Exosome Isolation Methods

Abstract

In certain exemplary embodiments, described herein are methods for isolating exosomes from biological fluids, such as those containing casein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to co-pending U.S. Provisional Patent Application No. 63 / 152,784, entitled "Exosome Isolation Method," filed February 23, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] Statement on Federally Sponsored Research This invention was made with Government support under Grants HL161237, HL056728, and HL141855 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] The subject matter disclosed herein relates generally to the isolation and preparation of exosomes. [Background technology]

[0004] Exosomes are membrane-bound nanovesicles released by cells and function as an evolutionarily conserved mechanism of long-distance intercellular signaling (Boulanger, 2017). In humans and other mammals, exosomes are secreted into the extracellular environment by almost all cell types and are abundant in most biological fluids, including blood, lymph, urine, and milk (Gyorgy, BS (2011). Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cellular and molecular life sciences, 2667). Exosomes are relatively uniform and small in size, ranging from 50 to 150 nm in diameter, and show preferential expression of many proteins, including CD81, CD9, and syntenin, but not others, such as calnexin (Vlassov, AM (2012). Exosomes: Current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. BBA-General Subjects, 940-947). That said, exosome composition can vary substantially, reflecting the cell type and / or physiological state from which they are secreted (Blaser MC, AE (2018). Roles and Regulation of Extracellular Vesicles in Cardiovascular Mineral Metabolism. Front Cardiovasc Med).Exosome cargo includes lipids, proteins, and nucleotide sequences (e.g., microRNAs), which may be encapsulated internally or present as external moieties such as receptors or adhesion molecules on the vesicle membrane (Rana, SZ (2011). Exosome target cell selection and the importance of exosomal tetraspanins: a hypothesis. Biochem Soc Trans, 559-562). The ability of exosomes to transport and protect biological signaling molecules in vivo has revealed their potential as a drug delivery platform, attracting the attention of the pharmaceutical industry. This attraction is further strengthened by the unique ability of certain exosome populations to cross tissue boundaries such as the skin barrier (Carrasco E,S.-HG(2019).The role of extracellular vesicles in Cutaneous Remodeling and Hair Follicle Dynamics.Int J Mol Sci,2758), blood-brain-barrier (Yang T,MP(2015).Exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio rerio.Pharm Res,2003-2014), and gut-blood barrier (Vashisht M,RP(2017).Curcumin encapsulated in milk exosomes resists human digestion and possesses enhanced intestinal permeability in vitro.Applied Biochem Biotechnol,993-1007).Exosomes also appear to evade immune surveillance and have been reported to be immunologically well tolerated when introduced autologously between individuals and species (Antes TJ, MR (2018). Targeting extracellular vesicles to injured tissue using membrane cloaking and surface display. J Nanobiotechnology) - further stimulating interest in their potential clinical application as a novel means to improve the safety of drug delivery. A limitation to the clinical and practical use of exosomes is the lack of availability of cost-effective purification methods, especially for large quantities. Thus, there is a need for improved methods of exosome isolation, especially methods that can be scaled to industrial-scale exosome preparations.

[0005] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. Summary of the Invention

[0006] In certain exemplary embodiments herein, a method is described for isolating exosomes (also called small extracellular vesicles) from a biological fluid, the method comprising the steps of centrifuging the biological fluid under conditions suitable for separating fat from one or more other components of the biological fluid, removing the separated fat from the biological fluid, centrifuging the remaining biological fluid one or more times after step (b) and skimming significantly separated fat after each centrifugation of step (c), filtering the remaining biological fluid after step (c) and optionally performing one or more ultracentrifugation steps after (d), and optionally performing one or more ultracentrifugation steps after (d) or (e). and optionally, after (e), chelating the divalent cations with about 10 mM to about 100 mM EDTA at about 30-42 degrees Celsius for about 15-120 minutes; and after (f), optionally performing tangential flow filtration to obtain a retentate, which is optionally ultracentrifuged by one or more ultracentrifugation steps or stored at -80°C, and optionally fractionating the retentate by column separation after the retentate has been ultracentrifuged or stored at -80°C, wherein the method includes step (e) or step (g), but not both.

[0007] In certain exemplary embodiments, divalent cation chelation occurs at about 30 mM EDTA.

[0008] In certain exemplary embodiments, divalent cation chelation occurs at about 37 degrees Celsius.

[0009] In certain exemplary embodiments, chelation of divalent ions at about 37 degrees occurs for 60 minutes.

[0010] In certain exemplary embodiments, the biological fluid is mammalian milk.

[0011] In certain exemplary embodiments, the biological fluid is not pasteurized.

[0012] In certain exemplary embodiments, steps (a) and (b) are repeated a combined number of times from 1 to 5.

[0013] In certain exemplary embodiments, steps (a), (b), (c), (d), (e), (g), or any combination thereof, are performed at about 4 degrees Celsius.

[0014] In certain exemplary embodiments, (a) comprises centrifuging the biological fluid at about 2,000-3,000 rcf. In certain exemplary embodiments, (a) comprises centrifuging the biological fluid at about 2,500 rcf.

[0015] In certain exemplary embodiments, step (a) is repeated 1 to 3 times.

[0016] In certain exemplary embodiments, (b) comprises a first centrifugation followed by a second centrifugation. In certain exemplary embodiments, the first centrifugation comprises centrifuging the remaining biological fluid at about 13,500-15,500 rcf for about 45-75 minutes. In certain exemplary embodiments, the first centrifugation comprises centrifuging the remaining biological fluid at about 14,500 rcf for about 60 minutes. In certain exemplary embodiments, a second centrifugation is performed on the biological fluid remaining after the first centrifugation, the second centrifugation being performed at about 24,800-26,800 rcf for about 45-75 minutes. In certain exemplary embodiments, a second centrifugation is performed on the biological fluid remaining after the first centrifugation, the second centrifugation being performed at about 25,800 rcf for about 60 minutes. In certain exemplary embodiments, the second centrifugation is repeated 1-3 times, each repeat being performed on the remaining biological fluid from the immediately preceding centrifugation.

[0017] In certain exemplary embodiments, (d) comprises filtering the remaining biological fluid through one or more filters in series ranging from about a 0.45 micron filter to about a 0.22 micron filter, in certain exemplary embodiments, (d) comprises filtering the remaining biological fluid through an about 0.45 micron filter, followed by filtering the remaining biological fluid through an about 0.22 micron filter.

[0018] In certain exemplary embodiments, (e) includes two or more successive ultracentrifugation steps, each step being performed on the remaining biological fluid from the previous ultracentrifugation. In certain exemplary embodiments, (e) includes an ultracentrifugation step performed at about 45,000-55,000 rcf, an ultracentrifugation step performed at about 65,000-75,000 rcf, an ultracentrifugation step performed at about 90,000-110,000 rcf, or a combination thereof. In certain exemplary embodiments, (e) includes an ultracentrifugation step performed at about 50,000 rcf, an ultracentrifugation step performed at about 70,000 rcf, an ultracentrifugation step performed at about 100,000 rcf, or a combination thereof. In certain exemplary embodiments, one or more of the one or more ultracentrifugation steps are each performed for about 45-75 minutes. In certain exemplary embodiments, one or more of the one or more ultracentrifugation steps are each performed for about 60 minutes.

[0019] In certain exemplary embodiments, (e) comprises a final ultracentrifugation step carried out at about 115,000-145,000 rcf for about 90-150 minutes, the resulting fluid is discarded, and the remaining pellet is resuspended in a suitable volume of a suitable solution prior to (f). In certain exemplary embodiments, (e) comprises a final ultracentrifugation step carried out at about 130,000 rcf for about 120 minutes, the resulting fluid is discarded, and the remaining pellet is resuspended in a suitable volume of a suitable solution prior to (f).

[0020] In certain exemplary embodiments, the tangential flow filtration of (g) is performed using an ultrafiltration membrane having a cutoff in the range of about 250 kDa to about 750 kDa. In certain exemplary embodiments, the tangential flow filtration of (g) is performed using a 250 kDa ultrafiltration membrane.

[0021] In certain exemplary embodiments, the tangential flow filtration of (g) is performed at a flow rate of about 5-15 mL per minute. In certain exemplary embodiments, the tangential flow filtration of (g) is performed at a flow rate of about 10 mL per minute.

[0022] In certain exemplary embodiments, in step (g), when the amount of remaining biological fluid reaches about 10 percent of the starting volume before tangential flow filtration, the retentate is diafiltered with a suitable buffer.

[0023] In certain exemplary embodiments, the method further comprises ultracentrifuging the retentate when the retentate reaches about 20 percent of the starting diafiltration volume. In certain exemplary embodiments, the ultracentrifugation is performed at about 115,000-145,000 rcf at about 4 degrees Celsius for about 90-150 minutes. In certain exemplary embodiments, the ultracentrifugation is performed at about 130,000 rcf at about 4 degrees Celsius for about 120 minutes.

[0024] In certain exemplary embodiments, the method does not include ultracentrifuging the retentate when the retentate reaches about 20 percent of the starting diafiltration volume. In certain exemplary embodiments, the retentate is stored at -80°C after the retentate reaches about 20 percent of the starting diafiltration volume and prior to column separation.

[0025] In certain exemplary embodiments, the method produces an exosome concentrate that is at least 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or at least 20 percent of the starting amount of milk.

[0026] In certain exemplary embodiments, the method further comprises loading the exosomes of the preparation resulting from the method of any one of the preceding claims with one or more cargoes.

[0027] Certain exemplary embodiments described herein include formulations, and in some cases pharmaceutical formulations, which are produced at least in part or in whole by the methods described herein. In certain exemplary embodiments, one or more exosomes are loaded with one or more cargoes.

[0028] In certain exemplary embodiments, methods are described herein that include administering to a subject in need thereof a formulation comprising exosomes and / or a formulation described herein, such as a formulation produced by the exosome isolation methods described herein, in certain exemplary embodiments, the one or more cargoes are therapeutic cargoes.

[0029] These and other aspects, objects, features and advantages of the exemplary embodiments will become apparent to those of ordinary skill in the art upon consideration of the following detailed description of the exemplary embodiments. [Brief description of the drawings]

[0030] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention can be utilized, and is accompanied by the following drawings. [Figure 1] Overview of the steps in one embodiment of an ultracentrifugation-based method for isolating exosomes from milk. [Diagram 2] Overview of the steps in one embodiment of a tangential flow filtration-based method for isolating exosomes from milk. [Diagram 3]A-E are embodiments of an ultracentrifugation-based protocol for exosome purification. Figure 3A shows successive nanodrop fractions collected during this SEC filtration step, with protein concentration in mg / ml shown on the y-axis. Figure 3B shows Western blots for exosomal markers CD-81, CD-9, and syntenin, as well as non-exosomal markers casein, Arf6 (a microvesicle marker), and calnexin (a cell membrane marker). The peak exosomal SEC fraction occurs between fractions 8 and 9. Contaminating proteins, including casein, become predominant from fraction 12 onwards. Lysate from HeLa cells is included as a comparative control. Figure 3C shows Nanosight Tracker analysis data for exosome isolates. Figure 3D shows negative stain electron microscopy of the final exosome isolates and (Figure 3E) negative stain electron microscopy of the final exosome isolates and (Figure 3E) negative stain electron microscopy of the casein isolates. [Figure 4] A-D: Summary of the analysis of the TFF-based protocol. Figure 4A: Nanodrops resulting from the optimized TFF-UC protocol show associated exosome and protein fractions. Figure 4B: Western blot analysis of exosome markers CD-81, CD-9 and syntenin, as well as interfering protein marker casein, microvesicle markers Arf6 and calnexin. These results indicate high purity of exosomes without interfering proteins or microvesicles. (Figure 4C) Nanosight Tracker analysis data of the exosome isolate. Negative staining electron microscopy of the final isolate showing (Figure 4D) ultra-dense accumulation of exosomes in the peak SEC fraction and (Figure 4D) high levels of casein macrostructures in the peak protein fraction, concentrations as indicated by NTA analysis. [Diagram 5]Calcein uptake into isolated milk exosomes. Peak exosome containing SEC fractions generated by the TFF-based method were diluted 1:10 in Hepes buffer. Images show uptake following 1, 2, 3, and 4 hours of incubation with Calcein-AM. Dye uptake suggests that extracellular vesicles contain esterase activity and can retain de-esterified calcein molecules. [Figure 6] Representative photographs and TEM images showing the isolation of milk exosomes. The top left photographic image shows the starting volume of milk (1 L) and the bottom left photographic image shows a typical post-isolation volume of approximately 125 mL of exosome concentrate obtained by TFF-based exosome isolation. The TEM image on the right shows a representative high magnification image of TFF-isolated exosomes. The inset TEM image shows a high magnification of standard exosomes. The representative TEM image is after SEC and exosomes contain fraction number 8.5 from Figure 4A. [Figure 7] Figures 7A and 7B are schematics of exosome loss during ultracentrifugation protocols. Large amounts of exosomes are lost during UC protocols, highlighting the need to reduce the UC spin and incorporate TFF into the procedure. Figure 7A shows a histogram of the 100,000 rcf pellet separated by SEC with a negative stained EM image of the peak fractions 8.0-9.0 shown below. Figure 7B shows a histogram of the 70,000 rcf pellet separated by SEC with a negative stained EM image of the peak fractions 8.0-9.0 shown below. [Figure 8] Summary of exosome loss during ultracentrifugation protocol after a 130,000 RCF spin. A large number of exosomes were left in the supernatant at the end of the UC protocol, highlighting the need for TFF reduction of interfering proteins to optimize efficiency. [Figure 9]A-D show the effect of various aspects of temperature-assisted chelation on exosome samples. Raw "gold standard" conventional UC isolated exosome inserts show high casein micelle structure. Figure 9B shows the complete "gold standard" conventional isolation combined with EDTA treatment without utilizing either SEC or TFF filtration. Figure 9C shows the results from the modified UC protocol of the present disclosure without EDTA treatment at 37°C for 1 hour and SEC filtration. Figure 9D shows the results from the modified UC protocol of the present disclosure treated with 30 mM EDTA at room temperature (20°C) and SEC filtration. [Figure 10] Treatment with 10–100 mM EDTA at 37 °C not only results in overall exosome loss, but also damages the exosome ultrastructure as shown in the black box, reducing the efficiency of exosome isolation. [Figure 11A] FIG. 1 shows an embodiment of an ultracentrifugation method for isolating exosomes from human breast milk. A graph is shown showing the concentration of exosomes or protein (x-axis) in each fraction in mg / mL (y-axis). [Figure 11B] Isolation of exosomes from human breast milk by an embodiment of the ultracentrifugation method. Nanosight Tracker analysis data of exosome isolates is shown. [Figure 11C] Isolation of exosomes from human breast milk by an embodiment of the ultracentrifugation method. High magnification TEM images of isolated exosomes (FIG. 11C) and representative TEM images of different exosome fractions (FIG. 11D) are shown. [Figure 11D] Isolation of exosomes from human breast milk by an embodiment of the ultracentrifugation method. High magnification TEM images of isolated exosomes (FIG. 11C) and representative TEM images of different exosome fractions (FIG. 11D) are shown. [Figure 12] Representative TEM images showing the effect of post-SEC storage on isolated exosomes. Storage conditions are specified below the TEM images. [Figure 13]A and B are representative TEM images of isolated exosomes after storage prior to SEC. Figure 13A shows freshly isolated exosomes not stored at -80°C. Figure 13B shows isolated exosomes after TFF and prior to SEC stored at -80°C for 6 months prior to SEC.

[0031] The figures herein are for illustrative purposes only and are not necessarily drawn to scale. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Before describing the present disclosure in detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, the preferred methods and materials are now described.

[0034] All publications and patents cited herein are for the purpose of disclosing and describing the methods and / or materials for which the publications are cited. All such publications and patents are incorporated herein by reference as if each individual publication or patent was specifically and individually incorporated by reference. Such incorporation by reference is expressly limited to the methods and / or materials described in the cited publications and patents and does not extend to the lexicographical definitions of the cited publications and patents. Lexicographical definitions in cited publications and patents that are not expressly repeated in this application should not be treated as such and should not be read as defining terms that appear in the appended claims. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the scope of the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, dates of publication provided may be different from the actual publication dates and may need to be independently confirmed.

[0035] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0036] When a range is expressed, a further embodiment includes from the one particular value and / or to the other particular value. When a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit, and any other stated or intervening value in that stated range, is encompassed in the disclosure, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded value in the stated range. When a stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the disclosure. For example, when a stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the disclosure, for example, the expression "from x to y" includes the range from "x" to "y", as well as greater than "x" and less than "y". The ranges may also be expressed as upper limits, such as "about x, y, z, or less," and should be interpreted to include the specific ranges of "about x," "about y," and "about z," as well as the ranges "less than x," "less than y," and "less than z." Similarly, the phrase "about x, y, z, or more" should be interpreted to include the specific ranges of "about x," "about y," and "about z," as well as the ranges "greater than x," "greater than y," and "greater than z." Additionally, the phrase "about 'x' to 'y'," where "x" and "y" are numerical values, includes "about 'x' to about 'y'."

[0037] It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It will be further understood that each endpoint of a range expresses meaning not only in relation to the other endpoint, but also independently of the other endpoint. It is also understood that there are several values ​​disclosed herein, and that each value is also disclosed herein as "about" that particular value in addition to the value itself. For example, if a value of "10" is disclosed, then "about 10" is also disclosed. Ranges can be expressed herein as from "about" one particular value and / or to "about" another particular value. Similarly, when values ​​are expressed as approximations, using the antecedent "about," it will be understood that the particular value forms a further aspect. For example, if a value of "about 10" is disclosed, then "10" is also disclosed.

[0038] It should be understood that such range formats are used for convenience and brevity, and thus should be interpreted flexibly to include not only the numerical values ​​explicitly recited as range limitations, but also all individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were explicitly specified. For example, a numerical range of "about 0.1% to 5%" should be interpreted to include not only the values ​​of about 0.1% to about 5% explicitly recited, but also the individual values ​​(e.g., about 1%, about 2%, about 3%, and about 4%) and subranges (e.g., about 0.5% to about 1.1%, about 5% to about 2.4%, about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible subranges) within the indicated range. General definition

[0039] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms and techniques in molecular biology can be found in Molecular Cloning: A Laboratory Manual, 2 ndedition(1989)(Sambrook,Fritsch,and Maniatis);Molecular Cloning:A Laboratory Manual,4 th edition(2012)(Green and Sambrook);Current Protocols in Molecular Biology (1987)(F.M.Ausubel et al.eds.);the series Methods in Enzymology(Academic Press,Inc.):PCR 2:A Practical Approach(1995)(M.J.MacPherson,B.D.Hames,and G.R.Taylor eds.):Antibodies,A Laboratory Manual(1988)(Harlow and Lane,eds.):Antibodies A Laboratory Manual,2 ndedition 2013(EAGreenfield ed.);Animal Cell Culture(1987)(RIFreshney,ed.);Benjamin Lewin,Genes IX,published by Jones and Bartlet,2008(ISBN 0763752223);Kendrew et al.(eds.),The Encyclopedia of Molecular Biology,published by Blackwell Science Ltd.,1994(ISBN 0632021829); Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley&Sons(New York, NY1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4thed., John Wiley & Sons (New York, NY1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2 nd This can be found in the 2011 edition.

[0040] As used herein, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include both singular and plural referents.

[0041] As used herein, "about," "approximately," "substantially," and the like, when used in connection with a measurable variable, such as a parameter, amount, time duration, and the like, are meant to encompass variations within experimental error and within a range of from a particular value (which may be determined, for example, by a given data set, art-recognized standards, and / or with, for example, a given confidence interval (e.g., a 90%, 95%, or greater confidence interval from the mean)) to the extent that such variations are appropriate to carry out the disclosed invention, such as variations of + / -10% or less, + / -5% or less, + / -1% or less, and + / -0.1% or less from a specified value. As used herein, the terms "about," "approximately," "or about," and "substantially" may mean that the amount or value in question may be an exact value, or a value that will produce results or effects equivalent to those described or taught in the claims herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not, and need not be, exact, but may be approximate and / or larger or smaller, as appropriate, to reflect tolerances, conversion factors, rounding, measurement errors, and the like, and other factors known to those of ordinary skill in the art that would produce equivalent results or effects. In some circumstances, it may not be possible to reasonably determine a value that would produce equivalent results or effects. Generally, amounts, sizes, formulations, parameters, or other quantities or characteristics are "about," "approximately," or "nearly," whether or not expressly stated as such. When "about," "approximately," or "or about" is used before a quantitative value, it is understood that the parameter also includes the specific quantitative value itself, unless otherwise specified.

[0042] As used herein, the terms "optionally" or "optionally" mean that the subsequently described event or circumstance may or may not occur, and the description includes cases where the event or circumstance occurs and cases where the event or circumstance does not occur.

[0043] The recitation of numerical ranges by endpoints includes all values ​​and fractions subsumed within each range, as well as the recited endpoints.

[0044] As used herein, a "biological sample" may include whole cells and / or live cells and / or cell debris. A biological sample may include (or be derived from) a "biological fluid." The invention encompasses embodiments in which the biological fluid is selected from amniotic fluid, aqueous humor, vitreous biological fluid, bile, serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudate, feces, female semen, gastric acid, gastric juice, lymph, mucus (including nasal mucus, phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rhubarb, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vomit, and mixtures of one or more thereof. Biological samples include cell cultures, biological fluids, and cell cultures from biological fluids. Biological fluids may be obtained from a mammal, for example, by puncture or other collection or sampling procedures.

[0045] "Subject," "individual," or "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, livestock, game animals, and pets. Also included are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro.

[0046] Various embodiments are described below. It should be noted that a particular embodiment is not intended to be exhaustive or limiting to the broader aspects discussed herein. An aspect described in connection with a particular embodiment is not necessarily limited to that embodiment, but may be practiced in any other embodiment. Throughout this specification, reference to "one embodiment," "an embodiment," or "an example embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present disclosure. Thus, the appearance of the phrases "in one embodiment," "in an embodiment," or "in an example embodiment" in various places throughout this specification do not necessarily all refer to the same aspect, although they may. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments. Furthermore, some embodiments described herein include some features included in other embodiments but not other features, meaning that combinations of features of different embodiments are within the scope of the invention. For example, in the appended claims, any of the claimed embodiments may be used in any combination.

[0047] All publications, published patent documents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, published patent document, or patent application was specifically and individually indicated to be incorporated by reference. overview

[0048] Exosomes are membrane-bound nanovesicles released by cells and function as an evolutionarily conserved mechanism of long-distance intercellular signaling (Boulanger, 2017). In humans and other mammals, exosomes are small extracellular vesicles secreted into the extracellular environment by almost all cell types and are abundant in most biological fluids, including blood, lymph, urine, and milk (Gyorgy, BS 2011).Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cellular and molecular life sciences, 2667). Exosomes are relatively uniform and small in size, ranging from 50 to 150 nm in diameter, and show preferential expression of many proteins, including CD81, CD9, and syntenin, but not others, such as calnexin (Vlassov, AM (2012).Exosomes: Current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. BBA-General Subjects, 940-947). That said, exosome composition can vary substantially, reflecting the cell type and / or physiological state in which they are secreted (Blaser MC, AE (2018). Roles and Regulation of Extracellular Vesicles in Cardiovascular Mineral Metabolism. Front Cardiovasc Med.). Exosomal cargo includes lipids, proteins, and nucleotide sequences (e.g., microRNAs), which can be encapsulated internally or present as external moieties such as receptors and adhesion molecules on the vesicle membrane (Rana, SZ (2011). Exosome target cell selection and the importance of exosomal tetraspanins: a hypothesis. Biochem Soc Trans, 559-562).The ability of exosomes to transport and protect biological signaling molecules in vivo has attracted the attention of the pharmaceutical industry, as they have been shown to be useful as a drug delivery platform. This attraction is further strengthened by the unique ability of certain exosome populations to cross tissue boundaries such as the skin barrier (Carrasco E,S.-HG(2019).The role of extracellular vesicles in Cutaneous Remodeling and Hair Follicle Dynamics.Int J Mol Sci,2758), blood-brain-barrier (Yang T,MP(2015).Exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio rerio.Pharm Res,2003-2014), and gut-blood barrier (Vashisht M,RP(2017).Curcumin encapsulated in milk exosomes resists human digestion and possesses enhanced intestinal permeability in vitro.Applied Biochem Biotechnol,993-1007). Exosomes also appear to evade immune surveillance and have been reported to be immunologically well tolerated when introduced autologously between individuals and species (Antes TJ, MR (2018). Targeting extracellular vesicles to injured tissue using membrane cloaking and surface display. J Nanobiotechnology) - further stimulating interest in their potential clinical application as a novel means to improve the safety of drug delivery.

[0049] There are numerous methods to isolate exosomes from biological fluids, but the current "gold standard" technique is based on ultracentrifugation (UC). These methods usually include differential centrifugation steps and / or density gradient UC-based separation. However, the ability to generate large amounts of exosomes is limited by the need for multiple UC steps and the fact that only small amounts of UC can be spun. Furthermore, it remains that the shear forces applied during repeated UC spins may adversely affect the structural integrity of exosomes (Taylor DD, SS (2015). Methods of isolating extracellular vesicles impact downstream analyses of their cargoes. Methods, 3-10). Other techniques that are less physically stringent during exosome isolation include ultrafiltration, tangential flow filtration (TFF), size exclusion chromatography (SEC), and polyethylene glycol precipitation-based methods.The use of each is well documented, and the majority of groups use a combination of these approaches rather than one method alone (Pin Li,MK(2017).Progress in exosome isolation techniques.Theranostics,789-804;Escudier.(2005).Vaccination of metastatic melanoma patients with autologous dendritic cell derived exosomes:results of the first phase I clinical trial.J transl.med,1-13;Rood.(2010).Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome.Kidney Int,810-816; and An M.,WJ(2018).Comparison of an optimized ultracentrifugation method versus size-exclusion chromatography for isolation of exosomes from human serum.J Proteome Res,3599-3605).

[0050] Thus, a clear limitation to the clinical and practical use of exosomes is the existence of cost-effective purification methods, especially for large quantities, and there is a need for improved exosome isolation methods, especially methods that are scalable to industrial-scale exosome preparations.

[0051] That said, the embodiments disclosed herein can provide methods and techniques for exosome isolation and / or purification, particularly milk exosomes, that can provide large-scale yields of exosomes. Other compositions, compounds, methods, features, and / or advantages of the present disclosure will become apparent to one of ordinary skill in the art upon review of the following figures and detailed description, and examples. All such additional compositions, compounds, methods, features, and advantages are intended to be included in this description and are within the scope of the present disclosure.

[0052] Exosome Isolation Methods In recent years, it has become recognized that mammalian milk is rich in exosomes and may be a large-scale source of these small extracellular vesicles. Bovine milk is produced in large quantities by the dairy industry, widely consumed, and generally well immunologically tolerated by humans. In addition, milk exosomes have been reported to enter the blood circulation from the intestine and migrate to various organs, including the brain, heart, intestine, and lungs (Wolf T, BS (2015). The intestinal transport of bovine milk exosomes is mediated by endocytosis in human colon carcinoma Caco-2 cells and rat small intestinal IEC-6 cells. J Nutr, 2201-2206), properties that could provide the basis for oral administration of exosomes loaded with cargo (including but not limited to therapeutic agents). That being said, milk contains a diverse mixture of proteins, minerals, lipids, and other macromolecules. The complexity of milk components poses significant challenges in the purification of these extracellular vesicles. Casein proteins are the major components of milk, accounting for approximately 80% of the total milk protein. Casein aggregates with calcium phosphate and other milk proteins into large colloidal complexes called casein micelles. These micelles are about 10 nm in diameter and can further coalesce to form larger coagulated structures (Bhat, MT (2016). Casein Proteins: structural and functional aspects. Intech).Casein micellar aggregates are believed to bind exosomes via hydrostatic interactions, trapping them and preventing their separation from contaminating milk proteins; an observation confirmed by transmission electron microscopy (TEM) analysis of milk-derived exosome preparations (Sedykh SE, BE (2020). Milk Exosomes: Isolation, Biochemistry, Morphology, and Perspectives of Use. In CJ De Bona AG, Extracellular Vesicles and their importance in human health. Intech Open). As a result, current methods for isolating high-purity exosomes from milk are limited by contaminating proteins such as casein. Some embodiments herein describe a novel method for isolating exosomes from milk proteins in high yields using CaCl2+ at specific temperatures. 2+ Methods are described that include incorporating chelation of glycerol, glycerol, and other divalent cations. In some embodiments, a casein micelle solubilization step can be included in methods involving UC-based and / or TFF and SEC filtration steps for exosome isolation, thereby providing the basis for large-scale production of high-quality purified exosomes from milk.

[0053] In certain exemplary embodiments, a method of isolating exosomes from a biological fluid is described, comprising the steps of: (a) centrifuging the biological fluid under conditions suitable for separating fat from one or more other components of the biological fluid; (b) removing the separated fat from the biological fluid; (c) centrifuging the remaining biological fluid one or more times after step (b) and skimming significantly separated fat after each centrifugation of step (c); (d) filtering the remaining biological fluid after step (c); (e) optionally performing one or more ultracentrifugation steps after (d); and (f) after (d) or optionally (e), centrifugation with ethylenediaminetetraacetic acid (EDTA) and / or 1,2-bis(o- chelating divalent cations with other chelating agents, including, but not limited to, (aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), sodium citrate, and nitrophen; and (g)(f) optionally followed by tangential flow filtration to obtain a retentate, which is optionally ultracentrifuged by one or more ultracentrifugation steps or stored at -80°C, and optionally fractionating the retentate by column separation after the retentate has been ultracentrifuged or stored at -80°C, the method including step (e) or step (g), but not both. See, e.g., Figures 1-2.

[0054] In some embodiments, dialysis can be used in the method of reducing ion concentration. In some embodiments, the chelating agent is a divalent cation chelating agent. In some embodiments, the chelating agent is EDTA and / or other chelating agents, including but not limited to BAPDTA, EGTA, sodium citrate, nitrophen, included at equivalent chelation concentrations. In some embodiments, the concentration of the chelating agent can range from about 10 mM to about 100 mM. In some embodiments, the chelating agent is EDTA and is present at about 30 mM. In some embodiments, the divalent cation chelation occurs at about 37 degrees Celsius. In some embodiments, the divalent cation chelation occurs at about 37 degrees Celsius and is performed for about 60 minutes.

[0055] In certain examples, an embodiment is a method of isolating exosomes from a biological fluid, comprising the steps of: (a) centrifuging the biological fluid under conditions suitable for separating fat from one or more other components of the biological fluid; (b) removing the separated fat from the biological fluid; (c) centrifuging the remaining biological fluid one or more times after step (b) and skimming significantly separated fat after each centrifugation of step (c); (d) filtering the remaining biological fluid after step (c); (e) optionally performing one or more ultracentrifugation steps after (d); and (f) removing exosomes from (d) or optionally from (c) or optionally ... After (e), optionally chelating divalent cations with EDTA at about 30 mM at about 30-42 degrees Celsius for about 15-120 minutes; and after (g)(f), optionally tangential flow filtration to obtain a retentate, which is optionally ultracentrifuged by one or more ultracentrifugation steps or stored at -80°C, and optionally fractionating the retentate by column separation after the retentate is ultracentrifuged or stored at -80°C, the method including step (e) or step (g), but not both.

[0056] In certain examples, embodiments are methods of isolating exosomes from a biological fluid, comprising the steps of: (a) centrifuging the biological fluid under conditions suitable to separate fat from one or more other components of the biological fluid; (b) removing the separated fat from the biological fluid; (c) centrifuging the remaining biological fluid one or more times after step (b) and skimming significantly separated fat after each centrifugation of step (c); (d) filtering the remaining biological fluid after step (c); (e) optionally performing one or more ultracentrifugation steps after (d); and (f) filtering the remaining biological fluid after (d) or (c) optionally performing one or more ultracentrifugation steps after (d) or (d) or (b). and optionally after (e), chelating divalent cations with EDTA at about 30 mM at about 37 degrees Celsius for about 60 minutes; and after (g)(f), optionally tangential flow filtration to obtain a retentate, which is optionally ultracentrifuged by one or more ultracentrifugation steps or stored at -80°C, optionally fractionating the retentate by column separation after the retentate is ultracentrifuged or stored at -80°C, the method including step (e) or step (g), but not both.

[0057] In some embodiments, (f) is performed at about 30°C, 30.5°C, 31°C, 31.5°C, 32°C, 32.5°C, 33°C, 33.5°C, 34°C, 34.5°C, 35°C, 35.5°C, 36°C, 36.5°C, 37°C, 37.5°C, 38°C, 38.5°C, 39°C, 39.5°C, 40°C, 40.5°C, 41°C, 41.5°C, or about 42°C.

[0058] In some embodiments, (f) is about 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 41 minutes, 42 minutes, 43 minutes, 44 minutes, 45 minutes, 46 minutes, 47 minutes, 48 ​​minutes, 49 minutes, 50 minutes, 51 minutes, 52 minutes, 53 minutes, 54 minutes, 55 minutes, 56 minutes, 57 minutes, 58 minutes, 59 minutes, 60 minutes, 61 minutes, 62 minutes, 63 minutes, 64 minutes, 65 minutes, 66 minutes, 67 minutes, 68 minutes, 69 minutes, 70 minutes, 71 minutes, 72 minutes, 73 minutes, 74 minutes, 75 minutes, 76 minutes, 77 minutes, 78 minutes, 79 minutes, 80 minutes, 81 minutes, 82 minutes, 83 minutes, 84 minutes, 85 minutes, 86 minutes, 87 minutes, 88 minutes, 89 minutes, 90 minutes, 91 minutes, 92 minutes, 93 minutes, 94 minutes, 95 minutes, 96 minutes, 97 minutes, 98 minutes, 99 minutes, 100 minutes, 101 minutes, 102 minutes, 103 minutes, 104 minutes, 1 2 minutes, 53 minutes, 54 minutes, 55 minutes, 56 minutes, 57 minutes, 58 minutes, 59 minutes, 60 minutes, 61 minutes, 62 minutes, 63 minutes, 64 minutes, 65 minutes, 66 minutes, 67 minutes, 68 minutes, 69 minutes, 70 minutes, 71 minutes, 72 minutes, 73 minutes, 74 minutes, 75 minutes, 76 minutes, 77 minutes, 78 minutes, 79 minutes, 80 minutes, 81 minutes, 82 minutes, 83 minutes, 84 minutes, 85 minutes, 86 minutes, 87 minutes, 88 minutes, 89 minutes, 90 minutes, 91 minutes, 92 minutes, 93 minutes, 94 minutes, 95 minutes, 96 minutes, 97 minutes, 98 minutes, 99 minutes, 100 minutes, 101 minutes, 10 2 minutes, 103 minutes, 104 minutes, 105 minutes, 106 minutes, 107 minutes, 108 minutes, 109 minutes, 110 minutes, 111 minutes, 112 minutes, 113 minutes, 114 minutes, 115 minutes, 116 minutes, 117 minutes, 118 minutes, 119 minutes, 120 minutes, 121 minutes, 122 minutes, 123 minutes, 124 minutes, 125 minutes, 126 minutes, 127 minutes, 128 minutes, 129 minutes, 130 minutes, 131 minutes, 132 minutes, 133 minutes, 134 minutes, 135 minutes, 136 minutes, 137 minutes, 138 minutes, 139 minutes, 140 minutes, 141 minutes, 142 minutes , 143 minutes, 144 minutes, 145 minutes, 146 minutes, 147 minutes, 148 minutes, 149 minutes, 150 minutes, 151 minutes, 152 minutes, 153 minutes, 154 minutes, 155 minutes, 156 minutes, 157 minutes, 158 minutes, 159 minutes, 160 minutes, 161 minutes, 162 minutes, 163 minutes, 164 minutes, 165 minutes, 166 minutes, 167 minutes, 168 minutes, 169 minutes, 170 minutes, 171 minutes, 172 minutes, 173 minutes, 174 minutes, 175 minutes, 176 minutes, 177 minutes, 178 minutes, 179 minutes, or up to about 180 minutes.

[0059] In some embodiments, the concentration of chelating agents such as EDTA, BAPDTA, EGTA, sodium citrate, nitropen and / or the like is about 0 mM, 10.5 mM, 11 mM, 11.5 mM, 12 mM, 12.5 mM, 13 mM, 13.5 mM, 14 mM, 14.5 mM, 15 mM, 15.5 mM, 16 mM, 16.5 mM, 17 mM, 17.5 mM, 18 mM, 18.5 mM, 19 mM, 19.5 mM, 20 mM, 20.5 mM, 21 mM, 21.5 mM, 22 mM, 22.5 mM, 23 mM, 23.5 mM, 24 mM, 24.5 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, 56 mM, 57 mM, 58 mM, 59 mM, 60 mM, 61 mM, 62 mM, 63 mM, 64 mM, 65 mM, 66 mM, 67 mM, 68 mM, 69 mM, 70 mM, 71 mM, 72 mM, 73 mM, 74 mM, 75 mM, 76 mM, 77 mM, 78 mM, 79 mM, 80 mM, 81 mM, 82 mM, mM, 25.5mM, 26mM, 26.5mM, 27mM, 27.5mM, 28mM, 28.5mM, 29mM, 29.5mM, 30mM , 30.5mM, 31mM, 31.5mM, 32mM, 32.5mM, 33mM, 33.5mM, 34mM, 34.5mM, 35mM, 35 .5mM, 36mM, 36.5mM, 37mM, 37.5mM, 38mM, 38.5mM, 39mM, 39.5mM, 40mM, 40.5 mM, 41mM, 41.5mM, 42mM, 42.5mM, 43mM, 43.5mM, 44mM, 44.5mM, 45mM, 45.5mM, 46mM, 46.5mM, 47mM, 47.5mM, 48mM, 48.5mM, 49mM, 49.5mM, 50mM, 50.5mM, 51 mM, 51.5mM, 52mM, 52.5mM, 53mM, 53.5mM, 54mM, 54.5mM, 55mM, 55.5mM, 56mM, 56.5mM, 57mM, 57.5mM, 58mM, 58.5mM, 59mM, 59.5mM, 60mM, 60.5mM, 61mM, 61 .5mM, 62mM, 62.5mM, 63mM, 63.5mM, 64mM, 64.5mM, 65mM, 65.5mM, 66mM, 66.5m M, 67mM, 67.5mM, 68mM, 68.5mM, 69mM, 69.5mM, 70mM, 70.5mM, 71mM, 71.5mM, 72mM, 72.5mM, 73mM, 73.5mM, 74mM, 74.5mM, 75mM, 75.5mM, 76mM, 76.5mM, 77m M, 77.5mM, 78mM, 78.5mM, 79mM, 79.5mM, 80mM, 80.5mM, 81mM, 81.5mM, 82mM, 82.5mM, 83mM, 83.5mM, 84mM, 84.5mM, 85mM, 85.5mM, 86mM, 86.5mM, 87mM, 87.5mM, 88mM, 88.5mM, 89mM, 89.5mM, 90mM, 90.5mM, 91mM, 91.5mM, 92mM, 92.5mM, 93mM, 93.5mM, 94mM, 94.5mM, 95mM, 95.5mM, 96mM, 96.5mM, 97mM, 97.5mM, 98mM, 98.5mM, 99mM, 99.5mM, up to and / or about 100mM.

[0060] In certain exemplary embodiments, the biological fluid contains casein. In certain exemplary embodiments, the biological fluid is mammalian milk. In certain exemplary embodiments, the biological fluid is unpasteurized. In some embodiments, the mammalian milk is unpasteurized. In some embodiments, the mammalian milk is pasteurized. In some embodiments, the mammalian milk is cow's milk, sheep's milk, pig's milk, camel's milk, horse's milk, Capra's milk, human milk, and / or the like.

[0061] In certain exemplary embodiments, steps (a) and (b) are repeated 1 to 5 times combined. In some embodiments, steps (a) and (b) are repeated 1, 2, 3, 4 or 5 times combined.

[0062] In certain exemplary embodiments, steps (a), (b), (c), (d), (e), (g), or any combination thereof, are performed at or about 4 degrees Celsius.

[0063] In certain exemplary embodiments, (a) includes centrifuging the biological fluid at about 2,500 rcf. In certain exemplary embodiments, (a) includes centrifuging the biological fluid at about 2,000-3,000 rcf. In certain exemplary embodiments, (a) includes centrifuging the biological fluid at about 2000 rcf, 2010 rcf, 2020 rcf, 2030 rcf, 2040 rcf, 2050 rcf, 2060 rcf, 2070 rcf, 2080 rcf, 2090 rcf, 2100 rcf, 2110 rcf, 2120 rcf, 2130 rcf, 2140 rcf, 2150 rcf, 2160 rcf, 2170 rcf, 2180 rcf, 2190 rcf, 2200 rcf, 2210 rcf, 2220 rcf, 2230 rcf, 2240 rcf, 2250 rcf, 2260 rcf, 2270 rcf, 2280 rcf, 2290 rcf, 2300 rcf, 2310 rcf, 2320 rcf, 2330 rcf, 2340 rcf, 2350 rcf, 2360 rcf, 2370 rcf, 2380 rcf, 2390 rcf, 2400 rcf, 2410 rcf, 2420 rcf, 2430 rcf, 2440 rcf, 2450 rcf, 2460 rcf, 2470 rcf rcf, 2240rcf, 2250rcf, 2260rcf, 2270rcf, 2280rcf, 2290rcf, 2300rcf, 2310rcf, 2320rcf, 2330rcf, 2340rcf, 2350rcf, 2360rc f, 2370rcf, 2380rcf, 2390rcf, 2400rcf, 2410rcf, 2420rcf, 2430rcf, 2440rcf, 2450rcf, 2460rcf, 2470rcf, 2480rcf, 2490rcf, 2 500rcf, 2510rcf, 2520rcf, 2530rcf, 2540rcf, 2550rcf, 2560rcf, 2570rcf, 2580rcf, 2590rcf, 2600rcf, 2610rcf, 2620rcf, 263 0rcf, 2640rcf, 2650rcf, 2660rcf, 2670rcf, 2680rcf, 2690rcf, 2700rcf, 2710rcf, 2720rcf, 2730rcf, 2740rcf, 2750rcf, 2760r cf, 2770rcf, 2780rcf, 2790rcf, 2800rcf, 2810rcf, 2820rcf, 2830rcf, 2840rcf, 2850rcf, 2860rcf, 2870rcf, 2880rcf, 2890rcf, 2900rcf, 2910rcf, 2920rcf, 2930rcf, 2940rcf, 2950rcf, 2960rcf, 2970rcf, 2980rcf, 2990rcf, ~3000rcf.

[0064] In certain exemplary embodiments, step (a) is repeated 1-3 times. In some embodiments, step (a) is repeated 1-3 times.

[0065] In certain exemplary embodiments, (b) comprises a first centrifugation followed by a second centrifugation. In certain exemplary embodiments, the first centrifugation comprises centrifuging the remaining biological fluid at about 14,500 rcf for about 60 minutes. In certain exemplary embodiments, the first centrifugation comprises centrifuging the remaining biological fluid at about 13,500 rcf to about 15,500 rcf for about 45 to 75 minutes. In some embodiments, the first centrifugation centrifuges the remaining biological fluid at about 13500rcf, 13550rcf, 13600rcf, 13650rcf, 13700rcf, 13750rcf, 13800rcf, 13850rcf, 13900rcf, 13950rcf, 14000rcf, 14050rcf, 14100rcf, 14150rcf, 14200rcf, 14250rcf, 14300rcf, 14350rcf, 14400rcf, 14450rcf, 14500rcf, 14550rcf, 14600rcf, 14650rcf, 14700rcf, 14750rcf, 14800rcf, 14850rcf, 14900rcf, 14950rcf, 15000rcf, 15050rcf, 15100rcf, 15150rcf, 15200rcf, 15250rcf, 15300rcf, 15350rcf, 15400rcf, 15450rcf, or about 15500rcf for about 45 minutes, 4 The method includes centrifuging for 6 minutes, 47 minutes, 48 ​​minutes, 49 minutes, 50 minutes, 51 minutes, 52 minutes, 53 minutes, 54 minutes, 55 minutes, 56 minutes, 57 minutes, 58 minutes, 59 minutes, 60 minutes, 61 minutes, 62 minutes, 63 minutes, 64 minutes, 65 minutes, 66 minutes, 67 minutes, 68 minutes, 69 minutes, 70 minutes, 71 minutes, 72 minutes, 73 minutes, 74 minutes, or about 75 minutes.

[0066] In certain exemplary embodiments, a second centrifugation is performed on the biological fluid remaining after the first centrifugation, the second centrifugation is performed at about 25,800 rcf for about 60 minutes. In certain exemplary embodiments, a second centrifugation is performed on the biological fluid remaining after the first centrifugation, the second centrifugation is performed at about 24,800 to about 26,800 rcf for about 45 to about 75 minutes. In some embodiments, a second centrifugation is performed on the biological fluid remaining after the first centrifugation, the second centrifugation being at about 24800rcf, 24850rcf, 24900rcf, 24950rcf, 25000rcf, 25050rcf, 25100rcf, 25150rcf, 25200rcf, 25250rcf, 25300rcf, 25350rcf, 25400rcf, 25450rcf, 25500rcf, 25550rcf, 25600rcf, 25650rcf, 25700rcf, 25750rcf, 25800rcf, 25850rcf, 25900rcf, 25950rcf, 26000rcf, 26050rcf, 26100rcf, 26150rcf, 26200rcf, 26250rcf, 26300rcf, 26350rcf, 26400r cf, 26450rcf, 26500rcf, 26550rcf, 26600rcf, 26650rcf, 26700rcf, 26750rcf, 2680 At 0 rcf, it will be performed for approximately 45 minutes, 46 minutes, 47 minutes, 48 ​​minutes, 49 minutes, 50 minutes, 51 minutes, 52 minutes, 53 minutes, 54 minutes, 55 minutes, 56 minutes, 57 minutes, 58 minutes, 59 minutes, 60 minutes, 61 minutes, 62 minutes, 63 minutes, 64 minutes, 65 minutes, 66 minutes, 67 minutes, 68 minutes, 69 minutes, 70 minutes, 71 minutes, 72 minutes, 73 minutes, 74 minutes, or approximately 75 minutes.

[0067] In certain exemplary embodiments, the second centrifugation is repeated one to three times, with each repetition being performed on the remaining biological fluid from the immediately preceding centrifugation. In certain exemplary embodiments, the second centrifugation is repeated one, two or three times, with each repetition being performed on the remaining biological fluid from the immediately preceding centrifugation.

[0068] In certain exemplary embodiments, (d) includes filtering the remaining biological fluid through one or more filters in series ranging from about a 0.45 micron filter to about a 0.22 micron filter. In some embodiments, each filter in the series is independently selected from a 0.22 micron, 0.23 micron, 0.24 micron, 0.25 micron, 0.26 micron, 0.27 micron, 0.28 micron, 0.29 micron, 0.3 micron, 0.31 micron, 0.32 micron, 0.33 micron, 0.34 micron, 0.35 micron, 0.36 micron, 0.37 micron, 0.38 micron, 0.39 micron, 0.4 micron, 0.41 micron, 0.42 micron, 0.43 micron, 0.44 micron, or 0.45 micron filter. In some embodiments, the number of filters in the series ranges from 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 filters in series. In some embodiments, the filters in the series are all the same size cutoff. In some embodiments, at least two filters in the series have the same size cutoff. In some embodiments, at least two filters in the series have different size cutoffs. In some embodiments, all filters in the series have different size cutoffs. In some embodiments, the size exclusion decreases from large to small along the filters in the series. For example, in a series of three filters, the first filter may be a 0.45 micron filter, the second filter a 0.3 micron filter, and the last filter a 0.22 filter. Other configurations of filters in series will be understood in light of the description herein. In some embodiments, the filters in the series are all the same material. In some embodiments, the filters in the series are all different materials. In some embodiments, at least two filters in the series are all the same material. In some embodiments, at least two filters are made of different materials.Exemplary filters include, but are not limited to, membrane filters (e.g., polyethersulfone membrane filters, polyvinylidene fluoride membrane filters, cellulose membrane filters, mixed cellulose ester membrane filters, cellulose acetate membrane filters, cellulose nitrate membrane filters, polyamide membrane filters, polycarbonate membrane filters, polytetrafluoroethylene membrane filters, polypropylene membrane filters, nitrocellulose membrane filters, and / or the like), fiberglass or bead filters, and / or the like.

[0069] In certain exemplary embodiments, (d) comprises filtering the remaining biological fluid through about a 0.45 micron filter, followed by filtering the remaining biological fluid through about a 0.22 micron filter.

[0070] In certain exemplary embodiments, (e) includes two or more successive ultracentrifugation steps, each step performed on the remaining biological fluid from the previous ultracentrifugation. In certain exemplary embodiments, (e) includes 2-10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) serial ultracentrifugation steps, each step performed on the remaining biological fluid from the previous ultracentrifugation. In certain exemplary embodiments, (e) includes an ultracentrifugation step performed at about 50,000 rcf, an ultracentrifugation step performed at about 70,000 rcf, an ultracentrifugation step performed at about 100,000 rcf, or a combination thereof. In certain exemplary embodiments, (e) includes an ultracentrifugation step performed at about 45,000 to about 55,000 rcf, an ultracentrifugation step performed at about 65,000 to about 75,000 rcf, an ultracentrifugation step performed at about 90,000 to about 110,000 rcf, or a combination thereof. In certain exemplary embodiments, (e) is from about 45,000 to about 55,000 rcf (e.g., about 45000 rcf, 45100 rcf, 45200 rcf, 45300 rcf, 45400 rcf, 45500 rcf, 45600 rcf, 45700 rcf, 45800 rcf, 45900 rcf, 46000 rcf, 461 00rcf, 46200rcf, 46300rcf, 46400rcf, 46500rcf, 46600rcf, 46700rcf, 46800rcf, 4690 0rcf, 47000rcf, 47100rcf, 47200rcf, 47300rcf, 47400rcf, 47500rcf, 47600rcf, 47700 rcf, 47800rcf, 47900rcf, 48000rcf, 48100rcf, 48200rcf, 48300rcf, 48400rcf, 48500r cf, 48600rcf, 48700rcf, 48800rcf, 48900rcf, 49000rcf, 49100rcf, 49200rcf, 49300rc f, 49400rcf, 49500rcf, 49600rcf, 49700rcf, 49800rcf, 49900rcf, 50000rcf, 50100rcf , 50200rcf, 50300rcf, 50400rcf, 50500rcf, 50600rcf, 50700rcf, 50800rcf, 50900rcf,51000rcf, 51100rcf, 51200rcf, 51300rcf, 51400rcf, 51500rcf, 51600rcf, 51700rcf, 51800rcf, 51900rcf, 52000rcf, 52100rcf, 52200rcf, 52300rc f, 52400rcf, 52500rcf, 52600rcf, 52700rcf, 52800rcf, 52900rcf, 53000rcf, 53100rcf, 53200rcf, 53300rcf, 53400rcf, 53500rcf, 53600rcf, 53700r cf, 53800rcf, 53900rcf, 54000rcf, 54100rcf, 54200rcf, 54300rcf, 54400rcf, 54500rcf, 54600rcf, 54700rcf, 54800rcf, 54900rcf, or about 55000rcf), ultracentrifugation steps performed at about 65,000 to about 75,000rcf (e.g., about 65000rcf, 65100rcf, 65200rcf, 65300rcf, 65400rcf, 65500rcf, 65600rcf, 65700rcf, 65800rcf, 65900rcf, rcf, 66000rcf, 66100rcf, 66200rcf, 66300rcf, 66400rcf, 66500rcf, 66600rcf, 66700rcf, 66800rcf, 66900rcf, 67000rcf, 67100rcf, 67200rcf, 673 00rcf, 67400rcf, 67500rcf, 67600rcf, 67700rcf, 67800rcf, 67900rcf, 68000rcf, 68100rcf, 68200rcf, 68300rcf, 68400rcf, 68500rcf, 68600rcf, 68 700rcf, 68800rcf, 68900rcf, 69000rcf, 69100rcf, 69200rcf, 69300rcf, 69400rcf, 69500rcf, 69600rcf, 69700rcf, 69800rcf, 69900rcf, 70000rcf, 7 0100rcf, 70200rcf, 70300rcf, 70400rcf, 70500rcf, 70600rcf, 70700rcf, 70800rcf, 70900rcf, 71000rcf, 71100rcf, 71200rcf, 71300rcf, 71400rcf,71500rcf, 71600rcf, 71700rcf, 71800rcf, 71900rcf, 72000rcf, 72100rcf, 72200rcf, 72300rcf, 72400rcf, 72500rcf, 72600rcf, 72700rcf, 72800rc f, 72900rcf, 73000rcf, 73100rcf, 73200rcf, 73300rcf, 73400rcf, 73500rcf, 73600rcf, 73700rcf, 73800rcf, 73900rcf, 74000rcf, 74100rcf, 74200r cf, 74300rcf, 74400rcf, 74500rcf, 74600rcf, 74700rcf, 74800rcf, 74900rcf, or about 75000rcf), ultracentrifugation steps performed at about 90,000 to about 110,000rcf (e.g., 90000rcf, 90100rcf, 90200rcf, 90300rcf, 90400rcf, 90500rcf, 90600rcf, 90700rcf, 90800rcf, 90900rcf, 91000rcf, 91100rcf, 91200rcf, 91300rcf, 91400rcf, rcf, 91500rcf, 91600rcf, 91700rcf, 91800rcf, 91900rcf, 92000rcf, 92100rcf, 92200rcf, 92300rcf, 92400rcf, 92500rcf, 92600rcf, 92700rcf, 928 94 200rcf, 94300rcf, 94400rcf, 94500rcf, 94600rcf, 94700rcf, 94800rcf, 94900rcf, 95000rcf, 95100rcf, 95200rcf, 95300rcf, 95400rcf, 95500rcf, 9 5600rcf, 95700rcf, 95800rcf, 95900rcf, 96000rcf, 96100rcf, 96200rcf, 96300rcf, 96400rcf, 96500rcf, 96600rcf, 96700rcf, 96800rcf, 96900rcf,97000rcf、97100rcf、97200rcf、97300rcf、97400rcf、97500rcf、97600rcf、97700rcf、97800rcf、97900rcf、98000rcf、98100rcf、98200rcf、98300rcf、98400rcf、98500rcf、98600rcf、98700rcf、98800rcf、98900rcf、99000rcf、99100rcf、99200rcf、99300rcf、99400rcf、99500rcf、99600rcf、99700rcf、99800rcf、99900rcf、100000rcf、100100rcf、100200rcf、100300rcf、100400rcf、100500rcf、100600rcf、100700rcf、100800rcf、100900rcf、101000rcf、101100rcf、101200rcf、101300rcf、101400rcf、101500rcf、101600rcf、101700rcf、101800rcf、101900rcf、102000rcf、102100rcf、102200rcf、102300rcf、102400rcf、102500rcf、102600rcf、102700rcf、102800rcf、102900rcf、103000rcf、103100rcf、103200rcf、103300rcf、103400rcf、103500rcf、103600rcf、103700rcf、103800rcf、103900rcf、104000rcf、104100rcf、104200rcf、104300rcf、104400rcf、104500rcf、104600rcf、104700rcf、104800rcf、104900rcf、105000rcf、105100rcf、105200rcf、105300rcf、105400rcf、105500rcf、105600rcf、105700rcf、105800rcf、105900rcf、106000rcf、106100rcf、106200rcf、106300rcf、106400rcf、106500rcf、106600rcf、106700rcf、106800rcf、106900rcf、107000rcf、107100rcf、107200rcf、107300rcf, 107400rcf, 107500rcf, 107600rcf, 107700rcf, 107800rcf, 107900rcf, 108000rcf , 108100rcf, 108200rcf, 108300rcf, 108400rcf, 108500rcf, 108600rcf, 108700rcf, 108800rcf , 108,900 rcf, 109,000 rcf, 109,100 rcf, 109,200 rcf, 109,300 rcf, 109,400 rcf, 109,500 rcf, 109,600 rcf, 109,700 rcf, 109,800 rcf, 109,900 rcf, or about 110,000 rcf), or any combination thereof. In certain exemplary embodiments, one or more of the one or more ultracentrifugation steps are each performed for about 60 minutes. In certain exemplary embodiments, one or more of the one or more ultracentrifugation steps are each performed for about 45-75 minutes. In certain exemplary embodiments, one or more of the one or more ultracentrifugation steps are performed for about 45 minutes, 46 minutes, 47 minutes, 48 ​​minutes, 49 minutes, 50 minutes, 51 minutes, 52 minutes, 53 minutes, 54 minutes, 55 minutes, 56 minutes, 57 minutes, 58 minutes, 59 minutes, 60 minutes, 61 minutes, 62 minutes, 63 minutes, 64 minutes, 65 minutes, 66 minutes, 67 minutes, 68 minutes, 69 minutes, 70 minutes, 71 minutes, 72 minutes, 73 minutes, 74 minutes, or about 75 minutes, respectively.

[0071] In certain exemplary embodiments, (e) includes a final ultracentrifugation step carried out at about 130,000 rcf for about 120 minutes, the resulting fluid being discarded, and the remaining pellet being resuspended in a suitable volume of a suitable solution prior to (f). In certain exemplary embodiments, (e) includes a final ultracentrifugation step carried out at about 115,000 to about 145,000 rcf for about 90 to 150 minutes, the resulting fluid being discarded, and the remaining pellet being resuspended in a suitable volume of a suitable solution prior to (f). In certain exemplary embodiments, (e) is from about 115,000 to about 145,000 rcf (e.g., 115000 rcf, 115100 rcf, 115200 rcf, 115300 rcf, 115400 rcf, 115500 rcf, 115600 rcf, 115700 rcf, 115800 rcf, 115900 rcf, 116000 rcf, 116100 rcf, 116200 rcf, 116300 rcf, 116400 rcf , 116500rcf, 116600rcf, 116700rcf, 116800rcf, 116900rcf, 117000rcf, 117100rcf, 117200rcf, 117300rcf, 117400 rcf, 117500rcf, 117600rcf, 117700rcf, 117800rcf, 117900rcf, 118000rcf, 118100rcf, 118200rcf, 118300rcf, 118 400rcf, 118500rcf, 118600rcf, 118700rcf, 118800rcf, 118900rcf, 119000rcf, 119100rcf, 119200rcf, 119300rcf , 119400rcf, 119500rcf, 119600rcf, 119700rcf, 119800rcf, 119900rcf, 120000rcf, 120100rcf, 120200rcf, 120300 rcf, 120400rcf, 120500rcf, 120600rcf, 120700rcf, 120800rcf, 120900rcf, 121000rcf, 121100rcf, 121200rcf, 121 300rcf, 121400rcf, 121500rcf, 121600rcf, 121700rcf, 121800rcf, 121900rcf, 122000rcf, 122100rcf, 122200rcf,122300rcf、122400rcf、122500rcf、122600rcf、122700rcf、122800rcf、122900rcf、123000rcf、123100rcf、123200rcf、123300rcf、123400rcf、123500rcf、123600rcf、123700rcf、123800rcf、123900rcf、124000rcf、124100rcf、124200rcf、124300rcf、124400rcf、124500rcf、124600rcf、124700rcf、124800rcf、124900rcf、125000rcf、125100rcf、125200rcf、125300rcf、125400rcf、125500rcf、125600rcf、125700rcf、125800rcf、125900rcf、126000rcf、126100rcf、126200rcf、126300rcf、126400rcf、126500rcf、126600rcf、126700rcf、126800rcf、126900rcf、127000rcf、127100rcf、127200rcf、127300rcf、127400rcf、127500rcf、127600rcf、127700rcf、127800rcf、127900rcf、128000rcf、128100rcf、128200rcf、128300rcf、128400rcf、128500rcf、128600rcf、128700rcf、128800rcf、128900rcf、129000rcf、129100rcf、129200rcf、129300rcf、129400rcf、129500rcf、129600rcf、129700rcf、129800rcf、129900rcf、130000rcf、130100rcf、130200rcf、130300rcf、130400rcf、130500rcf、130600rcf、130700rcf、130800rcf、130900rcf、131000rcf、131100rcf、131200rcf、131300rcf、131400rcf、131500rcf、131600rcf、131700rcf、131800rcf、131900rcf、132000rcf、132100rcf、132200rcf、132300rcf、132400rcf、132500rcf、132600rcf、132700rcf、132800rcf、132900rcf、133000rcf、133100rcf、133200rcf、133300rcf、133400rcf、133500rcf、133600rcf、133700rcf、133800rcf、133900rcf、134000rcf、134100rcf、134200rcf、134300rcf、134400rcf、134500rcf、134600rcf、134700rcf、134800rcf、134900rcf、135000rcf、135100rcf、135200rcf、135300rcf、135400rcf、135500rcf、135600rcf、135700rcf、135800rcf、135900rcf、136000rcf、136100rcf、136200rcf、136300rcf、136400rcf、136500rcf、136600rcf、136700rcf、136800rcf、136900rcf、137000rcf、137100rcf、137200rcf、137300rcf、137400rcf、137500rcf、137600rcf、137700rcf、137800rcf、137900rcf、138000rcf、138100rcf、138200rcf、138300rcf、138400rcf、138500rcf、138600rcf、138700rcf、138800rcf、138900rcf、139000rcf、139100rcf、139200rcf、139300rcf、139400rcf、139500rcf、139600rcf、139700rcf、139800rcf、139900rcf、140000rcf、140100rcf、140200rcf、140300rcf、140400rcf、140500rcf、140600rcf、140700rcf、140800rcf、140900rcf、141000rcf、141100rcf、141200rcf、141300rcf、141400rcf、141500rcf、141600rcf、141700rcf、141800rcf、141900rcf、142000rcf、142100rcf、142200rcf、142300rcf, 142400rcf, 142500rcf, 142600rcf, 142700rcf, 142800rcf, 142900rcf, 143000rcf, 143 100rcf, 143200rcf, 143300rcf, 143400rcf, 143500rcf, 143600rcf, 143700rcf, 143800rcf, 143900 rcf, 144000rcf, 144100rcf, 144200rcf, 144300rcf, 144400rcf, 144500rcf, 144600rcf, 144700rcf, 144800rcf, 144900rcf, or about 145000rcf) for about 90 to 150 minutes (e.g., 90 minutes, 91 minutes, 92 minutes, 93 minutes, 94 minutes, 95 minutes, 96 minutes, 97 minutes, 98 minutes, 99 minutes, 100 minutes, 101 minutes, 102 minutes, 103 minutes, 104 minutes, 105 minutes, 106 minutes, 107 minutes, 108 minutes, 109 minutes, 110 minutes, 111 minutes, 112 minutes, 113 minutes, 114 minutes, 115 minutes, 116 minutes, 117 minutes, 118 minutes, 119 minutes, 120 minutes, 121 minutes, 122 minutes, 123 minutes, 124 minutes, 125 minutes, 126 minutes, 127 minutes, 128 minutes, 129 minutes, 130 minutes, 131 minutes (f) includes a final ultracentrifugation step performed for about 130 minutes (131 minutes, 132 minutes, 133 minutes, 134 minutes, 135 minutes, 136 minutes, 137 minutes, 138 minutes, 139 minutes, 140 minutes, 141 minutes, 142 minutes, 143 minutes, 144 minutes, 145 minutes, 146 minutes, 147 minutes, 148 minutes, 149 minutes, or about 150 minutes), the resulting liquid being discarded and the remaining pellet being resuspended in a suitable amount of a suitable solution prior to (f).

[0072] In certain exemplary embodiments, the tangential flow filtration of (g) is carried out using an ultrafiltration membrane having a molecular weight cut-off of about 250 kDa to about 750 kDa. In some embodiments, the cleavage region may be about 250 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 300 kDa, 310 kDa, 320 kDa, 330 kDa, 340 kDa, 350 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa, 400 kDa, 410 kDa, 420 kDa, 430 kDa, 440 kDa, 450 kDa, 460 kDa, 470 kDa, 480 kDa, 490 kDa, 500 kDa, 51 The molecular weight cut-off of an ultrafiltration membrane having a molecular weight cut-off of 0 kDa, 520 kDa, 530 kDa, 540 kDa, 550 kDa, 560 kDa, 570 kDa, 580 kDa, 590 kDa, 600 kDa, 610 kDa, 620 kDa, 630 kDa, 640 kDa, 650 kDa, 660 kDa, 670 kDa, 680 kDa, 690 kDa, 700 kDa, 710 kDa, 720 kDa, 730 kDa, 740 kDa, or about 750 kDa.

[0073] In certain exemplary embodiments, the tangential flow filtration of (g) is performed at a flow rate of about 10 mL per minute. In certain exemplary embodiments, the tangential flow filtration of (g) is performed at a flow rate ranging from about 5 mL to 15 mL per minute. In some embodiments, the tangential flow filtration of (g) is performed at a flow rate of about 5 mL / min, about 5.5 mL / min, about 6 mL / min, about 6.5 mL / min, about 7 mL / min, about 7.5 mL / min, about 8 mL / min, about 8.5 mL / min, about 9 mL / min, about 9.5 mL / min, about 10 mL / min, about 10.5 mL / min, about 11 mL / min, about 11.5 mL / min, about 12 mL / min, about 12.5 mL / min, about 13 mL / min, about 13.5 mL / min, about 14 mL / min, about 14.5 mL / min, or about 15 mL / min.

[0074] In certain exemplary embodiments, in step (g), when the amount of remaining biological fluid reaches about 10 percent of the starting volume before tangential flow filtration, the retentate is diafiltered with a suitable buffer.

[0075] In certain exemplary embodiments, the method further comprises ultracentrifuging the retentate when the retentate reaches about 20 percent of the starting diafiltration volume. In certain exemplary embodiments, ultracentrifugation of the retentate is performed at about 4 degrees Celsius and about 130,000 rcf for about 120 minutes. In certain exemplary embodiments, ultracentrifugation of the retentate is performed at a concentration of about 115,000 to about 145,000 rcf (e.g., 115000 rcf, 115050 rcf, 115100 rcf, 115150 rcf, 115200 rcf, 115250 rcf, 115300 rcf, 115350 rcf, 115400 rcf, 115450 rcf, 115500 rcf, 115550 rcf, 115600 rcf, 115650 rcf, 115700 rcf, 115750 rcf, 115800 rcf, cf, 115850rcf, 115900rcf, 115950rcf, 116000rcf, 116050rcf, 116100rcf, 116150rcf, 116200rcf, 116250rcf, 116300rcf, 116350 rcf, 116400rcf, 116450rcf, 116500rcf, 116550rcf, 116600rcf, 116650rcf, 116700rcf, 116750rcf, 116800rcf, 116850rcf, 11690 0rcf, 116950rcf, 117000rcf, 117050rcf, 117100rcf, 117150rcf, 117200rcf, 117250rcf, 117300rcf, 117350rcf, 117400rcf, 117 450rcf, 117500rcf, 117550rcf, 117600rcf, 117650rcf, 117700rcf, 117750rcf, 117800rcf, 117850rcf, 117900rcf, 117950rcf, 11 8000rcf, 118050rcf, 118100rcf, 118150rcf, 118200rcf, 118250rcf, 118300rcf, 118350rcf, 118400rcf, 118450rcf, 118500rcf, 1 18550rcf, 118600rcf, 118650rcf, 118700rcf, 118750rcf, 118800rcf, 118850rcf, 118900rcf, 118950rcf, 119000rcf, 119050rcf,119100rcf、119150rcf、119200rcf、119250rcf、119300rcf、119350rcf、119400rcf、119450rcf、119500rcf、119550rcf、119600rcf、119650rcf、119700rcf、119750rcf、119800rcf、119850rcf、119900rcf、119950rcf、120000rcf、120050rcf、120100rcf、120150rcf、120200rcf、120250rcf、120300rcf、120350rcf、120400rcf、120450rcf、120500rcf、120550rcf、120600rcf、120650rcf、120700rcf、120750rcf、120800rcf、120850rcf、120900rcf、120950rcf、121000rcf、121050rcf、121100rcf、121150rcf、121200rcf、121250rcf、121300rcf、121350rcf、121400rcf、121450rcf、121500rcf、121550rcf、121600rcf、121650rcf、121700rcf、121750rcf、121800rcf、121850rcf、121900rcf、121950rcf、122000rcf、122050rcf、122100rcf、122150rcf、122200rcf、122250rcf、122300rcf、122350rcf、122400rcf、122450rcf、122500rcf、122550rcf、122600rcf、122650rcf、122700rcf、122750rcf、122800rcf、122850rcf、122900rcf、122950rcf、123000rcf、123050rcf、123100rcf、123150rcf、123200rcf、123250rcf、123300rcf、123350rcf、123400rcf、123450rcf、123500rcf、123550rcf、123600rcf、123650rcf、123700rcf、123750rcf、123800rcf、123850rcf、123900rcf、123950rcf、124000rcf、124050rcf、124100rcf、124150rcf、124200rcf、124250rcf、124300rcf、124350rcf、124400rcf、124450rcf、124500rcf、124550rcf、124600rcf、124650rcf、124700rcf、124750rcf、124800rcf、124850rcf、124900rcf、124950rcf、125000rcf、125050rcf、125100rcf、125150rcf、125200rcf、125250rcf、125300rcf、125350rcf、125400rcf、125450rcf、125500rcf、125550rcf、125600rcf、125650rcf、125700rcf、125750rcf、125800rcf、125850rcf、125900rcf、125950rcf、126000rcf、126050rcf、126100rcf、126150rcf、126200rcf、126250rcf、126300rcf、126350rcf、126400rcf、126450rcf、126500rcf、126550rcf、126600rcf、126650rcf、126700rcf、126750rcf、126800rcf、126850rcf、126900rcf、126950rcf、127000rcf、127050rcf、127100rcf、127150rcf、127200rcf、127250rcf、127300rcf、127350rcf、127400rcf、127450rcf、127500rcf、127550rcf、127600rcf、127650rcf、127700rcf、127750rcf、127800rcf、127850rcf、127900rcf、127950rcf、128000rcf、128050rcf、128100rcf、128150rcf、128200rcf、128250rcf、128300rcf、128350rcf、128400rcf、128450rcf、128500rcf、128550rcf、128600rcf、128650rcf、128700rcf、128750rcf、128800rcf、128850rcf、128900rcf、128950rcf、129000rcf、129050rcf、129100rcf、129150rcf、129200rcf、129250rcf、129300rcf、129350rcf、129400rcf、129450rcf、129500rcf、129550rcf、129600rcf、129650rcf、129700rcf、129750rcf、129800rcf、129850rcf、129900rcf、129950rcf、130000rcf、130050rcf、130100rcf、130150rcf、130200rcf、130250rcf、130300rcf、130350rcf、130400rcf、130450rcf、130500rcf、130550rcf、130600rcf、130650rcf、130700rcf、130750rcf、130800rcf、130850rcf、130900rcf、130950rcf、131000rcf、131050rcf、131100rcf、131150rcf、131200rcf、131250rcf、131300rcf、131350rcf、131400rcf、131450rcf、131500rcf、131550rcf、131600rcf、131650rcf、131700rcf、131750rcf、131800rcf、131850rcf、131900rcf、131950rcf、132000rcf、132050rcf、132100rcf、132150rcf、132200rcf、132250rcf、132300rcf、132350rcf、132400rcf、132450rcf、132500rcf、132550rcf、132600rcf、132650rcf、132700rcf、132750rcf、132800rcf、132850rcf、132900rcf、132950rcf、133000rcf、133050rcf、133100rcf、133150rcf、133200rcf、133250rcf、133300rcf、133350rcf、133400rcf、133450rcf、133500rcf、133550rcf、133600rcf、133650rcf、133700rcf、133750rcf、133800rcf、133850rcf、133900rcf、133950rcf、134000rcf、134050rcf、134100rcf、134150rcf、134200rcf、134250rcf、134300rcf、134350rcf、134400rcf、134450rcf、134500rcf、134550rcf、134600rcf、134650rcf、134700rcf、134750rcf、134800rcf、134850rcf、134900rcf、134950rcf、135000rcf、135050rcf、135100rcf、135150rcf、135200rcf、135250rcf、135300rcf、135350rcf、135400rcf、135450rcf、135500rcf、135550rcf、135600rcf、135650rcf、135700rcf、135750rcf、135800rcf、135850rcf、135900rcf、135950rcf、136000rcf、136050rcf、136100rcf、136150rcf、136200rcf、136250rcf、136300rcf、136350rcf、136400rcf、136450rcf、136500rcf、136550rcf、136600rcf、136650rcf、136700rcf、136750rcf、136800rcf、136850rcf、136900rcf、136950rcf、137000rcf、137050rcf、137100rcf、137150rcf、137200rcf、137250rcf、137300rcf、137350rcf、137400rcf、137450rcf、137500rcf、137550rcf、137600rcf、137650rcf、137700rcf、137750rcf、137800rcf、137850rcf、137900rcf、137950rcf、138000rcf、138050rcf、138100rcf、138150rcf、138200rcf、138250rcf、138300rcf、138350rcf、138400rcf、138450rcf、138500rcf、138550rcf、138600rcf、138650rcf、138700rcf、138750rcf、138800rcf、138850rcf、138900rcf、138950rcf、139000rcf、139050rcf、139100rcf、139150rcf、139200rcf、139250rcf、139300rcf、139350rcf、139400rcf、139450rcf、139500rcf、139550rcf、139600rcf、139650rcf、 、139700rcf、139750rcf、139800rcf、139850rcf、139900rcf、139950rcf、140000rcf、140050rcf、140100rcf、140150rcf、140200rcf、140250rcf、140300rcf、140350rcf、140400rcf、140450rcf、140500rcf、140550rcf、140600rcf、140650rcf、140700rcf、140750rcf、140800rcf、140850rcf、140900rcf、140950rcf、141000rcf、141050rcf、141100rcf、141150rcf、141200rcf、141250rcf、141300rcf、141350rcf、141400rcf、141450rcf、141500rcf、141550rcf、141600rcf、141650rcf、141700rcf、141750rcf、141800rcf、141850rcf、141900rcf、141950rcf、142000rcf、142050rcf、142100rcf、142150rcf、142200rcf、142250rcf、142300rcf、142350rcf、142400rcf、142450rcf、142500rcf、142550rcf、142600rcf、142650rcf、142700rcf、142750rcf、142800rcf、142850rcf、142900rcf、142950rcf、143000rcf、143050rcf、143100rcf、143150rcf、143200rcf、143250rcf、143300rcf、143350rcf、143400rcf、143450rcf、143500rcf、143550rcf、143600rcf、143650rcf、143700rcf、143750rcf、143800rcf、143850rcf、143900rcf、143950rcf、144000rcf、144050rcf、144100rcf、144150rcf、144200rcf、144250rcf、144300rcf、144350rcf、144400rcf、144450rcf、144500rcf、144550rcf、144600rcf、144650rcf、144700rcf, 144750rcf, 144800rcf, 144850rcf, 144900rcf, 144950rcf, or about 145000rcf), for about 90 minutes to about 150 minutes, for example, (about 90 minutes, 91 minutes, 92 minutes, 93 minutes, 94 minutes, 95 minutes, 96 minutes, 97 minutes, 98 minutes, 99 minutes, 100 minutes, 101 minutes, 102 minutes, 103 minutes, 104 minutes, 105 minutes, 106 minutes, 107 minutes, 108 minutes, 109 minutes, 110 minutes, 111 minutes, 112 minutes, 113 minutes, 114 minutes, 115 minutes, 116 minutes, 117 minutes, 118 minutes, 119 minutes, 120 minutes, 121 minutes, 122 minutes, 123 minutes, 124 minutes, 125 minutes, 126 minutes, 127 minutes, 128 minutes, 129 minutes, 130 minutes, 131 minutes, 132 minutes, 133 minutes, 134 minutes, 135 minutes, 136 minutes, 137 minutes, 138 minutes, 139 minutes, 140 minutes, 141 minutes, 142 minutes, 143 minutes, 144 minutes, 145 minutes, 146 minutes, 147 minutes, 148 minutes, 149 minutes, or about 150 minutes) at about 4 degrees Celsius.

[0076] In certain exemplary embodiments, the retentate is not ultracentrifuged prior to optional fractionation of the retentate. In these embodiments, the retentate is stored at about -80°C prior to fractionation, which is optionally performed by column separation.

[0077] Optional fractionation of the retentate can be performed by any suitable method, including but not limited to column separation (based on size, charge, affinity, avidity, or other method or separation strategy). The fraction containing the exosomes can be retained.

[0078] In certain exemplary embodiments, the method produces an exosome concentrate that is at least 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or at least 20 percent of the starting volume of a biological fluid, such as milk. In certain exemplary embodiments, the methods produce an exosome concentrate that is about 7 percent, 7.5 percent, 8 percent, 8.5 percent, 9 percent, 9.5 percent, 10 percent, 10.5 percent, 11 percent, 11.5 percent, 12 percent, 12.5 percent, 13 percent, 13.5 percent, 14 percent, 14.5 percent, 15 percent, 15.5 percent, 16 percent, 16.5 percent, 17 percent, 17.5 percent, 18 percent, 18.5 percent, 19 percent, 19.5 percent, or about 20 percent of the starting volume of a biological fluid, such as milk.

[0079] Loaded with exosomes In certain exemplary embodiments, the method further comprises loading the exosomes of the formulation resulting from the methods described herein with one or more cargoes. The exosomes can be loaded by any suitable method. An exemplary method for loading milk exosomes, such as those prepared by the methods described herein, is any of the methods described in International Patent Application Publication WO2020 / 028439, particularly pages 83-87.

[0080] Representative cargo Milk exosomes can be loaded with suitable or desired cargo. In some embodiments, the cargo(s) is a therapeutic compound or molecule. Exemplary cargoes include, but are not limited to, DNA, RNA, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, hormones, immunomodulatory agents, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatory agents, antihistamines, anti-infective agents, radiosensitizers, chemotherapeutic agents, imaging agents, immunogens, anti-cancer agents, any combination thereof, and / or the like.

[0081] In certain exemplary embodiments, the cargo is a peptide, including but not limited to, ACT-11 peptide. In certain exemplary embodiments, the cargo is a peptide, including but not limited to, ACT-11-I peptide. Other peptide cargoes include those described in International Patent Application Publication WO2020 / 028439, particularly pages 67-82, 85, and 106-111. In some embodiments, the cargo compound is esterified, as described in International Patent Application Publication WO2020 / 028439, pages 81-86. In some embodiments, the cargo compound has multiple esterifications, as described in International Patent Application Publication WO2020 / 028439, pages 81-86.

[0082] Exemplary hormones include, but are not limited to, amino acid derived hormones (e.g., melatonin and thyroxine), small peptide and protein hormones (e.g., thyrotropin releasing hormone, vasopressin, insulin, growth hormone, luteinizing hormone, follicle stimulating hormone, and thyroid stimulating hormone), eicosanoids (e.g., arachidonic acid, lipoxins, prostaglandins), purines (e.g., ATP), enzymes (e.g., creatine), steroid hormones (e.g., estradiol, testosterone, tetrahydrotestosterone, cortisol).

[0083] Exemplary immunomodulatory agents include, but are not limited to, prednisone, azathioprine, 6-MP, cyclosporine, tacrolimus, methotrexate, interleukins (e.g., IL-2, IL-7, and IL-12), cytokines (e.g., interferons (e.g., IFN-a, IFN-β, IFN-ε, IFN-K, IFN-ω, and IFN-γ), granulocyte colony-stimulating factor, and imiquimod), chemokines (e.g., CCL3, CCL26, and CXCL7), cytosine phosphate-guanosine, oligodeoxynucleotides, glucans, antibodies, and aptamers).

[0084] Exemplary antipyretics include, but are not limited to, nonsteroidal anti-inflammatory drugs (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), aspirin and related salicylates (e.g., choline salicylate, magnesium salicylate, and sodium salicylate), paracetamol / acetaminophen, metamizole, nabumetone, phenazone, quinine.

[0085] Exemplary anti-anxiety agents include, but are not limited to, benzodiazepines (e.g., alprazolam, bromazepam, chlordiazepoxide, clonazepam, clorazepate, diazepam, flurazepam, lorazepam, oxazepam, temazepam, triazolam, and tofisopam), serotonergic antidepressants (e.g., selective serotonin reuptake inhibitors, tricyclic antidepressants, and monoamine oxidase inhibitors), temglycoluril, favomotisole, selanc, bromantane, emoxipine, azapirones, barbiturates, hydroxyzine, pregabalin, isovaleric acid, and beta-blockers.

[0086] Exemplary antipsychotics include benperidol, bromperidol, droperidol, haloperidol, moperone, pipamperone, timiperone, fluspirilene, penfluridol, pimozide, acepromazine, chlorpromazine, cyamemazine, dixyrazine, fluphenazine, levomepromazine, mesoridazine, perazine, pericyazine, perphenazine, pipotiazine, prochlorperazine, promazine, promethazine, prothipendyl, thioproperazine, thioridazine, trifluoperazine, triflupromazine, chlorprothixene, clopenthixol, flupenthixol, thiothixene, zuclopenthixol, clothiapine, loxapine, prothipepine, ... These include, but are not limited to, ndilu, carpipramine, clocapramine, molindone, mosapramine, sulpiride, veralipride, amisulpride, amoxapine, aripiprazole, asenapine, clozapine, blonanserin, iloperidone, lurasidone, melperone, nemonapride, olanzapine, paliperidone, perospirone, quetiapine, remoxipride, risperidone, sertindole, trimipramine, ziprasidone, zotepine, alstony, bifeprunox, bitopertine, brexpiprazole, cannabidiol, cariprazine, pimavanserin, pomaglumetad methionyl, babicaserin, zanomeline, and diclonapine.

[0087] Exemplary analgesics include, but are not limited to, paracetamol / acetaminophen, nonsteroidal anti-inflammatory agents (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), opioids (e.g., morphine, codeine, oxycodone, hydrocodone, dihydromorphine, pethidine, buprenorphine), tramadol, norepinephrine, flupirtine, nefopam, orphenadrine, pregabalin, gabapentin, cyclobenzaprine, scopolamine, methadone, ketobemidone, piritramide, aspirin and related salicylates (e.g., choline salicylate, magnesium salicylate, sodium salicylate).

[0088] Exemplary antispasmodics include, but are not limited to, mebeverine, papaverine, cyclobenzaprine, carisoprodol, orphenadrine, tizanidine, metaxalone, methocarbamol, chlorzoxazone, baclofen, dantrolene, baclofen, tizanidine, and dantrolene. Suitable anti-inflammatory drugs include, but are not limited to, prednisone, nonsteroidal anti-inflammatory drugs (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), and immunoselective anti-inflammatory derivatives (e.g., submandibular peptide-T and its derivatives).

[0089] Exemplary antihistamines include H1 receptor antagonists (e.g., acrivastine, azelastine, bilastine, brompheniramine, buclizine, bromodiphenhydramine, carbinoxamine, cetirizine, chlorpromazine, cyclizine, chlorpheniramine, clemastine, cyproheptadine, desloratadine, dexbromophenylamine, dexchlorpheniramine, dimenhydrinate, dimethindene, diphenhydramine, doxylamine, ebastine, embramine, fexofenadine, hydroxyzine, These include, but are not limited to, levocetirizine, loratadine, meclizine, mirtazapine, olopatadine, orphenadrine, phenindamine, pheniramine, phenyltoloxamine, promethazine, pyrilamine, quetiapine, rupatadine, tripelennamine, triprolidine), H2-receptor antagonists (e.g., cimetidine, famotidine, lafutidine, nizatidine, ranitidine, and roxatidine), tritoqualine, catechin, cromoglycate, nedocromil, and p2-adrenergic agonists).

[0090] Exemplary anti-infective agents include amebicides (e.g., nitazoxanide, paromomycin, metronidazole, tinidazole, chloroquine, miltefosine, amphotericin b, and iodoquinol), aminoglycosides (e.g., paromomycin, tobramycin, gentamicin, amikacin, kanamycin, and neomycin), anthelmintics (e.g., pyrantel, mebendazole, ivermectin, praziquantel, albendazole, thiabendazole, oxamniquine), antifungals (e.g., azole antifungals (e.g., itraconazole, fumaricin, furanol ... leuconazole, posaconazole, ketoconazole, clotrimazole, miconazole, voriconazole), echinocandins (e.g., caspofungin, anidulafungin, and micafungin), griseofulvin, terbinafine, flucytosine, and polyenes (e.g., nystatin, amphotericin b), antimalarials (e.g., pyrimethamine / sulfadoxine, artemether / lumefantrine, atovaquone / proquanil, quinine, hydroxychloroquine, mefloquine, chloroquine, doxycycline, pyrimethamine, and haloperidol). Fantrine), antituberculous drugs (e.g., aminosalicylic acid salts, isoniazid / rifampin, isoniazid / pyrazinamide / rifampin, bedaquiline, isoniazid, ethambutol, rifampin, rifabutin, rifapentine, capreomycin, and cycloserine), antiviral drugs (e.g., amantadine, rimantadine, abacavir / lamivudine, emtricitabine / tenofovir, cobicistat / elvitegravir / emtricitabine / tenofovir, efavirenz / emtricitabine / tenofovir, abaca vir / lamivudine / zidovudine, lamivudine / zidovudine, emtricitabine / tenofovir, emtricitabine / lopinavir / ritonavir / tenofovir, interferon alfa-2v / ribavirin, peginterferon alfa-2b, maraviroc, raltegravir, dolutegravir, enfuvirtide, foscarnet, fomivirsen, oseltamivir, zanamivir, nevirapine, efavirenz, etravirine, rilpivirine, delavirdine, nevirapine, entecavir, ramivudine, adefovir, sovosbuvir, didanosine, tenofovir,abacavir, zidovudine, stavudine, emtricitabine, zalcitabine, telbivudine, simeprevir, boceprevir, telaprevir, lopinavir / ritonavir, fosamprenavir, darunavir, ritonavir, tipranavir, atazanavir, nelfinavir, amprenavir, indinavir, saquinavir, ribavirin, valacyclovir, acyclovir, famciclovir, ganciclovir, and valganciclovir), carbapenems (e.g., doripenem, meropenem, ertapenem, and cilastatin / imipenem), cephalosporins (e.g., , cefadroxil, cephradine, cefazolin, cephalexin, cefepime, cefraroline, loracarbef, cefotetan, cefuroxime, cefprozil, loracarbef, cefoxitin, cefaclor, ceftibuten, ceftriaxone, cefotaxime, cefpodoxime, cefdinir, cefixime, cefditoren, cefizoxime, and ceftazidime), glycopeptide antibiotics (e.g., vancomycin, dalbavancin, oritavancin, and telavancin), glycylcyclines (e.g., tigecycline), leprosy drugs (e.g., cefadroxil, cephradine, cefazolin, cefazolin, cephalexin, cefepime, cefraroline, loracarbef, cefotetan, cefuroxime, cefprozil, loracarbef, cefoxitin, cefaclor, ceftibuten, ceftriaxone, cefotaxime, cefpodoxime, cefdinir, cefixime, cefditoren, cefizoxime, and ceftazidime), e.g., clofazimine and thalidomide), lincomycin and its derivatives (e.g., clindamycin and lincomycin), macrolides and their derivatives (e.g., telithromycin, fidaxomicin, erythromycin, azithromycin, clarithromycin, dirithromycin, and troandomycin), linezolid, sulfamethoxazole / trimethoprim, rifaximin, chloramphenicol, fosfomycin, metronidazole, aztreonam, bacitracin, penicillins (amoxicillin, ampicillin, bacampicillin, bacillin ... Cialis, carbenicillin, piperacillin, ticarcillin, amoxicillin / clavulanic acid, ampicillin / sulbactam, piperacillin / tazobactam, clavulanic acid / ticarcillin, penicillin, procaine penicillin, oxacillin, dicloxacillin, nafcillin), quinolones (e.g., lomefloxacin, norfloxacin, ofloxacin, moxifloxacin, ciprofloxacin, levofloxacin, gemifloxacin, moxifloxacin, cinoxacin, nalidixic acid, enoxacin, grepafloxacin, gatifloxacin,Antimicrobials include, but are not limited to, antimicrobials such as trovafloxacin, and sparfloxacin, sulfonamides (e.g., sulfamethoxazole / trimethoprim, sulfasalazine, sulfasoxazole), tetracyclines (e.g., doxycycline, demeclocycline, minocycline, doxycycline / salicylic acid, doxycycline / omega-3 polyunsaturated fatty acids, and tetracycline), and urinary anti-infectives (e.g., nitrofurantoin, methenamine, fosfomycin, cinoxacin, nalidixic acid, trimethoprim, and methylene blue).

[0091] Exemplary chemotherapeutic agents include paclitaxel, brentuximab vedotin, doxorubicin, 5-FU (fluorouracil), everolimus, pemetrexed, melphalan, pamidronate, anastrozole, exemestane, nelarabine, ofatumumab, bevacizumab, belinostat, tositumomab, carmustine, bleomycin, bosutinib, busulfan, alemtuzumab, irinotecan, vandetanib, bicalutamide, lomustine, daunorubicin, clofarabine, cabozantinib, dactinomycin, ramucirumab, cytarabinose, and the like. Bin, Cytoxan, Cyclophosphamide, Decitabine, Dexamethasone, Docetaxel, Hydroxyurea, Dacarbazine, Leuprolide, Epirubicin, Oxaliplatin, Asparaginase, Estramustine, Cetuximab, Vismodegib, Asparaginase Erwinia, Chrysanthemin, Amifostine, Etoposide, Flutamide, Toremifene, Fulvestrant, Letrozole, Degarelix, Pralatrexate, Methotrexate, Floxuridine, Obinutuzumab, Gemcitabine, Afatinib, Imatinib, Mesylate , carmustine, eribulin, trastuzumab, altretamine, topotecan, ponatinib, idarubicin, ifosfamide, ibrutinib, axitinib, interferon alfa-2a, gefitinib, romidepsin, ixabepilone, ruxolitinib, cabazitaxel, ado-trastuzumab emtansine, carfilzomib, chlorambucil, sargramostim, cladribine, mitotane, vincristine, procarbazine, megestrol, trametinib, mesna, strontium-89 chloride, mechlorethamine, mitomycin , busulfan, gemtuzumab ozogamicin, vinorelbine, filgrastim, pegfilgrastim, sorafenib, nilutamide, pentostatin, tamoxifen, mitoxantrone, pegaspargase, denileukin diftitox, alitretinoin, carboplatin, pertuzumab, cisplatin, pomalidomide, prednisone, aldesleukin, mercaptopurine, zoledronic acid, lenalidomide, rituximab, octreotide, dasatinib, regorafenib, histrelin, sunitinib, siltuximab, omacetaxine,These include, but are not limited to, thioguanine (thioguanine), dabrafenib, erlotinib, bexarotene, temozolomide, thiotepa, thalidomide, BCG, temsirolimus, bendamustine hydrochloride, triptorelin, arsenic trioxide, lapatinib, valrubicin, panitumumab, vinblastine, bortezomib, tretinoin, azacitidine, pazopanib, teniposide, leucovorin, crizotinib, capecitabine, enzalutamide, ipilimumab, goserelin, vorinostat, idelalisib, ceritinib, abiraterone, epothilone, tafluposide, azathioprine, doxifluridine, vindesine, and all-trans retinoic acid.

[0092] Suitable radiosensitizers include, but are not limited to, 5-fluorouracil, platinum analogs (e.g., cisplatin, carboplatin, and oxaliplatin), gemcitabine, DNA topoisomerase I targeted drugs (e.g., camptothecin derivatives (e.g., topotecan and irinotecan)), epidermal growth factor receptor blockade family agents (e.g., cetuximab, gefitinib), farnesyltransferase inhibitors (e.g., L-778-123), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), bFGF and VEGF targeted agents (e.g., bevazucimab and thalidomide), NBTXR3, nimoral, sodium trans crocetinate, NVX-108, and combinations thereof. See, e.g., Kvols, LK., J Nucl Med 2005;46:187S-190S.

[0093] Examples of immunogens carried as cargo or attached to the outer surface of isolated exosomes can include keyhole limpet hemocyanin (KLH), Concorepus concorepus hemocyanin (CCH) (also known as blue carrier immunogenic protein), bovine serum albumin (BSA), ovalbumin (OVA), and antigens used to generate an immune response against disease-causing pathogens such as diphtheria, tetanus, pertussis, measles, mumps, rubella, hepatitis A, hepatitis B, meningococcal disease (e.g., meningitis), human papillomavirus, chickenpox, rabies, influenza, rotovirus, HIV, malaria, and coronavirus diseases.

[0094] Milk exosome preparations and kits Also described herein are pharmaceutical formulations that can contain an amount, an effective amount, and / or a minimum effective amount, and / or a therapeutically effective amount of one or more milk exosomes, such as cargo-loaded milk exosomes, as described in more detail elsewhere herein, and a pharma- ceutically acceptable carrier or excipient. In certain exemplary embodiments, described herein are formulations comprising exosomes, the formulations being generated at least in part by any one of the methods described elsewhere herein, such as in any one of the preceding paragraphs and / or in the Examples below. In certain exemplary embodiments, described herein are methods comprising administering to a subject a formulation described elsewhere herein, such as in any one of the preceding paragraphs and / or in the Examples below. In some embodiments, the formulation administered to the subject includes any milk exosomes described elsewhere herein and / or prepared by the methods described elsewhere herein. In some embodiments, the milk exosomes are cargo-loaded milk exosomes.

[0095] In some embodiments, the subject to whom milk exosomes or formulations thereof are administered has a disease or disorder. Exemplary diseases or disorders include, but are not limited to, cancer, viral infection, bacterial infection, parasitic infection, external and internal wounds and tissue damage, cancer, ischemic and / or hypoxic injury (e.g., myocardial infarction, ischemic injury wound and / or stroke), multiple sclerosis, psoriasis, scleroderma, acne, eczema, or diseases of the skin and / or connective tissue, cardiac diseases or disorders, neurodegenerative diseases or disorders, neuropathy, atherosclerosis, epithelial permeability and / or vascularization (e.g., angiogenesis or vasculogenesis), respiratory distress syndrome (RDS), reperfusion injury, cutaneous vascular scars or malformations, macular degeneration, neovascularization of the choriocapillaris through Bruch's membrane, diabetic retinopathy, (inflammatory and inflammation-related diseases and disorders), and radiation dermatitis.

[0096] The wound may be a chronic wound or a wound that does not appear to heal completely. For example, a wound that does not heal within 3 months is said to be chronic. Chronic wounds include diabetic foot ulcers, ischemic ulcers, venous ulcers, venous leg ulcers, venous stasis, arterial, pressure, vasculitic, infectious, bedsores, burns, traumatic ulcers, gangrenous and mixed ulcers. Chronic wounds include wounds characterized by chronic inflammation, defects and excessive granulation tissue differentiation, and failure of re-epithelialization and wound closure, and longer repair times. Chronic wounds may include ocular ulcers, including corneal ulcers. Use of the disclosed invention in wound healing and tissue regeneration may include humans, agricultural animals, sports animals and pet animals.

[0097] Tissue injuries include, for example, cuts, abrasions, pressure wounds, stretch marks, lacerations, crush injuries, bite wounds, abrasions, bullet wounds, blast wounds, body piercings, puncture wounds, surgical wounds, surgical interventions, medical interventions, host rejection following cell, tissue, or organ transplantation, pharmaceutical effects, pharmaceutical side effects, pressure sores, radiation injuries, radiation illnesses, cosmetic skin injuries, visceral injuries, disease processes (e.g., asthma, cancer), infectious diseases, infectious agents, developmental processes, maturational processes (e.g., acne), genetic abnormalities, developmental abnormalities, environmental toxins, allergens, scalp injuries, facial injuries, jaw injuries, genital injuries, joint injuries, excretory organ injuries, foot injuries, finger injuries, toe injuries, bone injuries, eye injuries, corneal injuries, muscle injuries, adipose tissue injuries, lung injuries, airway injuries, hernias, anal injuries, impingement injuries, ear injuries, skin injuries, abdominal injuries. , retinal injury, eye injury, corneal injury, arm injury, leg injury, movement disorder, back injury, birth injury, premature birth injury, toxic bite, sting, barrier function injury, endothelial barrier function injury, epithelial barrier function injury, tendon injury, ligament injury, cardiac trauma, heart valve injury, vascular system injury, cartilage injury, lymphatic system injury, craniocerebral trauma, dislocation, esophageal perforation, fistula, nail injury, foreign body, fracture, frostbite, hand injury, heat stress injury, laceration, neck injury, self-amputation, shock, traumatic soft tissue injury, spinal cord injury, spinal cord injury, sprain, contusion, tendon injury, ligament injury, cartilage injury, chest injury, dental injury, trauma, nervous system injury, burn, thermal injury, wind burn, sunburn, chemical burn, aging, aneurysm, stroke, surgical radiation injury, gastrointestinal injury, infarction, or ischemic injury.

[0098] Cardiac diseases and disorders include, but are not limited to, myocardial infarction, cardiomyopathies (e.g., hypertrophic cardiomyopathy), arrhythmias, and congestive heart failure. The regenerative effects of the provided compositions may result in beneficial changes in cardiac membrane excitability and ionic transients. There are a variety of different types of arrhythmias that can cause abnormal cardiac function in humans. Arrhythmias include bradycardia, tachycardia, alternans, automaticity defects, reentrant arrhythmias, fibrillation, atrioventricular nodal arrhythmias, atrial arrhythmias and induced beats, long QT syndrome, short QT syndrome, Brugada syndrome, atrial premature beats, wandering atrial pacemaker, multifocal atrial tachycardia, atrial flutter, atrial fibrillation, supraventricular tachycardia, atrioventricular nodal reentrant tachycardia is the most common cause of paroxysmal supraventricular tachycardia, junctional rhythm, junctional tachycardia, premature junctional complex, Wolff-Parkinson-White syndrome, These include, but are not limited to, Lown-Gannon-Levin syndrome, premature ventricular contractions (PVCs), also known as ventricular ectopic beats, alternans and discordant alternans, accelerated intrinsic ventricular rhythm, monomorphic ventricular tachycardia, polymorphic ventricular tachycardia, ventricular fibrillation, first degree heart block manifested as PR prolongation, second degree heart block, type 1 second degree heart block, type 2 second degree heart block, third degree heart block, and some accessory pathway disorders (e.g., Wolff-Parkinson-White syndrome (WPW)).

[0099] Neurodegenerative diseases and disorders include, but are not limited to, dementia, Alzheimer's disease, Parkinson's disease and related PD diseases, amyotrophic lateral sclerosis (ALS), motor neuron diseases, schizophrenia, spinocerebellar ataxia, prion diseases, spinal muscular atrophy (SMA), multiple sclerosis, epilepsy and other seizure disorders, and Huntington's disease.

[0100] Inflammatory and inflammation-related diseases and disorders may be asthma, eczema, sinusitis, atherosclerosis, arthritis (including but not limited to rheumatoid arthritis), inflammatory bowel disease, cutaneous and systemic mastocytosis, psoriasis, and multiple sclerosis. As used herein, the term "inflammatory disorder" may include diseases or disorders caused or exacerbated at least in part by inflammation, which is generally characterized by increased blood flow, edema, immune cell activation (e.g., proliferation, cytokine production, enhanced phagocytosis), heat, redness, swelling, pain, and / or loss of function in affected tissues and organs. Causes of inflammation may be due to physical injury, chemicals, microorganisms, tissue necrosis, cancer, or other pathogens or conditions.

[0101] Inflammatory disorders include acute inflammatory disorders, chronic inflammatory disorders, and recurrent inflammatory disorders. Acute inflammatory disorders are generally relatively short-term, lasting from about a few minutes to about a day or two, but may last several weeks. Acute inflammatory disorders are characterized by increased blood flow, exudation of biological fluids and plasma proteins (edema), and migration of white blood cells, such as neutrophils. Chronic inflammatory disorders generally last for a longer period, for example, weeks to months to years or more, and are associated histologically with the presence of lymphocytes and macrophages, and proliferation of blood vessels and connective tissue. Recurrent inflammatory disorders include disorders that recur after a period of time, or that occur periodically. Some inflammatory disorders are classified into one or more categories. Exemplary inflammatory diseases include, but are not limited to, atherosclerosis, arthritis, inflammation-driven cancer, asthma, autoimmune uveitis, adaptive immune responses, dermatitis, multiple sclerosis, diabetic complications, osteoporosis, Alzheimer's disease, cerebral malaria, hemorrhagic fever, autoimmune disease, and inflammatory bowel disease. In some embodiments, the inflammatory disorder is an autoimmune disorder, which in some aspects is selected from lupus, rheumatoid arthritis, and autoimmune encephalomyelitis.

[0102] In some embodiments, the inflammatory disorder is a brain-associated inflammatory disorder. The term "brain-associated inflammatory" disorder is used herein to refer to a subset of inflammatory disorders that are at least partially caused, originating or exacerbated by inflammation in the brain of a subject.

[0103] As used herein, a "pharmaceutical formulation" refers to a combination of a pharma- ceutically acceptable carrier or excipient with an active agent, compound, or ingredient, making the composition suitable for in vitro, in vivo, or ex vivo diagnostic, therapeutic, or prophylactic use. As used herein, a "pharmaceutically acceptable carrier or excipient" refers to a carrier or excipient that is generally safe, non-toxic, and not biologically or otherwise undesirable, useful for preparing a pharmaceutical formulation, including carriers or excipients that are acceptable for veterinary and human pharmaceutical use. As used herein and in the claims, a "pharmaceutically acceptable carrier or excipient" includes both and one or more of such carriers or excipients. When a cargo is present, the cargo may optionally be present in the pharmaceutical formulation as a pharma- ceutically acceptable salt. In some embodiments, the pharmaceutical formulation may include one or more milk exosomes, such as cargo-loaded milk exosomes, as described in more detail elsewhere herein, such as an active ingredient.

[0104] In some embodiments, the cargo is present as a pharma- ceutically acceptable salt of the active ingredient. As used herein, a "pharma-ceutically acceptable salt" refers to any acid or base addition salt whose counterion is non-toxic to a subject administered pharmaceutical amounts of the salt. Suitable salts include, but are not limited to, hydrochloride, iodide, nitrate, bisulfate, phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, naphthalenesulfonate, propionate, malonate, mandelate, malate, phthalate, and pamoate salts.

[0105] The pharmaceutical formulations described herein can be administered to a subject in need thereof via any suitable method or route to a subject in need thereof. Suitable routes of administration include auricular (otic), buccal, conjunctival, dermal, dental, electroosmotic, intracervical, intrasinusoidal, intratracheal, intraintestinal, epidural, extraamniotic, extracorporeal intraarticular, hemodialytic, infiltrative, interstitial, intraperitoneal, intraamniotic, intraarticular, intrabiliary, intrabronchial, intracystic, intracardiac, intracartilaginous, intracaudal, intracavitary, intracoelastic, intracerebral, intracerebral, intravascular ... In the cisterna, between the corneas, in the crown (tooth), in the crown, in the body, in the corpus cavernosum, in the skin, in the disc, in the tube, in the duodenum, in the dura, in the epidermis, in the esophagus, in the stomach, in the gums, in the ileum, in the lesion, in the cavity, in the lymph, in the medulla, in the meninges, in the muscle, in the eye, in the ovary, in the pericardium, in the abdominal cavity, in the pleura, in the prostate, in the lung, in the sinus, in the spinal cord, in the joint bursa, in the olecranon tendon capsule, in the testis, in the medullary cavity, These include, but are not limited to, intrathoracic, intraductal, intratumoral, intratympanic, intrauterine, intravascular, intravenous, intravenous bolus, intravenous infusion, intraventricular, intravesical, intravitreal, iontophoretic, lavage, laryngeal, nasal, nasogastric, occlusive dressing technique, ocular, oral, oropharyngeal, other, parenteral, transdermal, periarticular, epidural, perineural, periodontal, rectal, respiratory (inhalation), retrobulbar, soft tissue, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, tympanic, ureteral, urethral, ​​and / or vaginal administration, and / or any combination of the above routes of administration, which typically depend on the disease being treated and / or the active ingredient(s) and / or cargo.

[0106] One or more milk exosomes, such as cargo-loaded milk exosomes, as described in more detail elsewhere herein, can provide a component, such as an active ingredient or agent, in a pharmaceutical formulation to a subject in need thereof. Thus, also described are pharmaceutical formulations containing one or more milk exosomes, such as mosquito-loaded milk exosomes, as described in more detail elsewhere herein, including a cargo in the form of a pharma- ceutically acceptable salt. Suitable salts include, but are not limited to, hydrochloride, iodide, nitrate, bisulfate, phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, naphthalenesulfonate, propionate, malonate, mandelate, malate, phthalate, and pamoate salts.

[0107] As used herein, an "agent" refers to any substance, compound, molecule, etc. that can be biologically active or that can induce a biological and / or physiological effect on a subject to which it is administered. As used herein, an "active agent" or "active ingredient" refers to a substance, compound, molecule that is biologically active or that induces a biological or physiological effect on a subject to which it is administered. In other words, an "active agent" or "active ingredient" refers to a component or components of a composition to which the effect of the composition is contributed in whole or in part. An agent may be a primary active agent, in other words, the component(s) of a composition to which the effect of the composition is contributed in whole or in part. An agent may be a secondary active agent, in other words, the component(s) of a composition to which an additional and / or other portion of the effect of the composition is contributed. In some embodiments, the active agent is milk exosomes or cargo-loaded milk exosomes. In some embodiments, the active agent includes or is the cargo of cargo-loaded milk exosomes.

[0108] In certain embodiments, the milk exosomes are prepared by any of the methods described elsewhere herein. In certain embodiments, the cargo-loaded milk exosomes are as described and / or prepared by any of the methods described elsewhere herein.

[0109] In some embodiments, the milk exosomes or formulations thereof are included in a material for administering the milk exosomes or formulations thereof to a subject. Non-limiting examples of materials include those used to treat wounds, such as bandages, stemistrips, sutures, staples, or grafts (e.g., skin grafts). Other exemplary materials include medical devices or implants (or components thereof). Non-limiting examples of medical implants include, but are not limited to, prosthetic limbs, breast implants, penile implants, testicular implants, artificial eyes, facial implants, artificial joints, heart valve prostheses, vascular prostheses, dental prostheses, facial prostheses, tilting disc valves, caged ball valves, artificial ears, artificial noses, pacemakers, cochlear implants, stents, shunts, catheters, filters, meshes, fillers (e.g., fat and dermal fillers), and skin substitutes (e.g., porcine xenografts / pigskin, BIOBRANE, cultured keratinocytes), and / or the like.

[0110] Pharmaceutically acceptable carriers and secondary ingredients and agents Pharmaceutical preparations can include pharmaceutically acceptable carriers.Suitable pharmaceutically acceptable carriers include, but are not limited to, water, milk and dairy products (e.g., casein, ice cream, custard, creamer, etc.), salt solution, alcohol, gum arabic, vegetable oil, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, flavor oils, fatty acid esters, hydroxymethylcellulose, and polyvinylpyrrolidone (which do not adversely react with the active composition).

[0111] The pharmaceutical preparations may be sterilized and, if desired, mixed with agents which do not deleteriously react with the active compounds, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and / or aromatic substances.

[0112] In some embodiments, the pharmaceutical formulation can also include an effective amount of a secondary active agent, including, but not limited to, a biological agent or molecule, including, but not limited to, for example, a polynucleotide, an amino acid, a peptide, a polypeptide, an antibody, an aptamer, a ribozyme, a hormone, an immunomodulatory agent, an antipyretic, an anxiolytic, an antipsychotic, an analgesic, an anticonvulsant, an anti-inflammatory agent, an antihistamine, an anti-infective agent, a chemotherapeutic agent, and any combination thereof. Effective dose

[0113] In some embodiments, the amount of the primary active agent (e.g., milk exosomes, cargo-loaded milk exosomes, and / or cargo) and / or any secondary agent may be an effective amount, a minimum effective amount, and / or a therapeutically effective amount. As used herein, an "effective amount" refers to the amount of the primary agent and / or any secondary agent included in the pharmaceutical formulation that achieves one or more therapeutic or desired effects. As used herein, a "minimum effective" amount refers to the minimum amount of the primary agent and / or any secondary agent included in the pharmaceutical formulation that achieves one or more therapeutic or desired effects. As used herein, a "therapeutically effective amount" refers to the amount of the primary agent and / or any secondary agent included in the pharmaceutical formulation that achieves one or more therapeutic effects.

[0114] As described elsewhere herein, the effective amount, minimum effective amount, and / or therapeutically effective amount of the primary and any secondary active agents included in the pharmaceutical formulation may be in the range of about 0 to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 9 , 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 pg, ng, μg, mg, or g, or any number or subrange within these ranges.

[0115] In some embodiments, the effective amount, minimum effective amount, and / or therapeutically effective amount are each about 0 to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, The effective concentration, minimum effective concentration, and / or therapeutically effective concentration may be an amount in the range of 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 pM, nM, μM, mM, or M, or any number or subrange within these ranges.

[0116] In other embodiments, the effective amount, minimum effective amount, and / or therapeutically effective amount of the primary and any secondary active agents may be about 0 to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, The amount may be in the range of 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 IU, or any number or subrange therein.

[0117] In some embodiments, the primary and / or any secondary active agents present in the pharmaceutical formulation are present at a concentration of from about 0 to 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0. 78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.9~1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9% w / w, v / v, or w / v, or any number or subrange therein.

[0118] In some embodiments where the cells or cell populations are present in the pharmaceutical formulation (e.g., as a secondary active agent), the effective amount of cells ranges from about 1 or 2 cells to 1×10 1 / mL, 1 × 10 20 / mL or more, e.g., about 1 × 10 1 / mL, 1 × 10 2 / mL, 1 × 10 3 / mL, 1 × 10 4 / mL, 1 × 10 5 / mL, 1 × 10 6 / mL, 1 × 10 7 / mL, 1 × 10 8 / mL, 1 × 10 9 / mL, 1 × 10 10 / mL, 1 × 10 11 / mL, 1 × 10 12 / mL, 1 × 10 13 / mL, 1 × 10 14 / mL, 1 × 10 15 / mL, 1 × 10 16 / mL, 1 × 10 17 / mL, 1 × 10 18 / mL, 1 × 10 19 / mL, ~ / or approximately 1 × 10 20 / mL, or any number or subrange within these ranges.

[0119] In some embodiments, the amount or effective amount (particularly when infectious particles are delivered (e.g., virus or virus-like particles as a primary or secondary agent, e.g., as cargo)), the effective amount of viral particles can be expressed as a titer (plaque forming units per unit volume) or MOI (multiplicity of infection). In some embodiments, an effective amount is about 1 x 10 per pL, nL, μL, mL, or L. 1 Particles ~ 1 x 10 per pL, nL, µL, mL, or L 20 / particle or more, e.g., approximately 1 x 10 per pL, nL, μL, mL, or L 1 , 1×10 2 , 1X10 3 , 1X10 4 , 1×10 5 , 1×10 6 , 1×107 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , 1×10 18 , 1×10 19 ~ / 1×10 20 In some embodiments, the effective titer is about 1×10 per pL, nL, μL, mL, or L. 1 Convert units to pL, nL, μL, mL, or 1 x 10 per L 20 / conversion unit or more, e.g., approximately 1 x 10 per pL, nL, μL, mL, or L 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , 1×10 18 , 1×10 19 , ~ / or about 1×10 20It can be a conversion unit or any number or subrange within these ranges. In some embodiments, the MOI of the pharmaceutical formulation is in the range of about 0.1 to 10 or more, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4. 9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10 or more, or any number or subrange within these ranges.

[0120] In some embodiments, the amount or effective amount of one or more active agents described herein included in the pharmaceutical formulation may range from about 1 pg / kg to about 10 mg / kg based on the body weight of a subject in need thereof, or the average body weight of a particular patient population to which the pharmaceutical formulation can be administered.

[0121] In embodiments in which a secondary agent is included in the pharmaceutical formulation, the effective amount of the secondary active agent will vary depending on, among other things, the secondary agent, the primary agent, the route of administration, the age of the subject, the disease, and the stage of the disease, as would be common knowledge to one of ordinary skill in the art.

[0122] If optionally present in the pharmaceutical formulation, the second active agent can be included in the pharmaceutical formulation or can be present as a separate compound or pharmaceutical formulation that can be administered simultaneously or sequentially with the compound, its derivative, or its pharmaceutical formulation.

[0123] In some embodiments, the effective amount of a secondary active agent, if any, is about 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 120, 12 , 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9% w / w, v / v, or w / v, or any numeric value or subrange therein. In additional embodiments, the effective amount of the second active agent is about, but not zero, 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 2 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9% w / w, v / v, or w / v, or any numeric value or subrange therein.

[0124] Dosage form In some embodiments, the pharmaceutical formulations described herein can be provided in a dosage form. The dosage form can be administered to a subject in need thereof. The dosage form can effectively produce a particular concentration, such as an effective concentration, at a predetermined site in a subject in need thereof. As used herein, a "dose", "unit dose", or "dosage amount" can refer to a physically separate unit suitable for use in a subject, each unit containing a predetermined amount of a primary active agent, and optionally a secondary active ingredient present, and / or a pharmaceutical formulation thereof calculated to produce a desired response in association with its administration. In some embodiments, the predetermined site is proximal to the site of administration. In some embodiments, the predetermined site is distal to the site of administration. In some cases, the dosage form contains a greater amount of one or more active ingredients present in the pharmaceutical formulation than the final intended amount required to reach a particular region or location in the subject, to compensate for loss of the active ingredients via first and second pass metabolism.

[0125] The dosage form can be adapted for administration by any suitable route. Suitable routes include, but are not limited to, oral (including buccal or sublingual), rectal, ocular, inhalation, intranasal, topical (including buccal, sublingual, or transdermal), vaginal, parenteral, subcutaneous, intramuscular, intravenous, intranasal, and intradermal. Other suitable routes are described elsewhere herein. Such formulations can be prepared by any method known in the art.

[0126] Dosage forms suitable for oral administration can be individual dosage units such as capsules, pellets or tablets, powders or granules, solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips, or oil-in-water or water-in-oil liquid emulsions. In some embodiments, pharmaceutical formulations suitable for oral administration also include one or more agents that flavor, preserve, color, or aid in the dispersion of the pharmaceutical formulation. Dosage forms prepared for oral administration can be in the form of a solution that can be delivered as a foam, spray, or solution. The dosage form can be administered to a subject in need thereof. If desired, the dosage forms described herein can be microencapsulated.

[0127] The dosage form can also be prepared to extend or sustain the release of any component. In some embodiments, the compounds, molecules, compositions, vectors, vector systems, cells, or combinations thereof described herein can be the component whose release is delayed. In some embodiments, the primary active agent is the component whose release is delayed. In some embodiments, any secondary active agent can be the component whose release is delayed. Suitable methods for delaying the release of a component include, but are not limited to, coating or embedding the component in a material such as a polymer, wax, gel, etc. Delayed release dosage forms can be prepared as described in standard references such as "Pharmaceutical dosage form tablets," eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989), "Remington-The science and practice of pharmacy," 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and "Pharmaceutical dosage forms and drug delivery systems," 6th Edition, Ansel et al., (Media, PA: Williams and Wilkins, 1995). These references provide information on excipients, materials, equipment, and processes for preparing delayed release dosage forms of tablets and capsules, as well as tablets and pellets, capsules, and granules. The delayed release can be from about 1 hour to about 3 months or more.

[0128] Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, acrylic acid polymers and copolymers, as well as methacrylic resins commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

[0129] Coatings can be formed using different ratios of water-soluble polymers, water-insoluble polymers, and / or pH-dependent polymers, with or without water-insoluble / water-soluble non-polymeric excipients, to produce the desired release profile. Coatings are performed either as dosage forms (matrix or simple), including but not limited to tablets (compressed with or without coated beads), capsules (with or without coated beads), beads, particulate compositions, "neat" formulated in suspension, or as sprinkle dosage forms.

[0130] Optionally, the dosage form described herein can be liposomal. In these embodiments, the first active ingredient(s) and / or any secondary active ingredient(s) and / or optionally its pharma- ceutically acceptable salt are incorporated into liposome. Thus, in the embodiment in which the dosage form is liposomal, the pharmaceutical preparation is a liposomal preparation. The liposomal preparation can be administered to a subject in need thereof.

[0131] Dosage forms suitable for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils. In some embodiments for the treatment of the eye or other external tissues, such as the mouth or skin, the pharmaceutical formulation is applied as a topical ointment or cream. When formulated into an ointment, the primary active ingredient, any secondary active ingredient, and / or optionally its pharma-ceutically acceptable salts can be formulated with a paraffinic or water-miscible ointment base. In other embodiments, the primary and / or secondary active ingredients can be formulated into a cream with an oil-in-water cream base or a water-in-oil cream base. Dosage forms suitable for topical administration in the mouth include lozenges, pastilles, and mouthwashes.

[0132] Dosage forms suitable for nasal or inhalation administration include aerosols, solutions, suspension drops, gels, or dry powders. In some embodiments, the primary active ingredient, any secondary active ingredient, and / or optionally a pharma- ceutically acceptable salt thereof may be in a dosage form suitable for inhalation, in a form having a reduced particle size obtained or obtainable by micronization. In some embodiments, the particle size of the size-reduced (e.g., micronized) compound or its salt or solvate is within a range of about 0.5 to about 10 microns D measured by a suitable method known in the art. 50 The dosage form is defined by the value. Suitable dosage forms for administration by inhalation also include particle dusts or mists. Suitable dosage forms in which the carrier or excipient is a liquid for administration as a nasal spray or nasal drops include aqueous or oily solutions / suspensions of the active (primary and / or secondary) ingredients, which can be generated by various types of metered-dose pressurized aerosols, nebulizers, or inhalers. Nasal / inhalation formulations can be administered to subjects in need thereof.

[0133] In some embodiments, the dosage form is an aerosol formulation suitable for administration by inhalation. In some of these embodiments, the aerosol formulation optionally comprises a solution or fine suspension of a first active ingredient, a second active ingredient, and / or its pharma- ceutically acceptable salt, and a pharma-ceutically acceptable aqueous or non-aqueous solvent. The aerosol formulation may be provided in single or multiple doses in sterile form in a sealed container. In some of these embodiments, the sealed container is a single-dose or multi-dose aerosol dispenser (e.g., a metered-dose inhaler) with a nasal or metered-dose valve intended to be discarded after the contents of the container are exhausted.

[0134] When the aerosol dosage form is contained in an aerosol dispenser, the dispenser contains a suitable propellant under pressure, such as compressed air, carbon dioxide, or an organic propellant, including but not limited to hydrofluorocarbons. In other embodiments, the aerosol formulation dosage form is contained in a pump sprayer. The pressurized aerosol formulation can also contain a solution or suspension of the first active ingredient, any secondary active ingredient, and / or a pharma-ceutically acceptable salt thereof. In further embodiments, the aerosol formulation also contains co-solvents and / or modifiers incorporated, for example, to improve the stability and / or taste and / or particulate mass characteristics (amount and / or profile) of the formulation. The administration of the aerosol formulation can be once a day or several times a day, for example, 2, 3, 4, or 8 times a day, with 1, 2, 3 or more doses being delivered each time. The aerosol formulation can be administered to a subject in need thereof.

[0135] In some dosage forms suitable and / or adapted for inhalation administration, the pharmaceutical formulation is a dry powder inhalable formulation. In addition to the primary active agent, any secondary active ingredient, and / or optionally a pharma- ceutically acceptable salt thereof, such dosage forms may contain a powder base such as lactose, glucose, trehalose, mannitol, and / or starch. In some of these embodiments, the primary active agent, any secondary active ingredient, and / or optionally a pharma- ceutically acceptable salt thereof, are in a form with reduced particle size. In further embodiments, a performance modifier such as L-leucine or another amino acid, cellobiose octaacetate, and / or a metal salt of stearic acid such as magnesium stearate or calcium stearate. In some embodiments, the aerosol formulation is arranged such that each metered dose of the aerosol contains a predetermined amount of active ingredient, such as one or more of the compositions, compounds, vector(s), molecules, cells, and combinations thereof described herein. Wherein.

[0136] The dosage form suitable for vaginal administration can be provided as pessary, tampon, cream, gel, paste, foam or spray formulation.The dosage form suitable for rectal administration includes suppository or enema.Vaginal preparation can be administered to the subject who needs it.

[0137] Formulations adapted for parenteral administration and / or for injection may include aqueous and / or non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostats, solutes that render the composition isotonic with the subject's blood, and aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickening agents. Formulations suitable for parenteral administration may be provided in single-unit dose or multi-unit dose containers, including, but not limited to, sealed ampoules or vials. The doses may be lyophilized and resuspended in a sterile carrier to reconstitute the dose prior to administration. In some embodiments, extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Parenteral formulations may be administered to subjects in need thereof.

[0138] In some embodiments, the dosage form contains a predetermined amount of a first active agent, an optional secondary active ingredient, and / or optionally a pharma- ceutically acceptable salt thereof per unit dose. In one embodiment, the predetermined amount of the first active agent, the secondary active ingredient, and / or optionally a pharma- ceutically acceptable salt thereof can be an effective amount, a minimally effective amount, and / or a therapeutically effective amount. In other embodiments, the predetermined amount of the first active agent, the secondary active agent, and / or optionally a pharma- ceutically acceptable salt thereof can be an appropriate fraction of the effective amount of the active ingredient. Co-therapy and combination therapy

[0139] In some embodiments, the pharmaceutical formulation(s) described herein are part of a combination treatment or therapy. The combination treatment may include the pharmaceutical formulations described herein and an additional treatment modality. The additional treatment modality may be chemotherapy, biological therapy, surgery, radiation, dietary modification, environmental modification, physical activity modification, and combinations thereof.

[0140] In some embodiments, the co- or combination therapy further includes, but is not limited to, polynucleotides, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, hormones, immunomodulatory agents, antipyretics, anxiolytics, antipsychotics, analgesics, anticonvulsants, anti-inflammatory agents, antihistamines, anti-infective agents, radiosensitizers, chemotherapeutic agents, imaging agents, immunogens, anti-cancer agents, or any combination thereof.

[0141] Administration of pharmaceutical preparations The pharmaceutical formulations or dosage forms thereof described herein can be administered hourly, daily, monthly, or yearly (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times hourly, daily, monthly, or yearly). In some embodiments, the pharmaceutical formulations or dosage forms thereof described herein can be administered continuously for a period ranging from minutes to hours, or even days. Devices and dosage forms effective for providing continuous administration of the pharmaceutical formulations described herein are known in the art and described herein. In some embodiments, the first one or several initial doses administered can be higher doses than subsequent doses. This is commonly referred to in the art as a loading dose and a maintenance dose, respectively. In some embodiments, the pharmaceutical formulations can be administered in tapered (increasing or decreasing) doses over time to gradually wean a subject off the pharmaceutical formulation or gradually introduce the pharmaceutical formulation to a subject.

[0142] As mentioned above, the dosage form may contain a predetermined amount of the primary active agent, any secondary active agent, and / or pharma-ceutically acceptable salts thereof, as appropriate, per unit dose. In some of these embodiments, the predetermined amount may be an appropriate fraction of the effective amount of the active ingredient. Thus, such unit doses may be administered once or multiple times daily, monthly, or yearly (e.g., 1, 2, 3, 4, 5, 6, or more times daily, monthly, or yearly). Such pharmaceutical formulations may be prepared by any method well known in the art.

[0143] When a joint therapy or multiple pharmaceutical preparations are delivered to a subject, different therapies or preparations can be administered sequentially or simultaneously.Sequential administration is administration in which a significant time elapses between administrations, for example, about 15, 20, 30, 45, 60 minutes or more.The time between administrations in sequential administration can be about hours, days, months, or even years, depending on the active agent present in each administration.Concurrent administration refers to administering two or more preparations at the same time or substantially the same time (for example, within a few seconds or only a few minutes apart), and it is intended that the preparations are administered at the same time.

[0144] kit Any of the compounds, compositions, formulations, and / or exosomes described herein, or combinations thereof, may be provided as a combination kit. In some embodiments, the kit includes one or more filters, tubes, devices, etc., used to prepare milk exosomes according to the methods described herein. As used herein, the term "combination kit" or "kit of parts" refers to a compound, composition, formulation, particle, cell, and any additional components used to package, sell, trade, deliver, and / or administer the combination of multiple elements or a single element, such as an active ingredient, contained therein. Such additional components include, but are not limited to, packages, syringes, blister packages, bottles, and the like. When one or more of the compounds, compositions, formulations, particles, cells, or combinations thereof (e.g., drugs) contained in the kit described herein are administered simultaneously, the combination kit can include the active agents in a single formulation, such as a pharmaceutical formulation (e.g., tablet), or in separate formulations. When the compounds, compositions, formulations, particles, and cells described herein, or combinations thereof and / or kit components are not administered simultaneously, the combination kit can include each agent or other component in a separate pharmaceutical formulation. The separate kit components may be included in a single package or in separate packages within the kit.

[0145] In some embodiments, the combination kit also includes instructions printed on or otherwise included in the tangible expression medium. The instructions can provide information regarding the compounds, compositions, formulations, particles, and / or exosomal contents described herein, or any combination thereof contained therein, safety information regarding the compounds, compositions, formulations (e.g., pharmaceutical formulations), particles, and / or exosomal contents described herein, or any combination thereof contained therein, information regarding dosage, instructions for use, and / or recommended treatment regimen(s) of the compound(s) and / or pharmaceutical formulation(s) contained therein. In some embodiments, the instructions provide instructions on how to prepare milk exosomes according to methods described elsewhere herein. In some embodiments, the instructions can provide instructions for administering the compounds, compositions, formulations, particles, and cells, or combinations thereof described herein to a subject in need thereof. EXAMPLES

[0146] Having thus far described embodiments of the present disclosure, the following examples generally describe some additional embodiments of the present disclosure. The embodiments of the present disclosure are described in conjunction with the following examples and corresponding text and figures, without the intention of limiting the embodiments of the present disclosure to this description. On the contrary, it is intended to cover all alternatives, modifications, and equivalents that fall within the spirit and scope of the present disclosure. The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to practice the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric pressure. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0147] Example 1 Isolation of exosomes from bovine milk result Optimized ultracentrifugation-based isolation protocol Two different protocols were optimized to isolate purified exosomes from milk. The critical step in each protocol was the timing of chemical solubilization of casein micellar structures by divalent cation chelation using 30 mM EDTA for 1 h at 37 °C. The first protocol is referred to as the ultracentrifugation (UC)-based method (Figure 1). The second incorporated tangential flow filtration (TFF) and is henceforth referred to as the TFF-based method (Figure 2). In the UC-based method, high levels of exosome yield and purity were achieved by placing a primary chelation step before the final Sepharose column (SEC) filtration step (Figures 3A-3E). The histogram in Figure 3A shows successive fractions collected during SEC filtration, with the y-axis showing protein concentration (mg / ml) measured by Nanodrop. Western blotting showed signals for exosome markers CD81, CD9, and syntenin in association with the absence of bands corresponding to calnexin (cell membrane marker), Arf6 (microvesicle marker), and casein in the peak exosome SEC fractions, i.e., fractions 8.5 and 9.0 (Figure 3B). Exosome protein markers were absent in later SEC fractions 15–18 (e.g., Figure 3B). The nanoparticle tracking analysis (NTA) graph in Figure 3C was measured from fraction 8.5, which was the most abundant exosome as shown by negative staining transmission electron microscopy (TEM) (Figure 3D). NTA analysis showed approximately 1 × 10 exosomes per mL. 13The concentration of particles showed particle sizes consistent with exosomes (mode ~133 nm). TEM images in Figure 3D show negative staining of densely packed exosomes in the peak SEC fractions. Figure 3E shows the casein macrostructure in fraction 17, with typical casein micelles found in these fractions shown in the inset at higher magnification. Taken together, TEM, NTA, and Western blotting confirmed the presence of very dense, pure, and ultrastructurally definitive exosomes in peak SEC fractions 8.0-9.0, with low levels of protein signal and particulate matter corresponding to casein and casein micelle aggregates, which are seen in later SEC fractions. Of note, from a typical starting volume of 1000 mL of milk at the start of a UC-based protocol, these final ultra-dense exosome concentrates constitute an average of 75 mL (+ / - 10 mL), or 7.5% of the starting volume of milk (Figure 6).

[0148] It is believed that shear forces applied during UC can adversely affect the structure of exosomes (MogiK,2018) (Lasser C,2012). Our observations also suggest that UC can affect the yield and purity of exosomes from milk. The composition of the pellets from the 70,000RCF and 100,000RCF spins, along with the TFF-enriched supernatant after the 130,000RCF spin of the UC-based method, are shown in Figures 7A-7B and 8, respectively. SEC filtration of these samples followed by TEM shows the presence of large numbers of exosomes in all of these samples. As these pellets and supernatants are discarded, the optimized UC-based method loses the large number of extracellular vesicles present in these fractions. Furthermore, because the levels of contaminating casein in the peak fractions resulting from SEC were higher compared to the optimized method, it is not possible to skip these low-speed spins and move directly to the 130,000rcf spin (data not shown).

[0149] Optimized TFF-based isolation protocol The loss of exosomes shown in Figures 7A-7B and 8 in the UC-based method led us to explore TFF as an alternative approach. The optimized protocol for TFF-based exosome isolation developed from our studies is summarized in Figure 2. Histograms obtained from the protein concentrations of successive SEC fractions after TFF are shown in Figure 4A, with protein concentration in mg / mL shown on the Y-axis. Although the results of the TFF-based protocol were similar in many respects to the UC-based method, we found that the casein solubilization step, optimally placed before the TFF filtration, resulted in a final yield approximately 100% greater than the UC-based method. Western blotting of the peak exosome SEC fractions showed the presence of exosome markers CD81, CD9 and syntenin, along with the absence of microvesicle markers calnexin and Arf6, and a significant reduction in casein compared to later SEC fractions, e.g., fraction 17 (Figure 4B). The NTA analysis shown is from peak fraction 8.5 and shows 100 nm mode with a concentration of over 1 x 1013 particles per mL of final solution (Figure 4C). The TEM image below the histogram (Figure 4D) shows a highly concentrated and pure exosome solution in the peak SEC fractions (8.5 and 9.0), but TEM of subsequent SEC fractions showed high levels of casein micelle aggregates (Figure 4E). The TFF-based protocol provided an average of 200 ml (+ / - 10 mL) of exosome concentrate in the peak fractions per 1000 ml of milk, i.e., 20% of the starting volume of milk.

[0150] Figure 5 shows confocal optical sections of calcein-labeled exosomes in peak SEC fractions generated by the TFF-based method and suspended in 1:10 diluted Hepes buffer. Images show uptake resulting from 1, 2, 3, and 4 h of incubation with Calcein-AM, a dye that is nonfluorescent until activated by deesterification. The patchy fluorescent signal suggests that extracellular vesicles in our exosome concentrates contain esterase activity and can retain deesterified calcein molecules. The level of calcein signal became stronger with longer incubations, suggesting cumulative retention of the dye and structural / functional integrity of the isolated exosomes. Similar patterns of calcein fluorescence and retention were observed with exosomes isolated using the UC-based protocol (data not shown).

[0151] Impact of deviations from the optimized protocol For comparison, Figure 9A shows the typical TEM negative staining appearance of exosomes isolated using the UC-based method, which does not undergo the final casein solubilization and SEC filtration steps as performed in the optimized protocol. The exosomes are ultrastructurally evident, but are significantly less dense than in the optimized protocol. The exosomes are also enriched in casein micelles. Figures 9B-9D show other examples of suboptimal results in terms of exosome density and casein contamination when the SEC filtration (Figure 9B), divalent cation chelation (Figure 9C) and / or 37°C temperature (Figure 9D) aspects of our optimized protocol are omitted. When divalent cation chelation or incubation at 37°C temperature was not performed, exosome density in the peak SEC fractions was reduced and casein contamination was increased, as evidenced by the ultrastructural presence of casein micelles and 20-25 kDa gel bands corresponding to casein in these same SEC fractions. We also investigated the impact of casein solubilization at EDTA concentrations below 30 mM, temperatures below 37 °C, incubation periods shorter than 60 min, and performing the casein solubilization step at various stages of the protocol other than pre-SEC or pre-TFF for the UC- and TFF-based methods, respectively. All these deviations from the optimal protocol resulted in peak SEC fractions exhibiting lower exosome density and increased casein contamination levels, similar to those shown in Figures 9A-9D (data not shown). Incubation in 30 mM EDTA for up to 2 h, while not detrimental, did not appear to provide any further benefit in terms of exosome yield and solubilization, while EDTA concentrations above 30 mM resulted in blebbing distortions in the exosome ultrastructure (Figure 10).

[0152] Consideration Variation in the purity of exosomes produced by different isolation protocols, including differences in the presence of EV subtypes and contaminating proteinaceous aggregates, are issues that impede progress in the field (Vaswani K., 2019). Contamination issues are of particular concern when isolating exosomes from milk, where casein micelles and higher-order polymeric structures containing casein routinely co-precipitate during purification of exosome fractions (Yamauchi M, 2019). This example demonstrates, at a minimum, an approach that significantly reduces the burden of contaminating proteins in exosome isolates. Central to this approach is the strategic deployment of a divalent cation chelation treatment at 37 °C that promotes solubilization of casein micelle structures. When this 1-hour treatment is used at specific time points in the TFF- and UC-based protocols described herein, highly efficient separation of exosomes from casein-containing aggregates can be achieved. Deviations from the optimized protocol, including the use of EDTA at concentrations less than 30 mM and incubation at temperatures less than 37° C., as well as deploying a chelation step at method stages other than those specified in FIGS. 1-2, result in isolated exosomes with lower purity and higher levels of contamination with milk proteins.

[0153] A further obstacle to the field is the currently limited ability to produce exosomes cheaply and efficiently on a large scale. Typically, large amounts of biofluid or tissue (e.g., plasma, urine, adipose tissue), or culture medium are required to start with, and even then, the yield of the final exosome isolate tends to be modest (Lasser C, 2012). This method allows the generation of large amounts of purified exosomes at high density from milk in a series of cost-effective and simple steps. Indeed, the extent to which exosomes make up a significant portion of milk by volume was an unexpected outcome of our study. The fact that milk is packed to this extent with extracellular vesicles, many of which have been reported in the literature to contain miRNAs and other molecules with communication or signaling potential (Golan-Gerstl R., 2017), provides an intriguing new insight into the developmental guidance functions and nutritional functions of mammalian nursing.

[0154] The difference in absolute yields obtained from the TFF- and UC-based methods is noteworthy. The ultra-high density accumulation of exosomes in the peak SEC fractions obtained from the TFF-based method (e.g., Figure 6) corresponds to approximately 20% of the starting volume of milk. The UC-based method still results in an excellent exosome enrichment at approximately 7.5% of the starting volume (Figures 3A-3E). That being said, our EM analysis suggests that the lower yield of the UC-based method may be due to the lower efficiency of this protocol, as shown in Figures 7-8. Considering the greater yield obtained with the TFF-based method, the TFF-based method appears to be the preferred approach. This preference is reinforced by the Western blotting and NTA results showing that the purity and particle density per unit volume of exosomes generated by the two methods are comparable. An additional consideration is that protocols incorporating TFF separation may be inherently more scalable than protocols relying on multiple UC steps and may ultimately be the basis for the production of exosomes from milk at an industrial scale.

[0155] The large quantities of pure exosomes generated by these methods provide a sufficient basis for ongoing experimental and methodological testing, including the development of technological approaches to load exosomes with cargo such as small and large molecule drugs, peptides, and miRNAs. Safe and effective drug delivery in animal models has been demonstrated for exosome-carried drugs including doxorubicin (Yang T., 2015), (Tian T., 2014), curcumin (Zhuang X., 2011), and paclitaxel (Agrawal AK, 2017), as well as siRNA (Alvarez-Erviti L., 2011) and miRNA (Wang F., 2018), (Momen-Heravi F., 2014). Techniques reported in the literature for loading exosomes with exogenous molecules include electroporation, sonication, freeze-thaw, extrusion and membrane saponification (Momen-Heravi F., 2014), (Haney MJ, 2015). Although somewhat effective, a drawback of such techniques is the damage to the exosome membrane, which reduces drug retention and effective delivery of therapeutic cargo to cells. Results of EM analysis of exosome fractions from various iterations of our isolation protocol suggest that the exosome membrane may be sensitive to mechanical and chemical disruption (e.g., Figures 9A-9D and 10). Furthermore, a novel and relatively gentle approach to drug loading is suggested by the calcein retention assay in Figure 5, suggesting that uptake and retention of exogenous molecules in exosomes may be enhanced by adding ester groups to the loaded molecules. Data from short therapeutic peptides based on the carboxyl terminus of connexin 43 (C×43) (Jiang J., 2019) suggest that esterification could be a strategy for exosomal drug loading. An embodiment in which a cargo compound is esterified to facilitate exosomal uptake is described on pages 81-86 of International Patent Application Publication WO2020 / 028439.

[0156] Exosomes have been utilized as drug delivery devices by many groups, combined with systems such as ultrasound targeted microbubble destruction (Sun W, 2019), as simple drug carriers for neurological diseases (Yang T., 2015), and engineered with designer cells to specifically target cell populations for direct delivery (Kojima R, 2018). The most promising method to utilize exosomes as DDDs is to engineer the exosome surface after isolation to specifically upregulate markers of interest for specific delivery of exosomal cargo (Si Y, 2020). Other groups have simply applied exosomes with loaded cargo orally, demonstrating a simple administration method that is highly effective for delivery of pharmaceutical cargo (Agrawal AK, 2017). Exosomes as DDDs have been extensively reviewed in previous reviews (Vader P, 2016). The methods described and demonstrated herein can facilitate the production of industrially and clinically relevant quantities of exosomes.

[0157] Additional methods Exosome isolation – Ultracentrifugation-based method Figure 1 summarizes the steps of the optimized UC-based protocol. Unpasteurized milk at 4 °C was obtained from Homestead Creamery of Wirtz, VA. All subsequent steps leading up to chelation and temperature treatment were performed at 4 °C. Milk was transferred to sterile large polypropylene centrifuge tubes (Thermo Scientific, 75007585) and centrifuged at 5,000 rcf (Sorval Legend X1R centrifuge with Sorval TX-400 75003629 rotor) for 30 min. Fat (cream) was removed by decanting from the supernatant (SN) or by whisking it off with filter paper. The remaining SN was transferred to a new container and the pellet was discarded. These steps were repeated 2-3 times to ensure skimming. The milk was then transferred to a 250 mL centrifuge vessel (Nalgene) and centrifuged for 60 minutes at 14,500 rcf (Beckmann Coulter Avanti J-26XP centrifuge with JLA16.25 rotor). The SN was then transferred to a 250 mL polypropylene vessel (Beckmann) and centrifuged for 60 minutes at 22,600 rcf (Beckmann Coulter Avanti J-26XP centrifuge with JLA16.25 rotor). After each centrifugation, the SN was decanted and the pellet discarded, removing all visible fat. This centrifugation, fat removal and SN decanting step was repeated 3-4 times at 22,600 rcf. The SN were then successively filtered through 0.45 μm and 0.22 μm filters (Millipore), transferred to Beckmann 355631 ultracentrifuge tubes, and centrifuged at 56,000 rcf (Beckmann Coulter Avanti J-26XP centrifuge with JA25.5 rotor) for 60 min. After these low speed centrifugations, the pellet was discarded and the SN was transferred to new 355631 Beckmann tubes and centrifuged at 70,000 rcf (Beckmann Coulter Optima L-100XP ultracentrifuge with SW.32.Ti rotor) for 60 min.The SN was then transferred to a new Beckmann 355631 tube and centrifuged at 100,000rcf (Beckmann Coulter Optima L-100XP ultracentrifuge with SW.32.Ti rotor) for 60 min. The resulting SN was further centrifuged at 130,000rcf (Beckmann Coulter Optima L-100XP ultracentrifuge with SW.32.Ti rotor) for 120 min. The resulting pellet was then dissolved in 2-3 ml of Hepes buffer (100 mM NaCl, 4 mM KCl, 20 mM Hepes, pH 6.7) overnight at 4 °C (pellet 10% by volume). The following morning, the solution was triturated and aliquoted in 500 µL. These aliquots were stored at -80 °C until further use. After thawing on ice, EDTA was added to the aliquots to a concentration of 30 mM, and the solution was incubated at 37 °C for 60 min. The solution was then passed through an IZON qEV Original 70 nm Sepharose column (1006881). The protein concentration of the resulting fractions was analyzed using a Nanodrop2000c running Nanodrop2000 software by 260 / 280 spectrophotometry using standard methods and Hepes buffer as blank control solution. After protein quantification, samples were aliquoted and stored at -80°C until further use.

[0158] Exosome Isolation – A Tangential Flow Filtration-Based Protocol Figure 2 summarizes the steps of the optimized TFF-based protocol. Unpasteurized milk at 4°C was obtained from Homestead Creamery of Wirtz, VA, transferred to sterile large polypropylene centrifuge tubes (Thermo Scientific, 75007585) and processed with the same low speed centrifugation and fat skimming steps as the UC-based method described above until filtration of the resulting SN through Millipore 0.45um and 0.22um filters. The resulting solution was then treated with 30mM EDTA for 60 minutes at 37°C. After processing, the solution was filtered using a Repligen KrosFlo TFF system on a 500kDa MidiKros TFF filter (Repligen) at 10mL / min. When the solution reached approximately 10% of the starting volume, the solution was further diafiltered with approximately 10 volumes of standard Hepes buffer composition as in the UC-based method. The solution was then stored at -80°C prior to column separation when this TFF retentate reached approximately 20% of the starting volume. In another example, after the TFF retentate reached approximately 20% of the starting volume, the solution was transferred to a Beckmann 355631 tube and ultracentrifuged at 130,000 rcf for 120 min at 4° C. using a SW.32.Ti rotor in a Beckmann Coulter Optima L-100XP ultracentrifuge. The resulting pellet was resuspended in approximately 10% of the starting volume (approximately 2.5 mL) of buffer and allowed to dissolve in this solution overnight at 4° C. The next morning, the solution was triturated and aliquoted in 500 μL volumes. After storage at −80° C. or ultracentrifugation of the retentate, the solution was separated on an IZON qEV Original 70 nm Sepharose column (1006881) and the resulting fractions were analyzed by Nanodrop and spectrophotometry as described in the UC-based method above. After protein quantification, the samples were aliquoted in fractions and stored at −80° C. until further use.

[0159] Gel electrophoresis and Western blotting To prepare samples for electrophoresis, samples were mixed with Lamelli's sample buffer (BioRad) containing 0.05% beta-mercaptoethanol (Thermo Fisher). Samples were then boiled for 5 min and 6.25 mg of protein was loaded into each lane of a 4%-20% Biorad unstained gel (BioRad-5678093). Electrophoresis was performed for 40 min at 200 V in standard running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) in a Biorad module (CRITERION Cell 135BR0030876). Stain-free gels were then imaged using a 5-min activation imaging on a Bio-Rad imager (BioRad Universal Hood III731BR00622). Transfer of proteins from the gel was performed using a Bio-Rad Transblot Turbo onto a PVDF (Millipore IPFL00010) membrane in standard transfer buffer (25 mM Tris, 192 mM glycine, 0.01% SDS) at 25 V, 1.0 A for 30 min. The membrane was then dried at room temperature (RT) for 1 h to allow protein fixation. The PVDF transfer membrane was then rehydrated with methanol, washed with distilled water, and blocked with 3% fish skin gelatin extract (FSE) (Thermo Fisher) in TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.6) for 1 h at RT. Overnight incubation of primary antibodies was performed according to the manufacturer's instructions, diluted in 3% FSE in TBST, and left at 4 °C overnight. Antibodies used: CD81 (Cell Signaling Technology, 56039S), TSG-101 (Invitrogen, MA1-23296), CD9 (Novus, NB500-494), Calnexin (EMD Millipore, AB2301), casein (Abcam, ab166596), ARF6 (Novus, NBP1-58310), Syntenin-1 (Santa Cruz, SC-100336), and relevant secondary antibodies: anti-mouse (Jackson Immuno, 715-035-150) and anti-rabbit (Southern Biotechnology, 4050-05).The membrane was then washed five times in TBST for 5 min at room temperature on an orbital shaker (VWR model 100 10M0219G) to remove unbound antibody. After washing, the membrane was incubated in secondary antibody diluted 1:20,000 in 1:1 TBST:3% FSE for 1 h at room temperature, then washed five times in TBST for 5 min on a shaker. The blot membrane was then activated with Thermo Scientific Pico Activation Buffer (Thermo Scientific) for 5 min and imaged on a Biorad imager under chemical detection settings. Bands were quantified using densitometric analysis in ImageLab version 6.1 (BioRad).

[0160] Nanosight Tracker Analysis Nanosight Tracker Analysis (NTA) analysis was performed on a Nanosight NS300. Exosome concentrates obtained after SEC were diluted 1:10 in Hepes buffer and bath sonicated for 1 min at room temperature in a Branson 2510 bath sonicator to reduce sample aggregation. Exosomes were then diluted (1:1000–1:10,000 depending on the sample), added to a 1 mL syringe, loaded onto a syringe pump and loaded into the NTA flow cell. Each sample was analyzed with three consecutive 1-min video recordings using a 405 nm laser at a constant flow rate set at 10 in the NTA software (version 3.4). The flow rate was set in the software and is unit-free. All videos were analyzed collectively in the NTA software and data were collected and stored in raw format.

[0161] Confocal microscopy analysis The SEC exosome concentrate was diluted 1:10 in Hepes buffer and incubated with 10 μM Calcein-AM (Thermo Fisher Scientific C1430) at 37 °C for 1, 2, 3 or 4 h intervals. After incubation, extravesicular dye was removed with a Sepharose G50 column (USA Scientific 1415-1601), pre-equilibrated with Hepes buffer. 6 μL of this solution was then dispensed onto a microscope slide (Premiere 75 × 25 × 1 mm, 9105) and cover-slipped (Fisherbrand, 12541A). Calcein intensity and dye retention were monitored directly on particles suspended in this solution by optical sectioning using a 63x objective (oil, 1.4 NA) on a Leica SP8 confocal microscope equipped with a 488 laser, HyD, 1 AU, scanning frequency of 700 Hz with 6 fields per slide.

[0162] Transmission electron microscopy – negative staining Formvar-coated 200 mesh copper grids (Electron Microscopy Sciences, FCF200-CU) were glow discharged for 1 min at 0.29 mBAR in a Pelco glow discharge unit (Pelco). 0.1% poly-L-lysine was applied to the grids for 1 min, after which excess solution was removed with Whatman #1 filter paper. The grids were washed twice with 10 μL of milli-Q water, and excess liquid was removed with filter paper. The grids were then allowed to dry overnight at room temperature. Samples were loaded by applying 10 μL of prepared SEC exosome concentrate to the grids for 5 min. Excess solution was blotted with filter paper, and samples were negatively stained with 10 μL of uranium-less stain (Electron Microscopy Sciences, 22409) for 1 min at room temperature. Excess stain was then blotted. The grids were then allowed to dry overnight at room temperature before being imaged with a transmission electron microscope (TEM). Imaging of negatively stained preparations was performed on an FEI Tecnai G20 Biotwin TEM at 120 kV and images were captured using an Eagle 4K HS camera.

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[0210] Example 2 - Isolation of exosomes from human breast milk This example at least demonstrates the isolation of milk exosomes from human breast milk using the ultracentrifugation method described elsewhere herein (see, e.g., FIG. 1). After milk collection, milk samples were stored at -80°C for 6 months prior to isolating exosomes. Human milk exosomes were isolated using a UC-based isolation method. See, e.g., FIG. 11 and related discussion elsewhere herein. FIGS. 11A-11D show the results of exosome isolation from human breast milk by one embodiment of the ultracentrifugation method. FIG. 11A shows a graph depicting the concentration of exosomes or protein (x-axis) in each fraction in mg / mL (y-axis). FIG. 11B shows Nanosight Tracker analysis data of exosome isolates. FIGS. 11C-11D show high magnification TEM images of isolated exosomes (FIG. 11C) and representative TEM images of various exosome fractions (FIG. 11D).

[0211] Example 3. Effect of pre- and post-SEC storage on isolated milk exosomes This example evaluates the impact of pre-SEC and post-SEC storage on milk exosomes. Two separate analyses were used: pre-SEC storage and pre-SEC storage. Without wishing to be bound by theory, pre-SEC storage is important as it is believed that exosomes are more stable prior to gel separation. For the results shown in FIG. 12, samples were stored at -80°C for up to 3 months after TFF and prior to SEC. SEC was then performed and samples were stored at 4°C for the period indicated below each representative TEM image in FIG. 12. For the results shown in FIG. 13A-13B, samples were either not stored at -80°C at all (FIG. 13A) or were fresh samples that were stored at -80°C after TFF but were stored for 6 months prior to SEC processing (FIG. 13B). The TEM of the 6-month sample at least indicates that long-term storage prior to SEC processing is possible. It is contemplated that the use of cryoprotectants including sucrose, lactose, or trehalose could also be used to store our isolated exosomes at -80°C. ***

[0212] Various modifications and variations of the described methods, pharmaceutical compositions and kits of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific embodiments, it should be understood that the invention as described in the claims is capable of further modifications and should not be unduly limited to such specific embodiments. Indeed, various modifications of the described methods for carrying out the invention that are apparent to those skilled in the art are intended to be within the scope of the present invention. This application is generally intended to cover any variations, uses, or adaptations of the present invention in accordance with the principles of the present invention, and it will be understood that such departures from the present disclosure are within the scope of known or customary practice to which the invention pertains and may be applied to the essential features described above.

[0213] Further attributes, features, and embodiments of the present invention may be understood by reference to the following numbered aspects of the disclosed invention. Reference to the disclosure of any of the foregoing aspects is applicable to any preceding numbered aspect and any combination of any number of the preceding aspects, as recognized by appropriate prior disclosure in any combination of the preceding aspects that may be practiced. The following numbered aspects are provided: 1. A method for isolating exosomes from a biological fluid, comprising: a. centrifuging the biological fluid under conditions suitable to separate fat from one or more other components of the biological fluid; b. removing the separated fat from the biological fluid; c. centrifuging the remaining biological fluid after step (b) one or more times and skimming any significantly separated fat after each centrifugation of step (c); d. filtering the remaining biological fluid after step (c); e. optionally performing one or more ultracentrifugation steps after (d); f. chelating divalent cations with about 10 mM to about 100 mM EDTA at about 30-42 degrees Celsius, optionally for about 15-120 minutes after (d) or optionally (e); g. after (f), optionally performing tangential flow filtration to obtain a retentate, which is optionally ultracentrifuged by one or more ultracentrifugation steps or stored at -80°C, and optionally separating fractions of the retentate by column separation after the retentate has been optionally ultracentrifuged or stored at -80°C, The method includes step (e) or step (g), but not both. 2. The method of embodiment 1, wherein chelation of divalent cations occurs with about 30 mM EDTA. 3. The method of embodiment 1 or 2, wherein the chelation of the divalent cation occurs at about 37 degrees Celsius. 4. The method of any one of aspects 1 to 3, wherein the biological fluid is mammalian milk. 5. The method of any one of aspects 1-4, wherein the biological fluid is not pasteurized. 6. The method of any one of aspects 1 to 5, wherein steps (a) and (b) are repeated 1 to 5 times in combination. 7. The method of any one of aspects 1-6, wherein steps (a), (b), (c), (d), (e), (g), or any combination thereof, are carried out at about 4 degrees Celsius. 8. The method of any one of aspects 1 to 7, wherein (a) comprises centrifuging the biological fluid at about 2,000 to 3,000 rcf. 9. The method of any one of aspects 1-8, wherein (a) comprises centrifuging the biological fluid at about 2,500 rcf. 10. The method of any one of aspects 1-9, wherein step (a) is repeated 1 to 3 times. 11. The method of any one of aspects 1-10, wherein (b) comprises a first centrifugation followed by a second centrifugation. 12. The method of embodiment 11, wherein the first centrifugation comprises centrifuging the remaining biological fluid at about 13,500-15,500 rcf for about 45-75 minutes. 13. The method of embodiment 11 or 12, wherein the first centrifugation comprises centrifuging the remaining biological fluid at about 14,500 rcf for about 60 minutes. 14. The method of any one of aspects 11 to 13, wherein the second centrifugation is performed on the biological fluid remaining after the first centrifugation, and the second centrifugation is performed at about 24,800 to 26,800 rcf for about 45 to 75 minutes. 15. The method of embodiment 14, wherein the second centrifugation is performed on the biological fluid remaining after the first centrifugation, and the second centrifugation is performed at about 25,800 rcf for about 60 minutes. 16. The method according to any one of aspects 11 to 15, wherein the second centrifugation is repeated 1 to 3 times, each repetition being performed on the remaining biological fluid from the immediately preceding centrifugation. 17. The method of any one of aspects 1-16, wherein (d) comprises filtering the remaining biological fluid through one or more filters in series ranging from about a 0.45 micron filter to about a 0.22 micron filter. 18. The method of embodiment 17, wherein (d) comprises filtering the remaining biological fluid through an about 0.45 micron filter, followed by filtering the remaining biological fluid through an about 0.22 micron filter. 19. The method of any one of aspects 1-18, wherein (e) comprises two or more successive ultracentrifugation steps, each step being performed on the remaining biological fluid from the previous ultracentrifugation. 20. The method of embodiment 19, wherein (e) comprises an ultracentrifugation step performed at about 45,000-55,000 rcf, an ultracentrifugation step performed at about 65,000-75,000 rcf, an ultracentrifugation step performed at about 90,000-110,000 rcf, or a combination thereof. 21. The method of embodiment 19 or 20, wherein (e) comprises an ultracentrifugation step performed at about 50,000 rcf, an ultracentrifugation step performed at about 70,000 rcf, an ultracentrifugation step performed at about 100,000 rcf, or a combination thereof. 22. The method of any one of aspects 19-21, wherein one or more of the one or more ultracentrifugation steps are each carried out for about 45 to 75 minutes. 23. The method of any one of aspects 19-22, wherein one or more of the one or more ultracentrifugation steps are each carried out for about 60 minutes. 24. The method of any one of aspects 19-23, wherein (e) comprises a final ultracentrifugation step carried out at about 115,000-145,000 rcf for about 90-150 minutes, the resulting fluid being discarded, and the remaining pellet being resuspended in a suitable volume of a suitable solution prior to (f). 25. The method of any one of aspects 1-24, wherein (e) comprises a final ultracentrifugation step carried out at about 130,000 rcf for about 120 minutes, the resulting fluid being discarded, and the remaining pellet being resuspended in a suitable volume of a suitable solution prior to (f). 26. The method of any one of aspects 1 to 25, wherein the tangential flow filtration in (g) is carried out using an ultrafiltration membrane having a cutoff in the range of about 250 kDa to about 750 kDa. 27. The method of any one of aspects 1 to 26, wherein the tangential flow filtration in (g) is carried out using an ultrafiltration membrane of about 250 kDa. 28. The method of any one of aspects 1 to 27, wherein the tangential flow filtration of (g) is performed at a flow rate of about 5 to 15 mL per minute. 29. The method of any one of aspects 1-28, wherein the tangential flow filtration of (g) is performed at a flow rate of about 10 mL per minute. 30. The method of any one of aspects 1-29, wherein in step (g), when the amount of remaining biological fluid reaches about 10 percent of its starting volume before tangential flow filtration, the retentate is diafiltered with a suitable buffer. 31. The method of embodiment 30, further comprising ultracentrifuging the retentate when the retentate reaches about 20 percent of the starting diafiltration volume. 32. The method of claim 31, wherein the ultracentrifugation is carried out at about 4 degrees Celsius and about 115,000-145,000 rcf for about 90-150 minutes. 33. The method of embodiment 31 or 32, wherein the ultracentrifugation is carried out at about 4 degrees Celsius and about 130,000 rcf for about 120 minutes. 34. The method of embodiment 30, wherein the retentate is stored at -80°C after the retentate reaches about 20 percent of the starting diafiltration volume and prior to column separation. 35. The method of any one of aspects 1-34, wherein said method produces an exosome concentrate that is at least 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or at least 20 percent of the starting amount of milk. 36. The method according to any one of aspects 1 to 35, wherein the method further comprises loading exosomes of the formulation resulting from the method according to any one of the preceding claims with one or more cargoes. 37. A formulation comprising exosomes, the formulation being at least partially produced by a method according to any one of aspects 1 to 36. 38. The formulation of embodiment 37, wherein one or more of said exosomes are loaded with one or more cargoes. 39. A method comprising administering the formulation described in embodiment 38 to a subject in need thereof. 40. The method of embodiment 39, wherein the one or more cargoes are therapeutic cargoes.

Claims

1. 1. A method for isolating exosomes from a biological fluid, comprising: a. centrifuging the biological fluid under conditions suitable to separate fat from one or more other components of the biological fluid; b. removing the separated fat from the biological fluid; c. centrifuging the remaining biological fluid after step (b) one or more times and skimming any significantly separated fat after each centrifugation of step (c); d. filtering the remaining biological fluid after step (c); e. Optionally, performing one or more ultracentrifugation steps after (d); f. chelating divalent cations with about 10 mM to about 100 mM EDTA at about 30-42 degrees Celsius, optionally for about 15-120 minutes after (d) or optionally (e); g. after (f), optionally performing tangential flow filtration to obtain a retentate, wherein the retentate is optionally ultracentrifuged by one or more ultracentrifugation steps or stored at −80° C., and optionally separating fractions of the retentate by column separation after the retentate has been optionally ultracentrifuged or stored at −80° C.; Including, The method includes step (e) or step (g), but not both.

2. Chelation of divalent cations occurs at about 30 mM EDTA, or Optionally, the chelation of divalent cations occurs at about 37 degrees Celsius; or 10. The method of claim 1, optionally wherein the biological fluid is mammalian milk, and optionally wherein the biological fluid is not pasteurized.

3. Repeating steps (a) and (b) combined 1 to 5 times; or 10. The method of claim 1, wherein optionally steps (a), (b), (c), (d), (e), (g), or any combination thereof, are performed at about 4 degrees Celsius.

4. (a) comprises centrifuging the biological fluid at about 2,000-3,000 rcf; or Optionally, (a) comprises centrifuging the biological fluid at about 2,500 rcf; or 10. The method of claim 1, wherein optionally step (a) is repeated 1 to 3 times.

5. (b) comprises a first centrifugation followed by a second centrifugation; and Optionally, the first centrifugation comprises centrifuging the remaining biological fluid at about 13,500-15,500 rcf for about 45-75 minutes; or Optionally, the first centrifugation comprises centrifuging the remaining biological fluid at about 14,500 rcf for about 60 minutes; or Optionally, the second centrifugation is repeated 1 to 3 times, each repetition being performed on the remaining biological fluid from a previous centrifugation; or Optionally, the second centrifugation is performed on the biological fluid remaining after the first centrifugation, the second centrifugation is performed at about 24,800-26,800 rcf for about 45-75 minutes; and 10. The method of claim 1, optionally wherein the second centrifugation is performed on the biological fluid remaining after the first centrifugation, the second centrifugation being performed at about 25,800 rcf for about 60 minutes.

6. (d) comprises filtering the remaining biological fluid through one or more filters in series ranging from about a 0.45 micron filter to about a 0.22 micron filter; and 10. The method of claim 1, optionally wherein (d) comprises filtering the remaining biological fluid through an about 0.45 micron filter, followed by filtering the remaining biological fluid through an about 0.22 micron filter.

7. (e) comprises two or more successive ultracentrifugation steps, each step being performed on the remaining biological fluid from a previous ultracentrifugation; and Optionally, (e) comprises an ultracentrifugation step performed at about 45,000-55,000 rcf, an ultracentrifugation step performed at about 65,000-75,000 rcf, an ultracentrifugation step performed at about 90,000-110,000 rcf, or a combination thereof; or Optionally, (e) comprises an ultracentrifugation step performed at about 50,000 rcf, an ultracentrifugation step performed at about 70,000 rcf, an ultracentrifugation step performed at about 100,000 rcf, or a combination thereof; or Optionally, one or more of said one or more ultracentrifugation steps are carried out for about 45-75 minutes each; or 10. The method of claim 1, wherein optionally one or more of the one or more ultracentrifugation steps are performed for about 60 minutes each.

8. (e) comprises a final ultracentrifugation step carried out at about 115,000-145,000 rcf for about 90-150 minutes, the resulting fluid being discarded and the remaining pellet being resuspended in a suitable volume of a suitable solution prior to (f); or 10. The method of claim 1, wherein (e) optionally comprises a final ultracentrifugation step carried out at about 130,000 rcf for about 120 minutes, the resulting fluid being discarded and the remaining pellet being resuspended in a suitable volume of a suitable solution prior to (f).

9. (g) the tangential flow filtration is performed using an ultrafiltration membrane having a cutoff in the range of about 250 kDa to about 750 kDa; or Optionally, the tangential flow filtration of (g) is performed using a 250 kDa ultrafiltration membrane; or Optionally, the tangential flow filtration of (g) is performed at a flow rate of about 5 to 15 mL per minute; or 10. The method of claim 1, wherein optionally the tangential flow filtration of (g) is performed at a flow rate of about 10 mL per minute.

10. In step (g), when the amount of the remaining biological fluid reaches about 10 percent of its starting volume before tangential flow filtration, the retentate is diafiltered with a suitable buffer; and Optionally, after the retentate reaches about 20 percent of the starting diafiltration volume and prior to column separation, the retentate is stored at −80° C.; or Optionally, when the retentate reaches about 20 percent of the starting diafiltration volume, ultracentrifuging the retentate; and Optionally, the ultracentrifugation is performed at about 4 degrees Celsius and about 115,000-145,000 rcf for about 90-150 minutes; or 10. The method of claim 1, optionally wherein the ultracentrifugation is performed at about 4 degrees Celsius and about 130,000 rcf for about 120 minutes.

11. the method produces an exosome concentrate that is at least 7.5 percent, 8 percent, 8.5 percent, 9 percent, 9.5 percent, 10 percent, 10.5 percent, 11 percent, 11.5 percent, 12 percent, 12.5 percent, 13 percent, 13.5 percent, 14 percent, 14.5 percent, 15 percent, 15.5 percent, 16 percent, 16.5 percent, 17 percent, 17.5 percent, 18 percent, 18.5 percent, 19 percent, 19.5 percent, or at least 20 percent of the starting amount of milk; or 11. The method of claim 1, optionally further comprising loading one or more cargoes onto exosomes of the formulation obtained from the method of any one of claims 1 to 10.

12. 12. A formulation comprising exosomes, said formulation being at least partially produced by the method of any one of claims 1 to 11.

13. The formulation of claim 12, wherein one or more of the exosomes are loaded with one or more cargoes.

14. 14. A method comprising administering to a subject in need thereof the formulation of claim 13.

15. 15. The method of claim 14, wherein the one or more cargoes are therapeutic cargoes.

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