A composition comprising extracellular vesicles for use in the treatment or prevention of cancer, in particular for inducing apoptosis in cancer cells, in particular osteosarcoma

Abstract

A composition comprising extracellular vesicles for use in the treatment or prevention of cancer, in particular for inducing the apoptosis in cancer cells, in particular osteosarcoma, wherein the composition is obtainable according to a process comprising the following steps:  Culturing of stem cells, preferably adipose-derived stem cells;  Differentiating the stem cells, preferably osteogenically or chondrogenically differentiating the stem cells;  Adding of a particulate material, preferably gelatin beads, to obtain a scaffold-free multi-dimensional cell culture;  Collecting the liquid containing extracellular vesicles from the scaffold-free multi- dimensional cell culture;  Subjecting the liquid containing the extracellular vesicles to tangential flow filtration (TFF).

Description

[0001] A COMPOSITION COMPRISING EXTRACELLULAR VESICLES FOR USE IN THE TREATMENT OR PREVENTION OF CANCER, IN PARTICULAR FOR INDUCING APOPTOSIS IN CANCER CELLS, IN PARTICULAR OSTEOSARCOMA

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a method for composition comprising extracellular vesicles for use in the treatment or prevention of cancer, in particular for inducing the apoptosis in cancer cells, in particular osteosarcoma, wherein the composition is obtainable according to a process using tangential flow cytometry (TFF).

[0004] BACKGROUND

[0005] Tangential flow filtration has already been described in conjunction with the isolation of the exosome from stem cells derived cell cultures.

[0006] US2023075630A1 to John Hopkins University and the University of Washington discloses extracellular vesicles-based agents useful for the treatment of neuropathic disorders. TFF is described with ultracentrifugation and ultrafiltration as separation methods for the vesicles.

[0007] US2023233615A1 to GWO XI Stem Cell discloses a pharmaceutical composition for treating Alzheimer’s disease. First adipose-derived stem cells are cultured in two culturing steps. Then, the extracellular vesicles are separated by first collecting the culture solution. The extracellular vesicles are separated from the culture solution by tangential flow filtration.

[0008] US2022133803A1 to Vitti Labs discloses a method for isolating mesenchymal stem cell exosomes from liquid, cell-free mesenchymal stem cell cultures. A first tangential flow filtration (TFF) with a pore size of 0.1 to 1 microns, preferably 0.65 is described followed by a second TFF with a pore size of 30 kD to 600 kD. Optionally, inter intermediate TFF can be performed with a pore size of 0,45 microns to 2 microns. But this method requires a complex bioreactor, see Figure 4A.

[0009] WQ2021149047A1 to Stem Cell Medicine discloses anti-aging cosmetic compositions prepared from adipose-derived stem cells. The protein fraction is obtained by concentrating and filtering the medium of the stem cell culture using tangential flow filtration (TFF) with a molecular weight cutoff 1 kDa or 3kDa. WO2020112694A9 to Arytha Biosciences, discloses nanoparticles containing a cellular membrane.

[0010] WO2Q22112528A1 to Novadip Biosciences discloses cellular and / or extracellular extracts obtained from a scaffold-free 3-dimensional culture of mature cells and a particulate material for preventing and / or treating cancer. The mature cells secrete the neosynthesized extracellular matrix. Both the mature cells and the particulate material are embedded in the neo-synthesized extracellular matrix. No bioreactor is required as the cells are cultured in flasks. The exosome is separated through ultracentrifugation. But ultracentrifugation leads to an increased purity. That means that less extracellular components are present in the isolated exosome.

[0011] SHORT DESCRIPTION OF THE INVENTION

[0012] The inventors surprisingly found that the exosome isolated through Tangential Flow Filtration (TFF) is enriched in extracellular components such as factors and micro RNAs as compared to ultracentrifugation.

[0013] The exosome is obtained from a scaffold-free biomaterial obtained through addition of particles to differentiated stem cells and the subsequent generation of neo-synthesized extracellular matrix through the differentiated stem cells.

[0014] The inventors further showed that the enriched exosome increases apoptosis in cancer cells.

[0015] Accordingly, a first aspect of the present invention is a process for the production of a composition comprising extracellular vesicles, comprising the following steps:

[0016] ■ Culturing of stem cells, preferably adipose-derived stem cells;

[0017] ■ Differentiating the stem cells, preferably osteogenically or chondrogenically differentiating the stem cells;

[0018] ■ Adding of a particulate material, preferably gelatin beads, to obtain a scaffold-free multi-dimensional cell culture;

[0019] ■ Collecting the liquid containing extracellular vesicles from the scaffold-free multi-dimensional cell culture; Subjecting the liquid containing the extracellular vesicles to tangential flow filtration (TFF).

[0020] In another aspect, the TFF device comprises a filtration membrane having a pore size ranging from 10 nm to 100 nm, preferably from 25 nm to 75 nm, even more preferably from preferably from 40 nm to 60 nm.

[0021] In another aspect, the number of extracellular vesicles in the composition comprising the extracellular vesicles or obtainable according to the process of the present invention is from 1.1011to 1.1013, preferably from 5.1011to 1.1013and even more preferably 5.1011to 5.1012per ml of the composition as measured by nanoparticle tracking analysis (NTA).

[0022] In another aspect, the protein concentration in the composition of the invention is from 1000 micrograms / mL to 15000 micrograms / mL, preferably from 2000 micrograms / mL to 15000 micrograms / mL, even more preferably from 2000 micrograms / mL to 10000 micrograms / mL.

[0023] In another aspect, the composition has a purity index as measured by the number of extracellular vesicles per microgram protein of from 1 .107to 1.109, preferably from 5.107to 1 .109, even more preferably from 5.107to 5.108.

[0024] In another aspect, the TFF is used to reduce the amount or level of liquid from the composition comprising the extracellular vesicles.

[0025] In another aspect, the TFF is used to remove from 50% to 99.9999%, preferably from 70% to 99.9999%, even more preferably from 90% to 99.9999% of the liquid from the composition.

[0026] In another aspect, the differentiated cells neo-synthesize an extracellular matrix through the addition of the particulate material.

[0027] In another aspect, the mature cells and the particulate material are embedded in the neosynthesized extracellular matrix.

[0028] In another aspect, the differentiated cells are selected from the group consisting of osteoblasts, osteocytes, chondroblasts, chondrocytes, keratinocytes, myofibroblasts, epithelial cells, endothelial cells, adipocytes, neural cells, and precursors thereof, and preferably are soft tissue cells, chondroblasts or osteoblasts.

[0029] In another aspect, the particulate material is selected from the group consisting of: ■ an organic material, including demineralized bone matrix (DBM), gelatin, agar / agarose, alginates chitosan, chondroitin sulfate, collagen, elastin, or elastin-like peptides (ELP), fibrinogen, fibrin, fibronectin, proteoglycans, heparan sulfate proteoglycans, hyaluronic acid, polysaccharides, laminins and cellulose derivatives, preferably gelatin;

[0030] ■ a calcium compound including particles of calcium phosphate, and even more preferably hydroxyapatite (HA) and / or [3-tricalcium phosphate ( - TCP);

[0031] ■ a polymer, including polyanhydrides, polylactic acid (PI_A), poly(lactic- co- glycolicacid) (PLGA), polyethylene oxide / polyethylene glycol (PEO / PEG), poly(vinyl alcohol) (PVA), fumarate-based polymers such as, for example polypropylene fumarate) (PPF) or polypropylene fumarate-co-ethylene glycol) (P(PF-co-EG)), oligopolypthylene glycol) fumarate) (OPF), poly(aldehyde guluronate) (PAG), polyp- vinyl pyrrolidone) (PNVP), or combinations thereof;

[0032] ■ a gel, including a self-assembling oligopeptide gel, a microgel, a nanogel, a particulate gel, a hydrogel, a thixotropic gel, a xerogel, a responsive gel, or combinations thereof;

[0033] ■ a creamer; and

[0034] ■ any combinations thereof.

[0035] Another aspect of the present invention is the use of the composition comprising extracellular vesicles obtained according to the process of the present invention for the treatment or prevention of cancer, in particular for inducing the apoptosis in cancer cells, in particular osteosarcoma, wherein the composition is obtained according to a process according to any of the preceding claims.

[0036] Another aspect of the present invention is a composition comprising extracellular vesicles for use in the treatment or prevention of cancer, in particular for inducing the apoptosis in cancer cells, in particular osteosarcoma, wherein the composition is obtainable according to a process comprising the following steps:

[0037] Culturing of stem cells, preferably adipose-derived stem cells; ■ Differentiating the stem cells, preferably osteogenically or chondrogenically differentiating the stem cells;

[0038] ■ Adding of a particulate material, preferably gelatin beads, to obtain a scaffold-free multi-dimensional cell culture;

[0039] ■ Collecting the liquid containing extracellular vesicles from the scaffold-free multi-dimensional cell culture;

[0040] ■ Subjecting the liquid containing the extracellular vesicles to tangential flow filtration (TFF).

[0041] In another aspect of the composition for use according of the present invention, the TFF device comprises a filtration membrane having a pore size ranging from 10 nm to 100 nm, preferably from 25 nm to 75 nm, even more preferably from preferably from 40 nm to 60 nm.

[0042] In another aspect of the composition for use according of the present invention, the number of extracellular vesicles in the composition comprising the extracellular vesicles or obtainable according to the process of the present invention is from 1.1011to 1.1013, preferably from 5.1011to 1 .1013and even more preferably 5.1011to 5.1012per ml of the composition as measured by nanoparticle tracking analysis (NTA).

[0043] In another aspect of the composition for use according of the present invention, the number of extracellular vesicles in the liquid containing extracellular vesicles prior to the TFF step is from 1.108to 1.1010, preferably from 5.108to 5.109and even more preferably 7.108to 3.109per ml of the composition as measured by nanoparticle tracking analysis (NTA).

[0044] In another aspect of the composition for use according of the present invention, the protein concentration in the composition of the invention is from 1000 micrograms / mL to 15000 micrograms / mL, preferably from 2000 micrograms / mL to 15000 micrograms / mL, even more preferably from 2000 micrograms / mL to 10000 micrograms / mL.

[0045] In another aspect of the composition for use according of the present invention, the composition has a purity index as measured by the number of extracellular vesicles per microgram protein of from 1.107to 1.109, preferably from 5.107to 1.109, even more preferably from 5.107to 5.108. In another aspect of the composition for use according of the present invention, the TFF is used to reduce the amount or level of liquid from the composition comprising the extracellular vesicles.

[0046] In another aspect of the composition for use according of the present invention, the TFF is used to remove from 50% to 99.9999%, preferably from 70% to 99.9999%, even more preferably from 90% to 99.9999% of the liquid from the composition.

[0047] In another aspect of the composition for use according of the present invention, the differentiated cells neo-synthesize an extracellular matrix through the addition of the particulate material.

[0048] In another aspect of the composition for use according of the present invention, the mature cells and the particulate material are embedded in the neo-synthesized extracellular matrix.

[0049] In another aspect of the composition for use according of the present invention, the differentiated cells are selected from the group consisting of osteoblasts, osteocytes, chondroblasts, chondrocytes, keratinocytes, myofibroblasts, epithelial cells, endothelial cells, adipocytes, neural cells, and precursors thereof, and preferably are soft tissue cells, chondroblasts or osteoblasts.

[0050] In another aspect of the composition for use according of the present invention, the particulate material is selected from the group consisting of:

[0051] ■ an organic material, including demineralized bone matrix (DBM), gelatin, agar / agarose, alginates chitosan, chondroitin sulfate, collagen, elastin, or elastin-like peptides (ELP), fibrinogen, fibrin, fibronectin, proteoglycans, heparan sulfate proteoglycans, hyaluronic acid, polysaccharides, laminins and cellulose derivatives; preferably gelatin;

[0052] ■ a calcium compound including particles of calcium phosphate, and even more preferably hydroxyapatite (HA) and / or [3-tricalcium phosphate ( - TCP);

[0053] ■ a polymer, including polyanhydrides, polylactic acid (PI_A), poly(lactic- co- glycolicacid) (PLGA), polyethylene oxide / polyethylene glycol (PEO / PEG), poly(vinyl alcohol) (PVA), fumarate-based polymers such as, for example polypropylene fumarate) (PPF) or polypropylene fumarate-co-ethylene glycol) (P(PF-co-EG)), oligopolypthylene glycol) fumarate) (OPF), poly(aldehyde guluronate) (PAG), polyp- vinyl pyrrolidone) (PNVP), or combinations thereof;

[0054] ■ a gel, including a self-assembling oligopeptide gel, a microgel, a nanogel, a particulate gel, a hydrogel, a thixotropic gel, a xerogel, a responsive gel, or combinations thereof;

[0055] ■ a creamer; and

[0056] ■ any combinations thereof.

[0057] DETAILED DESCRIPTION OF THE INVENTION

[0058] Extracellular extract

[0059] Extracellular extract as opposed to cellular extracts means any compound or element secreted or otherwise produced and transported outside the cell.

[0060] Exosome and extracellular vesicles

[0061] The terms extracellular vesicles (EVs), exosome, exosome-like or extracellular vesicles or nanoparticles are used interchangeably to designate particles released from cells upon fusion of an intermediate endocytic compartment, the multivesicular body (MVB), with the plasma membrane. In other words, exosomes, exosome-like or extracellular vesicles correspond to the intraluminal vesicles that are released into the extracellular space.

[0062] In one embodiment, the composition of the present invention are substantially free of differentiated cells, in particular free of differentiated viable or non-viable cells. In one embodiment, the exosomes comprises less than 0.1 w%, preferably less than 0.01 w%, and even more preferably less than 1 w% of viable or non-viable differentiated cells as compared to the total weight of the composition.

[0063] In one embodiment, the composition comprises differentiated viable or non-viable cells in a content of 0.001 w% to 1 w%, preferably from 0.001 w% to 0.1 w%, even more preferably from 0.001 w% to 0.01 w% as compared to the total weight of the composition.

[0064] Size of the extracellular vesicles

[0065] Usually the size of the extracellular vesicles is determined by NTA. Extracellular extract

[0066] The term extracellular extract refers to material that has been actively and / or passively transported - for example by secretion, exocytosis, or leakage - out of the cells. As used herein, an extracellular extract may include, but be not limited to, metabolites; nucleic acids, including miRNAs; polypeptides, including transcription factors, growth factors, components of the extracellular matrix, and lipids.

[0067] Enriched exosome - Nanoparticle tracking analysis

[0068] The composition comprising extracellular vesicles obtained by the process of the present invention differentiates from the known process through the yield, the protein concentration and the protein index.

[0069] Preferably the composition of the invention is a liquid composition.

[0070] In one embodiment, the concentration of extracellular vesicles is measured by nanoparticle tracking analysis (NTA) as the number of particles per volume of the final product (liquid).

[0071] In one embodiment, the protein content of the composition of the present invention is increased as compared to exosomes derived from 2D cultures, or exosomes isolated with UC2 protocol.

[0072] The yield in particles is measured by the NTA.

[0073] In a preferred embodiment, the process according to any one of the preceding claims, the number of extracellular vesicles in the composition comprising the extracellular vesicles or obtainable according to the process of the present invention is from 1.1011to 1.1013, preferably from 5.1011to 1 .1013and even more preferably 5.1011to 5.1012per ml of the composition as measured by nanoparticle tracking analysis (NTA).

[0074] In a preferred embodiment, the number of extracellular vesicles in the liquid containing extracellular vesicles prior to the TFF step is from 1 .108to 1.1010, preferably from 5.108to 5.109and even more preferably 7.108to 3.109per ml of the composition as measured by nanoparticle tracking analysis (NTA). Protein concentration

[0075] In one embodiment, the EV purity of the composition of the present invention is decreased as compared to ultracentrifugation. The purity index of each sample is established as ratio particles / proteins in the final volume.

[0076] In another aspect, the protein concentration in the composition of the invention is from 1000 micrograms / mL to 15000 micrograms / mL, preferably from 2000 micrograms / mL to 15000 micrograms / mL, even more preferably from 2000 micrograms / mL to 10000 micrograms / mL.

[0077] In a preferred embodiment, the total protein concentration is measured by BCA. miR-27a-3p, miR-34b-5p, miR-34c-5p, miR-125a-5p

[0078] In one embodiment, the composition of the invention comprises micro-RNAs (miRNAs) with tumor suppressive properties, in particular miR-27a-3p, miR-34b-5p, miR-34c-5p, miR-125a-5p.

[0079] In one embodiment, the level of miRNAs with tumor suppressive properties, in particular miR-27a-3p, miR-34b-5p, miR-34c-5p, miR-125a-5p are increased by at least 100 %, preferably by at least 125 %, even more preferably by at least 150 %, even more preferably by at least 200 % as compared to the number of miRNA molecules per ml liquid obtained after ultracentrifugation of three-dimensional culture.

[0080] In one embodiment, the level of miRNA with tumor suppressive properties , in particular miR-27a-3p, miR-34b-5p, miR-34c-5p, miR-125a-5p are increased by 100 w% to 3000 w%, preferably by 200 w% to 3000 w%, even more preferably by 300 w% to 3000 w%, as compared to to the number of miRNA molecules per ml liquid obtained after two- dimensional cell culture and TFF.

[0081] Purity

[0082] In one embodiment, the purity index of the composition of the present invention thanks to the TFF isolation is decreased as compared to the purity index after ultracentrifugation.

[0083] In one embodiment, the purity is measured by the purity index, i.e. the number of particles or extracellular vesicles per microgram protein.

[0084] In one embodiment, the purity index of each sample is established as ratio particles or extracellular vesicles to proteins in the composition comprising extracellular vesicles of the invention, i.e. after TFF. The inventors surprisingly found that “less pure” EVs derived from after TFF isolation are more efficient in the treatment of cancer as they trigger the tumor cell apoptosis compared to the EVs derived from the same upstream process but isolated using ultracentrifugation.

[0085] In a preferred embodiment, the composition of the invention has a purity index as measured by the number of extracellular vesicles per microgram protein of from 1 .107to 1 .109, preferably from 5.107to 1.109, even more preferably from 5.107to 5.108.

[0086] Stem cells

[0087] In one embodiment, the differentiated cells are derived from stem cells, such as pluripotent stem cells (PSCs), for example embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) or adult stem cells such as hematopoietic stem cells (HSCs), skin stem cells (SSCs), neural stem cells (NSCs), and mesenchymal stem cells (MSCs). MSCs are present in multiple tissues, including BM, adipose tissue, peripheral blood, and placenta.

[0088] In one embodiment, the differentiated cells are selected from the group comprising or consisting of osteoblasts, osteocytes, chondroblasts, chondrocytes, keratinocytes, myofibroblasts, epithelial cells, endothelial cells, adipocytes, neural cells, and precursors thereof, and preferably are soft tissue cells, chondroblasts or osteoblasts.

[0089] In a preferred embodiment, the differentiated cells are derived from adipose-derived stem cells (ASCs). Differentiated cells

[0090] The differentiated cells are embedded in the 3-dimensional neo-synthesized extracellular matrix.

[0091] In one embodiment, the differentiated cells are osteo-differentiated, skin-differentiated or chondro-differentiated cells. That means that differentiated cells can promote bone, skin and / or cartilage formation, and / or maintain existing bone, skin and / or cartilage in a healthy physiological condition.

[0092] In one embodiment, the differentiated cells are selected from the group comprising or consisting of osteoblasts, osteocytes, chondroblasts, chondrocytes, keratinocytes, myofibroblasts, epithelial cells, endothelial cells, adipocytes, neural cells, and precursors thereof, and preferably are soft tissue cells, chondroblasts or osteoblasts.

[0093] In one embodiment, the differentiated cells are myofibroblastic cells.

[0094] In another embodiment, the differentiated cells are endothelial differentiated cells.

[0095] In another embodiment, the differentiated cells are epithelial differentiated cells. In another embodiment, the differentiated cells are adipogenic differentiated cells.

[0096] In another embodiment, the differentiated cells are neural differentiated IPCSs.

[0097] Size ratio exosomes to differentiated cells

[0098] In one embodiment, the size ratio of exosomes to differentiated cells is from to , preferably from to , and even more preferably from to .

[0099] 3-dimensional (3D) neo-synthesized extracellular matrix (ECM)

[0100] The 3-dimensional neo-synthesized extracellular matrix is an extracellular matrix that the differentiated cells surprisingly secrete when the particulate material is added.

[0101] The 3-dimensional neo-synthesized extracellular matrix serves as scaffold. Consequently, no external scaffold needs to be added.

[0102] The differentiated cells are embedded in the 3-dimensional neo-synthesized extracellular matrix.

[0103] The differentiated cells and the particulate material are embedded in a neo- synthesized extracellular matrix.

[0104] Following the multi-dimensional induction through the addition of the particulate material to the differentiated cells, the differentiated cells and the particulate material are embedded in the neo-synthesized extracellular matrix.

[0105] Culturing

[0106] In one embodiment, the culturing is performed in flasks.

[0107] In a preferred embodiment, no bioreactor or external scaffold is required.

[0108] Differentiating

[0109] Differentiation of the stem cells can for example be done by the method described in Example 1 .

[0110] Adding of a particulate material

[0111] The particulate material is usually added by sprinkling over the differentiated cells.

[0112] Importantly, the particulate material does not serve as an external scaffold but induces the multi-dimensional expansion of the differentiated cells through the biosynthesis of the extracellular matrix by the differentiated cells (neo-synthesized extracellular matrix). Tangential Flow Filtration (TFF):

[0113] Tangential flow filtration means an isolation method using a tangential flow filtration device, such as the HansaBioMed. The pore is chosen to separate EVs from the supernatant of from the scaffold-free multi-dimensional cell culture.

[0114] In one embodiment, the pore size is from 10 nm to 100 nm, preferably from nm to nm even more preferably from nm to nm.

[0115] Scaffold-free

[0116] Consequently, the composition of the present invention is free of external scaffolds, i.e. scaffold-free. The particulate material is sprinkled on the differentiated cells to induce the multi-dimensional growth of the cell culture.

[0117] That means that no external, three-dimensional scaffold is used to which the cells attach.

[0118] Particulate material, size and concentration

[0119] The term particulate material as used herein refers to a solid material in the form of particles.

[0120] Within the scope of the invention, particulate material includes organic materials, such as, e.g., demineralized bone matrix (DBM) and gelatin; ceramic materials; polymers, such as, e.g., polyanhydrides; gel, such as, e.g., hydrogel; and any combination thereof.

[0121] The particulate material is preferably selected from the group consisting of:

[0122] ■ an organic material, including demineralized bone matrix (DBM), gelatin, agar / agarose, alginates chitosan, chondroitin sulfate, collagen, elastin or elastinlike peptides (ELP), fibrinogen, fibrin, fibronectin, proteoglycans, heparan sulfate proteoglycans, hyaluronic acid, polysaccharides, laminins and cellulose derivatives;

[0123] ■ calcium compound;

[0124] ■ a polymer, including polyanhydrides, polylactic acid (PLA), poly(lactic- co-glycolic acid) (PLGA), polyethylene oxide / polyethylene glycol (PEO / PEG), poly(vinyl alcohol) (PVA), fumarate-based polymers such as, for example polypropylene fumarate) (PPF) or polypropylene fumarate-co-ethylene glycol) (P(PF-co-EG)), oligopolypthylene glycol) fumarate) (OPF), poly(aldehyde guluronate) (PAG), polyp- vinyl pyrrolidone) (PNVP), or combinations thereof; ■ a gel, including a self-assembling oligopeptide gel, a microgel, a nanogel, a particulate gel, a hydrogel, a thixotropic gel, a xerogel, a responsive gel, or combinations thereof; or

[0125] ■ -a creamer; and

[0126] ■ any combinations thereof.

[0127] The particulate material preferably is gelatin, even more preferably gelatin beads.

[0128] In one embodiment, the gelatin of the invention is animal gelatin, preferably mammal gelatin, more preferably porcine gelatin.

[0129] As used herein, the term porcine gelatin may be replaced by pork gelatin or pig gelatin. A commercially available example is Cultispher.

[0130] In one embodiment, the gelatin is porcine skin gelatin.

[0131] In certain embodiments, the particulate material, in particular the gelatin is in the form of particles.

[0132] In one embodiment, the particles, in particular the gelatin particles or beads, have a volumetric mean diameter ranging from about 50 micrometers to about 1 ,000 micrometers as measured by laser diffraction granulometry, preferably with a Malvern Mastersizer.

[0133] Within the scope of the invention, the expression “from about 50 micrometers to about 1 ,000 micrometers ” encompasses 50 micrometers, 60 micrometers , 70 micrometers , 80 micrometers , 90 micrometers, 100 micrometers, 150 micrometers, 200 micrometers, 250 micrometers, 300 micrometers, 350 micrometers, 400 micrometers, 450 micrometers, 500 micrometers, 550 micrometers, 600 micrometers, 650 micrometers, 700 micrometers, 750 micrometers, 800 micrometers, 850 micrometers, 900 micrometers, 950 micrometers and 1 ,000 micrometers.

[0134] In one embodiment, the particulate material, preferably gelatin is added at a concentration ranging from about 0.1 cm3to about 5 cm3for a 150 cm2vessel, preferably from about 0.5 cm3to about 4 cm3, more preferably from about 0.75 cm3to about 3 cm3.

[0135] In one embodiment, gelatin is added at a concentration ranging from about 1 cm3to about 2 cm3for a 150 cm2vessel.

[0136] In one embodiment, gelatin is added at a concentration of about 1 cm3, 1.5 cm3or 2 cm3for a 150 cm2vessel. Within the scope of the invention, the expression “0.1 cm3to about 5 cm3” encompasses 0.1 cm3, 0.2 cm3, 0.3 cm3, 0.4 cm3, 0.5 cm3, 0.6 cm3, 0.7 cm3, 0.8 cm3, 0.9 cm3, 1 .0 cm3, 1 .5 cm3, 2.0 cm3, 2.5 cm3, 3.0 cm3, 3.5 cm3, 4.0 cm3, 4.5 cm3and 5.0 cm3.

[0137] In one embodiment, the particulate material is embedded in the secreted neo-synthesized extracellular matrix.

[0138] In one embodiment, the particles are larger than about 50 pm, preferably larger than about 100 pm as measured by laser diffraction granulometry, preferably with a Malvern Mastersizer.

[0139] Manufacturing process

[0140] The compositions may be manufactured in a scaffold-free process as shown in Figure 1 comprising:

[0141] ■ Stem cell provision;

[0142] ■ Stem cell proliferation;

[0143] ■ Differentiation, in particular osteogenic or chondrogenic differentiation;

[0144] ■ Particles sprinkling; and

[0145] ■ 3D-structure formation.

[0146] Medical use

[0147] In one embodiment, the composition of the invention is used for the treatment or the prevention of cancer.

[0148] In one embodiment, the composition of the invention is used for the induction of apoptosis of cancer cells.

[0149] Secreted proteins with anti-cancer activity

[0150] Examples of proteins with anti-cancer activity secreted by the differentiated cells present in the neo-synthesized extracellular matrix and implicated in the positive regulation of cell death pathways in osteosarcoma (OS) treatment such as apoptosis, autophagy and necroptosis, include:

[0151] Cancer

[0152] The compositions of the present inventions are useful in the treatment or prevention of cancer. In one embodiment, the compositions of the present invention are useful in one or more of the following treatments:

[0153] ■ inhibiting the viability of cancer cells,

[0154] ■ inhibiting the proliferation of cancer cells;

[0155] ■ inhibiting the migration of cancer cells;

[0156] ■ inhibiting the cell colony formation of cancer cells; or

[0157] ■ any combination thereof.

[0158] In one embodiment, the cancer is a solid cancer selected from the group consisting of or comprising of a bone cancer, a brain cancer, a skin cancer, a breast cancer, a cancer of the central nervous system, a cancer of the cervix, a cancer of the upper aero digestive tract, a colorectal cancer, an endometrial cancer, a germ cell cancer, a bladder cancer, a kidney cancer, a laryngeal cancer, a liver cancer, a lung cancer, a neuroblastoma, an esophageal cancer, an ovarian cancer, a pancreatic cancer, a pleural cancer, a prostate cancer, a retinoblastoma, a small intestine cancer, a soft tissue sarcoma, a stomach cancer, a testicular cancer and a thyroid cancer, and preferably is bone cancer or any metastases thereof or skin cancer.

[0159] Secreted miRNAs with anti -cancer activity

[0160] Examples of miRNAs with anti-cancer activity secreted by the differentiated cells and present in the neo-synthesized extracellular matrix are: hsa-miR-210-3p, hsa-miR-409-3p, hsa-let-7a-5p, hsa-miR-29b-3p, hsa-miR-30e-3p, hsa-let-7b-5p, hsa-miR-3184-3p, hsa- miR-92a-3p, hsa-miR-320a, hsa-miR-24-3p, hsa-let-7d-5p, hsa-miR-193b-5p, hsa-miR- 361 -3p, hsa-miR-199a-5p, hsa-miR-25-3p, hsa-miR-181a-5p, hsa-miR-151a-3p, hsa- miR-214-3p, hsa-miR-193a-5p, hsa-miR-30c-5p, hsa-miR-154-5p, hsa-let-7f-5p, hsa- miR-199a-3p, hsa-miR-664b-3p, hsa-miR-664a-5p, hsa-miR-3607-5p, hsa-miR-29a-3p, hsa-miR-27a-3p, hsa-miR-92b-3p, hsa-miR-199b-3p, hsa-miR-342-3p, hsa-miR-320b, hsa-miR-1291 , hsa-let-7e-5p, hsa-miR-130a-3p, hsa-miR-3651 , hsa-miR-103b, hsa-miR- 1273g-3p, hsa-miR-30a-3p, hsa-miR-664b-5p, hsa-miR-34a-3p, hsa-miR-125a-5p, hsa- miR-145-5p, hsa-miR-664a-3p, hsa-miR-140-5p, hsa-miR-21 -5p, hsa-miR-28-3p, hsa- miR-98-5p, hsa-miR-3609, hsa-let-7i-5p, hsa-miR-93-5p, hsa-miR-146b-5p, hsa-miR- 374c-3p, hsa-miR-125b-5p, hsa-miR-34a-5p, hsa-miR-337-3p, hsa-miR-10a-5p, hsa-let- 7g-5p, hsa-miR-222-3p, hsa-miR-4449, hsa-miR-22-3p, hsa-miR-191-5p, hsa-miR-3074- 5p, hsa-miR-6516-3p, hsa-miR-4668-5p, hsa-miR-574-3p, hsa-miR-424-5p, hsa-let-7i-3p, hsa-miR-24-2-5p, hsa-miR-199b-5p, hsa-miR-424-3p, hsa-miR-103a-3p, hsa-miR-29b-1- 5p, hsa-miR-423-5p, hsa-miR-328-3p, hsa-miR-324-5p, hsa-miR-335-5p, hsa-miR-574- 5p, hsa-miR-17-5p, hsa-miR-660-5p, hsa-miR-425-5p, hsa-miR-23b-3p, hsa-miR-23a-3p, hsa-miR-185-5p, hsa-miR-4461 , hsa-miR-196a-5p, hsa-let-7d-3p, hsa-miR-374b-5p, hsa- miR-127-3p, hsa-let-7c-5p, hsa-miR-423-3p, hsa-miR-196b-5p, hsa-miR-221-3p, hsa- miR-382-5p, hsa-miR-619-5p, hsa-miR-3613-5p, hsa-miR-3653-5p, hsa-miR-19b-3p, hsa-miR-99b-5p, hsa-miR-376c-3p, hsa-miR-99b-3p, hsa-miR-663b, hsa-miR-495-3p, hsa-miR-454-3p, and a combination thereof.

[0161] Accordingly, in another aspect, the composition of the present invention comprises one or more of above-mentioned miRNA(s).

[0162] Preferably, the composition of the present invention comprises one or more of miRNA(s) selected from a group consisting of: miRNA selected from the group consisting of MiR- 140, miR-199a, miR-34a, miR-335 and miR-505.

[0163] Criteria and conventions for miRNA identification and nomenclature have been described in Ambros et al. (A uniform system for microRNA annotation. RNA 2003 9(3):277-279). The miRNAs sequences may be retrieved from the miRbase database (http: / / www.mirbase.org / ) or the miRDB database (http: / / www.mirdb.org / ).

[0164] In practice, the RNAs profile may be assessed by any suitable method known in the art, or any method adapted therefrom.

[0165] Illustratively, RNA may be extracted, e.g. by the mean of commercial kit (such as miRNeasy kit from Qiagen®); and further sequenced, e.g. by the mean of a high- throughput sequencing system (such as NextSeq 500 system from Illumina®).

[0166] Illustratively, one may use the Qiazol lysis reagent (Qiagen®, Hilden, Germany) and a Precellys homogenizer (Bertin® instruments, Montigny-le-Bretonneux, France). RNAs may be purified using Rneasy mini kit (Qiagen®, Hilden, Germany) with an additional on column DNase digestion according to the manufacturer’s instruction.

[0167] Quality and quantity of RNA may be determined using a spectrophotometer (Spectramax® 190, Molecular Devices®, California, USA). cDNA may be synthesized from 0.5pg of total RNA using RP RNA first strand kit (Qiagen®, Hilden, Germany) for genes expression profiles though customized PCR arrays (Customized Human Osteogenic and angiogenic RP Profiler Assay - Qiagen®, Hilden, Germany). The ABI Quantstudio 5 system (Applied Biosystems®) and SYBR Green ROX Mastermix (Qiagen®, Hilden, Germany) may be used for detection of the amplification product. Quantification may be obtained according to the AACT method. The final result of each sample may be normalized to the means of expression level of housekeeping genes (e.g. ACTB, B2M and GAPDH).

[0168] Liquid injection

[0169] In another preferred embodiment, the composition is administered as an injection or in the form of an injectable liquid or an injectable suspension.

[0170] Accordingly, another aspect of the invention is an injectable liquid comprising the composition of the present invention.

[0171] Method of treatment

[0172] Another aspect of the present invention is a method of treatment or prevention of cancer comprising the administration of the composition of the present invention.

[0173] Pharmaceutically acceptable carrier

[0174] In another aspect, the composition of the present invention comprises pharmaceutically acceptable vehicles or carriers.

[0175] As used herein, pharmaceutically acceptable carrier refers to any solvent, dispersion medium, coating, antibacterial and / or antifungal agent, isotonic and absorption delaying agent and the like.

[0176] The pharmaceutically acceptable carrier may comprise one or more ingredient(s) selected in a group of additives, polypeptides, amino acids, lipids, and carbohydrates.

[0177] Among carbohydrates, one may cite sugars, including monosaccharides, di-, tri- tetra- and oligosaccharides, derivatized sugars such as alditols, aldolic acids, esterified sugars.

[0178] Examples of suitable pharmaceutically acceptable vehicles may include polypeptides such as, e.g., gelatin, casein, and the like.

[0179] SHORT DESCRIPTION OF THE DRAWINGS

[0180] Figure 1 shows a schematic outline of mesenchymal stem cell culturing methods and exosome collection methods of the present invention. Stem cells are first cultured and proliferated and then differentiated, preferably osteogenically differentiated. Then a particulate material, preferably gelatin beads, are sprinkled on the differentiated cells to obtain a three-dimensional cell culture. The supernatant is then collected and subjected to the tangential flow filtration (TFF) to obtain the composition of the present invention comprising extracellular vesicles..

[0181] Figure 2 shows a schematic of the two different protocols for the exosome isolation. Supernatants of 3D-structures were precleared by centrifugation first at 400g for 5 minutes at room temperature, and then at 2,000g for 20 min at 4°C. For one part of the pre-clarified supernatant, exosomes were isolated by a standard ultracentrifugation (UC2) protocol while on the second part a tangential flow filtration (TFF) protocol was applied. In case of UC2 protocol two steps of ultracentrifugation at 110,000g for 120 min were applied whereas in case of the TFF protocol the clarified supernatant was subjected to ultrafiltration in a TFF system with the use of a TFF-Evs filter (HansaBioMed, 50nm pores size) followed by a concentration step using a centrifugal concentrator (Am icon).

[0182] Figure 3 shows the compositions comprising EVs / exosomes derived from 2D cell cultures in proliferation medium (MP) vs EVs / exosomes derived from the 3D structures (EXO2) produced as described in Example 1 , after isolation using either ultracentrifugation (UC2) or TFF protocols according to the methods described in Figure 2.

[0183] Statistical significances were tested by Kruskal-Walli’ s test. *, p- O.05 and ns, not significant. Black dots: values of individual batches (biological replicates). Each chart represents the average value of the data from all batches tested. MP: EVs / exosomes derived from 2D cell cultures in proliferation medium; EXO2: EVs / exosomes derived from the 3D structures; UC2: isolation protocol based on ultracentrifugation; TFF: isolation protocol based on tangential flow filtration.

[0184] Figure 3A shows the yield of isolated particles at the end of the manufacturing process. All the values represent the number of isolated vesicles normalized to the initial supernatant collected from the respective cultures. A significant enrichment of the exosomes yield was observed for 3D-derived exosomes (EXO2), compared to exosomes derived from 2D cultures (MP), regardless the isolation method. A better yield was observed in case of TFF protocol.

[0185] Figure 3B Figure 3B shows the protein concentration in the respective compositions. All the values represent the mg of total protein per mL of the final volume in each compositions. A significant enrichment of the protein content was observed for 3D-derived exosomes (EXO2) isolated with TFF protocol, compared to exosomes derived from 2D- cultures (MP), or exosomes isolated with UC2 protocol.

[0186] Figure 3C shows the purity index for each compositions. All the values represent the No of particles per mg of total protein. A significant enrichment of the EVs purity was observed for 3D-derived exosomes (EXO2) isolated with UC2 protocol, compared to exosomes derived from 2D-cultures (MP), or exosomes isolated with TFF protocol.

[0187] Figure 4 shows the particle size in nm. The average particle size of all samples was found in the range of the expected size for exosomes, regardless the isolation method. Significant batch consistency was observed.

[0188] Statistical significances were tested by ANOVA-I. *, p- O.05 and ns, not significant. Black dots: values of individual batches (biological replicates). Each chart represents the average value of the data from all batches tested. MP: EVs / exosomes derived from 2D cell cultures in proliferation medium; EXO2: EVs / exosomes derived from the 3D structures; UC2: isolation protocol based on ultracentrifugation; TFF: isolation protocol based on tangential flow filtration.

[0189] Figure 5 shows the tetraspanin marker expression profile in each compositions. A clear enrichment of the tetraspanin levels was observed for 3D-derived exosomes (EXO2), compared to exosomes derived from 2D-cultures (MP). Significant higher levels of CD63 tetraspanins were observed in EXO2 when UC2 isolation method applied compared to TFF.

[0190] Statistical significances were tested by ANOVA-I when values passed the normality tests (a =0.05) or by Kruskal-Walli ’ s test when values did not pass the normality tests (a=0.05). *, p - O.05; **; p^0.01 , ***; p- 0.001 ****, p^0.0001 ; ns, not significant. Black dots: values of individual batches (biological replicates). Each chart represents the average value of the data from all batches tested. MP: EVs / exosomes derived from 2D cell cultures in proliferation medium; EXO2: EVs / exosomes derived from the 3D structures; UC2: isolation protocol based on ultracentrifugation; TFF: isolation protocol based on tangential flow filtration.

[0191] Figure 5A shows the CD63 levels detected by ELISA in each composition.

[0192] Figure 5B shows the CD81 levels detected by ELISA in each composition.

[0193] Figure 5C shows the CD9 levels detected by ELISA in each composition.

[0194] Figure 6 shows the unsupervised clustering results of the miRNAs detected in the different compositions using Next-generation sequencing (NGS). RPM (Reads per million mapped reads) normalized gene expression data were used to perform hierarchical clustering. miRNAs that were found with less than 5 RPM in 50% of the samples are prefiltered and removed from the dataset. MP: EVs / exosomes derived from 2D cell cultures in proliferation medium; EXO2: EVs / exosomes derived from the 3D structures; UC2: isolation protocol based on ultracentrifugation; TFF: isolation protocol based on tangential flow filtration.

[0195] Figure 6A shows the heatmap obtained from the unbiased miRNA transcriptom ic profiling. Sample dendrogram on miRNA data displayed a clear separation between 2D (MP) and 3D (EXO2) exosome samples.

[0196] MP, EXO2 and the isolation methods, TFF and UC2, are colored coded. The miRNA clustering tree is shown on the left, and the sample clustering tree is shown at the top. The color scale at the top right illustrates the relative expression level of miRNAs, with red indicating a higher expression level and blue a lower expression level.

[0197] Figure 6B shows principal component analysis (PCA) on the miRNA transcriptom ic data displaying a clear separation between 2D (MP) and 3D (EXO2) exosome samples.

[0198] MP and Exo-2 are colored coded, and the isolation method is indicated by the symbol.

[0199] Figure 7 shows the proteomic profiling obtained from the different tested compositions, HRMTMmass spectrometry for the unbiased quantification of all detectable peptides and proteins in each sample. The 3-dimensional, scaffold-free, extracellular matrix (ECM) seem to differentiate and stabilize the protein cargo of exosomes ensuring higher batch- to-batch consistency. MP: EVs / exosomes derived from 2D cell cultures in proliferation medium; EXO2: EVs / exosomes derived from the 3D structures; UC2: isolation protocol based on ultracentrifugation; TFF: isolation protocol based on tangential flow filtration. Figure 7A shows the heatmap derived from the unsupervised clustering of the proteomic data obtained from the tested compositions. Sample dendrogram on proteomic data displayed a clear separation between 2D (MP) and 3D (EXO2) exosome samples.

[0200] MP, EXO2 and the isolation methods, TFF and UC2, are colored coded. .

[0201] Figure 7B shows the total number of identified proteins in each tested composition, displaying lower variability in the number of total protein No identified in the 3D-derived exosomes (EXO2) compared to the exosomes derived from 2D cultures (MP). .

[0202] Figure 8 shows the potency assessment for anti-tumor activity of 3D-derived exosomes (EXO2) in comparison with exosomes from 2D culture (MP) in 143B osteosarcoma cells. A significant decrease of the viability / proliferation of 143B cells was highlighted when the cells were treated with the higher dose of ultracentrifugation (UC2)-isolated (with 74.60 ± [33.57] % viability inhibition) or tangential flow filtration (TFF)-isolated (with 76.75 ± [35.08] % viability inhibition) EXO2 samples, while a dose-response effect was observed. In addition, the 3-dimensional, scaffold-free, neosynthesized extracellular matrix (ECM) seems to improve the anti-tumor activity of the secreted exosomes (MP vs EXO2).

[0203] Graphs represent the percentage of cell viability normalized to untreated cells. 0.1 pM doxorubicin (Doxo 0.1 mM) was used as positive control. Red line represents 100% of viability. Statistical significances were tested by ANOVA-I when values passed the normality tests (a =0.05) or by Kruskal-Walli ’ s test when values did not pass the normality tests (a=0.05). *, p- O.05; ***, p^0.001 ****, p^0.0001 ; ns, not significant. Black dots: values of individual batches (biological replicates). Each chart represents the average value of the data from all batches tested. MP: EVs / exosomes derived from 2D cell cultures in proliferation medium; EXO2: EVs / exosomes derived from the 3D structures; UC2: isolation protocol based on ultracentrifugation; TFF: isolation protocol based on tangential flow filtration.

[0204] Figure 8A shows the viability of 143B cells after 144h of treatment with two doses (1 E+08 and 1 E+09 No particle / mL) of 3D-derived (EXO2) or2D-derived exosomes (MP) that have been isolated with UC2 protocol.

[0205] Figure 8B shows the viability of 143B cells after 144h of treatment with two doses (1 E+08 and 1 E+09 No particle / mL) of 3D-derived (EXO2) or2D-derived exosomes (MP) that have been isolated with TFF protocol. Figure 9 shows the impact on the viability of A673 Ewing sarcoma cells when the cells were treated for 144h with two doses (1 E+08 and 1 E+09 No particle / mL) of 3D-derived (EXO2) exosomes that have been isolated with tangential flow filtration (TFF) isolation method. A significant decrease of the viability / proliferation of A673 cells was highlighted when the cells were treated with the higher dose of TFF-isolated EXO2 sample (with 90.75± [19.50] % viability inhibition), while a dose-response effect was observed.

[0206] Graphs represent the percentage of cell viability normalized to untreated cells. 0.1 pM doxorubicin (Doxo 0.1 mM) was used as positive control. Red line represents 100% of viability. Statistical significances were tested by ANOVA-I. **, p- 0.01 ; ns, not significant. Black dots: values of individual batches (biological replicates). Each chart represents the average value of the data from all batches tested. TFF: isolation protocol based on tangential flow filtration.

[0207] Figure 10 shows the impact on the viability of non-tumor human bone marrow mesenchymal stem cells (hBM-MSCs) when the cells were treated for 144h with two doses (1 E+08 and 1 E+09 No particle / mL) of 3D-derived (EXO2) exosomes that have been isolated with different isolation methods: ultracentrifugation (UC2) or tangential flow filtration (TFF). UC2-isolated EXO2 significantly impacted the viability of hBM-MSC (76.20±[32.68]% viability inhibition for 1 E+08 particles / mL and 71.40±[39.20]% viability inhibition for 1 E+09 particles / mL), whereas TFF-isolated EXO2 did not show any significant cytotoxic effect on these non-tumor cells .

[0208] Graphs represent the percentage of cell viability normalized to untreated cells. 0.1 pM doxorubicin (Doxo 0.1 pM) was used as positive control. Red line represents 100% of viability. Statistical significances were tested by ANOVA-I. **, p- O.01 ; ***, p- 0.001 ; ns, not significant. Black dots: values of individual batches (biological replicates). Each chart represents the average value of the data from all batches tested. UC2: isolation protocol based on ultracentrifugation; TFF: isolation protocol based on tangential flow filtration.

[0209] Figure 11 shows the induction of apoptosis in osteosarcoma 143B cell line upon the treatment the following compositions: exosomes from 2D-cultures (MP), exosomes derived from the 3D scaffold-free structures (EXO-2). A clear induction of early apoptosis was observed after the treatment with EXO-2 in 143B cells, which appeared to be significantly higher than the one observed in case of EXO2 derived from 2D-culture. Graphs representing the fold induction in the treated samples normalized to untreated cells. 1 .0 and 5.0 pM doxorubicin were used as positive control. MP: EVs / exosomes derived from 2D cell cultures in proliferation medium; EXO2: EVs / exosomes derived from the 3D structures; UC2: isolation protocol based on ultracentrifugation; TFF: isolation protocol based on tangential flow filtration.

[0210] Figure 11 A shows the results for the exosomes isolated with UC2.

[0211] Figure 11 B shows the results for the exosomes isolated with TFF.

[0212] EXAMPLES

[0213] Example 1: Manufacturing of the composition of the invention

[0214] NVDM2 is an allogenic / off-the shelf product, currently developed by Novadip Biosciences: and is derived from the autologous product NVD002 process. NVD-002 is produced from human Adipose-derived Stem Cells (hASCs) differentiated into osteogenic cells combined with gelatine particles derived from porcine skin (Cultispher) to produce a « scaffold-free » 3D grafts.

[0215] 1.1 Upstream process:

[0216] The manufacturing process of the active substance starts after the tissue procurement as shown in Figure 1 comprising:

[0217] ■ Stem cell collection;

[0218] ■ Stem cell proliferation;

[0219] ■ Osteogenic differentiation;

[0220] ■ Particles sprinkling; and

[0221] ■ 3D-structure formation

[0222] The upstream process consists of the following 3 phases:

[0223] 1.1.1 Upstream process - phase 1 :

[0224] Isolation of the stromal vascular fraction (SVF) cells from the adipose tissue and subsequent expansion of human adipose-derived stem cells (hASCs) up to passage p3 / p4 in proliferative medium (MP).The cells are expanded to cell stocks. The cell stock is cryopreserved.

[0225] 1.1.2 Upstream process - phase 2: Manufacturing of three-dimensional cell product which includes the following steps: Thawing of ASCs: The first step of NVDM2 cell products manufacturing process is the cell thawing at P4 from the cell stock. Cells are thawed and inoculated into 150- cm2flasks in proliferation medium and rinsed the day after.

[0226] Proliferation phase: After that, the cells proliferate until they reach a confluency 70% and ^100%, before performing the passage P4 / P5.

[0227] Passage P4 / P5 and osteogenic induction phase: At the passage P4 / P5, the cells collected from all the flasks are pooled and then seeded in T 150cm2flasks with re- closable lid (“TPP” flask) in osteogenic differentiation medium (MD) at a cell seeding density between 0,5*104 cell / cm2and 0,8*104 cell / cm2

[0228] When cells reach a confluency and at least one osteoid nodule (un-mineralized, organic portion of the bone matrix that forms prior to the maturation of bone tissue) is observed in each flask, the addition of gelatin beads can be launched.

[0229] Addition of Cultispher particles: After being exposed to the osteogenic differentiation medium (MD), the culture vessels containing the confluent monolayer of adherent osteogenic cells are sprinkled with gelatin beads .

[0230] 1.1.3 Three-dimensional induction phase:

[0231] Few days after the addition of the gelatin beads, the differentiated cells and the particles dispersed become progressively entombed in mineralizing the neosynthesized extracellular matrix. At this point, the differentiated cells and gelatin beads particles start forming a large 3-dimensional patch (or few smaller patches) of partially mineralized brownish-yellow moldable putty detaching from each culture vessels.

[0232] The formation of the final three-dimensional cell product formation is obtained at the end of a maturation period. This cell product (CP) is frozen before downstream processing.

[0233] Downstream process: Exosome seperation through TFF

[0234] The supernatant obtained from the scaffold-free multi-dimensional cell culture is subjected to TFF.

[0235] The pore size of the TFF is chosen to allow for an efficient separation of the extracellular vesicles from the supernatant of the scaffold-free multi-dimensional cell culture. An exemplary process of the present process is shown in Figures 1 to 3. The TFF may be combined with further separation or concentration steps, such as centrifugation.

[0236] Example 2: Potency Bioassays to assess the anti-tumor activity of the product on Osteosarcoma cell lines.

[0237] The anti-tumor activity of matrisome was tested on three stable osteosarcoma cells lines: 143B, LI2OS and SaOS-2. Human bone marrow mesenchymal cells (hBM-MSCs) were also used in the study to investigate whether the potential anti-proliferative effect of matrisome is targeted only to OS cells.

[0238] Cell Culture

[0239] 143B cells were maintained in MEM medium containing 10% foetal bovine serum (FBS), with penicillin, streptomycin and amphotericin B. LI2OS, SaOS-2 and hBM-MSC were maintained in DMEM medium containing 10% fetal bovine serum (FBS), with penicillin, streptomycin and amphotericin B.

[0240] Viability bioassays

[0241] Cell viability was measured using CCK-8 assay

[0242] For this assay the Cell Counting Kit-8 Kit was used (Sigma-Aldrich). This viability assay is based on the reduction of tetrazolium salt (WST-8) by dehydrogenases within cells which gives an orange product soluble in the cell culture medium. This coloured product (formazan) is measured using a spectrophotometer. The formazan is directly proportional with the number of living cells presents in the cell culture.

[0243] Cells were seeded in 96-wells plates and allowed to attach for 24h.

[0244] After O / N incubation, the medium was removed, and the cells were incubated for 144h in the presence of the different doses of the test item . Doxorubicin treatment was also applied to each cell line as a reference compound to demonstrate that the system is able to detect inhibition of viability. The treatments were carried out in triplicates. After 144h of incubation, the 96-wells plate was removed from the incubator, washed with 2X PBS and 200pl of medium containing 20pl of CCK8 reagent was added in each well. The plate was incubated at 37°C for 2 hour and then absorbance at 450 nm was measured. Transcriptomics analysis

[0245] The exosomes obtained by the scaffold-free 3D cultures are highly enriched in miRNAs, in particular miR-27a-3p, miR-34b-5p, miR-34c-5p, miR-125a-5p with tumor suppressive activity. Transcriptomics analysis

[0246] The exosomes obtained by the scaffold-free 3D-cultures are highly enriched in miRNAs with tumor suppressive activity, in particular miR-27a-3p, miR-34b-5p, miR-34c-5p, miR- 125a-5p.

[0247] MP: EVs / exosomes derived from 2D cell cultures in proliferation medium; EXO2: EVs / exosomes derived from the 3D structures; UC2: isolation protocol based on ultracentrifugation; TFF: isolation protocol based on tangential flow filtration.

[0248] Table 1 shows the list of miRNAs that were found at least 2-fold enriched in the exosomes derived from the 3D structures (EXO2) compared to the exosomes from the 2D-cultures (MP), when tangential flow filtration (TFF) isolation protocol was used. Table 2: shows the list of miRNAs that were at least 2-fold enriched in the exosomes derived from the 3D-structures (EXO2) compared to the exosomes from the 2D-cultures (MP), when ultracentrifugation (UC2) isolation protocol was used.

[0249]

[0250] Table 1

[0251]

[0252] Table 2

Claims

CLAIMS1. A composition comprising extracellular vesicles for use in the treatment or prevention of cancer, in particular for inducing the apoptosis in cancer cells, in particular osteosarcoma, wherein the composition is obtainable according to a process comprising the following steps:■ Culturing of stem cells, preferably adipose-derived stem cells;■ Differentiating the stem cells, preferably osteogenically or chondrogenically differentiating the stem cells;■ Adding of a particulate material, preferably gelatin beads, to obtain a scaffold-free multi-dimensional cell culture;■ Collecting the liquid containing extracellular vesicles from the scaffold-free multi-dimensional cell culture;■ Subjecting the liquid containing the extracellular vesicles to tangential flow filtration (TFF).

2. The composition for use according to claim 1 , wherein the TFF device comprises a filtration membrane having a pore size ranging from 10 nm to 100 nm, preferably from 25 nm to 75 nm, even more preferably from preferably from 40 nm to 60 nm.

3. The composition for use according to any one of the preceding claims, the number of extracellular vesicles in the composition comprising the extracellular vesicles or obtainable according to the process of the present invention is from 1.1011to 1.1013, preferably from 5.1011to 1.1013and even more preferably 5.1011to 5.1012per ml of the composition as measured by nanoparticle tracking analysis (NTA).

4. The composition for use according to any one of the preceding claims, the number of extracellular vesicles in the liquid containing extracellular vesicles prior to the TFF step is from 1.108to 1.1010, preferably from 5.108to 5.109and even more preferably 7.108to 3.109per ml of the composition as measured by nanoparticle tracking analysis (NTA).

5. The composition for use according to any one of the preceding claims, wherein the protein concentration in the composition of the invention is from 1000 micrograms / mL to 15000 micrograms / mL, preferably from 2000 micrograms / mLto 15000 micrograms / mL, even more preferably from 2000 micrograms / mL to 10000 micrograms / mL.

6. The composition for use according to any one of the preceding claims, wherein the composition has a purity index as measured by the number of extracellular vesicles per microgram protein of from 1.107to 1.109, preferably from 5.107to 1 .109, even more preferably from 5.107to 5.108.

7. The composition for use according to any one of the preceding claims, wherein the TFF is used to reduce the amount or level of liquid from the composition comprising the extracellular vesicles.

8. The composition for use according to any one of the preceding claims, wherein the TFF is used to remove from 50% to 99.9999%, preferably from 70% to 99.9999%, even more preferably from 90% to 99.9999% of the liquid from the composition.

9. The composition for use according to any one of the preceding claims, wherein the differentiated cells neo-synthesize an extracellular matrix through the addition of the particulate material.

10. The composition for use according to any one of the preceding claims, wherein the mature cells and the particulate material are embedded in the neo-synthesized extracellular matrix.

11. The composition for use according to any one of the preceding claims, wherein the differentiated cells are selected from the group consisting of osteoblasts, osteocytes, chondroblasts, chondrocytes, keratinocytes, myofibroblasts, epithelial cells, endothelial cells, adipocytes, neural cells, and precursors thereof, and preferably are soft tissue cells, chondroblasts or osteoblasts.

12. The composition for use according to any one of the preceding claims, wherein the particulate material is selected from the group consisting of:■ an organic material, including demineralized bone matrix (DBM), gelatin, agar / agarose, alginates chitosan, chondroitin sulfate, collagen, elastin, or elastin-like peptides (ELP), fibrinogen, fibrin, fibronectin, proteoglycans, heparan sulfate proteoglycans, hyaluronic acid, polysaccharides, laminins and cellulose derivatives; preferably gelatin;■ a calcium compound including particles of calcium phosphate, and even more preferably hydroxyapatite (HA) and / or [3-tricalcium phosphate ( - TCP);■ a polymer, including polyanhydrides, polylactic acid (PI_A), poly(lactic- co- glycolic acid) (PLGA), polyethylene oxide / polyethylene glycol (PEO / PEG), poly(vinyl alcohol) (PVA), fumarate-based polymers such as, for example polypropylene fumarate) (PPF) or polypropylene fumarate-co-ethylene glycol) (P(PF-co-EG)), oligopolypthylene glycol) fumarate) (OPF), poly(aldehyde guluronate) (PAG), polyp- vinyl pyrrolidone) (PNVP), or combinations thereof;■ a gel, including a self-assembling oligopeptide gel, a microgel, a nanogel, a particulate gel, a hydrogel, a thixotropic gel, a xerogel, a responsive gel, or combinations thereof;■ a creamer; and ■ any combinations thereof.

Images