Mesenchymal stromal cell secretome composition

Abstract

A composition comprising a. mesenchymal cell or mesenchymal stromal cell (collectively MSC) extracellular vesicles (EV) isolated from the secretome of MSCs; and extracellular matrix (ECM) components. The composition may comprise less than 3x10-9 µg / EV particle of proteins derived from the secretome and at least 0.5x10-11 µg / EV particle of the ECM components. The ECM content may at least 0.5x10-10 µg ECM / EV particle, in which case the EV and the ECM may be isolated from the secretome of MSCs; and the amount of at least one of albumin, thrombospondin-1, and thrombospondin-2 is at least 5 times lower per EV particle than in the secretome. Methods for making the compositions and uses thereof.

Description

[0001] MESENCHYMAL STROMAL CELL SECRETOME COMPOSITION TECHNICAL FIELD

[0002] The present invention relates to methods for obtaining a composition which comprises extracellular vesicles (EVs) and components from the extracellular matrix (ECM) from cells such as mesenchymal stromal cells (MSCs). The invention further relates to the composition obtained by such a method, and to the use of said composition in the field of medicine, such as in the treatment or prophylaxis of medical conditions, such as inflammatory diseases, ischemic heart disease, acute respiratory distress syndrome and scarring / fibrosis caused by diseases, aging or medical operations / procedures including plastic surgery.

[0003] BACKGROUND

[0004] Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are immune modulatory cells with angiogenetic, anti-apoptotic and anti-fibrotic properties1. These cells are clinically accepted, easy to harvest from healthy donors, can be used in allogeneic settings and used today for treatment of various systemic inflammatory conditions including Acute Respiratory Distress Syndrome (ARDS)2'3. Severely ill patients with ARDS in respirator and in need of extracorporeal membrane oxygenation have been treated with BM-MSCs, where the allogeneic BM-MSCs effectively down-regulated the inflammatory response, reduced apoptosis of alveolar epithelial cells and prevented scar formation with long-term preservation of lung function and physical capacity2'3. The safe profile of BM-MSCs together with their immune modulatory characteristics would make these cells a perfect biological drug for treatment of inflammatory diseases, for instance consequences of an acute myocardial infarction (AMI), but their clinical use is mainly limited by logistical problems. The cells demand special equipment for storage and preparation for injection after thawing that is not usually available in majority of hospitals. Thus, it is complicated to use BM-MSCs for treatment of acute conditions since one needs to plan the thawing and delivery process. Another problem is that the viability of the BM-MSCs is affected by the freezing / thawing process, which together with the timing might explain the reduced effect of BM-MSCs in treatment of inflammatory conditions in multi-center studies.

[0005] The effect of BM-MSCs is, to a large extent, mediated by secreted extracellular vesicles (EVs)4. EVs are small spherical (usually from 30 to 300 nanometers in diameter), lipid bilayer-enclosed(membrane) vesicles that are released by the majority if not all cell types of human body. EVs contain receptors on its surface and some RNA species and proteins inside. EVs can be stored for long periods of time at – 20OC with a moderate loss (compared to the cells) of their efficacy and they do not need special preparation before injection into patients. This means that the EVs are the main candidate for replacement of the cumbersome MSC-based therapies, since the EVs are immune modulatory and can be produced under controlled conditions in vitro, easily distributed to hospitals and injected at any needed time. For decades, the EV community has tried to develop methods for isolation of clean samples of EVs from other molecules that originate from the source cells and extracellular environment, because the impurities may cause undesirable side-effects and the consensus among the community is "the cleaner the better".

[0006] Current methods and procedures of handling EVs and using these in the field of medicine are associated with a number of problems, namely - the EVs are rapidly cleared from the cite of injections and from the circulation system; there is also a significant decrease in activity of clean EVs after freeze / thaw cycles, and significant loss of the EVs may occur due to adhesion to surfaces of storage containers and medical devices such as syringes, tubes, etc.

[0007] A standard method according to the state of the art to address the problem is to add a protective sample buffer to EV preparations. However, the protective sample buffer can have adverse effects in patients and adds costs to the final product.

[0008] The ECM is a network consisting of extracellular macromolecules and minerals, such as collagen, enzymes, glycoproteins and hydroxyapatite. Besides EVs, cells are source of many other molecules in the extracellular space (the impurities that are mentioned above). The cells produce and secrete proteins, lipids, RNA and DNA molecules either as organized biological processes or as a result of cellular death (cellular debris). All the molecules secreted by cells are collectively named "secretome". As stromal cells, MSCs are characterized by high levels of expression of ECM proteins that are the main component of the stroma and basement membranes. Many of the ECM proteins are large in size and undergo formidable post-translational modification to withstand the aggressive extracellular milieu.

[0009] Standard methods of EV isolation imply in vitro culturing of cells in a cell culture medium; collection of the medium which contains EVs and other molecules produced by the cells (thismedium is termed a "conditioned medium"); and purification of as clean a sample of the EVs as possible.

[0010] SUMMARY OF THE INVENTION

[0011] The inventors have surprisingly found that by keeping at least a part of the secretome, the therapeutic properties of the EVs may be augmented.

[0012] The present invention provides a composition wherein extracellular vesicles are mixed with components of the extracellular matrix (ECM).

[0013] It has now been surprisingly found that the removal of at least some of the ECM:s from the secretome render the EVs less stable during handling and storage. The inventors have shown that the presence of ECM:s slows down the clearing of the EVs from the site of injections and from the circulation system, and lessens the decrease in activity of clean EVs after freeze / thaw cycles and also decreases the loss of the EVs due to adhesion to surfaces of storage containers and medical devices such as syringes, vials, or tubing.

[0014] The present invention consequently provides a method for purifying extracellular vesicles from the secretome which have been obtained from a cell culture, such as mesenchymal stromal cells. It should be noted that the purifying method does not remove all components of the secretome.

[0015] The invention further provides a composition obtained from such a method of purification, and the use of said composition in the treatment of various diseases.

[0016] The present invention relates to the following itemized embodiments. The subject matter disclosed in the items below should be regarded disclosed in the same manner as if the subject matter were disclosed in patent claims.

[0017] 1. A composition comprising:

[0018] a. mesenchymal stromal cell (MSC) extracellular vesicles (EV) isolated from the secretome of MSCs; and

[0019] b. extracellular matrix (ECM) components,

[0020] wherein the composition comprises:

[0021] i. less than 3x10-9µg / EV particle of proteins derived from the secretome; andii. at least 0.5x10-11µg / EV particle of the ECM components.

[0022] 2. A composition comprising:

[0023] a. mesenchymal stromal cell (MSC) extracellular vesicles (EV); and

[0024] b. extracellular matrix (ECM),

[0025] wherein:

[0026] i. the ECM content is at least 0.5x10-10µg ECM / EV particle;

[0027] ii. the EV and the ECM are isolated from the secretome of MSCs; and iii. wherein the amount of at least one of albumin, thrombospondin-1, and thrombospondin-2 is at least 5 times lower per EV particle than in the secretome.

[0028] 3. The composition according to item 1 or 2, wherein the composition comprises at least 0.5x10-11µg / EV particle of at least one ECM component selected from collagen I, collagen II, collagen IV, laminin-521, laminin-421, laminin-111 and hyaluronic acid.

[0029] 4. The composition according to any of the preceding items, wherein the composition comprises less than 3x10-9µg / EV particle of ECM components derived from the secretome.

[0030] 5. The composition according to any of the preceding items, wherein the composition comprises less than 3x10-9µg / EV particle of ECM components.

[0031] 6. The composition according to any of the preceding items, wherein the composition comprises less than 3x10-9µg / EV particle of total protein.

[0032] 7. The composition according to any one of the preceding items, wherein the composition comprises less than 1x10-8µg / EV particle of glucosaminoglycans including but not limited to hyaluronic acid, heparan sulfate, heparin and keratan sulfate.

[0033] 8. The composition according to any one of the preceding items, wherein the composition comprises less than 1x10-8µg / EV particle of any one of: collagen I, collagen II, collagen IV, laminin-521, laminin-421, laminin-111, hyaluronic acid.9. The composition according to any one of the preceding items, wherein the composition comprises any one of collagen I, collagen II, collagen IV, laminin-521, laminin-421, laminin-111 and hyaluronic acid in an amount of less than 5x10-9µg / EV particle.

[0034] 10. The composition according to any one of the preceding items, wherein the composition comprises any one of collagen I, collagen II, collagen IV, laminin-521, laminin-421, laminin-111 and hyaluronic acid in an amount of less than 2x10-9µg / EV particle.

[0035] 11. The composition according to any one of the preceding items, wherein the composition comprises any one of collagen I, collagen II, collagen IV, laminin-521, laminin-421, laminin-111 and hyaluronic acid in an amount of less than 1x10-9µg / EV particle.

[0036] 12. The composition according to any one of the preceding items, wherein the composition comprises at least 1x109EVs / ml, or in an increasing order of preference, 1x1010EVs / ml, 1x1011EVs / ml, 5x1011EVs / ml.

[0037] 13. The composition according to any one of the preceding items, wherein the composition comprises at least 5x1010EVs / ml.

[0038] 14. The composition according to any one of the preceding items, wherein the EVs and / or the ECMs are isolated using ultracentrifugation, tangential flow filtering, anion exchange, gel filtration, precipitation, affinity isolation on magnetic beads or a resin, or a combination thereof.

[0039] 15. The composition according to any one of the preceding items, wherein the composition contains less than 0.1x10-10µg / EV particle of at least one of albumin, thrombospondin-1 or thrombospondin-2.

[0040] 16. The composition according to anyone of the preceding items, wherein at least 10 wt.% of the ECM is made up of: polysaccharides such as glycans, glycosaminoglycans, heparan sulfate or hyaluronic acid, and ECM proteins chosen from: collagens, laminins, fibronectin, elastin, nidogens, fibrillin, proteoglycans, and glycoproteins.The composition according to any one of the preceding items, wherein at least 1 wt.% of the ECM is made up of: collagens, laminins and hyaluronic acid.

[0041] The composition according to any one of the preceding items, wherein the ECM are chosen from the group of hyaluronic acid and proteins having at least 80% sequence identity to the proteins taken from the group consisting of: collagen chosen from the group of SEQ ID NO 1 to 30; laminin chosen from the group of SEQ ID NO 31 to 42; nidogen chosen from SEQ ID NO 43 or 44; fibrillin chosen from the group of SEQ ID NO 45, 46 and 47, elastin of SEQ ID NO 48, fibronectin of SEQ ID NO 49, Cartilage oligomeric matrix protein P49747 of SEQ ID NO 50, and Basement membrane-specific heparan sulfate proteoglycan core protein P98160 of SEQ ID NO 51.

[0042] The composition according to any one of the preceding items, wherein at least 1 wt.% of the ECM consists of one or more molecules taken from the group consisting of: Collagen I, laminin-521, laminin-421, and hyaluronic acid.

[0043] The composition according to any one of the preceding items, wherein the EVs carry at least one EV marker chosen from the group of CD63, CD81, and CD9, and the ECMs are chosen from the group consisting of collagen I, collagen IV, laminin-521, laminin-421, laminin-111, and hyaluronic acid.

[0044] The composition according to any one of the preceding items, wherein the ECM molecules are hydrophobic proteins.

[0045] The composition according to any one of the preceding items, wherein at least 1 wt.% of the ECM consists of hyaluronic acid.

[0046] A composition obtainable by the method according to any one of items 30-38.

[0047] The composition according to any one of the preceding items for use as a medicament. The composition for use according to item 24, for use in the treatment or prophylaxis of a medical condition selected from the group consisting of: ischemic and nonischemic heart failure including heart failure with preserved ejection fraction and heart failure with reduced ejection fraction; heart insufficiency; myocardial infarction; coronary artery disease; cardiomyopathy including hypertrophic, restrictive and dilated; Takotsubo syndrome; amyloidosis; aortic disorders including aneurysm,dissection, ulcers and hematoma; hypertension; congenital heart disease; myocarditis; valve dysfunction; acute respiratory distress syndrome (ARDS); critical illness myopathy (CIM); ventilator induced diaphragm muscle dysfunction (VIDD); graft-versus-host disease (GvHD); solid organ rejection; rejection of cell, organ or tissue transplants; preservation of organs and tissues intended for transplantations; inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis; rheumatoid diseases such as arthritis; inflammation-driven or immunologically induced diseases such as multiple sclerosis, ALS, sarcoidosis, idiopathic pulmonary fibrosis, psoriasis, dermatitis, or eczema; allergies such as allergies to food, animals, plants, medicines, chemicals, metals, or dust; autoimmune diseases such pemphigus, type 1 diabetes, systemic lupus erythematosus (SLE), multiple sclerosis (MS), or Guillain-Barre syndrome; diabetes type 2; tumor necrosis factor (TNF) receptor- associated periodic syndrome (TRAPS); deficiency of the interleukin-1 receptor antagonist (DIRA); endometriosis; autoimmune hepatitis; scleroderma; myositis; stroke; acute spinal cord injury; vasculitis; organ failure such as kidney failure, liver failure, lung failure, or heart failure; cancer including lung cancer and skin cancer; during operations with medical devices including but not limited to heart pumps, pace makers, mechanical and biological valves, stents, artificial vessels including artificial blood vessels, and others; scar formation and fibrosis of various kinds including but not limited scaring / fibrosis caused by diseases and medical operations / procedures; chronic inflammatory conditions of various origin including low grade inflammation; and burns including thermal and chemical burns.

[0048] 26. The composition for use according to item 24, for use in the treatment or prophylaxis of a cardiovascular or respiratory disease.

[0049] 27. The composition for use according to item 26, wherein the cardiovascular disease is ischemia reperfusion injury of the heart.

[0050] 28. The composition for use according to any of items 26-27, wherein the composition is administered to a patient in need via percutaneous coronary intervention (PCI) for implantation of a stent.

[0051] 29. A use of a composition according to any one of the preceding items, in a cosmetic method.A method for obtaining a composition comprising EVs and ECM from mesenchymal stromal cell (MSC) secretome, said method comprising the steps of:

[0052] a) fractionating a MSC secretome such that some fractions are enriched in EVs and other fractions enriched in ECM components;

[0053] b) analyzing the fractions for enrichment of at least one EV marker chosen from the group consisting of CD63, CD81, and CD9, and for enrichment of at least one ECM component, chosen from the group consisting of a collagen, collagen I, collagen II, collagen IV, a laminin, laminins containing laminin alpha 5 chain, nidogens, laminins containing laminin alpha 4 chain, elastin, fibronectin, proteoglycans, glycosaminoglycans, hyaluronic acid, glycoproteins, and heparan sulfate;

[0054] c) combining fractions enriched in EVs with fractions enriched in the ECM component into a composition comprising EVs and ECMs,

[0055] by selecting the fractions for the composition such that the obtained composition is depleted in at least one of albumin, thrombospondin-1 or thrombospondin-2, such that it contains 80% less of at least one of albumin, thrombospondin-1 or thrombospondin-2 per EV particle, compared to the secretome prior to fractionation.

[0056] The method according to item 30, wherein the EV marker chosen is CD63.

[0057] The method according to item 30 or 31, wherein the ECM component chosen is collagen I.

[0058] The method according to any one of items 30-32, wherein the obtained composition is depleted in at least two of: albumin, thrombospondin-1 and thrombospondin-2. The method according to any one of items 30-33, wherein the obtained composition is depleted in all three of: albumin, thrombospondin-1 and thrombospondin-2.

[0059] The method according to any one of items 30-34, wherein the fractionation is at least in part performed using a hydrophobic interaction chromatography resin and the elution of molecules from the resin is performed by a gradient of salts.36. The method according to any one of items 30-35, wherein the fractionation is at least in part performed using an ion exchange resin and the elution of molecules from the resin is performed by a gradient of salts.

[0060] 37. The method according to any one of items 30-36, wherein the fractionation is at least in part performed using a size exclusion chromatography.

[0061] 38. The method according to any one of items 30-37, wherein the fractionation is at least in part performed using a stationary phase being a reverse phase chromatography stationary phase and the elution of molecules from the stationary phase is performed by ionic liquids or solvents.

[0062] DEFINITIONS

[0063] " Extracellular vesicles or EVs" are lipid-membrane enclosed vesicles that are generated by vast majority of cells1. EVs contain proteins, nucleic acids and lipids and act as important intercellular communicators, which is facilitated by receptors on the membrane of EVs1'2. Term "extracellular vesicles" includes exosomes, microvesicles and apoptotic bodies that represent the major subtypes of EVs. In preferred embodiments, the term refers to exosomes only. Exosomes are produced inside the cells, released via the endosomal pathway and range between approximately 30 and 150 nm in diameter. Microvesicles are budding from cell plasma membrane and range between approximately 50 and 1000 nm. Apoptotic bodies are released during cell death, contain various parts of the cell and range between approximately 50 and 5000 nm. According to the present invention, the extracellular vesicles contain at least one marker chosen from CD63, CD81, and CD9. More specifically, and for the purposes of the present invention, the term EVs refers to a population of vesicles isolated from the secretome of mesenchymal stromal cells (MSCs). Preferably, the EVs in this population have a diameter in the range of 30-150 nm. Furthermore, for the purposes of determining the number of EV particles as recited in the claims, the concentration of EV particles is determined by Nanoparticle Tracking Analysis. A suitable method for this determination is performed using a NanoSight 3000 device (Malvern Panalytical, UK), as further described in the Examples herein.The term "multipotent stem cells" refers to the ability of such cells: (i) to give rise to one or more types of somatic cells (fully differentiated cells) and (ii) their significant proliferation potential. The term "multipotent progenitor cells" refers to multipotent cells that are direct predecessors of somatic cells. The term "multipotent" means the ability to differentiate into discrete cell types or only one cell type of somatic cells. However, unlike stem cells, progenitor cells have a limited proliferation potential.

[0064] The term " Mesenchymal stromal cells" or " MSCs" refers to cells that comply with the following definition: (1) expression of certain cell membrane markers CD73, CD90 and CD105; (2) lack of expression of CDllb, CD14, CD34, CD45, CD19, CD79a and HLA-DR; (3) plastic adherence; and (4) trilineage multipotency (ability to differentiate into osteoblasts, chondrocytes and adipocytes) in in vitro and in vivo tests9. MSCs can be obtained from many, if not all, tissues and organs of the body such as bone marrow, Wharton's jelly, fat tissue, oral cavity, the heart and teeth10. Alternatively, the MSCs can be differentiated from stem cells or transdifferentiated from other types of cells. For instance, MSCs can be differentiated from human pluripotent cells.

[0065] The term "biologically active" or "active" EVs refers to their ability to significantly improve the function of organs and tissues in in vivo models of diseases, particularly inflammatory diseases.

[0066] The term "conditioned medium" refers to cell culture medium that has been in contact with cells and contains factors produced by the cells.

[0067] The term "secretome" refers to the the entire set of molecules— including proteins, growth factors, cytokines, enzymes, and extracellular vesicles (like exosomes)— that are secreted by a cell, tissue, or organism into the extracellular space. Conditioned medium contains the secretome of the cultured cells, but may also contain additional components already present in the medium prior to culture, or components added during culture. The present invention relates in particular to the secretome of MSCs.

[0068] The term "extracellular matrix" or " ECM" refers to macromolecules that are produced in the cells and transported into extracellular space either via specific mechanisms or after cellular death. The ECM macromolecules can be found in the extracellular space. The ECM consists of proteins, glycoproteins, proteoglycans, polysaccharides, glycosaminoglycans and the like thatreside in the extracellular space. More specifically, and for the purposes of the present invention, the term ECM refers either to the heterogeneous mixture of macromolecules isolated from the secretome of mesenchymal stromal cells (MSCs) or to isolated macromolecules that normally reside in the secretome of MSCs. This mixture and the isolated macromolecules include, but is not limited to, proteins and polysaccharides such as collagens, laminins, fibronectin, elastin, nidogens, fibrillin, proteoglycans, and glycosaminoglycans. Matrisome DB (MatrisomeDB: 2023 updates of the ECM protein knowledge database. Shao X, Gomez CD, Kapoor N, Considine JM, Gao Y, Naba A. Nucleic Acids Research, 2022, gkac1009) is a reference online database containing detailed information on ECM molecules.

[0069] For the purposes of quantifying the ECM content according to the present invention, the mass of ECM in micrograms (μg) may be determined as the total protein concentration of the composition (i.e. considering any protein within the EVs negligible). If applicable, the amount any non-ECM proteins added to the composition may be subtracted. A suitable and preferred method for this determination is the Bicinchoninic Acid (BCA) Protein Assay. This assay can be performed using a commercial kit, such as the Pierce™ BCA Protein Assay Kits (ThermoFisher Scientific), according to the manufacturer's instructions, as is well understood by a person skilled in the art and as further described in the Examples herein. If desired, individual ECM components may be quantified using standard methods, e.g. immunochemical methods such as ELISA, or chromatographic methods such as quantitative LC-MS.

[0070] The term "motif" refers to any part of a protein or a large molecule.

[0071] Matrigel or EHS-matrix refer synonymously to a soluble mixture of ECM proteins that contains approximately 40% of Collagen IV and 60% of laminin-111, derived from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma. It is available commercially from Corning (Matrigel™).

[0072] Sequence identity expressed in percentage is defined as the value determined by comparing two optimally aligned sequences over a comparison window, wherein a portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yieldthe percentage of sequence identity. Unless indicated otherwise, the comparison window is the entire length of the sequence being referred to. In this context, optimal alignment is the alignment produced by the BLASTP algorithm as implemented online by the US National Center for Biotechnology Information (see The NCBI Handbook [Internet], Chapter 16), with the following input parameters: Word length=3, Matrix=BLOSUM62, Gap cost=11, Gap extension cost=1.

[0073] A reference to a product or method "comprising" certain features should be interpreted as meaning that it includes those features, but that it does not exclude the presence of other features, as long as they do not render the invention unworkable. In reference to the compounds or compositions according to the invention, the term "consisting essentially of" means that specific further components can be present, namely those not materially affecting the essential characteristics of the compound or composition.

[0074] DETAILED DESCRIPTION

[0075] Any chromatography method that separates molecules according to differences in their surface hydrophobicity and / or involves fractionation of the eluate can be used to simultaneously isolate EVs and ECM for example, but not limited to, hydrophobic interaction chromatography (HIC) and reversed phase chromatography (RPC). The purification of the EVs and the hydrophobic part of the secretome may also be performed by any chromatographical method that involves fractionation of the eluate for example, but not limited to, gel filtration and ion exchange chromatography, and the various fractions may then be combined into one composition. A successful isolation should preferably decrease the amount of Albumin, Thrombospondin-1 and / or Thrombospondin-2 per one EV at least five times in the final preparation compared to the initial amount in the conditioned medium. The amount of proteins can be quantitatively compared using Western bloting or mass-spectrometry based proteomics.

[0076] Compositions

[0077] It is an object of the present invention to overcome the above-mentioned problems and provide a biologically active and stable composition of EVs for use in medicine.It has been surprisingly found that a composition comprising extracellular vesicles (EVs) and at least a fraction of the extracellular matrix (ECM) overcomes the above-mentioned problems. The compositions according to the invention are stable and maintain their capacity of being biologically active upon storage, and the adherence by EVs to various surfaces is mitigated.

[0078] Thus, in one aspect, the invention provides a composition comprising mesenchymal stromal cell (MSC) extracellular vesicles (EV); and extracellular matrix (collectively ECM).

[0079] In one embodiment, the composition comprises or consists of extracellular vesicles (EV), isolated from mesenchymal stromal cells; and extracellular matrix molecules (ECM).

[0080] In a further embodiment, the composition comprises or consists of EVs and / or ECM isolated using ultracentrifugation, tangential flow filtering, anion exchange, gel filtration, precipitation, affinity isolation on magnetic beads or a resin, or a combination thereof.

[0081] Thus, in a first aspect, the present invention provides a composition comprising:

[0082] a. mesenchymal stromal cell (MSC) extracellular vesicles (EV); and

[0083] b. extracellular matrix (ECM),

[0084] wherein the ECM content is at least 0.5x10-10μg ECM / EV particle (preferably at least 1x10-10μg), wherein the EV and the ECM are isolated from the secretome of MSCs, wherein the amount of at least one of albumin, thrombospondin-1, and thrombospondin-2 in the composition is at least 5 times lower per EV particle (or, in increasing order of preference, at least 6, 7, 8, 9, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times lower per EV particle) than in the secretome, from which the composition was isolated.

[0085] The first aspect may also refer to a composition comprising:

[0086] a. mesenchymal stromal cell (MSC) extracellular vesicles (EV) isolated from the secretome of MSCs; and

[0087] b. extracellular matrix (ECM) components, optionally isolated from the secretome of MSCs,

[0088] wherein the composition comprises:i. less than 5x10-9μg / EV particle (preferably less than 3x10-9μg / EV, more preferably less than 2x10-9μg / EV) of proteins derived from the secretome; and

[0089] ii. at least 0.5x10-11µg / EV particle of the ECM components.

[0090] In certain embodiments, the composition comprises at least 0.5x10-11µg / EV particle of at least one ECM component selected from collagen I, collagen II, collagen IV, laminin-521, laminin-421, laminin-111 and hyaluronic acid.

[0091] In certain embodiments, the composition comprises less than 2x10-9μg / EV particle (preferably less than 1x10-9μg / EV, more preferably less than 0.5x10-9μg / EV) of ECM components derived from the secretome.

[0092] In certain embodiments, the composition comprises less than 2x10-9μg / EV particle (preferably less than 1x10-9μg / EV, more preferably less than 0.5x10-9μg / EV) of ECM components.

[0093] In certain embodiments, the composition comprises less than 2x10-9μg / EV particle (preferably less than 1x10-9μg / EV, more preferably less than 0.5x10-9μg / EV) of total protein.

[0094] In certain embodiments, the composition comprises less than 1x10-8μg / EV particle of hyaluronic acid.

[0095] In certain embodiments, the composition comprises less than 1x10-8μg / EV particle of glucosaminoglycans including but not limited to hyaluronic acid, heparan sulfate, heparin, and keratan sulfate.

[0096] In certain embodiments, the composition contains less than 1x10-12μg of albumin, thrombospondin-1, and / or thrombospondin-2, per EV particle, or in increasing order of preference, less than 1x10-11μg, 5x10-11μg, 1x10-10μg, 5x10-10μg or 1x10-9μg.

[0097] In certain embodiments, said amount refers to the combined amount of albumin and thrombospondin-1. In certain embodiments, said amount refers to the combined amount of albumin, and thrombospondin-2. In certain embodiments, said amount refers to the combined amount of thrombospondin-2 and thrombospondin-2. In certain embodiments,said amount refers to the combined amount of albumin, thrombospondin-1 and thrombospondin-2.

[0098] In certain embodiments, the composition contains less than 1x10-12μg of at least one of albumin, thrombospondin-1, and thrombospondin-2, per EV particle, or in increasing order of preference, less than 1x10-11μg, 5x10-11μg, 1x10-10μg, 5x10-10μg or 1x10-9μg. In certain embodiments, the composition contains less than 1x10-12μg of any one of albumin, thrombospondin-1, and thrombospondin-2, per EV particle, or in increasing order of preference, less than 1x10-11μg, 5x10-11μg, 1x10-10μg, 5x10-10μg or 1x10-9μg.

[0099] In certain embodiments, the composition comprises anyone of: (i) a collagen such as collagen I, collagen II or collagen IV; (ii) a laminin such as laminin-521, laminin-421 or laminin-111; and (iii) hyaluronic acid in an amount of less than lxlO-8pg / EV particle or in an increasing order of preference, less than 5xl0-9pg / EV particle, less than 2xl0-9pg / EV particle or less than 1x10’9pg / EV particle.

[0100] In certain embodiments, the composition comprises any one of: collagen I, collagen IV, laminin-521, laminin-421, laminin-111 and hyaluronic acid in an amount of less than 1x10-8μg / EV particle or in an increasing order of preference, less than 5x10-9μg / EV particle, less than 2x10-9μg / EV particle or less than 1x10-9μg / EV particle.

[0101] In certain embodiments, the composition comprises at least 1x109EVs / ml, or in an increasing order of preference, 1x1010EVs / ml, 1x1011EVs / ml or 5x1011EVs / ml.

[0102] According to the invention, a pharmaceutical composition may comprise various pharmaceutically acceptable constituents, such as solvents, buffers, carriers, stabilizers, preservatives, etc. The term "pharmaceutically acceptable" means being useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes being useful for veterinary use as well as human pharmaceutical use.

[0103] In one embodiment the composition contains less than 20 weight % of albumin, thrombospondin-1, and / or thrombospondin-2 per one EV particle, compared to the amount of albumin, thrombospondin-1, and / or thrombospondin-2 per one EV particle in the secretome prior to isolation / purification. In certain embodiments, the weight percentage ofalbumin, thrombospondin-1, and / or thrombospondin-2 per EV particle is less than 18%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%, in increasing order of preference.

[0104] In a preferred embodiment, the composition contains at least one ECM chosen from the group consisting of proteins and / or polysaccharides.

[0105] In one embodiment, the invention provides a composition according to the above, wherein at least 10 wt.% (or in an increasing order of preference, at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 95, 98, 99 or 100 wt.%) of the ECM is made up of the types of molecules chosen from: polysaccharides such as glycans, glycosaminoglycans, heparan sulfate or hyaluronic acid, and ECM proteins chosen from: collagens, laminins, fibronectin, elastin, nidogens, fibrillin, proteoglycans, and glycoproteins.

[0106] In one embodiment, the invention provides a composition according to the above, wherein at least 1 wt.% (or in an increasing order of preference, at least 2, 3, 4, 5, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 85, 90, 95, 98, 99 or 100 wt.%) of the ECM consists of: collagens, laminins, nidogens, fibrillin, proteoglycans, glycosaminoglycans, glycoproteins, polysaccharides, heparan sulfate, hyaluronic acid, fibronectin, and elastin, preferably of: collagens, laminins and hyaluronic acid.

[0107] The amino acid sequences of these are shown in Table 1. The ECM proteins may be native proteins or recombinant proteins.

[0108] In one embodiment the ECMs are chosen from the group of hyaluronic acid and proteins having at least 80% sequence identity (or in an increasing order of preference, at least 90, 95, 98, 99 or 100%) to the proteins taken from the group consisting of: collagen chosen from the group of SEQ ID NO 1 to 30; laminin chosen from the group of SEQ ID NO 31 to 42; nidogen chosen from SEQ ID NO 43 or 44; fibrillin chosen from the group of SEQ ID NO 45, 46 and 47, elastin of SEQ ID NO 48, fibronectin of SEQ ID NO 49, Cartilage oligomeric matrix protein P49747 of SEQ ID NO 50, and Basement membrane-specific heparan sulfate proteoglycan core protein P98160 of SEQ ID NO 51.

[0109] Table 1 Sequence Listing

[0110] Collagen alpha-1(I) chain P02452 SEQ ID NO 1

[0111] Collagen alpha-2(I) chain P08123 SEQ ID NO 2Collagen alpha-1(II) chain P02458 SEQ ID NO 3 Collagen alpha-l(VII) chain Q02388 SEQ ID NO 4 Collagen alpha-l(XIX) chain Q14993 SEQ ID NO 5 Collagen alpha-l(VIII) chain P27658 SEQ ID NO 6 Collagen alpha-l(XX) chain Q9P218 SEQ ID NO 7 Collagen alpha-l(XXIV) chain Q17RW2 SEQ ID NO 8 Collagen alpha-l(XXII) chain Q8NFW1 SEQ ID NO 9 Collagen alpha-l(X) chain Q03692 SEQ ID NO 10 Collagen alpha-l(XXVII) chain Q8IZC6 SEQ ID NO 11 Collagen alpha-l(XIII) chain Q5TAT6 SEQ ID NO 12 Collagen alpha-l(XXVIII) chain Q2UY09 SEQ ID NO 13 Collagen alpha-l(XI) chain P12107 SEQ ID NO 14 Collagen alpha-l(XII) chain Q99715 SEQ ID NO 15 Collagen alpha-l(XVI) chain Q07092 SEQ ID NO 16 Collagen alpha-1(III) chain P02461 SEQ ID NO 17 Collagen alpha-1(V) chain P20908 SEQ ID NO 18 Collagen alpha-1(IX) chain P20849 SEQ ID NO 19 Collagen alpha-1(VI) chain P12109 SEQ ID NO 20 Collagen alpha-2(VI) chain P12110 SEQ ID NO 21 Collagen alpha-3(VI) chain P12111 SEQ ID NO 22 Collagen alpha-1(XXI) chain Q96P44 SEQ ID NO 23 Collagen alpha-1(XIV) chain Q05707 SEQ ID NO 24 Collagen alpha-1(XXVI) chain Q96A83 SEQ ID NO 25 Collagen alpha-1(IV) chain P02462 SEQ ID NO 26 Collagen alpha-2(IV) chain P08572 SEQ ID NO 27Collagen alpha-4(IV) chain P53420 SEQ ID NO 28 Collagen alpha-1(XXV) chain Q9BXS0 SEQ ID NO 29 Collagen alpha-1(XVIII) chain P39060 SEQ ID NO 30 Laminin subunit gamma-1 P11047 SEQ ID NO 31 Laminin subunit beta-1 P07942 SEQ ID NO 32 Laminin subunit alpha-2 P24043 SEQ ID NO 33 Laminin subunit alpha-1 P25391 SEQ ID NO 34 Laminin subunit beta-2 P55268 SEQ ID NO 35 Laminin subunit gamma-3 Q9Y6N6 SEQ ID NO 36 Laminin subunit alpha-4 Q16363 SEQ ID NO 37 Laminin subunit alpha-5 015230 SEQ ID NO 38 Laminin subunit beta-4 A4D0S4 SEQ ID NO 39 Laminin subunit alpha-3 Q16787 SEQ ID NO 40 Laminin subunit beta-3 Q13751 SEQ ID NO 41 Laminin subunit gamma-2 Q13753 SEQ ID NO 42 Nidogen-1 P14543 SEQ ID NO 43 Nidogen-2 Q14112 SEQ ID NO 44 Fibrillin-1 P35555 SEQ ID NO 45 Fibrillin-2 P35556 SEQ ID NO 46 Fibrillin-3 Q75N90 SEQ ID NO 47 Elastin P15502 SEQ ID NO 48 Fibronectin P02751 SEQ ID NO 49 Cartilage oligomeric matrix

[0112] protein P49747 SEQ ID NO 50 Basement membrane-specificheparan sulfate proteoglycan core

[0113] protein P98160 SEQ ID NO 51

[0114] In certain embodiments, at least 1 wt.% (or in an increasing order of preference, at least 2, 3, 4, 5, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 95, 98, 99 or 100 wt.%) of the ECM consists of one or more types of molecules taken from the group consisting of: Collagen I, laminin-521, laminin-421, and hyaluronic acid.

[0115] In certain embodiments, the composition of the first aspect is obtained via direct mixing isolated MSC EVs and purified MSC ECM molecules according to the above.

[0116] In certain embodiments, the EVs carry at least one EV marker chosen from the group of CD63, CD81, and CD9, and the ECMs are chosen from the group consisting of collagen I, collagen IV, laminin-521, laminin-421, laminin-111, and hyaluronic acid.

[0117] In certain embodiments the ECM molecules are hydrophobic proteins.

[0118] In one embodiment, in a composition according to the above, at least 1 wt.% of the ECM consists of hyaluronic acid or a synthetic molecule consisting of several linked hyaluronic acid molecules or a synthetic molecule that includes motifs with 80% similarity to hyaluronic acid. In one embodiment, the composition contains EVs and Matrigel, collagen I (Col I), laminin-521 (LN521), laminin-421 (LN421) and hyaluronic acid (Hyal).

[0119] In a preferred embodiment, the ECM in the composition is one or more molecules taken from the group consisting of: Collagen I, laminin-521, laminin-421, and hyaluronic acid.

[0120] In one aspect, the composition is obtained via direct mixing purified EVs with the ECM molecules listed above. Preferably, the amount of albumin, thrombospondin-1, and / or thrombospondin-2 per one EV particle, is less than 20% (or in increasing order of preference less than 18%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%) compared to the amount of albumin, thrombospondin-1, and / or thrombospondin-2 per one EV particle in the secretome. The amount of these proteins in the secretome can be determined by standard methods such as ELISA or Western blotting.

[0121] In one embodiment, composition contains EVs and ECM, wherein the EVs carry at least one EV marker chosen from the group of CD63, CD81, and CD9, and the ECMs are chosen from thegroup consisting of collagen I, collagen IV, laminin-521, laminin-421, laminin-111, and hyaluronic acid.

[0122] In a preferred embodiment, at least one of the ECM molecules is hyaluronic acid.

[0123] In certain embodiments, the composition of the first aspect is obtained or obtainable by the method of the fourth aspect.

[0124] Medical and non-medical uses

[0125] In the second aspect, the invention provides the composition according to the first or the fourth aspect for use in medicine or as a medicament.

[0126] In a third aspect, the invention provides the composition according to the first aspect for use in the treatment or prophylaxis of a medical condition selected from the group consisting of: ischemic and non-ischemic heart failure including heart failure with preserved ejection fraction and heart failure with reduced ejection fraction; heart insufficiency; myocardial infarction; coronary artery disease; cardiomyopathy including hypertrophic, restrictive and dilated; Takotsubo syndrome; amyloidosis; aortic disorders including aneurysm, dissection, ulcers and hematoma; hypertension; congenital heart disease; myocarditis; valve dysfunction; acute respiratory distress syndrome (ARDS); critical illness myopathy (CIM); ventilator induced diaphragm muscle dysfunction (VIDD); graft-versus-host disease (GvHD); solid organ rejection; rejection of cell, organ or tissue transplants; preservation of organs and tissues intended for transplantations; inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis; rheumatoid diseases such as arthritis; inflammation-driven or immunologically induced diseases such as multiple sclerosis, ALS, sarcoidosis, idiopathic pulmonary fibrosis, psoriasis, dermatitis, or eczema; allergies such as allergies to food, animals, plants, medicines, chemicals, metals, ordust; autoimmune diseases such pemphigus, type 1 diabetes, systemic lupus erythematosus (SLE), multiple sclerosis (MS), or Guillain-Barre syndrome; diabetes type 2; tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS); deficiency of the interleukin-1 receptor antagonist (DIRA); endometriosis; autoimmune hepatitis; scleroderma; myositis; stroke; acute spinal cord injury; vasculitis; organ failure such as kidney failure, liver failure, lung failure, or heart failure; cancer including lung cancer and skin cancer; during operations with medical devices including but not limited to heart pumps, pace makers, mechanical and biological valves, stents, artificial vessels including artificial blood vessels, and others; scar formation and fibrosis of various kindsincluding but not limited scaring / fibrosis caused by diseases, aging and medical operations / procedures; chronic inflammatory conditions of various origin including low grade inflammation; and burns including thermal and chemical burns.

[0127] In a preferred embodiment, the invention provides the composition according to the first or the fourth aspect for use in the treatment or prophylaxis of a cardiovascular or respiratory disease.

[0128] In a preferred embodiment, the invention provides the composition according to the first or the fourth aspect for use in the treatment or prophylaxis of ischemia reperfusion injury of the heart.

[0129] In a preferred embodiment, the invention provides the composition according to the first or the fourth aspect, for use in plastic surgery and cosmetics. Also provided is a use of a composition according to the first or the fourth aspect, in a cosmetic method, in a cosmetic preparation and / or for cosmetic purposes. For avoidance of any doubt, cosmetic uses are non-therapeutic. They relate to uses aimed at achieving an aesthetic, rather than therapeutic, effect. The cosmetic uses include but are not limited to skin rejuvenation and improving the aesthetic appearance of skin.

[0130] Needless to say, in the medical and non-medical uses detailed above, the compositions of the present invention may diluted, co-administered or mixed with pharmaceutically acceptable vehicles, pharmaceutically acceptable excipients or additional pharmaceutically acceptable substances or compositions.

[0131] The disclosures of the composition "for use" in a therapeutic purpose, method or application also disclose a corresponding method of treatment and a corresponding use in the manufacture of a medicament for the treatment, for the purposes disclosed herein.

[0132] Isolation methods and obtainable compositions

[0133] In a fourth aspect, the invention provides a method for obtaining a composition comprising EVs and ECMs, wherein both the EVs and the ECM are isolated from the secretome of stromal cells, preferably the secretome of mesenchymal stromal cells (MSCs).In certain embodiments of the fourth aspect, the invention provides a method for obtaining a composition comprising EVs and ECM, said method comprising the steps of:

[0134] a) fractionating a MSC secretome such that some fractions are enriched in EVs and other fractions enriched in ECM components;

[0135] b) analyzing the fractions for enrichment of at least one EV marker chosen from the group consisting of CD63, CD81, and CD9, and for enrichment of at least one ECM component, chosen from the group consisting of a collagen, collagen I, collagen II, collagen IV, a laminin, laminins containing laminin alpha 5 chain, nidogens, laminins containing laminin alpha 4 chain, elastin, fibronectin, proteoglycans, glycosaminoglycans, hyaluronic acid, glycoproteins, and heparan sulfate; c) combining fractions enriched in EVs with fractions enriched in the ECM component into a composition comprising EVs and ECMs,

[0136] by selecting the fractions for the composition such that the obtained composition is depleted in at least one of albumin, thrombospondin-1, or thrombospondin-2, such that it contains 80% less (or in an increasing order of preference 85%, 90%, 95%, 98%, 99% or 100% less) of at least one of albumin, thrombospondin-1, orthrombospondin- 2 per EV particle, compared to the secretome prior to fractionation.

[0137] In certain embodiments of the fourth aspect, the invention provides a method for obtaining a composition comprising EVs and ECMs, said method comprising the steps of;

[0138] a) fractionating a MSC secretome such that some fractions are enriched in EVs and other fractions enriched in ECMs;

[0139] b) eluting molecules from the resin or stationary phase in fractions;

[0140] c) analyzing the fractions for enrichment of at least one EV marker chosen from the group consisting of CD63, CD81, and CD9, and at least one ECM molecule, chosen from the group consisting of collagen, collagen I, collagen II, collagen IV, laminins, laminins containing laminin alpha 5 chain, nidogens, laminins containing laminin alpha 4 chain, elastin, fibronectin, proteoglycans, glycosaminoglycans, hyaluronic acid, glycoproteins, and heparan sulfate, respectively;d) combining fractions containing EVs with fractions containing the at least one ECM molecule into a composition comprising EVs and ECMs,

[0141] by selecting the fractions for the combination such that the obtained composition is depleted in albumin, thrombospondin-1, and / or thrombospondin-2, such that it contains 80% less (or in an increasing order of preference 85%, 90%, 95%, 98%, 99% or 100% less) of albumin, thrombospondin-1, and / or thrombospondin-2 per EV particle, compared to the amount of albumin, thrombospondin-1, and / or thrombospondin-2 per EV particle in the secretome prior to fractionation.

[0142] The EV-marker analyzed for enrichment is preferably CD63. The ECM component / molecule analyzed for enrichment is preferably collagen I. Most preferably, the EV marker is CD63 and the ECM component is collagen I.

[0143] In certain embodiments, the obtained composition is depleted in at least two of: albumin, thrombospondin-1 and thrombospondin-2.

[0144] In certain embodiments, the obtained composition is depleted in albumin and thrombospondin-1.

[0145] In certain embodiments, the obtained composition is depleted in albumin and thrombospondin-2.

[0146] In certain embodiments, the obtained composition is depleted in thrombospondin-land thrombospondin-2.

[0147] In certain embodiments, the obtained composition is depleted in all three of: albumin, thrombospondin-1 and thrombospondin-2.

[0148] Ina preferred embodiment, the fractionation is at least in part performed using a hydrophobic interaction chromatography resin and the elution of molecules from the resin is performed by a gradient of salts.

[0149] In another embodiment is the fractionation at least in part performed using an ion exchange resin and the elution of molecules from the resin is performed by a gradient of salts.

[0150] In another embodiment is the fractionation at least in part performed using a size exclusion chromatography.In another embodiment is the fractionation at least in part performed using a stationary phase being a reverse phase chromatography stationary phase and the elution of molecules from the stationary phase is performed by ionic liquids or solvents.

[0151] In a preferred embodiment, the resin is a hydrophobic interaction chromatography resin and the elution of molecules from the resin is performed by a gradient of salts.

[0152] In a preferred embodiment the resin is an ion exchange resin and the elution of molecules from the resin is performed by a gradient of salts.

[0153] In a preferred embodiment the resin is a size exclusion resin and the elution of molecules from the resin is performed by application of the solvent.

[0154] The stationary phase may be a reverse phase chromatography stationary phase and the elution of molecules from the stationary phase is then performed by ionic liquids or solvents of salts.

[0155] The invention also provides a composition comprising EVs and ECMs which is obtainable or obtained by the method according to the fourth aspect.

[0156] BRIEF DESCRIPTION OF THE DRAWINGS

[0157] Figure 1. Retention of EVs after local injection into the heart. A) Comparison of the retention of the clean EVs with that of the EVs mixed with Matrigel, Collagen I (Col I), laminin-521 (LN521), laminin-421 (LN421), and hyaluronic acid (hyal). B) Comparison of the retention of the clean EVs with that of the EVs developed under the new method allowing simultaneous purification of the EVs and ECM (hydroph).

[0158] Figure 2. Expression of pro-inflammatory IL-6 in LPS-stimulated macrophages after treatment with clean EVs and various mixes of clean EVs with ECM molecules. A) Comparison of the expression without any treatment (No treat); after treatment with Matrigel without the EVs (Matrigel); after treatment with the clean EVs; and after treatment with the mixes of ECM molecules with the EVs (abbreviations as in the legends for Figure 1). B) Comparison of the expression after treatment with the clean EVs and the EVs developed under the new method allowing simultaneous purification of the EVs and ECM (hydroph).Figure 3. Loss of EVs on the walls of the storage containers. Bars demonstrate the relative percentage of the EVs recovered from the tubes after one hour incubation in relation to the initial number of EVs. A) Comparison of the losses of the clean EVs with that of the EVs mixed with Matrigel, Collagen I (Col I), laminin-521 (LN521), laminin-421 (LN421), and hyaluronic acid (hyal). B) Comparison of the losses of the clean EVs with that of the EVs developed under the new method allowing simultaneous purification of the EVs and ECM (hydroph).

[0159] Figure 4. Expression of pro-inflammatory IL-6 in LPS-stimulated macrophages after treatment with clean EVs, secretome and various mixes of clean EVs with ECM molecules. Comparison of the expression without any treatment (No treat); after treatment with LN521 alone without EVs (LN521); after treatment with isolated EVs containing reduced amount of ECM (EV, clean); after treatment with non-fractionated secretome containing EVs and ECMs (secretome); after treatment with the mixes of clean EVs with LN521 (EV, LN521); and the EVs combined with ECM purified from the secretome using hydrophobic chromatography (EV, hydroph) (abbreviations as in Figure 1).

[0160] EXAMPLES

[0161] Example 1: Mesenchymal stromal cell (MSC) culture

[0162] MSCs were cultured on standard cell-culture treated plates in low glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% of foetal bovine serum (FBS). All cultures were performed in humidified incubators at 37 °C, 5% CO2.

[0163] For passaging, the cells were washed twice with PBS and exposed to TrypLE Express enzyme for 8 minutes at 37 °C, 5% CO2. After that, the cells were gently detached from the surface using a scraper, pipetted to dissociate into single-cell suspension; and three volumes of the complete medium was added. Then, the cells were centrifuged at 280 g for 5 minutes, resuspended in the complete medium and replated at the concentration of 7xl03cells / cm2.

[0164] Example 2: Production of EVs

[0165] For the production of EVs, BM-MSCs were cultured until approximately 80 % confluency and carefully washed twice with PBS. After that, serum-free Opti-MEM™ medium (GIBCO, Carlsbad, CA, USA) was added and the cells were incubated for 48 hours at 37 °C, 5% CO2. Then, the conditioned medium was collected and centrifuged for 5 minutes at 300 g to remove living cells and, after that, for 100 minutes at 250 g to remove cellular debris.Example 3: Isolation of EVs

[0166] The centrifuged conditioned medium was sterile filtered and concentrated using TFF, KR2i TFF system (SpectrumLabs). Forthat, a modified polyethersulfone (mPES) hollow fiber filter with 300 kDa membrane pore size was used (MidiKros, 370 cm2surface area, SpectrumLabs). The flow rate was set to 100 ml / min. At first, the conditioned medium was concentrated to a volume of 75 ml in the container (100 ml total volume in the system) and then dialyzed with 1 liter of sterile filtered PBS. After the dialysis, the sample was concentrated to 30 ml and sterilized using a 0,22 μm filter. Thus, we obtained EV-enriched secretome (also referred here as secretome). After that, the sample was further purified by size exclusion chromatography column (iZON biosciences) according to the manufacturer's instruction to obtain clean EVs. To do that, the sample was passed through the column and fractionated. After that, the fractions were analyzed for the EV content using a West Blot analysis for at least one EV marker chosen from the group consisting of CD63, CD81, and CD9. The fractions containing EVs were pooled. Thus, we obtained clean EV.

[0167] Example 4: Determination of protein concentrations

[0168] The protein concentrations were determined using Pierce™ BCA Protein Assay Kits (ThermoFischer Scientific) according to the manufacturer's instructions.

[0169] Example 5: Nanoparticle tracking analysis

[0170] Concentration of particles (also referred here as concentration of EVs) and estimation of their size distribution was done using a nanoparticle tracking measurement such as NanoSight NS300 (Malvern Panalytical, UK). The instrument was equipped with a 488 nm laser and the software version NTA3.4. To obtain the mean value of size and the concentration, five 30-second videos were recorded. Movies were recorded in light scatter mode with camera level 13, and the software settings for the analysis were set for the screen gain to 2.5 and for the detection threshold to 2. The analyses were performed in triplicates. Determination of the protein and EV concentrations allowed us to further characterize the EV-enriched secretome and the clean EVs by calculation of protein amount per one EV. Only preparations of clean EVs with the concentration of proteins per EV below 3x10-9μg / EV were used in further experiments. Any preparations of clean EVs with higher concentrations of proteins per EVwere additionally purified using the size exclusion chromatography column as described above.

[0171] Example 6: Western Blotting

[0172] The samples were mixed with Laemmli Sample Buffer (Bio-Rad) under reducing conditions and warmed at 95° for 10 min. Then, 4-12% gradient gels were used for SDS electrophoresis and the proteins were transferred to polyvinylidene fluoride membranes using iBIot 2 (Invitrogen) according to the manufacturer's instructions. The membranes were first hybridized with the antibody of interest and then with the corresponding HRP-conjugated secondary antibodies. For visualization, chemoluminescent HRP-substrate from Amersham Biosciences was used and the figures were made using ChemiDoc imaging system (Bio-Rad).

[0173] Example 7: Transmission electron microscopy

[0174] A 5pl drop of the sample was placed on a formvar and carbon coated 200-mesh copper grid. The excess solution was removed by bloting with filter paper. The sample was then directly contrasted with 2% uranyl acetate. Excess of uranyl acetate was removed by bloting on filter paper. The contrasting step was repeated twice.

[0175] Dried grids were examined by Tecnai™ G2 Spirit BioTwin transmission electron microscope (Thermo Fisher / FEI) at 80 kV with an ORIUS SC200 CCD camera and Gatan Digital Micrograph software (both from Gatan Inc.).

[0176] Example 8: In vitro immunomodulation assay

[0177] The in vitro immunomodulation assay was based on macrophage cell line RAW 264.7. The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% of foetal bovine serum (FBS) at 37°C and 5% CO2 in a humidified incubator. RAW 264.7 cells were cultured on a 24-well plate until 80% confluency. After that, the medium was changed to DMEM without FBS and the cells were simultaneously stimulated with Lipopolysaccharide (LPS, 0.5 p.g / ml) to produce proinflammatory cytokines and treated with different EV preparations (with and without ECM). All the EV samples contained 3xl08EVs per well. After 18 hours of incubation, the supernatant was collected and centrifuged at 500 g for 10 minutes followed by 3000 g for 20 minutes to remove residual cells and cellular debris, respectively. Then, the supernatant was analyzed for expression of inflammation markers interleukin-6 (II-6) using the Enzyme-Linked Immunosorbent Assay (ELISA) kit (R& D Systems, Minneapolis, MN) according to the manufacturer's instructions.

[0178] Example 9: Development and isolation of Luciferase labelled EVs (Luc-EVs)

[0179] MSCs were grown until 80% confluency in 15 cm cell culture treated dishes. A total of 30 pg of pLEX-CD63-Nanoluc plasmid5were mixed with Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions and distributed on the 15 cm dish in dropwise manner. After approximately 16 hours, the medium was changed to Opti-MEM™ and the EVs were produced and isolated as described above.

[0180] Example 10: Mouse experiments

[0181] All the animal experiments described here were approved by the Uppsala Animal Ethics Committee, diary number 5.8.18-1434.2023. For EV in vivo pharmacokinetic studies, female NMRI mice were sedated with isoflurane, the heart was exposed and the Luc-EVs were injected intramyocardially (into the apex) with 2xl09Luc-EVs in 20 pL of PBS in four spots (approximately 5 pL per each spot). To collect the samples, the mice were sacrificed at different timepoint, the hearts were isolated and were immediately frozen at -80°C.

[0182] For the luciferase measurement, the hearts were lysed using Qiagen Tissue Lyser II according to the manufacturer's instructions. The tissue lysates were mixed with Nano-Gio substrate (Nano-Glow Luciferase Assay, Promega) and the luciferase intensity was measured using a Luminometer machine (Promega).

[0183] Example 11: ECM molecules

[0184] Human laminin-521 and laminin-421 were purchased from BioLamina AB, Sweden. Matrigel and human Collagen I were purchased from Corning. Hyaluronic Acid was purchased from Biorbyt.

[0185] Example 12: Retention of EVs with ECM after intramyocardial injection

[0186] To study the effects of extracellular matrix (ECM) molecules on EVs, we prepared combinations ofclean EV (protein content 1.87xl0-9pg / EV) isolated from mesenchymal stromal cells (MSCs) as described above with Matrigel, collagen I (Col I), laminin-521 (LN521), laminin-421 (LN421) and hyaluronic acid (Hyal) taken at concentrations 1x1010pg / EV. We injected the EV without addition (clean EVs) and the mixes into the myocardium of mice and compared the retentionof the EVs in the heart measured by luminescence intensity at the time of the injection and 15 minutes later (Fig. 1A). The mixes with ECM molecules allowed significantly higher retention of the EVs after local injections. Injection of the ECM molecules alone into myocardium did not cause any luminescence.

[0187] Example 13: Increased activity of clean EVs with ECM over same EVs without additional ECM in in vitro immunomodulation assay

[0188] To test the ability of the EVs to immunomodulate, we studied the effects of the clean EVs (protein content 1.87xl0-9pg / EV) and the mixes on the expression of proinflammatory cytokine IL-6 in LPS-stimulated macrophages (Fig. 2A). The mixes with ECM molecules allowed significantly decrease the expression of IL-6 in comparison with control (no treatment, only PBS was added) and with treatment with the clean EVs. The treatment with of the ECM molecules alone failed to decrease the expression of IL-6 (Fig. 2A).

[0189] Example 14: Simultaneous isolation of EVs and ECM from conditioned media

[0190] Since EVs and ECM share hydrophobicity, that feature was used for simultaneous isolation of EVs and ECM that is clean from other contaminants produced by the parental cells. For the purification, the conditioned medium from the MSCs (as described above) was dialyzed to exchange buffer to the high salts buffer needed for binding of hydrophobic molecules to the stationary phase. After that, the MSC secretome (content of the conditioned medium produced by MSCs) was applied to the stationary phase (for instance a column) of the chosen chromatography method for binding. The stationary phase was then extensively washed by the high salts buffer. After that, the molecules were eluted from the stationary phase by gradual decrease in the salt content and the eluate was collected in fractions. The fractions were then analyzed for the presence of CD63 (EV marker) and Collagen I (ECM marker), for instance by using Western Bloting with antibodies against CD63 and Collagen I. The fractions containing CD63 or Collagen I or both were collected, dialyzed to exchange the buffer to a desired one for subsequent application for instance phosphate-buffered saline (PBS) and characterized according to the criteria for EVs6to obtain EVs with ECM from the hydrophobic fraction (hydrophEV).

[0191] A successful isolation should decrease the amount of Albumin, Thrombospondin-1 and Thrombospondin-2 per one EV at least five times in the final preparation compared to theinitial amount in the conditioned medium. The amounts of proteins can be quantitatively compared using Western blotting or mass-spectrometry based proteomics.

[0192] To test the efficacy of hydrophEV, mouse EV retention and in vitro immunomodulation assays were performed according to examples 12 and 13. The hydrophEV exhibited higher retention after local injection and higher ability to decrease the expression of pro-inflammatory IL-6 in macrophages (Fig. IB and Fig. 2B).

[0193] Example 15: Decrease in losses of EVs with ECM on surfaces of storage containers.

[0194] To study the effects of extracellular matrix (ECM) molecules on EVs, combinations of the EV isolated from mesenchymal stromal cells (MSCs) were prepared as described above with Matrigel, collagen I (Col I), laminin-521 (LN521), laminin-421 (LN421) and hyaluronic acid (Hyal) taken at amounts of 10 pg per lxlO11EV (1x1010pg / EV). All the EV preparations were taken at the same initial concentration lxlO11particles (EVs) / ml. To test the retention of the EVs on the walls of storage containers, 100 pl of clean EVs and the mixes were placed in 1.5 ml tubes (four repeats per each EV preparation) and incubated for one hour at room temperature with constant shaking on an orbital shaker (10 rotation per minute). After that, the tubes were briefly centrifuged to collect the sample and the EV concentrations were determined using the method described above. The number of the EVs retained by the surfaces was significantly higher for clean EVs in comparison with that for the mixes (Fig. 3A). Similarly, the hydrophEV isolated using the new method, exhibited significantly lower loss of the EVs in comparison with that for the clean EVs (Fig. 3B).

[0195] Example 16: Increased activity of EVs with ECM over the secretome in in vitro immunomodulation assay

[0196] To test the ability of the EVs to immunomodulate, we compared the effects of the clean EVs (protein content 1.87xl0-9pg / EV), secretome (protein content 1.06xl0-8pg / EV) and different preparations of the clean EVs with ECM on the expression of proinflammatory cytokine IL-6 in LPS-stimulated macrophages (Fig. 4). The ability to immunomodulate (decrease the expression of IL-6) was decreased in the clean EVs in comparison with the secretome, but, surprisingly, the addition of ECM to clean EVs or co-isolation of the EVs with ECM led to even higher ability to immunomodulate in comparison with the secretome. The treatment with ofthe ECM molecules alone failed to decrease the expression of IL-6 (see " LN521" in Fig 4, " Matrigel" in Fig. 2A).

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[0203] 6. Lbtvall, J. et al. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. Journal of Extracellular Vesicles.3 (1), 10.3402 / jev.v3.26913, doi: 10.3402 / JEV. V3.26913 (2014).

Claims

1. CLAIMS1. A composition comprising:a. mesenchymal stromal cell (MSC) extracellular vesicles (EV) isolated from the secretome of MSCs; andb. extracellular matrix (ECM) components,wherein the composition comprises:i. less than 3x10-9µg / EV particle of proteins derived from the secretome; andii. at least 0.5x10-11µg / EV particle of the ECM components.

2. A composition comprising:a. mesenchymal stromal cell (MSC) extracellular vesicles (EV); and b. extracellular matrix (ECM),wherein:i. the ECM content is at least 0.5x10-10µg ECM / EV particle;ii. the EV and the ECM are isolated from the secretome of MSCs; and iii. wherein the amount of at least one of albumin, thrombospondin-1, and thrombospondin-2 is at least 5 times lower per EV particle than in the secretome.

3. The composition according to claim 1 or 2, wherein the composition comprises at least 0.5x10-11µg / EV particle of at least one ECM component selected from collagen I, collagen II, collagen IV, laminin-521, laminin-421, laminin-111 and hyaluronic acid.

4. The composition according to any of the preceding claims, wherein the composition comprises less than 3x10-9µg / EV particle of ECM components derived from the secretome.

5. The composition according to any of the preceding claims, wherein the composition comprises less than 3x10-9µg / EV particle of ECM components.

6. The composition according to any of the preceding claims, wherein the composition comprises less than 3x10-9µg / EV particle of total protein.

7. The composition according to any one of the preceding claims, wherein the composition comprises less than 1x10-8µg / EV particle of glucosaminoglycans including but not limited to hyaluronic acid, heparan sulfate, heparin and keratan sulfate.

8. The composition according to any one of the preceding claims, wherein the composition comprises less than 1x10-8µg / EV particle of any one of: collagen I, collagen II, collagen IV, laminin-521, laminin-421, laminin-111, hyaluronic acid.

9. The composition according to any one of the preceding claims, wherein the composition comprises any one of collagen I, collagen II, collagen IV, laminin-521, laminin-421, laminin-111 and hyaluronic acid in an amount of less than 5x10-9µg / EV particle.

10. The composition according to any one of the preceding claims, wherein the composition comprises any one of collagen I, collagen II, collagen IV, laminin-521, laminin-421, laminin-111 and hyaluronic acid in an amount of less than 2x10-9µg / EV particle.

11. The composition according to any one of the preceding claims, wherein the composition comprises any one of collagen I, collagen II, collagen IV, laminin-521, laminin-421, laminin-111 and hyaluronic acid in an amount of less than 1x10-9µg / EV particle.

12. The composition according to any one of the preceding claims, wherein the composition comprises at least lxlO9EVs / ml, or in an increasing order of preference, lxlO10EVs / ml, lxlO11EVs / ml, 5xlOnEVs / ml.

13. The composition according to any one of the preceding claims, wherein the composition comprises at least 5x1010EVs / ml.

14. The composition according to any one of the preceding claims, wherein the EVs and / or the ECMs are isolated using ultracentrifugation, tangential flow filtering, anionexchange, gel filtration, precipitation, affinity isolation on magnetic beads or a resin, or a combination thereof.

15. The composition according to any one of the preceding claims, wherein the composition contains less than 0.1x10-10µg / EV particle of at least one of albumin, thrombospondin-1 or thrombospondin-2.

16. The composition according to any one of the preceding claims, wherein at least 10 wt.% of the ECM is made up of: polysaccharides such as glycans, glycosaminoglycans, heparan sulfate or hyaluronic acid, and ECM proteins chosen from: collagens, laminins, fibronectin, elastin, nidogens, fibrillin, proteoglycans, and glycoproteins.

17. The composition according to any one of the preceding claims, wherein at least 1 wt.% of the ECM is made up of: collagens, laminins and hyaluronic acid.

18. The composition according to any one of the preceding claims, wherein the ECM are chosen from the group of hyaluronic acid and proteins having at least 80% sequence identity to the proteins taken from the group consisting of: collagen chosen from the group of SEQ ID NO 1 to 30; laminin chosen from the group of SEQ ID NO 31 to 42; nidogen chosen from SEQ ID NO 43 or 44; fibrillin chosen from the group of SEQ ID NO 45, 46 and 47, elastin of SEQ ID NO 48, fibronectin of SEQ ID NO 49, Cartilage oligomeric matrix protein P49747 of SEQ ID NO 50, and Basement membrane-specific heparan sulfate proteoglycan core protein P98160 of SEQ ID NO 51.

19. The composition according to any one of the preceding claims, wherein at least 1 wt.% of the ECM consists of one or more molecules taken from the group consisting of: Collagen I, laminin-521, laminin-421, and hyaluronic acid.

20. The composition according to any one of the preceding claims, wherein the EVs carry at least one EV marker chosen from the group of CD63, CD81, and CD9, and the ECMs are chosen from the group consisting of collagen I, collagen IV, laminin-521, laminin- 421, laminin-111, and hyaluronic acid.

21. The composition according to any one of the preceding claims, wherein the ECM molecules are hydrophobic proteins.

22. The composition according to any one of the preceding claims, wherein at least 1 wt.% of the ECM consists of hyaluronic acid.

23. A composition obtainable by the method according to any one of claims 30-38.

24. The composition according to any one of the preceding claims for use as a medicament.

25. The composition for use according to claim 24, for use in the treatment or prophylaxis of a medical condition selected from the group consisting of: ischemic and nonischemic heart failure including heart failure with preserved ejection fraction and heart failure with reduced ejection fraction; heart insufficiency; myocardial infarction; coronary artery disease; cardiomyopathy including hypertrophic, restrictive and dilated; Takotsubo syndrome; amyloidosis; aortic disorders including aneurysm, dissection, ulcers and hematoma; hypertension; congenital heart disease; myocarditis; valve dysfunction; acute respiratory distress syndrome (ARDS); critical illness myopathy (CIM); ventilator induced diaphragm muscle dysfunction (VIDD); graft-versus-host disease (GvHD); solid organ rejection; rejection of cell, organ or tissue transplants; preservation of organs and tissues intended for transplantations; inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis; rheumatoid diseases such as arthritis; inflammation-driven or immunologically induced diseases such as multiple sclerosis, ALS, sarcoidosis, idiopathic pulmonary fibrosis, psoriasis, dermatitis, or eczema; allergies such as allergies to food, animals, plants, medicines, chemicals, metals, or dust; autoimmune diseases such pemphigus, type 1 diabetes, systemic lupus erythematosus (SLE), multiple sclerosis (MS), or Guillain-Barre syndrome; diabetes type 2; tumor necrosis factor (TNF) receptor- associated periodic syndrome (TRAPS); deficiency of the interleukin-1 receptor antagonist (DIRA); endometriosis; autoimmune hepatitis; scleroderma; myositis; stroke; acute spinal cord injury; vasculitis; organ failure such as kidney failure, liver failure, lung failure, or heart failure; cancer including lung cancer and skin cancer; during operations with medical devices including but not limited to heart pumps, pace makers, mechanical and biological valves, stents, artificial vessels including artificial blood vessels, and others; scar formation and fibrosis of various kinds including but not limited scaring / fibrosis caused by diseases and medical operations / procedures;chronic inflammatory conditions of various origin including low grade inflammation; and burns including thermal and chemical burns.

26. The composition for use according to claim 24, for use in the treatment or prophylaxis of a cardiovascular or respiratory disease.

27. The composition for use according to claim 26, wherein the cardiovascular disease is ischemia reperfusion injury of the heart.

28. The composition for use according to any of claims 26-27, wherein the composition is administered to a patient in need via percutaneous coronary intervention (PCI) for implantation of a stent.

29. A use of a composition according to any one of the preceding claims, in a cosmetic method.

30. A method for obtaining a composition comprising EVs and ECM from mesenchymal stromal cell (MSC) secretome, said method comprising the steps of:a) fractionating a MSC secretome such that some fractions are enriched in EVs and other fractions enriched in ECM components;b) analyzing the fractions for enrichment of at least one EV marker chosen from the group consisting of CD63, CD81, and CD9, and for enrichment of at least one ECM component, chosen from the group consisting of a collagen, collagen I, collagen II, collagen IV, a laminin, laminins containing laminin alpha 5 chain, nidogens, laminins containing laminin alpha 4 chain, elastin, fibronectin, proteoglycans, glycosaminoglycans, hyaluronic acid, glycoproteins, and heparan sulfate;c) combining fractions enriched in EVs with fractions enriched in the ECM component into a composition comprising EVs and ECMs,by selecting the fractions for the composition such that the obtained composition is depleted in at least one of albumin, thrombospondin-1 or thrombospondin-2, such that it contains 80% less of at least one of albumin, thrombospondin-1 or thrombospondin-2 per EV particle, compared to the secretome prior to fractionation.

31. The method according to claim 30, wherein the EV marker chosen is CD63.

32. The method according to claim 30 or 31, wherein the ECM component chosen is collagen I.

33. The method according to any one of claims 30-32, wherein the obtained composition is depleted in at least two of: albumin, thrombospondin-1 and thrombospondin-2.

34. The method according to any one of claims 30-33, wherein the obtained composition is depleted in all three of: albumin, thrombospondin-1 and thrombospondin-2.

35. The method according to any one of claims 30-34, wherein the fractionation is at least in part performed using a hydrophobic interaction chromatography resin and the elution of molecules from the resin is performed by a gradient of salts.

36. The method according to any one of claims 30-35, wherein the fractionation is at least in part performed using an ion exchange resin and the elution of molecules from the resin is performed by a gradient of salts.

37. The method according to any one of claims 30-36, wherein the fractionation is at least in part performed using a size exclusion chromatography.

38. The method according to any one of claims 30-37, wherein the fractionation is at least in part performed using a stationary phase being a reverse phase chromatography stationary phase and the elution of molecules from the stationary phase is performed by ionic liquids or solvents.

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