Placental culture to isolate exosomes

Abstract

Several methods are provided to produce, isolate and characterize exosomes recovered from a cultured placenta or portion thereof. Alternatively described herein facilitate the production, isolation and characterization of exosomes that can be used as biotechnological tools and therapeutic agents. Also provided herein are exosome populations derived from a culture of a placental organ or a culture of a portion of the placenta. Compositions comprising the population of exosomes and methods of using the compositions for treating a subject are also provided.

Description

[0001] This application is a divisional application of a patent application with application number 201880079103.8 and application date November 16, 2018.

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 587,335, filed on November 16, 2018, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] Methods are provided for producing, isolating and characterizing exosomes from cultured placenta or parts thereof. The alternatives described herein facilitate the production, isolation and characterization of exosomes that can be used as biotechnological tools and therapeutic agents. Background Art

[0004] Exosomes are nanoscale double lipid membrane vesicles secreted from living cells that play an important role in intercellular communication. During human pregnancy, the placenta plays a central role in regulating physiological homeostasis and supporting fetal development. Extracellular vesicles and exosomes secreted by the placenta are known to facilitate communication between the placenta and maternal tissues to maintain maternal-fetal tolerance. Exosomes contain active biological agents, including lipids, cytokines, microRNAs, mRNAs and DNA, as well as proteins that can be presented on the surface of exosomes. Exosomes are believed to be useful in many therapeutic approaches, including immunomodulation, promotion of angiogenesis, and for drug delivery. More methods that allow the isolation of large quantities of exosomes are clearly needed. Summary of the invention

[0005] Aspects of the invention relate to methods for producing, isolating, and characterizing exosomes from cultured placenta or parts thereof. The methods described herein facilitate the production, isolation, and characterization of exosomes that can be used as biotechnology tools and therapeutic agents. Preferred alternatives include:

[0006] 1. A method for isolating exosomes from a placenta or a portion thereof, the method comprising:

[0007] a) contacting the placenta or a portion thereof, preferably a cultured placenta or a portion thereof, with a first culture medium; and

[0008] b) obtaining a first fraction comprising a population of exosomes from said placenta or a portion thereof;

[0009] c) optionally, contacting the placenta or a portion thereof with a second culture medium and obtaining a second fraction comprising a population of exosomes from the placenta or a portion thereof;

[0010] d) optionally, contacting the placenta or a portion thereof with a third culture medium and obtaining a third fraction comprising a population of exosomes from the placenta or a portion thereof; and

[0011] e) optionally separating the exosome population from the first fraction, the second fraction and / or the third fraction using an antibody or a binding portion thereof specific for a marker or peptide present on the desired exosome population, preferably by sequential centrifugation and / or affinity chromatography, wherein the antibody or the binding portion thereof is immobilized on a substrate such as a membrane, a resin, a bead or a vessel.

[0012] 2. The method of alternative 1, wherein the placenta or portion thereof further comprises amniotic membrane.

[0013] 3. The method of alternative embodiment 2, wherein the placenta or a portion thereof is a human placenta or a portion thereof.

[0014] 4. A method as described in any of the aforementioned alternatives, wherein the first culture medium, the second culture medium and / or the third culture medium are in contact with the placenta or a portion thereof for at least 45 minutes, such as 45 minutes or 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours or 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days or 20 days or any amount of time within a range defined by any two of the aforementioned time points.

[0015] 5. A method as described in any of the aforementioned alternatives, wherein the first culture medium, the second culture medium and / or the third culture medium are in contact with the placenta or a portion thereof for at least 7 days, 14 days, 28 days, 35 days or 42 days or any amount of time within the range defined by any two of the aforementioned time points.

[0016] 6. A method as described in any of the previous alternatives, wherein the placenta or part thereof has been minced, ground or enzymatically treated.

[0017] 7. A method as described in any of Alternatives 1-5, wherein the placenta or portion thereof is substantially flat or sheet-like and has been decellularized and substantially dry, and wherein the method further comprises contacting a fluid containing exogenous cells with the decellularized placenta or portion thereof so as to inoculate the decellularized placenta or portion thereof with the exogenous cells, and wherein the contacting of the cells with the decellularized placenta or portion thereof is performed prior to contacting the decellularized placenta or portion thereof with a first culture medium.

[0018] 8. A method as described in Alternative Embodiment 7, wherein the exogenous cells are obtained from a subject different from the donor subject of the placenta or part thereof.

[0019] 9. The method of alternative 7 or 8 wherein the fluid comprises ascites, blood or plasma.

[0020] 10. The method of Alternative 7 or 8, wherein the cells are from an organ.

[0021] 11. The method of Alternative 10, wherein the cells are from the liver, kidney, lung or pancreas.

[0022] 12. The method of Alternative 7 or 8, wherein the cell is an immune cell.

[0023] 13. The method of alternative 12, wherein the cell is a T cell or a B cell.

[0024] 14. The method of any of the preceding alternatives, wherein the first culture medium comprises phosphate buffered saline (PBS).

[0025] 15. The method of Alternative Embodiment 9, wherein the first fraction, the second fraction, or the third fraction comprises exosomes from ascites, blood, or plasma.

[0026] 16. The method of Alternative 10, wherein the first fraction, the second fraction, or the third fraction comprises exosomes from organ cells.

[0027] 17. The method of Alternative 11 wherein the cells are from the liver, kidney, lung or pancreas.

[0028] 18. A method as described in any of the preceding alternatives, wherein the second culture medium comprises growth factors.

[0029] 19. A method as described in any of the preceding alternatives, wherein the third culture medium comprises a chelating agent.

[0030] 20. The method of alternative 19 wherein the chelating agent is a phosphonate, BAPTA tetrasodium salt, BAPTA / AM, bis-Notrophen™ reagent tetrasodium salt, EGTA / AM, pyridoxal isonicotinic acid hydrazide, N,N,N′,N′-tetrakis-(2-pyridylmethyl)ethylenediamine, 6-bromo-N′-(2-hydroxybenzylidene)-2-methylquinoline-4-carbohydrazide, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl ester), (ethylenedinitrilo)tetraacetic acid (EDTA), Edathamil, ethylenedinitrilotetraacetic acid, ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid or ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid tetrasodium salt (EGTA), or any combination thereof.

[0031] 21. The method of any of Alternatives 19 or 20, wherein the chelating agent is EDTA or EGTA or a combination thereof.

[0032] 22. A method as described in any of alternatives 19-21, wherein the chelator is provided in the third culture medium at a concentration of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM, or at a concentration within a range defined by any two of the foregoing concentrations.

[0033] 23. A method as described in any of alternatives 19-22, wherein the concentration of EDTA in the third culture medium is provided at a concentration of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM, or at a concentration within a range defined by any two of the foregoing concentrations.

[0034] 24. The method of any of the preceding alternatives, wherein the third culture medium comprises a protease.

[0035] 25. The method of Alternative 24 wherein the protease is trypsin, collagenase, chymotrypsin or carboxypeptidase or any combination thereof.

[0036] 26. The method of Alternative 25 or 25 wherein the protease is trypsin.

[0037] 27. A method as described in Alternative Embodiment 24, wherein the protease is provided in the third culture medium at a concentration of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM, or at a concentration within a range defined by any two of the foregoing concentrations.

[0038] 28. The method of any of the preceding alternatives, wherein the method further comprises contacting the placenta or portion thereof with additional multiple culture media, wherein the contacting results in obtaining multiple fractions comprising exosomes.

[0039] 29. The method of alternative 28, wherein the first culture medium, the second culture medium, the third culture medium or the additional culture medium comprises glucose.

[0040] 30. The method of Alternative 28 or 29 wherein said first culture medium, said second culture medium, said third culture medium or said additional culture medium comprises GM-CSF.

[0041] 31. The method of any of Alternatives 28-30, wherein the first culture medium, the second culture medium, the third culture medium or the additional culture medium comprises serum.

[0042] 32. The method of any one of Alternatives 28-31, wherein the first culture medium, the second culture medium, the third culture medium or the additional culture medium comprises DMEM.

[0043] 33. The method of any one of Alternatives 28-32, wherein the first culture medium, the second culture medium, the third culture medium or the additional culture medium comprises an AHR antagonist.

[0044] 34. The method of Alternative 33, wherein the AHR antagonist is SR1.

[0045] 35. The method of alternative embodiment 34 wherein the concentration of SR1 is 1 nM, 10 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM or 1 mM, or any other concentration within the range defined by any two of the foregoing values.

[0046] 36. A method as described in any of the aforementioned alternatives, wherein the first culture medium is contacted with the placenta or a portion thereof while maintaining a temperature of 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C or 40°C or a temperature within a range defined by any two of the foregoing temperatures.

[0047] 37. A method as described in any of the preceding alternatives, wherein the second culture medium is contacted with the placenta or a portion thereof while maintaining a temperature of 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C or 40°C or a temperature within a range defined by any two of the preceding temperatures.

[0048] 38. A method as described in any of the aforementioned alternatives, wherein the third culture medium is contacted with the placenta or a portion thereof while maintaining a temperature of 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C or 40°C, or a temperature within a range defined by any two of the aforementioned values.

[0049] 39. A method as described in any of alternatives 28-38, wherein the additional multiple culture media are contacted with the placenta or a portion thereof while maintaining a temperature of 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C or 40°C or a temperature within a range defined by any two of the foregoing values.

[0050] 40. The method of any of the preceding alternatives, wherein the first perfusion, the second perfusion, or the third perfusion or the additional plurality of culture media comprises an antibiotic.

[0051] 41. The method of any of the preceding alternatives, wherein the exosomes are isolated from the first fraction, the second fraction, and / or the third fraction or fractions by a method comprising the steps of:

[0052] (a) passing the first fraction, the second fraction and / or the third fraction or the plurality of fractions through a tissue filter;

[0053] (b) subjecting the filtrate collected in (a) to a first centrifugation to produce a cell pellet and a first supernatant;

[0054] (c) centrifuging the first supernatant a second time to produce a second supernatant; and

[0055] (d) centrifuging the second supernatant for a third time to produce an exosome pellet; and optionally,

[0056] (e) resuspending the exosomes in a solution.

[0057] 42. The method of any of the preceding alternatives, wherein the exosomes comprise CD63, CD63-A, perforin, Fas, TRAIL or granzyme B or any combination thereof.

[0058] 43. The method of alternative 42, wherein the exosomes comprise CD63A.

[0059] 44. The method of any of the preceding alternatives, wherein the exosomes comprise signaling molecules.

[0060] 45. The method of any of the preceding alternatives, wherein the exosomes comprise cytokines, mRNA or miRNA.

[0061] 46. ​​The method of any of the preceding alternatives, further comprising isolating the exosomes by affinity chromatography, wherein the affinity chromatography is selective for removing exosomes comprising viral antigens, viral proteins, bacterial antigens, bacterial proteins, fungal antigens, or fungal proteins.

[0062] 47. The method of any of the preceding alternatives, further comprising isolating the exosomes by one or more additional affinity chromatography steps, wherein the one or more additional chromatography steps are selective for removing exosomes comprising inflammatory markers and / or tumor markers.

[0063] Also provided is a composition comprising exosomes derived from human placenta, wherein the exosomes are positive for CD1c, CD20, CD24, CD25, CD29, CD2, CD3, CD8, CD9, CD11c, CD14, CD19, CD31, CD40, CD41b, CD42a, CD44, CD45, CD49e, CD4, CD56, CD62P, CD63, CD69, CD81, CD86, CD105, CD133-1, CD142, CD146, CD209, CD326, HLA-ABC, HLA-DRDPDQ, MCSP, ROR1, SSEA-4, or a combination thereof.

[0064] The exosomes described herein include specific markers. Such markers may, for example, be suitable for identifying the exosomes and for distinguishing the exosomes from other exosomes (e.g., exosomes not derived from the placenta). In certain embodiments, such exosomes are positive for one or more markers, for example, as determined by flow cytometry, for example, by fluorescence activated cell sorting (FACS). In addition, the exosomes provided herein can be identified based on the absence of certain markers. The presence or absence of such markers can be determined using methods known in the art, such as fluorescence activated cell sorting (FACS).

[0065] In some embodiments, the exosomes are positive for CD1c, CD20, CD24, CD25, CD29, CD2, CD3, CD8, CD9, CD11c, CD14, CD19, CD31, CD40, CD41b, CD42a, CD44, CD45, CD49e, CD4, CD56, CD62P, CD63, CD69, CD81, CD86, CD105, CD133-1, CD142, CD146, CD209, CD326, HLA-ABC, HLA-DRDPDQ, MCSP, ROR1, and SSEA-4. In some embodiments, the exosomes are positive for 2, 3, 4, 5, 6, 7, 8, 9, 10 or more markers selected from the group consisting of CD1c, CD20, CD24, CD25, CD29, CD2, CD3, CD8, CD9, CD11c, CD14, CD19, CD31, CD40, CD41b, CD42a, CD44, CD45, CD49e, CD4, CD56, CD62P, CD63, CD69, CD81, CD86, CD105, CD133-1, CD142, CD146, CD209, CD326, HLA-ABC, HLA-DRDPDQ, MCSP, ROR1, and SSEA-4.

[0066] In some embodiments, the exosomes are CD3-, CD11b-, CD14-, CD19-, CD33-, CD192-, HLA-A-, HLA-B-, HLA-C-, HLA-DR-, CD11c-, or CD34-. In some embodiments, the exosomes are CD3-, CD11b-, CD14-, CD19-, CD33-, CD192-, HLA-A-, HLA-B-, HLA-C-, HLA-DR-, CD11c-, and CD34-.

[0067] In some embodiments, the exosomes comprise non-coding RNA molecules. In some embodiments, the RNA molecules are microRNAs. In some embodiments, the microRNAs are selected from the group consisting of the microRNAs in Table 7 and combinations thereof. In some embodiments, the microRNA is selected from the group consisting of hsa-mir-26b, hsa-miR-26b-5p, hsa-mir-26a-2, hsa-mir-26a-1, hsa-miR-26a-5p, hsa-mir-30d, hsa-miR-30d-5p, hsa-mir-100, hsa-miR-100-5p, hsa-mir-21, hsa-miR-21-5p, hsa-mir-22, hsa-miR-22-3p, hsa-mir-99b, hsa-miR-99b-5p, hsa-mir-181a-2, hsa-mir-181a-1, hsa-miR-181a-5p and combinations thereof.

[0068] In some embodiments, the exosomes comprise a cytokine selected from the group consisting of the cytokines in Table 3 and combinations thereof.

[0069] In some embodiments, the exosomes comprise a cytokine receptor selected from the group consisting of the cytokine receptors in Table 4 and combinations thereof.

[0070] In some embodiments, the exosomes comprise a protein selected from the group consisting of proteins in Table 6 and combinations thereof. In some embodiments, the exosomes comprise a protein selected from the group consisting of cytoplasmic aconitate hydratase, cell surface glycoprotein MUC18, protein arginine N-methyltransferase 1, guanine nucleotide binding protein G(s) subunit alpha, Cullin-5, calcium binding protein 39, glucosidase 2 subunit beta, intracellular chloride channel protein 5, brain semaphorin-3B, 60S ribosomal protein L22, spliceosomal RNA helicase DDX39B, transcription activator protein Pur-α, programmed cell death protein 10, BRO1 domain-containing protein BROX, kynurenine-ketoglutarate aminotransferase 3, laminin subunit alpha-5, ATP binding cassette subfamily E member 1, syntaxin binding protein 3, proteasome subunit beta-7 type, and combinations thereof.

[0071] In some embodiments, the exosomes comprise at least one marker molecule, the level of the at least one marker molecule being at least twice the level of exosomes derived from mesenchymal stem cells, umbilical cord blood, or placental perfusate. In some embodiments, the exosomes comprise at least one marker molecule, the level of the at least one marker molecule being at least twice the level of exosomes derived from mesenchymal stem cells, umbilical cord blood, and placental perfusate.

[0072] In some embodiments, the exosomes are isolated from culture medium of a whole placenta culture. In some embodiments, the exosomes are isolated from culture medium of a whole culture comprising a placental lobule or a portion of a placenta.

[0073] In some embodiments, the exosomes are produced by the methods of the present invention. In some embodiments, the composition is in a form suitable for intravenous administration. In some embodiments, the composition is in a form suitable for local injection. In some embodiments, the composition is in a form suitable for topical administration. In some embodiments, the composition is in a form suitable for ultrasonic delivery.

[0074] Also provided is a method of increasing the proliferation of an immune cell, the method comprising contacting the cell with a composition as described in any one of claims 48-65.

[0075] In some embodiments, the immune cell is a T cell.

[0076] In some embodiments, the immune cell is a NK cell.

[0077] In some embodiments, the immune cells are CD34+ cells.

[0078] Also provided is a method of inhibiting proliferation of cancer cells, the method comprising contacting the cells with a composition of the present invention.

[0079] Also provided is a method of promoting angiogenesis or blood vessel formation in the subject, the method comprising administering to the subject a composition of the invention.

[0080] Also provided are methods of modulating the immune system of the subject, the methods comprising administering to the subject a composition of the invention.

[0081] Also provided are methods of repairing diseased or damaged tissue in a subject, the methods comprising administering to the subject a composition of the invention.

[0082] Also provided are methods of treating cancer in a subject, the methods comprising administering to the subject a composition of the invention.

[0083] In some embodiments of the above methods, the subject is a human.

[0084] Also provided herein are compositions comprising exosomes. Such compositions generally do not comprise placental cells from which the exosomes are derived. In addition, such compositions generally do not comprise cell culture supernatants from cell cultures from which the exosomes are derived.

[0085] In certain embodiments, the purified exosomes are formulated into a pharmaceutical composition suitable for administration to a subject in need. In certain embodiments, the subject is a human. The pharmaceutical composition containing placental-derived exosomes provided herein can be formulated for topical, systemic, subcutaneous, parenteral, intravenous, intramuscular, topical, oral, intradermal, transdermal or intranasal administration to a subject in need. In a certain embodiment, the pharmaceutical composition containing placental-derived exosomes provided herein is formulated for topical administration. In a certain embodiment, the pharmaceutical composition containing placental-derived exosomes provided herein is formulated for systemic subcutaneous administration. In a certain embodiment, the pharmaceutical composition containing placental-derived exosomes provided herein is formulated for parenteral administration. In a certain embodiment, the pharmaceutical composition containing placental-derived exosomes provided herein is formulated for intramuscular administration. In a certain embodiment, the pharmaceutical composition containing placental-derived exosomes provided herein is formulated for topical administration. In a certain embodiment, the pharmaceutical composition containing placental-derived exosomes provided herein is formulated for oral administration. In a certain embodiment, the pharmaceutical composition containing placental-derived exosomes provided herein is formulated for intradermal administration. In a certain embodiment, the pharmaceutical composition containing placental-derived exosomes provided herein is formulated for transdermal administration. In a certain embodiment, the pharmaceutical composition containing placental-derived exosomes provided herein is formulated for intranasal administration. In a specific embodiment, the pharmaceutical composition containing placental-derived exosomes provided herein is formulated for intravenous administration.

[0086] In another aspect, provided herein is a use of the exosomes described herein and / or a pharmaceutical composition comprising the exosomes.

[0087] In a specific embodiment, the exosomes described herein and / or pharmaceutical compositions comprising exosomes are used to treat and / or prevent diseases and / or disorders of subjects in need. In a specific embodiment, the exosomes described herein and / or pharmaceutical compositions comprising exosomes are used to promote angiogenesis and / or vascularization of subjects in need. In another specific embodiment, the exosomes described herein and / or pharmaceutical compositions comprising exosomes are used to regulate the immune activity of subjects in need (e.g., increase immune response or reduce immune response). In another specific embodiment, the exosomes described herein and / or pharmaceutical compositions comprising exosomes are used to repair tissue damage in subjects in need, such as tissue damage caused by acute or chronic injury.

[0088] In another specific embodiment, the exosomes of the sources described herein and / or the pharmaceutical compositions comprising exosomes are used in methods for treating and / or preventing diseases and / or disorders of subjects in need. In another embodiment, the pharmaceutical compositions comprising exosomes described herein are used in methods for treating diseases and / or disorders of subjects in need. In another embodiment, the pharmaceutical compositions comprising exosomes described herein are used in methods for preventing diseases and / or disorders of subjects in need. In a specific embodiment, the pharmaceutical compositions comprising exosomes described herein are used in methods for promoting angiogenesis and / or vascularization of subjects in need. In another specific embodiment, the pharmaceutical compositions comprising exosomes described herein are used in methods for regulating the immune activity of subjects in need (e.g., increasing immune response or reducing immune response). In another specific embodiment, the pharmaceutical compositions comprising exosomes described herein are used in methods for repairing tissue damage (e.g., tissue damage caused by acute or chronic injury) of subjects in need.

[0089] In another specific embodiment, the exosomes and / or pharmaceutical compositions comprising exosomes described herein are used as cell protectants. In another aspect, the exosomes and / or pharmaceutical compositions comprising exosomes described herein are provided in the form of a kit suitable for pharmaceutical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1 Schematic diagram showing the process for culturing cells for exosome isolation.

[0091] Figure 2A-2CShown are 3 pExo isolates whose size distribution was analyzed by NanoSight.This work was performed and reported by SBI Inc. (System Bioscience Inc.) using contract services (www.systembio.com / services / exosome-services / ).

[0092] Figures 3A-3C The MACSPlex kit was used to detect exosomes from placental perfusate ( Figure 3B ) and cord blood serum-derived exosomes ( Figure 3C ) compared with pExo(N=12)( Figure 3A ) protein markers present on the surface.

[0093] Figure 4 Functional pathways of proteins identified in the placental exosome population are shown.

[0094] Figure 5 Common and unique proteins identified in the three placental exosome samples are shown.

[0095] Figure 6 It was shown that pExo promoted the migration of human dermal fibroblasts in a transwell system.

[0096] Figure 7 It was shown that pExo promoted the migration of human umbilical cord endothelial cells.

[0097] Figure 8 It is shown that pExo stimulates the proliferation of HUVECs.

[0098] Fig. 9 pExo is shown to stimulate proliferation of human CD34+ cells.

[0099] Fig.10 pExo is shown to stimulate colony formation of human CD34+ cells.

[0100] Fig.11 It was shown that pExo inhibited the proliferation of SKOV3 cancer cells.

[0101] Fig.12 It was shown that pExo inhibited the proliferation of A549 cancer cells.

[0102] Fig.13 It was shown that pExo inhibited the proliferation of MDA321 cancer cells.

[0103] Fig.14 It was shown that pExo did not affect the proliferation of CD3+ T cells in culture.

[0104] Fig.15It was shown that pExo increased the expression of the activation marker CD69 in UBC T CD3+ cells.

[0105] Fig.16 pExo was shown to increase the expression of the activation marker CD69 in adult PBMC T CD3+ cells.

[0106] Fig.17 pExo was shown to increase CD56+ NK cells in PBMCs. DETAILED DESCRIPTION

[0107] 5.1. Placenta-derived exosomes

[0108] As described below, the placenta-derived exosomes described herein can be selected and identified by their morphology and / or molecular markers. The placenta-derived exosomes described herein are different from exosomes known in the art, such as chorionic mesenchymal stem cell-derived exosomes, such as the exosomes described in Salomon et al., 2013, PLOS ONE, 8:7, e68451. Therefore, the term "placenta-derived exosomes" as used herein is not meant to include exosomes obtained or derived from chorionic mesenchymal stem cells.

[0109] In certain embodiments, a population of placenta-derived exosomes described herein does not comprise cells, e.g., nucleated cells, e.g., placental cells.

[0110] 5.1.1. Placenta-derived exosome markers

[0111] Placenta-derived exosomes described herein contain markers that can be used to identify and / or separate the exosomes. These markers can be, for example, proteins, nucleic acids, sugar molecules, glycosylated proteins, lipid molecules, and can exist in monomer, oligomer and / or polymer form. In certain embodiments, the marker is produced by cells derived from exosomes. In certain embodiments, the marker is provided by cells derived from exosomes, but the cells do not express the marker at a higher level. In a specific embodiment, when compared with control marker molecules, compared with the origin cells, the markers of the exosomes described herein are higher in exosomes. In another specific embodiment, compared with from another cell type (e.g., chorionic mesenchymal stem cells and preadipocyte mesenchymal stem cells described in Salomon et al., 2013, PLOS ONE, 8:7, e68451), the markers of the exosomes described herein are enriched in the exosomes, wherein the exosomes are separated by the same method.

[0112] The three-dimensional structure of the exosome allows the marker to be retained on the surface of the exosome and / or contained in the exosome. Similarly, the marker molecule may be partially present in the exosome, partially present on the outer surface of the exosome and / or across the phospholipid bilayer of the exosome. In a specific embodiment, the marker associated with the exosome described herein is a protein. In certain embodiments, the marker is a transmembrane protein, which is anchored in the exosome phospholipid bilayer, or anchored across the exosome phospholipid bilayer, so that part of the protein molecule is in the exosome, while part of the same molecule is exposed to the outer surface of the exosome. In certain embodiments, the marker is completely contained in the exosome. In another specific embodiment, the marker associated with the exosome described herein is a nucleic acid. In certain embodiments, the nucleic acid is a non-coding RNA molecule, such as a microRNA (miRNA).

[0113] 5.1.1.1. Surface markers

[0114] The exosomes described herein contain surface markers that allow their identification and can be used to separate / obtain substantially pure cell exosome populations without their cells of origin and other cells and non-cellular material. Methods for determining the composition of exosome surface markers are known in the art. For example, exosome surface markers can be detected by fluorescence activated cell sorting (FACS) or Western blotting.

[0115] In certain embodiments, the exosomes described herein comprise greater amounts of surface markers as determined by, for example, FACS, compared to exosomes known in the art.

[0116] 5.1.1.2. Output

[0117] The exosomes described herein can be isolated according to the methods described herein, and their yields can be quantified. In a specific embodiment, the exosomes described herein are isolated at a concentration of about 0.5-5.0 mg per liter of culture medium (e.g., culture medium with or without serum). In another specific embodiment, the exosomes described herein are isolated at a concentration of about 2-3 mg per liter of culture medium (e.g., culture medium containing serum). In another specific embodiment, the exosomes described herein are isolated at a concentration of about 0.5-1.5 mg per liter of culture medium (e.g., culture medium lacking serum).

[0118] 5.1.2. Storage and preservation

[0119] The exosomes described herein can be preserved, ie, placed under conditions that allow for long-term storage or conditions that inhibit degradation of the exosomes.

[0120] In certain embodiments, the exosomes described herein can be stored in a composition comprising a buffer at an appropriate temperature after being collected according to the methods described above. In certain embodiments, the exosomes described herein are stored frozen, for example, at about -20°C or about -80°C.

[0121] In certain embodiments, the exosomes described herein can be cryopreserved, for example, in a small container, such as an ampoule (e.g., a 2 mL vial). In certain embodiments, the exosomes described herein are cryopreserved at a concentration of about 0.1 mg / mL to about 10 mg / mL.

[0122] In certain embodiments, the exosomes described herein are cryopreserved at a temperature of about -80°C to about -180°C. The cryopreserved exosomes may be transferred to liquid nitrogen before thawing for use. In some embodiments, for example, once the ampoules reach about -90°C, they are transferred to a liquid nitrogen storage area. Cryopreservation may also be performed using a controlled rate freezer. The cryopreserved exosomes may be thawed at a temperature of about 25°C to about 40°C prior to use.

[0123] In certain embodiments, the exosomes described herein are stored at a temperature of about 4°C to about 20°C for a short period of time (eg, less than two weeks).

[0124] 5.2. Composition

[0125] Also provided herein are compositions, such as pharmaceutical compositions, comprising the exosomes provided herein. The compositions described herein can be used to treat certain diseases and conditions in a subject (e.g., a human subject) where treatment with exosomes is beneficial.

[0126] In certain embodiments, in addition to comprising exosomes provided herein, the compositions described herein (e.g., pharmaceutical compositions) further comprise a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" means approved by a federal regulatory agency or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias for use in animals and more specifically in humans. As used herein in the context of a pharmaceutically acceptable carrier, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle administered with a pharmaceutical composition. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" JP Remington and AR Gennaro, 1990, 18th edition.

[0127] In certain embodiments, the compositions described herein further comprise one or more buffers, such as saline, phosphate buffered saline (PBS), Dulbecco's PBS (DPBS), and / or sucrose phosphate glutamate buffer. In other embodiments, the compositions described herein do not comprise a buffer. In certain embodiments, the compositions described herein further comprise plasmalyte.

[0128] In certain embodiments, the compositions described herein further comprise one or more salts, such as sodium chloride, calcium chloride, sodium phosphate, monosodium glutamate, and aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), or a mixture of such aluminum salts). In other embodiments, the compositions described herein do not comprise a salt.

[0129] The compositions described herein may be included in a container, pack, or dispenser together with instructions for administration.

[0130] The compositions described herein can be stored prior to use, for example, the compositions can be stored frozen (eg, at about -20°C or at about -80°C); stored under refrigerated conditions (eg, at about 4°C); or stored at room temperature.

[0131] 5.2.1. Formulation and route of administration

[0132] The amount of exosomes or compositions described herein that will be effective for therapeutic use in the treatment and / or prevention of a disease or disorder will depend on the nature of the disease and can be determined by standard clinical techniques. The exact dose of exosomes or compositions thereof to be administered to a subject will also depend on the route of administration and the severity of the disease or disorder to be treated, and should be determined according to the judgment of the practitioner and the circumstances of each subject. For example, the effective dose may vary depending on the mode of administration, the target site, the patient's physiological state (including age, weight, and health status), whether the patient is human or animal, other drugs administered, and whether the treatment is preventive or therapeutic. The therapeutic dose is optimally titrated to optimize safety and efficacy.

[0133] The administration of exosomes or compositions thereof described herein can be carried out via various routes known in the art. In certain embodiments, exosomes or compositions thereof described herein are administered by local, systemic, subcutaneous, parenteral, intravenous, intramuscular, topical, oral, intradermal, transdermal or intranasal administration. In a specific embodiment, the administration is via intravenous injection. In a specific embodiment, the administration is via subcutaneous injection. In a specific embodiment, the administration is topical administration. In another specific embodiment, the exosomes or compositions thereof are administered in a preparation comprising an extracellular matrix. In another specific embodiment, the exosomes or compositions thereof are administered in combination with one or more additional delivery devices (e.g., stents). In another specific embodiment, the exosomes or compositions thereof are administered locally, for example, at or around the site of the area to be treated with the exosomes or compositions, such as hypoxic tissue (e.g., for the treatment of ischemic diseases) or draining lymph nodes.

[0134] 5.3. Usage

[0135] 5.3.1. Treatment of diseases that benefit from angiogenesis

[0136] The exosomes and compositions thereof described herein promote angiogenesis and are therefore useful for treating diseases and conditions that benefit from angiogenesis. Thus, provided herein are methods of promoting angiogenesis in subjects in need thereof using exosomes or compositions thereof described herein. As used herein, the term "treatment" encompasses the cure, remedy, improvement, reduction in severity, or reduction in the time course of a subject's disease, disorder, or condition or any parameter or symptom thereof. In a specific embodiment, the subject treated according to the methods provided herein is a mammal, such as a human.

[0137] In one embodiment, provided herein is a method of inducing angiogenesis or vasculogenesis in a subject, the method comprising administering to the subject an exosome or a composition thereof provided herein. Thus, the methods provided herein can be used to treat diseases and conditions that benefit from increased angiogenesis / angiogenesis in a subject. Examples of such diseases / illnesses that benefit from increased angiogenesis and can therefore be treated with the exosomes and compositions described herein include, but are not limited to, myocardial infarction, congestive heart failure, peripheral arterial disease, critical limb ischemia, peripheral vascular disease, hypoplastic left heart syndrome, diabetic foot ulcers, venous ulcers, or arterial ulcers.

[0138] In one embodiment, provided herein is a method for treating, for example, a subject with blood flow interruption in the peripheral vascular system, the method comprising administering to the subject an exosome provided herein or a composition thereof. In a specific embodiment, provided herein is a method comprising treating a subject with ischemia with an exosome provided herein or a composition thereof. In certain embodiments, ischemia is peripheral arterial disease (PAD), for example, critical limb ischemia (CLI). In certain other embodiments, ischemia is peripheral vascular disease (PVD), peripheral arterial disease, ischemic vascular disease, ischemic heart disease or ischemic nephropathy.

[0139] 5.3.2. Patient groups

[0140] In certain embodiments, the exosomes described herein are administered to a subject in need of treatment for any disease or disorder described herein. In another embodiment, the compositions described herein are administered to a subject in need of treatment for any disease or disorder described herein. In certain embodiments, the subject is a human.

[0141] In a specific embodiment, the exosomes or compositions described herein are administered to a subject (eg, a human) in need of treatment to increase angiogenesis and / or vascularization.

[0142] 5.4.Medicine box

[0143] Provided herein is a pharmaceutical pack or kit comprising one or more containers filled with one or more ingredients of the pharmaceutical composition described herein (i.e., a composition comprising exosomes described herein). Optionally, associated with such one or more containers may be a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of drugs or biological products, the notice reflecting the agency's approval of the manufacture, use, or sale for human administration.

[0144] The kits described herein can be used in the above methods. The compositions described herein can be prepared in a form that is easy to administer to an individual. For example, the composition can be contained in a container suitable for medical use. Such a container can be, for example, a sterile plastic bag, a flask, a jar, or other container from which the composition can be easily dispensed. For example, the container can be a blood bag or other plastic, medically acceptable bag suitable for intravenous administration of liquids to a recipient.

[0145] Exemplary Placenta Culture

[0146] The placenta is a reservoir of cells, including stem cells, such as hematopoietic stem cells (HSC) and non-hematopoietic stem cells. This article describes a method for isolating exosomes from a placenta or a portion thereof, which is cultured in a bioreactor. Exosomes are secreted by cells during the culture process, and the exosomes are secreted into the culture medium, which facilitates further processing and isolation of the exosomes. Exosomes can also be isolated from the placenta or a portion thereof at different culture stages (e.g., at different time points, and different perfusates can be used at each recovery step). Once in culture, the exosomes can be isolated using, for example, centrifugation, commercially available exosome isolation kits, lectin affinity and / or affinity chromatography (e.g., using an immobilized binding agent, such as a binding agent attached to a substrate, that is specific for small Rab family GTPases, annexins, flotillins, Alix, Tsg101, ESCRT complexes, CD9, CD37, CD53, CD63, CD63A, CD81, CD82), Hsp70, Hsp90, epithelial cell adhesion molecule (EpCam), perforin, TRAIL, granzyme B, Fas, one or more cancer markers (e.g., Fas ligand, CD24, EpCAM, EDIL3, fibronectin, survivin, PCA3, TMPRSS2:ERG, Glypican-1, TGF-β1, MAGE 3 / 6, EGFR, EGFRvIII, CD9, CD147, CA-125, EpCam and / or CD24) or one or more inflammatory or pathogenic markers (such as: viral, fungal or bacterial proteins or peptides, including but not limited to α-synuclein, HIV or HCV proteins, tau, β-amyloid protein, TGF-β, TNF-α, fetuin-A and / or CD133) are specific) to further separate exosomes. The isolated exosomes can be used as therapeutic agents, diagnostic agents and as biotechnology tools.

[0147] "Exosomes" as described herein are vesicles present in many and possibly all eukaryotic fluids, including ascites, blood, urine, serum and breast milk. They may also be referred to as extracellular vesicles. Exosomes are double lipid membrane vesicles secreted from living cells, which play an important role in intercellular communication. Exosomes are produced by cells (such as stem cells, epithelial cells), and a subtype of exosomes (defined as matrix-bound nanovesicles (MBVs)) is present in an extracellular matrix (ECM) bioscaffold (non-fluid). The reported diameter of the exosomes is between 30 and 100 nm, which is larger than low-density lipoprotein (LDL), but much smaller than, for example, red blood cells. When multivesicular bodies fuse with the plasma membrane, exosomes can be released from cells, or directly released from the plasma membrane.

[0148] Exosomes have been shown to have specialized functions and play a key role in processes such as coagulation, intercellular signaling, and waste management. Extracellular vesicles and exosomes secreted by the placenta are known to contribute to communication between the placenta and maternal tissues to maintain maternal-fetal tolerance. Exosomes isolated from human placental explants have been shown to have immunomodulatory activity. It has also been shown that stem cell-derived exosomes reduce neuroinflammation by inhibiting the activation of astrocytes and microglia, and may promote neurogenesis by targeting neurogenic niches, both of which contribute to neural tissue repair and functional recovery after TBI. (Reviewed in Yang et al. 2017, Frontiers in Cellular Neuroscience). Exosomes derived from human embryonic mesenchymal stem cells also promote osteochondral regeneration (Zhang et al. 2016, Osteoarthritis and Cartilage). Exosomes carrying functional Fas ligands and Trail molecules secreted by the human placenta have been shown to transmit apoptosis in activated immune cells, indicating that fetal exosomes mediate immune immunity. (Ann-Christin Stenqvist et al., Journal of Immunology, 2013, 191: doi: 10.4049).

[0149] Exosomes contain active biological agents, including lipids, cytokines, microRNAs, mRNAs, and DNA. They can also act as a medium for intercellular communication via genetic material and / or protein transfer. Exosomes can also contain cell type-specific information that can reflect the function or physiological state of the cell. Therefore, there is increasing interest in the development of clinical and biological applications of exosomes.

[0150] Thus, exosomes isolated from human placenta or a portion thereof using the methods described herein (optionally including characterizing the exosomes (e.g., by identifying the presence or absence of one or more proteins or markers on the exosomes)) can be used to stimulate immunomodulatory, anti-fibrotic environments and / or pro-regenerative effects. Thus, exosomes isolated from human placenta or a portion thereof using the methods described herein can be selected (e.g., based on the presence or absence of markers on the exosomes), purified, frozen, lyophilized, packaged and / or distributed as a therapeutic product and / or biotechnological tool.

[0151] In some alternatives, it may be beneficial to identify exosomes with tumor markers or peptides, pathogenic markers or peptides (such as viral, fungal or bacterial markers or peptides), and / or inflammatory markers (such as inflammatory peptides) so that such exosomes can be removed from the exosome population (e.g., by affinity chromatography using a binding molecule, such as an antibody or binding portion thereof, that is specific for such tumor markers or peptides, pathogenic markers or peptides, and / or inflammatory markers or peptides). Thus, in some alternatives, for example, a first population of exosomes is isolated from a human placenta or a portion thereof by the methods described herein, and once the first population of exosomes is isolated, this population of exosomes is further processed using a substrate having an immobilized antibody or binding portion thereof (e.g., a membrane, resin, bead or vessel having the immobilized antibody or binding portion thereof) to remove one or more subpopulations of exosomes, wherein the immobilized antibody or binding portion thereof is specific for a marker or peptide present on the subpopulation of exosomes that are selected for further isolation, such as one or more tumor markers or peptides, pathogenic markers or peptides (e.g., viral, fungal or bacterial markers or peptides) and / or inflammatory markers or inflammatory peptides. In some alternatives, a first population of exosomes isolated from a human placenta or a portion thereof by the methods described herein is contacted with a substrate having an immobilized antibody or a binding portion thereof (e.g., a membrane, resin, bead, or vessel having the immobilized antibody or a binding portion thereof), wherein the immobilized antibody or a binding portion thereof is specific for one or more cancer markers such as: Fas ligand, CD24, EpCAM, EDIL3, fibronectin, survivin, PCA3, TMPRSS2:ERG, Glypican-1, TGF-β1, MAGE 3 / 6, EGFR, EGFRvIII, CD9, CD147, CA-125, EpCam, and / or CD24, so as to separate a second population of exosomes from the first population of exosomes based on affinity for the immobilized antibody or a binding portion thereof. In some alternatives, a first population of exosomes isolated from a human placenta or a portion thereof by the methods described herein is contacted with a substrate having an immobilized antibody or a binding portion thereof (e.g., a membrane, resin, bead, or vessel having the immobilized antibody or a binding portion thereof), wherein the immobilized antibody or a binding portion thereof is specific for one or more inflammatory markers or pathogenic markers such as: viral, fungal, or bacterial proteins or peptides, including but not limited to α-synuclein, HIV or HCV proteins, tau, β-amyloid, TGF-β, TNF-α, fetuin-A, and / or CD133, or a portion thereof, so as to separate a second population of exosomes from the first population of exosomes based on affinity for the immobilized antibody or a binding portion thereof.

[0152] In some alternatives, the exosome population isolated and / or selected by the methods described herein has a marker or peptide that can be used as a therapeutic agent, such as perforin and / or granzyme B, which have been shown to mediate anti-tumor activity in vitro and in vitro (J Cancer 2016; 7(9): 1081-1087); or Fas, which has been found in exosomes that exert cytotoxic activity against target cancer cells. (Theranostics 2017; 7(10): 2732-2745). Thus, in some alternatives, a first population of exosomes isolated from a human placenta or a portion thereof by the methods described herein is contacted with a substrate having an immobilized antibody or a binding portion thereof (e.g., a membrane, resin, bead, or vessel having the immobilized antibody or a binding portion thereof), wherein the immobilized antibody or a binding portion thereof is specific for perforin, TRAIL and / or granzyme B and / or Fas, and a second population of exosomes is isolated from the first population of exosomes based on the affinity of the immobilized antibody or a binding portion thereof for perforin, TRAIL and / or granzyme B and / or Fas. In some alternatives, a population of exosomes is isolated that contains CD63 RNA and / or a desired microRNA. In some alternatives, affinity chromatography or immunoassays are used to isolate and / or characterize exosome populations after isolation, wherein the exosome populations comprise markers or peptides such as small Rab family GTPases, annexins, floccin, Alix, Tsg101, ESCRT complexes, CD9, CD37, CD53, CD63, CD63A, CD81, CD82), Hsp70, Hsp90), and / or epithelial cell adhesion molecule (EpCam). As described in detail above, in some alternatives, the first population of exosomes isolated from a human placenta or a portion thereof by the methods described herein is contacted with a substrate having an immobilized antibody or a binding portion thereof (e.g., a membrane, resin, bead, or vessel having the immobilized antibody or a binding portion thereof), wherein the immobilized antibody or a binding portion thereof is directed against small Rab family GTPases, annexin, vesin, Alix, Tsg101, ESCRT complex, CD9, CD37, CD53, CD63, CD63A, CD81, CD8 2), Hsp70, Hsp90) and / or epithelial cell adhesion molecule (EpCam), and the second exosome population is separated from the first exosome population based on the affinity of the immobilized antibody or its binding portion to small Rab family GTPase, membrane annexin, floating flank protein, Alix, Tsg101, ESCRT complex, CD9, CD37, CD53, CD63, CD63A, CD81, CD82), Hsp70, Hsp90) and / or epithelial cell adhesion molecule (EpCam).In other alternatives, a population of exosomes isolated from a human placenta or a portion thereof by the methods described herein is contacted with an antibody or binding portion thereof specific for one or more of small Rab family GTPases, annexins, vacuole proteins, Alix, Tsg101, ESCRT complex, CD9, CD37, CD53, CD63, CD63A, CD81, CD82, Hsp70, Hsp90 and / or epithelial cell adhesion molecule (EpCam), and binding of the antibody or binding portion thereof is detected with a second binding agent having a detectable agent that binds to the antibody or binding portion thereof (e.g., using an ELISA or blotting procedure) to confirm the presence of small Rab family GTPases, annexins, vacuole proteins, Alix, Tsg101, ESCRT complex, CD9, CD37, CD53, CD63, CD63A, CD81, CD82), Hsp70, Hsp90 and / or epithelial cell adhesion molecule (EpCam) in the isolated population of exosomes.

[0153] As used herein, "isolation" is a process used to separate exosomes from other materials. Exosomes can be isolated by high centrifugal forces in a centrifuge, using commercially available kits (e.g., SeraMir Exosome RNA Purification Kit (SBIsystem biosciences), Whole Exosome Purification and RNA Isolation (Combination Kit) Norgen BioTek Corp.) and using lectin affinity or affinity chromatography, with a binding agent (e.g., an antibody or a binding portion thereof) specific for a marker or peptide on the exosome, such as the marker or peptide mentioned above (e.g., for small Rab family GTPases, annexins, vespertin, Alix, Tsg101, ESCRT complex, CD9, CD37, CD53, CD63, CD63A, CD81, CD82), Hsp70, Hsp90, epithelial cell adhesion molecule (EpCam), perforin, TRAIL, granzyme B, Fas, one or more cancer markers (e.g., Fas ligand, CD24, EpCAM, EDIL3, fibronectin, survivin, PCA3, TMPRSS2:ERG, Glypican-1, TGF-β1, MAGE 3 / 6, EGFR, EGFRvIII, CD9, CD147, CA-125, EpCam and / or CD24) or one or more inflammatory or pathogenic markers (such as viral, fungal or bacterial proteins or peptides, including but not limited to α-synuclein, HIV or HCV proteins, tau, β-amyloid protein, TGF-β, TNF-α, fetuin-A and / or CD133)).

[0154] As described herein, "placenta" is an organ in the uterus of a pregnant eumammalia mammal that nurtures and maintains the fetus through the umbilical cord. As described herein, the placenta can be used as a bioreactor for obtaining exosomes. In some alternatives, the decellularized placenta can be used as a scaffold and a bioreactor having an exogenous cell population (e.g., a cell population that has been inoculated onto and cultured with the decellularized placenta) to obtain an exosome population from the cells, the exosome population being cell-specific. Therefore, in some alternatives, the decellularized placenta is inoculated with a regenerative cell population (e.g., a cell population comprising stem cells and / or endothelial cells and / or progenitor cells), and the regenerative cell population is cultured on the decellularized placenta in a bioreactor, and centrifugation, commercially available exosome separation kits, lectin affinity and / or affinity chromatography are used to apply a marker or peptide on the exosome such as the marker or peptide mentioned above with a specific binding agent (e.g., an antibody or its binding portion) (e.g., to small Rab family GTPases, annexins, floaters protein, Alix, Tsg101, ESCRT complex, CD9, CD37, CD53, CD63, CD63A, CD81, CD82), Hsp70, Hsp90, epithelial cell adhesion molecule (EpCam), perforin, TRAIL, granzyme B, Fas, one or more cancer markers (e.g., Fas ligand, CD24, EpCAM, EDIL3, fibronectin, survivin, PCA3, TMPRSS2:ERG, glypican-1, TGF-β1, MAGE The invention relates to separating cell-specific exosomes from the cultured cells by using a binding agent specific for one or more inflammatory or pathogenic markers (such as EGFR, EGFRvIII, CD9, CD147, CA-125, EpCam and / or CD24) or one or more inflammatory or pathogenic markers (such as viral, fungal or bacterial proteins or peptides, including but not limited to α-synuclein, HIV or HCV proteins, tau, β-amyloid protein, TGF-β, TNF-α, fetuin-A and / or CD133)).

[0155] As used herein, "ascites" is excess fluid in the space between the membranes lining the abdomen and abdominal organs (the peritoneal cavity). Ascites can be a source of exosomes.

[0156] As used herein, "plasma" is the liquid portion of blood and lymph that accounts for about half of the volume of blood. Plasma does not contain cells, and unlike serum, plasma does not clot. Plasma contains antibodies and other proteins. Plasma can be a source of exosomes.

[0157] Provided herein are several methods for culturing cells to produce a large number of exosomes. The culture medium used to recover or isolate exosomes can be provided with one or more nutrients, enzymes, or chelating agents. Chelating agents can be used to promote the release of exosomes from cultured cells. Without limitation, the chelating agent used in some of the methods may include a phosphonate, BAPTA tetrasodium salt, BAPTA / AM, bis-Notrophen™ reagent tetrasodium salt, EGTA / AM, pyridoxal isonicotinic acid hydrazide, N,N,N′,N′-tetrakis-(2-pyridylmethyl)ethylenediamine, 6-bromo-N′-(2-hydroxybenzylidene)-2-methylquinoline-4-carbohydrazide, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl ester), (ethylenedinitrilo)tetraacetic acid (EDTA), Edathamil, ethylenedinitrilotetraacetic acid, ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, or ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), or any combination thereof. The chelating agent can be provided in a concentration of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM or in a concentration within the range defined by any two of the aforementioned concentrations. As shown herein, the presence of one or more chelating agents in the culture medium unexpectedly enhances the recovery of exosomes from the placenta cultured in the bioreactor. The culture medium for culturing and / or recovering exosomes may also have a protease, which can further enhance the release of exosomes. In some alternatives, the protease provided in the culture medium is trypsin, collagenase, chymotrypsin or carboxypeptidase. In alternative embodiments, the protease is provided in the culture medium at a concentration of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM, or at a concentration within a range defined by any two of the foregoing concentrations. One or more sugars may also be added to the culture medium for culturing and / or recovering exosomes. In some alternative embodiments, the sugar added to the culture medium is glucose. It is contemplated that the presence of glucose in the culture medium enhances the release of exosomes. In alternative embodiments, glucose is provided in the culture medium at a concentration of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM, or at a concentration within a range defined by any two of the foregoing concentrations.The culture medium may also contain growth factors, cytokines, or one or more drugs, such as GM-CSF, serum, and / or an AHR antagonist.

[0158] Methods for collecting exosomes from the placenta or a portion thereof

[0159] An exemplary method for recovering exosomes from the placenta is described in Figure 1 As shown in . The source for exosome isolation can be from umbilical cord plasma; when the placenta is used as a bioreactor for exosome generation, PRP, placental perfusate (PS), placental tissue culture (PTS), placental organ culture (PO) or exogenous cells can be placed in the placenta or part thereof. By one method, the placenta or part thereof is collected (#200010323, collected on 9 / 25 / 2017). The placenta is contacted with a culture medium or perfused with a standard PSC-100 collection method and collected as PS-1 (9 / 26 / 2017). The placenta or part thereof is incubated in a fume hood for at least 4 hours. The placenta or part thereof is contacted with a culture medium (RPMI culture medium) or perfused with 500 mL RPMI basal culture medium (1% antibiotics) and collected as PS-2. The placenta or part thereof is then incubated overnight in a fume hood and covered. The placenta or part thereof is contacted with 750 mL of saline solution or perfused with 750 mL of saline solution and collected as PS-3. The samples were then shipped to the laboratory for analysis (Warren). PS1, PS2 and PS3 were analyzed by FACS on the same day after RBC lysis.

[0160] For the analysis, the placental tissue was cut into 1x1x1 cm in size and placed in 100 mL of solution (all containing 1% P&S) in a T75 flask (about 1 / 8 placenta each). Four solutions were tested: A: DMEM medium; B: PBS; C: PBS+5mMEDTA; D: PBS+0.025% trypsin-EDTA. Then it was incubated overnight (O / N) in a 37°C incubator.

[0161] The supernatant was then harvested, passed through a tissue filter and centrifuged at 400 g to harvest the cells (pellet). The supernatant after the first centrifugation was then centrifuged for exosome isolation (3000 g centrifugation broth > 10,000 g centrifugation broth: 100,000 g pellet)

[0162] The collected cells were also used for FACS analysis. Cell samples were in several buffers (A = PTS1; B = PTS2; C = PTS-3, D = PTS4). Exosomes were recovered and then assayed to identify the presence of exosomal markers, thereby confirming that exosomes were obtained and isolated by the procedure.

[0163] Characterization of exosome populations isolated from placenta bioreactors using ELISA and protein assays

[0164] The fractions of the supernatant from the placenta bioreactor are collected by the above method, and the fractions are filtered. The supernatant is then centrifuged at 400g x 10 minutes to collect cells. After the first centrifugation, a second centrifugation is performed at 3000g x 30 minutes to precipitate the cell debris. A third centrifugation is performed at 10,000g x 1 hour to precipitate the microvesicles. Then a fourth centrifugation is performed at 100,000g x 1.5 hours to precipitate the exosomes. The centrifuge tube containing the precipitated exosomes is then inverted on paper to drain the residual liquid. The exosome precipitate is then dissolved in an appropriate volume of sterile PBS (e.g., 2.0 mL) to dissolve the precipitate, and the solution containing the exosomes is then aliquoted in a sterile Eppendorf tube and frozen in a -20°C / -80°C freezer. The presence of the exosome-specific marker CD63A in the exosomes is then determined using ELISA-63A and a protein quantification kit.

[0165] As shown, PRP, placental perfusate and placental tissue contain exosome populations that are CD63+ and can be effectively separated by ultracentrifugation. To separate exosomes, the culture supernatant was first filtered through a tissue filter and centrifuged several times as described above to obtain exosomes, which were then frozen. For ELISA detection of exosomes, anti-CD63 antibodies were used. In the assay, the sample was diluted 1:1 with exosome binding buffer (60uL+60uL). CD63+ exosomes were effectively separated by this procedure.

[0166] Characterization of exosomes

[0167] Exosomes can contain proteins, peptides, RNA, DNA and cytokines. Methods such as miRNA sequencing, surface protein analysis (MACSPlex Exosome Kit, Miltenyi), proteomics, functional studies (enzyme assays, in vitro wound healing assay (scratch assay), exosome-induced cell proliferation (human keratinocytes or fibroblasts) (compared to 5 known stimuli), exosome-induced collagen production (human keratinocytes or fibroblasts): compared to TGFb, including serum and non-serum controls, ELISA for procollagen 1C peptide, exosome-induced inhibition of inflammatory cytokines: responding cell types include human keratinocytes or human fibroblasts, and comparison with lyophilized heat-killed bacteria or LPS) can be performed.

[0168] In some alternatives, the isolated exosomes were concentrated with a 100Kda Vivaspin filter (Sartorius), washed once with PBS, and approximately 40uL was recovered. The concentrated exosome population was mixed with 10uL of 5XRIPA lysis buffer containing 1x protease inhibitor cocktail (Roche) and vortexed, then sonicated for 5 minutes at 20°C in a water sonicator (UltrasonicCleaner, JSP). After sonication, the tubes were incubated on ice for 20 minutes with intermittent mixing. Next, the mixture was centrifuged at 10,000g for 10 minutes at 4°C. The separated clarified lysate was transferred to a fresh tube. The protein amount was measured with a BCA kit, and 10ug protein was loaded per lane for western blotting, and the target protein was determined using antibodies.

[0169] In another alternative, exosome labeling and cellular uptake (e.g., HEK293T) were examined. An aliquot of frozen eluted exosomes was resuspended in 1 mL PBS and labeled using the PKH26 fluorescent cell linker kit (Sigma-Aldrich). A 2x PNK26 dye solution (4uL dye in 1 mL diluent C) was prepared and mixed with 1 mL exosome solution to a final dye concentration of 2x10e-6M. The immediate sample was mixed for 5 minutes and the staining was terminated by adding 1% BSA to capture the Excel PKH26 dye. The labeled exosomes were transferred to a 100-Kda Vivaspin filter and centrifuged at 4000g, then washed twice with PBS, and approximately 50uL of sample was recovered for analysis of exosome concentration using NTA and then stored at -80C. PBS was used as a negative control for the labeling reaction. For uptake studies, HEK293T cells were seeded in 8-well chamber slides (1x10e4 / well) using regular culture medium. After 24 hours, the slides were washed twice with PBS and incubated with DMEM-exo-free FBS (10%) for 24 hours. Thereafter, fresh DMEM medium (containing 10% exo-free PBS (200uL)) each labeled with an exosome sample corresponding to 2x10e9 exosomes was added to each well and incubated in a cell incubator for 1.5 hours. After incubation, the slides were washed twice with PBS (500ul) and fixed with 4% paraformaldehyde solution at room temperature for 20 minutes. The slides were washed twice with PBS (500uL), dried, and mounted using ProLong Gold 4 anti-fading agent with DAPI. The cells were visualized using an Axioskop microscope (Zeiss)

[0170] High-yield isolation of exosomes from cultured postpartum human placentas

[0171] Perfusion of postpartum human placentas with full donor consent was obtained. The residual blood on the placenta was rinsed off with a large amount of sterile saline, and then cultured in a 5-L bioreactor with a serum-free medium supplemented with antibiotics, and cultured in a 37°C incubator (5% CO2), and rotated alternately under refrigeration for an extended period of time up to 4 days. The supernatant of the culture medium was processed by continuous centrifugation at 3000g and 10,000g to precipitate tissues, cells, and microvesicles. Exosomes were precipitated by ultracentrifugation at 100,000g from the supernatant centrifuged at 10,000g and dissolved with sterile PBS. The yield of exosomes was quantified by BCA protein assay.

[0172] Supernatants from placental organ cultures were processed as described in Methods for Isolation of Exosomes. ELISA assay using anti-CD63A antibody showed that the isolated exosomes contained CD63A protein, a specific protein marker for exosomes. It is estimated that one placenta cultured in 1 liter of culture medium produces approximately 40 mg of exosomes in 24 hours, or approximately 1x10 13 CD63A-positive exosome particles. These placental organ-derived exosomes were further characterized, including the expression of CD9 and CD81, size, and functional activity.

[0173] In another set of experiments, with full donor consent, postpartum human placenta was perfused to isolate exosomes with different concentrations of EDTA. Serum-free medium supplemented with antibiotics and different concentrations of EDTA (e.g., 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mM or within the range defined by any two of the aforementioned concentrations) was perfused into the placenta via a peristaltic pump at a constant rate through the umbilical vein, and cultured for another 24-48 hours under controlled conditions. After cultivation, 750 mL of physiological medium containing the amount of EDTA used was perfused at a controlled rate. Exosomes were then separated by continuous centrifugation and ultracentrifugation, confirmed by CD63A ELISA assay, and quantified by BCA protein assay, all as described above. It will be shown that the concentration of EDTA in the culture medium for recovering exosomes affects the amount of exosomes recovered from the placenta cultured in the bioreactor.

[0174] Another alternative

[0175] In some alternatives, a method for isolating exosomes from a placenta or a portion thereof is provided. The method comprises: a) contacting the placenta or a portion thereof with a first culture medium; b) obtaining a first fraction comprising exosomes from the placenta or a portion thereof; c) contacting the placenta or a portion thereof with a second culture medium; d) obtaining a second fraction comprising exosomes from the placenta or a portion thereof; e) contacting the placenta or a portion thereof with a third culture medium; f) obtaining a third fraction comprising exosomes from the placenta or a portion thereof; and optionally, isolating the exosomes from the first fraction, the second fraction, and / or the third fraction. In some alternatives, the method further comprises contacting the placenta or a portion thereof with an additional culture medium; and obtaining multiple steps of additional fractions comprising exosomes from the placenta or a portion thereof. These two steps may be repeated multiple times. Preferably, the placenta or a portion thereof is cultured and / or maintained in a bioreactor. In some alternatives, the placenta or a portion thereof comprises amniotic membrane. In some alternatives, the placenta or a portion thereof is a human placenta or a portion thereof. In some alternatives, the first culture medium, the second culture medium and / or the third culture medium are in contact with the placenta or a portion thereof for at least 45 minutes, such as 45 minutes or 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours or any time amount within the range defined by any two of the aforementioned time points. In some alternatives, the first culture medium, the second culture medium and / or the third culture medium are in contact with the placenta or a portion thereof for at least 7 days, 14 days, 28 days, 35 days or 42 days or any time amount within the range defined by any two of the aforementioned time points. In some alternatives, the placenta or a portion thereof has been chopped, ground or treated with an enzyme (such as collagenase and / or protease).

[0176] In some alternatives, placenta or its part is provided in the form of a substantially flat or sheet-like scaffold material, and the placenta or its part has been decellularized and optionally substantially dried. The decellularized placenta or its part is used as a scaffold to accommodate exogenous cells, such as a homogeneous cell colony (for example, including stem cells, endothelial cells and / or regenerative cells of progenitor cells) obtained from cell culture or primary separation procedures. The method also includes passing a fluid or a fluid containing cells to be inoculated into the decellularized placenta or its part. Once cells are established, the exosomes produced by the cells are recovered and separated using the above-mentioned procedure. In some alternatives, the fluid containing cells to be inoculated on the decellularized placenta or its part is ascites, blood or plasma. In some alternatives, the cells are from organs. In some alternatives, the cells are from liver, kidney, lung or pancreas. In some alternatives, the cells are immune cells. In some alternatives, the cells are T cells or B cells.

[0177] In some alternatives, the first culture medium comprises phosphate buffered saline (PBS). In some alternatives, the second culture medium comprises a growth factor. In some alternatives, the third culture medium comprises a chelating agent. In some alternatives, the chelating agent is EDTA, EGTA, a phosphonate, BAPTA tetrasodium salt, BAPTA / AM, bis-Notrophen™ reagent tetrasodium salt, EGTA / AM, pyridoxal isonicotinic acid hydrazide, N,N,N′,N′-tetrakis-(2-pyridylmethyl)ethylenediamine, 6-bromo-N′-(2-hydroxybenzylidene)-2-methylquinoline-4-carbohydrazide, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl ester), (ethylenedinitrilo)tetraacetic acid, EDTA, Edathamil, ethylenedinitrilotetraacetic acid, ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid or ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid tetrasodium salt or any combination thereof. In some alternatives, the chelating agent is EDTA or EGTA or a combination thereof. In alternatives, the chelating agent is provided in the third culture medium at a concentration of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM or at a concentration within the range limited by any two of the aforementioned concentrations. In some alternative embodiments, the concentration of EDTA in the third culture medium is provided at a concentration of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM, or at a concentration within a range defined by any two of the foregoing concentrations.

[0178] In some alternatives, the third substratum comprises a protease. In some alternatives, the protease is trypsin, collagenase, chymotrypsin or carboxypeptidase or its mixture. In some alternatives, the protease is trypsin. In alternatives, the protease is provided in the third substratum with a concentration of 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM or 100mM or with a concentration in the range limited by any two of the aforementioned concentrations.

[0179] In some alternatives, the method further comprises contacting the placenta or a portion thereof with a plurality of additional culture media, wherein the contacting is such that a plurality of fractions comprising exosomes are obtained. In some alternatives, the first culture medium, the second culture medium, the third culture medium, or an additional culture medium comprises glucose. In some alternatives, the first culture medium, the second culture medium, the third culture medium, or an additional culture medium comprises GM-CSF. In some alternatives, the first culture medium, the second culture medium, the third culture medium, or an additional culture medium comprises serum. In some alternatives, the first culture medium, the second culture medium, the third culture medium, or an additional culture medium comprises DMEM. In some alternatives, the first culture medium, the second culture medium, the third culture medium, or an additional culture medium comprises an AHR antagonist. In some alternatives, the AHR antagonist is SR1. In some alternatives, the concentration of SR1 is 1 nM, 10 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM or 1 mM, or any other concentration within the range defined by any two of the foregoing values.

[0180] In some alternatives, the first culture medium is contacted with the placenta or a portion thereof while maintaining a temperature of 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, or 40°C, or a temperature within a range defined by any two of the foregoing temperatures. In some alternatives, the second culture medium is contacted with the placenta or a portion thereof while maintaining a temperature of 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, or 40°C, or a temperature within a range defined by any two of the foregoing temperatures. In some alternatives, the third culture medium is contacted with the placenta or a portion thereof while maintaining a temperature of 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, or 40°C, or a temperature within a range defined by any two of the foregoing values. In some alternatives, the additional plurality of culture media are contacted with the placenta or portion thereof while maintaining a temperature of 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C or 40°C, or a temperature within a range bounded by any two of the foregoing values.

[0181] In some alternatives, the first culture medium, the second culture medium, the third culture medium, or the additional culture media comprises an antibiotic.

[0182] In some alternatives, the exosomes are isolated from the first fraction, the second fraction, and / or the third fraction or the plurality of fractions by a method comprising:

[0183] (a) passing the first fraction, the second fraction and / or the third fraction or the plurality of fractions through a tissue filter;

[0184] (b) subjecting the filtrate collected in (a) to a first centrifugation to produce a cell pellet and a first supernatant;

[0185] (c) centrifuging the first supernatant a second time to produce a second supernatant; and

[0186] (d) centrifuging the second supernatant for a third time to produce an exosome pellet; and optionally,

[0187] (e) The exosomes are resuspended in the solution.

[0188] In some alternatives, the isolated exosome population comprises exosomes having CD63, CD63-A, perforin, Fas, TRAIL, or granzyme B, or a combination thereof. In some alternatives, the isolated exosome population comprises exosomes containing signaling molecules. In some alternatives, the isolated exosome population comprises exosomes comprising cytokines, mRNA, or miRNA.

[0189] In some alternatives, the method further comprises isolating the exosomes by affinity chromatography, wherein the affinity chromatography is selective for removing exosomes comprising viral antigens, viral proteins, bacterial antigens or proteins, fungal antigens or proteins.

[0190] In some alternatives, the method further comprises isolating the exosomes by an alternative or additional affinity chromatography step, wherein the alternative or additional affinity chromatography step is selective for removing exosomes containing inflammatory proteins. In some alternatives, the method further comprises enriching the exosome population containing anti-inflammatory biomolecules.

[0191] In some alternatives, exosomes produced by any of the embodiments herein are provided. In some alternatives, the exosomes are from ascites, blood, or plasma. In some alternatives, the exosomes are from cells of an organ. In some alternatives, the exosomes are from immune cells. In some alternatives, the exosomes are from T cells or B cells.

[0192] Those skilled in the art will appreciate that, in general, the terms used herein and particularly in the appended claims (e.g., the bodies of the appended claims) are generally intended to be "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "including but not limited to," etc.). Those skilled in the art will further understand that if a specific number of a claim recitation is intended, such intent will be explicitly stated in the claim, and in the absence of such a statement, no such intent is present. For example, to aid understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be interpreted as implying that the introduction of a claim recitation by the indefinite article "a / an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one / or more" or "at least one" and an indefinite article such as "a / an" (e.g., a / an should be interpreted to mean "at least one" or "one / or more"); this is also true for the use of definite articles used to introduce claim recitations. Furthermore, even if a specific number of an introduced claim recitation is explicitly recited, one skilled in the art will still recognize that such recitation should be interpreted to mean at least one of the recited number (e.g., the straightforward recitation of "two recitations" without other modifiers means at least two recitations, or means two or more recitations). Furthermore, where a convention similar to “at least one of A, B, and C, etc.” is used, generally such a syntactic structure is intended to mean the convention in the sense of one skilled in the art (e.g., “a system having at least one of A, B, and C” would include, but not be limited to, systems having only A, only B, only C, both A and B, both A and C, both B and C, and / or both A, B, and C, etc.). Where a convention similar to “at least one of A, B, or C, etc.” is used, generally such a syntactic structure is intended to mean the convention in the sense of one skilled in the art (e.g., “a system having at least one of A, B, or C” would include, but not be limited to, systems having only A, only B, only C, both A and B, both A and C, both B and C, and / or both A, B, and C, etc.). One skilled in the art will further understand that, whether in the specification, claims, or drawings, virtually any disjunctive word and / or phrase presenting two or more alternative terms should be understood to contemplate the possibility of including one of the terms, either of the terms, or both of the terms.For example, the phrase "A or B" will generally be understood to include the possibilities of "A" or "B" or "A and B."

[0193] Example

[0194] 6.1. Example 1: Culture of human placenta

[0195] Receive human placenta and wash with sterile PBS or saline solution to remove blood. The placenta is then cultured as a whole organ in a vessel in a large container with a volume of 500mL or 1000mL of DMEM medium supplemented with antibiotics and 2mM EDTA. In different alternatives, the placenta can be cut into different sizes and placed in a culture container. Culture at 37°C in a cell culture incubator containing 5% CO2. The culture time is 4 to 8 hours, and the supernatant of the culture is used to isolate exosomes. New culture medium is added at each harvest time point (e.g., every 8 hours or every 12 hours), and the placental organs and tissues are cultured for at least 5 days.

[0196] 6.2. Example 2: Isolation and purification of placental exosomes

[0197] The supernatant of the culture was centrifuged at 3,000g for 30 minutes to precipitate the cells and tissue debris. The supernatant was then centrifuged at 10,000g for 1 hour, and the precipitate (small cell debris and organelles) was discarded. The supernatant was then centrifuged at 100,000g for 2 hours. The resulting precipitate is the exosome. The exosome precipitate can be further purified by the following method: resuspended with different volumes of sterile PBS, and centrifuged again at 100,000 for 2 hours, and then the final precipitate was resuspended with sterile PBS. The resuspended exosomes were filtered through a syringe filter (0.2um) and aliquoted at -80°C in different volumes of 300uL to 1mL.

[0198] Characterization of placental exosomes by size. Size distribution was analyzed by nanoparticle tracking assay. Three representative samples of pExo were sized using NanoSight. Each isolate had an average size of 117, 101, and 96, respectively, consistent with reported exosome sizes. Results are shown in Figure 2A – Figure 2C middle.

[0199] 6.3. Example 3: pExo markers by FACS analysis

[0200] Using MACSPlex exosome kit (Miltenyi Biotec, catalog number 130-108-813), the protein markers of pExo are analyzed according to the scheme provided by the kit. In short, 120uL of pExo isolates are incubated overnight at room temperature with 15uL of exosome capture beads. After washing once with 1mL washing solution, exosomes are incubated with exosome detection reagents CD9, CD63 and CD81 mixture, and incubated for another 1 hour. After two washings, samples are analyzed with FACS (BD Canto10). A total of 37 protein markers (Table 1) are included in this kit, excluding mIgG1 and REA controls.

[0201] Table 1: List of protein markers used to detect pExo in the MACSPlex Exosome Kit

[0202]

[0203] pExo samples were identified as highly positive for the following protein markers, including CD1c, CD9, CD20, CD24, CD25, CD29, CD2, CD3, CD8, CD9, CD11c, CD14, CD19, CD31, CD10, CD41b, CD42a, CD44, CD45, CD19c, CD4, CD15, CD19c, CD4, CD56, CD62P, CD83, CD69, CD81, CD86, CD105, CD133-1, CD142, CD148, HLA-ABC, HLA-DRDPDQ, MSCP, ROR1, SSEA-4. pExo had very low levels of CD209 (2.6%). Human placental perfusate obtained by perfusing the vascular system of the placenta with saline solution without culture in a culture medium and a cell culture incubator was also used to isolate exosomes, and marker protein expression was analyzed by the same method. Perfusate-derived exosomes also express high levels of most markers found in pExo, but have significantly lower CD11c (2.0%), MCSP (3.4%), and SSEA-4 (3.5%) compared to pExo. pExo also has significantly higher levels of CD142 and CD81 compared to placental perfusate exosomes. Umbilical cord blood serum was also used to isolate exosomes, and protein expression was analyzed by the same method. Umbilical cord blood serum-derived exosomes are also positive for most protein markers, but generally show lower levels of protein expression for each of these markers. Specifically, compared to pExo, umbilical cord blood serum exosomes have lower levels of CD56 (1.4%), CD3 (0.3%), and CD25 (3.9%). Umbilical cord serum exosomes also had higher MSCP protein levels compared to pExo. These data suggest that cultured placental tissue produces a unique population of exosomes compared to uncultured placenta and umbilical cord serum. The results for pExo samples compared to umbilical cord serum-derived exosomes and placental perfusate exosomes are shown in Figure 3A – Figure 3C and Table 2.

[0204] Table 2

[0205]

[0206]

[0207] 6.4 Example 4: Cytokines and Growth Factors of pExo Samples

[0208] The content of cytokines in pExo samples was analyzed using a MiltiPlex Luminex kit containing 41 different cytokines. The following table shows the data of cytokines detected on 15 different pExo preparations. The data show that pExo contains significant levels of cytokines (average> 50pg / mL), including FGF2, G-CSF, fractalkine, GDGF-AA / BB, GRO, IL-1RA, IL-8, VEGF and RANTES. pExo also contains detectable levels of other cytokines (5pg / mL to 49pg / mL), including EGF, Flt-3L, IFNa3, MCP-3, PDGF-AA, IL-15, sCD40L, IL6, IP-10, MCP-1, MIP-α, MIP-1β and TNF-α.

[0209] Table 3: Cytokines detected in pExo preparations

[0210]

[0211] The presence of soluble cytokine receptors in pExo (11 samples) was also analyzed by multiple Luminex analysis. The data are shown in the table below. The data show that pExo contains high levels (>100pg / mL) of sEFGR, sgp-130, sIL-1R1, sTNFR1, sTNFRII, sVEGRR1, sVEGFR1, sVEGFR3 and sCD30, and sIL-2Ra, sIL-6R, sRAGE (>10ng / mL) were also detected in some samples. Data shown as < were not detected and were considered negative.

[0212] Table 4

[0213]

[0214] 6.5 Example 5: Proteomic Analysis of Placental Exosomes

[0215] Three pExo samples were subjected to proteomic analysis. Submitted samples were solubilized using a sonic probe (QSonica) with the following settings: amplitude 40%, pulse 10x on 1 sec, off 1 sec. Protein concentration was determined by Qubit fluorometry. 10ug of each sample was processed by SDS page and the purified protein was trypsin digested. Table 5 shows the total proteins identified from each sample. A total of 1814 proteins were identified in these samples. Table 6 shows the identification and gene ID of the top identified proteins in the pExo samples. Additional data are in Figure 4 and Figure 5 Shown in.

[0216] Table 5

[0217]

[0218] Table 6

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226] 6.6. Example 6: RNA analysis of placental exosomes

[0227] The RNA profiles of three pExo samples were analyzed by sequencing. In short, RNA was extracted from pExo samples, converted into cDNA and sequenced. The sequencing data was then compared with the database to identify the type and characteristics of each sequencing data. Table 7 shows the overall overview of RNA sequencing results. The RNA in pExo contains tRNA, microRNA and other categories of non-coding RNA. MicroRNA is the second most abundant RNA in the composition of pEXO samples. A total of 1500 different microRNAs were identified in these three pExo samples. Some are commonly present in all three samples, and some are uniquely present in one or both of the samples. The gene IDs of the most abundant microRNAs as well as the relative frequency and abundance are shown. It is known that microRNA plays an important role in intercellular communication function.

[0228] Table 7

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236] 6.7. Example 7: Placental exosomes promote migration of human dermal fibroblasts (HDF)

[0237] The cytokine profile showed that pExo included chemotactic growth factors, suggesting that pExo should have the function of promoting cell migration. To examine this, a transwell migration assay was set up as follows: 750uL of DMEM basal medium (serum-free) was placed on the bottom chamber of a transwell (24-well) plate and 50uL of pExo was added. The same volume of PBS as the control was added. 1x10e5 HDFs were seeded on the top chamber of the transwell (8um pores). After 6 to 24 hours, the cells on the top chamber of the transwell were removed with a cotton swab. The transwell was then fixed in a solution containing 1% ethanol in PBS and then stained with 1% crystal violet dissolved in 1% ethanol-PBS. The migrated cells were visualized using a microscope. The data shows an example of the results of HDFs migrating to the lower side of the Transwell, while significantly fewer cells migrated through the wells in the PBS control Transwell. The studies show that pExo can promote the migration of human dermal fibroblasts. See Figure 6 .

[0238] 6.8. Example 8: Placental exosomes promote migration of human umbilical cord blood endothelial cells (HUVEC)

[0239] A transwell migration assay was also set up as follows: 750uL of DMEM basal medium (serum-free) was placed on the bottom chamber of a transwell (24-well) plate and 50uL of pExo was added. The same volume of PBS as for the control was added. 2x10e5 HUVECs expressing GFP protein were seeded on the top chamber of the transwell (8um wells). After 6 to 24 hours, the migrated wells were directly visualized using an inverted fluorescence microscope (AMG). The study demonstrated that all three pExo samples tested promoted the migration of HUVECs in all three replicate wells. Complete medium for HUVECs was used as a positive control with significant cell migration, and PBS was used as an additional control with significantly less cell migration compared to complete medium or pExo test wells. See Figure 7 .

[0240] 6.9. Example 9: Placental exosomes stimulate proliferation of HUVEC

[0241] The cytokine profile of pExo shows that it has several growth factors (PGDF-AA, BB, VEGF) known to be involved in the growth of HUVEC. In order to examine the effect of pExo on the growth and proliferation of HUVEC. HUVEC expressing GFP were seeded in 96-well plates (transparent bottom and non-transparent walls) at 1x10e4 cells in 100uL complete HUVEC growth medium. After 2 hours of inoculation, the cells attached to the bottom of the wells. Then 25uL of different pExo samples (N=6 for each sample) were added to the wells. The fluorescence intensity of the plate was then evaluated using a microplate reader (Synergy H4, excitation 395nm / emission 509nm) on days 0 and 2 after inoculation. As Fig.13 As shown, the complete medium showed higher GFP signal (an indicator of cell number) from day 0 to day 2. The PBS control diluted 50% in the complete medium showed slight growth compared to the complete medium. All eight different pExo samples showed higher GFP growth on day 2. See Figure 8 .

[0242] 6.10. Example 10: Placental exosomes stimulate proliferation and colony formation of human CD34+ cells

[0243] To test the effect of pExo on the proliferation of hematopoietic stem cells, human umbilical cord blood CD34+ cells (prepared in-house) were thawed and cultured in an expansion medium containing a mixture of 10% SCF, Flt-3, KL (medium A) and 10% FCS-IMDM at 1x10e4 / cell / mL (N=4). 25uL of PBS or 25uL of pExo samples (two pExo samples were tested) were added to the culture wells. After one week of culture, the total number of cells in each well was counted, and the percentage of CD34+ cells in the culture was evaluated by flow cytometry (FACS) using an anti-CD34 antibody. The total number of CD34+ cells was calculated as the total number of cells in the well and the percentage of CD34+ cells in the culture. The results showed that both pExo-treated cultures had significantly higher numbers of CD34+ cells compared to PBS control cultures. The effect of pExo on CD34+ cells was also tested in colony forming unit cultures (CFU). CFU cultures were established with MethoCult H4434 medium (Stem Cell Technologies) and pExo or PBS was added at 50uL / mL. After two weeks of culture, the total number of CFU in each 35-mm dish was counted (N=3). The data showed that the number of CFU was significantly higher in the presence of pExo compared to the PBS control culture. See Figure Fig. 9 and Fig.10 .

[0244] 6.11. Example 11: Inhibition of cancer cell proliferation

[0245] MicroRNA data and cytokine data indicate that pExo has activity in inhibiting cancer cell proliferation. pExo samples were used to examine their effects on the growth of SKOV3 (human ovarian cancer cell line) in 96-well plates. Such SKOV3 cells were engineered to express luciferase, therefore, measuring luciferase activity is an indicator of cell growth. A total of 8 different pExo samples were used. 2000 SKOV3 cells were added to a 96-well plate in 100uL growth medium (DMEM-10% FCS). After 2 hours, 40uL of pExo was added to the wells (N=6) and supplemented with 60uL of growth medium. 40uL of PBS was used as a control. Complete medium conditions were to add 100uL of medium to the wells. After 2 days of culture in an incubator, luciferase activity was measured by lysing the cells with a luciferase assay kit (Promega) and measuring luciferase activity by luminescence emission using a microplate reader (Synergy H4). The data show that at each cell concentration, the pExo-treated cultures had a significantly lower Luminex index compared to the PBS control. This data suggests that pExo inhibits the growth of SKOV3 cells. Fig.11 .

[0246] A549 cancer cell line (human lung cancer cell line) was seeded in a 96-well plate (Xiceligence) at 1500 cells / well. 24 hours after seeding, pExo was added to the growth medium (100uL) at three different doses (5uL, 25 and 50uL). The same amount of PBS was added as a control. From the first to the third day after seeding, the growth of cells can be monitored by software that reflects the adhesion of cells on the wells. The data show that the growth of cells in the presence of pExo (as shown as a standardized cell index, is significantly lower in the presence of pExo compared to the PBS control. The growth curves are each the average cell index from three independent wells. See Fig.12 .

[0247] pExo samples were used to examine their effect on the growth of MDA231 (human breast cancer cell line) at different cell doses in 96-well plates. This MDA231 cell was engineered to express luciferase, so measuring luciferase activity is an indicator of cell growth. MDA231-luciferase with different cell numbers was inoculated into 96-well plates (triplicate) and 25uL of pExo#789 was added. After 2 days of culture in an incubator, luciferase activity was measured by lysing the cells using a luciferase assay kit (Promega) and by a microplate reader (Synergy H4) using luminescence emission. The data showed that at each cell concentration, the pExo-treated culture had a significantly lower Luminex index compared to the PBS control. This data shows that pExo inhibits the growth of MDA231 cells. See Fig.13 .

[0248] 6.12. Example 12: Placental exosomes regulate the activation and differentiation of immune cells

[0249] To examine the effects of pExo on immune cells, human cord blood T cells were labeled with PKH fluorescent dye and then incubated with pExo or PHA as stimulation. After 5 days of culture in RPMI + 10% FCS, cells were analyzed by FACS using antibodies that can distinguish total T cells and subtypes of different types of T cells, including CD4, CD8, CD69, CD27. The data showed that in the presence of pExo, the MFI of CD3+ cells was similar to that of control cultures, indicating that pExo alone did not affect the proliferation activity of T cells. Under PHA stimulation, the MFI was significantly reduced, indicating that in the presence of both PHA and pExo, cells proliferated, and the MFI was similar to that of PHA alone, indicating that cell proliferation was not affected by the presence of pExo. It was found that CD69+ cells were significantly higher in cells treated with pExo, and CD69+ cells were significantly increased in CD3+ cells (T cells), indicating that T cell activation was increased by pExo. This observation was found in both cord blood T cells and PBMC cells. In addition, pExo was found to increase the percentage of CD56+ cells (NK cells) in PBMCs. Fig.14 , Fig.15 , Fig.16 and Fig.17 .

[0250] 6.13. Example 13: Yield of exosomes from cultured placenta, placental perfusate and PRP (umbilical cord serum)

[0251] Placental perfusate and PRP (umbilical cord serum) were isolated by the same method as cultured human placental tissue. The table below shows that the yield of exosomes from placental perfusate and PRP was significantly lower than that from cultured placenta.

[0252] Table 8

[0253]

[0254] discuss:

[0255] The subject methods are capable of producing large quantities of exosomes with unique and advantageous properties. The exosomes have been shown to contain a number of proteins and RNAs that are believed to be biologically active due to the demonstrated functions of the exosomes. The exosomes express a number of cell surface markers that can serve as binding partners, e.g., as receptors or ligands, and thereby allow targeting of such biological activity to desired cell types.

[0256] The data presented herein demonstrate the utility of the exosomes for a variety of indications, such as those described in Table 9.

[0257] Table 9

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269] Equivalent solution:

[0270] The scope of the present disclosure is not limited by the specific embodiments described herein. In fact, various modifications of the subject matter provided herein, in addition to those described, will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

[0271] Various publications, patents and patent applications are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims

1. A method for isolating exosomes from a placenta or a portion thereof, the method comprising: a) contacting a placenta or a portion thereof, preferably a cultured placenta or a portion thereof, with a first culture medium; as well as b) obtaining a first fraction comprising a population of exosomes from said placenta or a portion thereof; c) optionally, contacting the placenta or a portion thereof with a second culture medium and obtaining a second fraction comprising a population of exosomes from the placenta or a portion thereof; d) optionally, contacting the placenta or a portion thereof with a third culture medium and obtaining a third fraction comprising a population of exosomes from the placenta or a portion thereof; and e) optionally separating the exosome population from the first fraction, the second fraction and / or the third fraction using an antibody or a binding portion thereof specific for a marker or peptide present on the desired exosome population, preferably by sequential centrifugation and / or affinity chromatography, wherein the antibody or the binding portion thereof is immobilized on a substrate such as a membrane, a resin, a bead or a vessel.

2. The method of claim 1, wherein the placenta or portion thereof further comprises amniotic membrane.

3. The method of claim 2, wherein the placenta or portion thereof is a human placenta or portion thereof.

4. The method of any of the preceding claims, wherein the first culture medium, the second culture medium and / or the third culture medium is in contact with the placenta or a portion thereof for at least 45 minutes, such as 45 minutes or 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours or 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days or 20 days or any amount of time within a range defined by any two of the aforementioned time points.

5. A method as described in any of the preceding claims, wherein the first culture medium, the second culture medium and / or the third culture medium are in contact with the placenta or a portion thereof for at least 7 days, 14 days, 28 days, 35 days or 42 days, or any amount of time within a range defined by any two of the aforementioned time points.

6. A method as claimed in any preceding claim, wherein the placenta or part thereof has been minced, ground or enzymatically treated.

7. The method of any one of claims 1-5, wherein the placenta or portion thereof is substantially flat or sheet-like and has been decellularized and substantially dry, and wherein the method further comprises contacting a fluid comprising exogenous cells with the decellularized placenta or portion thereof so as to seed the decellularized placenta or portion thereof with the exogenous cells, and wherein the contacting of the cells with the decellularized placenta or portion thereof has been performed prior to contacting the decellularized placenta or portion thereof with a first culture medium.

8. The method of claim 7, wherein the exogenous cells are obtained from a subject different from the donor subject of the placenta or portion thereof.

9. The method of claim 7 or 8, wherein the fluid comprises ascites, blood or plasma.

10. The method of claim 7 or 8, wherein the cells are from an organ.

11. The method of claim 10, wherein the cells are from liver, kidney, lung or pancreas.

12. The method of claim 7 or 8, wherein the cell is an immune cell.

13. The method of claim 12, wherein the cell is a T cell or a B cell.

14. The method of any one of the preceding claims, wherein the first culture medium comprises phosphate buffered saline (PBS).

15. The method of claim 9, wherein the first fraction, the second fraction, or the third fraction comprises exosomes from ascites, blood, or plasma. 16 . The method of claim 10 , wherein the first fraction, the second fraction, or the third fraction comprises exosomes from organ cells.

17. The method of claim 11, wherein the cells are from liver, kidney, lung or pancreas.

18. The method of any one of the preceding claims, wherein the second culture medium comprises growth factors.

19. The method of any one of the preceding claims, wherein the third culture medium comprises a chelating agent.

20. The method of claim 19, wherein the chelating agent is a phosphonate, BAPTA tetrasodium salt, BAPTA / AM, bis-Notrophen™ reagent tetrasodium salt, EGTA / AM, pyridoxal isonicotinic acid hydrazide, N,N,N′,N′-tetrakis-(2-pyridylmethyl)ethylenediamine, 6-bromo-N′-(2-hydroxybenzylidene)-2-methylquinoline-4-carbohydrazide, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl ester), (ethylenedinitrilo)tetraacetic acid (EDTA), Edathamil, ethylenedinitrilotetraacetic acid, ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid or ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid tetrasodium salt (EGTA), or any combination thereof.

21. The method of any one of claims 19 or 20, wherein the chelating agent is EDTA or EGTA or a combination thereof.

22. The method of any one of claims 19-21, wherein the chelator is provided in the third culture medium at a concentration of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM, or at a concentration within a range defined by any two of the foregoing concentrations.

23. The method of any one of claims 19-22, wherein the concentration of EDTA in the third culture medium is provided at a concentration of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM, or at a concentration within a range defined by any two of the foregoing concentrations.

24. The method of any one of the preceding claims, wherein the third culture medium comprises a protease.

25. The method of claim 24, wherein the protease is trypsin, collagenase, chymotrypsin or carboxypeptidase or any combination thereof.

26. The method of claim 25 or 25, wherein the protease is trypsin.

27. The method of claim 24, wherein the protease is provided in the third culture medium at a concentration of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM, or at a concentration within a range defined by any two of the foregoing concentrations.

28. The method of any one of the preceding claims, wherein the method further comprises contacting the placenta or portion thereof with additional multiple culture media, wherein the contacting results in obtaining multiple fractions comprising exosomes.

29. The method of claim 28, wherein the first culture medium, the second culture medium, the third culture medium or the additional culture medium comprises glucose.

30. The method of claim 28 or 29, wherein the first culture medium, the second culture medium, the third culture medium or the additional culture medium comprises GM-CSF.

31. The method of any one of claims 28-30, wherein the first culture medium, the second culture medium, the third culture medium or the additional culture medium comprises serum.

32. The method of any one of claims 28-31, wherein the first culture medium, the second culture medium, the third culture medium or the additional culture medium comprises DMEM.

33. The method of any one of claims 28-32, wherein the first culture medium, the second culture medium, the third culture medium or the additional culture medium comprises an AHR antagonist.

34. The method of claim 33, wherein the AHR antagonist is SR1.

35. The method of claim 34, wherein the concentration of SR1 is 1 nM, 10 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM or 1 mM, or any other concentration within the range defined by any two of the foregoing values.

36. A method as described in any of the preceding claims, wherein the first culture medium is contacted with the placenta or a portion thereof while maintaining a temperature of 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C or 40°C, or a temperature within a range defined by any two of the foregoing temperatures.

37. A method as described in any of the preceding claims, wherein the second culture medium is contacted with the placenta or a portion thereof while maintaining a temperature of 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C or 40°C, or a temperature within a range defined by any two of the foregoing temperatures.

38. A method as described in any of the preceding claims, wherein the third culture medium is contacted with the placenta or a portion thereof while maintaining a temperature of 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C or 40°C, or a temperature within a range defined by any two of the foregoing values.

39. The method of any one of claims 28-38, wherein the additional plurality of culture media are contacted with the placenta or a portion thereof while maintaining a temperature of 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C or 40°C, or a temperature within a range defined by any two of the foregoing values.

40. The method of any one of the preceding claims, wherein the first perfusion, the second perfusion, or the third perfusion or the additional plurality of culture media comprises an antibiotic.

41. The method of any one of the preceding claims, wherein the exosomes are isolated from the first fraction, the second fraction, and / or the third fraction or fractions by a method comprising the steps of: (a) passing the first fraction, the second fraction and / or the third fraction or the plurality of fractions through a tissue filter; (b) subjecting the filtrate collected in (a) to a first centrifugation to produce a cell pellet and a first supernatant; (c) centrifuging the first supernatant a second time to produce a second supernatant; as well as (d) centrifuging the second supernatant for a third time to produce an exosome pellet; and optionally, (e) resuspending the exosomes in a solution.

42. The method of any of the preceding claims, wherein the exosomes comprise CD63, CD63-A, perforin, Fas, TRAIL, or granzyme B, or any combination thereof.

43. The method of claim 42, wherein the exosomes comprise CD63A.

44. The method of any one of the preceding claims, wherein the exosomes comprise signaling molecules.

45. The method of any one of the preceding claims, wherein the exosomes comprise cytokines, mRNA, or miRNA.

46. ​​The method of any of the preceding claims, further comprising isolating the exosomes by affinity chromatography, wherein the affinity chromatography is selective for removing exosomes comprising viral antigens, viral proteins, bacterial antigens, bacterial proteins, fungal antigens, or fungal proteins.

47. The method of any one of the preceding claims, further comprising isolating the exosomes by one or more additional affinity chromatography steps, wherein the one or more additional chromatography steps are selective for removing exosomes comprising inflammatory markers and / or tumor markers.

48. A composition comprising exosomes derived from human placenta, wherein the exosomes are positive for CD1c, CD20, CD24, CD25, CD29, CD2, CD3, CD8, CD9, CD11c, CD14, CD19, CD31, CD40, CD41b, CD42a, CD44, CD45, CD49e, CD4, CD56, CD62P, CD63, CD69, CD81, CD86, CD105, CD133-1, CD142, CD146, CD209, CD326, HLA-ABC, HLA-DRDPDQ, MCSP, ROR1, SSEA-4, or a combination thereof.

49. The composition of claim 48, wherein the exosomes are positive for CD1c, CD20, CD24, CD25, CD29, CD2, CD3, CD8, CD9, CD11c, CD14, CD19, CD31, CD40, CD41b, CD42a, CD44, CD45, CD49e, CD4, CD56, CD62P, CD63, CD69, CD81, CD86, CD105, CD133-1, CD142, CD146, CD209, CD326, HLA-ABC, HLA-DRDPDQ, MCSP, ROR1, and SSEA-4.

50. The composition of claim 48, wherein the exosomes are positive for 2, 3, 4, 5, 6, 7, 8, 9, 10 or more markers selected from the group consisting of CD1c, CD20, CD24, CD25, CD29, CD2, CD3, CD8, CD9, CD11c, CD14, CD19, CD31, CD40, CD41b, CD42a, CD44, CD45, CD49e, CD4, CD56, CD62P, CD63, CD69, CD81, CD86, CD105, CD133-1, CD142, CD146, CD209, CD326, HLA-ABC, HLA-DRDPDQ, MCSP, ROR1, and SSEA-4.

51. The composition of any one of claims 48-50, wherein the exosomes are CD3-, CD11b-, CD14-, CD19-, CD33-, CD192-, HLA-A-, HLA-B-, HLA-C-, HLA-DR-, CD11c-, or CD34-.

52. The composition of any one of claims 48-50, wherein the exosomes are CD3-, CD11b-, CD14-, CD19-, CD33-, CD192-, HLA-A-, HLA-B-, HLA-C-, HLA-DR-, CD11c-, and CD34-.

53. The composition of any one of claims 48-52, wherein the exosomes comprise non-coding RNA molecules.

54. The composition of claim 53, wherein the RNA molecule is a microRNA.

55. The composition of claim 54, wherein the microRNA is selected from the group consisting of the microRNAs in Table 7 and combinations thereof.

56. The composition of claim 54, wherein the microRNA is selected from the group consisting of hsa-mir-26b, hsa-miR-26b-5p, hsa-mir-26a-2, hsa-mir-26a-1, hsa-miR-26a-5p, hsa-mir-30d, hsa-miR-30d-5p, hsa-mir-100, hsa-miR-100-5p, hsa-mir-21, hsa-miR-21-5p, hsa-mir-22, hsa-miR-22-3p, hsa-mir-99b, hsa-miR-99b-5p, hsa-mir-181a-2, hsa-mir-181a-1, hsa-miR-181a-5p and combinations thereof.

57. The composition of any one of claims 48-56, wherein the exosomes comprise cytokines selected from the group consisting of the cytokines in Table 3 and combinations thereof.

58. The composition of any one of claims 48-57, wherein the exosomes comprise a cytokine receptor selected from the group consisting of the cytokine receptors in Table 4 and combinations thereof.

59. The composition of any one of claims 48-58, wherein the exosomes comprise a protein selected from the group consisting of the proteins in Table 6 and combinations thereof.

60. The composition of any one of claims 48-58, wherein the exosomes comprise a protein selected from the group consisting of cytoplasmic aconitate hydratase, cell surface glycoprotein MUC18, protein arginine N-methyltransferase 1, guanine nucleotide binding protein G(s) subunit alpha, Cullin-5, calcium binding protein 39, glucosidase 2 subunit beta, intracellular chloride channel protein 5, brain semaphorin-3B, 60S ribosomal protein L22, spliceosomal RNA helicase DDX39B, transcription activator protein Pur-α, programmed cell death protein 10, BRO1 domain-containing protein BROX, kynurenine-ketoglutarate aminotransferase 3, laminin subunit alpha-5, ATP binding cassette subfamily E member 1, syntaxin binding protein 3, proteasome subunit beta 7 type, and combinations thereof.

61. The composition of any one of claims 48-60, wherein the exosomes comprise at least one marker molecule at a level at least twice that of exosomes derived from mesenchymal stem cells, umbilical cord blood, or placental perfusate.

62. The composition of any one of claims 48-60, wherein the exosomes have at least one marker molecule, and the level of the at least one marker molecule is at least twice that of exosomes derived from mesenchymal stem cells, umbilical cord blood, and placental perfusate.

63. The composition of any one of claims 48-62, wherein the exosomes are isolated from culture medium of whole placenta culture.

64. The composition of any one of claims 48-62, wherein the exosomes are isolated from culture medium comprising a whole culture of placental lobules or a portion of a placenta.

65. The composition of any one of claims 48-64, wherein the exosomes are produced by the method of any one of claims 1-47.

66. The composition of any one of claims 48-65, in a form suitable for intravenous administration.

67. The composition of any one of claims 48-65, in a form suitable for local injection.

68. The composition of any one of claims 48-65, in a form suitable for topical administration.

69. The composition of any one of claims 48-65, in a form suitable for ultrasonic delivery.

70. A method of increasing the proliferation of immune cells, the method comprising contacting the cells with a composition as described in any one of claims 48-65.

71. The method of claim 70, wherein the immune cell is a T cell.

72. The method of claim 70, wherein the immune cell is a NK cell.

73. The method of claim 70, wherein the immune cells are CD34+ cells.

74. A method of inhibiting the proliferation of cancer cells, the method comprising contacting the cells with a composition as described in any one of claims 48-65.

75. A method of promoting angiogenesis or vascularization in the subject, the method comprising administering to the subject the composition of any one of claims 48-65.

76. A method of modulating the subject's immune system, the method comprising administering to the subject a composition as described in any one of claims 48-65.

77. A method of repairing diseased or damaged tissue in a subject, the method comprising administering to the subject a composition as described in any one of claims 48-65.

78. A method of treating cancer in a subject, the method comprising administering to the subject a composition as described in any one of claims 48-65.

79. The method of any one of claims 75-78, wherein the subject is a human.

Images