WO2021198954A1

WO2021198954A1 - Exosome-enriched extracellualr vesicles isolation, method and uses thereof

Info

Publication number: WO2021198954A1
Application number: PCT/IB2021/052700
Authority: WO
Inventors: Ioannis Sotiropoulos; Ana Patrícia AMORIM GOMES; Bruno COSTA DA SILVA; Nuno SOUSA
Original assignee: Universidade Do Minho; B Acis - Associação Ciencia Inovação E Saude - Braga; Fundação D Anna De Sommer Champalimaud E Dr Carlos Montez Champalimaud
Priority date: 2020-03-31
Filing date: 2021-03-31
Publication date: 2021-10-07

Abstract

The present disclosure relates to a method of obtaining an exosome-enriched preparation of extracellular vesicles (EVs) with reduced contaminants such as large vesicles, immature vesicles, subcellular organelles and/or debris. Specifically, the present disclosure describes a method of isolating spontaneously released EVs, including exosomes by using conditioning medium.

Description

EXOSOME-ENRICHED EXTRACELLUALR VESICLES ISOLATION,

METHOD AND USES THEREOF

Technical field

[0001] The present disclosure relates to a method of obtaining an exosome-enriched preparation of extracellular vesicles (EVs) with reduced contaminants such as large vesicles, immature vesicles, subcellular organelles and/or debris. Specifically, the present disclosure describes a method of isolating spontaneously released EVs, including exosomes by using conditioning medium.

Background

[0002] Current state of the art's brain extracellular vesicles (EVs) and exosomes isolation method relies on the digestion of brain tissue which can lead to contamination with immature intracellular vesicles. Moreover, current methods are unable to distinguish between these immature vesicles and released exosomes as they share similar molecular fingerprints. Currently, there is no solution for this contamination. There is thus a need for an alternative to the digestion-based method of isolating EVs and exosomes from brain tissue.

[0003] Kang Li et al "Cushioned-density gradient ultracentrifugation (C-DGUC): a refined and high-performance method for the isolation, characterization & use of EVs and exosomes" discloses a method of obtaining EVs by utilizing 60% iodixanol cushion during nanoparticle concentration step followed by density gradient ultracentrifugation. The disclosed method does not discriminate between EVs subtypes and larger vesicles.

[0004] Pérez-González et al discloses a method of isolating EVs and exosomes by gentle dissociation of brain tissue to free the brain extracellular space, followed by sequential low-speed centrifugations, filtration, and ultracentrifugation. To further purify the isolated extracellular vesicles, they are separated using a sucrose step gradient. The method disclosed yields pure EVs preparations free of large vesicles, subcellular organelles or debris. However, this method is unable to remove/discriminate exosomes from immature vesicles.

[0005] Document US 2014/0105817 A1 discloses a method of isolating EVs from human cerebral spinal fluid involving ultracentrifugation with a discontinuous sucrose density gradient.

[0006] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.

General Description

[0007] The present disclosure relates to a method of obtaining an exosome-enriched preparation of extracellular vesicles (EVs) with reduced contaminants such as large vesicles, immature vesicles, subcellular organelles and/or debris. Specifically, the present disclosure describes a method of isolating EVs including exosomes by using conditioning medium.

[0008] The method of the present disclosure surprinsigly allows the release of entire exosome without destroying it. The extract obtained by the present method is an enriched exosome medium wherein the exosome are intact.

[0009] The use of conditioning medium comprising L-glutamine medium supplement, and antibiotic-antimitotic allows the release of the exosomes by the tissue and the collection of intact exosome. Therefore, the exosomes obtained by the present disclosure has a higher quality and are intact.

[0010] The method of the present disclosure allows an exosome-enriched preparation to be obtained, wherein such exosome-enriched preparation has a high concentration of EVs with the appropriate characteristics (e.g. size between 50-120nm, protein profile).

[0011] An aspect of the present disclosure relates to a method of obtaining an exosome- enriched preparation of extracellular vesicles (EVs) by isolating EVs and exosomes released through the physiological exocytosis process. The method of the present disclosure does not involve a tissue digestion step.

[0012] The method of the present disclosure has the advantage of reducing contamination of the isolated EVs and exosomes by intracellular vesicles that may released by brain tissue digestion.

[0013] In contrast, the gold standard for brain EVs and/or exosome isolation uses digestion of brain tissue as a way to obtain EVs. The current gold standard method may lead to contamination of the EVs/exosome fraction with immature vesicles and other contaminants as aforemenitoned; if the digestion process had not been utilized, these immature vesicles might not be secreted by the brain cells/tissue.

[0014] An aspect of the present disclosure relates to the use of a neurobasal medium for isolated exosome-enriched extracellular vesicle preparation from brain tissue, wherein the medium comprises L-glutamine medium supplement, and antibiotic- antimitotic.

[0015] In an embodiment, the antibiotic-antimitotic composition is penicillin, streptomycin, amphotericin B, or mixtures thereof.

[0016] In an embodiment, the antibiotic-antimitotic composition is 10,000 units/mL of penicillin, 10,000 μg/mL of streptomycin, and 25 μg/mL of Amphotericin B, preferably Gibco Amphotericin B.

[0017] In an embodiment, the L-glutamine medium supplement is 1% glutamax.

[0018] In an embodiment, the isolated EVs/exosomes is structurally intact, preferably the majority of the isolated EVs/exosomes is structurally intact.

[0019] In an embodiment, 75% of the isolated EVs/exosomes comprises a size from 50- 150 nm. Preferably 80% of the isolated EVs/exosomes comprises a size from 50-150 nm.

[0020] The size measurement of the exosome-enriched extracellular vesicles/exossomes was performed by different methods. In this disclosure the EV/exossomes size was obtained by cryo-electron microscopy/ electron microscopy/ nanoparticle tracking analysis. Transmission electron microscopy provides a fast overview of the samples, enabling a good screening for sample preparation. However, it does not mainting the structure of the visualized structures. Thus, cryo-electron microscopy was used to acquire micrographs of undamaged vesicles, providing a more accurate way to analyze size of the seen vesicles. Lastly, nanotracking particle analysis is a state-of-the-art technique known in the EV field to acquire easily and fast measurements of size of particles and concentration of said particles.

[0021] In an embodiment, the isolated EVs/exosomes fraction is without contaminants such as large vesicles, immature vesicles, subcellular organelles or debris.

[0022] In an embodiment, the medium supplement is L-glutamine supplement, preferably 1% L-alanine-L-glutamine dipeotide (such us glutamax™).

[0023] Glutamax ™ is a dipeptide, L-alanine-L-glutamine, which is more stable in aqueous solutions and does not spontaneously degrade.

[0024] In an embodiment, the sucrose cushion is a solution comprising Heavy Water, sucrose and Tris-base.

[0025] An aspect of the present disclosure relates to the method for isolation of exosome-enriched extracellular vesicle preparation comprising the following steps: obtaining a brain tissue sample; incubating the brain tissue sample in a neurobasal incubating medium; wherein the medium comprises L-glutamine medium supplement, and antibiotic- antimitotic; isolating the obtained exosome-enriched extracellular vesicles by ultra- centrifuging.

[0026] In an embodiment, incubation of the brain tissue in the neurobasal incubating medium is at a temperature from 30 °C to 40 °C, preferably 37 °C.

[0027] In an embodiment, the step of isolating exosome-enriched extracellular vesicles by ultracentrifugation comprises the following step: centrifuge the medium at 500g for 10 minutes at 10°C; collect the supernatant and discard the pellet; centrifuge the supernatant at 3000g for 20 minutes at 10°C; transfer the supernatant to an ultracentrifuge tube; centrifuge the supernatant at 12000g for 20 minutes at 10°C; collect the supernatant and discard the pellet; centrifuge the supernatant at 100000g for 1 hour 10 minutes at 10°C; remove the supernatant and collect the pellet; resuspend the pellet in 500ul of PBS and pass it through a size exclusion chromatography column (namely, qEV column (qEVoriginal/70nm, Izon science ltd)); optionally collect the desired fraction and add PBS until 16mL to obtain a resuspension; add 4ml of sucrose solution to an ultracentrifuge tube, add the resuspension to the ultracentrifuge tube, 1 ml at a time until all the resuspension has been added to the tube in order to obtain a first mixture; centrifuge the mixture at 100000g for 1 hour 10 minutes at 10°C; collect 4ml of the supernatant using a syringe and mix the supernatant with 16 ml of phosphate buffer solution (PBS) in order to obtain a second mixture; centrifuge the second mixture at 100000g; resuspend the pellet.

[0028] In an embodiment, incubation of the second mixture is in 5% CO₂.

[0029] In an embodiment, the first supernatant is centrifuged at 3000g for 20 minutes at 10°C.

[0030] In an embodiment, the second supernatant is centrifuged at 12000g for 20 minutes at 10°C.

[0031] In an embodiment, the third supernatant is centrifuged at 100000g for 1 hour 10 minutes at 10°C.

[0032] In an embodiment, the first mixture is resuspended and goes through a size exclusion chromatography and/or the second mixture is centrifuged at 100000g for 1 hour 10 minutes at 10°C. [0033] In an embodiment, the third extract is centrifuged at 100000g ON at 10°C.

Brief Description of the Drawings

[0034] The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of invention.

[0035] Figure 1a and c are schematic representations of the presently disclosed protocol for isolating an exosome-enriched EVs preparations from brain tissue/cells compared with the state-of-the-art protocol. Figure 1b is a schematic representation of the intracellular pathway of EVs biogenesis.

[0036] Figure 2 is a schematic representation of the conditions used for the tests conducted on 3 different conditioning media.

[0037] Figure 3 shows the result of the cortical EVs and/or exosome isolation yield of the 3 different conditioning media.

[0038] Figure 4 shows micrographs of electron microscopy negative staining (a) as well as cryo-electron microscopy (b) of the two methods followed by a comparison of the percentage of small vesicles, the size of the vesicles (based on electron microscope micrographs) isolated in the state-of-the-art protocol and the presently disclosed method.

[0039] Figure 5 shows the size distribution (size obtained by cryo-electron microscopy microphotos) of the isolated vesicles of the state-of-the-art protocol and the presently disclosed method.

[0040] Figure 6 shows the results of Nanoparticle Tracking Analysis (NTA) for EVs of mouse brain (a-e) as well as human brain (f-i) collected by of the the state-of-the-art method and the presently disclosed method showing that the method of the present diclsoure leads to an exosome-enriched yield (size 50-150nm).

[0041] Figures 7a and b show that the proteomic profile and western blot validation of the EVs isolated by the method of the present disclosure which is highly enriched in proteins related to extracellular exosome, vesicles production and their biogenesis. In addition, Figures 7c-k show the flow cytometry analysis of the relative abundance of endosomal tetraspanins (markers of exosomes) in mouse brain EVs isolated from the presently disclosed method. Also, Figures 7l-m show the relative abundance of endosomal tetraspanins (markers of exosomes) and particles in EVs of mouse brain EVs isolated from the presently disclosed method (data obtained by NanoView IF-based technique).

[0042] Figures 8a-g show the results of Nanoparticle Tracking Analysis (NTA) where the method of the present disclosure detects a reduction of mouse brain EVs under pharmacological treatment with GW4869, a well-characterized blocker of exosome biogenesis (comparison of GW4869 treatment vs. vehicle). Figures 8h-i show that injection of brain EVs (isolated by method of the present disclosure and labelled by the dye Dill), at the dendrites of hippocampal granule cells [located at the outer molecular layer (OML) of the dentate gyrus (DG)] moved down at the soma of the granule cells [located at Granular molecular layer (GCL)] supporting the functionality of the EVs isolated by the method of the present disclosure.

[0043] Figure 9 shows the results of Nanoparticle Tracking Analysis (NTA) where the method of the present disclosure detects EVs from different brain areas (entorhinal cortex, hippocampus, rets of cortex) with different weight (entorhinal cortex < hippocampus < rest of cortex).

[0044] Figure 10 shows the extra data of the flow cytometry technique related to negative controls and the relative abundance of each one of the endosomal tetraspanins (markers of exosomes) in mouse brain EVs isolated from the presently disclosed method.

[0045] Figures 11a-f show the results of Nanoparticle Tracking Analysis (NTA) where the method of the present disclosure detects an increase of mouse brain EVs under pharmacological treatment with Picrotoxin (PTX), a well-characterized enhancer of neuronal activity (via inhibition of GABAergic neurons). Figure 11g shows extra data of the negative controls of the EVs injection in the mouse brain (see Fig 8h) e.g. injection of vehicle (PBS) or injection of dye Dill alone (without exosomes) which exhibit no Dil signal or no movement of Dil signal, respectively.

[0046] Figure 12 shows that results of the brain tissue analysis at the end the protocol of the the method of the present disclosure showing that, in contrast to 40 hr of brain tissue incubation or 16 hrs incubation with H202, our method-endosed 16hrs incubation of brain tissue does not significantly affect its viability (measurement of total of alive cells) or produce any markers of toxicity (e.g. cytochrome c) when compared to incubation of brain tissue for 0hrs.

Detailed Description

[0047] The present disclosure relates to a method of obtaining an exosome-enriched extracellular vesicles (EVs) preparation with reduced contaminants such as large vesicles, immature vesicles, subcellular organelles and/or debris. Specifically, the present disclosure describes a method of isolating EVs including exosomes by using conditioning medium.

[0048] An aspect of the present disclosure relates to a method of obtaining an exosome-enriched EVs preparations by isolating EVs and/or exosomes released during their secretion through the physiological exocytosis process. The method of the present disclosure does not involve a tissue digestion step.

[0049] The method of the present disclosure has the advantage of preventing contamination of the isolated EVs and/or exosomes by immature intracellular vesicles and other contamintants.

[0050] In an embodiment, the method of isolating EVs and/or exosomes comprises the use of a culture medium (for example, Neurobasal [Gibco # 21103049] with 1% glutamax (for example Gibco #35050038) and 1% antbiotic-antimycotic (for example Gibco # 15240062) in order to isolate spontaneously-released EVs including exosomes from brain tissue. [0051] In an embodiment, EVs and/or exosomes were isolated using the method of the present disclosure - spontaneous release protocol. Figure 1a is a brief schematic representation showing a comparison between the spontaneous release protocol and the existing (state-of-the-art protocol) brain digestion protocol used for isolating EVs/exosomes from brain tissue/cells. In contrast to the state-of-the-art protocol (gold standard protocol; left part of the Figure 1a) which involves the digestion of tissue that may result in contamination of the EVs/exosome yield, the new protocol (right part of Figurela) is based on the spontaneous release of EVs and exosomes by the brain tissue. Figure 1b is a schematic representation of the intracellular pathways involved in the biogenesis of EVs. Figure 1c is a more detailed representation of the state-of-the-art protocol and the method of the present disclosure.

[0052] In an embodiment, different conditioning media were used to incubate the brain tissue (cortex tissue). Table 1 shows the contents of the different media used for exosome-enriched EVs isolation.

[0053] Table 1 - contents of the different media used

[0054] aCSF recipe (mM): 119 NaCI, 2.5 KCI, 1.2 NaH2PO4, 24 NaHCO3, 12.5 glucose, 2 MgSO4.7H2O and 2 CaCI2.2H2O (~300 mOsm L^-1, pH 7.2-7.4 )

[0055] Figure 2 is a schematic representation of the conditions used for the tests conducted on 3 different conditioning media. The left bar corresponds to EVs isolated from tissue incubated in aCSF, the middle bar represents the EVs of the tissue that was incubated in Hibernate-A while the right bar shows the EVs that were isolated from the tissue incubated in Neurobasal

[0056] In an embodiment, tissue sample was prepared according to the following steps: Obtaining a sample of brain tissue; Add tissue to a container comprising basal medium, medium supplement and antibiotic-antimicotic;

Let the tissue and medium incubate;

Collect the medium;

Add protease and phosphatase inhibitors to the medium.

[0057] In an embodiment, the basal medium is neurobasal medium.

[0058] In an embodiment, the medium supplement is a L-glutamine supplement, preferably 1% glutamax.

[0059] In an embodiment, tissue sample was prepared according to the following steps:

- Add tissue to a container comprising culture medium (for example Neurobasal [Gibco # 21103049] with 1% glutamax [Gibco #35050038] and 1% anti-anti [Gibco # 15240062]);

- Let the tissue and culture medium incubate at 37 °C, for 16 hours, in 5% CO₂; and thereafter

- Collect the medium;

- Add protease and phosphatase inhibitors to the medium.

[0060] In an embodiment, the method for isolated exosome-enriched extracellular vesicle preparation from tissue prepared based on the preceding paragraph comprises the following steps:

Centrifuge the medium at 500g for 10 minutes at 10°C;

Collect the supernatant and discard the pellet;

Centrifuge the supernatant at 3000g for 20 minutes at 10°C;

Transfer the supernatant to an ultracentrifuge tube;

Centrifuge the supernatant at 12000g for 20 minutes at 10°C;

Collect the supernatant and discard the pellet;

Centrifuge the supernatant at 100000g for 1 hour 10 minutes at 10°C; Remove the supernatant and collect the pellet; optionally 500ul of PBS and pass it through a size exclusion chromatography column (namely, qEV column (qEVoriginal/70nm, Izon science ltd)); collect the desired fraction and add PBS until 16mL to obtain a resuspension; Add 4ml of sucrose solution to an ultracentrifuge tube, add the resuspension to the ultracentrifuge tube 1 ml at a time until all the resuspension has been added to the tube in order to obtain a mixture;

Centrifuge the mixture at 100000g for 1 hour 10 minutes at 10°C;

Collect 4ml of the supernatant using a syringe and mix the supernatant with 16 ml of phosphate buffer solution (PBS) in order to obtain a second mixture; Centrifuge the second mixture at 100000g;

Resuspend the pellet.

[0061] In an embodiment, ultracentrifugation with sucrose cushion is used to purify and isolate EVs/exosomes.

[0062] In an embodiment, the sucrose cushion comprises a solution of 30% sucrose in heavy water.

[0063] In an embodiment, comparison of the cortical EVs/exosome isolation yield of 3 different conditioning medium was conducted. Nanoparticle Tracking Analysis (Nanosight) analysis was conducted to determine the yield obtained. Figure 3 shows the result of the cortical EVs/exosome isolation yield of the 3 different conditioning media. Here the result shows that incubation in Neurobasal allows a bigger yield. Comparison between the state-of-the-art protocol and the presently disclosed method was done by electroph microscope and cryo-electorn microscopy (Figure 4). Figure 4 shows that the disclose method allows a preparation with much higher percentage of vesicles bigger that 50nm than the state-of-the-art method, that has a considerable amount of smaller vesicles that may reflect contamination with smaller vesicles (not the intended EV population). Altogether, these data suggest that the new protocol provides exosome- enriched EVs yield. Similarly, further support of exosome-enriched EVs yield is shown at Figure 5 where detailed distribution of EVs per diameter show that the new protocol exhibit increased number of EVs between 50-100nm. [0064] Figure 5 shows the size distribution of the isolated vesicles of the state-of-the- art protocol and the presently disclosed method (data obtained by cryo-electorn microscopy).

[0065] In an embodiment, Figure 6 shows the results of the release protocol for brain EVs collection leding to an exosome-enriched yield.

[0066] Figure 6 shows nanoparticle tracking analysis (NTA) of the mode size (Figure 6a), average size (Figure 6b), and size distribution (Figure 6c) of particles between the state- of-the-art method and the presently disclosed method; grey area highligts that size range of exosomes (5-150nm). Figure 6d shows the percentage of particles in the size range of exosomes (grey area) and bigger (>150nm). The release method of the present disclosure results in more particles in the exosome range being isolated. Figure 6e shows the correlation between protein (measure by micro BSA) and number of particles (measured by NTA). The spontaneous release method of the present disclosure exhibits a stronger correlation, indicating less free protein in the EVs yield. (N_digestion=7, N_release=8; each sample represents the pooling of the cortex of 2 ½ mice). Figures 6f-j show data from EVs isolated from human frontal cortex using the the state-of-the-art method and the presently disclosed method. Nanoparticle tracking analysis (NTA) of the mode size (Figure 6f), average size (Figure 6g), and size distribution (Figure 6h) of particles. Figure 6j shows the percentage of particles in the size range of exosomes (grey area) and bigger (>150nm). Similarly, the release method of the present disclosure results in more particles in the exosome range (brain samples N_diges=3, N_releas=3) being isolated. All numerical data represented as mean ± SEM. (*P < 0.05).

[0067] In an embodiment, Figure 7 shows the proteomic analysis results of EVs isolated through the release protocol being enriched in extracellular vesicles-associated proteins.

[0068] Figure 7a shows the cellular component bioinformatic analysis of the EVs yield of release protocol showing enrichment in the categories related to exosomes. Figure 7b shows western blot validation and representative blots of different exosome-related proteins in the EVs yield of spontaneous release protocol. CD81 and flotillin, proteins associated with EVs, as well as EEA-1 which points to endocytic origin of the isolated vesicles were detected. The lack of cytochrome c attests to the absence of contaminants. In figures 7c-k EVs from the cortex, hippocampus and entorhinal cortex of one wild type mouse were pooled together and subjected to fluorescent antibody labeling and flow cytometry analysis. Density and dot plots show the violet size scatter (vSSC) in function of the fluorescent signal of vSSC-triggered events in samples containing FITC-tagged nanoscale-sized beads (Figure 7c), a mixture of APC-tagged anti- CD81, anti-CD9, and CD63 antibodies (Figure 7d; figures 10a-g), EVs simultaneously labeled with APC-tagged anti-CD81, anti-CD9 and/or CD63 antibodies in the absence (Figure 7e) or presence of NP40 detergent (Figure 7f) and overlayed with EVs isolated from the conditioned media of cpVenus-HEK cells by ultracentrifugation (Figure 7g), and EVs individually labeled with the three APC-tagged antibodies overlayed (Figure 7h; Figures 10d-f for the individual dot plots). Figures 7i-k shows EVs from the cortex of three wild type mice (N=3) were subjected to the individual or simultaneous-labeling with PE-CD81 and/or APC-CD9 antibodies, followed by flow cytometry analysis of vSSC- triggered events. Results from one sample are shown here. In figure 7i is a dot plot overlay showing the PE vs. APC fluorescence signal of individually (PE-CD81, yellow; APC- CD9, red) or simultaneously (green) labeled EVs. The coincidence of multiple events due to swarming was assessed by analyzing a mixture of individually-labeled EVs (turquoise). Figures 7j are density plots showing the vSSC of simultaneously-labeled EVs in function of PE or APC fluorescence, respectively. Shaded gates designated based on the background signal of negative controls are indicated, as well as the total amount of events within (turquoise gate: background; yellow gate: PE⁺ events; red gate: APC⁺ events). Figure 7k presents histograms showing the relative abundance of gated events in function of PE or APC fluorescence, with total events shown in grey. Figures 7l-m is an ExoView analysis of EVs isolated by the release protocol (the presently disclosed method). Figure 7l shows the size distribution of particles positive for CD81 or CD9. Figure 7m shows the total number of particles positive for CD81 or CD9 of 3 different EVs samples. [0069] In an embodiment, examples of the protein detected in proteomic analysis are shown in Table 2 below.

[0070] In an embodiment, Figure 8 shows that the method of the present disclosure detects drug-driven changes to extracellular vesicles production while it also shows that the EVs by the presently disclosed method are functional.

[0071] Figure 8a is a schematic representation of the inhibiting effect of the drug GW4869 on intracellular pathway of exosome biogenesis. Figures 8b-d show nanoparticle tracking analysis (NTA) of the mode size (Figure 8b), average size (Figure 8c), and size distribution (Figure 8d) of particles isolated by the presently disclosed method in vehicle- and GW4869-treated brain tissue. In gray, the size range of exosomes (50-150nm). Figure 8e shows the percentage of particles in the size range of exosomes (grey area) and bigger (>150nm). There is a significant decrease in the release of vesicles upon GW4869 treatment when compared to vehicle. Figure 8f shows the number of particles normalized to original tissue weight, detected by NTA. Figure 8g shows the protein in EV fraction normalized to original tissue weight, detected by Micro BCA™ Protein Assay Kit. Incubation with 20uM GW4869 reduces the amount of particles and protein detected. (N_veh=5, N_GW4869=3. Each sample represents the pooling of the cortex of 2 ½ mice). All numerical data shown as mean ± SEM. (*P < 0.05; **P < 0.01).

[0072] Figure 8h is a schematic representation of the experimental design where exosomes isolated through the release protocol (the presently disclosed method) were labeled and injected back to the outer molecular layer of the (dorsal) hippocampus of wild type (brain) mice. Figure 8i shows representative confocal microscopy photos. One day after the injection of Dil-labelled exosomes (red) in the OML, the EV-cargo is detected at the point of injection (green square: OML; yellow square: DG). After 4 weeks, the Dil-labelled exosomes were detected in the granular cell layer (GCL) of dentate gyrus suggesting the uptake of labeled-exosomes from dendrites of granule cells (located at the OML) and their intraneuronal travel to soma (located at GCL).

[0073] In an embodiment, Figure 9 shows that the exosome isolation method of the present dislcosure improves the quality of the isolated vesicles cortical samples.

[0074] Figures 9a-e shows that the method of the present disclosure isolates EVs from small and pathologically relevant areas, i.e., entorhinal and hippocampus. NTA analysis of particle mode size (Figure 9a), average size (Figure 9b), and size distribution of EVs (Figure 9c). Figure 9d shows the percentage of particles within the 50-150nm size range and bigger than 150nm. Figure 9e shows the protein in EVs fraction normalized to original tissue weight, detected by Micro BCA™ Protein Assay Kit. Figure 9f shows the total number of particles normalized to original tissue weight detected by NTA. (N_entorhinal=4, N_hippocampus=4, N_cortex=8, Each sample represents the pooling of the entorhinal cortex and hippocampus of 5 mice and the cortex of 2 ½ mice).

[0075] In an embodiment, Figure 10 shows control samples and data for the flow cytometry analysis of the relative abundance of endosomal tetraspanins in EVs isolated by the presently disclosed method from mouse brain tissue.

[0076] Figures 10a-f shows EVs from the brain of one wild type mouse subjected to fluorescent antibody labelling and flow cytometry analysis. Density and dot plots show the violet size scatter (vSSC) in function of the fluorescent signal of fluorescence- triggered events in samples containing water (Figure 10a), EVs incubated with APC- tagged isotype controls (Figure 10b, c), and EVs individually labelled with FITC-tagged anti-CD81, anti-CD9 and/or CD63 antibodies (Figure 10d-f). Figure 10d-f) Percentage of events within shaded gates designated based on the background signal of negative controls is indicated. A color-coded size range based on the vSSC of FITC-tagged beads is included on the right of the panel for the size comparison of events. Figure 10 g) Overlay of negative controls evaluating the specificity of PE detection used in figure 5g- h and j.

[0077] Figure 11 shows that the method of the present disclosure detects changes (increase) to EVs production and isolates functional extracellular vesicles.

[0078] Figures 11 a-f shows cortical samples which were incubated with 100uM Picrotoxin, which inhibits GABAergic (inhibitory) neurons creating an excitatory brain state and thus, trigerring the release of EVs. Figures 11a-e show nanoparticle tracking analysis (NTA) of the mode size (Figure 11a), average size ( Figure 11b) and size distribution (Figure 11c) of particles. In gray, the size range of exosomes (50-150nm). Figure 1id shows the percentage of particles in the size range of exosomes (grey area) and bigger (>150nm). There is a significant increase in number of exosome-related particles upon picrotoxin treatment. Figure 11 e shows the number of particles normalized to original tissue weight, detected by NTA. Figure 11f shows the protein in EV fraction normalized to original tissue weight, detected by Micro BCA™ Protein Assay Kit. Incubation with 100uM picrotoxin increases the amount of particles detected. (N_veh=3, N_picrotoxin=3; Each sample represents the pooling of the cortex of 2 ½ mice). All numerical data shown as mean ± SEM (*P < 0.05)

[0079] Figures 11g show the control groups for the exosome injection in mouse brain showing the correct site of injection and further validate the functionality of the isolated EVs. Figure 11g shows that PBS injection has no detection of red (dye Dill) signal, as well as injection of dye Dill that went through the complete EV isolation protocol. Figures 11 shows Dil dye injected directly in the OML is detected locally at 1-day post-surgery and is not taken up by the neurons of the OML and therefore is not detected in the GCL after 4-weeks period. [0080] Figure 12 shows that incubation of tissue for 16 hours does not significantly affect its viability.

[0081] Figure 12a shows representative blots of western blot analysis from brain tissue after the end of the the presently disclosed method (which includes tissue incubation for 16 hours). Figure 12b shows that incubation for 16 hours does not affect the expression of CytC, (Figure 12c) nor neuron-specific enolase (both related to neuronal death), in comparison with similar tissue incubation for 40 hours or simialr inculabtion with neurotoxic compound (1%Η₂O₂). Figure 12d shows Trypan blue-based counting of cells isolated from incubated tissue. Figure 12e shows that, contrast to 40 hours or H₂O₂ treatments, there is no increase in dead cells following incubation for 16 hours (N=3).

[0082] The above described embodiments are combinable.

[0083] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0084] The following claims further set out particular embodiments of the disclosure.

Claims

C L A I M S

1. Use of a neurobasal medium for isolated exosome-enriched extracellular vesicles

(EV) or for isolated exossomes preparation, wherein the medium comprises L- glutamine medium supplement, and antibiotic-antimitotic.

2. The use according to the previous claim wherein the L-glutamine medium supplement is 1% L-alanine-L-glutamine dipeptide.

3. The use according to the previous claim wherein the antibiotic-antimiotic is select from a list select of the list penicillin, streptomycin, Amphotericin B, or mixtures thereof.

4. The use according to any of the previous claims wherein the isolated EV/exosome is structurally intact, preferably the majority of the isolated EV/exosome is structurally intact.

5. The use according to any of the previous claims wherein 75% of the isolated EV/exosomes comprises a size from 50-150 nm.

6. The use according to any of the previous claims wherein 80% of the isolated EV/exosomes comprises a size from 50-150 nm.

7. The use according to any of the previous claims wherein the isolated EVs/exosomes fraction is without contaminants such as large vesicles, immature vesicles, subcellular organelles or debris.

8. The method according to the previous claim wherein the medium supplement is L- glutamine supplement, preferably 1% L-alanine-L-glutamine dipeptide.

9. The use according to any of the previous claims wherein the sucrose cushion is a solution comprising Heavy Water, sucrose and Tris-base.

10. A method for isolation of exosome-enriched extracellular vesicle preparation comprising the following steps: obtaining a brain tissue sample; incubating the brain tissue sample in a neurobasal incubating medium; wherein the medium comprises L-glutamine medium supplement, and antibiotic-antimitotic; isolating the obtained exosome-enriched extracellular vesicles by ultra- centrifuging.

11. The method according to any of the previous claims, wherein incubation of the brain tissue in the neurobasal incubating medium is at a temperature from 30 °C to 40 °C, preferably 37 °C.

12. The method according to the any of the previous claims wherein the step of isolating exosome-enriched extracellular vesicles/exossomes by ultra-centrifugation comprises the following step: centrifuge the medium at 500g for 10 minutes at 10°C; collect the supernatant and discard the pellet; centrifuge the supernatant at 3000g for 20 minutes at 10°C; transfer the supernatant to an ultracentrifuge tube; centrifuge the supernatant at 12000g for 20 minutes at 10°C; collect the supernatant and discard the pellet; centrifuge the supernatant at 100000g for 1 hour 10 minutes at 10°C; remove the supernatant and collect the pellet; resuspend the pellet in 16 ml of PBS to obtain a resuspension and pass it through a size exclusion chromatography colunn; add 4ml of sucrose solution to an ultracentrifuge tube , add the resuspension to the ultracentrifuge tube 1 ml at a time until all the resuspension has been added to the tube in order to obtain a first mixture; centrifuge the mixture at 100000g for 1 hour 10 minutes at 10°C; collect 4ml of the supernatant using a syringe and mix the supernatant with 16 ml of phosphate buffer solution (PBS) in order to obtain a second mixture; centrifuge the second mixture at 100000 g; resuspend the pellet.

13. The method according to any of the previous claims, wherein incubation of the second mixture is in 5% CO₂.

14. The method according to any of the previous claims wherein the first supernatant is centrifuged at 3000g for 20 minutes at 10°C.

15. The method according to any of the previous claims wherein the second supernatant is centrifuged at 12000g for 20 minutes at 10°C.

16. The method according to any of the previous claims wherein the third supernatant is centrifuged at 100000g for 1 hours 10 minutes at 10°C.

17. The method according to any of the previous claims wherein the first mixture is resuspended and goes through a size exclusion chromatography and/or the second misture is centrifuged at 100000g for 1 hour 10 minutes at 10°C.

18. The method according to any of the previous claims wherein the third extract is centrifuged at 100000g at 10°C.