Composition comprising extracellular vesicles, and the preparation thereof

Abstract

The present invention relates to a process for preparing a medical composition comprising extracellular vesicles, the process 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 medical composition comprising the isolated extracellular vesicles.

Description

[0001] COMPOSITION COMPRISING EXTRACELLULAR VESICLES, AND THE PREPARATION THEREOF

[0002] 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.

[0003] BACKGROUND OF INVENTION

[0004] The importance of biological autologous materials in the healing process has been well documented. Two biological autologous materials have been shown to be directly implicated in the formation of the structure of blood clots, which provide a haemostatic barrier whose role is to ensure haemostasis and seal the wound: (1) fibrin, which derives from the separation of plasma fibrinogen into two strands through the action of thrombin, and (2) the activated membranes of platelets. The wound healing process is generally presented as the succession of a coagulation phase, an inflammatory process and a regeneration process. The coagulation phase (blood clotting or clot formation) is a complex process whereby thrombin, formed during activation of the coagulation pathway generates insoluble cross-linked fibrils of the protein fibrin and causes the platelets to aggregate irreversibly. The resulting platelet-fibrin clot is an effective barrier against loss of blood from the vascular system and also serves as a scaffold for subsequent repair of the lining of the blood vessel.

[0005] Platelet-rich plasma (PRP) 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.

[0006] As well as the various growth factors secreted by platelets, extracellular vesicles (EVs) such as exosomes derived from platelets have recently emerged having potential therapeutic benefits in their own right. EVs are membranous, subcellular structures released by any cell type. EVs enable cell-to-cell communication, the effects of which can prevent disease, or conversely lead to a pathological state, depending on the nature of the producing cell, and how EV production is activated. The potential therapeutic effects of EVs derived from cell cultures of stem cells and immune cells have been studied most intensively, but research has been hindered by the high regulatory requirements of ex vivo cell expansion. The use of platelet-derived EVs is therefore attractive from the point of view of safety and regulatory concerns, because necessary volumes of platelets can be obtained from whole blood donations, avoiding the need for ex vivo cell expansion.

[0007] Platelet-derived EVs (pEVs) share a number of the functions of platelets, but have been shown to have more potent effects than platelets in a number of capacities. Furthermore, unlike platelets, pEVs such as exosomes can cross tissue barriers, therefore could have therapeutic effects in tissues where platelets are rarely found. The main fields in which the effects of pEVs have been evaluated include injuries, neurogenesis, muscle regeneration, angiogenesis, biomaterials, bone regeneration and osteoarthritis. pEVs have also been postulated to play a key role in homeostatic processes, and rat models studies suggest that pEVs prevent uncontrolled blood loss and hemorrhagic shock (Antich-Rossello et al., 2021).

[0008] However, the process of separating and isolating pEVs, and EVs in general, and exosomes in particular, is not currently standardized. The isolation methodology used can alterthe nature of EVs, and it can be difficult to obtain highly pure EV samples because lipoproteins are usually co-isolated with EVs (Antich-Rossello et al., 2021). This leads to a lack of reproducibility, which has hindered the development of EVs as therapeutic and diagnostic agents.

[0009] Thus, there is a need to develop a process for the isolation and purification of EVs, in particular pEVs, which produces highly pure and functional EVs in a reproducible manner.

[0010] SUMMARY OF THE INVENTION

[0011] According to a first aspect of the present invention, there is provided a process for preparing a medical composition comprising extracellular vesicles, the process 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 medical composition comprising the isolated extracellular vesicles. In a preferred embodiment of this aspect of the invention, the bodily fluid is whole blood, the enriched fraction is platelet-rich plasma, and the medical composition comprises platelet- derived extracellular vesicles, in particular platelet-derived exosomes. Preferably, the density gradient medium is a thixotropic gel with density between about 1 .045 g / cm3and about 1 .055 g / cm3.

[0012] According to a second aspect of the invention, there is provided a process for preparing a biological sample for analysis for the presence of extracellular vesicles, the process 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.

[0013] In a preferred embodiment of this aspect of the invention, the bodily fluid is whole blood, the enriched fraction is platelet-rich plasma, and the biological sample comprises platelet-derived extracellular vesicles, in particular platelet-derived exosomes. Preferably, the density gradient medium is a thixotropic gel with density between about 1 .045 g / cm3and about 1 .055 g / cm3.

[0014] DETAILED DESCRIPTION

[0015] Platelet-rich plasma (PRP) is typically produced by centrifugation of whole blood to separate the platelets from the red blood cells. A bone marrow PRP can be obtained by the same process. Additional centrifugation or separation steps may be used to remove the platelet poor plasma. As the cellular composition of PRP, and hence its therapeutic effect, is strongly dependent on the methodology and device used to prepare the PRP, standardization of production of PRP is essential.

[0016] Medical devices for obtaining standardized PRP have been developed, including devices of use in an automated procedure in a closed circuit. For example, centrifugation tubes produced by Regen Lab and described in W02008 / 023026A2, WO2011 / 110948A2,

[0017] WO2013 / 0613092A2, WO2016 / 083549A2, WO2019 / 155391 A1 , WO2021 / 198312A1 and WO2022 / 269035A1 (all incorporated by reference herein in their entirety). The present inventors have discovered that PRP prepared using a centrifugation container as described herein, containing a density gradient medium, is particularly suitable as a source of platelets for isolating platelet-derived extracellular vesicles (pEVs), such as platelet-derived exosomes. PRP prepared using a centrifugation container as described herein, using the processes described herein, contains healthy, undamaged populations of cells, from which highly pure pEVs can be isolated, in a reproducible manner. The high purity of the EVs makes them particularly suitable for therapeutic applications, and for use in quantitative analytical techniques.

[0018] Thus, in one aspect of the present invention is provided a process for preparing a medical composition comprising extracellular vesicles, the process 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)).

[0019] In all of the process embodiments described herein, suitably step a-1) is directly followed by step a-2) (i.e. there are no intervening steps). Suitably step a-2) is directly followed by step a- 3) (i.e. there are no intervening steps). Suitably step a-3) is directly followed by step b-1) (i.e. there are no intervening steps). In embodiments wherein step b-2) is present, suitably step b- 1) is directly followed by step b-2) (i.e. there are no intervening steps).

[0020] In a preferred embodiment is provided 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)). Suitably, the density gradient medium is a thixotropic gel with density between about 1 .045 g / cm3and about 1 .055 g / cm3.

[0021] As mentioned in the background of invention, while EVs may have potential therapeutic applications, their presence can be indicative of a pathological state. As such, 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. As such, 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.

[0022] Thus, in another aspect of the present invention is provided a process for preparing a biological sample for analysis for the presence of extracellular vesicles, the process 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)).

[0023] In a preferred embodiment is provided a process for preparing a biological sample for analysis for the presence of 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 biological sample comprising the isolated platelet-derived extracellular vesicles (suitably of step b-1)).

[0024] Suitably, the density gradient medium is a thixotropic gel with density between about 1 .045 g / cm3and about 1 .055 g / cm3.

[0025] In certain embodiments of the processes above, 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.

[0026] As described in the background of invention, 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.

[0027] In one embodiment, the medical composition or biological sample isolated by the process of the invention comprises exosomes. In the case of whole blood, the medical composition or biological sample isolated by the process of the invention comprises platelet-derived exosomes. In this embodiment, 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. Typically, exosomes have a diameter of between about 30 nm and about 150 nm.

[0028] EVs carry a cargo of proteins, nucleic acids, lipids, metabolites and even organelles from the parent cell. They inherit the contents and characteristics of their parent cells which may contain relevant information that can be used for disease detection, and the rate of EV secretion can be changed in various conditions, hence their utility in diagnostic, prognostic and monitoring applications. In the process of the invention, 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). Thus, 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.

[0029] When the bodily fluid is whole blood, red blood cells (erythrocytes) move to the bottom of the container (below the density gradient medium) and platelets move above the red blood cells, in plasma which contains higher concentrations of platelets (and typically other cell components) compared with the whole blood that was centrifuged. Thus, when the bodily fluid is whole blood, the enriched fraction is platelet-rich plasma. Put another way, the enriched fraction is plasma which is enriched in platelets.

[0030] In the process of the invention, 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. In one embodiment, 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. Suitably, 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.

[0031] When the bodily fluid is whole blood, the centrifugation at least separates the red blood cells from the remaining plasma components. Further separation of the plasma components may occur, depending on the centrifugation conditions and the density gradient medium. When the bodily fluid is bone marrow, the centrifugation at least separates a fraction depleted in stem cells from a fraction which is enriched in stem cells e.g. mesenchymal stem cells. In one embodiment, the centrifugation in step a-2) is the only centrifugation step in the process forming the enriched fraction.

[0032] As mentioned above, following centrifugation in step a-2) an enriched fraction is formed above the density gradient medium. In step a-3) at least a portion of the enriched fraction is collected. In certain embodiments, it may be beneficial to remove a portion of the enriched fraction before proceeding with step a-3). For example, when the bodily fluid is whole blood, the enriched fraction is platelet-rich plasma. However, following centrifugation the platelet-rich plasma (enriched fraction) is not uniformly distributed, and the platelets are typically concentrated just above the density gradient medium, with a layer of platelet-poor plasma (PPP) above this layer i.e. at the proximal end of the centrifugation container. 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.

[0033] Thus, in one embodiment, 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)).

[0034] The at least a portion of the enriched fraction is typically removed from the proximal end of the centrifugation container. In this embodiment, step a-3) is collecting at least a portion of the remaining enriched fraction (i.e. the even more concentrated, or enriched fraction).

[0035] In embodiments wherein the bodily fluid is whole blood, in one embodiment, 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)).

[0036] The portion of the platelet-rich plasma fraction that is removed and discarded corresponds to at least a portion of the platelet poor plasma (PPP) layer. In this embodiment, 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). In these embodiments, suitably step a-2a) is directly followed by step a-2£>) (i.e. there are no intervening steps).

[0037] In one embodiment, the centrifugation container of the present invention has been sterilized. Preferably, 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.

[0038] In the processes of the invention, 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).

[0039] Techniques that separate EVs based on their size include size exclusion chromatography (SEC), microfiltration, ultrafiltration (UF), flow field-flow fractionation, hydrostatic filtration dialysis (HFD) and microfluidics.

[0040] 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. Alternatively, 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.

[0041] 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.

[0042] Flow field-flow fractionation (FFF) is another size-based fractionation technique for EV separation, with asymmetrical flow field-flow fractionation (AsFIFFF / AF4) being the most popular FFF technique.

[0043] Hydrostatic filtration dialysis (HFD) 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.

[0044] Techniques that separate EVs based on their charge include ion-exchange techniques such as anion-exchange chromatography (AIEC), metal-affinity based ion exchange, electrophoresis, diaelectrophoresis and ion concentration polarisation.

[0045] 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. In general, 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.

[0046] In electrophoresis and dielectrophoresis, EVs and their subpopulations can be separated based on their electrophoretic mobilities, giving additional information on characteristics of charged EVs that is not possible with size- and density-based techniques.

[0047] Techniques that separate EVs based on their affinity rely on highly selective and specific interactions between proteins or receptors found on the EV membrane and their corresponding ligands / antigens, such as antibodies. In a typical affinity-based isolation technique, the ligands are immobilized or conjugated onto / into a variety of solid media, such as magnetic beads or polymeric materials (e.g. agarose beads). Broadly speaking there are two groups of affinity-based isolation: affinity chromatography and immunocapture. The most widely used affinity-based technique relies on immunoaffinity capture by using antibodies against EV surface proteins. The antibodies are typically covalently coupled to magnetic beads via biotinylation. In general, bound EVs are either eluted from the beads using appropriate reagents, or bead-EV complexes can be used directly for further analysis (e.g. immunoassay).

[0048] Techniques that separate EVs based on their density include ultracentrifugation, density gradient centrifugation (DGC) and rate-zonal centrifugation techniques. The techniques involve the use of an ultracentrifuge, a device that using centrifugal force to separate the components of a fluid. The fluid is spun at very high speeds within a container, with denser particles moving outward in the radial direction (towards the bottom of the container). In the case of DGC, centrifugation occurs in a tube containing a density gradient medium. In one embodiment, step b-1) involves a step of ultracentrifugation (see Example 2). In one embodiment is provided a process for preparing a medical composition comprising extracellular vesicles, the process 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.

[0049] In one embodiment, is provided a process for preparing a biological sample for analysis for the presence of extracellular vesicles, the process 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 biological sample comprising the isolated extracellular vesicles.

[0050] In both of these embodiments, step a-3) is optional, in the case that the centrifugation container used in steps a-1 and a-2) is suitable for use in the ultracentrifugation process of step b-1). Typically, however, the at least a portion of the enriched fraction will be transferred to a different centrifugation container.

[0051] The ultracentrifugation force used to isolate the EVs will depend on the type of bodily fluid / cell extract being used. In one embodiment, 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. In one embodiment, 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. In one embodiment, 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.

[0052] Thus, in one embodiment, 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.

[0053] Suitably, the further ultracentrifugation process of step b-1 £>) is carried out at a greater force that the ultracentrifugation process of step b-1 a). In one embodiment, 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. In one embodiment, 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.

[0054] 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.

[0055] Thus, in one embodiment, 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.

[0056] In 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. In one embodiment, 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. In one embodiment, the centrifugation container used in the ultracentrifugation process of step b-1) comprises a density gradient medium. In another embodiment, 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.

[0057] In all of the process embodiments described herein, 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.

[0058] 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.

[0059] 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.

[0060] 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).

[0061] In one embodiment, 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.

[0062] 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.

[0063] In one embodiment, the material collected in step a-1) is a bodily fluid. In one embodiment, the bodily fluid is whole blood or a blood product (such as plasma or serum). In this embodiment, the extracellular vesicles to be isolated are typically platelet-derived extracellular vesicles.

[0064] In another embodiment, 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.

[0065] In all cases, the bodily fluid or cell extract contains cells from which EVs are formed, for example whole blood contains platelets, from which platelet-derived EVs are formed, and bone marrow contains stem cells, from which stem cell-derived EVs are formed.

[0066] For the avoidance of doubt, the medical composition or biological sample prepared according to a process of the invention contains a higher concentration of EVs than the original bodily fluid or cell extract. Thus, the process of the invention described herein prepares a medical composition or biological sample which is enriched in EVs.

[0067] In one embodiment, the bodily fluid or cell extract is autologous. In the context of the present invention, 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.

[0068] In another embodiment, the bodily fluid or cell extract is allogenic. In the context of the present invention, allogenic means a method using bodily fluid, tissue and / or cells from one or more third parties for use on a different recipient.

[0069] In embodiments where the material collected in step a-1) is 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. In one embodiment, 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.

[0070] 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. In a centrifugation container such as a tube, 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. Thus, 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.

[0071] Suitably, the density gradient medium is a thixotropic gel. The term “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. 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). When the centrifugation container (e.g. tube) also contains a bodily fluid or cell supernatant (e.g. whole blood), during centrifugation 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)). As the thixotropic gel becomes more fluid under centrifugal force, its position in the container such as a container (e.g. tube) (along with the other components) is reflected by its density. 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.

[0072] 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. Preferably the density gradient medium comprises, consists essentially of or consists of a thixotropic gel. In one embodiment, the density gradient medium is a thixotropic gel.

[0073] In one embodiment, the density gradient medium (e.g. thixotropic gel) has a density between about 1.010 g / cm3and about 1.095 g / cm3, such as about 1.010 g / cm3, about 1.015 g / cm3, about 1.020 g / cm3, about 1.025 g / cm3, about 1.030 g / cm3, about 1.035 g / cm3, about 1.040 g / cm3, about 1.045 g / cm3, about 1.050 g / cm3, about 1.055 g / cm3, about 1.060 g / cm3, about 1.065 g / cm3, about 1.070 g / cm3, about 1.075 g / cm3, about 1.080 g / cm3, about 1.085 g / cm3, about 1.090 g / cm3, or about 1.095 g / cm3. All densities of density gradient media (e.g. thixotropic gels) described herein are measured at 25 °C at atmospheric pressure.

[0074] In one embodiment, the density gradient medium (e.g. thixotropic gel) has a density between about 1.045 g / cm3and about 1.095 g / cm3, such as between about 1.050 g / cm3and about 1.095 g / cm3, or between about 1.055 g / cm3and about 1.095 g / cm3. In whole blood, platelets (also known as thrombocytes) typically have a density of around 1 .040 g / cm3. As such, when centrifuged with a density gradient medium with density between about 1 .045 and about 1 .095 g / cm3, (i.e. a density gradient medium with higher density), following centrifugation all or the majority of the platelets will reside above the layer of density gradient medium. As such, using a density gradient medium with density between about 1.045 g / cm3and about 1.095 g / cm3facilitates the preparation of platelet-rich plasma (PRP).

[0075] In one embodiment, the density gradient medium (e.g. thixotropic gel) has a density between about 1.070 g / cm3and about 1.090 g / cm3, such as between about 1.075 g / cm3and about 1 .090 g / cm3, about 1.080 g / cm3and about 1 .090 g / cm3, between about 1.070 g / cm3and about 1.080 g / cm3, or between about 1.075 g / cm3and about 1.080 g / cm3, e.g. about 1.075 g / cm3. In another embodiment, the density gradient medium (e.g. thixotropic gel) has a density between about 1.045 g / cm3and about 1.075 g / cm3, such as between about 1.045 g / cm3and about 1 .055 g / cm3, between about 1 .050 g / cm3and about 1 .070 g / cm3, or between about 1.050 g / cm3and about 1.060 g / cm3, e.g. about 1.055 g / cm3.

[0076] In whole blood, white blood cells (leukocytes) typically have a density of between about 1.060 g / cm3and about 1.085 g / cm3. Thus, when centrifuged with a density gradient medium (e.g. thixotropic gel) with density between about 1.045 g / cm3and about 1.055 g / cm3(i.e. density gradient medium with lower density), following centrifugation all or the majority of leukocytes will reside below the layer of density gradient medium. As such, using a density gradient medium with density between about 1.045 g / cm3and about 1.055 g / cm3facilitates the preparation of leukocyte-poor platelet-rich plasma (LP-PRP).

[0077] Monocytes and lymphocytes (types of leukocytes which are also known as agranulocytes) have density of between about 1 .060 g / cm3and about 1 .075 g / cm3. As such, when centrifuged with a density gradient medium (e.g. thixotropic gel) with density between about 1.070 g / cm3and about 1.090 g / cm3(i.e. density gradient medium with higher density), following centrifugation all or the majority of monocytes and lymphocytes will reside above the layer of density gradient media. Basophils, neutrophils and eosinophils (types of leukocytes which are also known as granulocytes) have density of between about 1.072 g / cm3and about 1.10 g / cm3. As such, using a density gradient medium with density between about 1 .070 g / cm3and about 1.090 g / cm3facilitates 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). Even though enhanced in agranulocytes, this PRP is considered overall to be leukocyte-poor PRP (LP-PRP).

[0078] In one embodiment, the density gradient medium (e.g. thixotropic gel) has a density between about 1.020 g / cm3and about 1.050 g / cm3, such as between about 1.025 g / cm3and about 1.040 g / cm3, e.g. about 1.030 g / cm3. Essentially all cellular constituents in whole blood have a density that is greater than 1.050 g / cm3, therefore using a density gradient medium with density between about 1.020 g / cm3and about 1.050 g / cm3facilitates the preparation of essentially acellularized plasma.

[0079] Leukocytes can be divided into three main populations in blood: granulocytes (65%), lymphocytes (30%) and monocytes (5%). Monocytes and lymphocytes play a positive role in tissue healing. Monocytes differentiate into macrophages in tissue, and through their phagocytic activity clear the wound of dead cells and other debris. They are also involved in the resolution of inflammation (Brancato et al., 2011). Lymphocytes can secrete large amounts of growth factors that stimulate angiogenesis and new collagen deposition by fibroblasts (Schaffer et al., 1998). Granulocytes, on the other hand are pro-inflammatory cells containing potent destructive enzymes such as peroxidases, proteases and collagenases. The release of these molecules is crucial for fighting bacterial infection in open wounds; however, it has a deleterious effect in aseptic wounds. Thus, as granulocytes are the most abundant form of leukocyte, using a leukocyte-poor PRP is advantageous in certain applications to avoid undesired inflammatory reactions. For such applications, use of a density gradient medium (e.g. thixotropic gel) with density between about 1.045 g / cm3and about 1.055 g / cm3is preferred. Commercially available A-CP tubes and Regen BCT tubes (both manufactured by Regen Lab) contain thixotropic gels with such densities.

[0080] However, in certain applications retaining the monocytes and lymphocytes in the PRP could have potentially beneficial effects. For such applications, use of a density gradient medium (e.g. thixotropic gel) with density between about 1.060 g / cm3and about 1.085 g / cm3is preferred. This density allows specific depletion of granulocytes while maintaining a potentially beneficial population of monocytes and leukocytes. The PRP produced is mononuclear cellrich (monocytes and leukocytes being mononuclear cells), but is overall still considered to be leukocyte poor, because the total leukocyte concentration is below that of the whole blood prior to centrifugation. Commercially available RegenTHT tubes (also manufactured by Regen Lab) contain thixotropic gels within this density range.

[0081] Because of its ability to recover mononuclear cells with platelets and plasma, the centrifugation containers described herein can be used to process bone marrow aspirate. Like blood, bone marrow aspirate consists of a suspension of blood cells as well as stem cells, precursors and immature cells of the different cell lineages produced by the bone marrow in plasma. When bone marrow aspirate is centrifuged in a container of the invention comprising a density gradient medium (in particular a thixotropic gel), the nature of the density gradient medium can be selected (as described in detail above) in order that mature red and white blood cells as well as most immature blood cells migrate below the density gradient medium, while platelets and the mononuclear cell fraction remain above the density gradient medium and can be recovered within the plasma. The mononuclear cell fraction of bone marrow contains not only mature blood mononuclear cells but also hematopoietic progenitor cells and mesenchymal stem cells. Commercially available RegenTHT tubes have been shown to recover mesenchymal stem cells with high efficiency (> 87%) which is the highest yield when compared to other systems for bone marrow processing. Thus, in embodiments wherein the bodily fluid is bone marrow, the enriched fraction is enriched in stem cells e.g. mesenchymal stem cells. When the enriched fraction is platelet-rich plasma, suitably the density gradient medium is a thixotropic gel with density between about 1 .045 g / cm3and about 1 .055 g / cm3.

[0082] The thixotropic gel is typically a large polymer complex. In one embodiment, 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.

[0083] 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.

[0084] The polyoxyalkylene polyol preferably comprises hydroxyl group containing groups of formula 1 :

[0085] Suitably trioctyl trimellitate is tris(2-ethylhexyl) trimellitate.

[0086] Suitable hydrocarbonated resins include a cycloaliphatic hydrocarbon resin.

[0087] In one embodiment, 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. In a preferred embodiment, the thixotropic gel comprises trioctyl trimellitate and / or a hydrocarbon resin, and in particular comprises trioctyl trimellitate and a hydrocarbon resin.

[0088] In one embodiment, 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. In one embodiment, 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.

[0089] The thixotropic gel can contain components in addition to the oligomer or polymer. In one embodiment, in addition to the oligomer or polymer, the thixotropic gel further comprises one or more additives selected from the group consisting of tris(2-ethylhexyl)benzene- 1 ,2,4- tricarboxylate, silicon dioxide, a silane, a dichlorodimethyl-reaction product, a monomeric silica (such as dimethyl dichlorosilane), a phenolic compound (such as tetrakis (3- (3,5-di-tert-butyl- 4-hydroxyphenyl) propionate of pentaerythritol), a polyol (such as a polyalkylene polyol), a phosphite ester (such as the phosphite ester of tris(2,4-di-tert-butylphenyle)), and an azelate ester. In one embodiment, in addition to the oligomer or polymer, the thixotropic gel further comprises further comprises a phenol and / or a phosphite ester.

[0090] In one embodiment, the thixotropic gel comprises, consists essentially of or consists of silica dimethyl silylate, a polyoxyalkylene polyol, trioctyl trimellitate, or a hydrocarbonated resin, or any combination thereof.

[0091] In one embodiment, the thixotropic gel comprises trioctyl trimellitate and / or a hydrocarbon resin, and in particular comprises trioctyl trimellitate and a hydrocarbon resin. In this embodiment, the thixotropic gel optionally further comprises a phenol and / or a phosphite ester.

[0092] In one embodiment, the thixotropic gel comprises, consists essentially of, or consists of trioctyl trimellitate (in particular tris(2-ethylhexyl) trimellitate), a hydrocarbon resin (in particular a cycloaliphatic hydrocarbon resin), a monomeric silica (in particular dimethyl dichlorosilane), a polyoxyalkylene polyol, a phenolic compound and a phosphite ester (in particular the phosphite ester of tris(2,4-di-tert-butylphenyle)). In this embodiment, suitably trioctyl trimellitate is present in an amount between about 40 wt.% and about 60 wt.% (for example about 50.96 wt.%); the hydrocarbon resin present in an amount between about 30 wt.% and about 60 wt.% (for example about 43 wt.%); the silica is present in an amount between about 2 wt.% and about 10 wt.% (for example about 4.21 wt.%); the polyoxyalkylene polyol is present in an amount between about 1 wt.% and about 5 wt.% (for example about 1.73 wt.%); the phenolic compound is present in an amount between about 0 wt.% and about 1 wt.% (for example about 0.05 wt.%); and the phosphite ester is present in an amount between about 0 wt.% and about 0.06 wt.% (for example about 0.05 wt.%).

[0093] In one embodiment, the thixotropic gel comprises, consists essentially of or consists of trioctyl trimellitate (which is suitably present in an amount between about 35 wt.% and about 55 wt.%), silica (which is suitably present in an amount between about 2 wt.% and about 10 wt.%), a hydrocarbon resin (which is suitably present in an amount between about 20 wt.% and about 40 wt.%), an azelate ester (which is suitably present in an amount between about 10 wt.% and about 30 wt.%), and a phenol (which is suitably present in an amount between about 0 wt.% and about 1 wt.%).

[0094] In one embodiment, the thixotropic gel comprises trioctyl trimellitate (which is suitably present in an amount of about 50.96 wt.%), silica (which is suitably present in an amount of about 4.21 wt.%), hydrocarbon resin (which is suitably present in an amount of about 43 wt.%), an azelate ester (which is suitably present in an amount of about 15.82 wt.%), and a phenol (which is suitably present in an amount of about 0.05 wt.%).

[0095] In one embodiment, the thixotropic gel comprises, consists or consists essentially of trioctyl trimellitate, a monomeric silica (in particular dimethyl dicholorsilane), a hydrocarbon resin (in particular a cycloaliphatic hydrocarbon resin), a phenol (in particular Tetrakis (3- (3,5-di-tert- butyl-4-hydroxyphenyl) propionate of pentaerythritol) and a phosphite ester (in particular tris (2,4-di-tert-butylphenyl)phosphite).

[0096] In one embodiment, the thixotropic gel comprises, consists essentially of or consists of trioctyl trimellitate in an amount between about 40 wt.% and about 60 wt.%, a silica in an amount between about 2 wt.% and about 10 wt.%, a hydrocarbon resin in an amount between about 30 wt.% and about 60 wt.%; a phenol in the range of between about 0 wt.% and about 1 wt.%, and a phosphite ester in an amount between about 0 wt.% and about 0.06 wt.%.

[0097] In one embodiment, the thixotropic gel comprises, consists essentially of or consists of trioctyl trimellitate in an amount of about 52.26 wt.%, a silica in an amount of about 7.99 wt.%, a hydrocarbon resin in an amount of about 39.65 wt.%, a phenol in an amount of about 0.05 wt.%, and a phosphite ester in an amount of about 0.05 wt.%.

[0098] The density gradient medium (e.g. thixotropic gel), is present in the centrifugation container in an amount which is sufficient to form a robust layer between bodily fluid / cell extract constituents, thereby facilitating straightforward and accurate separation of the bodily fluid / cell extract constituents. In one embodiment, the centrifugation container of the invention comprises between about 0.1 mL and about 10 mL of (total) density gradient medium (e.g. thixotropic gel), in particular between about 0.1 mL and about 0.6 mL, such as about 0.3 mL.

[0099] In one embodiment, the density gradient medium (e.g. thixotropic gel) is insoluble in water. In one embodiment, the density gradient medium (e.g. thixotropic gel) is partially soluble in acetone. In one embodiment, the density gradient medium (e.g. thixotropic gel) is easily soluble in hexane. In one embodiment, the density gradient medium (e.g. thixotropic gel) has a viscosity of between about 400 Pa.s and about 700 Pa.s at 15 °C, such as between about 100 Pa.s and about 250 Pa.s at 25 °C, between about 30 Pa.s and about 100 Pa.s at 45 °C, or between about 10 Pa.s and about 80 Pa.s at 65 °C.

[0100] The centrifugation container used in step a-1) may contain one or more additional materials. Suitably, the centrifugation container is pre-filled with such materials.

[0101] In one embodiment, the centrifugation container further comprises an anticoagulant which is used to prevent coagulation during collection of the bodily fluid (in particular whole blood) and to maintain the enriched fraction (which in the case of whole blood is PRP) in a liquid state. The anticoagulant is suitably selected from the group consisting of sodium citrate, acid citrate dextrose (ACD), modified ACD, heparin or a salt thereof, ethylenediaminetetraacetic acid (EDTA) or a salt thereof, an iodo acetate salt, an oxalate salt, and a fluoride salt. The anticoagulant can be prepared as a solution in water, and can be wet sprayed on an inner wall of the centrifugation container. Alternatively, the anticoagulant can be a lyophilised material which is dry sprayed on an inner wall of the centrifugation container. In a preferred embodiment, the anticoagulant is sodium citrate. Suitably, the sodium citrate is a solution of sodium citrate in water. In one embodiment, the anticoagulant is present in the centrifugation container (e.g. tube) at a concentration of between about 0.05 M and about 0.15 M, such as between about 0.08 M and about 0.14 M, such as about 0.1 M. Suitably, the anticoagulant is located at a proximal end of the centrifugation container, in order that it comes into immediate contact with the bodily fluid (e.g. whole blood), following collection.

[0102] The centrifugation container may also contain an antifibrinolytic substance (e.g. a compound) which inhibits fibrinolysis. Antifibrinolytic substances prevent or reduce the activation of plasminogen to form plasmin, thereby preventing or reducing blood clot degradation. Antifibrinolytic substances are typically synthetic analogues of the amino acid lysine. In one embodiment, the antifibrinolytic substance is selected from the group consisting of tranexamic acid, aminocaproic acid and aprotinin, or any combination thereof. In a preferred embodiment, the antifibrinolytic substance is tranexamic acid. Suitably, the antifibrinolytic substance is 5 wt.% tranexamic acid (suitably in water). Reference to “an” antifibrinolytic substance is intended to encompass “at least one” antifibrinolytic substance, and combinations of antifibrinolytic substances are also envisaged. In one embodiment, the centrifugation container comprises between about 0.1 mL and about 1 mL of antifibrinolytic substance, such as between about 0.1 mL and about 0.6 mL, in particular between about 0.2 mL and about 0.5 mL, such as about 0.3 mL.

[0103] The centrifugation container may also contain a coagulation activator. A coagulation activator is an agent, for example a compound or an enzyme, that is able to trigger or activate coagulation of plasma and platelets aggregation, forming a clot, which may have the consistency of a gel. Reference to “a” coagulation activator is intended to encompass “at least one” coagulation activator, and combinations of coagulation activators are also envisaged. Typically, the coagulation activator is a compound or moiety which is a thrombin activator and / or a fibrinogen activator. In one embodiment, the coagulation activator is a compound selected from the group consisting of a calcium salt and thrombin. Suitably the calcium salt is selected from the group consisting of calcium gluconate, calcium carbonate, calcium sulphate, calcium saccharate and calcium chloride; or any combination thereof, and in particular is calcium gluconate. In one embodiment, the coagulation activator is a calcium salt (in particular calcium gluconate) in a solution of water at between about 1 wt.% and about 20 w.% e.g. between about 5 wt.% and about 15 wt.% such as about 10 wt.%. The calcium salt may comprise a mixture of calcium salt e.g. a combination of calcium gluconate and calcium saccharate. In one embodiment, the centrifugation container of the invention comprises between about 0.1 mL and about 1.0 mL of coagulation activator, in particular between about 0.1 mL and about 0.6 mL, such as about 0.3 mL.

[0104] The process of the invention may comprise an additional activation step, wherein cells in the bodily fluid or cell extract are activated such that they secrete EVs (such as exosomes) or greater quantities of EVs. Thus, in one embodiment, the process of the invention further comprises the step of x-1) cell activation. The activation step may be carried out on the bodily fluid or cell extract of step a-1), following the centrifugation process of step a-2), or following collection of at least a portion of the enriched fraction in step a-3) i.e. between steps a-3) and b-1).

[0105] In embodiments of the process using whole blood, the process of the invention may optionally comprise a step of platelet activation. In such embodiments, the process comprises an additional step between steps a-3) and b-1) of adding a platelet activator to the platelet-rich plasma fraction of step a-3). Suitable platelet activators include collagen, von Willebrand factor, adenosine diphosphate (ADP), thrombin, fibrinogen, fibronectin, serotonin and plateletactivating factor.

[0106] Cells in cell extracts can be activated by any suitable means. For example, lipopolysaccharide (LPS), B-cell receptor (BCR) agonists or Toll-like receptor (TLR) agonists can be used to activate CD19+B2 or B1a cells (WO2022 / 150056A1 , incorporated by reference herein in its entirety).

[0107] The centrifugation container may also contain a structural biomaterial. Reference to “a” structural biomaterial is intended to encompass “at least one” structural biomaterial, and combinations of structural biomaterials are also envisaged. Typical structural biomaterials include a glycosaminoglycan, a silk protein, fibroin, collagen, polysaccharide or a polylactide, or any combination thereof. In one embodiment, the structural biomaterial is a glycosaminoglycan (which can be derivatised), such as selected from the group consisting of chondroitin, chondroitin sulfate, dermatan, dermatan sulfate, heparin, heparan sulfate, heparosan, hyaluronan and hyaluronic acid. In one embodiment, the structural biomaterial is a polysaccharide (which can be derivatised), such as selected from the group consisting of sucrose, lactulose, lactose, maltose, trehalose, cellobiose, mannobiose, chitobiose, chitosan and cellulose. A glycosaminoglycan can be derivatised with one or more side chains, e.g. side chains comprising a polyalkylene glycol-containing residue.

[0108] In one embodiment, the structural biomaterial is a glycosaminoglycan such as heparosan or hyaluronic acid, and in particular is hyaluronic acid. The biological effects of hyaluronic acid are related to its molecular weight. In one embodiment, the hyaluronic acid is low molecular weight hyaluronic acid (LMW-HA), with molecular weight between about 400 kDa and about 1 ,000 kDa. In another embodiment, the hyaluronic acid is middle molecular weight hyaluronic acid, with molecular weight between about 1 ,000 kDa and about 1 ,800 kDa, such as between about 1 ,400 kDa and about 1 ,600 kDa, e.g. about 1 ,500 kDa. In another embodiment, the hyaluronic acid is high molecular weight hyaluronic acid (HMW-HA), with molecular weight greater than 1 ,800 kDa. Preferably, the hyaluronic acid in the centrifugation container of the invention has molecular weight between about 1 ,000 kDa and about 1 ,800 kDa, such as between about 1 ,400 kDa and about 1 ,600 kDa, e.g. about 1 ,500 kDa.

[0109] A mixture of hyaluronic acids with different molecular weights may also be used. Thus, in one embodiment, the hyaluronic acid is a mixture of hyaluronic acids comprising: a low molecular weight hyaluronic acid (LMW-HA), with molecular weight between about 400 kDa and about 1 ,000 kDa; and / or a middle molecular weight hyaluronic acid, with molecular weight between about 1 ,000 kDa and about 1 ,800 kDa, such as between about 1 ,400 kDa and about 1 ,600 kDa, e.g. about 1 ,500 kDa; and / or a high molecular weight hyaluronic acid (HMW-HA), with molecular weight greater than 1 , 800 kDa.

[0110] In one embodiment, the hyaluronic acid is cross-linked. In another embodiment, the hyaluronic acid is linear. The hyaluronic acid is preferably added as a solution in water, wherein the concentration of hyaluronic acid in the solution (added to the centrifugation container) is between about 1 wt.% and about 5 wt.%, such as between about 1.8 % and about 2.2 wt.%. In one embodiment, the centrifugation container comprises between about 1.0 mL and about 5.0 mL of the hyaluronic acid, in particular between about 1.5 mL and about 3 mL, such as about 2 mL. In this embodiment, suitably the fill volume of the centrifugation container is 10 mL or 15 mL, in particular 15 mL.

[0111] The centrifugation container is suitably a centrifugation tube or a centrifugation syringe, in particular a centrifugation tube, such as a blood fractionation tube.

[0112] In one embodiment, the centrifugation container is made of glass, for example borosilicate glass (in particular type 1 borosilicate which is pharma injectable). In another embodiment, the container is made of one or more materials selected from the group consisting of silicone, glass, modified polyamide (MPA), polyethylene terephthalate, synthetic copolymers, ceramic and glass-ceramics, bioartificial blends of natural and synthetic materials. It should be understood that “comprises” in this context means that the material of the centrifugation container is composed of the material, rather than simply containing / holding the material (such as it does for the antifibrinolytic substance etc). Suitably, the centrifugation container is depyrogenated.

[0113] The centrifugation container can be coated with one or more substances, for example silicone and / or polypropylene. The outer wall and / or the inner wall of the container can be coated. Suitably, the inner wall of the centrifugation container is coated.

[0114] In a preferred embodiment, the centrifugation container is made from glass (in particular borosilicate glass), and also contains modified polyamide (MPA) or polyethyleneterephthalate (PET). Suitably, an interior wall is coated with polypropylene. In one embodiment, the centrifugation container is closed to the atmosphere. In one embodiment, the centrifugation container is under vacuum. Having the container under vacuum allows a pre-determined amount of fluid (e.g. whole blood or bone marrow) to be aspirated into the container. The centrifugation container can be closed using any suitable means. Suitably, the centrifugation container (in particular a centrifugation tube) includes a plastic stopper, for example a stopper comprised of butyl rubber (for example bromobutyl rubber) or halo butyl rubber having a hardness of 40-60 Shore A. The centrifugation container preferably has a shelf life with stable vacuum of 18-24 months.

[0115] The centrifugation container has a proximal end and a distal end. The proximal end of the container is the end via which the material to be centrifuged (e.g. bodily fluid such as whole blood or bone marrow) is collected. The distal end of the container is the end which is opposite the proximal end of the container.

[0116] In one embodiment, the centrifugation container (preferably a centrifugation tube), has a fill volume of between about 1 mL and about 20 mL e.g. is a 10 ml_, 15 mL or 20 mL container (fill volume). The container may have a length (as measured between the aperture though which the bodily fluid or cell extract is collected, and the base of the container) of approximately 130 mm (for example 131.6 mm), and a width (as measured between opposed surfaces of the container) of approximately 15.5 mm. The container may have a length (as measured between the aperture though which the whole blood or bone marrow is collected and the base of the container) of approximately 130 mm (for example 129.5 mm), and a width (as measured between opposed surfaces of the container) of approximately 21.4 mm.

[0117] The present inventors have discovered that for processes of the invention for preparing a biological sample for analysis of the presence of EVs, it is preferable to use a centrifugation container with a relatively small fill volume, e.g. between about 1 mL and about 5 mL, e.g. about 2 mL.

[0118] As mentioned above, platelet-derived EVs such as platelet-derived exosomes share a number of the functions of platelets, but have been shown to have more potent effects than platelets in a number of capacities, and can cross tissue barriers that platelets cannot. Thus, medical compositions comprising EVs prepared according to the process of the invention have potential therapeutic utility. In one embodiment, the medical composition prepared according to the process of the invention is of use in therapy. EVs have therapeutic potential in their own right, but can also be used as biological vehicles to carry compounds such as peptides, proteins, lipids, nucleic acids (e.g. RNA or DNA) and carbohydrates.

[0119] Various methods can be used to load EVs such as exosomes with cargo, including incubation, transfection, physical treatment (e.g. sonication, extrusion, freeze-thaw treatment, electroporation, surfactant treatment and dialysis), in situ association and synthesis. Further details of studies investigating the delivery of therapeutic agent (e.g. the delivery of miRNA and siRNA via exosomes) are described in Akbar et al., 2022.

[0120] EVs such as exosomes can induce neuroprotective and neurorestorative effects by modulating gene, protein, and miR expression in their target cells and tissues. Exosomes from specific cells such as MSCs reduce inflammation and increase anigiogenesis, neurogenesis and white matter remodeling after stroke (Chen et al., 2018). In one embodiment, is provided the medical composition prepared according to the process of the invention, for use in treating stoke.

[0121] In one embodiment, is provided the medical composition prepared according to the process of the invention, for use in treating or preventing a joint disorder or condition. Suitably, the joint disorder or condition is selected from the group consisting of arthritis, gout, fibromyalgia, lupus, polymyalgia and rheumatica. Arthritis includes osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, cervical spondylitis, psoriatic arthritis, enteropathic arthritis, oligoarthritis, polyarthritis and secondary arthritis.

[0122] In one embodiment, is provided the medical composition prepared according to the process of the invention, for use in bone regeneration.

[0123] In one embodiment, is provided 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).

[0124] In one embodiment, is provided the medical composition prepared according to the process of the invention, for use in treating or preventing melasma. In one embodiment, is provided the cosmetic use of a medical composition prepared according to the process of the invention, for treating or preventing melasma. In one embodiment, is provided the medical composition prepared according to the process of the invention, for use as an anti-aging agent. In one embodiment, is provided the cosmetic use of a medical composition prepared according to the process of the invention, for antiaging.

[0125] In one embodiment, is provided the medical composition prepared according to the process of the invention, for use in organ transplantation.

[0126] In one embodiment, is provided the medical composition prepared according to the process of the invention, for use in the treatment or prevention of cancer (Franco et al., 2015).

[0127] In one embodiment, is provided 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.

[0128] In one embodiment, is provided the medical composition prepared according to the process of the invention, for use the treatment or prevention of coagulopathy, e.g. traumatic coagulopathy.

[0129] In one embodiment, is provided 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.

[0130] In the embodiments above, the medical composition is suitably autologous. However, in certain conditions which benefit from the administration of a healthy population of cells, it may be beneficial to prepare 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.

[0131] In one embodiment, is provided the medical composition prepared according to the process of the invention, for use as a biomaterial.

[0132] As discussed above, EVs are precise markers for the diagnosis, prognosis and monitoring of various pathologies. In such embodiments for “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|31- 42, total tau, p-T181 tau and p-S396 tau have been determined in the exosomes that are isolated from the plasma of AD patients compared with healthy candidates.

[0133] 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).

[0134] EVs have been used for the diagnosis of cardiovascular disease, in particular 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.

[0135] 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.

[0136] As noted above, 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. Thus, in one embodiment, is provided the medical composition prepared according to the process of the invention, for a cosmetic use e.g. for treating or preventing hair loss. As described in Example 4, platelet-derived extracellular vesicles prepared using the methods of the invention are particularly suitable for use in detecting markers such as DNA and proteins.

[0137] It should be noted that in the context of the present application, when referring to a range of between about “AA” and about “BB”, the point values of AA and BB are intended to be included as possible values in the range.

[0138] The word “comprise”, and variations such as “comprises” and “comprising” as used herein should be understood to mean the inclusion of the stated integer, step, group of integers or group of steps, but not to the exclusion of any other integer, step, groups of integers or group of steps.

[0139] The word “consisting of’ as used herein limits the scope of the integer, step, group of integers or group of steps to the specified integer, step, groups of integers or group of steps. The word “consisting essentially of’ as used herein limits the scope of the integer, step, group of integers or group of steps, and further integers, steps, groups of integers or groups of steps that do not materially affect the basic and novel characteristics of the invention.

[0140] The invention embraces all combinations of indicated integers, steps, groups of integers or groups of steps recited above. All patents and patent applications referred to herein are incorporated by reference in their entirety.

[0141] ABBREVIATIONS

[0142] ACD acid citrate dextrose

[0143] AD Alzheimer's disease

[0144] ADP adenosine triphosphate

[0145] AIEC anion-exchange chromatography

[0146] BCR B-cell receptor

[0147] DGC density gradient centrifugation

[0148] DLD deterministic lateral displacement

[0149] DNA deoxyribonucleic acid

[0150] EDTA ethylenediaminetetraacetic acid

[0151] EGF epidermal growth factor

[0152] EV extracellular vesicle

[0153] FFF flow field-flow fractionation HA hyaluronic acid

[0154] HFD hydrostatic filtration dialysis

[0155] HMW-HA high molecular weight hyaluronic acid

[0156] ILV intraluminal vesicle

[0157] LMW-HA low molecular weight hyaluronic acid

[0158] LP-PRP leukocyte-poor platelet-rich plasma

[0159] LPS lipopolysaccharide

[0160] MPA modified polyamide

[0161] MSC mesenchymal stem cells

[0162] MVB multivesicular body

[0163] PBS phosphate buffered saline

[0164] PCR polymerase chain reaction

[0165] PDGF platelet-derived growth factor

[0166] PEG polyethylene glycol

[0167] PET polyethyleneterephthalate

[0168] PPP platelet-poor plasma

[0169] PRP platelet-rich plasma

[0170] RNA ribonucleic acid

[0171] SEC size exclusion chromatography

[0172] TFF tangential flow filtration

[0173] TGF transforming growth factor

[0174] TLR Toll-like receptor

[0175] UF ultrafiltration

[0176] VEGF vascular endothelial growth factor

[0177] EXAMPLES

[0178] Example 1 - Preparation of platelet concentrate

[0179] Platelet-rich plasma was prepared using RegenKit® A-PRP (RegenLab) according to manufacturer recommendations. RegenKit® A-PRP contains RegenBCT® centrifugation tubes which contain a thixotropic gel with density between about 1.045 g / cm3and about 1 .055 g / cm3. Samples of whole blood were collected into the tubes and centrifuged according to the manufacturer recommendations, to form a fraction enriched in platelets.

[0180] Example 2 - Isolation of extracellular vesicles Platelet-rich plasma prepared according to Example 1 was subjected to sequential centrifugation in order to isolate platelet-derived extracellular vesicles. A first centrifugation was carried out (300 g for 5 min at 4 °C) in order to remove dead cells, followed by a second centrifugation (3,000 g for 30 min at 4 °C) in order to remove apoptotic bodies. A first ultracentrifugation was then carried out (10,000 g for 30 min at 4 °C) in order to remove macrovesicles; followed by a second ultracentrifugation (100,000 g for 180 min at 4 °C) as a final purification step. The platelet-derived extracellular vesicles were resuspended in endotoxin-free PBS and stored at -80 °C.

[0181] Example 3 - Sizing the extracellular vesicles

[0182] Extracellular vesicles were analysed using a NanoAnalyzer (ZetaView, Particle Metrix). Parameters such as size distribution, concentration and zeta potential were obtained following manufacturer recommendations.

[0183] Example 4 - Analysis of extracellular vesicles

[0184] Platelet-derived extracellular vesicles prepared according to Example 2 were quantitatively analysed for DNA using real-time quantitative PCR. It was found that DNA detection in platelet- derived extracellular vesicles prepared according to Example 2 was significantly more reproducible than when platelet-derived extracellular vesicles prepared according to other methods, in particular using centrifugation tubes with thixotropic gels of differing viscosities. Without wishing to be bound by theory, it is believed that in order to accurately detect material present in platelet-derived extracellular vesicles, the platelets must be intact and undamaged. Hence, the quality of platelets is a significant requirement for detecting the DNA cargo in platelet-derived extracellular vesicles. It has been found that using the tubes having thixotropic gel with density between about 1.045 g / cm3and about 1.055 g / cm3, platelet-derived extracellular vesicles which are particular suitable for use in DNA detection can be isolated.

[0185] REFERENCES

[0186] Akbar et al. Cells 2022, 11 , 186

[0187] Antich-Rossello et al. International Journal of Molecular Sciences 2021 , 22, 8580.

[0188] Brancato et al. Am. J. Pathol. 2011 , 178, 19-25.

[0189] Chen et al. Stroke 2018, 1083-1090.

[0190] Franco et al. Blood 2015, 125(5), 582-588.

[0191] Frazier et al. Biomolecules 2020, 10, 1373. de Freitas et al. Int. J. Mol. Sci. 2022, 23, 9952.

[0192] Marx et al. J. Oral Maxillofac. Surg. 2004, 62, 489-496 Nilforoushzadeh et al. Cell J. 2021 , 23(5), 576-583 Nolan et al. Platelets 2017, 28(3), 256-262. Liangsupree et al. Journal of Chromatography A 2021 , 461773 Schaffer et al. Br. J. Surg. 1998, 85, 444-460.

[0193] Shi et al. Theranostics 2021 , 11(13), 6616-6631.

[0194] Sjors et al. blood 2019, 133(22), 2359-2364.

[0195] Tan et al. Cell. Physiol. Biochem. 2016, 38, 2348-2365.

Claims

CLAIMS1. A process for preparing a medical composition comprising extracellular vesicles, the process 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 medical composition comprising the isolated extracellular vesicles.

2. The process for preparing a biological sample for analysis for the presence of extracellular vesicles, the process 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.

3. The process according to claim 1 , 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 to form a platelet-rich plasma fraction; a-3) collecting at least a portion of the platelet-rich plasma fraction; b-1) subjecting the collected platelet-rich plasma fraction to a process for isolating platelet- derived extracellular vesicles; b-2) preparing a medical composition comprising the isolated platelet-derived extracellular vesicles; wherein the density gradient medium is a thixotropic gel with density between about 1.045 g / cm3and about 1.055 g / cm3.

4. The process according to claim 2, for preparing a biological sample for analysis for the presence of 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 to form a platelet-rich plasma fraction; a-3) collecting at least a portion of the platelet-rich plasma fraction;b-1) subjecting the collected platelet-rich plasma fraction to a process for isolating platelet- derived extracellular vesicles; b-2) preparing a biological sample comprising the isolated platelet-derived extracellular vesicles; wherein the density gradient medium is a thixotropic gel with density between about 1.045 g / cm3and about 1.055 g / cm3.

5. The process according to claim 1 or claim 2, wherein step a-2) comprises the steps of: a-2a) centrifuging the centrifugation container to form an enriched fraction; a-2£>) removing and discarding at least a portion of the enriched fraction.

6. The process according to claim 3 or claim 4, wherein 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.

7. The process according to any one of claims 1 to 6, wherein the medical composition or biological sample comprises exosomes.

8. The process according to any one of claims 1 to 7, wherein the process for isolating extracellular vesicles is selected from the group consisting of size exclusion chromatography, microfiltration, ultrafiltration, flow field-flow fractionation, hydrostatic filtration dialysis, microfluidics, anion-exchange chromatography, metal-affinity based ion exchange, electrophoresis, diaelectrophoresis, ion concentration polarisation, affinity- based isolation, ultracentrifugation, density gradient centrifugation, a rate-zonal centrifugation technique, precipitation and flow cytometry; or a mixture thereof.

9. The process according to any one of claims 1 to 8, wherein the material collected in the centrifugation container comprising a density gradient medium in step a-1) is a bodily fluid.

10. The process according to claim 9, wherein the bodily fluid is selected from the group consisting of whole blood, a blood product (e.g. plasma or serum), 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.

11. The process according to any one of claims 1 , 2, 5, 7 or 8, wherein the material collected in the centrifugation container comprising a density gradient medium in step a-1) is a cell extract.

12. The process according to any one of claims 1 to 11 , wherein in step a-1) the density gradient medium is a thixotropic gel.

13. The process according to any one of claims 1 to 12, wherein the centrifugation container further comprises an anticoagulant, which is suitably selected from the group consisting of sodium citrate, acid citrate dextrose, modified acid citrate dextrose, heparin or a salt thereof, ethylenediaminetetraacetic acid or a salt thereof, an iodo acetate salt, an oxalate salt, and a fluoride salt, and in particular is sodium citrate.

14. The process according to any one of claims 1 to 13, wherein the centrifugation in step a-2) is performed at a force of between about 1500 g and about 2800 g.

15. The process according to any one of claims 1 to 14, wherein the centrifugation in step a-2) is performed for a period of time between about 3 minutes and about 40 minutes.

16. A medical composition comprising extracellular vesicles prepared according to any one of claims 1 , 3, or 5 to 15, for use in therapy.

17. A medical composition comprising extracellular vesicles according to claim 16, for use in the treating or preventing a joint disorder or condition.

18. A medical composition comprising extracellular vesicles according to claim 17, wherein the joint disorder or condition is selected from the group consisting of arthritis, gout, fibromyalgia, lupus, polymyalgia and rheumatica.

19. A medical composition comprising extracellular vesicles according to claim 18, wherein arthritis is selected from the group consisting of osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, cervical spondylitis, psoriatic arthritis, enteropathic arthritis, oligoarthritis, polyarthritis and secondary arthritis.

20. A medical composition comprising extracellular vesicles according to claim 16, for use in wound healing, wound sealing, tissue regeneration, cartilage regeneration, and / or bone regeneration.21 . A medical composition comprising extracellular vesicles according to claim 16, for use in treating or preventing melasma.

22. A medical composition comprising extracellular vesicles according to claim 16, for use in treating or preventing cancer.

23. A medical composition comprising extracellular vesicles for use according to any one of claims 16 to 22, wherein the extracellular vesicles have been modified to carry compounds selected from the group consisting of peptides, proteins, lipids, nucleic acids (e.g. RNA or DNA) and carbohydrates.

24. A biological sample comprising extracellular vesicles prepared according to any one of claims 2, 4, or 5 to 15, for use in diagnosis, prognosis or monitoring of a disease or disorder.

25. A biological sample for use according to claim 24, wherein the disease or disorder is selected from the group consisting of Alzheimer’s disease, cancer (including colon cancer, breast cancer and prostate cancer), cardiovascular disease (including atherosclerosis, actuate coronary syndromes, myocardial infarction and heart failure) and infectious diseases (including urinary tract infection, hepatitis and HIV).

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