Method for scalable production of therapeutic exosomes

Abstract

The present disclosure broadly relates to therapeutic exosomes and methods for production of exosomes. In particular, the present disclosure provides a method for large-scale production of exosomes for therapeutic application.

Description

METHOD FOR SCALABLE PRODUCTION OF THERAPEUTICEXOSOMESCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to Indian Provisional Patent Application 202441042610 filed on May 31, 2024, the content of which is incorporated herein by reference in its entirety for all purposes.FIELD OF INVENTION

[0002] The present disclosure broadly relates to therapeutic exosomes and methods for production of exosomes. In particular, the present disclosure provides a method for large-scale production of exosomes for therapeutic application.BACKGROUND

[0003] Exosomes, a specific subclass of the extracellular vesicles secreted by most cell types, play an important role in cell-cell communication by transporting diverse content or cargo molecules including protein, mRNA, miRNA, and DNA. The cargo molecules are responsible for the anti-fibrotic, anti-inflammatory and pro-regeneration therapeutic effects of exosomes in humans. Unfortunately, the utility of exosomes in both laboratory research and clinical diagnoses has been limited by absence of techniques to isolate, characterize and analyze the exosomes showing best regenerative potential.

[0004] Many scientists have therefore put efforts into improving exosome-detection technologies, especially in the combination of traditional biosensors and nano-based chip techniques. However, the establishment of these new techniques have been limited by various factors such as time, cost, sensitivity, and other conditions. Thus, the conventional methods that are available in the art do not allow the scale-up production of MSCs and its products for therapeutic applications. Moreover, relying on the existing conventional assays to test the functional efficacy of the number of exosomes, it becomes difficult to manually correlate the experimental data and functional efficacy of the exosome under clinical studies, and to draw a conclusion out of it.

[0005] Thus, there is a need in the art to provide a novel method that relies on scalable production of clinical grade therapeutic exosomes in identification of the target exosome variant with best regenerative potential at pre-clinical or clinical stage.SUMMARY

[0006] In an aspect of the present disclosure, there is provided a method for producing exosomes, comprising: (a) culturing mesenchymal stem cells (MSCs) in a collection medium, for a period of 2- 3 days, and filtering to obtain a conditioned medium of said MSCs; (b) concentrating the conditioned medium using Tangential flow filtration (TFF) to obtain a concentrated secretome, wherein the conditioned medium is concentrated to a volume in the range of 1 / 10 to 1 / 100, preferably 1 / 20; and known as D-Exo A (Designer Exosomes) (c) purifying by ultracentrifuging the concentrated secretome at 110,000 x g to 130,000 x g, preferably 100,000 x g, for a period of 2-4 hours, preferably 4 hours, at a temperature of 2-8°C, preferably 4°C, to obtain a concentrated purified secretome known as D / K-Exo B (D-Exo B); and (d)subjecting the concentrated purified secretome to density gradient centrifugation at 90,000 x g to 110,000 x g, preferably at 100,000 x g, for a period of 4-18 hours, preferably 8 hours, at a temperature of 2-8°C, preferably 4°C, to a fraction comprising the exosomes (K- Exo A), Kuragenx Exosomes.

[0007] In another aspect of the present disclosure, there is provided a method for producing exosomes, comprising: (a) culturing mesenchymal stem cells (MSCs) in a collection medium, for a period of 2- 3 days, and filtering to obtain a conditioned medium of said MSCs; (b) concentrating the conditioned medium using Tangential flow filtration (TFF) to obtain a concentrated secretome, wherein the conditioned medium is concentrated to a volume in the range of 1 / 10 to 1 / 100, preferably 1 / 20; and (c) purifying by subjecting the concentrated secretome to density gradient centrifugation at 90,000 x g to 110,000 x g, preferably 100,000 x g, for a period of 4 - 18 hours, preferably 8 hours, at a temperature of 2-8°C, preferably 4°C, to a fraction comprising the exosomes.

[0008] In another aspect of the present disclosure, there is provided a population of exosomes obtained by the method disclosed herein.

[0009] In another aspect of the present disclosure, there is provided a composition comprising the exosomes obtained by the method disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be betterunderstood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0011] FIG. 1 depicts a schematic representation of a method for producing the exosomes using P3 MSCs, according to embodiments herein.

[0012] FIG. 2 is a schematic representation depicting certain exosomal variants, according to embodiments herein.

[0013] FIGS. 3A-3C depict data for hBMMSCs and iMSCs, wherein FIG. 3 A depicts a comparison of cell morphology, FIG. 3B depicts growth kinetics for hBMMSCs, and FIG. 3C depicts growth kinetics for iMSCs, in which the iMSC showed spindle-like morphology at day 5, higher growth, and produced more exosomes, according to embodiments herein.

[0014] FIG. 4 depicts results of Immunofluorescence staining for positive sternness markers (CD73, CD90, and CD105) and negative marker (CD34), and stress marker (a-SMA) at Day 6 of culture for hBMMSCs (upper row) and iMSCs (lower row), according to embodiments herein.

[0015] FIG. 5 shows a flowchart schematically illustrating an embodiment of the exosome production method of the disclosure.

[0016] FIG. 6 shows an illustration of the relationship between D-Exo A, D / K-Exo B, and K-Exo A.

[0017] FIG. 7A shows representative phase contrast microscopic images (Scale bar: 100 pm; Magnification: 10X) showing dynamic plasticity of hBMMSCs between unprimed and DMF- primed conditions.

[0018] FIG. 7B showing representative phase contrast microscopic images of unprimed and primed hBMMSCs at Day 5.

[0019] FIG. 7C shows a plot tracking cell density in each of Day 1 to Day 7 for both unprimed (naive) and DMF-primed hBMMSCs.

[0020] FIG. 7D shows a plot tracking generation number in each of Day 1 to Day 7 for both unprimed (naive) and DMF-primed hBMMSCs.

[0021] FIGS. 8A and 8B, show results in an immunoblot analysis confirming expression of tetraspanin markers CD9, CD63, and CD81 in D-Exo A, D / K-Exo B, and K-Exo A samples.

[0022] FIGS. 9, 10 A, and 10B shows quantification of percentage wound closure over time, with administration of D-Exo A, D / K-Exo B, and K-Exo A.

[0023] FIGS. 11A-11B and 12A-12B show quantification of aSMA based on fluorescent image acquisition, with administration of D-Exo A, D / K-Exo B, and K-Exo

[0024] FIGS. 13A-13C and 14A-14C, which shows the increase on inflammatory or anti-inflammatory markers in THP-1 cells based on administration of D-Exo A, D / K- Exo B, and K-Exo A.

[0025] FIGS. 15A-15C show phase contrast images of HUVECs with a positive control or a primed K-Exo A.

[0026] FIGS. 15D-15F show phase contrast images of SH-SY5Y (Human neuroblastoma) cell line with a positive control or a primed K-Exo A

[0027] FIG. 15G is a bar graph summarizing the results suggested in the figured of FIGS 15A-15F

[0028] FIG. 16A-16D shows fluorescent images of HUVEC cells with phalloidin staining.

[0029] FIG. 16E-16G shows chart showing the effect of VEGF, sFLT-1, and K-Exo A in morphological aspects HUVEC growth.DETAILED DESCRIPTION

[0030] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.Definitions

[0031] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0032] The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0033] The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.

[0034] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps.

[0035] The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.

[0036] The term “exosomes”, as used herein, refers to membranous vesicles secreted by cells. The term “extracellular vesicles” (EVs) and “exosomes” are used interchangeably herein. Typically, exosomes comprise a plasma membrane enclosing an internal space, and are of a size ranging from 30 to 200 nm. In an embodiment of the present disclosure, the exosomes are therapeutically potent having a size in the range of 30 to 200 nm, 30 to 150 nm, or 100 to 150 nm. Typically, exosomes carry cargo molecules including proteins, mRNA, miRNA and DNA. Depending on the type of cell and stimuli, the features of the exosomes and cargo molecules may vary. Exosomes variants, according to embodiments herein, may be unprimed or primed exosome variants produced by unprimed or primed stem cells, respectively. Exosomes, according to embodiments herein, may be variants produced by non-immortalized or immortalized stem cells. Accordingly, the term “primed exosomes”, as used herein, refers to exosomes produced by primed cells. The term “unprimed exosomes”, as used herein, refers to exosomes produced by unprimed cells.

[0037] The term “primed cell” or “primed stem cells”, as used interchangeable herein, refers to stem cells that have been cultured in the presence of one or more stimuli (also referred to herein as priming agent). The term “unprimed cells” or “unprimed stem cells”, as used interchangeable herein, refers to stem cells that have been cultured in the absence of the stimuli or priming agents. Accordingly, the term “primed MSCs” refer to MSCs that have been cultured in the presence of any stimuli or priming agents. The term “unprimed MSCs” refer to MSCs that have been cultured in the absence of the stimuli or priming agents. Similar terms may also be used in respect of NPC and iPSC, for example: “unprimed NPCs” and “unprimed iPSCs”, respectively.

[0038] The term “priming agent”, as used herein, refers to a stimulant provided to growing stem cells. Stem cells primed with the priming agent have been observed to display enhanced regenerative, sternness, anti-inflammatory, and anti-fibrotic properties. The priming agents, according to embodiments herein, are selected from a chemical substance, secretome of unprimed cells, hypoxia, or a combination thereof.

[0039] Embodiments herein provide a method for producing exosomes. The method, according to embodiments herein, is scalable and time efficient. The method is highly productive, consistent and reproducible in producing exosomes of consistent size and markers, at higher yields. The method, according to embodiments herein, is suitable for large scale production of exosomes. Further, embodiments of the method as disclosed herein achieve primed and unprimed variants of exosomes, wherein the primed variants may further include single primed and combinatorial primed variants of exosomes. The method, as disclosed herein, achieves large-scale production of exosomes variants of therapeutic clinical grade. The method, as disclosed herein, achieves a population of tissue specific primed exosomes. The primed exosomes achieved by the method, according to the present disclosure, exhibit higher levels of pro-regenerative, anti- fibrotic and anti-inflammatory, re-innervation, and vascular pro-angiogenic factors, compared to exosomes from naive cells. In that, the primed exosomes carry higher levels of Hepatocyte growth factor (HGF), Nerve growth factor (NGF), and vascular endothelial factor (VEGF), as compared to unprimed exosomes, i.e. exosomes secreted by unprimed cells. Embodiments herein provide a composition comprising one or more exosome variants.Method

[0040] Embodiments herein provide a method for producing exosomes. The method, according to embodiments herein, comprises: (a) culturing mesenchymal stem cells (MSCs) in a collection medium to obtain a conditioned medium (CM) of the MSCs; (b) concentrating the conditioned medium using Tangential flow filtration (TFF) to obtain a concentrated secretome; and (c) purifying the concentrated secretome by one of or a combination of two more of the following purification processes: filtration (e.g. with a 0.22 pm pore filter unit), pelleting ultracentrifugation, and density gradient centrifugation. In some embodiments, purifying the concentrated secretome comprises processing the concentrated secretome by pelleting ultracentrifugation followed by density gradient centrifugation. FIG. 5 shows a flowchart 100 schematically illustratingan embodiment of the exosome production method of the disclosure. Various portion of the flowchart 100 will be referred to herein below to illustrate, by way of examples, aspects of exosome production methods of the disclosure.Culturing MCSs

[0041] The method, according to embodiments herein, includes a step of culturing mesenchymal stem cells (MSCs) to obtain a conditioned medium (CM) of the MSCs (block 110). In certain embodiments, the culturing of MSCs is performed in a collection medium for a growth period of 2-3 days. In certain embodiments, the culturing is performed at 37°C and exposure to 5% CO2. Collection medium, according to the present disclosure, refers to any suitable medium for collection of extracellular vesicles (EV), also referred to herein as EV collection medium. Various collection media are known and may be used in embodiments herein. Examples of collection media includes, but are not limited to, RoosterCollect™-EV, Oxium TMExo, and MSC NutriStem® XF Medium. In a particular embodiment, the collection medium is RoosterCollect™EV.Centrifugation

[0042] In certain embodiments, the CM after the growth period and collected is processed through centrifugation, with the pellet (comprising cell debris and other undesirable matter) being discarded and the supernatant (comprising, e.g., EVs and soluble protein) being kept to obtain a clarified CM 130. In certain embodiments, the centrifugation is a serial centrifugation phase 120 comprising two or more centrifugation steps, for example 2, 3, 4, or 5 centrifugation steps (e.g., blocks 121, 123 and 125 as shown in FIG. 5) to produce the clarified CM 130. Each centrifugation step may have a spin strength of between about 500 x g and about 15000 x g, with the pellet being discarded and the supernatant being kept at each step. In certain embodiments, the centrifugation steps may be characterized by increasing spin strength, so that increasingly smaller debris are removed at each step. The duration of each centrifugation step may be between 10 minutes (min) and 60 min, for example about 15 min, about 20 min, about 30 min, about 40 min, about 45 min, about 50 min, and about 60 min. In a particular embodiment, for example as schematically illustrated in FIG. 5, the serial centrifugation comprises three centrifugation steps: 750 x g optionally for 15 min (block 121), 2000 x g optionally for 15 min (block 123), and 10,000 x g optionally for 45 min (block 125). In certain embodiments, steps of the centrifugationphase 120 are performed at refrigerated temperatures, e.g. between about 2°C and about 8°C.Tangential Flow Filtration (TFF)

[0043] Tangential Flow Filtration (TFF), also known as crossflow filtration, is an ultrafiltration technique to separate and purify biomolecules such as proteins and nucleic acids, cellular components such as organelles or EVs, or cells. Unlike traditional dead-end filtration, where the fluid flows directly into the membrane, TFF involves the feed solution flowing tangentially across the surface of the membrane. This design minimizes membrane clogging and allows for continuous operation. During the process, smaller molecules and solvents pass through the membrane as permeate, while larger molecules are retained and either recirculated or collected as retentate. TFF systems typically include a membrane module, pump, reservoir, and flow control components. The method is valued for its efficiency, scalability, and gentle handling of sensitive biomolecules and cellular components, making it advantageous for applications such as exosome concentration.

[0044] In certain embodiments, the clarified CM of MSCs are processed to concentrate the exosomes using TFF (block 140) to produce a concentrated secretome. Concentration of the conditioned media using TFF to create the concentrated secretome, according to the present disclosure, may be performed to reduce the volume of the clarified CM to between about 1 / 10 and about 1 / 100 of the initial volume, between about 1 / 10 and about 1 / 75 of the initial volume, between about 1 / 10 and about 1 / 50 of the initial volume, between about 1 / 10 and about 1 / 25 of the initial volume, between about 1 / 15 and about 1 / 100 of the initial volume, between about 1 / 15 and about 1 / 75 of the initial volume, between about 1 / 15 and about 1 / 50 of the initial volume, between about 1 / 15 and about 1 / 25 of the initial volume, about 1 / 10 of the initial volume, about 1 / 15 of the initial volume, about 1 / 20 of the initial volume, about 1 / 25 of the initial volume, about 1 / 30 of the initial volume, about 1 / 40 of the initial volume, about 1 / 50 of the initial volume, about 1 / 60 of the initial volume, about 1 / 70 of the initial volume, about 1 / 75 of the initial volume, about 1 / 80 of the initial volume, about 1 / 90 of the initial volume, or about 1 / 100 of the initial volume. In an embodiment, the conditioned medium is concentrated to a volume of 1 / 20 of its initial volume. As note above, TFF is an ultrafiltration method used to filter biomolecules using ultrafiltration membranes. The TFF system of the present disclosure may comprise membrane filtersthat may be, but are not limited to, polyether sulfone (PES) membrane filters, modified polyether sulfone (mPES) membrane filters, poly sulfone (PS) membrane filters, and mixed cellulose ester (ME) membrane filters. In certain embedments, the membrane filter may be a hollow fiber filter or a cassette filter. In certain embodiments, the membrane filters may have a pore size in the range of between about 0.5 pm and about 0.8 pm, between about 0.4 pm and about 0.9 pm, between about 0.4 pm and about 0.7 pm, and between about 0.6 pm and about 0.9 pm, about 0.4 pm, about 0.5 pm, about 0.6 pm, about 0.7 pm, about 0.8 pm, or about 0.9 pm. In certain embodiments, the membrane filters may have a molecular weight cut-off in the range of between about 100 kDa and about 750 kDa, between about 80 kDa and about 800 kDa, between about 150 kDa and about 750 kDa, between about 200 kDa and about 750 kDa, between about 500 kDa and about 750 kDa, between about 80 kDa and about 400 kDa, between about 80 kDa and about 500 kDa, between about 100 kDa and about 600 kDa, between about 200 kDa and about 500 kDa, about 80 kDa, about 100 kDa, about 150 kDa, about 200 kDa, about 300 kDa, about 400 kDa, about 500 kDa, about 600 kDa, about 700 kDa, about 750 kDa, and about 800 kDa. In a particular embodiment, the clarified CM is concentrated using a TFF system comprising modified polyether sulfones (mPES) hollow fiber filter having 0.5 pm pore size and a 100 kDa molecular weight cut-off.

[0045] In certain embodiments, membrane filters may be washed with sterile PBS (pH 7.4), prior to its use in filtration. In certain embodiments, the input flow rate may be maintained at order keep the sheer force of the feed stream sufficiently low to reduce damage to cells or cellular components such as exosomes, e.g., below about 2000 s-1. In certain embodiments, the flow rate may be kept at between about 80 mL / min and about 100 mL / min, between about 80 mL / min and about 120 mL / min, between about 80 mL / min and about 120 mL / min, between about 70 mL / min and about 90 mL / min, between about 70 mL / min and about 100 mL / min, between about 70 mL / min and about HOmL / min, between about 70 mL / min and about 120 mL / min, between about 90 mL / min and about 100 mL / min, between about 90 mL / min and about HOmL / min, between about 90 mL / min and about 120mL / min. In a particular embodiment, the flow rate may be kept at between about 80 mL / min and about 100 mL / min.

[0046] In certain embodiments, following the concentration using TFF, the concentrated secretome may be diafiltrated (block 140). Diafiltration is a specialized form of TFF used to remove small molecules, salts, or solvents from a solution while retaining, e.g., exosomes or larger molecules such as proteins or nucleic acids. Indiafiltration, a retentate in the TFF system following TFF concentration (e.g. in the present invention the concentrated secretome) receives one or more rounds of fresh buffer or solvent while simultaneously removing permeate through the membrane filters. This process gradually washes out unwanted salts and small molecules, effectively exchanging the solution around the target molecules or cell components such as EVs without losing them. In certain embodiments, the diafiltration is repeated about 4-6 times, preferably 6 times. In certain embodiments, the buffer is phosphate buffered saline (PBS, for example: IX PBS).

[0047] In certain embodiments, a concentrated secretome 210 produced by the TFF system is provided as is, optionally sterilized with filtration through a 0.22 pm pore filter (block 151) in a Process A as schematically shown in FIG. 5 to produce a sterilized concentrated secretome (e.g. D-Exo A as described hereinbelow).

[0048] In certain embodiments, the concentrated secretome may be further processed through ultracentrifugation, as further described herein below, and shown by way of example as Process B and Process A in FIG. 5.Ultracentrifugation

[0049] Embodiments of the method, according to the present disclosure, includes a step of purifying the concentrated secretome to remove macromolecules such as nucleic acids and proteins through ultracentrifugation, to obtain a pellet or fraction enriched in exosomes. In certain embodiments, the ultracentrifugation may be a pelleting ultracentrifugation (in which a pellet that is formed, typically in the bottom of an ultracentrifugation tube, is kept and the supernatant is discarded) and / or a densitygradient ultracentrifugation (in which components are separated based on their buoyant density within a gradient medium, and specific fractions are collected, typically without forming a discrete pellet at the bottom).

[0050] Pelleting ultracentrifugation is a technique used to spin samples at high speed, for example, > 50,000 rpm, for high g force separations to form an exosome-enriched pellet. The method, according to embodiments herein, uses high g force in the range of 80,000 x g 150,000 x g. The time duration and temperature of pelleting ultracentrifugation may be in the range of 2-4 hours and 2-8°C, respectively. In certain embodiments, the method comprises purifying by pelleting ultracentrifuging the concentrated secretome at 110,000 x g to 130,000 x g, preferably 100,000 x g, for a period of 2-4 hours, preferably 4 hours, at a temperature of 2-8°C, preferably 4°C. Incertain embodiments, the pelleting ultracentrifugation step is performed after the TFF with the concentrated secretome and before density gradient centrifugation.

[0051] The density gradient centrifugation, according to embodiments herein, may be performed using sucrose gradient or iodixanol gradient. The iodixanol (IDX) gradient may be prepared by floating about 3 mL of 10% w / v IDX solution containing NaCl (150 mM) and 25 mM Tris:HCl (pH 7.4) over 3 ml of 55% w / v IDX solution. For sucrose gradient, 30% sucrose (IM) containing ultracentrifuge tube may be used. Centrifugation, in sucrose gradient or iodixanol gradient, may be performed at g force in the range of 90,000 x g to 110,000 x g for a period of 4-18 hours at a temperature of 2-8°C to obtain the fraction comprising exosomes. In an embodiment, the centrifugation in said density gradient centrifugation is performed at 100,000 x g for a period of 8 hours at a temperature of 4°C, to obtain the fraction comprising of purified exosomes. In an embodiment, the density gradient centrifugation is performed using iodixanol gradient. In another embodiment, the density gradient centrifugation is performed using sucrose gradient.

[0052] Once a density gradient centrifugation is completed, different constituents in concentrated secretome becomes situated at different positions within the gradient solution based on the respective densities of the components, and a desired subset of the constituents may be selected by obtaining a desired layer of gradient solution along the length of the centrifuge tube, which may be referred to as a “fraction”. Because of the density gradient, different fractions may be characterized by a density range. A fraction that is enriched in exosomes, or includes a desired subset of exosomes, may be referred as a “purified exosome fraction”.

[0053] In certain embodiments, the purified exosome fraction is characterized by a density range of between about 1.13g / mL and about 1.18g / mL, between about 1.13g / mL and about 1.19g / mL, between about 1.13g / mL and about 1 ,20g / mL, between about 1.12g / mL and about 1.18g / mL, between about 1.12g / mL and about 1.19g / mL, between about 1.12g / mL and about 1.20g / mL, or between about 1.13g / mL and about 1.18g / mL, in the iodixanol gradient or sucroce gradient. In a particular embodiment, the purified exosome fraction is characterized by a density range of between about 1.13g / mL and about 1.18g / mL in an iodixanol gradient.

[0054] Post-centrifugation in an iodixanol gradient, the gradient solution the ultracentrifugation tube is typically fractionated into twelve 1 mL fractions collected from the top to the bottom of the tube. The fractions are designated as “fraction 1”starting from the top of the tube through “fraction 12” at the bottom of the tube, so that, for example, “fraction 7” refers to the 1 mL fraction that is seventh from the top of the tube, and “fraction 9” refers to the 1 mL fraction that is ningth from the top of the tube. As such, different subset of constituents within the concentrated secretome may be obtained by obtaining one or more of a desired numbered fraction, e.g., selected from fractions 1 through 12.

[0055] In certain embodiments, a purified exosome fraction comprising desired exosomes obtained by the method of the disclosure, is fraction 8 (i.e., the 9thfraction from the top of the tube out of 12 fractions), fraction 9, or fraction 10, of an iodixanol gradient. In a particular embodiment, the purified exosome fraction is faction 9 of the iodixanol gradient

[0056] In certain embodiments, the concentrated secretome produced by TFF 140 may be further processed through a pelleting ultracentrifugation 161 (and optionally sterilized with a filtration step 163 through a 0.22 pm pore filter) to produce a purified exosomes sample 220 (e.g. D / K-Exo B as described hereinbelow), which is schematically shown as Process B in FIG. 5. In certain embodiments, the concentrated secretome may be further processed through a pelleting ultracentrifugation 171 followed by a density gradient centrifugation 173 (and optionally sterilized with a filtration step 175 through a 0.22 pm pore filter) to produce a purified exosomes fraction 230 (e.g. D / K-Exo B as described hereinbelow), which is schematically shown as Process C in FIG. 5.

[0057] The purified exosome fraction may further be subjected to washing. In certain embodiments, the purified exosome fraction may be is transferred into a fresh ultracentrifuge tube and washed with ice-cold 5mM sodium phosphate buffer (optionally containing 2.5mM Trehalose) and washed by centrifuging at about 100,000 x g for 4 hours at 4°C to remove the residual iodixanol or sucrose from the fractions. Accordingly, in a particular embodiment, the method comprises washing the purified exosome fraction by centrifuging at 90,000 x g to 110,000 x g, preferably 100,000 x g, for 2-6 hours, preferably 4 hours, at 2-8 °C, preferably 4 °C, to obtain purified exosomes as pellet. The pellet obtained may then be re-suspended in ice-cold 5mM sodium phosphate buffer containing 2.5mM Trehalose and stored at about -80°C.

[0058] In a particular embodiment, the purifying step of the method comprises washing said fraction by centrifuging at 100,000 x g for 4 hours at 4 °C, to obtain purified exosomes.

[0059] In a particular embodiment, the method for producing exosomes comprises: a) culturing mesenchymal stem cells (MSCs) in a collection medium, for a period of 2- 3 days, and filtering to obtain a conditioned medium of said MSCs; b) concentrating the conditioned medium using Tangential flow filtration (TFF) to obtain a concentrated secretome, wherein the conditioned medium is concentrated to a volume in the range of 1 / 10 to 1 / 100, preferably 1 / 20; c) purifying by subjecting the concentrated secretome to a pelleting ultracentrifugation at 110,000 x g to 130,000 x g, preferably 100,000 x g, for a period of 2-4 hours, preferably 4 hours, at a temperature of 2-8°C, preferably 4°C, to obtain a purified secretome; and subjecting the purified secretome to a density gradient centrifugation at 90,000 x g to 110,000 x g, preferably at 100,000 x g, for a period of 4-18 hours, preferably 8 hours, at a temperature of 2-8°C, preferably 4°C, to isolate a fraction comprising the purified exosomes.

[0060] In another embodiment of the present disclosure, the method for producing exosomes comprises: a) culturing mesenchymal stem cells (MSCs) in a collection medium, for a period of 2- 3 days, and filtering to obtain a conditioned medium of said MSCs; b) concentrating the conditioned medium using Tangential flow filtration (TFF) to obtain a concentrated secretome, wherein the conditioned medium is concentrated to a volume in the range of 1 / 10 to 1 / 100, preferably 1 / 20; and c) purifying by subjecting the concentrated secretome to density gradient centrifugation at 90,000 x g to 110,000 x g, preferably 100,000 x g, for a period of 4-18 hours, preferably 8 hours, at a temperature of 2-8°C, preferably 4°C, to isolate a purified exosome fraction comprising the exosomes.Priming MSCs

[0061] In an embodiment, the MSCs are primed MSCs. The primed cells, according to embodiments herein, may be obtained by culturing unprimed MSCs for a period of between about 20 and about 168 hours in the presence of at least one priming agent. In certain embodiments, the primed or unprimed MSCs may be immortalized.

[0062] It is known that the profile of cargo molecules achieved by primed MSCs and unprimed MSCs vary, and is dependent on the priming agent used. As shown below, and as observed by the present inventors, exosomes from primed MSCs of the disclosure show higher levels of pro-regenerative, anti-fibrotic and anti-inflammatory, re-innervation, and vascular pro-angiogenic factors, compared to exosomes fromunprimed cells. Further, and advantageously the exosomes produced by the method, according to embodiments herein, show elevated cargo levels.

[0063] In an embodiment, the MSCs are primed cell obtained by a method comprising i. expanding unprimed cells in an expansion medium for a period of 3 to 5 days to obtain expanded unprimed cell; ii. passaging a first portion of the expanded unprimed cells to a growth medium and culturing for a period of 2-3 days to obtain secretome of unprimed cells; and ii. culturing a second portion of the expanded unprimed cells in a priming medium for a period of 20 to 168 hours in the presence of at least one priming agent to obtain the primed cells, and washing the primed cells to remove the priming agent.

[0064] Expansion of unprimed cells, according to the present disclosure, may be performed by culturing unprimed cells in an expansion medium to obtain an expanded population of unprimed cells. The term “expand”, “expanding” or “expanded”, as used herein, refers to an increase in the number of cells in a population as compared to the number of cells in the initial population. For example: expansion may be 10, 15, 20, 50, 100, 150, 200, or 250-fold expansion as compared to the initial population of cells. Accordingly, an expansion of 10-folds is wherein about 1 million cells are expanded to about 10 million cells after culturing the cells in the expansion medium for a period of 4-5 days. Expansion of cells may be performed for a period of 1 to 6 days. In an embodiment, the expansion is performed for a period of 3 to 5 days, preferably 4 days. In an embodiment, the cells exhibit an expansion of 10 to 15 folds as compared to the initial population.

[0065] Expansion medium, according to the present disclosure, refers to any suitable medium capable of expanding stem cells. In an embodiment, the expansion medium is a xeno-free media formulated for growth and expansion of mesenchymal stem cells (MSCs). Various stem cell expansion media are known and may be used in embodiments herein. Examples of expansion media include, but are not limited to, RoosterNourish media, Cellartis® MSC Xeno-Free Culture Medium (Takara Biosciences), MSC NutriStem® XF Medium (by Sartorius), StemPro MSC SFM XenoFree (by Thermofisher), and StemXVivo Xeno-Free Human MSC Expansion Media (by R&D systems).

[0066] In an embodiment of the method, the expanded unprimed cells are cultured in a priming medium for a period of 20 to 168 hours in the presence of at least one priming agent to obtain primed cells. In another embodiment, the population of expanded unprimed cells is apportioned into first portion and second portion, wherein the firstportion of expanded unprimed cells is passaged and cultured in a growth medium, for a period of 2-3 days, to obtain secretome of unprimed cells. This secretome of unprimed cells thus obtained may further be used as a priming agent in priming of MSCs according to embodiments herein. The growth medium, according to embodiments herein, may be any suitable media composition used to culture and allow growth of MSCs. Examples of growth media includes, but is not limited to, RoosterNourish media, Cellartis® MSC Xeno-Free Culture Medium (Takara Biosciences), MSC NutriStem® XF Medium (by Sartorius), StemPro MSC SFM XenoFree (by Thermofisher), and StemXVivo Xeno-Free Human MSC Expansion Media (by R&D systems). Further, the second portion of expanded unprimed cells may be cultured in a priming medium for a period of 20 to 168 hours in the presence of at least one priming agent to obtain primed cells. The primed cells are then subjected to washing to remove the priming agent before culturing in the collection medium as described herein above.

[0067] Accordingly, in an embodiment, the method for producing exosomes comprises (a) culturing mesenchymal stem cells (MSCs) in a collection medium to obtain a conditioned medium of the MSCs, wherein the MSCs are primed MSCs; (b) concentrating the conditioned medium using Tangential flow filtration (TFF) to obtain a concentrated secretome; and (c) purifying by ultracentrifuging the concentrated secretome to obtain a purified secretome, and subjecting the purified secretome to density gradient centrifugation to obtain the exosomes. In another embodiment, the method for producing exosomes comprises (a) culturing mesenchymal stem cells (MSCs) in a collection medium to obtain a conditioned medium of the MSCs, wherein the MSCs are primed MSCs; (b) concentrating the conditioned medium using Tangential flow filtration (TFF) to obtain a concentrated secretome; and (c) purifying by subjecting the purified secretome to density gradient centrifugation to obtain the exosomes.

[0068] The conditioned media of the MSCs, according to embodiments herein, may be characterized by a marker selected from Nrf2, HIF-la, VEGF, sFLTl, IL- 10, SIRT1, COX-2, HIF-1, CXCR4, CCR2, VEGF, Angiopoietin, HGF, IDO, NGF, BDNF, SDFla, or combinations thereof.

[0069] Accordingly, in an embodiment, the method as disclosed herein comprises subjecting the conditioned medium of the MSCs to centrifugation to collect supernatant, wherein centrifugation is performed, e.g., at 750 x g for 15 minutes at 4 °C; 2,000 x g for 15 min at 4°C; and 10,000 x g for 45 min at 4°C.

[0070] FIG. 1 depicts a schematic representation of the method for producing the exosomes using P3 MSCs, according to embodiments herein.

[0071] In an embodiment, the MSCs are primed cells obtained by a method comprising i. expanding unprimed cells in an expansion medium for a period of 3 to 5 days to obtain expanded unprimed cell; ii. passaging a first portion of the expanded unprimed cells to a growth medium and culturing for a period of 2-3 days to obtain secretome of unprimed cells; and ii. culturing a second portion of the expanded unprimed cells in a priming medium for a period of 20 to 168 hours in the presence of at least one priming agent to obtain the primed cells, and washing the primed cells to remove the priming agent.

[0072] In another embodiment of the present disclosure, a method for producing exosomes comprises a) expanding unprimed MSCs, non-immortalized or immortalized, in an expansion medium for a period of 3 to 5 days to obtain expanded unprimed MSCs; b) passaging a first portion of the expanded unprimed MSCs to a growth medium and culturing for a period of 2-3 days to obtain secretome of unprimed MSCs; c) culturing a second portion of the expanded unprimed MSCs in a priming medium for a period of 20 to 168 hours in the presence of at least one priming agent to obtain the primed unprimed MSCs, and washing the primed cells to remove the priming agent; d) culturing the primed MSCs in a collection medium, for a period of 2- 3 days, and filtering to obtain a conditioned medium of said primed MSCs; e) concentrating the conditioned medium using Tangential flow filtration (TFF) to obtain a concentrated secretome, wherein the conditioned medium is concentrated to a volume in the range of 1 / 10 to 1 / 100, preferably 1 / 20; and f) purifying by ultracentrifuging the concentrated secretome at 110,000 x g to 130,000 x g, preferably 120,000 x g, for a period of 2-4 hours, preferably 3 hours, at a temperature of 2-8°C, preferably 4°C, to obtain a purified secretome; and subjecting the purified secretome to density gradient centrifugation at 90,000 x g to 110,000 x g, preferably at 100,000 x g, for a period of 4-18 hours, preferably 8 hours, at a temperature of 2-8°C, preferably 4°C, to a fraction comprising the exosomes.

[0073] In another embodiment of the present disclosure, a method for producing exosomes comprises a) expanding unprimed MSCs, non-immortalized or immortalized, in an expansion medium for a period of 3 to 5 days to obtain expanded unprimed MSCs; b) passaging a first portion of the expanded unprimed MSCs to a growth medium and culturing for a period of 2-3 days to obtain secretome of unprimed MSCs; c) culturing a second portion of the expanded unprimed MSCs in a priming medium for a period of 20 to 168 hours in the presence of at least one priming agent to obtain the primed unprimed MSCs, and washing the primed cells to remove the priming agent; d) culturing the primed MSCs in a collection medium, for a period of 2- 3 days, and filtering to obtain a conditioned medium of said primed MSCs; e) concentrating the conditioned medium using Tangential flow filtration (TFF) to obtain a concentrated secretome, wherein the conditioned medium is concentrated to a volume in the range of 1 / 10 to 1 / 100, preferably 1 / 20; and f) purifying by subjecting the concentrated secretome to density gradient centrifugation at 90,000 x g to 110,000 x g, preferably 100,000 x g, for a period of 4-18 hours, preferably 8 hours, at a temperature of 2-8°C, preferably 4°C, to a fraction comprising the exosomes.

[0074] The method, according to the present disclosure, may be performed using 3D- culture systems, for example: in a Bioreactor. In an embodiment, the method is performed in a bioreactor comprising microcarriers for cell attachment, wherein cell seeding density is in the range of 11,000-70,000 cells / mL, and area density of 2222- 9333 cells / cm2 of microcarrier surface. The cells (MSCs) may be seeded on to microcarriers by inoculating a suitable amount of cell suspension into the bioreactors. In an example, hMSCs are cultured on Corning® Low Concentration SynthemaxTM II microcarriers (by Corning) inside PBS-0.1 or 0.5 single-use Vertical-WheelTM bioreactors PBS Biotech, Camarillo, CA, USA. In -vessel harvest of the conditioned media may be performed following expansion of MSCs in the bioreactor for a period of 5-6 days. The cells are separated from the microcarriers and conditioned media collected on day 7 after the addition of collection media, for example: Rooster EV collect media, on Day 5. The conditioned media is then subjected to concentration using TFF to obtain concentrated secretome; and purification to obtain the exosomes.Mesenchymal stem cells (MSCs)

[0075] The method, according to the present disclosure, involves the use of Mesenchymal stem cells (MSCs) in producing the exosomes. In certain embodiments,MSCs, which may also be referred to herein as producer cells, may be cultured in a collection medium for a period of 2-3 days to allow collection of exosomes secreted by the MSCs into the collection medium. The collection medium conditioned with the exosomes, referred to herein as the “conditioned medium” or “conditioned medium of the MSCs”, may then subjected to concentration and purification to obtain the exosomes as described herein above.

[0076] The MSCs used for producing exosomes by the method, according to the present disclosure, may be MSCs derived from sources such as Induced pluripotent stem cells (iPSCs), Corneal limbal stem cells, Umbilical cord stem cells (UCMSCs), unrestricted somatic stem cells, Bone marrow stem cells (BMSCs), Wharton’s jelly stem cells (WJSCs), Dental pulp stem cells (DPSCs), and Adipose tissue stem cells (ADSCs).

[0077] In an embodiment, the MSCs are derived from the sources such as human bone marrow (BM), corneal limbal stem cells, umbilical cord (UC), unrestricted somatic stem cells, Wharton’s jelly (WJ), dental pulp (DP) and adipose tissue (AD), induced pluripotent stem cells (iPSCs). In another embodiment, the MSCs may be nonimmortalized or immortalized, or derived from non-immortalized or immortalized cells. The MSCs may be primed MSCs obtained by priming of unprimed cells using the priming agent disclosed herein. The primed MSCs may be primed using a single priming approach or a combinatorial priming approach, wherein the priming agent is at least one selected from a chemical substance, secretome of unprimed cells, and hypoxia. In an embodiment, the MSCs are chemical substance primed MSCs. In an embodiment, the MSCs are secretome primed MSCs selected from corneal stromal stem cell (CSSC)- derived secretome primed MSCs, NPC secretome primed MSCs. In an embodiment, the MSCs are secretome primed MSCs, wherein the MSCs are derived from sources such as human bone marrow (BM), umbilical cord (UC), Wharton’s jelly (WJ), and adipose tissue (AD).

[0078] The MSCs may be obtained from commercially available sources or healthy donors. Examples of commercial sources include, but are not limited to, STEMCELL technologies, Ossium Health, Applied Stem Cell, and Catalent.

[0079] In an embodiment, the MSCs are selected from induced pluripotent stem cell- derived MSCs (iMSCs), Corneal limbal stem cells derived MSCs, umbilical cord (UCMSCs) derived MSCs, Bone marrow derived MSCs (BMMSCs), Wharton’s jelly derived MSCs (WJMSCs), Dental pulp derived MSCs (DPMSCs), Adipose tissue derived MSCs (ADMSCs), or combination thereof. In an example, the MSCs areHuman BMMSCs (hBMMSCs) procured from healthy donors procured from RoosterBio Inc.

[0080] The MSCs may be obtained from a working cell bank established after multiple passages. In an example, a xeno-free working cell bank (passage 3 (P3)) of human pluripotent stem cells (iPSCs) from a donor is expanded in a planar culture. The cells may be seeded for the first passage (P3) at 2000-3000 cells / cm2, preferably 2500 cells / cm2in T225 cell culture flasks, in xeno-free RoosterNourish medium. The P3 cells may be expanded for a period of 3 to 5 days to obtain expanded unprimed cell which may further be cultured in a priming medium to obtain primed cell according to the embodiments described herein. Similar process may be performed for MSCs derived from other sources such as BMMSCs, UCMSCs, WJMSCs, DPMSCs and ADMSCs. Such variations and alternatives would be apparent to a person skilled in the art in light of the disclosure herein and is understood to be included within the scope of the present invention.

[0081] The MSCs for producing the exosomes by the method, according to the present disclosure, may be non-immortalized or immortalized. Various immortalization techniques are known in the art, any of which may be used to immortalize the MSCs. In an embodiment, the MSCs are maybe transfected with at least one immortalization genes selected from hTERT gene, SV40 T-antigen gene, or combination thereof, to express hTERT, SV40 T-antigen, or combination thereof. Viral or non-viral immortalization approaches are known and may be used in achieving the immortalized cell lines.

[0082] Accordingly, in an embodiment, the MSCs are immortalized cells expressing a protein selected from SV40 large T antigen (SV40LT), human telomerase reverse transcriptase (hTERT), or combination thereof. Immortalization of cells may be performed at any level, for example: immortalization may be performed of primed cells or unprimed cells, or of producer MSCs or MSCs sources such as iPSC, WJ stem cell, DP stem cells, AD stem cells, etc. Accordingly, in an embodiment, the MSCs are immortalized primed cells. In another embodiment, the MSCs are immortalized unprimed cells. In an embodiment, the MSCs are non-immortalized primed cells. In another embodiment, the MSCs are non-immortalized unprimed cells.

[0083] The MSCs, according to embodiments herein, are characterized by positive markers and negative markers. In an embodiment, the MSCs, primed or unprimed, nonimmortalized or immortalized, are characterized by at least one positive marker selectedfrom CD44, CD90, CD105, CD73, CD90, CD146, CD107a, CXCR4, LepR, CD98, CD141, Oct-4 / HLA-G, CD46, CD55, CD152, CD153, Fas ligand, CD205, CD106, CD24, CD54, CD59, CD243, P-glycoprotein, CD81, CD47, CD276, CD151, CD147, CD58, CD273, CD54, CD119, CD268, CD133, CD106, or combination thereof; or at least one negative marker selected from GSTT1, CD 142, CD49c, CD 143, CDKN2A pl6, CD13, CCL2 / CCL5 / IL-8 (low), EGF, PDGF, TGF-b2, b-FGF, CD34, CD45, CD31, CD80, CD86, or combination thereof.

[0084] Table 1 depicts the markers expressed by stem cells, such as BMMSCs, UCMSCS, WJMSCs.

[0085] Table 1:Priming agents

[0086] The priming agent, according to embodiments herein, may be selected from a chemical substance; secretome of unprimed cells; hypoxia; or a combination thereof. The present inventors have observed that the use of the one or more of the priming agents disclosed herein in priming the MSCs, achieves exosomes having enhanced properties such as elevated levels of cargo profiles, increased yield, consistent size, etc. Accordingly, the exosome profiles achieved using single priming approach and combinatorial priming approach are varied and improved which facilitate in providing next-generation therapeutically potent exosomes. The exosomes variants thus obtained are tunable and may be engineered to provide improved and effective therapeutic options.

[0087] In certain embodiments, the chemical substances of the disclosure include, e.g., small molecules having molecular weight less than about 800 Da and pathway inducers that upregulate specific cellular pathways of the producer cells. The chemical substances may be used in a single priming approach or in combination priming with secretome of unprimed cells and / or hypoxia, in various embodiments herein. In certain embodiments, the chemical substances of the disclosure are selected from Nicotinamide adenine dinucleotide (NAD), Nicotinamide mononucleotide (NMN), Nicotinamide riboside (NR), SRT-1720, SRT-2104, trans-Resveratrol, Methyl 2-(N-(5-bromo-4- fluoro-2-methylphenyl)sulfamoyl)-5-(3,5-dichlorophenylsulfonamido)benzoate (MDL 800; SIRT6), Isoquercetin, Fucoidan, 4-Octyl itaconate (Nrf2 activator), CDDO-Im (CAS No.: 443104-02-7) (Nrf2 activator), Dimethyl fumarate (DMF) (Nrf2 activator), Curcumin, Berberine, Luteolin, Quercetin, 5-aminoimidazole-4-carboxamide-l-P-D- ribofuranoside (AICAR), Thienopyridone (A-769662), Metformin, All-trans retinoic acid (ATRA), Rapamycin, ML228 (CAS No. 1357171-62-0) , Succinate, 5- Phenylalkoxypsoralen (Psora-4), 5-azacytidine (5-Aza), 4-N-[2-benzyl-7-(2- methyltetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl]cyclohexane-l,4-diamine (UM171), (4-(4-fluorophenyl)-2-(4-methylsulfmylphenyl)-5-(4-pyridyl)-imidazole) (SB203580), Fisetin, Atorvastatin, Valproic acid, Sphingosine- 1 -phosphate (SIP), Astaxanthin (ATX), Polyunsaturated fatty acids (e.g., Arachidonic acid, Eicosapentaenoic acid, Docosahexaenoic acid), or combinations thereof.

[0088] Table 2 illustrates a list of priming agents (small molecules) with exemplary working concentrations and exemplary durations of treatment, respectively.

[0089] Table 2:

[0090] The secretome of unprimed cells, according to the present disclosure, include macromolecules. The secretome of unprimed cells may be used as a standalone priming agent or in combination priming with one or more chemical substance and / or hypoxia, in various embodiments herein. In certain embodiments, the priming agent may be secretome of unprimed cells selected from mesenchymal stem cells (MSCs), mesenchymal stem cells derived from induced pluripotent stem cell-derived MSCs (iMSCs), corneal limbal stem cells, umbilical cord derived mesenchymal stem cells (UCMSCs), Bone marrow derived mesenchymal stem cells (BMMSCs), Wharton’s jelly derived mesenchymal stem cells (WJMSC), dental pulp mesenchymal stem cells (DPMSCs) and adipose tissue derived mesenchymal stem cells (ADMSCs), corneal stromal stem cell (CSSCs), neural progenitor cells (NPCs), liver progenitor cell, lung progenitor cell, or combinations thereof.

[0091] The unprimed cells for secretome production may be non-immortalized or immortalized. In an embodiment, the secretome is a secretome of non-immortalized unprimed cells. In an embodiment, the secretome is a secretome of immortalized unprimed cells. The secretome may be obtained from commercial sources or produced by methods known in the art. In an embodiment, the secretome of unprimed cells is obtained by culturing the unprimed cells for a period of 2-3 days to obtain the secretome.

[0092] Hypoxia is a priming agent according to the present disclosure. It may be used in a single priming approach or in combination priming with one or more chemical substance and / or secretome of unprimed cells, in various embodiments herein. Hypoxia is induced by methods generally known in the art. Hypoxia induces the expression ofHIF-la and HIF- la over expression further induces the expression of miRNA-15, miR- 16, miR-17, miR-31, , miR-126, miR-145, miR-221, miR-222, miR-320, miR-424, that are associated to angiogenesis capacity, thereby improving regeneration and survival ofMSCs.

[0093] The unprimed cells may be cultured in the presence of hypoxia wherein the oxygen concentration is reduced to as low as a concentration in the range of 0.5-10%. In an embodiment, the priming agent is hypoxia by exposure to oxygen, preferably at a concentration in the range of 0.5-10%.

[0094] In an embodiment, the method comprises culturing the expanded unprimed cells in a priming medium for a period of 20 to 168 hours in the presence of at least one priming agent selected from a chemical substance; secretome of unprimed cells, hypoxia, or combination thereof, to obtain the primed cells, and washing the primed cells to remove the priming agent. Exposure to the priming agent maybe for a period of 1-7 days, 1-6 days, 1-5 days, etc. The exposure time may vary depending on the priming agent. In an embodiment, the exposure time is in the range of 24 to 96 hours, 24 to 72 hours, 24 to 46 hours, etc. In another embodiment, the exposure time is in the range of 1-7 days, 1-6 days, 1-5 days, etc. Accordingly, in an embodiment, the method comprises culturing the expanded unprimed cells in a priming medium for a period of 24 to 96 hours, or at least 24 hours, in the presence of a chemical substance. In another embodiment, the method comprises culturing the expanded unprimed cells in a priming medium for a period of 1-5 days in the presence of secretome of unprimed cells. For example, wherein the priming agent is DMF, the cells are cultured in the priming medium for a period of 24 hours. Similarly, wherein the priming agent is secretome of CSSC, the cells are cultured in the priming medium for a period of 5 days.

[0095] The priming medium, according to embodiments herein, comprises a growth medium and one or more priming agents. The growth medium would depend and vary on the cell type of the unprimed cells used for producing the secretome. Any suitable growth medium for culturing stem cells may be used which may be supplemented with the chemical substance and / or secretome of unprimed cells, to obtain the priming medium. The growth medium, according to embodiments herein, may be any suitable media used to culture and allow growth ofMSCs. Examples of growth media includes, but is not limited to, RoosterNourish media, Cellartis® MSC Xeno-Free Culture Medium (Takara Biosciences), MSC NutriStem® XF Medium (by Sartorius), StemProMSC SFM XenoFree (by Thermofisher), and StemXVivo Xeno-Free Human MSC Expansion Media (by R&D systems), preferably RoosterNourish media.

[0096] The concentration of priming agents may be suited as per requirement. Table 2 illustrates the working range of the chemical substance in the priming medium. In an embodiment, the concentration of the secretome of unprimed cells in the priming medium is in the range of 20 to 25% v / v in respect of the medium. In an embodiment, the priming media comprises secretome of CSSC in an amount of 20-25% v / v of the media. In another embodiment, the priming media comprises secretome of hNPC in an amount of 20-25% v / v of the media.

[0097] In an embodiment, the priming agent is a combination of priming agents selected from: secretome of CSSC and Nrf2 activator; secretome of NPC; SIRT1 activator and Nrf2 activator in the presence of hypoxia; SRT2104 in presence of hypoxia; Nrf2 activator in the presence of hypoxia; ATRA inducer in the presence of hypoxia; and SIRT1 inducer and ATRA in the presence of hypoxia.Exosomes

[0098] The exosomes, according to embodiments herein, are suitable for therapeutic application. The exosomes produced by the method, according to embodiments herein, have been found to exhibit elevated levels of cargo molecule profiles. The method, according to embodiments herein achieves large-scale production of exosome variants of improved therapeutic potential. Further, the method and exosome variants are tunable to facilitate improved and effective therapeutic options. The exosome variants as disclosed herein show higher levels of pro-regenerative, anti-fibrotic and antiinflammatory, re-innervation, and vascular pro-angiogenic factors. Accordingly, embodiments herein provide a population of exosomes, or exosome variants, obtained by the method as disclosed herein. FIG. 2 is a schematic representation depicting the exosomal variants, according to embodiments herein.

[0099] The exosomes produced by the method, according to embodiments herein, are characterized by the presence of cargo molecule such as Nrf2, HIF-la, VEGF, sFLTl, IL- 10, SIRT1, COX-2, HIF-1, CXCR4, CCR2, VEGF, Angiopoietin, HGF, IDO, NGF, BDNF, SDFla, or combination thereof. In an embodiment, the exosomes comprise the cargo molecules selected from Nrf2, HIF-la, VEGF, sFLTl, IL- 10, SIRT1, COX-2, HIF-1, CXCR4, CCR2, VEGF, Angiopoietin, HGF, IDO, NGF, BDNF, SDFla, or combination thereof

[0100] In an embodiment, the exosomes comprise a cargo molecule selected from(a) at least one mRNA molecule encoding a protein selected from BMP 15, COL4A2, CXCR7, IL1RN, IRF6, ADAM15, ADM2, BCL6B, BDH2, CDC14B, CLEC2A, CRLF1, CTNNA1, EPX, FUT3 gene, IBSP gene, LTA4H, MAGED2, MSN, NIN, RAB5A, RBL-1, S100A13, SCNN1G, SENP2, TOPORS, hTERT, TGF-beta R2, or combination thereof; or(b) at least one miRNA molecule selected from miR-1246, miR-126, miR- 133b, miR-145, miR-146, miR-155, miR184, miR-205, miR-21, miR-23b, miR-29c, miR-455-3p, Let-7, IncRNA, Malatl, miR-122, miR-101, miR-127-3p, miR-130, miR- 150, miR-15a, miR-15b, miR-16, miR-181a, miR-195, miR-451a, miR-497, miR-145, miR-361-5p, miR-200b, miR-100, miR-29 (b and c), miR-22, miR-210, miR-lOb, miR- 126-5p, miR-9, miR-105, miR-214, miR-136, miR-140, miR-181-5p, miR-223, miR-494, miR-22, miR-lOb, miR-143, miR-378e, miR-499a-5p, miR-98-5p, miR-3202, miR-32-5p, miR-363-5p, miR-548j-3p, miR-219a-5p, miR-450a-5p, miR-516b, miR-495, miR-218-5p, miR-101, miR-132, miR-196a, miR-1246, miR-515-5p, miR-342- 5p, miR-574-5p, miR-4787-3p, miR-25-3p, miR-193a, miR-567, miR-548a-5p, miR- 483-3p, miR-24, miR-204, miR-509-3p, miR-424, miR-297, miR-92b, miR-346, miR- 150-5p, miR-204-p, miR-30a-5p, miR-487b, miR-34c-5p, miR-34b, miR-663, miR- 503, miR-181c, miR-595, miR-200a, miR-7-1, miR-149, miR-188-3p, miR125b-5p, miR-143-3p, miR-155-5p, miR-21-5p, miR-1910-3p, miR-124-3p, Let-7c, Let-7b-5p, miR-183-5p, miR-375, miR-99b, miR-34a, miR-122, miR-124, miR-127, miR-27b- 3p, miR-19a, miR-17, miR-31, miR-126, miR-145, miR-221, miR-222, miR-320, miR- 424, miR-144, or combination thereof.

[0101] Table 3 illustrates a list of protein biomarkers for characterizing the primed exosomes.

[0102] Table 3:

[0103] Table 4 illustrates a list of miRNA analyzed in exosome variants

[0104] Table 4:

[0105] Table 5 illustrates a list of mRNA analyzed in exosome variants

[0106] Table 5:

[0107] The exosomes may further be suitably formulated to produce a composition for therapeutic application. Accordingly, embodiments herein provide a composition comprising the exosomes. In an embodiment, the composition comprises the exosomes, or population thereof, obtained by the method disclosed herein; and a pharmaceutically acceptable carrier.ENUMERATED EMBODIMENTS

[0108] Provided herein are non-limited exemplary embodiments of the disclosure.Set I

[0109] Embodiment 1-1. A method for producing exosomes, comprising:(a) culturing mesenchymal stem cells (MSCs) in a collection medium, for a period of 2- 3 days, and filtering to obtain a conditioned medium of said MSCs;(b) concentrating the conditioned medium using Tangential flow filtration (TFF) to obtain a concentrated secretome, wherein the conditioned medium is concentrated to a volume in the range of 1 / 10 to 1 / 100, preferably 1 / 20 known as D-Exo A; and(c) purifying by ultracentrifuging the concentrated secretome at 100,000 x g to 130,000 x g, preferably 120,000 x g, for a period of 2-4 hours, preferably 4 hours, at a temperature of 2-8°C, preferably 4°C, to obtain a concentrated purified secretome (D / K-Exo B); and subjecting the purified secretome to density gradient centrifugation at 90,000 x g to 110,000 x g, preferably at 100,000 x g, for a period of 4-18 hours, preferably 8 hours, at a temperature of 2-8°C, preferably 4°C, to a fraction comprising the purified exosomes (K-Exo A).

[0110] Embodiment 1-2. A method for producing exosomes, comprising:(a) culturing mesenchymal stem cells (MSCs) in a collection medium, for a period of 2- 3 days, and filtering to obtain a conditioned medium of said MSCs;(b) concentrating the conditioned medium using Tangential flow filtration (TFF) to obtain a concentrated secretome, wherein the conditioned medium is concentrated to a volume in the range of 1 / 10 to 1 / 100, preferably 1 / 20; and(c) purifying by subjecting the concentrated secretome to density gradient centrifugation at 90,000 x g to 110,000 x g, preferably 100,000 x g, for a period of 4- 18 hours, preferably 8 hours, at a temperature of 2-8°C, preferably 4°C, to a fraction comprising the exosomes.[OHl] Embodiment 1-3. The method of Embodiment 1-1 or 1-2, wherein said MSCs are primed or unprimed cells, and optionally immortalized.

[0112] Embodiment 1-4. The method of any one of Embodiments I-I-l to 1-4, wherein said MSCs are selected from induced pluripotent stem cells derived MSCs (iMSCs), Corneal limbal stem cells derived MSCs, umbilical cord (UCMSCs) derived MSCs, Bone marrow derived MSCs (BMMSCs), Wharton’s jelly derived MSCs (WJMSC), Dental pulp derived MSCs (DPMSCs), Adipose tissue derived MSCs (ADMSCs), or combination thereof.

[0113] Embodiment 1-5. The method of Embodiment 1-3, wherein said MSCs are primed cells obtained by culturing unprimed stem cells for a period of 20 to 168 hours in the presence of at least one priming agent.

[0114] Embodiment 1-6. The method of Embodiment 1-3, wherein the MSCs are primed cells obtained by a method comprising: i. expanding unprimed cells, non-immortalized or immortalized, in an expansion medium for a period of 3 to 5 days to obtain expanded unprimed cell; ii. passaging a first portion of the expanded unprimed cells to a growth medium and culturing for a period of 2-3 days to obtain secretome of unprimed cells; and iii. culturing a second portion of the expanded unprimed cells in a priming medium for a period of 20 to 168 hours in the presence of at least one priming agent to obtain the primed cells, and washing the primed stem cells to remove the priming agent.

[0115] Embodiment 1-7. The method of Embodiment 1-6, wherein the priming agent is selected from a chemical substance; secretome of unprimed cells; hypoxia; or combination thereof, wherein the chemical substance is selected from Nicotinamide adenine dinucleotide (NAD), Nicotinamide mononucleotide (NMN), Nicotinamide riboside (NR), SRT- 1720, SRT-2104, / ra / rs-Resveratrol, Methyl 2-(N-(5-bromo-4-fluoro-2- methylphenyl)sulfamoyl)-5-(3,5-dichlorophenylsulfonamido)benzoate (MDL 800;SIRT6), Isoquyesercetin, Fucoidan, 4-Octyl itaconate (Nrf2 activator), CDDO-Im (Nrf2 activator), Dimethyl fumarate (DMF) (Nrf2 activator), Curcumin, Berberine, Luteolin, Quercetin, 5-aminoimidazole-4-carboxamide- 1 -P-D-ribofuranoside (AICAR), Thienopyridone (A-769662), Metformin, All-trans retinoic acid (ATRA), Rapamycin, ML228 (CAS No. 1357171-62-0) , Succinate, 5-Phenylalkoxypsoralen (Psora-4), 5-azacytidine (5-Aza), 4-N-[2-benzyl-7-(2-methyltetrazol-5-yl)-9H- pyrimido[4,5-b]indol-4-yl]cyclohexane-l,4-diamine (UM171), (4-(4-fluorophenyl)-2- (4-methylsulfinylphenyl)-5-(4-pyridyl)-imidazole) (SB203580), Fisetin, Atorvastatin, Valproic acid, Sphingosine- 1 -phosphate (SIP), Astaxanthin (ATX), or Polyunsaturated fatty acids (Arachidonic acid, Eicosapentaenoic acid, Docosahexaenoic acid, or combinations thereof, wherein the unprimed cells are, non-immortalized or immortalized cells, selected from, MSCs derived from induced pluripotent stem cells derived MSCs (iMSCs), Corneal limbal stem cells derived MSCs, umbilical cord (UCMSCs) derived MSCs, Bone marrow derived MSCs (BMMSCs), Wharton’s jelly derived MSCs (WJMSC), Dental pulp derived MSCs (DPMSCs), Adipose tissue derived MSCs (ADMSCs), Corneal stromal stem cell (CSSCs), Neural progenitor cells (NPCs), liver progenitor cell, lung progenitor cell, or combination thereof, wherein said hypoxia is by exposure to oxygen, preferably at a concentration in the range of 0.5-10%.

[0116] Embodiment 1-8. The method of Embodiment 1-3, wherein said MSCs are immortalized cells expressing a protein selected from SV40 large T antigen (SV40LT), human telomerase reverse transcriptase (hTERT), or combination thereof.

[0117] Embodiment 1-9. The method of Embodiment 1-1, wherein said method comprises subjecting said conditioned medium to centrifugation to collect supernatant, wherein centrifugation is performed at 750 x g for 15 minutes at 4 °C; 2,000 x g for 15 min at 4°C; and 10,000 x g for 45 min at 4°C.

[0118] Embodiment I- 10. The method of Embodiment 1-7, wherein said priming agent is a combination selected from: secretome of CSSC and Nrf2 activator; secretome of NPC; SIRT1 activator and Nrf2 activator in the presence of hypoxia; SRT2104 in presence of hypoxia; Nrf2 activator in the presence of hypoxia; ATRA inducer in the presence of hypoxia; SIRT1 inducer and ATRA in the presence of hypoxia.

[0119] Embodiment 1-11. The method of Embodiment 1-1 or 1-2, wherein said exosomes comprise a cargo molecule selected from Nrf2, HIF-la, VEGF, sFLTl, IL-10, SIRT1, COX-2, HIF-1, CXCR4, CCR2, VEGF, Angiopoietin, HGF, IDO, NGF, BDNF, SDFla, or combination thereof.

[0120] Embodiment 1-12. The method of Embodiment 1-1 or 1-2, wherein said conditioned medium is characterized by a marker selected from Nrf2, HIF-1 a, VEGF, sFLTl, IL- 10, SIRT1, COX-2, HIF-1, CXCR4, CCR2, VEGF, Angiopoietin, HGF, IDO, NGF, BDNF, SDFla, or combination thereof.

[0121] Embodiment 1-13. The method of Embodiment 1-1 or 1-2, wherein said MSCs are characterized by at least one positive marker selected from CD44, CD90, CD105, CD73, CD90, CD146, CD107a, CXCR4, LepR, CD98, CD141, Oct-4 / HLA- G, CD46, CD55, CD152, CD153, Fas ligand, CD205, CD106, CD24, CD54, CD59, CD243, P-gly coprotein, CD81, CD47, CD276, CD 151, CD 147, CD58, CD273, CD54, CD119, CD268, CD133, CD106, or combination thereof; or at least one negative marker selected from GSTT1, CD 142, CD49c, CD 143,CDKN2A pl6, CD13, CCL2 / CCL5 / IL-8 (low), EGF, PDGF, TGF-b2, b-FGF, CD34, CD45, CD31, CD80, CD86, or combination thereof.

[0122] Embodiment 1-14. The method of Embodiment 1-1 or 1-2, wherein said exosomes comprises a cargo molecule selected from a. at least one mRNA molecule encoding a protein selected from BMP 15, COL4A2, CXCR7, IL1RN, IRF6, ADAM15, ADM2, BCL6B, BDH2, CDC14B, CLEC2A, CRLF1, CTNNA1, EPX, FUT3 gene, IBSP gene, LTA4H, MAGED2, MSN, NIN, RAB5A, RBL-1, S100A13, SCNN1G, SENP2, TOPORS, hTERT, TGF-beta R2, or combination thereof; or b. at least one miRNA molecule selected from miR-1246, miR-126, miR-133b, miR-145, miR-146, miR-155, miR184, miR-205, miR-21, miR-23b, miR-29c, miR- 455-3p, Let-7, IncRNA, Malatl, miR-122, miR-101, miR-127-3p, miR-130, miR-150, miR-15a, miR-15b, miR-16, miR-181a, miR-195, miR-451a, miR-497, miR-145, miR- 361-5p, miR-200b, miR-100, miR-29 (b and c), miR-22, miR-210, miR-lOb, miR-126- 5p, miR-9, miR-105, miR-214, miR-136, miR-140, miR-181-5p, miR-223, miR-494, miR-22, miR-lOb, miR-143, miR-378e, miR-499a-5p, miR-98-5p, miR-3202, miR-32- 5p, miR-363-5p, miR-548j-3p, miR-219a-5p, miR-450a-5p, miR-516b, miR-495, miR- 218-5p, miR-101, miR-132, miR-196a, miR-1246, miR-515-5p, miR-342-5p, miR- 574-5p, miR-4787-3p, miR-25-3p, miR-193a, miR-567, miR-548a-5p, miR-483-3p, miR-24, miR-204, miR-509-3p, miR-424, miR-297, miR-92b, miR-346, miR-150-5p, miR-204-p, miR-30a-5p, miR-487b, miR-34c-5p, miR-34b, miR-663, miR-503, miR-181c, miR-595, miR-200a, miR-7-1, miR-149, miR-188-3p, miR125b-5p, miR-143- 3p, miR-155-5p, miR-21-5p, miR-1910-3p, miR-124-3p, Let-7c, Let-7b-5p, miR-183- 5p, miR-375, miR-99b, miR-34a, miR-122, miR-124, miR-127, miR-27b-3p, miR- 19a, miR-17, miR-31, miR-126, miR-145, miR-221, miR-222, miR-320, miR-424, miR-144, or combination thereof.Embodiment 1-15. The method of any one of Embodiment 1-1 or 1-2, wherein said TFF is performed using TFF membrane filter selected from polyether sulfones (PES), modified polyether sulfones (mPES), poly sulfones (PS), and mixed cellulose esters (ME), having pore size of 0.5 pm- 0.8 pm, and molecular weight cut-off in the range 100 kDa- 750 kDa.

[0123] Embodiment 1-16. The method of Embodiment 1-14, wherein sheer force of feed stream, in said TFF, is maintained below 2000 s-1, preferably having input flow rate in that range of 80 mL / min to lOOmL / min.

[0124] Embodiment 1-17. The method of Embodiment 1-1 or 1-2, wherein the density gradient centrifugation is by iodixanol gradient centrifugation or sucrose gradient centrifugation.

[0125] Embodiment 1-18. The method of Embodiment 1-1 or 1-2, wherein said exosomes are obtained as fraction (F9) with a density in the range of 1.13g / mL to 1.18g / mL in iodixanol gradient.

[0126] Embodiment 1-19. The method of Embodiment 1-1 or 1-2, wherein said purifying step (c) comprises washing said fraction by centrifuging at 90,000 x g to 110,000 x g, preferably 100,000 x g, for 3-6 hours, preferably 4 hours, at 2-8 °C, preferably 4 °C, to obtain purified exosomes.

[0127] Embodiment 1-20. The method of Embodiment 1-5 or 1-6, wherein said exosomes are tissue-specific primed exosomes.

[0128] Embodiment 1-21. The method of Embodiment 1-1 or 1-2, wherein said method is performed in a bioreactor comprising microcarriers for cell attachment, wherein cell seeding density is in the range of 11,000-70,000 cells / mL, and area density of 2222-9333 cells / cm2 of microcarrier surface.

[0129] Embodiment 1-22. A population of exosomes obtained by a method claimed in any one of claims 1-19, or 21.

[0130] Embodiment 1-23. A composition comprising the exosomes obtained by a method claimed in any one of claims 1-19, or 21.Set II

[0131] Embodiment II- 1. A method for producing exosomes, comprising:(a) culturing mesenchymal stem cells (MSCs) in a collection medium to obtain a conditioned medium (CM) of said MSCs;(b) concentrating the CM using Tangential flow filtration (TFF) to obtain a concentrated secretome.

[0132] Embodiment II-2. The method of Embodiment II- 1, wherein the MSCs are cultured in the collection medium for 2 or 3 days.

[0133] Embodiment II-3. The method of Embodiment II-l or Embodiment II-2 wherein the CM is concentrated using the TFF to reduce the volume of the CM to between about 1 / 10 and about 1 / 100 of the initial volume.

[0134] Embodiment II-4. The method of any one of Embodiments 1-1 through 1-3, further comprising (c) subjecting the concentrated secretome to a pelleting ultracentrifugation to obtain a purified secretome.

[0135] Embodiment II-5. The method of Embodiment II-4, wherein the pelleting ultracentrifugation is performed at a spin strength of between about 100,000 x g and about 130,000 x g for a duration of between about 2 hour and about 4 hours.

[0136] Embodiment II-6. The method of Embodiment II-4 or Embodiment II-5, further comprising (d) subjecting the purified secretome to a density gradient centrifugation.

[0137] Embodiment II-7. The method of Embodiment II-6, wherein the density gradient centrifugation is performed at at 90,000 x g to 110,000 x g for a period of 4- 18 hours.

[0138] Embodiment II-8. The method of any one of Embodiment II- 1 to Embodiment II-7, wherein the MSCs are primed by culturing the MSCs in the presence of at least one priming agent prior to the MSCs being cultured in the collection medium.

[0139] Embodiment II-9. The method of Embodiment II-8, wherein the MSCs are cultured in the presence of the at least one priming agent for a period of at least 20 hours.

[0140] Embodiment II- 10. The method of any one of Embodiment II-l to Embodiment II-4, wherein said MSCs are selected from the group consisting of: induced pluripotent stem cell-derived MSCs (iMSCs), corneal limbal stem cell-derived MSCs, umbilical cord-derived MSCs (UCMSCs), Bone marrow-derived MSCs(BMMSCs), Wharton’s jelly-derived MSCs (WJMSC), Dental pulp-derived MSCs (DPMSCs), Adipose tissue derived-MSCs (ADMSCs), and combinations thereof.

[0141] Embodiment II- 11. The method of Embodiment II-8, wherein the MSCs are primed by a method comprising: i. expanding unprimed cells in an expansion medium for a period of 3 to 5 days to obtain expanded unprimed cells; ii. passaging a first portion of the expanded unprimed MSCs to a growth medium and culturing for a period of 2-3 days to obtain secretome of unprimed cells; and iii. culturing a second portion of the expanded unprimed MSCs in a priming medium for a period of 20 to 168 hours in the presence of the at least one priming agent to obtain the primed MSCs, and washing the primed MSCs to remove the priming agent.

[0142] Embodiment 11-12. The method of Embodiment II- 11, wherein the priming agent is selected from a chemical substance; a secretome of unprimed cells; hypoxia; or combinations thereof.

[0143] Embodiment 11-13. The method of Embodiment 11-12, wherein the priming agent comprises the chemical substance, and wherein the chemical substance is selected from the group consisting of: Nicotinamide adenine dinucleotide (NAD), Nicotinamide mononucleotide (NMN), Nicotinamide riboside (NR), SRT-1720, SRT-2104, trans- Resveratrol, Methyl 2-(N-(5-bromo-4-fluoro-2-methylphenyl)sulfamoyl)-5-(3,5- dichlorophenylsulfonamido)benzoate (MDL 800; SIRT6), Isoquyesercetin, Fucoidan, 4-Octyl itaconate (Nrf2 activator), CDDO-Im (Nrf2 activator), Dimethyl fumarate (DMF) (Nrf2 activator), Curcumin, Berberine, Luteolin, Quercetin, 5-aminoimidazole- 4-carboxamide-l-P-D-ribofuranoside (AICAR), Thienopyridone (A-769662), Metformin, All-trans retinoic acid (ATRA), Rapamycin, ML228 (CAS No. 1357171- 62-0) , Succinate, 5 -Phenyl alkoxy psoralen (Psora-4), 5-azacytidine (5-Aza), 4-N-[2- benzyl-7-(2-methyltetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl]cyclohexane-l,4- diamine (UM171), (4-(4-fluorophenyl)-2-(4-methylsulfmylphenyl)-5-(4-pyridyl)- imidazole) (SB203580), Fisetin, Atorvastatin, Valproic acid, Sphingosine- 1 -phosphate (SIP), Astaxanthin (ATX), or Polyunsaturated fatty acids (Arachidonic acid, Eicosapentaenoic acid, Docosahexaenoic acid, and combinations thereof.

[0144] Embodiment 11-14. The method of Embodiment 11-12 or Embodiment 11-13, wherein the priming agent comprises the secretome of the unprimed cells, and whereinthe unprimed cells are selected from the group consisting of: MSCs derived from iMSCs, UCMSCs, BMMSCs, WJMSCs, DPMSCs, Corneal stromal stem cells (CSSCs), Neural progenitor cells (NPCs), liver progenitor cells, lung progenitor cells, and combinations thereof.

[0145] Embodiment 11-15. The method of Embodiment 11-12, wherein the priming agent is the hypoxia, and wherein said hypoxia is by exposure to reduced oxygen at a concentration in the range of 0.5-10%.

[0146] Embodiment 11-16. The method of Embodiment 11-12, wherein the priming agent is a combination selected from the group consisting of: a SIRT1 activator and a Nrf2 activator in the presence of hypoxia; SRT-2104 in the presence of hypoxia; a Nrf2 activator in the presence of hypoxia; ATRA in the presence of hypoxia; and a SIRT1 inducer and ATRA in the presence of hypoxia.

[0147] Embodiment 11-17. The method of Embodiment 11-12, wherein said priming agent is a combination selected from the group consisting of: a secretome of unprimed CSSCs and a Nrf2 activator; and a secretome of unprimed NPCs.

[0148] Embodiment 11-18. The method of any one of Embodiments II-8 to 11-15, wherein said MSCs are immortalized cells.

[0149] Embodiment 11-19. The method of Embodiment 11-16, wherein the immortalized cells are characterized by expression of a protein selected from the group consisting of: SV40 large T antigen (SV40LT), human telomerase reverse transcriptase (hTERT), or a combination thereof.

[0150] Embodiment 11-20. The method of any one of Embodiment II-l to 11-17, wherein said method comprises, prior to concentrating the CM using TFF, subjecting said CM to centrifugation to collect supernatant.

[0151] Embodiment 11-21. The method of Embodiment 11-19, wherein the centrifugation is serial centrifugation comprising a first centrifugation step performed at 750 x g, a second centrifugation performed at 2,000 x g, and a third centrifugation step performed at 10,000 x g.

[0152] Embodiment 11-22. The method of anyone of Embodiments II-l to 11-21, wherein said exosomes comprise a cargo molecule selected from the group consisting of: Nrf2, HIF-la, VEGF, sFLTl, IL- 10, SIRT1, COX-2, HIF-1, CXCR4, CCR2, VEGF, Angiopoietin, HGF, IDO, NGF, BDNF, SDFla, and combinations thereof.

[0153] Embodiment 11-23. The method of any one of Embodiments II-l to 11-22, wherein the CM is characterized by a marker selected from the group consisting of:Nrf2, HIF-la, VEGF, sFLTl, IL- 10, SIRT1, COX-2, HIF-1, CXCR4, CCR2, VEGF, Angiopoietin, HGF, IDO, NGF, BDNF, SDFla, and combinations thereof.

[0154] Embodiment 11-24. The method of any one of Embodiments II-l to 11-23, wherein the MSCs are characterized by at least one positive marker selected from the group consisting of: CD44, CD90, CD105, CD73, CD90, CD146, CD107a, CXCR4, LepR, CD98, CD141, Oct-4 / HLA-G, CD46, CD55, CD152, CD153, Fas ligand, CD205, CD106, CD24, CD54, CD59, CD243, P-glycoprotein, CD81, CD47, CD276, CD151, CD 147, CD58, CD273, CD54, CD 119, CD268, CD133, CD 106, and combinations thereof.

[0155] Embodiment 11-25. The method of any one of Embodiments II- 1 to 11-24, wherein the MSCs are characterized by at least one negative marker selected from the group consisting of: GSTT1, CD142, CD49c, CD143, CDKN2A pl6, CD13, CCL2 / CCL5 / IL-8 (low), EGF, PDGF, TGF-b2, b-FGF, CD34, CD45, CD31, CD80, CD86, and combinations thereof.

[0156] Embodiment 11-26. The method of any one of Embodiments II-l to 11-25, wherein the exosomes comprises a cargo molecule selected from at least one mRNA molecule encoding a protein selected from the group consisting of: BMP 15, COL4A2, CXCR7, IL1RN, IRF6, ADAM15, ADM2, BCL6B, BDH2, CDC14B, CLEC2A, CRLF1, CTNNA1, EPX, FUT3 gene, IBSP gene, LTA4H, MAGED2, MSN, NIN, RAB5A, RBL-1, S100A13, SCNN1G, SENP2, TOPORS, hTERT, TGF-beta R2, and combinations thereof.

[0157] Embodiment 11-27. The method of any one of Embodiments II-l to 11-26, wherein the exosomes comprises a cargo molecule selected firomat least one miRNA molecule selected from the group consisting of: miR-1246, miR-126, miR-133b, miR- 145, miR-146, miR-155, miR184, miR-205, miR-21, miR-23b, miR-29c, miR-455-3p, Let-7, IncRNA, Malatl, miR-122, miR-101, miR-127-3p, miR-130, miR-150, miR- 15a, miR-15b, miR-16, miR-181a, miR-195, miR-451a, miR-497, miR-145, miR-361- 5p, miR-200b, miR-100, miR-29 (b and c), miR-22, miR-210, miR-lOb, miR-126-5p, miR-9, miR-105, miR-214, miR-136, miR-140, miR-181-5p, miR-223, miR-494, miR- 22, miR-lOb, miR-143, miR-378e, miR-499a-5p, miR-98-5p, miR-3202, miR-32-5p, miR-363-5p, miR-548j-3p, miR-219a-5p, miR-450a-5p, miR-516b, miR-495, miR- 218-5p, miR-101, miR-132, miR-196a, miR-1246, miR-515-5p, miR-342-5p, miR- 574-5p, miR-4787-3p, miR-25-3p, miR-193a, miR-567, miR-548a-5p, miR-483-3p, miR-24, miR-204, miR-509-3p, miR-424, miR-297, miR-92b, miR-346, miR-150-5p,miR-204-p, miR-30a-5p, miR-487b, miR-34c-5p, miR-34b, miR-663, miR-503, miR- 181c, miR-595, miR-200a, miR-7-1, miR-149, miR-188-3p, miR125b-5p, miR-143- 3p, miR-155-5p, miR-21-5p, miR-1910-3p, miR-124-3p, Let-7c, Let-7b-5p, miR-183- 5p, miR-375, miR-99b, miR-34a, miR-122, miR-124, miR-127, miR-27b-3p, miR- 19a, miR-17, miR-31, miR-126, miR-145, miR-221, miR-222, miR-320, miR-424, miR-144, and combinations thereof.

[0158] Embodiment 11-28. The method of any one of Embodiments II-l to 11-27, wherein the TFF is performed using a TFF membrane filter selected from a polyether sulfones (PES) membrane filter, a modified polyether sulfones (mPES) membrane filter, a poly sulfones (PS) membrane filter, and a mixed cellulose esters (ME) membrane filter.

[0159] having pore size of 0.5 pm- 0.8 pm, and molecular weight cut-off in the range 100 kDa- 750 kDa.

[0160] Embodiment 11-29. The method of any one of Embodiments II-l to 11-28, wherein sheer force of feed stream, in said TFF, is maintained below about 2000 s-1.

[0161] Embodiment 11-30. The method of Embodiment 11-29, wherein the TFF is performed with an input flow rate between about 80 mL / min and about lOOmL / min.

[0162] Embodiment II-31. The method of any one of Embodiments II-6 to 11-30, wherein the density gradient centrifugation is iodixanol gradient centrifugation or sucrose gradient centrifugation.

[0163] Embodiment 11-32. The method of Embodiment 11-31 wherein said purified exosome fraction is obtained as a fraction in an iodixanol gradient having a density range between 1.13g / mL and 1.18g / mL.

[0164] Embodiment 11-33. The method of Embodiment 11-32 wherein said purified exosome fraction is obtained as fraction 9 in an iodixanol gradient

[0165] Embodiment 11-34. The method of any one of Embodiments II-6 to 11-33, further comprising washing the purified exosome fraction by centrifuging at 90,000 x g to 110,000 x g .

[0166] Embodiment 11-35. The method of any one of Embodiments II-l to 11-34 wherein the MSCs are cultured in a bioreactor comprising microcarriers for cell attachment, wherein cell seeding density is in the range of 11,000 to 70,000 cells / mL, and area density of microcarrier surface is in the range of 2222 to9333 cells / cm2.

[0167] Embodiment 11-36. A population of exosomes obtained by a method claimed in any one of Embodiments II-l to 11-35.

[0168] Embodiment 11-37. A composition comprising the exosomes obtained by a method claimed in any one of Embodiment II- 1 to 11-35.EXAMPLES

[0169] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.Materials used:

[0170] hBMMSCs: procured from the manufacturer (RoosterBio) with its recommended media (RoosterNourish Media XF, RoosterBio, Cat no: KT-016); IX PBS; TrypLE; Dulbecco's phosphate-buffered saline (DPBS); Rooster EV collect media: procured from RoosterBio, Cat no: M2001.Producer cells (MSCs):Source of MSCs

[0171] For the purpose of the present disclosure, mesenchymal stem cells (MSCs) were derived from the sources such as human bone marrow (BM), corneal limbal stem cells, umbilical cord (UC), unrestricted somatic stem cells, Wharton’s jelly (WJ), dental pulp (DP) and adipose tissue (AD), induced pluripotent stem cells (iPSCs), engineered cells, corneal stromal stem cell-derived (CSSC)-derived conditioned media primed MSCs were used for producing exosomes described herein. For exemplification, the stem cells derived from sources such as, human bone marrow (BM), umbilical cord (UC), Wharton’s jelly (WJ), adipose tissue (AD) are represented as B, U, W, A, respectively. Source of immortalized stem cell lines using non- Viral immortalized MSC cell lines:

[0172] (a) hTERT immortalized human bone marrow mesenchymal stem cells (hBMMSCs): Unprimed BMMSCs with clinically approved CD105+, CD90+, CD73+markers were used for producing unprimed exosomes. Primed exosomes wereproduced using primed BMMSCs primed using secretome of unprimed CSSCs, which was used for primed exosome production for avascular tissue regeneration. Clinically approved non-viral human telomerase enzyme reverse transcriptase (hTERT) induced immortalized BMMSCs were used for constant production of the exosomes.

[0173] (b) hTERT immortalized human Wharton’s jelly-derived MSC (WJMSCyUmbilical cord-derived MSC (UCMSC) cell line: UCMSCs with clinically approved CD166+, CD90+, CD73+markers & CD34', a-SMA' markers were selected and grown in xeno-free media and unprimed exosomes were produced from cells expressing the markers. Clinically approved non-viral hTERT induced immortalized UCMSCs were used for constant production of the exosomes.EXAMPLE 1: Production of unprimed exosomes from unprimed MSCs. a) Expanding imprinted hBMMSCs under xeno-free conditions

[0174] 10M human BMMSC (hBMMSC) cells (passage 2) (unprimed cells) were cultured as per the manufacturer’s protocol. Rooster Booster-MSC-Xeno-free and Rooster Basal-MSC were thawed at room temperature and transferred aseptically in a biosafety cabinet to reconstitute into a 500 mL media. The vial containing the hBMMSCs (stored in liquid nitrogen) was thawed in a 37°C water bath for 2-3 min. The contents were aseptically resuspended in the said culture media and centrifuged at 200xg for 10 min. The obtained pellet was redissolved in complete media and the initial cell count was recorded. The cells were seeded into CELLBIND T225 cm2 or Hyperflasks or Cell stacks flasks at a density of 2000-3000 cells / cm2 and incubated at 37°C with 5% CO2.

[0175] The cells were monitored for day 3 and the media was kept unchanged until 80% confluence i.e. (43000-50000) cells / cm2, when the cells were considered ready for harvest. The spent media was collected in sterile tubes for quenching TrypLE cell dissociation enzyme. The cells were washed with IX PBS followed by addition of TrypLE and incubated at 37°C for 5 min to detach the cells. An equal volume of fresh media was added to stop the TrypLE activity. The cell suspension was centrifuged at 200 x g for 10 min and the cell pellets were resuspended in fresh media, followed by cell counting. The cells were further diluted in Dulbecco's phosphate-buffered saline (DPBS) to get a count range of (0.1-1) x 106cells / mL and cryopreserved till further use. b) Culturing hBMMSCs to obtain a conditioned media of hBMMSCs

[0176] 10M hBMMSC cell vials were revived and observed since day 3. Cells were washed on day 4 / 5, depending on the cell density (43000-50000 cells / cm2), twice with 20 mL of PBS. The second wash was done at 37°C for 10 min in a 5% CO2 incubator and media was changed to Rooster EV collect media. After 48h of incubation, conditioned media was collected and the cells harvested were counted using a cell counter.

[0177] Collected conditioned media was immediately processed with the serial centrifugation steps to collect the supernatant: 750 x g for 15 min at 4°C, 2000 x g for 15 min at 4°C, 10,000 x g for 45 min at 4°C, followed by filtering using 0.22-micron sterile filter using PES membrane, to obtain a filtrate. c) Purification of exosomes without concentrating by TFF for comparative analysis

[0178] The filtrate was subjected to 120,000 x g for 3h at 4°C (UC step 1 - Purified secretome), and 100,000 x g for 8h at 4°C (UC step 2 - Purified Exosomes) using density gradient (iodixanol or sucrose) ultracentrifugation for the isolation of the exosomes.EXAMPLE 2: Production of primed exosomes from MSCs derived from iPSCs (iMSCs)Material used:

[0179] iPSCs; RoosterNourish medium: procured from RoosterBio, Cat no: M2001; DMF: procured from Sigma Aldrich Cat No. 242926; PBS procured from Gibco. Cat No. 10010023; T225 cell culture flasks, from Corning®: CellBIND T225 flasks or CellB IND Hyperflasks, Cat no. 10024 (500mL per flask); RoosterCollectEV: procured from RoosterBio, Cat no: M2001.

[0180] Procedure: A Xeno-Free working cell bank (WCB, passage 3) of human induced pluripotent stem cells (iPSCs) from one donor were thawed and expanded in planar culture. Briefly, thawed cells were seeded for the first passage (P3) at 2500 cells / cm2 in T225 cell culture flasks, in xeno-free RoosterNourish medium. A T75 flask was seeded to monitor confluence and the remainder of the cells of T225 flask were cryopreserved. After approximately five days of cell expansion, cells were harvested and passaged (P4) in two arms; (Arm 1 - control) 4 x T225 cell culture flasks layer and Arm 2 - DMF priming) 4 x T225, at 2500 cells / cm2 in growth medium. Arm 1 was grown in only a growth medium. On day 4 (P4) cells from Arm 2, were treated with100 pM DMF (Dimethyl fumarate; priming agent). After 24 hours of priming with DMF, the spent media was discarded, and the cells were washed with fresh PBS at room temperature, followed by a second wash with PBS and incubation for 10 mins at 37 °C. Fresh RoosterCollect EV was added to the vessels and cultivated for two days. On Day two of post-media exchange, the conditioned media (CM) containing exosomes from (Arm 7) 4 x T225, was pooled to generate a control CM. Similarly, the conditioned media (CM) containing exosomes from (Arm 2) 4 x T225, was pooled to generate CM of primed MSCs.

[0181] Arml (processing of Control)'. CM was clarified to remove bioburden by serial centrifugation steps (i.e. 750 x g for 15 min at 4°C, 2000 x g for 15 min at 4°C, 10,000 x g for 45 min at 4°C) followed by filtering using 0.22-micron sterile filter using PES membrane, to obtain a filtrate. This filtrate or clarified conditioned medium, also referred to herein as the “ crude secretome ” , was stored at 2-4°C before further processing. The clarified conditioned media was distributed in the ultracentrifuge tubes evenly before subjecting to 120,000 x g 3h at 4°C. The pellet was resuspended in ice- cold lOmM sterile filtered sodium phosphate buffer aseptically. The suspension was pooled to generate the purified secretome (UC1 step).

[0182] The purified secretome was then layered on iodixanol gradient and subjected to density gradient centrifugation at 100,000 x g for 8h at 4°C, to obtain a fraction comprising exosomes. Fractions 9 (see explanation regarding fraction 9 in Example 2 below) from multiple runs were isolated, pooled, and washed using ice-cold 2mM sterile filtered sodium phosphate buffer and centrifuged at 100,000 x g for 4h at 4°C to obtain a fraction pellet and remove the residual iodixanol. The fraction pellets were reconstituted in ice-cold 2mM sterile filtered sodium phosphate buffer containing 5mM sterile filtered Trehalose to generate Purified Exosome fractions and stored at -80°C.

[0183] Arm2 (preparation of DMF primed exosomes) '. The CM from the DMF -treated cells was processed separately. Briefly, CM was clarified to remove bioburden by serial centrifugation steps (i.e. 750 x g for 15 min at 4°C, 2000 x g for 15 min at 4°C, 10,000 x g for 45 min at 4°C

[0184] The clarified conditioned medium (CM) was stored at 2-4°C for 12-14 hours before further processing. The CM was concentrated using TFF (as described in Example 7) to obtain the concentrated secretome. The concentrated secretome obtained from the CM of iMSCs (DMF-primed or unprimed) through processing with TFF may be referred to herein, including in subsequent Examples, as “D-Exo A”.

[0185] The concentrated secretome D-Exo A was distributed in the ultracentrifuge tubes evenly before subjecting to pelleting ultracentrifugation performed at 100,000 x g for 4h at 4°C. The pellet was resuspended in ice-cold lOmM sterile filtered sodium phosphate buffer aseptically. The suspension was pooled to generate the purified secretome (Purification: UC1 step). The purified secretome obtained from the CM of iMSCs (DMF-primed or unprimed) that is processed with TFF and pelleting ultracentrifugation as describe in the present Example, may be referred to herein, including in subsequent Examples, as “D / K-Exo B”.

[0186] To isolate and characterize exosomes from the D / K-Exo B preparation, a discontinuous iodixanol (OptiPrep) density gradient ultracentrifugation protocol was employed. A step gradient was prepared by sequentially layering 3 mL of 55% iodixanol solution (w / v), 3 mL of 10% iodixanol, followed by 6 mL of the D / K-Exo B sample on top. The gradient was subjected to ultracentrifugation using a Beckman SW40 Ti swinging-bucket rotor at 100,000 * g for 8 hours at 4 °C.

[0187] Post-centrifugation, the gradient was fractionated into twelve (12) 1 mL fractions collected from the top of the tube to the bottom of the tube, with the top fraction being designated as fraction 1, through to the bottom fraction being designated as fraction 12. The refractive index of each fraction was measured and converted to density using a standard reference table provided by OptiPrep. The resulting density profile ranged from approximately 1.02 g / mL (fraction 1) to 2.00 g / mL (fraction 12).

[0188] We focused our analyses on fractions falling within density range of 1.13 g / mL to 1.18 g / mL. Fractions 8 through 12 were found to lie within or near this target density range. To further refine the selection, fractions 8, 9, and 10 were subjected to physicochemical characterization and in vitro potency assays.

[0189] Among these, fraction 9 demonstrated superior qualities in terms of cargo enrichment, enhanced bioactivity, and higher yield. Therefore, fraction 9 was selected as the fraction of choice for further study and analysis (see Examples below), and fraction 9 of the iodixanol gradient was obtained for further study:

[0190] Purified secretome D / K-Exo B was layered on iodixanol gradient and subjected to density gradient centrifugation (Purification: UC2 step) at 100,000 x g for 8h at 4°C, and fractions 9 from multiple runs were isolated, pooled, and washed using ice-cold 5mM sterile filtered sodium phosphate buffer and centrifuged at 100,000 x g for 4h at 4°C (Washing step) to remove the residual iodixanol from the fractions. The fraction pellets were reconstituted in ice-cold 5mM sterile filtered sodium phosphate buffercontaining 2.5mM sterile filtered Trehalose and stored at -80°C. This purified exosome fraction obtained from the CM of iMSCs (DMF-primed or unprimed) that is processed with TFF, pelleting ultracentrifugation, and density gradient centrifugation as described in the present Example, may be referred to herein, including in subsequent Examples, as “K-Exo A”.EXAMPLE 3: Production of secretome of Corneal Stromal Stem Cells (CSSCs) for priming MSCsMaterial used:

[0191] CSSCs procured from Eyebanks in India and US; MEM media procured from Gibco, Cat no: 11095080; Liberase procured from: Roche, Cat no: 5401119001; Human Platelet Lysate PLTGold® procured from Sartorius, Cat no: PLTGOLD100 GMP; Synthemax solution from Corning, Cat no: 3535; Tryple (IX, Gibco) Procedure: a) Culturing CSSCs under xeno-free conditions^.

[0192] Human donor-derived corneas were used to derive CSSCs using the protocol under xeno-free conditions. The corneas were washed with antibiotic fortified buffered saline (PBS) before extracting limbus which contains CSSCs. In aseptic conditions, a 360° limbal ring was excised using surgical instruments and washed with buffered saline and minced into smaller fragments. The minced tissue fragments were collected into incomplete media (MEM) and subjected to Liberase digestion by adding 20 pL of reconstituted Liberase at a concentration of 0.5 lU / pL to the tissue suspension. After 16 h of incubation, enzymatic digestion was stopped by adding 2 mL of complete media fortified with 2% human platelet lysate. The digested tissues were spun down at 200 x g for 5 min at room temperature, in saline supplemented with penicillin and streptomycin followed by various levels of passaging.

[0193] Passage 0 (P0): During passage 0, the digested explants were resuspended in 5 mL xenofree complete media (MEM + 2% HPL, IX ITS, 10 ng / mL EGF) and cultured in a T25 Coming CellBIND flask for 7-14 days. The media was changed every 3 days. For passage 0, flasks were coated with Coming Synthemax II-SC (125 pL of Synthemax solution from 1 mg / mL stock solution dissolved in cell culture grade water was added to the T25 flask and kept at room temperature for 2h for coating, after which the solution was removed. The flask was either used immediately or can be stored at 4°C for future use).

[0194] Passage 0 (P0): During passage 0, the digested explants were resuspended in 5 mL xenofree complete media (MEM + 2% HPL, IX ITS, 10 ng / mL EGF) and cultured in a T25 Coming CellBIND flask for 7-14 days. The media was changed every 3 days. For passage 0, flasks were coated with Coming Synthemax II-SC (125 pL of Synthemax solution from 1 mg / mL stock solution dissolved in cell culture grade water was added to the T25 flask and kept at room temperature for 2h for coating, after which the solution was removed. The flask was either used immediately or can be stored at 4°C for future use).

[0195] For secretome collection: a) Pl - Day 3 (50% media replenishing): Day 5 (Complete media change) b) P2 - Day 3 (50% media replenishing): Day 5 (Complete media change) c) P3 - Day 3 (50% media replenishing): Day 5 (Complete media change)

[0196] For the quality control, cells at Pl, P2 and P3 were characterized(Immunofluorescence imaging) using markers, such as, stem cell markers like CD90, CD73, and CD105, corneal cell specific marker like ABCG2, ABCB5 and negative markers like cr-SMA, CD34, p63cr. b) Collection of secretome from CSSC culture

[0197] From passages 1-2-3 as described above, every media change was accompanied by collection of the secretome from the flasks. The secretome was further pre-processed by centrifuging the media at 300 x g for 5 min to collect the supernatant. The supernatant was further centrifuged at 3000 x g for 20 min at 4°C followed by recentrifuging the supernatant at 13000 x g for 30 min at 4°C to collect the further processed supernatant. The media was double-filtered through a 0.45-micron filter and further using 0.22-micron filter in PES sterile filter membrane to collect the supernatant. ELISA based cell modulator characterization for HGF, NGF, VEGF, sFlt- 1, IDO was performed. The collected supernatant (conditioned media) was stored at - 80°C for long term storage.EXAMPLE 4: Production of secretome of Human neuro progenitor cells (hNPCs) for priming MSCsMaterials used:

[0198] Human NPCs procured from Applied StemCells SE-9211; Catalog Number ASE-9740MM.

[0199] Coating matrix: 80 pg / mL Matrigel®.Procedure:

[0200] The human hNPCs were thawed and cultured for the study. These cells express high levels of the neuronal lineage biomarker, Nestin. The NPC were further passaged in the medium (provided by Applied StemCells) and characterized for the presence of biomarkers (by using Nestin R&D systems MAB1259). The cell culture vessels were coated with a coating matrix, (80 pg / mL). The plates were incubated at room temperature for at least 1 hour before use. Thawing and Culturing of Cryopreserved NPCs: To thaw the cryopreserved NPCs, one vial was removed from the storage unit. The vial was immersed in the water bath (up to 2 / 3rd of the vial) and the cells were rapidly brought to the biological cabinet, and immediately the outer surface of the vial was thoroughly cleaned with 70% ethanol and wiped off with an autoclaved paper towel. The cells were removed from the vial, using a plOOO micropipette (or serological pipette) and transferred slowly, dropwise while swirling into a 15 mL conical tube containing 5 mL of pre-warmed NPC culture medium. The vial was washed with 1 mL medium from the 15 mL conical tube and transferred it back to the tube. The cells were centrifuged at 250 x g for 5 minutes at room temperature. The medium was aspirated very carefully using a vacuum (or pipette if preferred), leaving only a drop of liquid in the tube. Using a pl 000 micropipette, 1 mL of the pre-warmed NPC culture medium was added into the tube and gently resuspended cells by pipetting up and down 2-3 times. A 10 pL aliquot of the cell suspension was mixed with 10 pL of Trypan blue solution for cell counting purpose. After counting the cells, the coating material was aspirated from the pre-wared cell culture vessels, and the cells were seeded in the coating material at a density of 100,000 live cells / cm2 in NPC Culture Media. The cells were placed in the incubator (37°C / 5% CO2 / humidity control) for 5 days until the cells were confluent. Cells were characterized using biomarkers Sox-2, Nestin, Musashi, b-Tubulin and GFAP. The secretome of NPCs were collected for both Arms (i.e. Arm 1 and Arm 2) of the study.Single priming protocol of hBMMSCs with hN PC-derived secretome

[0201] Priming of hBMMSCs (passage 4 or 3 cells) at a seeding density of 2000-3000 cells / cm2was performed in media in culture flasks, supplemented with secretome (1%, 2%, 5%) of hNPC (human neuro progenitor cells), with a volume percentage in the range of 1-5% v / v (Day 0). hBMMSCs were expanded till 80-85% confluence was reached i.e. 43000-50000 cells / cm2and further shifted to EV collect media on day 5.500ml / flask of the conditioned media (EV collect media) was collected after 48h (day 7) and further used for exosome production.

[0202] A Xeno-Free working cell bank (WCB, passage 3) of hBMMSCs from one donor was thawed and expanded in planar culture. Briefly, thawed cells were seeded for the first passage (P3) at 2000-3000 cells / cm2 in T25 cell culture flasks from corning, supplemented with different concentration ranging from 1-5% hNPC derived secretome, in xeno-free RoosterNourish medium. One well in a 6-well plate was seeded to monitor confluence and the remainder of the cells in T75 flasks were cryopreserved. After approximately five days of cell expansion, cells were harvested and passaged (P4) in two arms (Arm 7) 4 x T25 cell culture flasks layer and (Arm 2) 4 x T25 at 2500 cells / cm2 in growth medium. Control Arm 1 was grown in only a growth medium. On day 4 (P4) cells from Arm 2, were treated with DMF. After 24 hours of priming with DMF, the spent media was discarded, and the cells were washed with fresh PBS at room temperature, followed by a second wash with PBS and incubation for 10 mins at 37 °C. Fresh RoosterCollect EV was added to the vessels and cultivated for two days. On day two of post-media exchange, the conditioned media (CM) containing EVs from (Arm 7) 4 x T25, was pooled to generate CM for downstream processing (DSP). Similarly, the conditioned media (CM) containing EVs from (Arm 2) 4 x T25, was pooled to generate CM for downstream processing (DSP).Downstream Process

[0203] Downstream Process of Arm P. CM was clarified to remove bioburden by serial centrifugation steps followed by sterile filtration to generate crude secretome. The clarified conditioned medium (CCM) or filtrate was stored at 2-4°C before further processing. The crude secretome was distributed in the ultracentrifuge tubes evenly before subjecting to 120,000 x g 3h at 4°C. The pellet was resuspended in ice-cold lOmM sterile filtered sodium phosphate buffer aseptically. The suspension was pooled to generate the purified secretome (UC1). The purified secretome was then layered on iodixanol gradient and subjected to centrifugation at 100,000 x g for 8h at 4°C. Fractions 9 were isolated, pooled, and washed using ice-cold 2mM sterile filtered sodium phosphate buffer and centrifuged at 100,000 x g for 4h at 4°C to remove the residual iodixanol from the fractions. The fraction pellets are reconstituted in ice-cold 2mM sterile filtered sodium phosphate buffer containing 5mM sterile filtered Trehalose to generate purified exosome fractions and stored at -80°C.

[0204] Downstream Process of Arm2'. The CM from the DMF -treated cells was processed separately. Briefly, CM was clarified to remove bioburden by serial centrifugation steps followed by sterile filtration to generate crude seer etome. The clarified conditioned medium (CCM) or filtrate was stored at 2-4°C before further processing. The crude secretome was distributed in the ultracentrifuge, tubes evenly before subjecting to 120,000 x g 3h at 4°C. The pellet was resuspended in ice-cold lOmM sterile filtered sodium phosphate buffer aseptically. The suspension was pooled to generate the Purified Secretome (UC1). The suspension was then layered on iodixanol gradient and subjected to centrifugation at 100,000 x g for 8h at 4°C. Fractions 9 were isolated, pooled, and washed using ice-cold 2mM sterile filtered sodium phosphate buffer and centrifuged at 100,000 x g for 4h at 4°C to remove the residual iodixanol from the fractions. The fraction pellets are reconstituted in ice-cold 2mM sterile filtered sodium phosphate buffer containing 5mM sterile filtered Trehalose to generate purified exosome fractions and stored at -80°C.EXAMPLE 5 Single priming protocol for Priming of stem cells5.1. Single priming protocol of hBMMSCs with CSSC secretome

[0205] Priming of hBMMSCs (passage 4 or passage 3 cells) at a seeding density of 2000-3000 cells / cm2was performed in media (in hyperflask), supplemented with secretome of corneal stromal stem cell (CSSC) (obtained as per Example 3), with a volume percentage in the range of 10-20% v / v (Day 0). hBMMSCs were expanded till 80-85% confluence was reached i.e. 43000-50000 cells / cm2and further shifted to EV collect media on day 5. 500ml / flask of the conditioned media (EV collect media) was collected after 48h (day 7) and further used for exosome production.5.2. Single priming protocol of hBMMSCs with Nrf2 activator

[0206] hBMMSC (passage 3 or 4 cells) at a seeding density of 2000-3000 cells / cm2were treated with Nrf2 activators (DMF or 4OI) in T225 cm2flasks. The cells were washed with PBS once they reached 70-80 % confluence (no media change until then) and replenished with fresh complete media having DMF (50 pM or 100 pM) or 4OI (50 pM or 100 pM) for 24h-72h, prior to switching to EV collect. On day 6 / 7, the cells were shifted to EV collect (40-45mL / flask) and incubated for 48h. The cells were harvested at a cell density of 43000-50000 cell / cm2, followed by collection of the conditioned media (40-45mL / flask) and processing for exosome production.EXAMPLE 6: Combinatorial priming protocol for Priming of stem cells6.1. Combinatorial priming of hBMMSCs with CSSC-derived conditioned media andNrf2 activator - (EXO VARIANT B’)

[0207] For priming of hBMMSC, the cells were thawed and seeded at (2000-3000) cells / cm2in T225 cm2flasks in a media supplemented with 10-20% CSSC-derived conditioned media. Once, hBMMSCs reached 70-80 % confluency (30000-40000 cells / cm2), the cells were washed in PBS and were then replenished with Nrf2 activator, DMF (50pM, 75pM, or lOOpM) or 4OI (50pM, 75pM, or 100 pM) in supplemented media for 24-72h prior to switching to EV collection media. At 70-80 % confluence, the cells were shifted to EV collect media and incubated for 48h, followed by collection of conditioned media (40-45mL / flask) at a harvesting density of 43000-50000 cells / cm2for exosome production.6.2. Combinatorial priming of UCMSCs / hBMMSCs with SIRT1 activator and Nrf2 activator in presence of hypoxia - (Exo variant B’)

[0208] SIRT1 activator (SRT2014 or RSV) induced UCMSC / hBMMSCs priming was performed in the presence of alternate hypoxia / normoxia cycles (8-20 cycles with an interval of 30-90 min), at an oxygen concentration of 0.5-10% for hypoxia and 14-22% for normoxia using a tri gas chamber-incubator set up. After the cells reached 80% confluence, it was treated with Nrf2 inducer (DMF, 4OI) for 24-72 h, prior to switching to extracellular vesicles (EV) collection media for exosome production.6.3. Combinatorial priming protocol of UCMSCs / hBMMSCs with SIRT1 activator (SRT2104 or Resveratrol) in presence of hypoxia - (Exo variant B’)

[0209] UCMSCs / hBMMSCs were cultured up to passage 5-6 and primed with SIRT1 activator (SRT2014 or RSV). Hypoxia priming was performed in presence of alternate hypoxia / normoxia cycles (8-20 cycles with an interval of 30-90 min), at an oxygen concentration of 0.5-10% for hypoxia and 14-22% for normoxia using a tri gas chamber-incubator set up. Once UCMSC / hBMMSCs reached 80% confluence, the cell survivability. HIF -la, HGF, VEGF and TNF-a expression was checked in the purified secretome and the derived exosome variants. The purified secretome and exosome profile was compared with UCMSC / hBMMSC derived exosomes where cells were maintained in normoxic condition.6.4. Combinatorial priming protocol of UCMSCs / hBMMSCs with NRF2 inducer (DMF or 4OI in presence of hypoxia)- (Exo variant B’)

[0210] UCMSCs / hBMMSCs were cultured up to passages 5-6. Hypoxia priming was performed in presence of alternate hypoxia / normoxia cycles (8-20 cycles with an interval of 30-90 min), at an oxygen concentration of 0.5-10% for hypoxia and 14-22% for normoxia using a tri gas chamber-incubator set up. Prior to shifting to EV collect, UCMSCs / hBMMSCs were treated with Nrf2 activator (DMF, 4OI) for 24-72 h. Purified secretome was characterized by detecting the levels of Nrf2, EUF-la, HGF, VEGF, sFLTl by ELISA. The primed exosome variant was characterized by ELISA for detecting the levels of exosomes cargo molecule expressions, such as Nrf2, HIF-la, VEGF, sFLTl, IL- 10, SIRT1.6.5. Combinatorial priming protocol of U CMSCs / hBMMSCs with A TRA inducer in presence of hypoxia- (Exo variant B’)

[0211] UCMSCs / hBMMSCs were cultured up to passage 5-6. Hypoxia priming was performed in presence of alternate hypoxia / normoxia cycles (8-20 cycles with an interval of 30-90 min), at an oxygen concentration of 0.5-10% for hypoxia and 14-22% for normoxia using a tri gas chamber-incubator set up. Prior to shifting to EV collect, UCMSC / hBMMSCs were treated with ATRA inducer (0.1 -500) pM for 24 -72 h. The purified secretome was checked for levels of COX-2, HIF-1, CXCR4, CCR2, VEGF, Ang-2 and Ang-4 by ELISA.

[0212] The primed exosome variant was characterized by ELISA detecting the levels of exosomes cargo molecule expressions, such as COX-2, HIF-1, CXCR4, CCR2, VEGF, Ang-2 and Ang-4.6.6. Combinatorial priming of UCMSCs / hBMMSCs with SIR T1 inducer and ATRA in presence of hypoxia- (Exo variant B’)

[0213] UCMSCs / hBMMSCs were cultured up to passage 5-6. SIRT1 activator (SRT2014 or RSV) induced UCMSC / hBMMSCs priming was performed in presence of alternate hypoxia / normoxia cycles (8-20 cycles with an interval of 30-90 min), at an oxygen concentration of 0.5-10% for hypoxia and 14-22% for normoxia using a tri gas chamber-incubator set up. At 80% confluence, the cells were treated with ATRA inducer (0.1 -500) pM for 24 -72h, after which the cells were shifted to EV collect. The conditioned media was collected after 48h and screened for levels of COX-2, HIF-1, CXCR4, CCR2, VEGF, Ang-2 and Ang-4 expressions using ELISA.

[0214] Conditioned media obtained post priming was screened for anti-inflammatory molecule expressions using ELISA, and used for exosome production. UCMSC / hBMMSC primed exosome variant was isolated from combinatorialpriming / single priming set. Primed variant of exosomes and the secretome was characterized by ELISA for detecting various cargo molecules.EXAMPLE 7: Concentration of the Conditioned media using TangentialFlow Filtration (TFF).The conditioned media collected from hBMMSCs, and UCMSCs (refer Example 5 and Example 6) was subjected to TFF. TFF was performed using a KrosFlo KR2i Tangential Flow Filtration System (Repligen). The conditioned media (500 mL) were filtered using sterile hollow fiber poly ethersulfone membranes with mPESlOO pm (D02-E100-05-N) (Repligen) with surface area 115cm2molecular weight cut-off pores to remove cell debris and free biomolecules, respectively. A TFF hollow-fiber membrane with molecular weight cutoff (MWCO) of 100 kDa (115 cm2surface area) was used. Filters were first washed with three times the volume of sterile PBS (pH 7.4), prior to filtration. The input flow rate was 80 mL / min in order to keep the shear force of the feed stream below 2000 s ' . Exosomes were concentrated to approximately 20 mL and diafiltrated six times. The final concentrated exosomes were then analyzed.EXAMPLE 8: Purification of exosomes from concentrated secretome

[0215] The concentrated secretome (obtained in Example 7) was subjected to purification. Purification was performed using two steps: (i) 2 step purification: by pelleting ultracentrifugation (PUC) followed by density gradient centrifugation; (ii) 1 step purification: by density gradient centrifugation using sucrose based cushion density ultracentrifugation or iodixanol density gradient ultracentrifugation. Purification of exosomes from conditioned media was done by following methods described below.(a) Purification by pelleting ultracentrifugation and density gradient centrifugation using sucrose-based cushion density ultracentrifugation (2 step purification)Materials used:

[0216] Sucrose procured from Sigma.

[0217] Procedure: The cells at 80% confhiency were washed in IX PBS (20 mL), followed by addition of EV collect media to the flasks and incubated for 48h at 37°C and 5% CO2. The conditioned media collected was subjected to pre-processing. The media was centrifuged at 750 x g for 15 min at 4°C, and the supernatant was collected.The supernatant was centrifuged at 2000 x g for 15 min at 4°C, the supernatant was collected. The supernatant was centrifuged at 10,000 x g for 45 min at 4°C and the supernatant was collected. The media was filtered through a 0.22-micron filter and stored at 4°C for short term storage (24 h) or -80°C for long term storage (1 month).

[0218] For processing the media (immediately or frozen), the conditioned media was centrifuged at 120,000 x g for 3h at 4 °C and the supernatant was removed carefully. A clear pellet at the bottom of the tube was resuspended in filtrate ice cold, particle free lOmM sodium phosphate buffer. The enriched exosomes were transferred on to 30% sucrose (IM) containing ultracentrifuge tube. The speed was set to 100,000 g for 8 h at 4°C and the acceleration and deceleration were set to one. The fractions were carefully collected, and the purified exosome fractions were washed individually in a filtrate ice cold particle free 2mM sodium phosphate buffer at 100,000 g for 4h at 4°C. The pellets were carefully resuspended in a filtrate ice cold particle free 2mM sodium phosphate buffer containing 5mM Trehalose. The exosomes obtained, were aliquoted, and stored at -80° C.(b) Purification by Density gradient centrifugation using pelleting ultracentrifugation (1 step purification)

[0219] To purify the exosomes using single-step centrifugation, the cells at 80% confluence were washed in IX PBS (20 mL). The PBS was discarded, and 40-45 mL / flask of EV collect media was added to the flasks, incubated for 48 h at 37°C and 5% CO2. The media was collected and proceeded with the pre-processing steps. The media was centrifuged at 750 x g for 15 min at 4°C, and the supernatant was collected. The supernatant was further centrifuged at 2000 x g for 15 min at 4°C, the supernatant was collected. The supernatant was centrifuged at 10,000 x g for 45 min at 4°C and the supernatant was collected. The media was further filtered through a 0.22-micron filter. The conditioned media was stored at 4°C for short term storage (24 h) or -80°C for long term storage (1 month). For processing the media (immediately or frozen), the conditioned media was subjected to pelleting ultracentrifugation at 120,000 x g for 3h at 4 °C and the supernatant was removed carefully. A clear pellet at the bottom of the tube was resuspended in a filtrate ice cold particle free lOmM sodium phosphate buffer and the aliquots are stored at -80 °C.(c) Purification by Pelleting ultracentrifugation and density gradient centrifugation using iodixanol density gradient ultracentrifugation (2 step purification)Material used:

[0220] lodixanol solution procured from Sigma, #D1556.

[0221] Procedure: At 80% confluence i.e., 43000-5000 cells / cm2, the cells were washed in IX PBS (20 mL) followed by addition of 40-45mL / flask of EV collect media to the flasks and incubating for 48 h at 37°C and 5% CO2. The media was collected and immediately proceeded with the pre-processing steps. The media was centrifuged at 750 x g for 15 min at 4°C, and the supernatant was collected. The supernatant was centrifuged at 2000 x g for 15 min at 4°C, the supernatant was collected. The supernatant was centrifuged at 10,000 x g for 45 min at 4°C and the supernatant was collected. The media was filtered through a 0.22-micron filter. The conditioned media was stored at 4°C for short term storage (24 h) or -80°C for long term storage (1 month). For processing the media (immediately or frozen), the conditioned media was subjected to pelleting ultracentrifutation at 120,000 x g for 3h at 4 °C and the supernatant was removed carefully. A clear pellet at the bottom of the tube was resuspended in filtrate ice cold, particle free lOmM sodium phosphate buffer.

[0222] An iodixanol (IDX) gradient was prepared by floating 3 mL of 10% w / v IDX solution containing NaCl (150 mM) and 25 mM Tris:HCl (pH 7.4) over 3 ml of 55% w / v IDX solution. Concentrated conditioned media (6 mL) was floated on the top of the IDX cushion and was ultracentrifuged using a Beckman Coulter SW 40 Ti rotor for 8 h at 100,000xg (4 °C). Twelve fractions (1 mL each) were collected from the top of the gradient on ice and each fraction was collected into pre-chilled 1.5 mL tubes. Fraction 9 was transferred into a fresh ultracentrifuge tube and washed with filtrate ice cold particle free 2mM sodium phosphate buffer and washed at 100,000 x g for 4 h in Optima XPN-100 ultracentrifuge using a Beckman Coulter Type 45 Ti rotor at 4°C. Supernatant was discarded and exosomes were re-suspended in a filtrate ice cold particle free 5mM sodium phosphate buffer containing 2.5mM Trehalose. Different aliquots were prepared with 50-100 pL and stored at -80°C for long term storage.EXAMPLE 9: Exosome production in 3D culture of producer cells

[0223] hMSCs were cultured on Corning® Low Concentration Synthemax™ II microcarriers (Corning) inside PBS-0.1 or 0.5 single-use Vertical-Wheel™ bioreactors (PBS Biotech, Camarillo, CA, USA). To seed the cells onto the microcarriers, a 1-6.3 mL cell suspension was inoculated into the bioreactor containing 30 mL of medium and 0.75-2.5 g of microcarriers, resulting in cell seeding density of 11,000-70,000cells / mL, and areal density of 2222-9333 cells / cm2of microcarrier surface. To facilitate attachment, the cells were incubated for 20 min at static condition, then gently shaken to re-distribute the cells and the microcarriers, and finally incubated for 20 more minutes to further achieve cell attachment to microcarriers. Our data showed that an average of 84% cell attachment could be achieved using this seeding method. At the end of the cell attachment duration, fresh culture medium was added to a total volume of 90 mL. The PBS-0.1 vessel was placed on its controller base in a 5% CO2 incubator and agitation was initiated at 25 rpm. On Day 3 of the bioreactor culture, an XF bioreactor feed, RoosterReplenish™-MSC-XF (RoosterBio), was added at 2% of the working volume and agitation speed was increased to 30 rpm.Bioreactor Sampling

[0224] Daily sampling of the bioreactor was performed to quantify cell numbers as well as the metabolite and waste profile to assess the kinetics of cell growth and media consumption. A 3 mL sample was taken daily while the cells / microcarriers were in suspension at 30 rpm agitation. The cell / microcarrier sample were left to sediment, the supernatant was isolated for medium analysis, and an equal volume of TrypLE was added to dissociate the cells from the microcarriers. Following dissociation, the number of cells was counted.

[0225] Visualization of cell attachment and cell / microcarrier aggregation was performed by obtaining a 1 mL sample from the bioreactor and visualizing it under light microscopy.In- Vessel Harvest

[0226] Following hMSC expansion in bioreactors for 5-6 days, an in-vessel harvest was performed. While bioreactor sampling provides the kinetics during culture, the most accurate quantification of total cell yield after the culture duration is obtained through complete in-vessel harvest, minimizing any potential errors from nonrepresentative sampling when large cell-microcarrier aggregates are present in the culture. After agitation is stopped and the cells / microcarriers settle to the bottom of the bioreactor, the medium was removed and the remaining cells / microcarrier solution were washed with PBS. TrypLE was added to the bioreactor at half of the working volume, and agitation was initiated (20-100 rpm for 20-40 min) in a 37°C incubator. Cells were then separated from the microcarriers using a 100-pm cell strainer, quenched with an equal volume of medium, and then counted to obtain the final harvest yield.Values are presented as concentrations (cells / mL) to emphasize the scalable process that was being developed.

[0227] The conditional media collected on Day 7 after the addition Rooster EV collect on Day 5 were further processed for exosome isolation and purification process. Once the exosomes are isolated and purified, they are analyzed for particle number, size, total protein estimation, tetraspanins detection and cargo characterization and in vitro functional validation.Tangential Flow Filtration for Highly Efficient Concentration:

[0228] Tangential flow filtration (TFF) was used for concentrating the conditioned media. KrosFlo Research 2i Tangential Flow Filtration System was used. Conditioned media (IL) was filtered using sterile hollow fiber polyethersulfone membranes with 0.5 pm and 100 kDa molecular weight cut-off pores to remove cell debris and free biomolecules, respectively. Filters were first washed with three times the volume of sterile PBS (pH 7.4), prior to filtration. The input flow rate was 80 mL / min in order keep the sheer force of the feed stream below 2000 s-1. EVs were concentrated to approximately 50 mL (from IL, thus 20X concentration) and diafiltrated six times in buffer (IX PBS). The concentrated secretome was then processed through iodixanol gradient centrifugation to obtain purified exosomes (refer Example 8 for iodixanol gradient centrifugation).EXAMPLE 10: Growth kinetics of hBMMSCs and iMSCs

[0229] Human bone marrow-derived mesenchymal stem cells (hBMMSCs) or induced pluripotent stem cell (iPSC)-derived MSCs (iMSCs) were grown in accordance with the procedure described in Example 2 above. Briefly, the cells were seeded at a density of 2500 cells / cm2in Coming CellBIND flasks and cultured in RoosterNourish™ media until Day 5. FIGS. 3A-3C depicts data for hBMMSCs and iMSCs, wherein FIG. 3A depicts a comparison of cell morphology at days 1-5, FIG. 3B depicts growth kinetics for hBMMSCs at days 1-5, and FIG. 3C depicts growth kinetics for iMSCs at days 1-5. The iMSCs showed spindle-like morphology at day 5, higher growth, and produced more exosomes, according to embodiments herein. As shown in FIG. 3C, hBMMSCs and iMSCs proliferate at a rapid rate through day 4, then plateau. The plateauing may largely coincides with with cells reaching confluence at around day 4, as shown for both cell types in Fig. 3 A.

[0230] FIG. 4 depicts results of Immunofluorescence staining for positive MSC markers (CD73, CD90, and CD 105) and negative MSC marker (hematopoietic marker CD34), and stress marker (a-SMA) at Day 6 of culture for hBMMSCs (upper row) and iMSCs (lower row), according to embodiments herein. The results confirm that both hBMMSCs and iMSCs maintain a MSC identity after proliferation.EXAMPLE 11: Growth kinetics of hBMMSCs with and without DMF priming

[0231] Human bone marrow-derived mesenchymal stem cells (hBMMSCs) were grown in accordance with the procedure described in Example 2 above. Briefly, the hBMMSCs were seeded at a density of 2500 cells / cm2in Coming CellBIND flasks and cultured in RoosterNourish™ media until Day 4, followed by treatment with 75pM dimethyl fumarate (DMF) for 24 hours.

[0232] FIG. 7A shows representative phase contrast microscopic images (Scale bar: 100 pm; Magnification: 10X) showing dynamic plasticity of hBMMSCs between unprimed and DMF- primed conditions at each day from Day 1 to Day 4, showing robust proliferation of hBMMSCs, achieving high cell density with no significant morphological changes. As shown in FIG. 7B showing representative phase contrast microscopic images of unprimed and primed hBMMSCs at Day 5, DMF treatment led to a slight reduction in cell growth in the DMF -primed cell cultures, as evidenced by a decrease in cell density compared to unprimed hBMMSCs. FIG. 7C shows a plot tracking cell density in each of Day 1 to Day 7 for both unprimed (naive) and DMF-primed hBMMSCs, and FIG. 7D shows a plot tracking generation number in each of Day 1 to Day 7 for both unprimed (naive) and DMF-primed hBMMSCs. Cell density and generation number were measured using Image J software at different time points for unprimed and DMF- primed hBMMSCs. Plotted values are average of 10 images per timepoint on that particular day. Despite this transient effect of DMF priming, the cells exhibited recovery in growth during subsequent culture in Rooster EV Collect media on Days 6 and 7, indicating dynamic plasticity of the DMF-primed hBMMSCs.

[0233] Immunofluorescence (IF) characterization of the hBMMSC producer cells from Days 4 to 7 in unprimed and 75 pM DMF-primed conditions substantiated the maintenance of hBMMSC functionality and phenotype. Sternness markers CD73, CD90, and CD 105 (not shown) were consistently expressed across all time points, indicating that hBMMSCs retained their mesenchymal phenotype throughout the process. No expression of the hematopoietic marker CD34 was observed (not shown), further validating the mesenchymal identity of the cells. Although DMF treatmentinduced transient expression of the stress marker aSMA on Day 5 (not shown), its levels declined on Days 6 and 7 to basal levels, indicating minimal long-term stress effects.EXAMPLE 12: Physicochemical and molecular characterization of D-ExoA, D / K-Exo B, and K-Exo A

[0234] With reference to Example 2, three different secretome / exosome preparations, prepared from CM from DMF-primed iMSCs, D-Exo A, D / K-Exo B, and K-Exo A, were characterized. D-Exo A is a concentrated secretome obtained by TFF of the CM of DMF- primed or unprimed (naive) iMSCs, as described in Example 2. D / K-Exo B is a purified secretome obtained from the CM of DMF-primed or unprimed (naive) iMSCs that is processed with TFF and pelleting ultracentrifugation (PUC) as describe in Example 2. K-Exo A is a purified exosome fraction obtained from the CM of DMF-primed or unprimed (naive) iMSCs that is processed with TFF, pelleting ultracentrifugation, and density gradient centrifugation (fractionation), as described in Example 2. The relationship between D-Exo A, D / K-Exo B, and K-Exo A is schematically shown in Fig. 6. As shown in the figure, K-Exo A may be viewed as high purified exosomes that form a subset of the constituents of D / K-Exo B, and D / K-Exo B may be viewed a subset of the constituents of less stringently purified D-Exo A.

[0235] Table 6 tabulates various physicochemical parameters regarding D-Exo A, D / K-ExoB, and K-Exo ATable 6:

[0236] As shown in Table 6, the values of the total yield of particles (e.g., exosomes) in billions per liter and the particles in billions per million cells indicate that each successive level of purification leads to loss of yield on the particles. However, the total protein in pg per billion particles went up with each successive level of purification.

[0237] All secretome / exosome preparations — D-Exo A, D / K-Exo B, and K-Exo A — were analyzed using nanoparticle tracking analysis (NTA) with the ZetaView Twin220 (Particle Metrix) to obtain size distribution metrics. Also as shown in Table 6, peak sizes were 116.6 nm for Primed D-Exo A ,136.6 nm for Primed D / K-Exo B, 138.3 nm for Primed K-Exo A indicating a distinct distribution between these subtypes, The DIO size indicates a diameter at which 10% of the particles in a sample are smaller, the D30 size indicates a diameter at which 30% of the particles in a sample are smaller), and the D50 size indicates a diameter at which 50% of the particles in a sample are smaller.. Each of these size distribution parameters indicate that each successive level of purification increased the prevalence of larger particles.The cargo profiling of the different secretome / exosome preparations were determined as follows: Equivalent numbers of exosomes from D-Exo A, D / K-Exo B, and K-Exo A samples were lysed using 1 x RIPA buffer supplemented with 1 x protease inhibitor cocktail (PIC) for 30 minutes. The resulting lysates were analyzed to quantify specific molecular cargoes, including HGF, NGF, IDO, VEGF, and sFlt-1 (as shown in Table 6), using commercial ELISA kits (R&D Systems), following the manufacturer’s protocols measuring the absorbance at 450nm. The concentrations of these cargo proteins were normalized and reported as picograms per milliliter (pg / mL) per 1 billion exosomes. Given the overall pattern of the relationships9 between the abundance of markers in D-Exo A, D / K-Exo B, and K-Exo A, the pg / ml / 10 particles of VEGF in K-Exo A was found to be unexpectedly low, indicating that the exosomepurification for obtaining K-Exo A is unexpectedly robust in selectively removing VEGF. This property indicates that K-Exo A may be especially advantageous for use in non -vascular tissue such as cornea, where a high abundance of angiogenic factors such as VEGF would be unwelcome.

[0238] Table 7 tabulates the total yield of particles (e.g., exosomes) and cost.Table 7

[0239] As can be seen in Table 7, the yield of particles (e.g., exosomes) get progressively worse with each successive purification, such that the yield of D-Exo A is 50 times that of K-Exo A. At least due to this reason, K-Exo A is also the most expensive to provide, in term of per billion particles

[0240] Reference is now made to FIGS. 8A-8B. The surface tetraspanin markers CD9, CD63, and CD81 were characterized by Western blot analysis D-Exo A, D / K-Exo B, and K-Exo A, as well as in cell lysate (CL) and Exosome-depleted media (EVD). Secretome / exosome preparations (D-Exo A, D / K-Exo B, and K-Exo A) containing 10 pg of total protein were lysed in 1 * RIPA buffer supplemented with 1 x protease inhibitor cocktail (PIC) for 30 minutes. Protein lysates were resolved by electrophoresis on 12% polyacrylamide gels and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 1% non-fat skim milk in TBST for 1 hour at room temperature, followed by overnight incubation at 4 °C with primary antibodies against CD9, CD63, and CD81. Detection was performed using a chemiluminescence detection kit (Cytiva). As shown in FIGS. 8A and 8B, the Immunoblot analysis confirmed the expression of tetraspanin markers CD9, CD63, and CD81 in D-Exo A, D / K-Exo B, and K-Exo A samples.EXAMPLE 13: In vitro assays with D-Exo A, D / K-Exo B, and K-Exo A

[0241] Therapeutic effects of D-Exo A, D / K-Exo B, and K-Exo A, were characterized with in vitro assays, including a scratch assay, an anti-fibrosis assay, an anti- inflammatory assay, and an angiogenesis assay.13.1. Scratch assay

[0242] The 2D Scratch assay was performed with iHCE (immortalized Human Corneal Epithelial cells) in a 48 well plate. Cells were seeded at 44,000 cells per well and a scratch wound was created using a pipette tip. The positive control for wound healing was media containing 50 ng / mL rhHGF (Human Hepatocyte Growth Factor Recombinant Protein), and a basal media negative control was also done. Exosome treatments (D-Exo A, D / K-Exo B, K-Exo A) were given at a concentration of 10 billion exosomes per million cells (10,000 exosomes per cell) in triplicates. The readout of the assay was phase contrast images of the treated wells captured periodically at Oh, 6h, lOh, 24h, 30h and 48h post the scratch wound (not shown). The wound area was quantified over time and the percentage wound closure was plotted, as shown in FIG. 9. Both D-Exo A and D / K-Exo B showed faster wound healing compared to basal (no treatment). Surprisingly, D-Exo A showed a slightly better wound closure than D / K- Exo B. As shown in FIGS. 10A and 10B, K-Exo A showed significantly better wound closure as compared to basal.13.2. Anti Fibrosis assay

[0243] Reference is made to FIGS. 11A-11B and 12A-12B. The Anti Fibrosis assay was performed with HDF (Human Dermal Fibroblast) cells in a 96 well plate. Cells were seeded at 2500 cells per well and human TGF pi (Transforming growth factor beta 1) was used as an inducer of fibrosis. The positive control for anti-fibrosis was media containing the inducer along with 50 ng / mL rhHGF (Human Hepatocyte Growth Factor Recombinant Protein), and the negative control was media containing just the inducer (TGF pi). Exosome treatments (D-Exo A, D / K-Exo B, and K-Exo A) were given at concentrations of 10 billion exosomes per million cells (10,000 exosomes per cell) in triplicates, along with the inducer. The readout of the assay was immunofluorescence staining for aSMA (alpha Smooth Muscle Actin), whose expression level is a measure of stress fiber (fibrosis) induced in the cell. As shown in FIGS. 11A and 11B, fluorescent image acquisition and quantification of aSMA intensities across all treatments showed that D-Exo A and D / K-Exo B, whether prepared from the CM from unprimed (naive) or DMF-primed iMSC, all showed significant reduction in TGF- pi induced stress fiber formation, with expression levels lower than the rhHGF-treated positive control, and comparable to the basal media treatment. That said, the D-Exo A and D / K-Exo B sourced from DMF-primed cells were more effective than their respective counterparts from unprimed (naive) cells. FIGS. 12A and 12B, whichcompared the effect of D-Exo A, D / K-Exo B, and K-Exo A from DMF-primed cells, shows that each had substantially the same anti-fibrotic properties, and all were, surprisingly, more potently anti-fibrotic than the rhHGF-treated positive control.13.3. Anti Inflammatory assay

[0244] Reference is made to FIGS. 13A-13C and 14A-14C, which shows the increase on inflammatory or anti-inflammatory markers in THP-1 cells based on administration of D-Exo A, D / K-Exo B, and K-Exo A. Cells were then stimulated with LPS (10 ng / mL) for 6 hours to induce an inflammatory response. THP-1 cells (Human Monocytes) were seeded at 0.5 x 106cells / well in 12-well plates and differentiated for 48 hours using RPMI media supplemented with 10% FBS, 0.05 mM P- mercaptoethanol, 1% Penicillin-Streptomycin, and 150 nM PMA, followed by a 48- hour rest period in complete medium without PMA. Cells were then stimulated with LPS (10 ng / mL) for 6 hours to induce an inflammatory response. Treatment groups included a blank control (basal media), negative control (LPS alone), positive control (LPS + rhHGF, 50 ng / mL), and experimental groups treated with LPS in combination with D-Exo A, D / K-Exo B, or K-Exo A (10,000 exosomes / cell) in triplicates. Culture supernatants were harvested 6 hours post-treatment and analyzed for interleukin-6 (IL- 6) and tumor necrosis factor-alpha (TNF-a), both pro-inflammatory cytokines, and interleukin- 10 (IL- 10), an anti-inflammatory cytokine, were quantified using commercially available ELISA kits (R&D Systems). Cytokine levels are presented as absolute concentrations in pg / mL.

[0245] Treatment with primed exosome variants, including D-Exo A, D / K-Exo B, and K-Exo A, significantly reduced pro-inflammatory cytokines IL-6 and TNF-a, as assessed by ELISA. Both naive and primed Exo variants exhibited anti-inflammatory effects in IL-6 assays, while TNF-a ELISA indicated greater efficacy of primed Exo variants over unprimed (naive) forms. Notably, all primed Exo variants studied increased IL-10 levels, further supporting their anti-inflammatory potential. Surprisingly, as shown in FIG> 14A, K-Exo A obtained from DMF-primed cells, was especially potent in reducing the LPS-induced increase in IL-6, when compared to D- Exo A, D / K-Exo B obtained from DMF-primed cells13.4. Neurogenesis assay

[0246] Reference is made to FIGS. 15A-15G. The Neurogenesis assay was performed with SH-SY5Y (Human neuroblastoma) cell line in a 24 well plate. Cells were seeded at 4500 cells per coverslip, and prior to the treatments were primed with 5pM RetinoicAcid (RA) for 48h along with gradual reduction of serum. The positive control for inducing neurite formation was 50 ng / mL rhBDNF (Human Brain-derived Neurotrophic Factor Recombinant Protein) and a basal media negative control was also done. Exosome treatment (K-Exo A obtained from DMF-primed cells) was given at a concentration of 5 billion exosomes per million cells (5000 exosomes per cell) in triplicates. The readout of the assay was phase contrast imaging and immunofluorescence staining for pill Tubulin which marks the neuronal bodies and neurites. In the phase contrast images of FIGS. 15A-15C, as well as the fluorescent images of 15D-15F, the administration of primed K-Exo A improved neurite outgrowth, As shown in FIG. 15G, the fluorescent image acquisition and quantification of neurite outgrowths showed that the treatment with primed K-Exo A - significantly increased neurite formation in cells compared to the basal media control.

[0247] 13.5. Angiogenesis assay

[0248] Reference is made to FIGS. 16A-16G. An angiogenesis assay qA performed using HUVECs (Human Umbilical Vein Endothelial cells) seeded on Matrigel -coated 96-well plates (50 pL / well). 15,000 HUVEC cells were seeded in basal media (devoid of growth factors) with the following four treatment conditions: basal media (FIG. 16A); added VEGF (20 ng / mL) as negative control (FIG. 16B), sFLT-1 (50 ng / mL) as positive control (FIG. 16C), and primed K- Exo A (FIG. 16D) at 10 billion exosomes per million cells (10,000 exosomes / cell) in triplicates. After 16 hours of incubation, cells were fixed and stained with phalloidin to visualize the cytoskeleton. Fluorescent images were captured and tube formation was quantified using the ‘Angiogenesis Analyzer’ plugin in Image J to evaluate angiogenic potential. Phalloidin staining highlighted the actin cytoskeleton, with extensive tubular networks observed in VEGF treated wells, and disrupted or minimal networks under sFLT-1. Primed K-Exo A treatments showed lack of tube formation with results similar to sFLT-1 treatments, thus indicating that primed K- Exo A is anti-angiogenic. FIG. 16E shows a plot of the number of junctions observed in phalloidin-stained HUVECs grown under the four treatment conditions noted above, FIG. 16F shows a plot of the total segment length observed in the phalloidin-stained HUVECs, and FIG. 16G shows a plot of the mean mesh size in the phalloidin-stained HUVECs. In all three plots (as shown in FIGS. 16E-16G), the effect of primed K-Exo A treatment was similar to that of sFLT-1 treatment, in having an anti -angiogenic effect.

Claims

CLAIM1. A method for producing exosomes, comprising:(a) culturing mesenchymal stem cells (MSCs) in a collection medium to obtain a conditioned medium (CM) of said MSCs;(b) concentrating the CM using Tangential flow filtration (TFF) to obtain a concentrated secretome.

2. The method of claim 1 , wherein the MSCs are cultured in the collection medium for 2 or 3 days.

3. The method of claim 1 or claim 2 wherein the CM is concentrated using the TFF to reduce the volume of the CM to between about 1 / 10 and about 1 / 100 of the initial volume.

4. The method of any one of claim 1-3, further comprising (c) subjecting the concentrated secretome to a pelleting ultracentrifugation to obtain a purified secretome.

5. The method of claim 4, wherein the pelleting ultracentrifugation is performed at a spin strength of between about 100,000 x g and about 130,000 x g for a duration of between about 2 hour and about 4 hours.

6. The method of claim 4 or claim 5, further comprising (d) subjecting the purified secretome to a density gradient centrifugation.

7. The method of claim 6, wherein the density gradient centrifugation is performed at at 90,000 x g to 110,000 x g for a period of 4-18 hours.

8. The method of any one of claims 1-7, wherein the MSCs are primed by culturing the MSCs in the presence of at least one priming agent prior to the MSCs being cultured in the collection medium.

9. The method of claim 8, wherein the MSCs are cultured in the presence of the at least one priming agent for a period of at least 20 hours.

10. The method of any one of claims 1 to 4, wherein said MSCs are selected from the group consisting of: induced pluripotent stem cell-derived MSCs (iMSCs), corneal limbal stem cell-derived MSCs, umbilical cord-derived MSCs (UCMSCs), Bone marrow-derived MSCs (BMMSCs), Wharton’s jelly-derived MSCs (WJMSC), Dental pulp-derived MSCs (DPMSCs), Adipose tissue derived-MSCs (ADMSCs), and combinations thereof.

11. The method of claim 8, wherein the MSCs are primed by a method comprising: i. expanding unprimed cells in an expansion medium for a period of 3 to 5 days to obtain expanded unprimed cells; ii. passaging a first portion of the expanded unprimed MSCs to a growth medium and culturing for a period of 2-3 days to obtain secretome of unprimed cells; and iii. culturing a second portion of the expanded unprimed MSCs in a priming medium for a period of 20 to 168 hours in the presence of the at least one priming agent to obtain the primed MSCs, and washing the primed MSCs to remove the priming agent.

12. The method of claim 11, wherein the priming agent is selected from a chemical substance; a secretome of unprimed cells; hypoxia; or combinations thereof.

13. The method of claim 12, wherein the priming agent comprises the chemical substance, and wherein the chemical substance is selected from the group consisting of: Nicotinamide adenine dinucleotide (NAD), Nicotinamide mononucleotide (NMN), Nicotinamide riboside (NR), SRT-1720, SRT-2104, / ra / rs-Resveratrol, Methyl 2-(N- (5-bromo-4-fluoro-2-methylphenyl)sulfamoyl)-5-(3,5- dichlorophenylsulfonamido)benzoate (MDL 800; SIRT6), Isoquyesercetin, Fucoidan, 4-Octyl itaconate (Nrf2 activator), CDDO-Im (Nrf2 activator), Dimethyl fumarate (DMF) (Nrf2 activator), Curcumin, Berberine, Luteolin, Quercetin, 5-aminoimidazole- 4-carboxamide-l-P-D-ribofuranoside (AICAR), Thienopyridone (A-769662), Metformin, All-trans retinoic acid (ATRA), Rapamycin, ML228 (CAS No. 1357171- 62-0) , Succinate, 5 -Phenyl alkoxy psoralen (Psora-4), 5-azacytidine (5-Aza), 4-N-[2- benzyl-7-(2-methyltetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl]cyclohexane-l,4- diamine (UM171), (4-(4-fluorophenyl)-2-(4-methylsulfmylphenyl)-5-(4-pyridyl)-imidazole) (SB203580), Fisetin, Atorvastatin, Valproic acid, Sphingosine- 1 -phosphate (SIP), Astaxanthin (ATX), or Polyunsaturated fatty acids (Arachidonic acid, Eicosapentaenoic acid, Docosahexaenoic acid, and combinations thereof.

14. The method of claim 12 or claim 13, wherein the priming agent comprises the secretome of the unprimed cells, and wherein the unprimed cells are selected from the group consisting of: MSCs derived from iMSCs, UCMSCs, BMMSCs, WJMSCs, DPMSCs, Corneal stromal stem cells (CSSCs), Neural progenitor cells (NPCs), liver progenitor cells, lung progenitor cells, and combinations thereof.

15. The method of claim 12, wherein the priming agent is the hypoxia, and wherein said hypoxia is by exposure to reduced oxygen at a concentration in the range of 0.5- 10%.

16. The method of claim 12, wherein the priming agent is a combination selected from the group consisting of: a SIRT1 activator and a Nrf2 activator in the presence of hypoxia; SRT-2104 in the presence of hypoxia; a Nrf2 activator in the presence of hypoxia; ATRA in the presence of hypoxia; and a SIRT1 inducer and ATRA in the presence of hypoxia.

17. The method of claim 12, wherein said priming agent is a combination selected from the group consisting of: a secretome of unprimed CSSCs and aNrf2 activator; and a secretome of unprimed NPCs.

18. The method of any one of claims 8-15, wherein said MSCs are immortalized cells.

19. The method of claim 16, wherein the immortalized cells are characterized by expression of a protein selected from the group consisting of: SV40 large T antigen (SV40LT), human telomerase reverse transcriptase (hTERT), or a combination thereof.

20. The method of any one of claims 1-17, wherein said method comprises, prior to concentrating the CM using TFF, subjecting said CM to centrifugation to collect supernatant.

21. The method of claim 19, wherein the centrifugation is serial centrifugation comprising a first centrifugation step performed at 750 x g, a second centrifugation performed at 2,000 x g, and a third centrifugation step performed at 10,000 x g.

22. The method of any one of claims 1 or 2, wherein said exosomes comprise a cargo molecule selected from the group consisting of: Nrf2, HIF-la, VEGF, sFLTl, IL- 10, SIRT1, COX-2, HIF-1, CXCR4, CCR2, VEGF, Angiopoietin, HGF, IDO, NGF, BDNF, SDFla, and combinations thereof.

23. The method of any one of claims 1-22, wherein the CM is characterized by a marker selected from the group consisting of: Nrf2, HIF-la, VEGF, sFLTl, IL-10, SIRT1, COX-2, HIF-1, CXCR4, CCR2, VEGF, Angiopoietin, HGF, IDO, NGF, BDNF, SDFla, and combinations thereof.

24. The method of any one of claims 1-23, wherein the MSCs are characterized by at least one positive marker selected from the group consisting of: CD44, CD90, CD105, CD73, CD90, CD146, CD107a, CXCR4, LepR, CD98, CD141, Oct-4 / HLA- G, CD46, CD55, CD152, CD153, Fas ligand, CD205, CD106, CD24, CD54, CD59, CD243, P-gly coprotein, CD81, CD47, CD276, CD 151, CD 147, CD58, CD273, CD54, CD119, CD268, CD133, CD106, and combinations thereof.

25. The method of any one of claims 1-24, wherein the MSCs are characterized by at least one negative marker selected from the group consisting of: GSTT1, CD 142, CD49c, CD143, CDKN2A pl6, CD13, CCL2 / CCL5 / IL-8 (low), EGF, PDGF, TGF-b2, b-FGF, CD34, CD45, CD31, CD80, CD86, and combinations thereof.

26. The method of any one of claims 1-25, wherein the exosomes comprises a cargo molecule selected from at least one mRNA molecule encoding a protein selected from the group consisting of: BMP15, COL4A2, CXCR7, IL1RN, IRF6, ADAM15, ADM2, BCL6B, BDH2, CDC14B, CLEC2A, CRLF1, CTNNA1, EPX, FUT3 gene, IBSP gene, LTA4H, MAGED2, MSN, NIN, RAB5A, RBL-1, S100A13, SCNN1G, SENP2, TOPORS, hTERT, TGF-beta R2, and combinations thereof.

27. The method of any one of claims 1-26, wherein the exosomes comprises a cargo molecule selected fromat least one miRNA molecule selected from the group consisting of: miR-1246, miR-126, miR-133b, miR-145, miR-146, miR-155, miR184, miR-205, miR-21, miR-23b, miR-29c, miR-455-3p, Let-7, IncRNA, Malatl, miR-122, miR-101, miR-127-3p, miR-130, miR-150, miR-15a, miR-15b, miR-16, miR-181a, miR-195, miR-451a, miR-497, miR-145, miR-361-5p, miR-200b, miR-100, miR-29 (b and c), miR-22, miR-210, miR-lOb, miR-126-5p, miR-9, miR-105, miR-214, miR-136, miR- 140, miR-181-5p, miR-223, miR-494, miR-22, miR-lOb, miR-143, miR-378e, miR- 499a-5p, miR-98-5p, miR-3202, miR-32-5p, miR-363-5p, miR-548j-3p, miR-219a-5p, miR-450a-5p, miR-516b, miR-495, miR-218-5p, miR-101, miR-132, miR-196a, miR- 1246, miR-515-5p, miR-342-5p, miR-574-5p, miR-4787-3p, miR-25-3p, miR-193a, miR-567, miR-548a-5p, miR-483-3p, miR-24, miR-204, miR-509-3p, miR-424, miR- 297, miR-92b, miR-346, miR-150-5p, miR-204-p, miR-30a-5p, miR-487b, miR-34c- 5p, miR-34b, miR-663, miR-503, miR-181c, miR-595, miR-200a, miR-7-1, miR-149, miR-188-3p, miR125b-5p, miR-143-3p, miR-155-5p, miR-21-5p, miR-1910-3p, miR- 124-3p, Let-7c, Let-7b-5p, miR-183-5p, miR-375, miR-99b, miR-34a, miR-122, miR-124, miR-127, miR-27b-3p, miR-19a, miR-17, miR-31, miR-126, miR-145, miR-221, miR-222, miR-320, miR-424, miR-144, and combinations thereof.

28. The method of any one of claims 1-27, wherein the TFF is performed using a TFF membrane filter selected from a polyether sulfones (PES) membrane filter, a modified polyether sulfones (mPES) membrane filter, a poly sulfones (PS) membrane filter, and a mixed cellulose esters (ME) membrane filter. having pore size of 0.5 pm- 0.8 pm, and molecular weight cut-off in the range 100 kDa- 750 kDa.

29. The method of any one of claims 1-28, wherein sheer force of feed stream, in said TFF, is maintained below about 2000 s-1.

30. The method of claim 29, wherein the TFF is performed with an input flow rate between about 80 mL / min and about lOOmL / min.

31. The method of any one of claims 6-30, wherein the density gradient centrifugation is iodixanol gradient centrifugation or sucrose gradient centrifugation.

32. The method of claim 31 wherein said purified exosome fraction is obtained as a fraction in an iodixanol gradient having a density range between 1.13g / mL and 1.18g / mL.

33. The method of claim 32 wherein said purified exosome fraction is obtained as fraction 9 in an iodixanol gradient.

34. The method of any one of claims 6-33, further comprising washing the purified exosome fraction by centrifuging at 90,000 x g to 110,000 x g .

35. The method of any one of claims 1-34 wherein the MSCs are cultured in a bioreactor comprising microcarriers for cell attachment, wherein cell seeding density is in the range of 11,000 to 70,000 cells / mL, and area density of microcarrier surface is in the range of 2222 to9333 cells / cm2.

36. A population of exosomes obtained by a method claimed in any one of claims 1-35.

37. A composition comprising the exosomes obtained by a method claimed in any one of claims 1-35.

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