while cellular fibronectin is produced by many cell types, including fibroblasts, endothelial cells, stem cells, muscle cells, and chondrocytes.

[0045] In wound healing, it has been reported that plasma fibronectin accumulates significantly in wounds after in vivo wounding, which is crucial for various functions of platelets, fibroblasts, and endothelial cells (e.g., adhesion, migration, and aggregation), highlighting that plasma fibronectin may serve as a suitable substrate for accelerating wound healing in vivo. Indeed, in animal models, a provisional matrix containing plasma fibronectin significantly supports the adhesion and migration of epidermal cells in the re-epithelialization process, indicating the clinical potential of plasma fibronectin in human wound healing and tissue repair.

[0046] Although they share many physical and immunological similarities, these two classes of fibronectins differ in their electrophoretic behavior, solubility, and biological activity (Tamkun et al., J. Biol. Chem. 258(7):4641-47(1983); Yamada et al., J. Cell Biol. 80:492-98(1979); Yamada et al. Biochemistry 16(25):2552-59(1977).

[0047] Furthermore, primary structural differences between plasma and cellular fibronectin have been found by peptide mapping (Hayashi et al. J. Biol. Chem. 256(21):11,292-11,300 (1981)) and immunological techniques (Atherton et al. Cell 25:133-41 (1981)). Recently, a divergent region encoding exactly one 90 amino acid type III structural repeat was identified in mRNA from human fibroblasts and two human tumor cell lines but was undetectable in human liver mRNA. Kornblihtt et al.EMBO J.4(7):1755-59(1985), Kornblihtt et al.,EMBO J.3(1):221-26(1984), Kornblihtt et al.,Nucleic Acids Res.12(14):5853-68(1984). Because plasma fibronectin is synthesized by hepatocytes, this extra type III repeat may be a unique domain of cellular fibronectin. Schwarzbauer et al., Proc. Natl. Acad. Sci. USA., 82:1424-28(1985); Kornblihtt et al., EMBO J. 3(1):221-26(1984); Kornblihtt et al., Nucleic Acids Res. 12(14):5853-68(1984). Further discussion of the differences between plasma and cellular fibronectin is provided in WS, et al., Fibrogenesis Tissue Repair 4,21(2011).

[0048] Cellular fibronectin is characterized by fibronectin splice variants that are not present in the circulating plasma fibronectin pool. These alternative splice variants result in the fibronectin sequence having extra domains (type III domains), called EIIIA and EIIIB (or EDA and EDB). White et al. The Journal of pathology. vol. 216(1): 1-14 (2008); White and Muro, IUBMB Life, 63: 538-546 (2011). Thus, in some embodiments, the cellular fibronectin is EDA+ and / or EDB+. In some embodiments, the cellular fibronectin is EDA+, EDB+, and / or V+. In some embodiments, the cellular fibronectin is EDA+. In some embodiments, the cellular fibronectin is EDB+. In some embodiments, the cellular fibronectin is V+. One or both type III domains may be incorporated. Cellular fibronectin may be a mixture of these isoforms.

[0049] Despite the great interest in the therapeutic application of fibronectin to promote wound healing, clinical trials to date have focused on plasma fibronectin. No clinical trials with cellular fibronectin have been reported so far, and clinical trials with plasma fibronectin have shown inconsistent results among patients with ocular diseases. For example, McCulley, JP. et al. reported that patients with persistent corneal epithelial defects unfortunately did not respond to plasma fibronectin treatment. McCulley JP, Horowitz B, Husseini ZM, Horowitz M. Trans Am Ophthalmol Soc. 1993; 91: 367-86; Discussion 386-90.

[0050] A growing number of studies have shown that growth factors can bind to FN at various sites. Thus, FN may act as an effective reservoir to retain growth factors and increase their local concentration in physiological microenvironments. For example, it has been reported that HGF associates into a multimeric complex that promotes cell migration by binding to HGF receptors and integrins as well as FN. Rahman,Salman et al.,BMC cell biology vol.6,1 8.17 Feb.2005. FN has also been shown to bind, via its C-terminal heparin-II domain (FN III), to various growth factors from the PDGF / VEGF and FGF families, as well as several growth factors from the transforming growth factor-β (TGF-β) and neurotrophin families. Although growth factors sequestered by FN are presumed to have local, sustained, and enhanced GF activity, it remains challenging to develop recombinant combination therapies using recombinant FN and growth factors that retain their endogenous biochemical and biophysical properties.

[0051] A.Definition The terms used in the claims and specification are defined as set forth below unless otherwise indicated. In the event of a direct conflict with a term used in the parent provisional patent application, the term used herein shall control.

[0052] As used herein, "isolated" refers to material that has been removed from its original environment and thus altered "by the hand of man" from its natural state.

[0053] As used herein, "enrichment" means to selectively concentrate or increase the amount of one or more materials by either eliminating unwanted materials or selecting and separating desired materials from a mixture (e.g., separating cells with a particular cell marker from a heterogeneous population of cells where not all cells in the population express the marker).

[0054] As used herein, the term "substantially purified" refers to a population of cells that is substantially homogeneous for a particular marker or combination of markers. Substantially homogeneous means at least 90%, preferably 95% homogeneous for a particular marker or combination of markers. As used herein, the term "pluripotent stem cells" refers to true stem cells, but can only differentiate into a limited number of types. For example, bone marrow contains pluripotent stem cells that generate all the cells of the blood, but may not be able to differentiate into other types of cells.

[0055] The term "animal-free" when referring to certain compositions, growth conditions, media, etc. described herein means that no non-human animal-derived materials, such as bovine serum, proteins, lipids, carbohydrates, nucleic acids, vitamins, etc., are used in 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 been present in or come into contact with the body or material of a non-human animal and are free of xenogenic contamination. 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.

[0056] The term "expanded" with respect to a cell composition means that the cell population comprises a significantly higher concentration of cells than obtained using previous methods. For example, the cell level per gram of amniotic tissue in an expanded composition of AMP cells is at least 50-fold and up to 150-fold higher than the number of cells in a primary culture after 5 passages, compared to about 20-fold doubling of such cells using previous methods. In another example, the cell level per gram of amniotic tissue in an expanded composition of AMP cells is at least 30-fold and up to 100-fold higher than the number of cells in a primary culture after 3 passages. Thus, the "expanded" population has at least a 2-fold and up to a 10-fold improvement in the number of cells per gram of amniotic tissue compared to previous methods. The term "expanded" is meant to include only situations where human intervention has been used to increase the number of cells.

[0057] As used herein, "conditioned medium" is a medium in which a particular cell or cell population has been cultured and then removed. When cells are cultured in medium, they may secrete cellular factors that may support or affect the behavior of other cells. Such factors include, but are not limited to, hormones, cytokines, extracellular matrix (ECM), proteins, vesicles, antibodies, chemokines, receptors, inhibitors, and granules. A medium containing cellular factors is a conditioned medium. Examples of methods for preparing conditioned medium are described in U.S. Pat. No. 6,372,494, which is incorporated herein by reference in its entirety. As used herein, conditioned medium also refers to conditioned medium or components such as proteins, for example, that are recovered and / or purified from MSC cells.

[0058] As used herein, the term "mesenchymal stem cell composition" or "MSC composition" refers to a conditioned medium derived from MSCs and optionally subjected to further processing. In some embodiments, "MSC secretome" can refer to a crude conditioned medium derived from MSCs. In some embodiments, "MSC secretome" can refer to a composition obtained from the crude conditioned medium after it has been subjected to further processing as described herein.

[0059] As used herein, the term "suspension" refers to a liquid that contains dispersed components, e.g., cytokines. The dispersed components may be fully solubilized, partially solubilized, suspended, or otherwise dispersed in the liquid. Suitable liquids include, but are not limited to, water, osmotic solutions such as salt and / or sugar solutions, cell culture media, and other aqueous or non-aqueous solutions.

[0060] "Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, e.g., 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, i.e., an α carbon attached to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, 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 that differs from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted one-letter codes.

[0061] An "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a given amino acid sequence (the amino acid sequence of the starting polypeptide) with a second, different, "replacement" amino acid residue. An "amino acid insertion" refers to the incorporation of at least one additional amino acid into a given amino acid sequence. An insertion usually consists of the insertion of one or two amino acid residues, although larger "peptide insertions", e.g., insertions of about 3 to about 5 or up to about 10, 15, or 20 amino acid residues, can now be made. The inserted residues 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 given amino acid sequence.

[0062] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms include amino acid polymers in which one or more amino acid residues are artificial chemical analogues of a corresponding naturally occurring amino acid, as well as naturally occurring and non-naturally occurring amino acid polymers.

[0063] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-stranded or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed 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 interchangeably with gene, cDNA, and mRNA encoded by a gene. A polynucleotide as used herein can be composed of any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. For example, a polynucleotide may be composed of single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA that may be single-stranded, or more typically double-stranded, or a mixture of single-stranded and double-stranded regions. Additionally, a polynucleotide may be composed of triple-stranded regions that contain RNA or DNA, or both RNA and DNA. A polynucleotide may 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; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.

[0064] As used herein, the term "secretome composition" refers to a composition that includes one or more substances secreted from a cell. In certain embodiments, a secretome composition may include 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 include one or more substances that are not secreted from a cell (e.g., culture medium, additives, nutrients, etc.). Some secretome compositions include only trace amounts or no one or more substances that are not secreted from a cell (e.g., culture medium, additives, nutrients, etc.).

[0065] As used herein, the terms "treatment," "treat," or "treating" and the like include any treatment of a human or non-human mammal (e.g., rodents, cats, dogs, horses, cattle, sheep, primates, and the like), including preventing a disease or condition from occurring in a subject who may be predisposed to, but has not yet been diagnosed as having, the disease or condition. It also includes suppressing (halting the onset), alleviating or ameliorating (causing regression) the disease, condition, and / or associated symptoms, or curing (permanently halting the onset or progression) of the disease, condition, and / or any associated symptoms. The terms "treatment", "treat" or "treating" as used herein include any treatment of a disease or condition in a mammal, particularly a human, including (a) preventing the occurrence of a disease or condition in a subject who may be predisposed to the disease or condition but has not yet been diagnosed as having it, (b) inhibiting the disease or condition, e.g., arresting its onset, (c) alleviating and / or ameliorating the disease or condition, e.g., causing regression of the disease or condition, or (d) curing the disease or condition, e.g., halting its onset or progression. The population of subjects treated by the methods of the invention includes subjects suffering from an undesirable condition or disease, as well as subjects at risk of developing a condition or disease. In some embodiments, "treatment" (also "treat" or "treating") refers to any administration of a therapy that results in partial or complete amelioration, improvement, reversal, inhibition, delay in onset, reduction in severity, and / or reduction in the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be 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 in addition, 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 as suffering from 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.

[0066] As used herein, a "wound" refers to any cause that disrupts normal tissue (internal and / or external tissue), including, but not limited to, traumatic injuries such as mechanical (e.g., contusion, penetration), thermal, chemical, electrical, radiation, impact and incisional injuries, elective wounds such as surgery and resultant incisional hernias, fistulas, etc.; acute, chronic, infected and sterile wounds, as well as wounds associated with pathology (e.g., eye contusion). Wounds are dynamic and the healing process is continuous, requiring a series of integrated and interrelated cellular processes that begin at the time of wounding and progress beyond initial wound closure until stable wound closure is reached. 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 improving, through some form of intervention, the natural cellular processes and humoral substances of tissue repair such that healing is faster and / or the resulting healed area has less scarring and / or the healed area has tissue strength approaching that of uninjured tissue and / or the wounded tissue achieves some degree of functional recovery.

[0067] As used herein, the terms "a" or "an" mean one or more than one or at least one.

[0068] As used herein, a "therapeutically effective" or "effective" dose or amount of a composition is an amount sufficient to produce a positive effect on a given medical condition. If not immediate, a therapeutically effective or effective dose or amount produces a noticeable or measurable effect on the health and well-being of a patient over a period of time.

[0069] As used herein, "pharmaceutical composition" refers to an effective amount of the composition described herein in combination with a delivery component. Pharmaceutical compositions can optionally contain other components, such as pharma- ceutical suitable carriers and excipients, that can facilitate administration of the composition and / or its individual components to a subject.

[0070] 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.

[0071] The term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound.

[0072] As used herein, the terms "mixing," "mixing," and the like, describe a mechanical process or treatment of the ingredients. For example, mixing can mean performing repeated cycles of pressing and folding or equivalent processing steps that result in strong compression and mixing of the provided hydrophobic matrix.

[0073] Adult stem cells can be harvested from a variety of adult tissues, including bone marrow, adipose, and dental pulp tissue. Although all adult stem cells are considered to be self-renewing cables and multipotent, their therapeutic functions vary depending on their origin. As a result, each type of adult stem cell has its own characteristics that make it suitable for specific diseases. Mesenchymal stem cells (MSCs) are non-hematopoietic (non-blood) stem cells isolated (derived) from the mesoderm, which are typically derived, multipotent, and capable of differentiating into a variety of tissues, including osteoblasts (e.g., bone cells), chondrocytes (e.g., cartilage cells), myocytes (e.g., muscle cells), and adipocytes (e.g., adipocytes that give rise to bone marrow adipose tissue). As used herein, "isolated" refers to cells that have been removed from their original environment. Stem cells produce factors that regulate or are important for regulating multiple biological processes, such as growth factors. Growth factors are agents, such as naturally occurring substances, that can stimulate cell growth and / or proliferation and / or cell differentiation. Generally, growth factors are proteins or steroid hormones. As used herein, terms such as "growth factor" and "factor" are used interchangeably, however, the term "biological factor" is not limited to growth factors.

[0074] Human mesenchymal stem cells (MSCs) can be characterized by a surface marker profile of CD45- / CD31- / CD73+ / CD90+ / CD105+ / CD44+ (or a suitable subset thereof). (See Bourin et al, Cytotherapy 15(6):641-648 (2013)). Additionally, 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 may be used in accordance with the present application includes CD45- / CD31- / CD73+ / CD90+ / CD105+. In another embodiment utilizing mouse stem cells, the stem cells are characterized by the Sca-1 marker instead of CD34, with the remaining markers remaining the same, defining what appears to be homologous to the human cells described above.

[0075] The phrase "conditioned medium" or "CM" refers to a medium that contains biological factors secreted by MSCs. It may also be referred to herein as "secretome", "MSC-CM", "MSC secretome" and / or "MSC-derived secretome". Also provided is a processed "conditioned medium" that contains biological factors secreted by MSCs and has been further processed, for example, by filtration, purification, and / or concentration procedures. The "conditioned medium" is obtained 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 that contains biological factors and fewer stem cells than were present prior to separation. The conditioned medium can be used in the methods described herein and is substantially free of stem cells (may contain a small percentage of stem cells) or free of stem cells. Biological factors that may be present in the conditioned medium include, but are not limited to, proteins (e.g., cytokines, chemokines, growth factors, enzymes), nucleic acids (e.g., miRNAs), lipids (e.g., phospholipids), polysaccharides, and / or combinations thereof. Any combination of these biological factors can be bound to the interior or surface of the extracellular vesicles (e.g., exosomes) or separated from the extracellular vesicles.

[0076] B. Compositions and Formulations The present invention meets this need by providing compositions comprising fibronectin, optionally one or more growth factors non-covalently attached to the fibronectin (FN), for use in such treatments, and methods of making such compositions.

[0077] In some embodiments, the FN is derived from a stem cell.

[0078] In some embodiments, the FN is FN derived from MSCs.

[0079] In some embodiments, the FN is MSC-secreted FN.

[0080] In some embodiments, the FN is cellular FN. In some embodiments, the cellular FN of the present invention is a mixture of alternative splicing variants / isoforms (such as EDA, EDB, and V+). In some embodiments, the cellular FN is EDA+. In some embodiments, the cellular FN is EDB+. In some embodiments, the cellular FN is EDA+ and EDB+. In some embodiments, the cellular fibronectin is V+.

[0081] In some embodiments, the cellular FN is cell-derived FN, where the FN is non-covalently attached to one or more growth factors.

[0082] In some embodiments, the compositions provided herein are obtained from conditioned medium. In some embodiments, the conditioned medium is obtained from a culture of mesenchymal stem cells (MSCs).

[0083] In some embodiments, the composition comprising FN is derived from MSC secretome (including processed MSC secretome).

[0084] In some embodiments, provided herein is 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).

[0085] In some embodiments, the FN composition comprises 0.1 ng / mL to 150,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 140,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 130,000 ng / ML of FN. In some embodiments, the FN composition comprises about 135,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 120,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 110,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 100,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 90,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 80,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 70,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 60,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 50,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 40,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 30,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 20,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 10,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 9,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 8,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 7,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 6,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 5,000 ng / ML of FN.In some embodiments, the FN composition comprises 0.1 ng / mL to 4,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 3,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 150,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 140,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 130,000 ng / ML of FN. In some embodiments, the FN composition comprises about 135,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 120,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 110,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 100,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 90,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 80,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 70,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 60,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 50,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 40,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 30,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 20,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 10,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 9,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 8,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 7,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 6,000 ng / ML of FN.In some embodiments, the FN composition comprises 50 ng / mL to 5,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 4,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 3,000 ng / ML of FN.

[0086] In some embodiments, the FN composition comprises 500ng / mL to 150,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 140,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 130,000ng / ML of FN. In some embodiments, the FN composition comprises about 135,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 120,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 110,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 100,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 90,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 80,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 70,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 60,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 50,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 40,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 30,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 20,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 10,000ng / ML of FN. In some embodiments, the FN composition comprises 500 ng / mL to 9,000 ng / ML of FN. In some embodiments, the FN composition comprises 500 ng / mL to 8,000 ng / ML of FN. In some embodiments, the FN composition comprises 500 ng / mL to 7,000 ng / ML of FN. In some embodiments, the FN composition comprises 500 ng / mL to 6,000 ng / ML of FN. In some embodiments, the FN composition comprises 500 ng / mL to 5,000 ng / ML of FN.In some embodiments, the FN composition comprises 500 ng / mL to 4,000 ng / ML of FN. In some embodiments, the FN composition comprises 500 ng / mL to 3,000 ng / ML of FN.

[0087] In some embodiments, the FN composition comprises 1000ng / mL to 150,000ng / ML of FN. In some embodiments, the FN composition comprises 1000ng / mL to 140,000ng / ML of FN. In some embodiments, the FN composition comprises 1000ng / mL to 130,000ng / ML of FN. In some embodiments, the FN composition comprises about 135,000ng / ML of FN. In some embodiments, the FN composition comprises 1000ng / mL to 120,000ng / ML of FN. In some embodiments, the FN composition comprises 1000ng / mL to 110,000ng / ML of FN. In some embodiments, the FN composition comprises 1000ng / mL to 100,000ng / ML of FN. In some embodiments, the FN composition comprises 1000ng / mL to 90,000ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 80,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 70,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 60,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 50,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 40,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 30,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 20,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 10,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 9,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 8,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 7,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 6,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 5,000 ng / ML of FN.In some embodiments, the FN composition comprises 1000 ng / mL to 4,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 3,000 ng / ML of FN.

[0088] In some embodiments, the FN composition comprises 50-5000 ng / mL of FN. In some embodiments, the FN composition comprises 50-4000 ng / mL of FN. In some embodiments, the FN composition comprises 100-4000 ng / mL of FN. In some embodiments, the FN composition comprises 150-3500 ng / mL of FN.

[0089] In some embodiments, the FN composition comprises 1000-70,000 ng / mL of FN. In some embodiments, the FN composition comprises 500-50,000 ng / mL of FN. In some embodiments, the FN composition comprises 1000-40,000 ng / mL of FN. In some embodiments, the FN composition comprises 1500-35,000 ng / mL of FN.

[0090] In some embodiments, the FN composition comprises about 0.5-50 g / mL of FN. In some embodiments, the FN composition comprises 5-45 ng / mL of FN. In some embodiments, the FN composition comprises 10-40 ng / mL of FN. In some embodiments, the FN composition comprises 15-35 ng / mL of FN. In some embodiments, the FN composition comprises 20-30 ng / mL of FN. In some embodiments, the cellular FN composition comprises about 25 ng / mL of FN.

[0091] In some embodiments, the FN composition further comprises one or more growth factors. In some embodiments, the FN composition comprising one or more growth factors is derived from the MSC secretome. In some embodiments, the cellular FN in the composition is attached to one or more growth factors. In some embodiments, the FN is non-covalently attached to one or more growth factors. In some embodiments, the one or more growth factors are selected from the group consisting of FGF (such as FGF-2 (also referred to as fibroblast growth factor-2)), PDGF (also referred to as platelet-derived growth factor), HGF (also referred to as hepatocyte growth factor), VEGF, TGFβ1 (also referred to as TGF beta 1 or transforming growth factor beta 1), TGFβ2 (also referred to as TGF beta 2 or transforming growth factor beta 2), IGF-1 (also referred to as insulin growth factor 1), IGF-2 (also referred to as insulin growth factor 2), NGF (also referred to as nerve growth factor), neurotrophins, and EGF (also referred to as epidermal growth factor).

[0092] In some embodiments, the FN composition further comprises FGF. In some embodiments, the FGF is FGF-2. In some embodiments, the FN composition further comprises FGF-2.

[0093] In some embodiments, the FN composition further comprises PDGF. In some embodiments, the composition further comprises at least about 0.1 ng / mL of PDGF.

[0094] In some embodiments, the FN composition further comprises HGF. In some embodiments, the FN composition comprises 0.1-10 ng / mL or 2.0+ / -0.3 ng / mL of HGF.

[0095] In some embodiments, the FN composition further comprises VEGF, in some embodiments, the VEGF is at a concentration of about 100-800 pg / mL or 304 + / - 44 pg / mL.

[0096] In some embodiments, the FN composition further comprises TGFβ1.

[0097] In some embodiments, the FN composition further comprises TGFβ2.

[0098] In some embodiments, the FN composition further comprises IGF-1, IGF-2, and EGF.

[0099] In some embodiments, the FN composition further comprises IGF-2.

[0100] In some embodiments, the FN composition further comprises NGF.

[0101] In some embodiments, the FN composition further comprises a neurotrophin.

[0102] In some embodiments, the FN composition further comprises EGF.

[0103] In some embodiments, the FN composition is formulated at a pH of about pH 4.5 to about pH 8. In some embodiments, the FN composition is formulated at a pH of about pH 4.7 to about pH 7.8. In some embodiments, the FN composition is formulated at a pH of about pH 5.0 to about pH 7.5. In some embodiments, the FN composition is formulated at a pH of about pH 5.5 to about pH 7.5. In some embodiments, the FN composition is formulated at a pH of about pH 6 to about pH 7.5.

[0104] In some embodiments, the FN composition is formulated at a pH of about pH 4.5, about pH 5.0, about pH 5.5, about pH 6.0, about pH 6.5, about pH 7.0, about pH 7.4, about pH 8.0. In some embodiments, the cellular FN 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.

[0105] In some embodiments, the FN composition is free of certain components. In some embodiments, the FN composition is free of certain components found in cell culture media. In some embodiments, the FN composition is free of one or more components selected from the group consisting of xenobiotic components (e.g., animal serum); phenol red; peptides and biomolecules <3 kDa; antibiotics; protein aggregates (e.g., protein aggregates >200 nm); cells; cell debris (cell debris does not include exosomes / extracellular vesicles (EVs); e.g., non-exosome, non-EV cell debris); hormones (e.g., hormones include, but are not limited to, insulin and / or hydrocortisone); and / or L-glutamine. In some embodiments, the FN composition is free of xenobiotic components. In some embodiments, the FN composition is free of phenol red. In some embodiments, the FN composition is free of peptides and biomolecules less than 3 kDa. In some embodiments, the FN composition is free of antibiotics. In some embodiments, the FN composition is free of protein aggregates (e.g., protein aggregates greater than 200 nm). In some embodiments, the FN composition is free of cells. In some embodiments, the FN composition does not include cellular debris (cellular debris does not include exosomes / EVs; e.g., non-exosome, non-EV cellular debris). In some embodiments, the FN composition does not include hormones (e.g., hormones include, but are not limited to, insulin and / or hydrocortisone). In some embodiments, the FN composition does not include L-glutamine.

[0106] In some embodiments, the FN composition 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 FN composition 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 FN composition further comprises sodium monophosphate / sodium diphosphate, mannitol, and trehalose, and the composition has a pH of about pH 7.4.

[0107] In some embodiments, the FN composition can include one or more additional agents including, but not limited to, glycine, glycerol, sodium chloride, potassium chloride, and / or dextrose. In some embodiments, the FN composition can include one or more additional agents selected from the group consisting of glycine, glycerol, sodium chloride, potassium chloride, and dextrose. In some embodiments, the FN composition can include one or more additional agents selected from the group consisting of glycine and glycerol, and dextrose. In some embodiments, the FN composition can include one or more additional agents selected from the group consisting of sodium chloride and potassium chloride.

[0108] In some embodiments, the FN composition is formulated in a buffer system. In some embodiments, the FN composition is formulated in a buffer system including, but not limited to, di / monosodium phosphate, sodium citrate / citric acid, citric acid / sodium citrate, boric acid / sodium tetraborate, and / or citric acid / disodium phosphate. In some embodiments, the FN 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 FN composition is formulated in a di / monosodium phosphate buffer system. In some embodiments, the FN composition is formulated in a sodium citrate / citric acid buffer system. In some embodiments, the FN composition is formulated in a boric acid / sodium citrate buffer system. In some embodiments, the FN composition is formulated in a boric acid / sodium citrate buffer system. In some embodiments, the FN composition is formulated in a boric acid / sodium tetraborate buffer system. In some embodiments, the FN composition is formulated in a citrate / disodium phosphate buffer system.

[0109] 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 cellular FN composition comprises disodium phosphate / citric acid, mannitol, and trehalose, and the composition has a pH of about pH 6.4.

[0110] In some embodiments, the cellular FN 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.

[0111] In some embodiments, the FN composition further comprises an adhesive, including, but not limited to, hypromellose, poloxamer 407, poloxamer 188, poloxomer 237, poloxomer 338, hypromellose (HPMC), HEC, polycarbophil, polyvinylpyrrolidone (PVP), PVA (polyvinyl alcohol), polyimide, sodium hyaluronate, gellan gum, poly(lactic-co-glycolic acid) (PLGA), polysiloxane, polyimide, carboxymethylcellulose (CMC), or hydroxypropylmethylcellulose (HPMC), hydroxymethylcellulose, hydroxyethylcellulose, sodium carboxymethylcellulose, fibrin glue, polyethylene glycol, and GelCORE. In some embodiments, the adhesive is hypromellose. In some embodiments, the adhesive is fibrin glue. In some embodiments, the adhesive is polyethylene glycol. In some embodiments, the adhesive is GelCORE (see Sani, et al., Science Advances, Vol. 5, no. 3 (2019)).

[0112] In some embodiments, the FN composition comprises (a) a processed conditioned medium comprising an MSC secretome generated by any one of the methods described herein, and (b) a polymer. In some embodiments, the cellular FN composition comprises a conditioned medium comprising an MSC secretome generated as described herein and a polymer. In some embodiments, the cellular FN composition comprises a processed conditioned medium comprising an MSC secretome generated as described herein and a polymer. In some embodiments, the polymer can be a biodegradable polymer from which the composition components can be released. In some embodiments, the polymer allows for sustained (slow) release of the components.

[0113] In some embodiments, the FN compositions provided herein are in the form of a therapeutic dressing (e.g., a polymer impregnated with a cellular FN 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.

[0114] In some embodiments, the FN compositions exhibit bio-permeability, e.g., ocular penetration, corneal penetration, and / or corneal penetration. In some embodiments, the FN compositions exhibit the ability to be absorbed by the eye. In some embodiments, the FN compositions exhibit inherent bio-permeability. In some embodiments, the FN compositions exhibit excipient-compatible bio-permeability. In some embodiments, the FN compositions exhibit bio-permeability due to up-regulation of smaller factors. In some embodiments, the FN compositions exhibit bio-permeability due to the presence of a biopreservative. In some embodiments, the FN compositions exhibit bio-permeability due to the presence of biopreservative benzalkonium chloride.

[0115] In some embodiments, the FN compositions exhibit a long half-life and / or have increased stability compared to other treatments. In some embodiments, the cellular FN compositions provided herein allow for the upregulation of proteins that allow for increased stability of the MSC secretome. In some embodiments, the cellular FN compositions provided herein allow for the upregulation of chaperone proteins to improve the stability of other proteins in the MSC secretome.

[0116] In some embodiments, the FN composition exhibits super-potency when administered to a subject in need thereof, in some embodiments, the FN composition allows for a therapeutic effect with one drop or one administration per day.

[0117] C. Method of Production / Manufacturing In some embodiments, the cellular FN provided herein is secreted by cells into conditioned medium. In some embodiments, the cells are stem cells (such as mesenchymal stem cells). In some embodiments, the conditioned medium is further processed to remove undesirable components to obtain the cellular FN composition.

[0118] In some embodiments, the conditioned medium from which the cellular FN composition (and thus the mesenchymal stem cell secreted factors) is derived can be obtained from mesenchymal stem cells taken from the patient or individual to be treated (patient in need thereof) or from another (donor) individual, i.e., from a young and / or healthy donor and / or commercially obtained mesenchymal stem cells. For example, MSCs obtained from the individual to be treated (autologous stem cells) or from a donor (allogeneic stem cells) can be used to generate the conditioned medium described herein, which can then be further processed into the cellular FN composition described herein.

[0119] According to the present invention, a method for generating a mesenchymal stem cell (MSC) secretome comprises: i. culturing mesenchymal stem cells (MSCs) in a first medium; ii. removing the initial medium from step (i) from the MSCs; iii. Washing the MSCs of step (ii); iv. Add the second medium and incubate for about 1 to 5 days; v. harvesting the second medium from step (iv) as conditioned medium; and vi. Processing the conditioned medium of step (v) into an MSC secretome composition as described herein.

[0120] In some embodiments, the culturing can be performed using a bioreactor system for culturing cells. In some embodiments, the culturing can be performed using a bioreactor system for culturing stem cells. In some embodiments, the culturing can be performed using a bioreactor system for culturing mesenchymal stem cells. In some embodiments, the culturing can be performed using a medium mixing technique. In some embodiments, the culturing can be performed using PBSVerticalWheel™ mixing technique (commercially available from PBS Biotech, Inc.).

[0121] In some embodiments, in step (iv), processing the conditioned medium of step (v) into a secretome composition comprises: a) filtering the conditioned medium harvested from step (v) to remove cell particles; b) concentrating the filtered conditioned medium from step (a); and c) buffer exchange with the formulation buffer; Includes.

[0122] 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.

[0123] 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).

[0124] 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.

[0125] In some embodiments, the centrifugation-based size exclusion technique uses a MW cutoff of 3 to 10 kDa. In some embodiments, the centrifugation-based size exclusion technique uses a MW cutoff of at least 3 kDa, at least 4 kDa, at least 5 kDa, at least 6 kDa, at least 7 kDa, at least 8 kDa, at least 9 kDa, at least 10 kDa, at least 11 kDa, at least 12 kDa, at least 13 kDa, at least 14 kDa, at least 15 kDa, at least 16 kDa. In some embodiments, a MW cutoff of at least 17 kDa MW cutoff, at least 18 kDa MW cutoff, at least 19 kDa MW cutoff, at least 20 kDa MW cutoff, at least 21 kDa MW cutoff, at least 22 kDa MW cutoff, at least 23 kDa MW cutoff, at least 24 kDa MW cutoff, at least 25 kDa MW cutoff, at least 26 kDa MW cutoff, at least 27 kDa MW cutoff, at least 28 kDa MW cutoff, at least 29 kDa MW cutoff, and / or at least 30 kDa MW cutoff may be used.

[0126] In some embodiments, the method produces an MSC secretome composition and / or formulation as described herein above. In some embodiments, the first and / or second culture medium is MSC medium and / or MSC-XF.

[0127] MSCs, or cells differentiated from MSCs, can be made to produce conditioned medium containing cellular FN and optional other desired secretome components, including, for example, desired cytokines and / or desired therapeutic properties as described herein. For example, secretomes can be produced from MSCs of super donor cell lines. Secretomes can also be produced from commercially obtained MSCs. In upcoming embodiments, allogeneic MSCs (and / or cells derived therefrom) and / or allogeneic MSC-derived secretome compositions can be prepared and stored for large groups of individuals. Allogeneic MSCs (and / or cells derived therefrom) and / or MSC-derived secretome compositions can be made in advance so that they are ready when people need them. In certain embodiments, MSCs (and / or cells derived therefrom) and / or MSC-derived secretome compositions can be processed to produce more concentrated solutions or compositions (e.g., mesenchymal stem cell-derived secretome compositions or MSC secretome compositions as described herein).

[0128] In some embodiments, the initial culture medium and the first culture medium are different. In some embodiments, the initial medium and the first medium are the same. Non-limiting examples of cell culture media or media useful for culturing MSCs to produce conditioned medium comprising the MSC secretome according to the present invention include hMSC Media Booster XFM, hMSC High Performance Basal Media, Minimum Essential Medium Eagle (MEME), ADC-1, LPM (without Bovine Serum Albumin), F10 (HAM), F12 (HAM), DCCM1, DCCM2, RPMI1640, BGJ Medium (with and without Fitton-Jackson Modification), StemPro, MSC Gro, MesenCult, NutriStem, Basal Medium Eagle (BME-with the addition of Earle's salt base), Dulbecco's Modified Eagle Medium (DMEM-with or without serum), Yamane, IMEM-20, Glasgow Modified Eagle Medium (GMEM), Leibovitz L-15 Medium, McCoy's 5A Medium, Medium Examples of suitable mediums include M199 (M199E-with Earle's salt base), Medium M199 (M199H-with Hank's salt base), Minimum Essential Medium Alpha (MEM-alpha), Minimum Essential Medium Eagle (MEM-E-with Earle's salt base), Minimum Essential Medium Eagle (MEM-H-with Hank's salt base) and Minimum Essential Medium Eagle (MEM-with NAA-lacking amino acids), but particularly Medium 199, CMRL1415, CMRL1969, CMRL1066, NCTC135, MB75261, MAB8713, DM145, Williams'G, Neuman & Tytell, Higuchi, MCDB301, MCDB202, MCDB501, MCDB401, MCDB411, MDBC153.A preferred medium for use in the present invention is MEM-alpha. These and other useful media are available from GIBCO (Grand Island, NY, USA) and Biological Industries (BetHaEmek, Israel), among others. Many of these media are summarized in Enzymology, Volume LVIII, "Cell Culture", pp. 6272, edited by William B. Jakoby and Ira H. Pastan, published by Academic Press, Inc.

[0129] In some embodiments, the cell culture medium for mesenchymal stem cells can be serum-free medium. In some embodiments, the cell culture medium for mesenchymal stem cells can be supplemented with serum. In some embodiments, the cell culture medium for mesenchymal stem cells can be supplemented with human platelet lysate. In some embodiments, the serum can include fetal bovine serum (FBS). In some embodiments, the cell culture medium for mesenchymal stem cells can be supplemented with serum, such as bovine or other species fetal serum. In some embodiments, the cell culture medium for mesenchymal stem cells can be supplemented with other components, such as mercaptoethanol and / or antibiotics, to promote cell growth and / or promote cell health. In some embodiments, the cell culture medium for mesenchymal stem cells is not supplemented with antibiotics.

[0130] In some embodiments, the percentage of oxygen is varied to promote cell growth and / or promote cell health. In some embodiments, the oxygen is in an amount of 5%, 10%, 15%, 20%, or 25% to promote cell growth and / or promote cell health. In some embodiments, the mesenchymal stem cells are grown under partial oxygen tension to promote cell growth and / or promote cell health. In some embodiments, the mesenchymal stem cells are grown under a low oxygen tension environment to promote cell growth and / or promote cell health.

[0131] In one aspect, the present invention is directed to a conditioned medium (CM) comprising biological factors secreted by mesenchymal stem cells, which may be referred to as conditioned medium comprising MSC secretome. The conditioned medium may be obtained by culturing mesenchymal stem cells in a medium as described herein and separating the resulting medium comprising mesenchymal stem cells and their secreted mesenchymal stem cell products (referred to as biological factors and / or secretome) into component parts of the conditioned medium containing secretome and mesenchymal stem cells grown in the conditioned medium. The separated conditioned medium comprises mesenchymal stem cell secretome and may be further processed and / or used according to the methods described herein, and is substantially free of mesenchymal stem cells (may contain low and / or trace amounts of stem cells) or free of mesenchymal stem cells. The MSC secretome is composed of various biological factors, such as hormones, cytokines, extracellular matrix, proteins, vesicles, antibodies, chemokines, receptors, inhibitors, granules, etc. As described herein, the conditioned medium or media containing the MSC secretome (CM or conditioned medium containing the MSC secretome) can be further processed to generate concentrated conditioned medium (pCM or enriched MSC secretome).

[0132] In some embodiments, the conditioned medium containing the MSC secretome or enriched MSC secretome is generated by culturing mesenchymal stem cells in a culture medium that replaces the culture medium in which the mesenchymal stem cells were cultured. In some embodiments, the resulting conditioned medium containing the MSC secretome is harvested (collected) and then processed to generate an enriched MSC secretome. In certain embodiments, processing the harvested conditioned medium containing the MSC secretome includes removal of some, most, or essentially all of the medium, or removal of some, most, or essentially all of selected components of the conditioned medium.

[0133] 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.

[0134] In one aspect, provided herein is a method for producing a processed conditioned medium containing cellular FN, the method comprising: (a) culturing stem cells in a cell culture medium, thereby producing a conditioned medium containing factors secreted by mesenchymal stem cells (e.g., a conditioned medium containing a mesenchymal stem cell secretome); (b) harvesting the conditioned medium, thereby producing a harvested conditioned medium (e.g., a harvested mesenchymal stem cell secretome); and (c) filtering the harvested conditioned medium (e.g., a harvested mesenchymal stem cell secretome) to produce a processed conditioned medium (mesenchymal stem cell secretome). In some embodiments, the stem cells of (a) are cultured (cultured) in a proliferation medium before being cultured in a medium that does not contain growth factors. Thus, in some embodiments, the method comprises: (a) culturing mesenchymal stem cells in a first growth medium; (b) replacing the first growth medium with a second growth medium and culturing the stem cells in the second growth medium, thereby producing a conditioned medium comprising the 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.

[0135] 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 that have been transferred from their original environment. MSCs can differentiate into mesodermal lineage cells, such as adipocytes, osteoblasts, and chondrocytes. MSCs have long, thin, small cell bodies with few cellular processes. The cell body contains a large round nucleus with prominent nucleoli, which are surrounded by finely dispersed chromatin particles, giving the nucleus a distinct appearance. The remainder of the cell body contains small amounts of the Golgi apparatus, rough endoplasmic reticulum, mitochondria, and polysomes. The long, thin cells are widely dispersed, and the adjacent extracellular matrix has some reticular fibrils, but lacks other types of collagen fibrils [Brighton, et al. 1991 The Journal of Bone and Joint Surgery 73(6):832-47]. The MSCs described herein may express the following molecular marker (protein molecules characteristic of the plasma membrane of a cell or cell type) profile: bone morphogenetic protein receptors 「1」 (BMPR + );CD34 + Scal + Lin ” ;CD44 + ;c-kit + ;Sca-1 + ;Thy-1 + ;NOTCH3;JAG1;ITGA11. MSCs express other cell type specific markers (stemcells.nih.gov; Kaltz,et al.2010 Exp Cell Res Oct1;316(16):2609-17, incorporated herein by reference). The MSCs described herein can be identified based on colony forming unit assays to detect the multipotent differentiation potential of MSCs (which cell types they give rise to). However, some differentiated cells (progenitor cells) can also be used.

[0136] i.FN Composition - Treatment In some embodiments, to obtain the FN composition, the conditioned medium containing the MSC secretome described herein may be collected and filtered and / or purified to remove cell particles and / or other deleterious components, in some embodiments. For example, the second culture medium from step (iv) is harvested as conditioned medium, as described in step (v) above. The filtration membrane used herein may be selected from any of those known in the art having a suitable membrane and configuration such that it can retain the desired MSC secretome components while passing cell particles and / or other deleterious components. Thus, any suitable membrane may be used that allows for retention of cells under selected hydrodynamic conditions while allowing deleterious components to pass for removal. In some embodiments, an upper pore size limit of about 5 microns and a lower limit of about 0.1 microns would be appropriate. In some embodiments, the filtration may be performed using a micropore filter. In some embodiments, the filtration may be performed using a 0.5 μm to 0.2 μm filter. In some embodiments, filtration can be performed using 0.5 μm, 0.45 μm, 0.4 μm, 0.35 μm, 0.3 μm, 0.25 μm, 0.22 μm, and / or 0.2 μm filters. In some embodiments, filtration can be performed using a 0.45 μm filter. In some embodiments, filtration can be performed using a 0.22 μm filter. In some embodiments, filtration / purification can be performed using a low protein binding polyvinylidene fluoride (PVDF) membrane. In some embodiments, filtration / purification can be performed using polyethersulfone (PES).

[0137] In some embodiments, filtration is by ultrafiltration. In some embodiments, the conditioned medium is filtered using a filter size of 3 kD (to achieve purification, desalting, and concentration in the processed conditioned medium of molecules larger than the filter size). In some embodiments, a filter size less than 3 kD is used to filter the conditioned medium, while in other embodiments, a filter size greater than 3 kD is used depending on the application for which the processed conditioned medium is to be used. In other embodiments, ultrafiltration of the harvested conditioned medium is performed using filters of different pore sizes (e.g., 2 kD, 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.

[0138] In some embodiments, harmful components in the growth support medium are removed by medium exchange, preferably via "cross-flow filtration". Cross-flow filtration refers to a filtration mode in which a suspension of MSC secretome cells flows substantially parallel to a filter that permeates the components of the suspension other than the cells. 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 structure of the filtration system, the flow conditions, and the properties of the fluid. Such cross-flow processes can also include hollow fiber filtration systems in some embodiments. See, for example, U.S. Pat. No. 5,053,334, which is incorporated herein by reference in its entirety.

[0139] In some embodiments, the FN composition may be further concentrated in the absence and / or after filtration. In some embodiments, the FN composition may be concentrated using hollow fiber tangential flow technology.

[0140] In some embodiments, the FN composition can be concentrated using a centrifugation-based size exclusion technique, for example, Amicon and / or Centricon can be used during the concentration step. In some embodiments, the size cutoff is a MW cutoff of 3-10 kDa. In some embodiments, the molecular weight cutoff for use during the centrifugation-based size exclusion technique concentration method is at least about 3 kDa, at least about 4 kDa, at least about 5 kDa, at least about 6 kDa, at least about 7 kDa, at least about 8 kDa, at least about 9 kDa, or at least about 10 kDa, or at least about 15 kDa, or at least about 20 kDa, or at least about 25 kDa, or at least about 30 kDa.

[0141] In some embodiments, the FN composition is concentrated about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, or about 100-fold. In some embodiments, the FN composition is concentrated about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, 95-fold, or about 100-fold compared to the conditioned medium prior to concentration.

[0142] In some embodiments, the FN composition is further buffer exchanged into a final formulation buffer after the concentration step. In some embodiments, the FN composition is further buffer exchanged into a final formulation buffer without the adhesive after the concentration step. In some embodiments, the buffer exchange comprises changing the buffer components of the FN composition. In some embodiments, the FN composition is not diluted during the buffer exchange step. In some embodiments, the FN composition is diluted to less than 1%, less than 5%, less than 10%, less than 15%, less than 20%, or less than 25% during the buffer exchange step.

[0143] In some embodiments, the FN composition is buffer exchanged after the concentration step such that all trace culture medium components are removed, hi some embodiments, the FN composition 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.

[0144] ii.FN composition-formulation In some embodiments, the FN composition comprises 0.1 ng / mL to 150,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 140,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 130,000 ng / ML of FN. In some embodiments, the FN composition comprises about 135,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 120,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 110,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 100,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 9,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 80,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 70,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 70,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 60,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 50,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 40,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 30,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 20,000 ng / ML of FN. In some embodiments, the FN composition comprises 0.1 ng / mL to 10,000 ng / mL of FN. In some embodiments, the FN composition comprises 50 to 5000 ng / mL of FN. In some embodiments, the FN composition comprises 100 to 4000 ng / mL of FN. In some embodiments, the FN composition comprises 150 to 3500 ng / mL of FN.

[0145] In some embodiments, the FN composition comprises 50 ng / mL to 150,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 140,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 130,000 ng / ML of FN. In some embodiments, the FN composition comprises about 135,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 120,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 110,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 100,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 90,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 80,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 70,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 60,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 50,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 40,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 30,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 20,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 10,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 9,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 8,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 7,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 6,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 5,000 ng / ML of FN. In some embodiments, the FN composition comprises 50 ng / mL to 4,000 ng / ML of FN.In some embodiments, the FN composition comprises between 50 ng / mL and 3,000 ng / ML of FN.

[0146] In some embodiments, the FN composition comprises 500ng / mL to 150,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 140,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 130,000ng / ML of FN. In some embodiments, the FN composition comprises about 135,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 120,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 110,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 100,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 90,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 80,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 70,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 60,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 50,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 40,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 30,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 20,000ng / ML of FN. In some embodiments, the FN composition comprises 500ng / mL to 10,000ng / ML of FN. In some embodiments, the FN composition comprises 500 ng / mL to 9,000 ng / ML of FN. In some embodiments, the FN composition comprises 500 ng / mL to 8,000 ng / ML of FN. In some embodiments, the FN composition comprises 500 ng / mL to 7,000 ng / ML of FN. In some embodiments, the FN composition comprises 500 ng / mL to 6,000 ng / ML of FN. In some embodiments, the FN composition comprises 500 ng / mL to 5,000 ng / ML of FN.In some embodiments, the FN composition comprises 500 ng / mL to 4,000 ng / ML of FN. In some embodiments, the FN composition comprises 500 ng / mL to 3,000 ng / ML of FN.

[0147] In some embodiments, the FN composition comprises 1000ng / mL to 150,000ng / ML of FN. In some embodiments, the FN composition comprises 1000ng / mL to 140,000ng / ML of FN. In some embodiments, the FN composition comprises 1000ng / mL to 130,000ng / ML of FN. In some embodiments, the FN composition comprises about 135,000ng / ML of FN. In some embodiments, the FN composition comprises 1000ng / mL to 120,000ng / ML of FN. In some embodiments, the FN composition comprises 1000ng / mL to 110,000ng / ML of FN. In some embodiments, the FN composition comprises 1000ng / mL to 100,000ng / ML of FN. In some embodiments, the FN composition comprises 1000ng / mL to 90,000ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 80,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 70,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 60,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 50,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 40,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 30,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 20,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 10,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 9,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 8,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 7,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 6,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 5,000 ng / ML of FN.In some embodiments, the FN composition comprises 1000 ng / mL to 4,000 ng / ML of FN. In some embodiments, the FN composition comprises 1000 ng / mL to 3,000 ng / ML of FN.

[0148] In some embodiments, the FN composition comprises 1000-70,000 ng / mL of FN. In some embodiments, the FN composition comprises 500-50,000 ng / mL of FN. In some embodiments, the FN composition comprises 1000-40,000 ng / mL of FN. In some embodiments, the FN composition comprises 1500-35,000 ng / mL of FN.

[0149] In some embodiments, the FN composition comprises about 0.5-50 g / mL of FN. In some embodiments, the FN composition comprises 5-45 ng / mL of FN. In some embodiments, the FN composition comprises 10-40 ng / mL of FN. In some embodiments, the FN composition comprises 15-35 ng / mL of FN. In some embodiments, the FN composition comprises 20-30 ng / mL of FN.

[0150] In some embodiments, the FN is at a concentration of any suitable value within the range provided above. In some embodiments, the FN composition comprises about 20 ng / mL of FN. In some embodiments, the FN composition comprises about 21 ng / mL of FN. In some embodiments, the FN composition comprises about 22 ng / mL of FN. In some embodiments, the FN composition comprises about 23 ng / mL of FN. In some embodiments, the FN composition comprises about 24 ng / mL of FN. In some embodiments, the FN composition comprises about 25 ng / mL of FN. In some embodiments, the FN composition comprises about 26 ng / mL of FN. In some embodiments, the FN composition comprises about 27 ng / mL of FN. In some embodiments, the FN composition comprises about 28 ng / mL of FN. In some embodiments, the FN composition comprises about 29 ng / mL of FN. In some embodiments, the FN composition comprises about 30 ng / mL of FN. In some embodiments, the FN composition comprises about 25 ng / mL of FN.

[0151] In some embodiments, the FN composition comprises about 0.5-20 ng / mL of FN. In some embodiments, the FN composition comprises about 3-8 ng / mL of FN. In some embodiments, the FN composition comprises about 3 ng / mL of FN. In some embodiments, the FN composition comprises about 3.5 ng / mL of FN. In some embodiments, the FN composition comprises 4 ng / mL of FN. In some embodiments, the FN composition comprises about 4.5 ng / mL of FN. In some embodiments, the FN composition comprises 5 ng / mL of FN. In some embodiments, the FN composition comprises about 5.5 ng / mL of FN. In some embodiments, the FN composition comprises 6 ng / mL of FN. In some embodiments, the FN composition comprises about 6.5 ng / mL of FN. In some embodiments, the FN composition comprises 7 ng / mL of cellular FN. In some embodiments, the FN composition comprises about 7.5 ng / mL of cellular FN. In some embodiments, the FN composition comprises about 8 ng / mL of cellular FN.

[0152] In some embodiments, the FN composition is prepared in a formulation containing about 2 mg to 3 mg / mL of monobasic sodium phosphate, hi some embodiments, the FN composition is prepared in a formulation containing about 4% to 5% / mL of monobasic sodium phosphate.

[0153] In some embodiments, the FN composition is prepared in a formulation comprising about 11 mg to 12 mg / mL of sodium phosphate, dibasic. In some embodiments, the FN composition is prepared in a formulation comprising about 21.5% to 23% / mL of sodium phosphate, dibasic.

[0154] In some embodiments, the FN composition is prepared in a formulation that includes about 11.5 mg to 13 mg / mL of mannitol. In some embodiments, the FN composition is prepared in a formulation that includes about 23% to 25% / mL of mannitol.

[0155] In some embodiments, the FN composition is prepared in a formulation that includes about 23 mg to 25 mg / mL of trehalose dihydrate, hi some embodiments, the FN composition is prepared in a formulation that includes about 46% to 48% / mL of trehalose dihydrate.

[0156] In some embodiments, the FN composition is prepared without hypromellose. In some embodiments, the FN composition is prepared in a formulation that optionally includes hypromellose. In some embodiments, the FN composition is prepared in a formulation that includes about 0.5 mg to 2 mg / mL of hypromellose. In some embodiments, the FN composition is prepared in a formulation that includes about 1% to 3% / mL of hypromellose.

[0157] In some embodiments, the FN composition is prepared in a formulation that includes hydrochloric acid and / or sodium hydroxide. In some embodiments, the FN composition is prepared in a formulation that includes hydrochloric acid. In some embodiments, the FN composition is prepared in a formulation that includes sodium hydroxide. In some embodiments, hydrochloric acid and / or sodium hydroxide are used to obtain the desired pH.

[0158] In some embodiments, the formulation comprises NaCl. In some embodiments, the formulation does not comprise NaCl. In some embodiments, the formulation does not comprise detectable levels of NaCl. In some embodiments, the formulation comprises MgCl2. In some embodiments, the formulation does not comprise MgCl2. In some embodiments, the formulation does not comprise detectable levels of MgCl2. In some embodiments, the formulation does not comprise either NaCl or MgCl2. In some embodiments, the formulation does not comprise detectable levels of either NaCl or MgCl2.

[0159] In some embodiments, the FN formulation is isotonic with tears (including, for example, tears of natural origin and synthetic tears or tear-like solutions).

[0160] In some embodiments, the FN composition is prepared in a formulation including the ingredients provided in Table 1 below. [Table 1] [Table 2]

[0161] In some embodiments, the FN composition comprises: [Table 3]

[0162] In some embodiments, the FN composition comprises: [Table 4]

[0163] In some embodiments, the FN composition does not include NaCl and includes the following: [Table 5]

[0164] In some embodiments, the FN composition does not include MgCl2 and includes the following: [Table 6]

[0165] In some embodiments, the FN composition does not include NaCl or MgCl2, and includes the following: [Table 7]

[0166] In some embodiments, the FN composition comprises 0.5-50 ng / ml FN, 2.28 mg / ml monobasic sodium phosphate, 10-12 mg / ml dibasic sodium phosphate, 11-13 mg / ml mannitol, 2-25 mg / ml trehalose dihydrate, and 0.5-2 mg / ml hypromellose. In some embodiments, the FN composition does not include NaCl and / or MgCl2.

[0167] In some embodiments, the FN composition comprises 0.5-50 ng / ml FN, 2.28 mg / ml monobasic sodium phosphate, 11.45 mg / ml dibasic sodium phosphate, 12.2 mg / ml mannitol, 24 mg / ml trehalose dihydrate, and 1 mg / ml hypromellose. In some embodiments, the FN composition does not include NaCl and / or MgCl2.

[0168] In some embodiments, the FN composition comprises 0.5-50 ng / ml FN, 1.31 mg / ml monobasic sodium phosphate, 4.5-7 mg / ml dibasic sodium phosphate, 5.5-7.5 mg / ml mannitol, 11-13 mg / ml trehalose dihydrate, and 0.1-1.5 mg / ml hypromellose. In some embodiments, the FN composition does not include NaCl and / or MgCl2.

[0169] In some embodiments, the FN composition comprises 0.5-50 ng / ml FN, 1.31 mg / ml monobasic sodium phosphate, 5.73 mg / ml dibasic sodium phosphate, 6.1 mg / ml mannitol, 12 mg / ml trehalose dihydrate, and 0.5 mg / ml hypromellose. In some embodiments, the FN composition does not include NaCl and / or MgCl2.

[0170] D. Assay Methods / Therapeutic Characteristics In some embodiments of the present invention, the FN composition is processed to achieve specific component ratios / concentrations as well as properties of the FN composition.

[0171] In some embodiments of the invention, the FN composition is processed to achieve specific potency performance criteria. In some embodiments, a buffer exchange step enhances the potency of the FN composition.

[0172] Extracellular vesicles are membrane-bound particles that carry a cargo of soluble and insoluble substances as described above. The term "extracellular vesicles" refers to a group of secreted or shed vesicles of various species. They are generally classified into the following subtypes: 1) microvesicles or shed microvesicles, which typically exhibit a size range of 50-1500 nm, 2) exosomes, which typically exhibit a size range of 30-120 nm, and 3) vesicles, which typically exhibit a size range of less than 500 nm (i.e., <500 nm). (See, e.g., WO2019016799, which is incorporated herein by reference in its entirety). In some embodiments, the FN composition can be analyzed for particle counting and / or to quantify extracellular vesicles (EVs) present in the secretome.

[0173] 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.

[0174] 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 1.0x10^5 / uL, 3.9x10^5 / uL, 4.0x10^5 / uL, 4.1x10^5 / uL, 4.2x10^5 / uL, 4.3x10^5 / uL, 4.4x10^5 / uL, 4.5x10^5 / uL, 4.6x10^5 / uL, 4.7x10^5 / uL, 4.8x10^5 / uL, 4.9x10^5 / uL, or approximately 5.0x10^5 / uL and average 110-120nm in diameter. 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 are present at concentrations of 1.0x10^5 / uL, 3.9x10^5 / uL, 4.0x10^5 / uL, 4.1x10^5 / uL, 4.2x10^5 / uL, 4.3x10^5 / uL, 4.4x10^5 / uL, 4.5x10^5 / uL, 4.6x10^5 / uL, 4.7x10^5 / uL, 4.8x10^5 / uL, 4.9x10^5 / uL, or approximately 5.0x10^5 / uL and average 112-116nm in diameter.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, 3.8x In some embodiments, the EVs are present at a concentration of about 3.8x10^5 / uL+ / -0.8x10^5 and have a mean diameter of 114 nm.

[0175] iii.FN composition-therapeutic properties In some embodiments, the FN composition of the present invention comprises cellular FN. In some embodiments, the FN composition comprises cellular FN. In some embodiments, the cellular FN of the present invention is a mixture of alternative splicing variants / isoforms (such as EDA, EDB, and V+). In some embodiments, the cellular FN is EDA+. In some embodiments, the cellular FN is EDB+. In some embodiments, the cellular FN is EDA+ and EDB+. In some embodiments, the cellular fibronectin is V+.

[0176] In some embodiments, the FN compositions of the present disclosure further comprise one or more growth factors.

[0177] In some embodiments, the FN compositions of the present disclosure exhibit a variety of therapeutic properties including, for example, anti-angiogenic properties (blood and / or lymphatic vessels), anti-fibrotic properties, anti-inflammatory properties, properties promoting cell migration, proliferation, cell adhesion, spreading, survival, and extracellular matrix (ECM) assembly and structure, mitogenic properties, and anti-oxidative stress / damage properties.

[0178] In some embodiments, the FN composition exhibits anti-fibrotic properties. In some embodiments, such anti-fibrotic properties can be assayed for using standard assays. In some embodiments, the presence of various factors and / or activities associated with the FN composition is indicative of anti-fibrotic properties. In some embodiments, factors exhibiting anti-fibrotic properties include one or more growth factors selected from the group consisting of FGF (such as FGF-2), PDGF, HGF, VEGF, TGFβ1, TGFβ2, IGF-1, IGF-2, NGF, neurotrophins, and EGF.

[0179] In some embodiments, the FN composition exhibits anti-inflammatory properties. In some embodiments, the FN composition inhibits inflammation. In some embodiments, the FN composition 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 FN composition prevents mast cell degranulation.

[0180] In some embodiments, the FN compositions promote cell migration and proliferation, including, for example, mitogenic and promotility activities. In some embodiments, the FN compositions promote mitogenic activity. In some embodiments, the FN compositions promote motility activity. In some embodiments, the FN compositions further comprise one or more growth factors selected from the group consisting of FGF (such as FGF-2), PDGF, HGF, VEGF, TGFβ1, TGFβ2, IGF-1, IGF-2, NGF, neurotrophins, and EGF, which provide additional cell migration and proliferation activities to the FN compositions.

[0181] In some embodiments, the FN composition further comprises an FGF (such as FGF-2), which provides the FN composition with additional cell migration and proliferation activity.

[0182] In some embodiments, the FN composition further comprises HGF, which provides the FN composition with additional cell migration and cell proliferation activity.

[0183] In some embodiments, the FN composition further comprises an anti-apoptotic agent, which confers cell migration and cell proliferation activity to the FN composition. In some embodiments, the FN composition comprises an anti-apoptotic agent (including but not limited to FGF-2, HGF, and IGF-1), which confers additional cell migration and cell proliferation activity to the FN composition. In some embodiments, the FN composition comprises an anti-apoptotic agent selected from the group consisting of FGF-2, HGF, and IGF-1, which confers additional cell migration and cell proliferation activity to the FN composition.

[0184] iv.FN Composition - Biophysical / Biochemical Properties Biochemical and biophysical characterization: In some embodiments, the present invention provides a method for characterization of FN compositions. In some embodiments, characterization of FN compositions includes 1) comprehensive and / or quantitative mapping of molecular entities in the FN composition, 2) measuring the contribution of selected factors to biological activity, and 3) measuring biophysical parameters. In some embodiments, various efficacy assays can be performed on the FN composition as described herein to determine the properties of the FN composition. In some embodiments, the FN composition can be subjected to comprehensive and / or quantitative mapping of molecular entities in the FN composition, 2) measuring the contribution of selected factors to biological activity, and 3) measuring biophysical parameters. In some embodiments, the characterization assays include, but are not limited to, biophysical assays, biochemical assays, and bioassays. In some embodiments, the characterization assays can include, but are not limited to, characterization of physical components, oxidative stress assays, safety analyses, stability assays, proliferation assays, migration assays, angiogenesis assays, differentiation / scarring assays, inflammation assays, and / or epithelial barrier integrity assays. In some embodiments, the characterization assay is selected from the group consisting of physical component characterization, oxidative stress assays, safety analyses, stability assays, proliferation assays, migration assays, angiogenesis assays, differentiation / scarring assays, inflammation assays, and / or epithelial barrier integrity assays.

[0185] Physical Component Characterization: In some embodiments, characterizing the FN composition includes methods using a combination of bioanalytical techniques. In some embodiments, characterizing the FN composition includes determining the physical components of the FN composition. In some embodiments, characterizing the FN composition includes the use of protein arrays, enzyme-linked immunosorbent assays (ELISAs), mass spectrometry, and immunoblotting. In some embodiments, characterizing the FN composition can be used to identify molecules within the FN composition. In some embodiments, protein arrays can be used to identify factors in the FN composition. In some embodiments, mass spectrometry can be used to determine the presence of one or more factors in the FN composition. In some embodiments, quantitative techniques can be used to measure the levels of one or more factors. In some embodiments, quantitative techniques such as ELISAs can be used to measure the levels of each factor.

[0186] In some embodiments, the FN composition comprises cellular FN. In some embodiments, the cellular FN of the present invention is a mixture of alternative splicing variants / isoforms (such as EDA, EDB, and V+). In some embodiments, the cellular FN is EDA+. In some embodiments, the cellular FN is EDB+. In some embodiments, the cellular FN is EDA+ and EDB+. In some embodiments, the cellular fibronectin is V+.

[0187] In some embodiments, the FN composition comprises an MSC secretome, which comprises protein factors and extracellular vesicles (EVs). In some embodiments, the cellular FN composition comprises trophic factors.

[0188] In some embodiments, secretomes are extracellular vesicles (EVs) with sizes ranging from 30-200 nm and up to 1x10 per mL. 8 ~5x10 9 Includes EVs.

[0189] In some embodiments, depletion studies can be performed to extract the individual contributions of key factors. In some embodiments, antibody-based pull-down methods can be used to remove defined factors from the FN composition. In some embodiments, depletion can be verified by Western blot and then evaluated by one or more bioassays, as described below. In some embodiments, depletion studies can be performed to evaluate the contributions of protein fractions and EV fractions.

[0190] Oxidative stress: In some embodiments, an oxidative stress prevention assay may be performed on the FN composition. In some embodiments, the FN composition prevents damage to the corneal epithelium. In some embodiments, the cellular FN composition reduces the presence of inflammation. In some embodiments, the FN composition reduces the presence of inflammation as determined by an increased presence of anti-inflammatory markers. In some embodiments, the FN composition reduces the presence of inflammation as determined by an increased presence of anti-inflammatory markers, such as IL-8.

[0191] Safety Characterization: In some embodiments, the FN composition can be evaluated for hemocompatibility and tested for sterility and pyrogen and endotoxin levels. In some embodiments, the FN composition can be evaluated for hemocompatibility. In some embodiments, the evaluation of hemocompatibility includes hemolysis and hemagglutination assays. In some embodiments, the FN composition does not exhibit adverse effects upon systemic exposure. In some embodiments, the FN composition does not exhibit adverse effects upon systemic exposure, such as severe eye burns. In some embodiments, the FN composition does not exhibit hemagglutination activity. In some embodiments, the FN composition does not induce hemolysis. In some embodiments, the FN composition does not induce hemolytic activity.

[0192] In some embodiments, the FN composition can be sterile so that it can be administered as part of a formulation. In some embodiments, the FN composition can be free or substantially free of endotoxins. In some embodiments, the FN composition can be free or substantially free of microorganisms.

[0193] Stability: In some embodiments, biophysical properties of the FN composition can be evaluated and / or determined. In some embodiments, fluorescence, static light scattering, and dynamic light scattering characterize protein stability metrics. In some embodiments, the following parameters can be measured to further characterize the secretome: thermal melting, thermal aggregation, delta G, and / or viscosity. In some embodiments, a thermal melting assay is used to determine the stability of the FN composition. In some embodiments, a thermal aggregation assay is used to determine the stability of the FN composition. In some embodiments, delta G is used as a measure to determine the stability of the FN composition. In some embodiments, viscosity is measured as a FN composition property. In some embodiments, viscosity is what determines the stability of the FN composition.

[0194] In some embodiments, biophysical metrics can be used to establish stability parameters to characterize different FN composition formulations.

[0195] In some embodiments, the FN composition is stable at -20°C, 4°C, and room temperature (20°C) for at least 7 days. In some embodiments, the FN composition is stable at -20°C, 4°C, and room temperature (20°C) for at least 14 days. In some embodiments, the FN composition 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 FN composition 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 FN composition 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 FN composition 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.

[0196] In some embodiments, the FN composition is stable for at least 7 days at about -20° C. In some embodiments, the FN composition is stable for at least 7 days at about 4° C. In some embodiments, the FN composition is stable for at least 7 days at about 20° C. In some embodiments, the FN composition is stable for at least 7 days at about 25° C. (room temperature).

[0197] In some embodiments, the FN composition is stable for at least 14 days at about -20°C. In some embodiments, the FN composition is stable for at least 14 days at about 4°C. In some embodiments, the FN composition is stable for at least 14 days at about 20°C (or room temperature). In some embodiments, the FN composition is stable for at least 14 days at about 25°C (room temperature).

[0198] Epithelial barrier integrity assay The corneal epithelium, more precisely the apical surface of the epithelium, is a major contributor to the overall barrier properties of the cornea, and changes to the corneal barrier are a sensitive factor in biocompatibility analysis. In some embodiments, the biophysical characteristics of the FN composition can be evaluated and / or determined, such as by an epithelial barrier integrity assay. In some embodiments, the epithelial barrier integrity assay is transepithelial electrical resistance (TEER). In some embodiments, the transepithelial electrical resistance (TEER) can be evaluated to measure the 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, the TEER can be measured using an epithelial volt-ohmmeter EVOMO and an EndOhm-12 chamber (World Precision, Sarasota, FL). In some embodiments, at the end of the procedure, the tissue can be used for tissue viability assessment using the following formula: % Barrier Integrity = 100x[TEER(treated tissue) / TEER(placebo control)]

[0199] In some embodiments, TEER can be used to evaluate the effect of topical application of FN composition on barrier integrity. In some embodiments, TEER can be used to evaluate the effect of topical application of FN composition on barrier integrity after corneal epithelial injury caused by topical exposure to nitrogen mustard (NM) utilizing EpiCorneal tissue model (MatTekCorp). In some embodiments, FN composition 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.

[0200] Bioassay In some embodiments, bioassays can be used to characterize FN compositions. In some embodiments, bioassays can relate to corneal wound healing: epithelial cell migration and proliferation, stromal cell differentiation (e.g., scarring), angiogenesis, and inflammation. In some embodiments, bioassays can be used to evaluate the ability of FN compositions to mediate corneal wound healing: epithelial cell migration and proliferation, stromal cell differentiation (scarring), angiogenesis, and inflammation. In some embodiments, the FN compositions provided and assayed herein include cellular FN. In some embodiments, the cellular FN of the present invention is a mixture of alternative splicing variants / isoforms (such as EDA, EDB, and / or V+). In some embodiments, the cellular FN is EDA+. In some embodiments, the cellular FN is EDB+. In some embodiments, the cellular FN is EDA+ and EDB+. In some embodiments, the cellular fibronectin is V+.

[0201] In some embodiments, FN compositions may be evaluated for their ability to promote wound healing (such as ocular wound healing). In some embodiments, FN compositions may be evaluated for their ability to promote proliferation and migration. In some embodiments, FN compositions may be evaluated for their ability to promote proliferation. In some embodiments, FN compositions may be evaluated for their ability to promote migration.

[0202] In some embodiments, the FN composition comprises cellular FN. In some embodiments, the FN composition promotes proliferation and / or migration. In some embodiments, the FN composition promotes ocular wound healing. In some embodiments, the FN composition promotes proliferation. In some embodiments, the FN composition promotes migration. In some embodiments, the FN composition promotes cell adhesion. In some embodiments, the FN composition promotes cell spreading. In some embodiments, the FN composition promotes cell survival. In some embodiments, the FN composition promotes proper assembly and / or construction of extracellular matrix (ECM). In some embodiments, the FN composition may be evaluated using a scratch assay to determine its ability to promote healing. In some embodiments, the FN composition may be evaluated using a transwell migration assay to determine its ability to promote proliferation. In some embodiments, the FN composition may be evaluated using a transwell migration assay to determine its ability to promote migration.

[0203] Scratch assay In some embodiments, the assay of the present invention may include a "scratch assay" (also called a "scratch wound assay", "scratch wound closure assay", "wound closure assay", or "wound healing assay"). In some embodiments, the FN composition promotes migration, and this promotion of migration is determined and / or examined using a "scratch assay". In some embodiments, the FN composition promotes proliferation, and this promotion of proliferation is determined and / or examined using a scratch assay. In general, the scratch assay method is based on the generation of an artificial gap, also called a "scratch", in a confluent cell monolayer. The "scratch" can be monitored to see whether cells at the edge of the newly created gap migrate towards 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)). In some embodiments, the scratch assay is used to screen candidates that have the ability to induce wound healing.

[0204] In one example embodiment, the scratch assay provided herein comprises: (a) providing a layer of cells; (b) introducing a wound gap / scratch into the layer of cells; (c) determining whether the wound gap heals / closes in the presence of the test composition, wherein the composition is administered to the cells either before or after step (b), and closure of the wound gap indicates that the test composition has the ability to induce ocular wound healing; Includes.

[0205] In some embodiments, the scratch assay is used with corneal cells. In some embodiments, the cells assayed are mammalian cells. In some embodiments, the cells assayed are human cells. In some embodiments, the scratch assay is used with retinal cells. In some embodiments, the scratch assay is used with epithelial cells. In some embodiments, the scratch assay is used with corneal keratocytes. In some embodiments, the scratch assay is used with fibroblasts. In some embodiments, the scratch assay is used with optic nerve cells. In some embodiments, the scratch assay is used with ganglion cells. In some embodiments, the scratch assay is used with retinal pigment epithelial cells. In some embodiments, the scratch assay is used with retinal pigment epithelial cells. In some embodiments, the scratch assay is used with lens epithelial cells. In some embodiments, the scratch assay is used with iris pigment epithelial cells. In some embodiments, the scratch assay is used with conjunctival fibroblasts. In some embodiments, the scratch assay is used with non-pigmented ciliary epithelial cells. In some embodiments, the scratch assay is used with trabecular meshwork cells. In some embodiments, the scratch assay is used with ocular choroidal fibroblasts. In some embodiments, the scratch assay is used with conjunctival epithelial cells.

[0206] In some embodiments, the cells being assayed form a confluent layer at the time one or more scratches (also referred to as "gaps" or "wounds") are introduced. In some embodiments, the confluent layer of cells being assayed is a monolayer.

[0207] In some embodiments, one or more scratches are introduced into the cell. In some embodiments, a single scratch is introduced. In some embodiments, multiple scratches are introduced into the cell. In some embodiments, one or more scratches are introduced into the cell chemically. In some embodiments, one or more scratches are introduced into the cell via a chemical burn. In some embodiments, one or more scratches are introduced into the cell via a pharmaceutical agent or compound. In one exemplary embodiment, the chemical burn is an alkali burn. In another exemplary embodiment, the chemical burn is a nitrogen mustard gas burn. In some embodiments, one or more scratches are introduced into the cell by mechanically disrupting a layer of cells. In some embodiments, one or more scratches are introduced by a heat shock. In some embodiments, one or more scratches are introduced into the cell via a laser pulse.

[0208] In some embodiments, the scratch introduced into the cell comprises a linear scratch. In some embodiments, the scratch introduced into the cell comprises a cross-hatch scratch. In some embodiments, the scratch introduced into the cell comprises a circular scratch. In some embodiments, the scratch introduced into the cell comprises a zigzag scratch. In some embodiments, the scratch introduced into the cell comprises a combination of one or more of the shapes described above.

[0209] In some embodiments, the size of the scratch introduced (i.e., the transverse length separating the migration fronts on either side of the wound / scratch) is about 0.01 mm to 10 mm. In some embodiments, the size of the scratch is about 0.01 mm to 0.1 mm, about 0.1 mm to 1 mm, about 1 mm to 10 mm, or any suitable value within these ranges.

[0210] In some embodiments, the test reagent / composition is administered to the cells after one or more scratches are introduced. In some embodiments, the test reagent / composition is administered to the cells before one or more scratches are introduced. In some embodiments, the test reagent / composition is a FN composition. In some embodiments, the test reagent / composition is a conditioned medium. In some embodiments, the test reagent / composition is a biopolymer (such as a protein). In some embodiments, the test reagent / composition is a pharmaceutical composition of one or more active compounds. In some embodiments, the test reagent / composition is screened for its ability to promote ocular wound healing.

[0211] In some embodiments, the test reagent / composition is concentrated before being administered to the cell. In some embodiments, the test reagent / composition is diluted before being administered to the cell. In some embodiments, the test reagent / composition is purified (e.g., buffer exchanged) before being administered to the cell. In some embodiments, the test reagent / composition is lyophilized before being administered to the cell. In some embodiments, the test reagent / composition administered to the cell comprises about 10-100 μg / mL of active ingredient. In some embodiments, the test reagent / composition administered to the cell comprises about 10-100 μg / mL of protein. In some embodiments, the test reagent / composition administered to the cell comprises about 10-90 μg / mL of protein. In some embodiments, the test reagent / composition administered to the cell comprises about 20-80 μg / mL of protein. In some embodiments, the test reagent / composition administered to the cell comprises about 30-70 μg / mL of protein. In some embodiments, the test reagent / composition administered to the cell comprises about 40-60 μg / mL of protein. In some embodiments, the test reagent / composition administered to a cell comprises about 45 μg / mL of protein. In some embodiments, the test reagent / composition administered to a cell comprises about 50 μg / mL of protein. In some embodiments, the test reagent / composition administered to a cell comprises about 55 μg / mL of protein.

[0212] In some embodiments, closure of one or more scratches indicates that the test reagent has the ability to promote ocular wound healing. In some embodiments, closure of one or more scratches indicates that the test reagent has the ability to promote cell proliferation. In some embodiments, closure of one or more scratches indicates that the test reagent has the ability to promote cell migration. In some embodiments, scratch closure is characterized as the total number of cells that have migrated into the scratch. In some embodiments, the total number of cells that have migrated into the scratch is measured by physical counting (image analysis software) with or without staining (colorimetric or fluorometric). In some embodiments, the amount of cells in the scratch is quantified spectroscopically by measuring the total number of cells that have migrated into the scratch by absorbance-based or fluorometric-based methods. In some embodiments, scratch closure is characterized as a percentage of wound closure or a separate mathematical value, i.e., the initial wound surface area minus the remaining scratch surface area at a particular time point divided by the initial scratch surface area. In some embodiments, scratch closure is characterized as the percentage of remaining scratch area, i.e., the total percentage minus the percentage of wound closure. In some embodiments, scratch closure is characterized as the size of the scratch (i.e., the transverse length separating the migration fronts on either side of the wound). In some embodiments, image analysis software is used to establish the migrating scratch front (or boundary) and measure the distance of the remaining scratch (e.g., expressed in pixels, um, etc.). In some embodiments, scratch closure is characterized as the surface area of ​​the scratch. In some embodiments, the surface area of ​​the scratch (e.g., in pixels^2 or μm^2) is determined by image analysis software. In some embodiments, scratch closure is characterized as a function of time. In some embodiments, scratch closure is characterized as the time it takes for all or a percentage (e.g., 50%) of the scratch to close.In some embodiments, the closure of the scratch is characterized as a rate (such as a cell-free surface area of ​​the scratch) as a function of time. In some embodiments, the rate measured is cell migration distance / migration time.

[0213] In some embodiments, the closure of one or more scratches is measured for about 1-5 days. In some embodiments, the closure of one or more scratches is measured for about 2-4 days. In some embodiments, the closure of one or more scratches is measured for about 2-3 days. In some embodiments, the closure of one or more scratches is measured for about 2 days. In some embodiments, the closure of one or more scratches is measured for about 3 days. In some embodiments, the closure of one or more scratches is measured at regular intervals. In some embodiments, the closure of one or more scratches is measured once a day. In some embodiments, the closure of one or more scratches is measured continuously.

[0214] In some embodiments, the FN compositions of the present invention induce ocular wound healing in a scratch assay provided herein. In some embodiments, the FN compositions of the present invention induce ocular wound healing in a scratch assay provided herein. In some embodiments, the FN compositions of the present invention increase wound closure in a scratch assay by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more. In some embodiments, the FN compositions of the present invention increase wound closure in a scratch assay by at least 1-fold. In some embodiments, the FN compositions of the present invention increase wound closure in a scratch assay by at least 2-fold. In some embodiments, the FN compositions of the present invention increase wound closure in a scratch assay by at least 3-fold. In some embodiments, the FN compositions of the present invention increase wound closure in a scratch assay by at least 4-fold. In some embodiments, the FN compositions of the present invention increase wound closure in a scratch assay by at least 5-fold. In some embodiments, the FN compositions of the present invention increase wound closure in a scratch assay by at least 6-fold. In some embodiments, the FN compositions of the present invention increase wound closure in a scratch assay by at least 7-fold. In some embodiments, the FN compositions of the present invention increase wound closure in a scratch assay by at least 8-fold. In some embodiments, the FN compositions of the present invention increase wound closure in a scratch assay by at least 9-fold. In some embodiments, the FN compositions of the present invention increase wound closure in a scratch assay by at least 10-fold. In some embodiments, the FN compositions of the present invention increase wound closure in a scratch assay by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more. In some embodiments, the FN compositions of the present invention increase wound closure in a scratch assay by at least 10%. In some embodiments, the FN compositions of the present invention increase wound closure in a scratch assay by at least 20%.In some embodiments, the FN compositions of the present invention increase wound closure in a scratch assay by at least 30%. In some embodiments, the FN compositions of the present invention increase wound closure in a scratch assay by at least 40%. In some embodiments, the FN compositions of the present invention increase wound closure in a scratch assay by at least 50%. In some embodiments, the FN compositions of the present invention increase wound closure in a scratch assay by at least 60%. In some embodiments, the FN compositions of the present invention increase wound closure in a scratch assay by at least 70%. In some embodiments, the FN compositions of the present invention increase wound closure in a scratch assay by at least 80%. In some embodiments, the FN compositions of the present invention increase wound closure in a scratch assay by at least 90%. In some embodiments, the FN compositions of the present invention increase wound closure in a scratch assay by at least 100%.

[0215] In some embodiments, the FN composition of the present invention induces ocular wound healing in a scratch assay provided herein. In some embodiments, the FN composition induces ocular wound healing in a scratch assay provided herein. In some embodiments, the FN composition increases wound closure in a scratch assay by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more. In some embodiments, the FN composition increases wound closure in a scratch assay by at least 1-fold. In some embodiments, the FN composition increases wound closure in a scratch assay by at least 2-fold. In some embodiments, the FN composition increases wound closure in a scratch assay by at least 3-fold. In some embodiments, the FN composition increases wound closure in a scratch assay by at least 4-fold. In some embodiments, the FN composition increases wound closure in a scratch assay by at least 5-fold. In some embodiments, the FN composition increases wound closure in a scratch assay by at least 6-fold. In some embodiments, the FN composition increases wound closure in a scratch assay by at least 7-fold. In some embodiments, the FN composition increases wound closure in a scratch assay by at least 8-fold. In some embodiments, the FN composition increases wound closure in a scratch assay by at least 9-fold. In some embodiments, the FN composition increases wound closure in a scratch assay by at least 10-fold. In some embodiments, the FN composition increases wound closure in a scratch assay by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more. In some embodiments, the FN composition increases wound closure in a scratch assay by at least 10%. In some embodiments, the FN composition increases wound closure in a scratch assay by at least 20%. In some embodiments, the FN composition increases wound closure in a scratch assay by at least 30%.In some embodiments, the FN composition increases wound closure in a scratch assay by at least 40%. In some embodiments, the FN composition increases wound closure in a scratch assay by at least 50%. In some embodiments, the FN composition increases wound closure in a scratch assay by at least 60%. In some embodiments, the FN composition increases wound closure in a scratch assay by at least 70%. In some embodiments, the FN composition increases wound closure in a scratch assay by at least 80%. In some embodiments, the FN composition increases wound closure in a scratch assay by at least 90%. In some embodiments, the FN composition increases wound closure in a scratch assay by at least 100%.

[0216] In some embodiments, at least 30 μg / mL of the test reagent is required to effect ocular wound healing in the scratch assays provided herein. In some embodiments, at least 35 μg / mL of the test reagent is required to effect ocular wound healing in the scratch assays provided herein. In some embodiments, at least 40 μg / mL of the test reagent is required to effect ocular wound healing in the scratch assays provided herein. In some embodiments, at least 45 μg / mL of the test reagent is required to effect ocular wound healing in the scratch assays provided herein. In some embodiments, at least 50 μg / mL of the test reagent is required to effect ocular wound healing in the scratch assays provided herein.

[0217] In some embodiments, the test reagent is FN. In some embodiments, the FN is cellular FN.

[0218] Transwell migration assay In some embodiments, the assay of the invention comprises a "Transwell migration assay" (also referred to as a "Transwell cell invasion assay" or "Transwell assay"). In some embodiments, a Transwell migration assay is used to evaluate the ability of a candidate agent on cell migration and / or proliferation. In some embodiments, a Transwell migration assay is used to evaluate the ability of a candidate agent on cell migration. In some embodiments, a Transwell migration assay is used to evaluate the ability of a candidate agent on cell proliferation. In some embodiments, a Transwell migration assay is used to screen candidates for their ability to induce cell migration / proliferation.

[0219] In one example embodiment, the transwell migration assay provided herein comprises: (a) adding cells to an upper chamber containing a membrane having holes, the cells being supplemented with basal medium in the absence of the test reagent / composition; (b) placing the upper chamber in a container containing a test reagent / composition, wherein a membrane having holes separates the corneal cells in the upper chamber from the test reagent / composition in the container; (c) incubating the cells; (d) measuring / quantifying cells that migrate through the membrane, said migration indicating that the test reagent / composition has the ability to induce cell migration and / or proliferation; Includes.

[0220] In some embodiments, the transwell migration assay is used for corneal cells. In some embodiments, the transwell migration assay is used for retinal cells. In some embodiments, the transwell migration assay is used for epithelial cells. In some embodiments, the transwell migration assay is used for keratocytes. In some embodiments, the transwell migration assay is used for fibroblasts. In some embodiments, the transwell migration assay is used for optic nerve cells. In some embodiments, the transwell migration assay is used for ganglion cells. In some embodiments, the transwell migration assay is used for retinal pigment epithelial cells. In some embodiments, the transwell migration assay is used for retinal pigment epithelial cells. In some embodiments, the transwell migration assay is used for lens epithelial cells. In some embodiments, the transwell migration assay is used for iris pigment epithelial cells. In some embodiments, the transwell migration assay is used for conjunctival fibroblasts. In some embodiments, the transwell migration assay is used for non-pigmented ciliary epithelial cells. In some embodiments, the transwell migration assay is used for trabecular meshwork cells. In some embodiments, the transwell migration assay is used for ocular choroidal fibroblasts. In some embodiments, the transwell migration assay is used for conjunctival epithelial cells.

[0221] In some embodiments, the upper chamber of the transwell migration assay is sealed by a membrane with holes. In some embodiments, the upper chamber is a glass chamber. In some embodiments, the upper chamber is a plastic chamber. In some embodiments, the upper chamber is a Boyden chamber.

[0222] In some embodiments, the membrane of the upper chamber is a polycarbonate membrane with a defined pore size. In some embodiments, the membrane is a basement membrane. In some embodiments, the average pore size of the membrane is below the size of the cells to be assayed. In some embodiments, the average pore size of the membrane is about 1-15 μm. In some embodiments, the average pore size of the membrane is about 3 μm. In some embodiments, the average pore size of the membrane is about 5 μm. In some embodiments, the average pore size of the membrane is about 8 μm. In some embodiments, the average pore size of the membrane is about 12 μm.

[0223] In some embodiments, the membrane of the upper chamber is pre-treated. In some embodiments, the membrane is pre-coated with one or more compounds or biopolymers that enhance cell attachment and / or proliferation. In some embodiments, the membrane is pre-coated with an extracellular matrix. In some embodiments, the membrane is pre-coated with collagen. In some embodiments, the membrane is pre-coated with fibronectin. In some embodiments, the membrane is pre-coated with laminin.

[0224] In some embodiments, the cells are added to the upper chamber, which contains a basal cell culture medium. In some embodiments, the basal cell culture medium in the upper chamber is serum-free. Examples of cell culture media include, but are not limited to, hMSC Media Booster XFM, hMSC High Performance Basal Media, Minimum Essential Medium Eagle (MEME), ADC-1, LPM (without bovine serum albumin), F10 (HAM), F12 (HAM), DCCM1, DCCM2, RPMI 1640, BGJ Medium (with and without Fitton-Jackson modification), StemPro, MSC Gro, MesenCult, NutriStem, Basal Medium Eagle (BME-with Earle's salt base), Dulbecco's Modified Eagle Medium (DMEM-with or without serum), Yamane, IMEM-20, Glasgow Modification Eagle Medium (GMEM), Leibovitz L-15 Medium, McCoy's 5A Medium, Medium M199 (M199E-with Earle's salt base), Medium Examples include M199 (M199H - with Hank's salt base), Minimum Essential Medium Alpha (MEM-alpha), Minimum Essential Medium Eagle (MEM-E - with Earle's salt base), Minimum Essential Medium Eagle (MEM-H - with Hank's salt base), and Minimum Essential Medium Eagle (MEM-NAA with essential amino acids), among many others, including medium199, CMRL1415, CMRL1969, CMRL1066, NCTC135, MB75261, MAB8713, DM145, Williams'G, Neuman & Tytell, Higuchi, MCDB301, MCDB202, MCDB501, MCDB401, MCDB411, MDBC153, among others.An example of a medium for use in the present invention is MEM-alpha. In some embodiments, the cells to be assayed are added to the upper chamber containing the gel. In some embodiments, the gel contains a biological matrix (such as an extracellular matrix).

[0225] In some embodiments, the upper chamber is suspended within a container that contains one or more test reagents / compositions. In some embodiments, the upper chamber and the container contain the same composition except for the one or more test reagents / compositions. In other embodiments, the upper chamber and the container contain different compositions in addition to the one or more test reagents / compositions. In some embodiments, the container contains one or more growth factors.

[0226] In some embodiments, the container is a reaction vessel. In some embodiments, the container is a well in a multi-well plate. In some embodiments, the container is a well in a multi-well plate (such as a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, or a 96-well plate).

[0227] In some embodiments, the test reagent / composition provided herein comprises a chemoattractant for the cells being assayed. In some embodiments, the test reagent / composition comprises a FN composition. In some embodiments, the test reagent / composition comprises a conditioned medium. In some embodiments, the test reagent / composition comprises a biopolymer (such as a protein). In some embodiments, the test reagent / composition comprises a pharmaceutical composition of one or more active compounds. In some embodiments, the test reagent / composition is screened for its ability to promote cell migration and / or proliferation.

[0228] In some embodiments, the test reagent / composition is present only in the container and is excluded from the upper chamber. In some embodiments, the test reagent / composition is present in both the upper chamber and the container. In one example embodiment, there is a concentration gradient of increasing concentration of the test reagent / composition from the upper chamber to the container.

[0229] In some embodiments, the test reagent / composition is concentrated prior to addition. In some embodiments, the test reagent / composition is diluted prior to addition. In some embodiments, the test reagent / composition is purified (e.g., buffer exchanged) prior to addition. In some embodiments, the test reagent / composition is lyophilized prior to addition. In some embodiments, the test reagent / composition in the container comprises about 10-100 μg / mL of active ingredient. In some embodiments, the test reagent / composition in the container comprises about 10-100 μg / mL of protein. In some embodiments, the test reagent / composition in the container comprises about 10-90 μg / mL of protein. In some embodiments, the test reagent / composition in the container comprises about 20-80 μg / mL of protein. In some embodiments, the test reagent / composition in the container comprises about 30-70 μg / mL of protein. In some embodiments, the test reagent / composition in the container comprises about 40-60 μg / mL of protein. In some embodiments, the test reagent / composition in the container comprises about 45 μg / mL of protein. In some embodiments, the test reagent / composition in the container comprises about 50 μg / mL of protein, hi some embodiments, the test reagent / composition in the container comprises about 55 μg / mL of protein.

[0230] In some embodiments, the cells are incubated in the upper chamber for about 6-72 hours. In some embodiments, the incubation time is about 12 hours-60 hours. In some embodiments, the incubation time is about 18 hours-48 hours. In some embodiments, the incubation time is any suitable value within these ranges. In some embodiments, the incubation time is about 6 hours. In some embodiments, the incubation time is about 18 hours. In some embodiments, the incubation time is about 24 hours. In some embodiments, the incubation time is about 30 hours. In some embodiments, the incubation time is about 36 hours. In some embodiments, the incubation time is about 42 hours. In some embodiments, the incubation time is about 48 hours.

[0231] In some embodiments, the total number of cells that have migrated through the membrane is measured by physical counting (image analysis software) with or without staining (colorimetric (e.g., calcein AM) or fluorometric (e.g., crystal violet)). In some embodiments, live cell stains are used to quantitate the migrated cells. In some embodiments, the cell mass is quantified spectrophotometrically, measuring the total number of cells that have migrated through the membrane by absorbance- or fluorometric-based methods. In some embodiments, the migrated cells are quantitated by flow cytometry.

[0232] In some embodiments, the FN compositions of the present invention exhibit the ability to induce ocular wound healing in a transwell migration assay provided herein. In some embodiments, the FN compositions of the present invention induce cell migration and / or proliferation in a transwell migration assay provided herein. In some embodiments, the FN compositions of the present invention induce cell migration in a transwell migration assay provided herein. In some embodiments, the FN compositions of the present invention induce cell proliferation in a transwell migration assay provided herein. In some embodiments, the FN compositions of the present invention exhibit the ability to induce ocular wound healing in a transwell migration assay provided herein. In some embodiments, the FN compositions of the present invention induce cell migration and / or proliferation in a transwell migration assay provided herein. In some embodiments, the FN compositions of the present invention induce cell migration in a transwell migration assay provided herein. In some embodiments, the FN compositions of the present invention induce cell proliferation in a transwell migration assay provided herein.

[0233] In some embodiments, the FN compositions of the present invention increase cell migration and / or proliferation in a transwell assay by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more. In some embodiments, the FN compositions increase cell migration and / or proliferation in a transwell assay by at least 1-fold. In some embodiments, the FN compositions increase cell migration and / or proliferation in a transwell assay by at least 2-fold. In some embodiments, the FN compositions increase cell migration and / or proliferation in a transwell assay by at least 3-fold. In some embodiments, the FN compositions increase cell migration and / or proliferation in a transwell assay by at least 4-fold. In some embodiments, the FN compositions increase cell migration and / or proliferation in a transwell assay by at least 5-fold. In some embodiments, the FN compositions increase cell migration and / or proliferation in a transwell assay by at least 6-fold. In some embodiments, the FN compositions increase cell migration and / or proliferation in a transwell assay by at least 7-fold. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 8-fold. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 9-fold. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 10-fold. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 10%. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 20%.In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 30%. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 40%. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 50%. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 60%. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 70%. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 80%. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 90%. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 100%.

[0234] In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 1-fold. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 2-fold. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 3-fold. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 4-fold. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 5-fold. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 6-fold. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 7-fold. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 8-fold. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 9-fold. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 10-fold. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 10%. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 20%.In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 30%. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 40%. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 50%. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 60%. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 70%. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 80%. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 90%. In some embodiments, the FN composition increases cell migration and / or proliferation in a transwell assay by at least 100%.

[0235] In some embodiments, at least 30 μg / mL of a test reagent / composition is required to induce cell migration and / or proliferation in a transwell migration assay provided herein. In some embodiments, at least 35 μg / mL of a test reagent / composition is required to induce cell migration and / or proliferation in a transwell migration assay provided herein. In some embodiments, at least 40 μg / mL of a test reagent / composition is required to induce cell migration and / or proliferation in a transwell migration assay provided herein. In some embodiments, at least 45 μg / mL of a test reagent / composition is required to induce cell migration and / or proliferation in a transwell migration assay provided herein. In some embodiments, at least 50 μg / mL of a test reagent / composition is required to induce cell migration and / or proliferation in a transwell migration assay provided herein.

[0236] In some embodiments, an endothelial cell tube formation assay may be performed on the FN composition. In some embodiments, the endothelial cell tube formation assay may show that the FN composition is not pro-angiogenic. In some embodiments, the endothelial cell tube formation assay provides a measure of the angiogenic potential of the FN composition. In some embodiments, the FN composition exhibits anti-angiogenic properties. In some embodiments, the FN composition is anti-angiogenic. In some embodiments, the endothelial cell tube formation assay provides a ratio of anti-angiogenic and pro-angiogenic signals. In some embodiments, a negative result of the endothelial cell tube formation assay will confirm that the anti:promotion ratio is high, ensuring that the FN composition does not promote angiogenesis. In some embodiments, a negative result of the endothelial cell tube formation assay will confirm that the anti:promotion ratio is high, ensuring that the FN composition does not promote CNV (choroidal neovascularization) or general angiogenesis. In some embodiments, an inhibition assay of TGFb (also referred to as TGF beta or TGFβ)-induced myofibroblast differentiation may be performed on the cellular FN composition. In some embodiments, an inhibition assay of TGFb-induced myofibroblast differentiation may be performed on the cellular FN composition to show that the FN composition inhibits scarring. In some embodiments, the FN composition prevents scarring. In some embodiments, the FN composition prevents scarring of corneal opacity. In some embodiments, the cellular FN composition has low angiogenic induction. In some embodiments, the FN composition has a reduced angiogenic response. In some embodiments, the FN composition has a reduced angiogenic potential. In some embodiments, the FN composition impairs and / or reduces the normal formation of blood vessels in the presence of angiogenesis-supportive medium. In some embodiments, the FN composition has a reduced angiogenic potential when compared to a control in which the FN composition is not treated. In some embodiments, the FN composition has a reduced angiogenic potential compared to a sample treated with serum-containing medium. In some embodiments, the FN composition attenuates the angiogenic response. In some embodiments, the FN composition reduces the angiogenic response induced by serum-free medium.In some embodiments, a reduced angiogenic response is induced by the FN composition when secretome and serum-containing medium (reduced or no angiogenic response) is compared to serum-containing medium (angiogenic response). In some embodiments, the angiogenic response is indicated by tube formation in a cell-based assay. In some embodiments, the angiogenic response is indicated by tube formation in an endothelial cell tube formation assay.

[0237] Differentiation / scarring: In some embodiments, the FN composition can be evaluated for its ability to prevent differentiation and prevent scarring. In some embodiments, the FN composition prevents and / or inhibits scarring. In some embodiments, the FN composition prevents scarring. In some embodiments, the FN composition reduces scarring compared to other standard treatments. In some embodiments, the FN composition prevents and / or inhibits differentiation. In some embodiments, the FN composition prevents and / or inhibits myofibroblast differentiation. In some embodiments, the FN composition reduces loss of corneal transparency. In some embodiments, the FN composition reduces loss of corneal transparency by preventing and / or inhibiting myofibroblast differentiation.

[0238] In some embodiments, the FN composition can be evaluated for its ability to modulate factors involved in differentiation. In some embodiments, the FN composition can be evaluated for its 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 cellular FN composition 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 FN composition induces a decrease in a factor that is upregulated during normal differentiation. In some embodiments, the FN composition induces an increase in a factor that is downregulated during normal differentiation. In some embodiments, the cellular FN composition induces a decrease in the expression of a factor such as SMA. In some embodiments, the FN composition induces a decrease in the expression of a factor such as SMA, indicating FN composition efficacy.

[0239] Angiogenesis: In some embodiments, the FN composition can be evaluated for its ability to prevent angiogenesis. In some embodiments, the FN composition prevents, inhibits, suppresses, and / or reduces angiogenesis. In some embodiments, the FN composition does not suppress or promote angiogenesis. In some embodiments, the FN composition can be evaluated for its ability to prevent angiogenesis. In some embodiments, the FN composition prevents, inhibits, suppresses, and / or reduces angiogenesis. In some embodiments, the FN composition suppresses angiogenesis.

[0240] In some embodiments, the FN composition can be further evaluated using a depletion assay. In some embodiments, the FN composition can deplete a specific factor. In some embodiments, the FN composition can deplete a specific factor, for example, but not limited to, TIMP1 and / or serpinE1. In some embodiments, the FN composition can deplete TIMP1 and / or serpinE1. In some embodiments, the FN composition can deplete TIMP1. In some embodiments, the FN composition can deplete serpinE1.

[0241] inflammation: In some embodiments, the FN compositions can be evaluated for their ability to prevent, inhibit, suppress, and / or reduce inflammation. In some embodiments, the FN compositions prevent, inhibit, suppress, and / or reduce inflammation. In some embodiments, the FN compositions suppress inflammation. In some embodiments, the FN compositions are characterized in vitro and / or in vivo to determine their ability to prevent, inhibit, suppress, and / or reduce inflammation. In some embodiments, the FN compositions prevent, inhibit, suppress, and / or reduce inflammation in vitro and / or in vivo. In some embodiments, the FN compositions prevent, inhibit, suppress, and / or reduce inflammation in vitro. In some embodiments, the FN compositions prevent, inhibit, suppress, and / or reduce inflammation in vivo. In some embodiments, tissue models can be used to characterize the prevention, inhibition, suppress, and / or reduction of inflammation in vitro. In some embodiments, 3D tissue models can be used to characterize the prevention, inhibition, suppress, and / or reduction of inflammation in vitro. In some embodiments, a nitrogen mustard (NM) gas burn model can be used to assess prevention, inhibition, suppression, and / or reduction of inflammation in vitro. In some embodiments, a nitrogen mustard (NM) gas burn model can be used to assess prevention, inhibition, suppression, and / or reduction of inflammation in vitro and can be used as a surrogate for in vivo conditions. In some embodiments, a cytokine profile can be determined in response to treatment and / or administration with the FN composition. In some embodiments, the levels of certain cytokines can be determined. In some embodiments, the levels of IL-8 can be determined. In some embodiments, the levels of IL-8 expression can be reduced in tissues treated with the FN composition. In some embodiments, the levels of IL-8 expression are reduced in tissues treated with the FN composition, indicating prevention, inhibition, suppression, and / or reduction of inflammation.

[0242] E. Treatment Methods The present disclosure also provides methods of treatment using FN compositions (such as cellular FN compositions) optionally comprising one or more growth factors selected from the group consisting of FGF (such as FGF-2), PDGF, HGF, VEGF, TGFβ1, TGFβ2, IGF-1, IGF-2, NGF, neurotrophins, and EGF. In particular, the FN compositions are used in the treatment of ocular conditions. In particular, the FN compositions are 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 FN compositions find use in the treatment of ocular conditions, including accelerating wound healing. In some embodiments, the FN compositions find use in the treatment of ocular conditions, including reducing scarring. In some embodiments, the FN compositions find use in the treatment of ocular conditions, including reducing inflammation. In some embodiments, the FN compositions find use in the treatment of ocular conditions, including reducing inflammation and thus promoting growth. In some embodiments, the FN compositions find use in the treatment of ocular conditions, such as reducing inflammation at the ocular surface. In some embodiments, the FN compositions find use in the treatment of ocular conditions, including reducing angiogenesis. In some embodiments, the FN compositions find use in the treatment of ocular conditions, including reducing angiogenesis in the cornea. In some embodiments, the FN compositions find use in the treatment of ocular conditions, including dry eye treatment (e.g., including treatment of severe dry eye, including where epithelial cells are damaged). In some embodiments, the FN compositions find use in the treatment of ocular conditions, such as restoring integrity to damaged ocular tissue. In some embodiments, the FN compositions find use in the treatment of ocular conditions, such as accelerating the healing of damaged ocular tissue. In some embodiments, the FN compositions find use in the treatment of ocular conditions, such as regeneration of damaged ocular nerve tissue. In some embodiments, the FN compositions find use in the treatment of ocular conditions, such as retinal pathology. In some embodiments, the FN compositions find use in the treatment of ocular conditions, such as regeneration of damaged ocular nerve tissue associated with PCED.In some embodiments, the FN compositions find use in treating ocular conditions such as PCED. In some embodiments, the FN compositions find use in treating ocular conditions such as inflammatory damage to the ocular surface. In some embodiments, the FN compositions find use in treating ocular conditions such as, for example, GvHD and / or Sjogren's syndrome.

[0243] In some embodiments, the ocular condition is selected from the group consisting of retinal pathology, chronic graft-versus-host disease (GvHD), Stevens-Johnson syndrome, ocular mucosa pemphigoid, persistent corneal epithelial defect (PCED), dry eye, ocular nerve tissue injury, and traumatic injury to the eye (such as a percussive injury, ocular contusion, or chemical burn).

[0244] In some embodiments, the FN compositions find use in accelerating wound healing. In some embodiments, the FN compositions find use in reducing scarring. In some embodiments, the FN compositions find use in reducing inflammation. In some embodiments, the FN compositions find use in reducing inflammation and therefore promoting growth. In some embodiments, the FN compositions find use in reducing inflammation at the ocular surface. In some embodiments, the FN compositions find use in reducing angiogenesis. In some embodiments, the FN compositions find use in reducing angiogenesis in the cornea. In some embodiments, the FN compositions find use in protecting and repairing retinal epithelial cells and retinal ganglion cells. In some embodiments, the FN compositions find use in inducing regeneration of the trabecular meshwork and reducing intraocular pressure.

[0245] In some embodiments, the FN composition is administered for the treatment of an ocular disease. In some embodiments, the method comprises administering a therapeutically effective amount of an FN composition as described herein to a patient in need thereof. In some embodiments, the FN composition is administered to a patient in need thereof to promote or induce ocular wound healing. In some embodiments, the FN composition is administered to a patient in need thereof to reduce and / or inhibit angiogenesis, reduce and / or inhibit scarring, promote and / or maintain vision, and / or increase wound closure rate (e.g., shorten wound closure time). In some embodiments, the FN composition is administered to a patient in need thereof to prevent, reduce, and / or inhibit angiogenesis. In some embodiments, the FN composition is administered to a patient in need thereof to prevent, reduce, and / or inhibit scarring reduction. In some embodiments, the FN composition is administered to a patient in need thereof to promote and / or maintain vision. In some embodiments, the FN composition is administered to a patient in need thereof to promote and / or maintain vision. In some embodiments, the FN composition is administered to promote and / or induce faster wound closure (e.g., shorten the time required for wound closure). In some embodiments, the FN composition prevents, reduces, and / or inhibits or does not promote angiogenesis and reduces scarring to promote vision preservation. In some embodiments, the FN composition is administered to a patient in need thereof to prevent, reduce, and / or inhibit angiogenesis and reduce scarring to promote vision preservation. In some embodiments, the FN composition prevents, reduces, and / or inhibits inflammation. In some embodiments, the FN composition is administered to a patient in need thereof to prevent, reduce, and / or inhibit inflammation.

[0246] In some embodiments, the FN composition is administered to treat visual dysfunction following traumatic injury to an ocular structure. In some embodiments, the treatment comprises administering a therapeutically effective amount of the FN composition as described herein to a patient in need thereof.

[0247] In some embodiments, the FN composition is administered for the treatment of traumatic injury of optic nerve degeneration after percussive injury. In some embodiments, the percussive injury to the eye is selected from the group consisting of an ocular contusion and a blunt injury to the eye. In some embodiments, the FN composition 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 the FN composition as described herein.

[0248] In some embodiments, the FN composition is administered to improve optic nerve degeneration following percussive injury to the eye. In some embodiments, the method for improving optic nerve degeneration comprises administering to a patient a therapeutically effective amount of an FN composition as described herein. In some embodiments, the percussive injury to the eye is selected from the group consisting of an ocular contusion and a blunt injury to the eye. In some embodiments, the percussive injury to the eye is an ocular contusion. In some embodiments, the percussive injury to the eye is a blunt injury to the eye.

[0249] Efficacy readouts may include, for example, a reduction in symptoms and / or a decrease in pathology, including an improvement in quality of life. In some embodiments, a reduction in symptoms and / or a decrease in pathology of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% indicates a therapeutic effect. In some embodiments, a reduction in inflammation of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% indicates a therapeutic effect. In some embodiments, a reduction in scarring of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% indicates a therapeutic effect. In some embodiments, a reduction in angiogenesis of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% indicates therapeutic efficacy.

[0250] In some embodiments, the disease or condition is an ocular disease or condition. In some embodiments, the disease or condition is visual dysfunction following trauma to an ocular structure. In some embodiments, the disease or condition is a percussive (e.g., blunt or non-blunt) injury to the eye. In some embodiments, the disease or condition is a burn, including a chemical burn, to the eye.

[0251] In some embodiments, the FN composition is administered to a specific target area. In some embodiments, the specific target area is the eye. In some embodiments, the FN composition is administered to a specific target area and is formulated to not spread to other surrounding areas.

[0252] In some embodiments, the FN composition is formulated to be administered to a specific target area and not spread to other surrounding areas.

[0253] In some embodiments, the FN compositions are administered to a specific target area and are 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.

[0254] In some embodiments, the FN composition is administered to the affected area immediately after the wound or injury. In some embodiments, the FN composition 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.

[0255] In some embodiments, the FN composition is administered topically. In some embodiments, the cellular FN composition is administered by subconjunctival injection. In some embodiments, the FN composition exhibits super-potency when administered to a subject in need thereof. In some embodiments, the FN composition is administered topically once, two, three, four, five, and / or up to six times per day. In some embodiments, the FN composition allows for a therapeutic effect with one drop or one administration per day. In some embodiments, one drop is administered one, two, three, four, five, or six times per day. In some embodiments, one drop is administered at intervals of one hour, two hours, three hours, or four hours. In some embodiments, one drop is administered at least once per day for one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, or ten weeks. In some embodiments, one drop is administered at least twice per day for one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, or ten weeks. In some embodiments, one drop is administered at least three times a day for one, two, three, four, five, six, seven, eight, nine, or ten weeks. In some embodiments, one drop is administered at least four times a day for one, two, three, four, five, six, seven, eight, nine, or ten weeks. In some embodiments, one drop is administered at least five times a day for one, two, three, four, five, six, seven, eight, nine, or ten weeks. In some embodiments, one drop is administered at least six times a day for one, two, three, four, five, six, seven, eight, nine, or ten weeks.

[0256] In some embodiments, the FN composition for use in the method of treatment further comprises a low level of VEGF, hi some embodiments, the FN composition for use in the method of treatment further comprises 1 pg / mL to 400 pg / mL of VEGF.

[0257] In some embodiments, the FN compositions for use in the treatment methods have a pH of about 4.7 to about 7.5.

[0258] In some embodiments, the FN compositions for use in the treatment methods are formulated with a buffer system selected from the group consisting of disodium phosphate / monosodium, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and citric acid / disodium phosphate.

[0259] In some embodiments, the FN composition for use in the method of treatment further comprises a tonicity modifying agent, hi some embodiments, the tonicity modifying agent is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin.

[0260] In some embodiments, the FN composition for use in the treatment method further comprises monosodium phosphate / di-sodium phosphate, mannitol, and trehalose, and the composition has a pH of about pH 7.4.

[0261] In some embodiments, the FN composition for use in the method of treatment further comprises a divalent cation. In some embodiments, the divalent cation is Mg2 + , Ca2 + , and Zn2 + is selected from the group consisting of:

[0262] In some embodiments, the FN 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.

[0263] In some embodiments, the FN composition for use in the treatment method further comprises an adhesive.

[0264] In some embodiments, the FN compositions for use in the methods of treatment do not contain one or more components selected from the group consisting of xenobiotic components, phenol red, peptides and biomolecules <3 kDa, antibiotics, protein aggregates >200 nm, cells, non-exosomes / non-extracellular vesicle cellular debris, hormones, and L-glutamine.

[0265] In some embodiments, the FN compositions for use in the methods of treatment comprise anti-angiogenic or anti-scarring factors.

[0266] F. Kit The kit can include a packaged FN composition or a conditioned medium for use in preparing a packaged FN composition as disclosed herein, and instructions for use. Additionally, the kit can include components for mixing to prepare a solution for use in treating the eye, and instructions for mixing and use.

[0267] The container may include at least one vial, well, test tube, flask, bottle, syringe, or other container means, containing the containerized FN composition or conditioned medium for use in preparing the FN composition, optionally appropriately aliquoted. If additional components are provided, the kit may include additional containers into which the components may be placed. Such containers may include injection or blow molded plastic containers into which the desired vials are held. The container and / or kit may include a label with instructions and / or warnings.

[0268] The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, Genbank sequences, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0269] The invention can provide kits comprising a panel of tests and / or assays for characterizing the MSC secretome, the panel comprising at least two characterization assays, the characterization assays being selected from the group consisting of physical component characterization, oxidative stress assays, safety analysis, stability assays, proliferation assays, migration assays, angiogenesis assays, differentiation / scarring assays, inflammation assays, and / or epithelial barrier integrity assays. In some embodiments, the panel of tests and / or assays identifies the MSC secretome as described herein.

[0270] The invention can provide kits comprising a panel of tests and / or assays for determining FN lot-to-lot consistency, the panel comprising one or more characterization assays selected from the group consisting of physical component characterization, oxidative stress assays, safety analyses, stability assays, proliferation assays, migration assays, angiogenesis assays, differentiation / scarring assays, inflammation assays, and / or epithelial barrier integrity assays. In some embodiments, the panel of tests and / or assays identifies a cellular FN composition as described herein. EXAMPLES

[0271] Example 1: Characterization of Fibronectin Identification of FN was performed in MSC secretomes first by mass spectrometry followed by quantification by ELISA. Detection of FN was performed in conditioned medium (i.e., medium harvested directly after removing cells) at concentrations of approximately 0.5-50 ng / mL.

[0272] Results from cell-based in vitro assays showed that: Depletion of FN significantly impaired wound closure (scratch wound assay).

[0273] Addition of MSC secretome promoted HCEC adhesion and spreading, depletion of FN impaired HCEC adhesion and spreading, and exogenous addition of FN promoted HCEC adhesion and spreading.

[0274] Exogenous addition of fibronectin promoted transwell migration of HCECs, whereas depletion of FN impaired transwell migration of HCECs.

[0275] Fibronectin stimulates the migration of human corneal epithelial cells. Exogenous addition of recombinant human fibronectin (1ug / mL) significantly stimulates cell migration through the transwell membrane after 36 hours compared to the negative control (serum-free medium).The bottom side of the transwell migration insert stained with gentian violet is shown (Figure 2).

[0276] Depletion of fibronectin impairs human corneal epithelial cell migration. Fibronectin immunodepleted MSC secretomes exhibit impaired migration compared to intact MSC secretomes in a transwell migration assay. The bottom side of a transwell migration insert stained with gentian violet is shown (Figure 3).

[0277] Depletion of fibronectin impairs in vitro wound closure of human corneal epithelial cells. Fibronectin immunodepleted MSC secretomes impair cell migration into the wound gap compared to intact MSC secretomes in a transwell migration assay: after 24 hours, the closure rate in MSC secretome treated wounds was 30±1.2%, in contrast to 16±1.9% in wounds treated with FN-depleted MSC secretomes (Figure 4).

[0278] Example 2: Fibronectin binds to growth factors Fibronectin was immunodepleted from MSC secretomes using an anti-fibronectin capture antibody and isolated using protein G-coupled magnetic beads. The lysate and bead fractions were assayed for HGF via ELISA. The beads were washed 3 times with PBS, resuspended in 100uL of PBS, and then heated at 80°C for 10 minutes. Samples were then diluted and measured by ELISA using recombinant HGF to generate a standard curve. See Figure 5 for HGF assay results in pg / mL.

[0279] Example 3: MSC secretome contains cellular fibronectin Immunological assays were used to analyze the MSC secretome. Results demonstrated that the MSC secretome contained cellular fibronectin, as supported by the detection of EDA+ and EDB+ fibronectin splicing variants.

[0280] Characterization of fibronectin in the secretome Secretomes isolated from MSCs were assessed by immunoblotting using antibodies specific for the EDA sequence, EDB+fibronectin, and general fibronectin (FN). Both anti-EDA and anti-EDB antibodies cross-reacted with fibronectin, indicating that the species present in the secretome was cellular fibronectin (Figure 6).

[0281] Detection of cellular EDA+fibronectin using a sandwich ELISA An ELISA standard curve for EDA+fibronectin (open circles) was established using recombinant cellular fibronectin and an EDA sequence-specific capture antibody. Anti-EDA antibodies were generated by including the EDA sequence used as an immunogen in the recombinant fibronectin used to generate the standard curve. MSC secretomes were assayed in an EDA sandwich ELISA, and cellular fibronectin was readily detected (blue dots) (Figure 7).

Claims

1. A composition comprising fibronectin (FN).

2. The composition according to claim 1, wherein the FN is FN derived from mesenchymal stem cells (MSCs).

3. The composition according to claim 1, wherein the FN is FN secreted by MSCs.

4. The composition according to claim 1, wherein the FN is cellular FN.

5. The composition according to claim 4, wherein the cellular FN is cell-derived FN, and the FN is non-covalently attached to one or more growth factors.

6. The composition according to claim 4, wherein the cellular FN is EDA+ and / or EDB+.

7. The composition according to claim 1, wherein the FN is obtained from a conditioned medium.

8. The composition according to claim 7, wherein the conditioned medium is obtained from mesenchymal stem cells (MSCs).

9. The composition according to claim 1, wherein the composition comprises an MSC secretome.

10. The composition according to claim 8, wherein the MSCs are derived from bone marrow.

11. The composition according to claim 1, further comprising one or more growth factors selected from the group consisting of FGF (such as FGF-2), PDGF, HGF, VEGF, TGFβ1, TGFβ2, IGF-1, IGF-2, NGF, neurotrophin, and EGF.

12. The composition according to claim 1, wherein the FN is non-covalently attached to one or more growth factors selected from the group consisting of FGF (such as FGF-2), PDGF, HGF, VEGF, TGFβ1, TGFβ2, IGF-1, IGF-2, NGF, neurotrophin, and EGF.

13. The composition according to claim 1, wherein the FN is at a concentration of about 0.5 to 50 ng / mL.

14. The composition according to claim 13, wherein the FN is at a concentration of about 25 ng / mL.

15. The composition according to claim 1, further comprising at least about 0.1 ng / mL of PDGF.

16. The composition according to claim 1, further comprising about 0.3 to 4.5 ng / mL of HGF.

17. The composition according to claim 1, further comprising about 1 pg / mL to 400 pg / mL of VEGF.

18. The composition according to claim 1, further comprising a tension modifier.

19. The composition according to claim 18, wherein the tensile modifier is selected from the group consisting of NaCl, KCl, mannitol, dextrose, sucrose, sorbitol, and glycerin.

20. The composition according to claim 1, wherein the composition contains 0.5 to 50 ng / ml of FN, 2.28 mg / ml of sodium monophosphate, 10 to 12 mg / ml of disodium phosphate, 11 to 13 mg / ml of mannitol, 2 to 25 mg / ml of trehalose dihydrate, and 0.5 to 2 mg / ml of hypromellose.

21. The composition according to claim 1, wherein the composition contains 0.5 to 50 ng / ml of FN, 2.28 mg / ml of sodium monophosphate, 11.45 mg / ml of disodium phosphate, 12.2 mg / ml of mannitol, 24 mg / ml of trehalose dihydrate, and 1 mg / ml of hypromellose.

22. The composition according to claim 1, wherein the composition contains 0.5 to 50 ng / ml of FN, 1.31 mg / ml of sodium monophosphate, 4.5 to 7 mg / ml of disodium phosphate, 5.5 to 7.5 mg / ml of mannitol, 11 to 13 mg / ml of trehalose dihydrate, and 0.1 to 1.5 mg / ml of hypromellose.

23. The composition according to claim 1, wherein the composition contains 0.5 to 50 ng / ml of FN, 1.31 mg / ml of sodium monophosphate, 5.73 mg / ml of disodium phosphate, 6.1 mg / ml of mannitol, 12 mg / ml of trehalose dihydrate, and 0.5 mg / ml of hypromellose.

24. The composition does not contain NaCl and / or MgCl ２ The composition according to claim 20, which does not contain

25. A pharmaceutical composition comprising the composition according to any one of claims 1 to 24 for the treatment of eye conditions.

26. The eye condition is selected from the group consisting of retinopathy, chronic graft-versus-host disease (GvHD), Stevens-Johnson syndrome, ocular mucous membrane pemphigoid, persistent corneal epithelial defect (PCED), dry eye, optic nerve tissue damage, and impact injury to the eye (such as impact injury, eye contusion, or chemical burn), the pharmaceutical composition according to claim 25.

27. Use of the composition according to any one of claims 1 to 24 for preparing a medicament for the treatment of eye conditions.

28. A method for preparing the composition according to any one of claims 1 to 24, comprising (a) Culturing stem cells in a cell culture medium, thereby generating a conditioned medium containing factors secreted by the stem cells; (b) Harvesting the conditioned medium, thereby obtaining a harvested conditioned medium; (c) Filtering the harvested conditioned medium to obtain a processed conditioned medium; The method comprising the above. **Claim 29** The method according to claim 28, further comprising concentrating the processed conditioned medium. **Claim 30** The method according to claim 28, wherein the processed conditioned medium is further subjected to buffer exchange with a formulation buffer. **Claim 31** The method according to claim 30, wherein the formulation buffer comprises one or more of disodium hydrogen phosphate / sodium dihydrogen phosphate, sodium citrate / citric acid, boric acid / sodium citrate, boric acid / sodium tetraborate, and citric acid / disodium hydrogen phosphate. **Claim 32** The method according to claim 28, wherein the stem cells are mesenchymal stem cells (MSCs). **Claim 33** The method according to claim 28, wherein the cell culture medium is serum-free. **Claim 34** Before step (a), the method further comprises: (i) Culturing the stem cells in a growth medium; (ii) Exchanging the growth medium with the cell culture medium of step (a). The method according to claim 28, comprising the above.

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