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WO2025073913A1 - Composition comprenant des vésicules extracellulaires et leur préparation - Google Patents

Composition comprenant des vésicules extracellulaires et leur préparation Download PDF

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Publication number
WO2025073913A1
WO2025073913A1 PCT/EP2024/077972 EP2024077972W WO2025073913A1 WO 2025073913 A1 WO2025073913 A1 WO 2025073913A1 EP 2024077972 W EP2024077972 W EP 2024077972W WO 2025073913 A1 WO2025073913 A1 WO 2025073913A1
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Prior art keywords
extracellular vesicles
centrifugation
platelet
density gradient
medical composition
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Antoine Turzi
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Regen Lab SA
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Regen Lab SA
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/19—Platelets; Megacaryocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0644—Platelets; Megakaryocytes

Definitions

  • the present invention relates to an improved and standardized process for isolating extracellular vesicles such as exosomes, in particular platelet-derived extracellular vesicles such as exosomes. Centrifugation containers for use in the improved process are also provided.
  • the isolated extracellular vesicles have potential utility as therapeutic agents, as biomarkers for diagnostic, prognostic and/or monitoring applications, and as research tools.
  • Platelet-rich plasma can be defined as an autologous concentrate of platelets in a small volume of plasma. It has been developed as an autologous biomaterial and has proven to be useful in the healing and regeneration of tissues (Marx et al., 2004). PRP not only comprises a platelet concentrate but also contains growth factors (such as platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor (TGF) and epidermal growth factor (EGF)) that are actively secreted by platelets and are known to have a fundamental role in the wound healing initiation process. PRP is used in various medical (both therapeutic and cosmetic) applications, in particular in wound and tissue healing.
  • PDGF platelet-derived growth factor
  • VEGF vascular endothelial growth factor
  • TGF transforming growth factor
  • EGF epidermal growth factor
  • a process for preparing a biological sample for analysis for the presence of extracellular vesicles comprising the steps of: a-1) collecting a bodily fluid or a cell extract in a centrifugation container comprising a density gradient medium; a-2) centrifuging the centrifugation container to form an enriched fraction; a-3) collecting at least a portion of the enriched fraction; b-1) subjecting the collected enriched fraction to a process for isolating extracellular vesicles; b-2) preparing a biological sample comprising the isolated extracellular vesicles.
  • the bodily fluid is whole blood
  • the enriched fraction is platelet-rich plasma
  • the biological sample comprises platelet-derived extracellular vesicles, in particular platelet-derived exosomes.
  • the density gradient medium is a thixotropic gel with density between about 1 .045 g/cm 3 and about 1 .055 g/cm 3 .
  • a process for preparing a medical composition comprising extracellular vesicles comprising the steps of: a-1) collecting a bodily fluid or a cell extract in a centrifugation container comprising a density gradient medium; a-2) centrifuging the centrifugation container (suitably of step a-1)) to form an enriched fraction; a-3) collecting at least a portion of the enriched fraction (suitably of step a-2)); b-1) subjecting the collected enriched fraction (suitably of step a-3)) to a process for isolating extracellular vesicles; b-2) preparing a medical composition comprising the isolated extracellular vesicles (suitably of step b-1)).
  • step a-1) is directly followed by step a-2) (i.e. there are no intervening steps).
  • step a-2) is directly followed by step a- 3) (i.e. there are no intervening steps).
  • step a-3) is directly followed by step b-1) (i.e. there are no intervening steps).
  • step b- 1) is directly followed by step b-2) (i.e. there are no intervening steps).
  • a process for preparing a medical composition comprising platelet-derived extracellular vesicles, the process comprising the steps of: a-1) collecting whole blood in a centrifugation container comprising a density gradient medium; a-2) centrifuging the centrifugation container (suitably of step a-1)) to form a platelet-rich plasma fraction; a-3) collecting at least a portion of the platelet-rich plasma fraction (suitably of step a-2)); b-1) subjecting the collected platelet-rich plasma fraction (suitably of step a-3)) to a process for isolating platelet-derived extracellular vesicles; b-2) preparing a medical composition comprising the isolated platelet-derived extracellular vesicles (suitably of step b-1)).
  • the density gradient medium is a thixotropic gel with density between about 1 .045 g/cm 3 and about 1 .055 g/cm 3 .
  • EVs may have potential therapeutic applications, their presence can be indicative of a pathological state.
  • EVs and their cargo can be used as a diagnostic tool.
  • the present inventors have discovered that the process of the invention provides highly pure EVs, in samples that are reproducible and can be used for quantitative analysis e.g. for DNA and proteins (see Example 4).
  • the isolated EVs have also been found to be particularly stable in solution.
  • EVs prepared using a centrifugation container as described herein containing a density gradient medium are particularly suitable as biomarkers in diagnostic, prognostic and/or monitoring applications.
  • the EVs prepared using a centrifugation container as described herein containing a density gradient medium are also useful for more general research purposes e.g. further investigations of the role of EVs in cellular communication, the role of subpopulations of EVs, discovering new subpopulations, all of which require improved EV isolation and separation techniques.
  • a process for preparing a biological sample for analysis for the presence of extracellular vesicles comprising the steps of: a-1) collecting a bodily fluid or a cell extract in a centrifugation container comprising a density gradient medium; a-2) centrifuging the centrifugation container (suitably of step a-1)) to form an enriched fraction; a-3) collecting at least a portion of the enriched fraction (suitably of step a-2)); b-1) subjecting the collected enriched fraction (suitably of step a-3)) to a process for isolating extracellular vesicles; b-2) preparing a biological sample comprising the isolated extracellular vesicles (suitably of step b-1)).
  • the density gradient medium is a thixotropic gel with density between about 1 .045 g/cm 3 and about 1 .055 g/cm 3 .
  • the process is for preparing a medical composition or a biological sample comprising platelets and platelet-derived extracellular vesicles. In other embodiments of the processes above, the process is for preparing a medical composition or a biological sample comprising platelet-derived extracellular vesicles, but essentially no platelets, or no platelets.
  • EVs are lipid-bilayer-delimited particles that are naturally released by all types of cells, but unlike a cell, cannot replicate. EVs are typically divided according to size and synthesis route into various subtypes including exosomes, microvesicles and apoptotic bodies. For the avoidance of doubt, in the context of the present invention, platelets are not considered to be EVs.
  • the medical composition or biological sample isolated by the process of the invention comprises exosomes.
  • the medical composition or biological sample isolated by the process of the invention comprises platelet-derived exosomes.
  • other EVs may also be isolated, but exosomes must be present.
  • Exosomes are produced in the endosomal compartment of most eukaryotic cells, and are formed through the inward budding of a late endosome, also known as a multivesicular body (MVB).
  • the intraluminal vesicles (ILVs) of the MVB bud inward into the endosomal lumen. If the MVB fuses with the plasma membrane of the cell surface, the ILVs are released as exosomes.
  • exosomes typically, exosomes have a diameter of between about 30 nm and about 150 nm.
  • step a-2 involves centrifuging the centrifugation container to form an enriched fraction.
  • the nature of the enriched fraction will depend on the material being centrifuged. Centrifugation separates the components of a solution based on their density, such that particles with a higher density move towards the bottom of the container (distal end), while those of a lower density move towards the top of the container (proximal end).
  • the “enriched fraction” can be described as a fraction which, following centrifugation, contains a higher concentration of particular cells than the bodily fluid or cell extract that was centrifuged.
  • the cells are cells from which EVs can be derived.
  • the process of the invention uses a centrifugation container containing a density gradient medium which separates components of the bodily fluid or cell extract based on their density.
  • the enriched fraction is defined as the fraction residing above the density gradient medium, following centrifugation.
  • the centrifugation in step a-2) is performed at a force which is sufficient, over a particular length of time, to form a barrier between the components of the bodily fluid that are desired to be separated e.g. when the bodily fluid is whole blood, this is the force/time which results in the density gradient medium separating the erythrocytes from the platelets and any other desirable whole blood components.
  • the centrifugation in step a-2) is performed at a force of between about 700 g and about 2800 g, e.g. at a force of between about 1500 g and about 2800 g, between about 1500 g and about 2500 g, such as between about 1500 g and about 2000 g.
  • the centrifugation in step a-2) is performed for a period of time between about 3 minutes and about 40 minutes, e.g. between about 3 minutes and about 15 minutes.
  • step a-3 It can be beneficial to remove a proportion of this PPP, before step a-3) e.g. removing between about half and about one third of the enriched fraction (from the top end of the centrifugation container, i.e. from the end furthest from the density gradient medium, the proximal end), thereby removing some or all of the PPP layer, leading to an enriched fraction which is even more concentrated, or enriched.
  • step a-2) comprises the steps of: a-2a) centrifuging the centrifugation container (suitably of step a-1)) to form an enriched fraction; a-2£>) removing and discarding at least a portion of the enriched fraction (suitably of step a- 2a)).
  • step a-3 is collecting at least a portion of the remaining enriched fraction (i.e. the even more concentrated, or enriched fraction).
  • step a-2) comprises the steps of: a-2a) centrifuging the centrifugation container (suitably of step a-1)) to form a platelet-rich plasma fraction; a-2£>) removing and discarding at least a portion of the platelet-rich plasma fraction (suitably of step a-2a)).
  • step a-3 is collecting at least a portion of the remaining platelet-rich plasma fraction (i.e. which is even more concentrated, or enriched, in platelets).
  • step a-2a is directly followed by step a-2£>) (i.e. there are no intervening steps).
  • the centrifugation container of the present invention has been sterilized.
  • the centrifugation container is steam sterilized, at a temperature greater than 100 °C, such as greater than 110 °C, greater than 115 °C, greater than 120 °C or greater than 121 °C.
  • the step of isolating EVs (such as platelet-derived EVs) from the enriched fraction (such as the platelet-rich plasma fraction) is carried out by any suitable method.
  • EV isolation techniques typically rely on separation of the EVs based on size, charge, affinity, density, and/or dispersity alteration. It may be necessary to use a combination of these techniques to reach the required level of EV purity (Liangsupree et al., 2021).
  • SEC size exclusion chromatography
  • UF ultrafiltration
  • HFD hydrostatic filtration dialysis
  • microfluidics microfluidics
  • SEC is a well-established technique to separate molecules based on their molecular size or hydrodynamic volumes.
  • a typical SEC system consists of a porous stationary phase for chromatographic separation with or without coupling to a pump for elution.
  • SEC may be particularly suitable for isolating heterogenous populations of EVs, followed by a second technique to separate the EV subpopulations.
  • a two-stage SEC process can be used to separate smaller EVs such as exosomes, e.g. a two-dimensional SEC which uses two SEC columns of different pore size.
  • UF is another well known technique, typically used to isolate EVs from relatively dilute solutions.
  • UF devices generally consist of a UF membrane inserted in a container.
  • UF includes centrifugal UF, tangential flow filtration (TFF) and hydrostatic filtration dialysis (HFD). Sequential UF steps can increase the purity of isolated EVs.
  • Hydrostatic filtration dialysis uses hydrostatic forces for isolation. Small particles diffuse through the membrane, while large ones remain in the tube.
  • Deterministic lateral displacement (DLD) pillar array is a microfluidic technique that separates EVs based on their trajectories in a pillar array.
  • ion-exchange techniques such as anion-exchange chromatography (AIEC), metal-affinity based ion exchange, electrophoresis, diaelectrophoresis and ion concentration polarisation.
  • AIEC anion-exchange chromatography
  • metal-affinity based ion exchange metal-affinity based ion exchange
  • electrophoresis electrophoresis
  • diaelectrophoresis ion concentration polarisation
  • Anion exchange techniques exploit the interactions between negatively charged EV membrane components whose charges have been determined by zeta potential and an anion exchanger with positively charged functional groups or cations.
  • bound EVs can be released by introducing high salt concentrations to increase the ionic strength of the buffer to promote the desorption of EVs from positively charged media.
  • a process for preparing a medical composition comprising extracellular vesicles comprising the steps of: a-1) collecting a bodily fluid or a cell extract in a centrifugation container comprising a density gradient medium; a-2) centrifuging the centrifugation container to form an enriched fraction; a-3) collecting at least a portion of the enriched fraction; b-1) subjecting the collected enriched fraction to an ultracentrifugation process for isolating extracellular vesicles; b-2) preparing a medical composition comprising the isolated extracellular vesicles.
  • the ultracentrifugation force used to isolate the EVs will depend on the type of bodily fluid/cell extract being used.
  • the ultracentrifugation process of step b-1) is carried out at a force of between about 3,000 g and about 500,000 g, such as between about 5,000 g and about 500,000 g, for example between about 3,000 g and 250,000 g e.g. between about 5,000 g and 150,000 g.
  • the ultracentrifugation process of step b-1) is performed at a force of between about 3,000 g and about 5,000 g e.g. about 4,000 g.
  • the ultracentrifugation process of step b-1) is performed at a force of between about 6,000 g and about 15,000 g e.g. about 10,000 g. In one embodiment, the ultracentrifugation process of step b-1) is performed at a force of between about 10,000 g and about 120,000 g e.g. about 100,000 g.
  • Step b-1) may involve more than one ultracentrifugation step e.g. may involve two or more, such as two ultracentrifugation steps. For example, a first ultracentrifugation may be carried out at a relatively lower force, and a second ultra centrifugation may be carried out at a relatively higher force.
  • step b-1) involves: b-1) subjecting the collected enriched fraction to a process for isolating extracellular vesicles, comprising: b-1 a) subjecting the collected enriched fraction (suitably of step a-3) to a first ultracentrifugation process; and b-1 £>) subjecting the fraction of step b-1 a) to a further ultracentrifugation process.
  • the further ultracentrifugation process of step b-1 £>) is carried out at a greater force that the ultracentrifugation process of step b-1 a).
  • the ultracentrifugation process of step b-1 a) is carried out at a force of between about 6,000 g and about 25,000 g, e.g. between about 7,500 g and about 15,000 g, such as about 10,000 g.
  • the ultracentrifugation process of step b-1 £>) is carried out at a force of between about 50,000 g and about 200,000 g, e.g. between about 5,000 g and about 125,000 g, such as about 100,000 g.
  • Step b-1) may involve one or more centrifugation steps prior to the ultracentrifugation step(s) e.g. to remove cells such as dead cells and apoptotic bodies.
  • step b-1) involves: b-1) subjecting the collected enriched fraction to a process for isolating extracellular vesicles, comprising: b-1 a) subjecting the collected enriched fraction (suitably of step a-3)) to one or more centrifugation processes; and b-1 £>) subjecting the fraction of step b-1 a) to one or more ultracentrifugation process.
  • step b-1 a) the centrifugation process/processes is/are typically carried out using a force of between about 250 g and about 4,000 g. Suitable forces for the ultracentrifugation process/processes of step b-1 £>) are described in embodiments above.
  • step b-1 a) involves a first centrifugation process at a force between about 250 g and about 750 g, e.g. about 300 g, followed by a further centrifugation process at a force between about 2,000 g and about 4,000 g, e.g. about 3,000 g.
  • the centrifugation container used in the ultracentrifugation process of step b-1) comprises a density gradient medium.
  • the centrifugation container used in the ultracentrifugation process of step b-1) comprises a density gradient medium which is a thixotropic gel. In another embodiment, the centrifugation container used in the ultracentrifugation process of step b-1) does not contain a density gradient medium. In another embodiment, the centrifugation container used in the ultracentrifugation process of step b-1) contains a density gradient medium which is not a thixotropic gel. In one embodiment where step b-1) comprises a centrifugation process (in addition to an ultracentrifugation process), the centrifugation container used in the centrifugation process comprises a density gradient medium, in particular a thixotropic gel. In another embodiment, the centrifugation container does not contain a density gradient medium, and in particular does not contain a thixotropic gel.
  • step b-2) is optional, if step b-1) of subjecting the enriched fraction to a process for isolating extracellular vesicles produces a medical composition/biological sample which is suitable for its intended use without any further processing i.e. the medical composition/biological sample is prepared simply by the EV isolation process.
  • Precipitation is a technique that separates EVs based on their dispersibility alteration.
  • a polymer such as polyethylene glycol (PEG) is used to form a mesh-like net that embeds EVs.
  • Charge-based precipitation techniques have also been developed to isolate EVs.
  • CN115200969A (incorporated by reference herein in its entirety) describes a method for separating EVs using a mixture of hydrophilic polymer, salt ion solutions and glycogen, together with centrifugation processes.
  • Flow cytometry uses light scattering to detect and measure physical and chemical characteristics of a population of cells or particles, and can also be used to separate EVs (Nolan et al., 2017).
  • the process (step b-1) for isolating extracellular vesicles is selected from the group consisting of size exclusion chromatography (SEC), microfiltration, ultrafiltration, flow field-flow fractionation, hydrostatic filtration dialysis (HFD), microfluidics, anion-exchange chromatography (AIEC), metal-affinity based ion exchange, electrophoresis, diaelectrophoresis, ion concentration polarisation, affinity-based isolation, ultracentrifugation (UF), density gradient centrifugation (DGC), a rate-zonal centrifugation technique, precipitation and flow cytometry; or a mixture thereof.
  • SEC size exclusion chromatography
  • HFD hydrostatic filtration dialysis
  • AIEC anion-exchange chromatography
  • metal-affinity based ion exchange metal-affinity based ion exchange
  • electrophoresis diaelectrophoresis
  • ion concentration polarisation affinity-based isolation
  • UF ultracentrifug
  • step a-1 The nature of the bodily fluid or cell extract that is collected in step a-1) of the processes of the invention will vary depending on the extracellular vesicles to be isolated, and also on the intended use of the medical composition or biological sample.
  • the bodily fluid is selected from the group consisting of urine, milk, saliva, nasal lavage, bronchoalveolar lavage fluid, synovial fluid, a cerebrospinal fluid, bone marrow, ascites, tear, stromal vascular fraction, semen, peritoneal dialysis effluent and amniotic fluid.
  • the bodily fluid or cell extract is autologous.
  • autologous (which may also be referred to as autogenic or autogenous) means a method wherein a single donor’s bodily fluid, tissue and/or cells are used and wherein the bodily fluid, tissue and/or cell extracted from this donor is intended for use on the same donor.
  • the bodily fluid or cell extract is allogenic.
  • allogenic means a method using bodily fluid, tissue and/or cells from one or more third parties for use on a different recipient.
  • a cell extract which may also be described as a cell culture or cell supernatant
  • reference to “a” cell extract is intended to encompass “at least one” cell extract, and combinations of cell extracts are also envisaged.
  • the cell extract (which is suitably autologous) is selected from an extract of keratinocytes, bone marrow, fibroblasts, periosteum or corneal cells, melanocytes and Langerhans cell, fat cells, muscle cells such as myoblasts and satellite cells, osteoblasts, chondrocytes, umbilical cord cells, stem cells, mesenchymal stem cells (MSCs), preadipocytes, pre-endothelial cells, Schwann cells or Achilles tendon cells, or any combination thereof.
  • Suitable processes for obtaining such cell extracts are described in W02008/023026A1 , which is incorporated by reference herein in its entirety.
  • the centrifugation container of the present invention comprises a density gradient medium.
  • Reference to “a” density gradient medium is intended to encompass “at least one” density gradient medium, and combinations of density gradient media are also envisaged.
  • Constituents of bodily fluids such as whole blood have specific densities, meaning that they can be separated from one another by gravity or by centrifugal force.
  • a primary function of the density gradient medium is to separate bodily fluid and cell supernatant components, on the basis of their density.
  • particles with a density which is higher than that of density gradient medium will move below the density gradient medium, and particles with a lower density will move above the density gradient medium.
  • the density gradient medium has a density which is intermediate to the density of particles in the bodily fluid or cell extract i.e. the bodily fluid or cell extract will contain particles of both higher density and lower density that the density gradient medium, such that the density gradient medium functions to separate at least a proportion of the bodily fluid or cell extract components.
  • the density gradient medium is preferably chemically inert to components deriving from the body, such as cells.
  • the density gradient medium is a thixotropic gel.
  • thixotropic gel is well known in the art, and refers to a gel that becomes more fluid as a result of agitation or pressure, in particular a gel having a viscosity which decreases as a result of agitation or pressure.
  • a thixotropic gel When used in a centrifugation container, a thixotropic gel is thick or solid under static conditions, but when subjected to centrifugal force becomes more fluid and can migrate within the container (e.g. tube).
  • the centrifugation container e.g. tube
  • the centrifugation container e.g. tube
  • a bodily fluid or cell supernatant e.g.
  • the components separate into layers depending on their density (those with a higher density moving towards the bottom of the container (e.g. tube), and those with a lower density moving towards the top of the container (e.g. tube)).
  • a container e.g. tube
  • the other components reflected by its density.
  • the thixotropic gel When centrifugation ends, the thixotropic gel regains its original thick or solid consistency, acting as a mechanical barrier between separate constituents having a density which is higher (these constituents will end up below the thixotropic gel) and those having a density which is lower (these constituents will end up above the thixotropic gel).
  • the mechanical barrier provided by the gel facilitates quick and easy separation of the different constituents, leading to greater consistency in the separation process, as human error is reduced or eliminated entirely.
  • the centrifugation container of the present invention can comprise a single density gradient media, or can comprise two, three, four or five density gradient media.
  • the density gradient medium comprises, consists essentially of or consists of a thixotropic gel.
  • the density gradient medium is a thixotropic gel.
  • the density gradient medium (e.g. thixotropic gel) has a density between about 1.045 g/cm 3 and about 1.095 g/cm 3 , such as between about 1.050 g/cm 3 and about 1.095 g/cm 3 , or between about 1.055 g/cm 3 and about 1.095 g/cm 3 .
  • platelets also known as thrombocytes
  • a density gradient medium with density between about 1 .045 and about 1 .095 g/cm 3 , (i.e.
  • Monocytes and lymphocytes (types of leukocytes which are also known as agranulocytes) have density of between about 1 .060 g/cm 3 and about 1 .075 g/cm 3 .
  • a density gradient medium e.g. thixotropic gel
  • density gradient medium i.e. density gradient medium with higher density
  • Basophils, neutrophils and eosinophils types of leukocytes which are also known as granulocytes
  • Basophils, neutrophils and eosinophils have density of between about 1.072 g/cm 3 and about 1.10 g/cm 3 .
  • using a density gradient medium with density between about 1 .070 g/cm 3 and about 1.090 g/cm 3 facilitates the preparation of agranulocyte-rich PRP (which is rich in monocytes and lymphocytes) while also being granulocyte poor (i.e. depleted levels of basophils, neutrophils and eosinophils).
  • this PRP is considered overall to be leukocyte-poor PRP (LP-PRP).
  • the thixotropic gel is typically a large polymer complex.
  • the thixotropic gel comprises, consists essentially of or consists of an oligomer or polymer selected from the group consisting of a polyolefin hydrocarbon oligomer, a polyester gel, an acrylic resin mixture, a silica (such as silica dimethyl silylate), a PEG-silica gel, a polyoxyalkylene polyol, trioctyl trimellitate, a hydrocarbonated resin, or any combination thereof.
  • Suitable polyoxyalkylene polyols include polyethylene glycol trimethylolpropane ether, polypropylene glycol trimethylolpropane ether, methyloxirane polymer with oxirane, ether with 2-ethyl-2-(hydroxymethyl)-1 ,3-propanediol; poly(oxyethylene) trimethylolpropane ether, poly(oxypropylene)trimethylolpropane ether, trimethylol propane, ethoxylated trimethylolpropane, propxylated trimethylol propane, or any combination thereof.
  • the polyoxyalkylene polyol preferably comprises hydroxyl group containing groups of formula 1 :
  • trioctyl trimellitate is tris(2-ethylhexyl) trimellitate.
  • Suitable hydrocarbonated resins include a cycloaliphatic hydrocarbon resin.
  • the thixotropic gel comprises two different polymers/oligomers selected from the group consisting of a polyolefin hydrocarbon oligomer, a polyester gel, an acrylic resin mixture, an oligomeric or polymeric silica (such as silica dimethyl silylate), a PEG-silica gel, a polyoxyalkylene polyol, trioctyl trimellitate, a hydrocarbonated resin.
  • the thixotropic gel comprises trioctyl trimellitate and/or a hydrocarbon resin, and in particular comprises trioctyl trimellitate and a hydrocarbon resin.
  • the thixotropic gel comprises three different polymers/oligomers selected from the group consisting of a polyolefin hydrocarbon oligomer, a polyester gel, an acrylic resin mixture, a silica (such as silica dimethyl silylate), a PEG-silica gel, a polyoxyalkylene polyol, trioctyl trimellitate, a hydrocarbon resin.
  • the thixotropic gel comprises four different polymers/oligomers selected from the group consisting of a polyolefin hydrocarbon oligomer, a polyester gel, an acrylic resin mixture, a silica (such as silica dimethyl silylate), a PEG-silica gel, a polyoxyalkylene polyol, trioctyl trimellitate, a hydrocarbon resin.
  • the medical composition prepared according to the process of the invention for use in wound healing, wound sealing, tissue regeneration, cartilage regeneration, and/or bone regeneration (Frazier et al., 2020; Shi et al., 2021).
  • the medical composition prepared according to the process of the invention for use in treating or preventing melasma.
  • the cosmetic use of a medical composition prepared according to the process of the invention, for treating or preventing melasma is provided the medical composition prepared according to the process of the invention, for use as an anti-aging agent.
  • the cosmetic use of a medical composition prepared according to the process of the invention, for antiaging is provided.
  • the medical composition prepared according to the process of the invention for use in organ transplantation.
  • the medical composition prepared according to the process of the invention for use in the treatment or prevention of cancer (Franco et al., 2015).
  • the medical composition prepared according to the process of the invention for use the treatment or prevention of postpartum haemorrhage, menorrhagia, trauma-associated haemorrhage, and surgical bleeding.
  • the medical composition prepared according to the process of the invention for use the treatment or prevention of coagulopathy, e.g. traumatic coagulopathy.
  • the medical composition prepared according to the process of the invention for use the treatment or prevention of post-inflammatory hyperpigmentation, dermal melanosis, rosacea, and telangiectasia.
  • the medical composition is suitably autologous.
  • an allogenic composition e.g. the addition of stem cell derived exosomes from a healthy donor could have a therapeutic application in certain pathological conditions.
  • the medical composition prepared according to the process of the invention for use as a biomaterial.
  • EVs are precise markers for the diagnosis, prognosis and monitoring of various pathologies.
  • a process for preparing a biological sample for analysis for the presence of extracellular vesicles it should be understood that this encompasses analysis of the presence of EVs and/or their characterization and/or quantitative analysis of EVs.
  • EVs are desirable markers for Alzheimer’s disease (AD), for instance increased levels of A
  • AD Alzheimer’s disease
  • EVs can also be used as markers for cancer.
  • Increased levels of certain miRNAs (let-7a, miR- 1 ,229, miR-1 ,246, miR-150, miR-21 , miR-223 and miR-23a) in exosomes have been found in early stages of colon cancer, and are considered to be biomarkers.
  • High presentation of miRNA141 and miRNA195 have been detected in circulating exosomes of early stage breast cancer patients.
  • the IncRNA content of exosomes can be indicative of cancer, for example lincRNA-P21 has been observed in the urine exosomes of prostate cancer patients (see also de Frietas et al., 2022 and Sjors et al., 2019).
  • miRNA-133a miRNA- 143/145, miRNA-150, miRNA-155, miRNA-214, miRNA-223 and miRNA320b have been identified in exosomes and considered as biomarkers of atherosclerosis.
  • miRNA- 1 and miRNA- 133a are altered in the circulatory system of patients with acute coronary syndromes and myocardial infarction, and exosomal miRNA with diagnostic potential are miRNA-34a, miRNA-146a, miRNA-92, miRNA-192 and miRNA-194 are associated with heart failure. See also Tan et al., 2016.
  • EVs can be used to diagnose and formulate a prognosis for infectious diseases.
  • Akt and CD9 have been found to increase in exosomes of patients with urinary tract infection, and can be identified as biomarkers.
  • Exosomal IncRNA-HEIH increases in chronic hepatitis C related hepatocellular carcinoma, and can be used as a biomarker.
  • Neurofilament-light, high mobility group box 1 and amyloid p are upregulated in circulating exosomes of HIV-infected patients and can indicated neuronal damage caused by the virus.
  • EVs have utility in cosmetic as well as therapeutic applications.
  • Nilforoushzadeh et al., 2021 describe a study introducing the potential positive effects of exosomes derived from adipose stem cells increasing the proliferation and survival of dermal papilla cells.
  • the medical composition prepared according to the process of the invention for a cosmetic use e.g. for treating or preventing hair loss.
  • platelet-derived extracellular vesicles prepared using the methods of the invention are particularly suitable for use in detecting markers such as DNA and proteins.

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Abstract

La présente invention concerne un procédé de préparation d'une composition médicale comprenant des vésicules extracellulaires, le procédé comprenant les étapes consistant : a-1) à collecter un fluide corporel ou un extrait cellulaire dans un récipient de centrifugation comprenant un milieu à gradient de densité; a-2) à centrifuger le récipient de centrifugation pour former une fraction enrichie; a-3) à collecter au moins une partie de la fraction enrichie; b-1) à soumettre la fraction enrichie collectée à un procédé d'isolement de vésicules extracellulaires; b-2) à préparer une composition médicale comprenant les vésicules extracellulaires isolées.
PCT/EP2024/077972 2023-10-05 2024-10-04 Composition comprenant des vésicules extracellulaires et leur préparation Pending WO2025073913A1 (fr)

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