Combining extracellular vesicles for characterization

Abstract

The invention provides a method for combining extracellular vesicles (10) from a biological sample (15) into a reconstituted vesicle (20), the method comprising: an isolation stage comprising isolating the extracellular vesicles (10) from the biological sample (15) to provide a prepared sample (115) comprising the extracellular vesicles; a drying stage comprising (a) providing a droplet (125) of the prepared sample (115) on a support (120) wherein the support (120) is electrically conductive (121), and (b) drying the droplet (125) to provide a multilayer (135) of dried extracellular vesicles (10); a reconstitution stage comprising (a) exposing the multilayer (135) to an aqueous reconstitution liquid (130), wherein the reconstitution liquid (130) comprises < 600 mM of ions, and (b) exposing the multilayer (135) to an AC electric field having a field strength selected from the range of 20-1500 V / m and a frequency selected from the range of 5 - 20 Hz, thereby providing the reconstituted vesicle (20), wherein the reconstituted vesicle (20) has a equivalent spherical diameter (DR) selected from the range of 2 - 50 pm; and a separation stage comprising removing the reconstituted vesicle (20) from the support (120).

Description

[0001] Combining extracellular vesicles for characterization

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a method for combining extracellular vesicles from a biological sample into a reconstituted vesicle. The invention further relates to a characterization method. The invention further relates to a system for combining extracellular vesicles from a biological sample into a reconstituted vesicle.

[0004] BACKGROUND OF THE INVENTION

[0005] Methods for characterizing extracellular vesicles are known in the art. For instance, FERGUSON et al., Single extracellular vesicle analysis for early cancer detection, Trends in Molecular Medicine, 2022, Vol. 28, No. 8, provides an overview of extracellular vesicles and the rationale for using them for early cancer detection, and describes technologies for single EV analysis (sEVA).

[0006] SUMMARY OF THE INVENTION

[0007] In the body, cells, including tumor cells, communicate with distant cells by secreting membrane-encapsulated vesicles comprising biological information into bodily fluids, such as into blood, urine, saliva, and cerebrospinal fluids. These vesicles are called Extracellular Vesicles (or “EVs”). Once taken up, the biological information carried by an EV can be used by a recipient cell, and may result in changes in the recipient cell, such as in favor of a tumor cell from which the EV originated.

[0008] The biological information carried by EVs may be indicative of a state of the cell from which the EV originated. For instance, EVs of different cell types may be distinguished, and EVs from diseased cells, such as from cancer cells, may be distinguished from healthy cells.

[0009] The membrane of EVs may resemble the membrane of the cell from which the EVs originate. Hence, a characterization of membrane properties of a cell may be approximated by characterization of a corresponding EV.

[0010] The prior art may describe methods for isolating EVs, for characterizing properties of EVs, and for distinguishing between EVs originating from healthy and unhealthy cells. However, the prior art methods may be limited in the possible analyses the EVs are subjected to. For instance, prior art methods may be unsuitable for an electrophysiological characterization of EVs.

[0011] The prior art may further describe methods for isolating cells from a (human) body by taking a biopsy and subsequently characterizing the cells. However, such methods are invasive, which may be detrimental to subjects, may require a preceding localization of (diseased) cells, and may only be suitable once a disease, e.g., a tumor, has substantially developed.

[0012] Hence, it is an aspect of the invention to provide an alternative method, which preferably further at least partly obviates one or more of above-described drawbacks. The present invention may have as object to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

[0013] In a first aspect, the invention may provide a method for combining extracellular vesicles from a biological sample into a reconstituted vesicle. In embodiments, the method may comprise an isolation stage, a drying stage, and a reconstitution stage. The isolation stage may comprise isolating the extracellular vesicles from the biological sample to provide a prepared sample comprising the extracellular vesicles. The drying stage may comprise providing a droplet of the prepared sample on a support (directly or indirectly). The drying stage may further comprise drying the droplet to provide a multilayer of dried extracellular vesicles. The reconstitution stage may comprise (a) exposing the multilayer to an aqueous reconstitution liquid, especially wherein the reconstitution liquid comprises < 600 mM of ions, and (b) exposing the multilayer to an AC electric field, wherein the AC electric field has a field strength selected from the range of 20 - 1500 V / m at a frequency selected from the range of 5 - 20 Hz, thereby providing the reconstituted vesicle. The reconstituted vesicle may especially have an equivalent spherical diameter selected from the range of 2 - 50 pm, such as from the range of 2 - 30 pm. The separation stage may comprise removing the reconstituted vesicle from the support.

[0014] Hence, the invention may provide a method for combining extracellular vesicles from a biological sample into a reconstituted vesicle, the method comprising an isolation stage comprising isolating the extracellular vesicles from the biological sample to provide a prepared sample to provide the extracellular vesicles; a drying stage comprising (a) providing a droplet of the prepared sample on a support, and (b) drying the droplet to provide a multilayer of dried extracellular vesicles; a reconstitution stage comprising (a) exposing the multilayer to an aqueous reconstitution liquid, wherein the reconstitution liquid comprises < 600 mM of ions, and (b) exposing the multilayer to an alternating current electric field, the alternating current electric field having a field strength selected from the range of 20 - 1500 V / m and a frequency selected from the range of 5 - 20 Hz, thereby providing the reconstituted vesicle, wherein the reconstituted vesicle has a equivalent spherical diameter selected from the range of 2 - 50 pm; and a separation stage comprising removing the reconstituted vesicle from the support.

[0015] Thereby, the method of the invention may provide a reconstituted vesicle from a plurality of extracellular vesicles. In particular, the reconstituted vesicle may have a larger size than typical EVs and may be suitable for downstream analysis requiring a minimal size exceeding typical EV sizes. For instance, the small size of EVs (diameters are typically tens to hundreds of nanometers) may hinder experiments such as patch clamp electrophysiology, which may require samples of at least a couple of micrometers in diameter.

[0016] For instance, the method of the invention may facilitate an electrophysiological characterization of EVs, which may be particularly useful to determine the properties of membrane proteins of EV, such as of ion channels. In cells, membrane proteins provide vital functions, including the transport of ions, small molecules, and water in response to internal and external signals, define the cell’s immunological identity, and facilitate intra- and intercellular communication. Further, membrane proteins are clinically attractive molecules to diagnose and treat afflictions, such as tumors, as the membrane protein composition of tumor cells may generally differ from their healthy counterparts, the changes depending on the stage of the tumor and the specific relevance of the protein for tumor cells. Yet further, membrane proteins are exposed to the extracellular environment and thus relatively easy to target. Membrane proteins being suitable and appropriate targets may be exemplified by an overrepresentation of membrane proteins as drug target among drugs approved by the American Food and Drug Administration (FDA).

[0017] In particular, since EVs represent part of tumor cells’ membrane proteins, it would be possible to study ion-conducting membrane proteins and receptors of tumor cells, i.e. potential drug targets, by measuring ion channel activities in the reconstituted vesicle by using patch clamp electrophysiology. In particular, the reconstituted vesicle, including the membrane proteins from the original EVs, may be exposed to candidate drugs before or during an electrophysiology measurement to assess the impact of the candidate drug on ion channel blockage. The testing of the impact of candidate drugs on cells to assess ion channels blockage may be a key method for drug discovery. The method of the invention may further facilitate testing drug candidates with respect to the membrane proteins present on the diseased cells, e.g. tumor cells, of a specific patient, i.e., the method may the testing of drug candidates on an individual level (personalized medicine). A further benefit of the method of the invention is that the biological sample may be obtained in a non-invasive manner or, depending on the sample type, with a relatively simple and safe procedure. Yet further, as the EVs are dispersed through the (human) body, an exact localization of a (disease) tissue of interest is not required in order to isolate corresponding EVs. The method may thus be suitable for characterization of EVs - and their corresponding cells - in an early stage of disease onset.

[0018] The invention may herein for explanatory purposes be primarily described in the context of EVs originating from tumor cells. It will be clear to the person skilled in the art, however, that the invention is not limited to such applications and may also be used for the generation of a reconstituted vesicle of EVs originating from healthy cells, such as of immune cells.

[0019] As described above, the invention may provide a method for combining extracellular vesicles from a biological sample into a reconstituted vesicle.

[0020] The term “extracellular vesicle” may herein refer to a lipid bilayer nanoparticles secreted by cells, such as by eukaryotic cells, or such as by prokaryotic cells. Cells, including tumor cells, may communicate with their neighbors and distant cells by secreting biological information in such extracellular vesicles. The extracellular vesicles may, for instance, comprise microvesicles, exosomes, membrane particles, membrane vesicles, and / or apoptotic bodies. In embodiments, the extracellular vesicles may especially comprise exosomes.

[0021] The term “biological sample” may herein refer to essentially any biological sample, such as a sample of a bodily fluid, or such as a sample of a body of water or of a biofilm. The biological sample may especially comprise a bodily fluid, such as a bodily fluid selected from the group comprising a blood sample, a urine sample, a saliva sample, a sputum sample, a stool sample, a lymph fluid sample, a pancreatic juice sample, a tear sample, an ascitic fluid sample, a pleural fluid sample, a thoracic fluid sample, a breast milk sample, and a cerebrospinal fluid sample.

[0022] The term “reconstituted vesicle” may herein refer to a reconstructed vesicle. In particular, the EVs may be dried and subsequently reconstituted as reconstituted vesicles by rehydration and exposure to an alternating current electric field (see below).

[0023] In embodiments, the method may comprise an isolation stage, a drying stage, and a reconstitution stage.

[0024] The isolation stage may comprise isolating the extracellular vesicles from the biological sample to provide a prepared sample. The prepared sample may especially be enriched in extracellular vesicles relative to the biological sample. The efficiency (and success) of the drying and reconstitution of the vesicles, as well as the subsequent analysis, may be affected by the amount of extracellular vesicles in the prepared sample, especially by the extracellular vesicle protein concentration and the extracellular vesicle lipid concentration in the prepared sample.

[0025] The terms “extracellular vesicle protein concentration” and “extracellular vesicle lipid concentration” may herein refer to the amount of proteins and lipids, respectively, thar are comprised by extracellular vesicles, i.e., excluding any potential free-floating lipid and / or protein.

[0026] In embodiments, the prepared sample may comprise an extracellular vesicle protein concentration selected from the range of 0.01 - 6 mg / mL, such as from the range of 0.02 - 3 mg / mL, especially from the range of 0.1 - 2 mg / mL, such as from the range of 0.2 - 1.6 mg / ml. If the protein concentration is low, e.g., (well) below 0.01 mg / ml, reconstituted vesicles may have few - or even no - membrane proteins, i.e., multiple reconstituted vesicles may need to be analyzed in order to characterize the EVs. However, if the protein concentration is high there will be many channels relative to the membrane area of the reconstituted vesicle (high current density), which may complicate the analysis of the membrane proteins, i.e., it may be more challenging to identify the membrane proteins and to characterize them. Hence, if the extracellular vesicle protein concentration in the prepared sample is above 3 mg / ml, especially above 6 mg / ml, the prepared sample may be diluted, especially to provide a prepared sample with an extracellular vesicle protein concentration in the range of 0.2 - 1.6 mg / ml. The dilution may especially be performed using an aqueous liquid, preferably with similar properties to the reconstitution liquid (see below), such as comprising < 600 mM of ions, especially with the reconstitution liquid.

[0027] In further embodiments, the prepared sample may comprise an extracellular vesicle lipid concentration selected from the range of 0.2 - 1.6 mg / mL, such as from the range of 0.4 - 1.2 mg / mL, especially from the range of 0.7 - 0.9 mg / mL. If the lipid concentration is low a provided dried multilayer (see below) may be relatively thin, e.g. < 5 layers, which may lead result in a relatively poor generation of reconstituted vesicles, particularly in the generation of few reconstituted vesicles. Further, higher field strengths may need to be applied to generate reconstituted vesicles from thin multilayers and the reconstitution may be slower. The higher field strength and longer duration may further affect the protein activity either directly or indirectly by lipid oxidation, which in turn may affect the structure and / or the activity of the ion channels. However, if the lipid concentration is high, thick multilayer vesicles or tubes may be generated, which may complicate, especially prevent, ion channel activity measurements. Furthermore, as ion channel activity may require ionic solutions on both side of the single bilayer, multilayer vesicles with limited amount of ions in between the layers may generate only a small amount of current before the ions are drained. Further, the analysis of membrane proteins may exclude any proteins embedded in the inner layers of a multilayer vesicle, i.e., these membrane proteins would be lost from the analysis, if the extracellular vesicle lipid concentration in the prepared sample is above 1.2 mg / ml, especially above 1.6 mg / ml, the prepared sample may be diluted, especially to provide a prepared sample with an extracellular vesicle lipid concentration in the range of 0.7 - 0.9 mg / ml. The dilution may especially be performed using an aqueous liquid, preferably with similar properties to the reconstitution liquid (see below), such as comprising < 600 mM of ions, especially with the reconstitution liquid.

[0028] If the extracellular vesicle lipid concentration is below a threshold value, e.g., below 0.7 mg / mL, such as below 0.4 mg / mL, especially below 0.2 mg / mL, synthetic lipids may be provided to complement the amount of lipids. The lipids may be complemented with the addition of liposomes to the prepared sample and / or by drying layers of extracellular vesicles and synthetic lipids on the same spot in the drying stage (see below).

[0029] Hence, in embodiments, the isolation stage may comprise supplementing the prepared sample with liposomes. The liposomes may then be dried and reconstituted along with the EVs, essentially providing an increased lipid content while (essentially) not influencing the composition of membrane proteins.

[0030] In further embodiments, the liposomes may have a distribution of equivalent spherical diameters approximating the distribution of EVs in the prepared sample. For homogeneity, it may be preferable for the liposomes to have similar sizes as the EVs. In embodiments, in the prepared sample, the liposomes may have a number average equivalent spherical diameter dcso. In further embodiments, in the prepared sample, the EVs may have a number average equivalent spherical diameter dsso, wherein dcso / dEso is selected from the range of 0.5 - 2.0, especially from the range of 0.75 - 1.5, such as from the range of 0.8 - 1.2. In further embodiments, d so may be selected from the range of 30 - 1000 nm, such as from the range of 50 - 500 nm, especially from the range of 80 - 200 nm.

[0031] The equivalent spherical diameter (or ESD) (or “spherical equivalent diameter”) of an (irregularly shaped) three-dimensional shape is the diameter of a sphere of equivalent volume. For a sphere, the diameter is the same as the equivalent spherical diameter. Would a sphere in an xyz-plane with a diameter D be distorted to any other shape (in the xyz-plane), without changing the volume, then the equivalent spherical diameter of that shape would be D. It will be clear to the person skilled in the art that the performance of membrane proteins may depend on the lipid composition of the membrane in which the membrane proteins are arranged. A characterization of the reconstituted vesicle may thus preferably be performed on a reconstituted vesicle having (approximately) the same lipid composition as the corresponding EVs. Hence, in embodiments, the lipid composition of the liposomes may be based on the (lipid composition of the) extracellular vesicles. Especially, the lipid composition of the liposomes may approximate the lipid composition of the extracellular vesicles. In embodiments, at least 80 wt% of the lipid composition of the liposomes may correspond to the lipid composition of the EVs, such as at least 90%, especially at least 95%. The lipid composition of EVs may vary between different cell types and based on a disease state, such as vary between a tumor cell and a (healthy) cell of the same cell type. In particular, healthy cells and tumor cells may differ in lipid composition. The lipid composition of an EV may depend on the lipid composition of the cell it originates from, but may differ therefrom. The following table indicates lipids with increase prevalence or decreased prevalence in various types of EVs. In embodiments, the prepared sample may relative to the biological sample be enriched in extracellular vesicles of a first type. For instance, the isolation stage may comprise specifically isolating extracellular vesicles of a first type, especially wherein the extracellular vesicles of a first type originate from a first cell type, e.g., lung cells or immune system cells, or a first disease type, e.g., tumor cells. For instance, in embodiments, the isolation stage may comprise specifically isolating EVs originating from lung cells from the biological sample. Alternatively, as a further example, the isolation stage may comprise specifically isolating extracellular vesicles originating from tumor cells from the biological sample. The EVs originating from different cell types may be distinguished based on biological markers, such as based on membrane proteins.

[0032] In embodiments, the isolation stage may comprise selectively isolating a first type of extracellular vesicles from the biological sample using affinity purification. For instance, the isolation stage may comprise exposing the biological sample to an affinity probe, such as an antibody or aptamer, selective for the first type of extracellular vesicles. The biological sample may, for instance, be passed along a resin, wherein the affinity probe is (physically or chemically) bound to the resin. Alternatively, for example, the biological sample may be (temporarily) exposed to beads, wherein the affinity probe is (physically or chemically) bound to the beads. Methods for affinity purification will be known to the person skilled in the art.

[0033] In embodiments wherein a biological sample is obtained from a subject, such as embodiments wherein the biological sample comprises a bodily fluid, the extracellular vesicle of the first type may typically correspond to a cell type of the subject, e.g., extracellular vesicles of a specific type of human cells, such as lung cells or heart cells. However, in embodiments, the extracellular vesicle of the first type may especially be microbial extracellular vesicles, such as bacterial extracellular vesicles. For instance, bacterial extracellular vesicles (or “BEVs”) of gut bacteria may penetrate the gut-vascular barrier to enter a (human) subject’s circulatory system. Possibly, the BEVs from gut microbes may further penetrate the choroid plexus vascular barrier Besides in stool samples, BEVs may, for instance, also have been found in blood samples. The characterization of microbial extracellular vesicles may provide further information on a health status of a subject, especially regarding a disease status.

[0034] In such embodiments, the extracellular vesicle protein concentration may specifically refer to proteins of extracellular vesicles of the first type.

[0035] In further embodiments, the first type of extracellular vesicles corresponds to tumor cells. In such embodiments, the isolation stage may, for instance, comprise selectively isolating the extracellular vesicles based on one or more of epithelial cell adhesion molecule EpCAM, a tetraspanin selected from the group comprising CD9, CD81, and CD63, and other surface proteins, such as EGFR. The epithelial cell adhesion molecule EpCAM may be overexpressed in carcinomas and may serve as a marker for tumor cells and, by extent, for extracellular vesicles originating from tumor cells.

[0036] In further embodiments, the first type of EVs may correspond to cancer cells of a specific cell type. In such embodiments, the isolation stage may comprise selectively isolating the EVs based on factors enriched in tumor cells of the specific cell type. The following table provides some examples of exosome targets that may be suitable for isolating tumor cells of a specific cell type:

[0037] In further embodiments, the first type of extracellular vesicles corresponds to immune cells, such as a dendritic cell, or such as a T-cell, or such as a natural killer cell. The characterization of a reconstituted vesicle formed from EVs from immune cells may facilitate the identification of an immune status, such as in the context of immunotherapy or immunomodulation.

[0038] As described above, the method may further comprise a drying stage. The drying stage may comprise providing a droplet of the prepared sample on a support, especially directly on the support, or especially indirectly on the support (see below). The support may especially be electrically conductive (for reconstitution and separation; see below). In embodiments, the support may thus comprise an electrically conductive material. For instance, in embodiments, the support may comprise indium tin oxide.

[0039] The drying stage may further comprise drying the droplet (on the support) to provide a multilayer of dried extracellular vesicles. In particular, the EVs within the droplet may dry on top of one another, thereby providing a multilayer of dried EVs. Without being bound by theory, it appears that the EVs may open during drying, resulting in each EV providing a single (bi)layer of the multilayer.

[0040] In embodiments, the droplet may have a droplet volume selected from the range of 0.5 - 5 pL, especially from the range of 1-3 pL, such as from the range of 1.5 - 2.5 pL. The droplet volume may especially be selected in view of the concentration of extracellular vesicles in the prepared sample. In particular, the droplet size may be selected to provide a desired number of layers in the multilayer, such as 5 - 10 layers.

[0041] The number of layers in the multilayer may affect the reconstitution in the reconstitution stage. If the number of layers in the multilayer is too low, the reconstituted vesicle may not form. If, however, the number of layers in the multilayer is too high, tubes and multilayer liposomes (layered like onions) may be formed instead of reconstituted vesicles.

[0042] Hence, in embodiments, (at least part of) the multilayer of dried extracellular vesicles comprises n layers of dried extracellular vesicles, wherein n is selected from the range of 3 - 20, such as from the range of 4 - 15, such as from the range of 5 - 10, or such as from the range of 8-12 layers. In further embodiments, n > 3, such as > 5, especially > 8, such as > 10. In further embodiments, n < 20, such as < 15, especially < 12, such as < 10. A multilayer of (about) 10 layers may be particularly suitable for the formation of the reconstituted vesicle.

[0043] The drying stage may further comprise providing a plurality of droplets to the support, especially directly, or especially indirectly. In particular, the drying stage may comprise providing the plurality of droplets in spatially separated spots, i.e., such that the droplets are spatially separated on the support. The spatial separation of the droplets may facilitate the reconstitution as water may enter between dried lipid layers via the empty space between droplets.

[0044] As described above, the drying stage may comprise drying the droplet (or droplets) on the support to provide a multilayer (or “multilayers”) of the extracellular vesicles.

[0045] In further embodiments, the drying stage may comprise drying the droplet such that the multilayer has a water content selected from the range of < 10 wt%, such as < 5 wt%, especially < 1%.

[0046] The drying stage may, in embodiments, comprise drying the droplet to air or to an inert gas, especially to an inert gas. In such embodiments, the drying stage may especially have a duration of 1 - 5 days, such as 2 - 4 days. A drying duration of multiple days may, however, be detrimental to the membrane proteins due to potential degradation and oxidation.

[0047] Hence, in further embodiments, the drying stage may comprise drying the droplet using vacuum drying. Vacuum drying may be a relatively quick drying method. However, a complete dehydration in vacuum may be detrimental to the membrane proteins in the EVs. Hence, in embodiments comprising vacuum drying the drying stage may especially comprise partially drying the droplets. For instance, in such embodiments, the drying stage may comprise (partially) drying the droplet at room temperature until a vapor pressure is in the range of 11.7 - 18 mmHg, especially in the range of 13.00 mmHg - 17.25 mmHg. The unit mmHg corresponds to 133.322 Pa, i.e., to 133.322 N / m2. For instance, in embodiments, the droplet may be dried in the presence of a saturated salt solution (in a vacuum chamber) such that some humidity may remain during the vacuum drying.

[0048] Further, in embodiments comprising vacuum drying, the drying stage may have a duration selected from the range of 1-6 hours, especially from the range of 2-4 hours.

[0049] It will be clear to the person skilled in the art that drying may be accelerated by exposing the droplet to an elevated temperature. However, an elevated temperature may also damage the extracellular vesicles, such as damage the membrane proteins, which may be detrimental to downstream analyses. Hence, in embodiments, the drying stage may be performed at a temperature selected from the range of 20 - 38 °C, such as at room temperature.

[0050] As mentioned above, a low extracellular vesicle lipid concentration in the prepared sample may be complemented with the addition of drying layers of extracellular vesicles and synthetic lipids on the same spot in the drying stage. Hence, in embodiments, the drying stage may comprise providing a dried synthetic lipid layer on the support, and subsequently providing the droplet of the prepared sample on the synthetic lipid layer, i.e., providing the droplet (indirectly) to the support by providing the droplet on the synthetic lipid layer.

[0051] In further embodiments, the drying stage may comprise (a) providing a droplet of the prepared sample to a spot and drying the droplet, and subsequently (b) providing a lipid droplet of a lipid composition to the spot and drying the lipid droplet. Alternatively, in further embodiments, the drying stage may comprise (a) providing a lipid droplet of a lipid composition to a spot and drying the lipid droplet, and subsequently (b) providing a droplet of the prepared sample to the spot and drying the droplet. Drying the lipid droplet first and then adding the prepared sample may provide the benefit that if any lipid remains on the support, the remaining lipid is more likely to be of synthetic origin, i.e., a larger proportion of the EVs, including potential membrane protein, may end up in the reconstituted vesicle.

[0052] Hence, the drying stage may comprise providing the droplets to the support and drying the droplets such that the dried multilayer is suitable for subsequent reconstitution into reconstituted vesicles. The drying stage may especially be directed to providing the multilayer with (1) a suitable thickness and (2) defects (or “empty space”) between droplets. In particular, the best results (in terms of reproducibility, reconstituted vesicle size, and the reconsititued vesicles comprising a single-bilayer) were obtained when samples were applied in 2 microliter spatially separated droplets on the support (e.g., on an ITO glass surface). After drying, the surface has more homogeneously formed lipid layers. If instead of individual droplets a high concentration of sample was applied in one spot, the dried lipids would form thicker lipid layers, and their rehydration would yield tubes and multilayer liposomes. These tube and multilayer liposome structures may be unsuitable for electrophysiological studies of membrane proteins.

[0053] As described above, the method may further comprise the reconstitution stage. The reconstitution stage may comprising exposing the multilayer to an aqueous reconstitution liquid (or ‘aqueous reconstitution solution’) and to an alternating current electric field (or ‘AC electric field) to provide a reconstituted vesicle. In particular, exposure of the multilayer to water or a sugar solution may cause the dried lipids to rehydrate and swell. In the meantime, the application of the AC electric field may vibrate the solution and may cause growing lipid bilayer blebs to fuse to their neighbors, resulting in the formation of reconstituted vesicles with tens of micrometers in diameter.

[0054] The aqueous reconstitution liquid may comprise water and one or more dissolved compounds. The dissolved compounds may affect the pH and osmolarity of the reconstitution liquid and may, for instance, affect the membrane potential during an analysis of the reconstituted particle (see below). Further, the properties of the aqueous reconstitution liquid may affect the formation of the reconstituted particle. For instance, (salt) ions may affect the size of the vesicle and excess ions may even prevent the formation of reconstituted vesicles. In particular, the (salt) ions may shield the surface charges on the phospholipids and may reduce inter membrane repulsive electrostatic forces, which may hamper their fusion during the electroformation process. In particular, the ion concentration may inversely correlated with an (average) size of the reconstituted vesicles. Hence, in embodiments, the reconstitution liquid may comprise < 800 mM of ions, such as < 600 mM of ions, especially < 300 mM of ions. In further embodiments, the reconstitution liquid may comprise < 60 mM of ions, such as < 30 mM of ions, especially < 10 mM of ions, such as < 5 mM of ions, including (essentially) 0 mM of ions. Ion concentrations of < 10 mM may be particularly suitable for the consistent formation of reconstituted vesicles having a suitable size range for analysis with patch clamp electrophysiology (see below). However, depending on the membrane protein, the presence of (salt) ions may affect the shape, and by extent the function, of a membrane protein. As (salt) ions are present under physiological conditions, it may be desirable to have some (salt) ion presence in the reconstitution liquid. Hence, in further embodiments, the reconstitution liquid may comprise > 0 mM of ions, especially > 5 mM of ions, such as > 10 mM of ions, especially > 20 mM of ions.

[0055] In further embodiments, the reconstitution liquid may comprise sugar, especially sucrose. The sugar may serve to adjust the osmolarity of the reconstitution liquid, and thus of the formed reconstituted vesicle, without raising the ion concentration. In particular, the sugar may serve to match the osmolarity of the reconstituted vesicle with an electrolyte used in a downstream analysis. For instance, ion channel activity may typically be tested at an osmolarity approximating a typical physiological osmolarity, such as at about 300 mOsm, i.e., with an electrolyte comprising (about) 300 mM of osmotically active compounds. In principle, any sugar may be used to raise the osmolarity. In embodiments, the sugar may comprise a sugar alcohol, especially sorbitol. In further embodiments, the sugar may be selected from the group comprising sucrose, glucose, fructose, galactose, maltose or lactose. The sugar may especially comprise one or more of sucrose, fructose or galactose, more especially (at least) sucrose. Sucrose may be particularly beneficial due to its relatively high density which - in aqueous iso- osmolar solutions - may facilitate sedimentation of the reconstituted vesicle at the bottom of an analysis chamber, which may simplify the downstream analysis. In particular, such sedimentation may be beneficial for patch clamp electrophysiology as the vesicles have to be ‘caught’ for analysis. Hence, in embodiments, the reconstitution liquid may comprise 100 - 400 mM of sugar, such as 150 - 350 mM of sugar, especially 200 - 300 mM of sugar. In further embodiments, the reconstitution liquid may comprise at least 100 mM of sugar, such as at least 150 mM of sugar, especially at least 200 mM of sugar, such as at least 250 mM of sugar. In further embodiments, the reconstitution liquid may comprise at most 500 mM of sugar, such as at most 400 mM of sugar, especially at most 350 mM of sugar, such as at most 300 mM of sugar, especially at most 250 mM of sugar.

[0056] The reconstitution liquid may further have a pH corresponding to the pH of the environment of cells from which the EVs originate, i.e., the reconstitution liquid may have a physiologically relevant pH for the EVs and the thereby comprised membrane proteins. In particular, the shape, and by extent the function, of a membrane-protein may be pH-dependent. Membrane proteins may thus be preferably characterized in an environment having a pH matching the physiological pH in which these membrane proteins operate. Although pH may typically be tightly controlled in the (human) body, the pH may vary for different organs. For instance, blood may typically have a pH of about 7.3 - 7.45, whereas lungs may typically have a pH of around 6.7, the stomach may typically have a pH of around 1.5 - 3.5, and the bladder may typically have a pH of about 4.5 - 8.

[0057] In particular, in embodiments wherein the extracellular vesicles are of a first cell type, the reconstitution liquid may have a pH selected based on a physiological pH of the first cell type. In further embodiments, the reconstitution liquid may have a pH selected from the range of 1.5 - 8, such as from the range of 4.5 - 8, especially from the range of 6 - 7.8, such as from the range of 7.2 - 7.6.

[0058] Hence, in specific embodiments, the reconstitution liquid may (a) comprise 200 - 300 mM of sugars, especially of sucrose, (b) comprise < 10 mM of ions, and (c) have a pH selected from the range of 5.5 - 8, especially from the range of 7.2 - 7.6.

[0059] The reconstitution stage may further comprise exposing the multilayer to an AC electric field. The AC electric field may have two related functions: (1) to facilitate rehydration of lipid bilayers by the reconstitution liquid in a well-controlled manner by affecting intermembrane electrostatic forces, and (2) to generate gentle mechanical agitation. Both of these functions may be important for lipid rehydration, fusion, and detachment from the surface. Generally, the rehydration of dried lipids may be facilitated by hydration forces, inter membrane electrostatic forces, and van der Waals forces. However, even when exposed to the reconstitution liquid, the multilayer of dried lipid bilayers will not form reconstituted vesicles with an equivalent spherical diameter of 2+ pm spontaneously; only some blisters and possibly some (very) small vesicles may form, which vesicles may typically remain bound to the support. The application of an AC electric field may affect inter membrane electrostatic forces and, as a result, lipid bilayers swell and form small vesicles (e.g., about 10 micrometer in diameter), which may come in contact with other swelling vesicles and fuse to each other, and at some point, detach from the lipid film. Then, the same process may occur for any remaining layer(s) of lipids. The AC electric field may also generate gentle vibrations, which helps facilitate the formation, fusion and detachment of vesicles from the support (e.g., from remaining lipid bilayers on the support).

[0060] In particular, the formation of the reconstituted vesicle may depend on the field strength and frequency of the AC electric field and on the duration of the application of the AC electric field.

[0061] In embodiments, the AC electric field may have a field strength selected from the range of 5 - 2500 V / m, especially from the range of 10 - 2000 V / m, such as from the range of 20 - 1500 V / m, especially from the range of 400 - 1100 V / m. The field strength may be selected in view of the number of layers in the multilayer (see below). In particular, a higher field strength may be selected for multilayers with fewer layers. For instance, a field strength < 1500 V / m may be selected for multilayers comprising about 10 layers, whereas a field strength of (about) 2000 V / m may be selected for multilayers comprising about 5 layers.

[0062] In further embodiments, the AC electric field may have a frequency selected from the range of 5 - 20 Hz, especially from the range of 10-12 Hz, or especially from the range of 5 - 10 Hz.

[0063] In principle, in the reconstitution stage, the AC electric field may directly be applied with a maximal field strength and frequency, e.g., with a field strength of 1100 V / m and a frequency of 10 Hz. However, it may be beneficial for the formation of the reconstituted vesicle - and for the therein comprised membrane proteins - to be gradual. Hence, in embodiments, the reconstitution stage may comprise ramping up the field strength and / or the frequency over time (or “during the reconstitution stage”), especially at least the field strength, or especially at least the frequency. The gradient in the field strength and / or the frequency may facilitate gradually forming the rehydrated vesicles and preventing damage to the membrane proteins present in the lipid bilayers.

[0064] In further embodiments, the reconstitution stage may be devoid of a ramp (or “gradient”) for the field strength and / or for the frequency, especially for the field strength and the frequency. Especially, in such embodiments, the field strength may be selected from the range of 0.4 - 1.5, such as from the range of 0.8 - 1.2. Further, in such embodiments, the frequency may be selected from the range of 6 - 20 Hz, such as from the range of 8 - 16 Hz. Such embodiments may facilitate a relatively fast generation of the reconstituted vesicle.

[0065] In specific embodiments, the reconstitution stage may comprise exposing the multilayer to an AC electric field having a field strength selected from the range of 20 - 1500 V / m and a frequency selected from the range of 5 - 20 Hz, especially from the range of 10-12 Hz, thereby providing the reconstituted vesicle.

[0066] The amount of time it takes to form the reconstituted vesicle may depend on several factors, including the composition of the reconstitution liquid, the empty space around the dried multilayer, and the properties of the AC electric field. In embodiments, the reconstitution stage may, for instance, have a duration selected from the range of 20 - 360 minutes, such as from the range of 30 - 240 minutes, especially from range of 60 - 240 minutes, such as from the range of 60 - 200 minutes. In further embodiments, the reconstitution stage may have a duration selected from the range of 60 - 200 min, wherein (during that duration) the AC electric field has a field strength selected from the range of 20 - 1100 V / m at a frequency selected from the range of 5 - 10 Hz.

[0067] The reconstitution stage may, in embodiments, be performed at a temperature above a phase transition temperature of the lipids in the EVs. In particular, if a temperature below phase transition temperature is during rehydration, the acyl chains may be extended and packed, e.g., more solid-like. If the lipid head groups are not properly hydrated and acyl chains are not fluid enough as in their final form, it may be detrimental to the membrane protein function. Above the transition temperature, the lipid acyl chains may be randomly oriented and more fluid like, which may be preferred as it may be more in line with lipid acyl chains in the EVs. For EVs obtained from the (human) body, a temperature above 37°C may typically be above the phase transition temperature. Hence, a temperature of (about) body temperature may be used, e.g., a temperature of 38 °C. In further embodiments, the reconstitution stage may be performed at a temperature selected from the range of 20 - 38 °C, such as at room temperature. In further embodiments, acyl chains in the multilayer may be a (median) phase transition temperature TA, wherein the reconstitution stage is performed at a temperature > TA, such as > TA + 1 °C, especially > TA + 2 °C, such as > TA + 5 °C. High temperature may, however, cause heat denaturation of membrane proteins. Hence, it may be preferable to select a temperature a bit above the phase transition temperature TA of the acyl chains. In further embodiments, the reconstitution stage may be performed at a temperature < TA + 20 °C, such as < TA + 15 °C, especially < TA + 10 °C, such as < TA + 5 °C.

[0068] As described above, the formation of the reconstituted vesicle may facilitate analyses requiring a particle size exceeding the (typical) extracellular vesicles. Hence, in embodiments, the reconstituted vesicle may be (substantially) larger than the extracellular vesicles (from which the reconstituted vesicle is formed). Hence, in embodiments the extracellular vesicles may have a number average equivalent spherical diameter dE5o, the reconstituted vesicle(s) may have an (number average) equivalent spherical diameter dnso, wherein dR5o / dE5o is selected from the range of > 5, such as > 10, especially > 100. In further embodiments, dR5o / dE5omay be selected from the range of 10 - 1000, such as from the range of 50 - 300.

[0069] In further embodiments, the reconstituted vesicle(s) may especially have a (number average) equivalent spherical diameter selected from the range of 1 - 60 pm, especially from the range of 1 - 50 pm, such as from the range of 2 - 50 pm, especially from the range of 2 - 30 gm, such as from the range of 5 - 30 gm, especially from the range of 10 - 30 pm. An equivalent spherical diameter of at least (about) 2 pm may facilitate patch clamp electrophysiology measurements (see below). Hence, in embodiments, the reconstituted vesicle may have an equivalent spherical diameter > 2 pm. Large vesicles may, however, also be relatively fragile, e.g., sensitive to (mechanical) perturbations. Hence, in embodiments, the reconstituted vesicle may especially have an equivalent spherical diameter < 50 pm, such as < 30pm.

[0070] The e.s.d. of extracellular vesicles may especially be determined using wide- field epi-fluorescence video microscopy with a fluorescent label. In particular, the EVs may be fluorescently labeled by adding a standard fluorescent lipid label into the EV suspension, e.g., 20 nM of Memglow™ 560, followed by microscopy. The e.s.d. of reconstituted vesicles may be determined by microscopy, e.g., by using a gridded chamber to determine the size distribution of a plurality of reconstituted vesicles.

[0071] When the reconstitution liquid is added (see above) into dried lipid films, water goes through the bilayers and through the defects (driven by hydration forces) and increases the interlayer separation from 0 to (about) 10 Angstrom. As a result of osmotic and electrostatic forces, repulsion may between the bilayers, which repulsion may overcome van der Waals attractions. At this stage, the bilayer may bend and reconstituted vesicles may be formed. Without being bound by theory, the ends of (a layer of) the multilayer may have exposed hydrophobic lipid acyl chains, which is energetically unfavorable in the aqueous reconstitution liquid, which may lead to line tension in the lipid bilayer, which may in turn decrease the length of the defects and, with the help of repulsive forces, may facilitate membrane bending and vesicle formation.

[0072] Depending on the parameters of the AC electric field, the reconstituted vesicle may be formed and released from the support (or from a lower layer of the multilayer) or may remain bound to the support during the reconstitution stage.

[0073] Hence, the method may further comprise a separation stage. The separation stage may comprise removing the reconstituted vesicle from the support.

[0074] In embodiments, the separation stage may comprise exposing the multilayer to (gentle) sonication to remove the reconstituted vesicle from the support, i.e., in embodiments the separation stage may comprise sonicating the support, especially the multilayer.

[0075] In further embodiments, the separation stage may especially comprise imposing a second alternating current electric field (or ‘second AC electric field) over the multilayer. The use of a second AC electric field may be particularly suitable for separation as an AC electric field is already used during reconstitution (the device can be re-used) and as a (second) AC electric field may facilitate gently separating the reconstituted vesicle from the support.

[0076] The second AC electric field may, in embodiments, especially have a higher field strength than the AC electric field. Further, in embodiments, the second AC electric field may have a lower frequency than the AC electric field. The reconstituted vesicle may be essentially made up of biological membranes, which are formed by phospholipids and are inherently overall negatively charged on their surface. After the formation of the reconstituted vesicle in the reconstitution stage, the application of a higher field strength (e.g., switching from 1100 V / m to 2000 V / m) in the separation stage will - in view of the surface charge - facilitate movement of surface attached reconstituted vesicles towards the anode due to the electrophoretic motion. Simultaneously lowering the frequency, e.g., from 12 Hz to 4 Hz may provide a (more) gentle vibration. Thereby, the reconstituted vesicles on the glass surface are detached in a controlled and relatively gentle way.

[0077] In further embodiments, the second AC electric field may have a field strength selected from the range of 1200 - 4000 V / m, such as from the range of 1600 - 3000 V / m, especially from the range of 1800 - 2200 V / m.

[0078] In further embodiments, the second AC electric field may have a frequency selected from the range of 1 - 12 Hz, especially from the range of 2 - 8 Hz, such as from the range of 3 - 6 Hz.

[0079] The duration of the separation stage may, typically, be lower than the duration of the reconstitution stage. Hence, in embodiments, the reconstitution stage may have a duration DR, the separation stage may have a duration Ds, wherein DR > Ds. In further embodiments, the separation stage have a duration selected from the range of 10 - 120 minutes, such as from the range of 15 - 90 minutes, especially from range of 20 - 60 minutes.

[0080] The separation stage may preferably - similarly to the reconstitution stage as described above be performed at a temperature above a phase transition temperature of the lipids in the EVs. In embodiments, the separation stage may be performed at a temperature selected from the range of 20 - 38 °C, such as at (about) room temperature. In further embodiments, acyl chains in the multilayer may be a (median) phase transition temperature TA, wherein the separation stage is performed at a temperature > TA, such as > TA + 1 °C, especially > TA + 2 °C, such as > TA + 5 °C. High temperature may, however, cause heat denaturation of membrane proteins. Hence, it may be preferable to select a temperature a bit above the phase transition temperature TA of the acyl chains. In further embodiments, the separation stage may be performed at a temperature < TA + 20 °C, such as < TA + 15 °C, especially < TA + 10 °C, such as < TA + 5 °C.

[0081] In specific embodiments, the separation stage may have a (separation) duration selected from the range of 10 - 60 min, wherein (for the duration) the separation stage comprises exposing the reconstituted vesicle to a second AC electric field having a second field strength selected from the range of 1600 - 3000 V / m at a second frequency selected from the range of 2 - 8 Hz.

[0082] In a further aspect, the invention may provide a characterization method for determining a parameter of a reconstituted vesicle (and a corresponding cell type). The characterization method may comprise a preparation stage, a measurement stage and an analysis stage. The preparation stage may comprise provide a reconstituted vesicle using the method of the invention (see above). The preparation stage may further comprise arranging the reconstituted vesicle (in an opening) between a first electrolyte and a second electrolyte, especially thereby physically separating the first electrolyte and the second electrolyte. In embodiments, the measurement stage may comprise controlling an (absolute) voltage or a current through (or “over”) the reconstituted vesicle and measuring an (induced) current or voltage, respectively. For instance, in embodiments, the measurement stage may comprise controlling an absolute voltage through (or “over”) the reconstituted vesicle in the range of 1 - 100 mV, recording a resulting (induced) current, and providing a related current signal. In further embodiments, the measurement stage may comprise controlling a current through (or “over”) the reconstituted vesicle in the range of 1 pA - 10 pA, recording a resulting voltage change, and providing a related voltage signal. The analysis stage may comprise determining the parameter (of the reconstituted vesicle) based on the current signal and / or the voltage signal.

[0083] Hence, the invention may provide a characterization method for determining a parameter of a reconstituted vesicle, the characterization method comprising a preparation stage comprising (I) providing the reconstituted vesicle using the method of the invention, and (II) arranging the reconstituted vesicle between a first electrolyte and a second electrolyte; a measurement stage comprising (I) controlling an absolute voltage through the reconstituted vesicle in the range of 1 - 100 mV, recording a resulting current, and providing a related current signal; and / or (II) controlling a current through the reconstituted vesicle in the range of 1 pA - 10 pA, recording a resulting voltage change, and providing a related voltage signal; and an analysis stage comprising determining the parameter based on the current signal and / or the voltage signal. The characterization method of the invention may facilitate characterizing reconstituted vesicles and, by extent, the extracellular vesicles from which the reconstituted vesicles were formed and thus, by extent, the cells from the extracellular vesicles originated. In particular, the characterization method may facilitate determining the identities and quantities of membrane proteins present in the reconstituted vesicle. Further, the characterization method may facilitate determining the influence of active compounds, such as drug candidated, on properties of the reconstituted vesicle, such as on properties of the membrane proteins. For instance, in the context of tumor cells, the characterization method may facilitated identifying druggable surface markers of the tumor cells from tumor-derived extracellular vesicles present in a subjects’ bodily fluids (blood, urine, saliva, etc) at a given time, especially without prior knowledge of the location of the tumor in the body, and especially without solid biopsy. Further, the characterization method may facilitate functionally testing the effect of drugs (i.e. already FDA approved, or else any other drug candidates) on the membrane proteins in the reconstituted vesicle through electrophysiology.

[0084] Hence, the invention may provide a characterization method for determining a parameter of a reconstituted vesicle. The parameter may be essentially any parameter that can be deduced from imposing a voltage or current over a membrane and measuring the induced current or voltage, respectively. The characterization method may be particularly suitable for identifying membrane proteins, such as ion channels, and for quantifying such proteins (see experiments below). Hence, in embodiments, the parameter may comprise a membrane protein parameter. Especially, in further embodiments, the parameter may comprise an ion channel parameter selected from the group comprising ion channel identity and ion channel abundance (for one or more types of ion channels).

[0085] The characterization method may, in embodiments, comprise a preparation stage, a measurement stage, and an analysis stage.

[0086] The preparation stage may comprise providing the reconstituted vesicle as defined above, especially using the method of the invention (see above).

[0087] The preparation stage may further comprise arranging the reconstituted vesicle between a first electrolyte and a second electrolyte, especially thereby physically separating the first electrolyte and the second electrolyte. In particular, the preparation stage may comprise arranging the reconstituted vesicle such that the first electrolyte and the second electrolyte are fluidically connected via (only) the reconstituted vesicle.

[0088] For instance, in embodiments, the preparation stage may comprise arranging the reconstituted vesicle at an opening, especially to (fully) cover the opening. In such embodiments, the first electrolyte may be arranged at a first side of the opening and the second electrolyte may be arranged at the second side of the opening. In further embodiments, a pipette, especially a pipette tip, may provide the opening. In particular, the preparation stage may comprise arranging the reconstituted vesicle at the opening (of the pipette tip) by suction (or ‘by providing a negative pressure’). In further embodiments, a solid state nanopore may provide the opening.

[0089] The characterization method may further comprise the measurement stage. The measurement stage may comprise (a) imposing a voltage across the (membranes of the) reconstituted vesicle and recording a resulting current and / or (b) imposing a current across the (membranes of the) reconstituted vesicle and recording a resulting voltage. It will be clear to the person skilled in the art that in embodiments comprising both the imposing of a voltage through the vesicle and the imposing of a current through the vesicle, and recording the other, that the imposing of the voltage and of the current will be temporally separated. Hence, the measurement stage may comprise first imposing the voltage (and recording the current) and then passing the current (and recording the voltage) or vice versa.

[0090] The imposed current and / or voltage may especially be selected to stimulate activation of transport of ions across membrane proteins, such as due to the opening of ion channels. The transport of ions may depend on the imposed voltage or current, as well as on the membrane proteins present in the reconstituted vesicle. As the transport of ions may be detected (via the induced current and / or voltage), and as the imposed voltage and / or current is known, the measurement may allow identification and quantification of the membrane proteins. The measurement stage may especially comprise imposing different (values of) voltage and / or current through the vesicle to stimulate the activation of different membrane proteins.

[0091] In embodiments, the measurement stage may comprise controlling a voltage through the reconstituted vesicle in the range of -150 - 150 mV, such as from the range of -100 - 100 mV, especially from the range of -80 - 80 mV, recording a resulting current, and providing a related current signal. In further embodiments, the measurement stage may comprise controlling an absolute voltage through the reconstituted vesicle in the range of 0.5 - 150 mV, such as from the range of 1 - 100 mV, especially from the range of 2 - 80 mV, recording a resulting current, and providing a related current signal.

[0092] In particular, in embodiments, the measurement stage may comprise imposing different voltages over the reconstituted vesicle. For instance, the measurement stage may comprise starting the imposed voltage at a first voltage value and increasing or decreasing the voltage towards a second voltage value (or vice versa), especially in a stepwise manner, or especially in a continuous manner. In embodiments, the first voltage value and the second voltage value may be of opposite sign. For instance, the first voltage value may be -90 mV and the second voltage value may be 90 mV, and the measurement stage may comprise taking steps of (about) 10 mV in the range of -90 mV - 90 mV.

[0093] In further embodiments, the first voltage value may be selected from the range of -0.5 - -150 mV, such as from the range of -1 - -100 mV, especially from the range of -2 - - 80 mV. In further embodiments, the second voltage value may be selected from the range of 0.5 - 150 mV, such as from the range of 1 - 100 mV, especially from the range of 2 - 80 mV. In further embodiments, the measurement stage may comprise imposing different voltage values over the reconstituted vesicle, wherein the voltage values are selected from the range of the first voltage value to the second voltage value. In further embodiments, the measurement stage may comprise steps of 1-30 mV, especially of 5-20 mV, between successive voltage values.

[0094] In further embodiments, the measurement stage may comprise controlling a current through the reconstituted vesicle in the range of 0.5 pA - 20 pA, especially in the range of 1 pA - 10 pA, such as in the range of 5 pA - 2 pA, recording a resulting voltage change, and providing a related voltage signal.

[0095] The term related current signal may herein refer to a signal that is related to the detected current. In particular, the related current signal may comprise raw and / or processed data related to the (detected) current. Similarly, the term related voltage signal may herein refer to a signal that is related to the detected voltage. In particular, the related voltage signal may comprise raw and / or processed data related to the (detected) voltage.

[0096] The measurement stage may (thus) especially comprise subjecting the reconstituted vesicle to patch clamping, especially voltage clamping, or especially current clamping.

[0097] Hence, both the ionic current passing through open ion channels and also the membrane potential across a reconstituted vesicle, generated by ionic concentration difference and ion channel composition of the vesicle, can be measured.

[0098] The detected (ionic) current may be informative regarding the ion conductance of ion channels in the reconstituted vesicle. In particular, ion conductance properties (e.g., type of ion that can go though, permeability and selectivity for a particular ion, opening and closing mechanism, kinetics of opening, closure, desensitization, and open dwell time) may be derived from current measurements. In particular, ion channels may be identifiable based on their ion conductance properties and, by extent, based on the measurements of (induced) currents. Further, the same ion conductance properties may be relevant for the assessment of candidate drugs. Hence, the imposing of voltage and the recording of (ionic) current may be particularly informative in the context of identifying membrane proteins in tumor cells and for the assessment of suitable drug candidates.

[0099] Hence, in embodiments, the measurement stage may comprise exposing the reconstituted vesicle to an active compound (or ‘active compound candidate’), such as to a drug (candidate). Generally, in such embodiments, the measurement stage may comprise performing measurements both before and after the addition of the active compound in order to assess the effect (if any) of the active compound. In particular, the measurement stage may comprise (a) imposing a voltage through the reconstituted vesicle and recording a resulting current during a measurement duration, wherein during at least part of the measurement duration the reconstituted vesicle is (essentially) unexposed to the active compound, and wherein during at least part of the measurement duration the reconstituted vesicle is exposed to the active compound, and (b) providing a related current signal.

[0100] The detected (induced) voltage (or “membrane potential”) may be informative regarding the total number of ion channels present in the reconstituted vesicle and regarding the ionic concentration difference between the inside and the outside of the reconstituted vesicle.

[0101] In embodiments, the characterization method may further comprise the analysis stage. The analysis stage may comprise determining the parameter based on the (related) current signal and / or the (related) voltage signal, especially at least based on the (related) current signal, or especially at least based on the (related) voltage signal. For instance, the analysis stage may comprise determining a parameter based on the procedure outlined in COLQUHOUN, Chapter 6 - Practical analysis of single channel records, Microelectrode Techniques, 1987, pages 101 - 139, which is hereby herein incorporated by reference. In further embodiments, the analysis stage may comprise determining a membrane protein parameter, especially an ion channel parameter, i.e., the parameter may comprise a membrane protein parameter, especially an ion channel parameter. Especially, in further embodiments, the parameter may comprise an ion channel parameter selected from the group comprising ion channel identity and ion channel abundance (for one or more types of ion channels).

[0102] The identification of ion channels may further facilitate determining suitable active compounds, e.g., suitable drug candidates. For instance, the identified ion channels may be looked up in an (online) database comprising information on (candidate) drugs and their (ion channel) targets. The online database may, for example, be selected from the group comprising DrugBank, ChEMBL, PubChem, DrugCentral, and the Therapeutic Target Database. Hence, in embodiments, the characterization method, especially the analysis stage, may comprise providing a list of drug candidates based on the ion channel parameter and an (online) database.

[0103] The characterization method may thus facilitate (a) identifying druggable tumor cell surface markers, (b) providing a list of potential drug candidates by matching drug targets with drug data bases, optionally using artificial intelligence and / or bioinformatics tools, and (c) the testing of (candidate) drugs against (patient-specific) tumor cell surface markers and identifying the most effective drug or drug combination.

[0104] These benefits may further facilitate (a) classifying patients into treatments groups, thereby providing a quicker identification of a suitable treatment and reducing the use of ineffective and potentially harmful drugs, (b) identifying suitable drug targets on a personal level is highly desired, (c) diagnosing a disease, e.g., the presence of a tumor, at an early stage as EVs are secreted at every stage of cancer, even before the tumor reaches a detectable size with conventional methods, and (d) monitoring a disease status during or after a medical treatment as effective treatments on a tumor may affect their surface markers, which can be detected via the corresponding EVs.

[0105] Patch clamp experiments and analyses may, for instance, be based on the procedures as outlined in SAKMANN and NEBER, Single-Channel Recording, Second Edition, 2009. ISBN-10: 1441912304.

[0106] In a further aspect, the invention may provide a system for combining extracellular vesicles from a biological sample into a reconstituted vesicle. The system may comprise one or more of a sample receiver, an isolation element, a support, a drying element, a liquid supply configured to host an aqueous reconstitution liquid, an electric field generator, and a control system. In embodiments, the sample receiver may be configured to receive a biological sample and to provide (at least part of) the biological sample to the isolation element. The control system may be configured to control other elements of the system, such as one or more of the isolation element, the drying element, the liquid supply, and the electric field generator. In particular, the control system may have an operational mode comprising an isolation stage, a drying stage, a reconstitution stage, and a separation stage. In the isolation stage the isolation element may (be configured to) isolate the extracellular vesicles from the biological sample to provide a prepared sample. In embodiments, the prepared sample may comprise a concentration of extracellular vesicles selected from the range of 0.02 - 3 mg / mL. In the drying stage, the drying element may (be configured to) receive at least part of the prepared sample from the isolation element. In the drying stage, the drying element may further be configured (a) to provide a droplet of the prepared sample on the support (in a hosting chamber), and (b) to dry the droplet to provide a multilayer of dried extracellular vesicles on the support. In the reconstitution stage, (a) the liquid supply may (be configured to) provide (at least part of) the aqueous reconstitution liquid to the support (in the hosting chamber), especially wherein the reconstitution liquid comprises < 600 mM of ions, and (b) the electric field generator may (be configured to) expose the multilayer to an AC electric field, the AC electric field having a field strength selected from the range of 20 - 1500 V / m at a frequency selected from the range of 5 - 20 Hz, thereby providing the reconstituted vesicle. In embodiments, the reconstituted vesicle may have an equivalent spherical diameter selected from the range of 2 - 50 pm, such as from the range of 2 - 30 pm. In the separation stage, the electric field generator may (be configured to) expose the multilayer to a second AC electric field to separate the reconstituted vesicle from the support. The second AC electric field may have a second field strength selected from the range of 1600 - 3000 V / m and a second frequency selected from the range of 2 - 8 Hz, especially for a (separation) duration selected from the range of 10 - 60 min.

[0107] Hence, the invention may further provide a system for combining extracellular vesicles from a biological sample into a reconstituted vesicle, the system comprising a sample receiver, an isolation element, a support, a drying element, a liquid supply configured to host an aqueous reconstitution liquid, an electric field generator, and a control system, wherein the sample receiver is configured to receive a biological sample and to provide the biological sample to the isolation element, wherein the control system has an operational mode comprising an isolation stage comprising the isolation element isolating the extracellular vesicles from the biological sample to provide a prepared sample, wherein the prepared sample comprises an extracellular vesicle protein concentration selected from the range of 0.02 - 3 mg / mL; a drying stage comprising (a) the drying element receiving at least part of the prepared sample from the isolation element and providing a droplet of the prepared sample on the support, and (b) the drying element drying the droplet to provide a multilayer of dried extracellular vesicles on the support; a reconstitution stage comprising (a) the liquid supply providing the aqueous reconstitution liquid to the support, wherein the reconstitution liquid comprises < 600 mM of ions, and (b) the electric field generator exposing the multilayer to an AC electric field, the AC electric field having a field strength selected from the range of 20 - 1500 V / m at a frequency selected from the range of 5 - 20 Hz, thereby providing the reconstituted vesicle, wherein the reconstituted vesicle has a equivalent spherical diameter selected from the range of 2 - 50 pm; and a separation stage comprising the electric field generator exposing the multilayer to a second AC electric field, the second AC electric field having a second field strength selected from the range of 1600 - 3000 V / m at a second frequency selected from the range of 2 - 8 Hz to separate the reconstituted vesicle from the support.

[0108] In particular, the control system may be configured to (have the system) execute the method of the invention.

[0109] In embodiments, the system may be integrated in a (compact) device, such as in a lab-on-a-chip device. Such device may provide a reliable and simple to use platform for combining EVs from a subject into a reconstituted vesicle and for characterizing the reconstituted vesicle. For instance, the device may be a sample-in-result-out device that takes in plasma / urine / saliva sample as an input and tells as the output which ion channels are in the (tumor) cells from which the EVs originated.

[0110] As described above, the system may comprise one or more of a sample receiver, an isolation element, a support, a drying element, a liquid supply configured to host an aqueous reconstitution liquid, an electric field generator, and a control system.

[0111] In embodiments, the system may comprise the sample receiver. The sample receiver may be configured to receive a biological sample. Especially, the sample receiver may comprise an inlet (or ‘receptacle’) for receiving the biological sample. The sample receiver may comprise a reception chamber configured to take up a specific amount of sample. In further embodiments, the sample receiver may be dedicated for a specific type of sample. For instance, the sample receiver may comprise a funnel or mouthpiece configured to receive a saliva sample. Alternatively, the sample receiver may, for example, comprise a lancet for collecting a blood sample by fingerstick.

[0112] In embodiments, the system may comprise the isolation element. The isolation element may be configured to isolate extracellular vesicles from a biological sample and to provide a prepared sample. The isolation element may especially be configured to contact the biological sample with (fixed) affinity probes targeting the EVs, such as specific aptamers or antibodies. The affinity probes may especially be configured to target extracellular vesicles of a first type (see above). For instance, in embodiments, the isolation element may comprise an isolation chamber comprising a resin, wherein the affinity probes are (chemically or physically) bound to the resin, and wherein the isolation element is configured to pass the biological sample through the resin to allow binding between the affinity probes and the EVs, and to subsequently pass an eluent through the resin to release the EVs, thereby providing the prepared sample, especially to the drying element. Especially, in embodiments, the isolation element may comprise an affinity purification element, wherein the affinity purification element is configured to selectively isolate a first type of extracellular vesicles from the biological sample, wherein the first type of extracellular vesicles corresponds to a first cell type or a first disease type. The affinity purification element may especially be configured to contact the biological sample with (fixed) affinity probes targeting the first type of extracellular vesicles.

[0113] As described above, the prepared sample may, in embodiments, comprise an extracellular vesicle protein concentration selected from the range of 0.01 - 6 mg / mL, such as from the range of 0.02 - 3 mg / mL, especially from the range of 0.1 - 2 mg / mL.

[0114] In further embodiments, the system may comprise the drying element and the support. The drying element may be configured to receive the prepared sample from the isolation element. The drying element may further be configured to provide a droplet of the prepared sample to a support, especially to an electrically conductive support. Especially, the drying element may be configured to provide a plurality of droplets to the support in spatially separated spots, i.e., with space between the spots where no droplets are applied.

[0115] In further embodiments, the drying element may be configured to dry the droplet to provide a multilayer of dried extracellular vesicles on the support. In particular, in embodiments, the drying element may be configured to dry the droplet using vacuum drying.

[0116] In embodiments, the system further comprises the liquid supply. The liquid supply may be configured to host the aqueous reconstitution liquid and to provide (at least part of) the aqueous reconstitution liquid to the support (during the reconstitution stage), especially to a hosting chamber comprising the support. The reconstitution liquid may, in embodiments, comprise < 800 mM of ions, such as < 600 mM of ions, especially < 300 mM of ions. In further embodiments, the reconstitution liquid may comprise < 60 mM of ions, such as < 30 mM of ions, especially < 10 mM of ions, such as < 5 mM of ions, including (essentially) 0 mM of ions. In further embodiments, the reconstitution liquid may comprise > 0 mM of ions, such as > 1 mM of ions, especially > 3 mM of ions.

[0117] In further embodiments, the reconstitution liquid may comprise 100 - 400 mM of sugar, such as 150 - 350 mM of sugar, especially 200 - 300 mM of sugar.

[0118] In further embodiments, the liquid supply may further be configured to host an eluent and to provide the eluent to the isolation element (during the isolation stage).

[0119] The system may, in embodiments, further comprise an electric field generator. The electric field generator may especially be configured to expose the support, especially the multilayer, to an alternating current electric field (‘AC electric field’) during the reconstitution stage and to a second AC electric field during the separation stage. The electric field generator may especially be configured to provide the AC electric field and the second AC electric field over the support.

[0120] In embodiments, during the reconstitution stage, the electric field generator may (be configured to) expose the multilayer to an AC electric field. The AC electric field may especially have a field strength selected from the range of 10 - 2000 V / m, such as from the range of 20 - 1500 V / m, especially from the range of 400 - 1100 V / m. In further embodiments, the AC electric field may have a frequency selected from the range of 5 - 20 Hz, especially from the range of 10-12 Hz, or especially from the range of 5 - 10 Hz.

[0121] In further embodiments, during the separation stage, the electric field generator may (be configured to) expose the multilayer to a second AC electric field. The second AC electric field may, in embodiments, especially have a higher field strength than the AC electric field. Further, in embodiments, the second AC electric field may have a lower frequency than the AC electric field. In further embodiments, the second AC electric field may have a field strength selected from the range of 1200 - 4000 V / m, such as from the range of 1600 - 3000 V / m, especially from the range of 1800 - 2200 V / m. In further embodiments, the second AC electric field may have a frequency selected from the range of 1 - 12 Hz, especially from the range of 2 - 8 Hz, such as from the range of 3 - 6 Hz.

[0122] The system may, in embodiments, further comprise the control system. As indicated above, the control system may be configured to control other elements of the system, such as the isolation element, the drying element, the liquid supply, and the electric field generator. The term “controlling” and similar terms herein may especially refer at least to determining the behavior or supervising the running of an element. Hence, herein “controlling” and similar terms may e.g. refer to imposing behavior to the element (determining the behavior or supervising the running of an element), etc., such as e.g. measuring, displaying, actuating, opening, shifting, changing temperature, etc.. Beyond that, the term “controlling” and similar terms may additionally include monitoring. Hence, the term “controlling” and similar terms may include imposing behavior on an element and also imposing behavior on an element and monitoring the element. The controlling of the element can be done with a control system. The control system and the element may thus at least temporarily, or permanently, functionally be coupled. The element may comprise the control system. In embodiments, the control system and the element may not be physically coupled. Control can be done via wired and / or wireless control. The term “control system” may also refer to a plurality of different control systems, which especially are functionally coupled, and of which e.g. one master control system may be a control system and one or more others may be slave control systems.

[0123] The system, especially the control system, may have an operational mode. The term “operational mode” may also be indicated as “controlling mode”. The system, or apparatus, or device (see further also below) may execute an action in a “mode” or “operational mode” or “mode of operation”. Likewise, in a method an action, stage, or step may be executed in a “mode” or “operation mode” or “mode of operation”. This does not exclude that the system, or apparatus, or device may also be adapted for providing another operational mode, or a plurality of other operational modes. Likewise, this does not exclude that before executing the mode and / or after executing the mode one or more other modes may be executed. However, in embodiments, a control system (see further also below) may be available, that is adapted to provide at least the operational mode. Would other modes be available, the choice of such modes may especially be executed via a user interface, though other options, like executing a mode in dependence of a sensor signal or a (time) scheme, may also be possible. The operational mode may in embodiments also refer to a system, or apparatus, or device, that can only operate in a single operational mode (i.e. “on”, without further tunability).

[0124] In further embodiments, the control system may comprise a fluid control system. The fluid control system may be configured to control the liquid supply. The fluid control system may further be configured to control the movement of fluid between the system components, such as (a) the movement of a biological sample from the sample receiver to the isolation element, (b) the movement of a prepared sample from the isolation element to the drying element, and / or (c) the movement of a solution comprising the reconstituted vesicle from the support, especially from the hosting chamber, to the measurement element.

[0125] In embodiments, the support may be movable. For instance, the system, especially the control system, may be configured to move the device between the drying stage and the reconstitution stage. Especially, the support may be movable between a first position and a second position, wherein the drying element is configured to access the support in the first position, and wherein the reconstitution element is configured to access the support in the second position.

[0126] In embodiments, the system may comprise a hosting chamber, wherein the support is arranged in the hosting chamber. In further embodiments, the drying element may be configured to provide the droplet to the support in the hosting chamber and to dry the droplet to form the multilayer while the support is in the hosting chamber. Further, in embodiments, the liquid supply may be configured to provide the aqueous reconstitution liquid to the hosting chamber. Yet further, the electric field generator may be configured to expose the support to the AC electric field and to the second AC electric field in the hosting chamber, i.e., while the support is in the hosting chamber.

[0127] The system may, in embodiments, further comprise one or more outlets for providing (intermediate) products. For instance, if only a subset of the isolated EVs are required for the downstream steps of the operational mode, the remainder of the isolated EVs may be removed from the system via a first outlet.

[0128] Hence, in embodiments, the system may comprise a first outlet, wherein the first outlet is configured to provide (a second part of) the prepared sample.

[0129] In further embodiments, the system may comprise a second outlet, wherein the second outlet is configured to provide the reconstituted vesicle.

[0130] The system may further comprise a measurement element for characterizing the reconstituted vesicle. The measurement element may especially comprise a patch clamp. In such embodiments, the operational mode may further comprise a measurement stage and an analysis stage.

[0131] In embodiments, the measurement element may be configured to receive the reconstituted vesicle during the separation stage, especially from the reconstitution chamber. The measurement element may be configured to arrange the reconstituted vesicle between a first electrolyte and a second electrolyte, especially thereby physically separating the first electrolyte and the second electrolyte. In particular, the measurement element may be configured to arrange the reconstituted vesicle such that the first electrolyte and the second electrolyte are fluidically connected via (only) the reconstituted vesicle. For instance, in embodiments, the measurement element may be configured to arrange the reconstituted vesicle at an opening, especially to (fully) cover the opening. Especially, the first electrolyte may be arranged at a first side of the opening and the second electrolyte may be arranged at the second side of the opening. In further embodiments, the measurement element may comprise a pipette providing the opening. In such embodiments, the measurement element may be configured to arrange the reconstituted vesicle at the opening (of the pipette) by applying suction (or ‘by providing a negative pressure’). In further embodiments, the measurement element may comprise a solid state nanopore providing the opening.

[0132] The first electrolyte and the second electrolyte may be provided by the liquid supply. Hence, in embodiments, the liquid supply may (further) be configured to host the first electrolyte and the second electrolyte and to provide the first electrolyte and the second electrolyte to the measurement element, especially during the measurement stage. The measurement element may especially be configured to impose a voltage through (or “over”) the openings, especially through (or “over”) the reconstituted vesicle, and to record a resulting (induced) current. Additionally or alternatively, the measurement element may be configured to impose a current through (or “over”) the openings, especially through (or “over”) the reconstituted vesicle, and to record a resulting (induced) voltage.

[0133] In embodiments, in the measurement stage, the measurement element may (be configured to) (a) control an absolute voltage through the reconstituted vesicle in the range of .5 - 150 mV, such as from the range of 1 - 100 mV, especially from the range of 2 - 80 mV, (b record a resulting current, and (c) provide a related current signal to the control system.

[0134] In further embodiments, in the measurement stage, the measurement element may (be configured to) (a) control a current through the reconstituted vesicle in the range of 0.5 pA - 20 pA, especially in the range of 1 pA - 10 pA, such as in the range of 5 pA - 2 pA, (b) record a resulting voltage change, and (c) provide a related voltage signal to the control system.

[0135] The control system may be configured to determine the parameter of the reconstituted vesicle based on the current signal and / or the voltage signal. In particular, in in the analysis stage, the control system may (be configured to) determine the parameter of the reconstituted vesicle based on the current signal and / or the voltage signal, especially (at least) based on the current signal, or especially (at least) based on the voltage signal.

[0136] In embodiments, the parameter may comprise a membrane protein parameter, especially an ion channel parameter selected from the group comprising ion channel identity and ion channel abundance (for one or more types of ion channels). In such embodiments, the control system may be configured to determine the membrane protein parameter, especially the ion channel parameter.

[0137] In further embodiments, the system may comprise a display, wherein the display is configured to display the (ion channel) parameter.

[0138] The term “stage” and similar terms used herein may refer to a (time) period (also “phase”) of a method and / or an operational mode. The different stages may (partially) overlap (in time). For example, the reconstitution stage may be initiated prior to the separation stage, but may, in embodiments, partially overlap in time therewith. Similarly, the measurement stage may be initiated prior to the analysis stage but may, in embodiments, (partially ) overlap in time therewith, i.e., analysis may occur in real-time along with the measurements. However, for example, the isolation stage may be completed prior to the drying stage which, in turn, may be completed prior to the reconstitution stage. It will be clear to the person skilled in the art how the stages may be beneficially arranged in time.

[0139] The embodiments described herein are not limited to a single aspect of the invention. For example, an embodiment describing the method may, for example, further relate to the system, especially to an operational mode of the system, or especially to the control system. Similarly, an embodiment of the system describing an operation of the system may further relate to embodiments of the method. In particular, an embodiment of the method describing an operation (of the system) may indicate that the system may, in embodiments, be configured for and / or be suitable for the operation. Similarly, an embodiment of the system describing actions of (a stage in) an operational mode may indicate that the method may, in embodiments, comprise those actions.

[0140] BRIEF DESCRIPTION OF THE DRAWINGS

[0141] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which: Fig.lA-B schematically depict embodiments of the method. Fig. 2A-B schematically depict embodiments of the characterization method. Fig. 3 schematically depicts an embodiment of the system. Fig. 4A- D schematically depict experimental results obtained with the method and characterization method of the invention. The schematic drawings are not necessarily on scale.

[0142] DETAILED DESCRIPTION OF THE EMBODIMENTS

[0143] Fig. 1A schematically depicts an embodiment of the method for combining extracellular vesicles 10 from a biological sample 15 into a reconstituted vesicle 20. In the depicted embodiment, the method comprises an isolation stage, a drying stage, a reconstitution stage, and a separation stage. The isolation stage comprises isolating the extracellular vesicles 10 from a biological sample 15 to provide a prepared sample 115, especially wherein the prepared sample 115 comprises an extracellular vesicle protein concentration selected from the range of 0.02 - 3 mg / mL. In the depicted embodiment, two biological samples are depicted, e.g., urine (top) and blood (bottom). In practice, generally a single biological sample may be used. The drying stage comprises providing a droplet 125 of the prepared sample 115 on a support 120, wherein the support is electrically conductive. In particular, in the depicted embodiment, the drying comprises providing a plurality of droplets 125 on the support 120 such that the droplets are spatially separated. The drying stage further comprises drying the droplet 125 to provide a multilayer 135 of dried extracellular vesicles 10, especially using vacuum drying. The multilayer 135 may comprise n layers 136, especially wherein n is selected from the range of 5-10. The reconstitution stage comprises exposing the multilayer 135 to (a) an aqueous reconstitution liquid 130, especially wherein the reconstitution liquid 130 comprises < 600 mM of ions, and (b) to an AC electric field having a field strength selected from the range of 20 - 1500 V / m at a frequency selected from the range of 5 - 20 Hz, thereby providing the reconstituted vesicle 20. The reconstituted vesicle 20 may especially have an equivalent spherical diameter DR selected from the range of 2 - 50 p, such as from the range of 2 - 30 pm. The separation stage may comprise removing the reconstituted vesicle 20 from the support 120.

[0144] In the depicted embodiment, the biological sample 15 may comprise a urine sample or a blood sample. In further embodiments, the biological sample 15 may comprise a bodily fluid selected from the group comprising a blood sample, a urine sample, a saliva sample, a sputum sample, a stool sample, a lymph fluid sample, a pancreatic juice sample, a tear sample, an ascitic fluid sample, a pleural fluid sample, a thoracic fluid sample, a breast milk sample, and a cerebrospinal fluid sample.

[0145] In the depicted embodiment, the isolation stage may comprise selectively isolating a first type 11 of extracellular vesicles 10 from the biological sample 15 using affinity purification, wherein the prepared sample 115 comprises the extracellular vesicles 10 of the first type 11.

[0146] Fig. IB schematically depicts two further embodiments of the method, differing in the formation of the multilayer 135 (see left side).

[0147] In one of the embodiments depicted in Fig. IB, a synthetic lipid layer 35 is formed on the support 120 prior to the providing of the droplet 125 to the support 120. Hence, in such embodiments, the drying stage may comprise providing a dried synthetic lipid layer 35 on the support 120, and subsequently providing the droplet 125 of the prepared sample 115 on the synthetic lipid layer 35. In further embodiments, the drying stage may comprise providing a (synthetic) liposome 31 to the support 120 and drying the liposome to form the synthetic lipid layer 35.

[0148] In the other embodiment depicted in Fig. IB, liposomes 31 and extracellular vesicles 10 are combined prior to the drying stage. In such embodiments, the drying stage may comprise providing a droplet 125 comprising extracellular vesicles 10 and liposomes 31 to the support 120 and drying the droplet 125 to provide the multilayer 135. Fig. IB further schematically depicts an embodiment of the characterization method for determining a parameter of a reconstituted vesicle 20. In embodiments, the characterization method may comprise arranging the reconstituted vesicle 20 between a first electrolyte 141 and a second electrolyte 142, such as at an opening 140 separating the first electrolyte 141 and the second electrolyte 142. In Fig. IB, the reconstituted vesicle 20 is arranged at an opening 140 of a pipette tip between a first electrolyte 141 and a second electrolyte 142. The characterization method may further comprise (a) controlling an absolute voltage through the reconstituted vesicle 20 in the range of 1 - 100 mV, recording a resulting current, and providing a related current signal; and / or (b) controlling a current through the reconstituted vesicle 20 in the range of 1 pA - 10 pA, recording a resulting voltage change, and providing a related voltage signal. In further embodiments, the characterization method may further comprise determining the parameter based on the current signal and / or the voltage signal. In embodiments, the parameter may comprise an ion channel parameter selected from the group comprising ion channel identity and ion channel abundance.

[0149] Fig. 2A schematically depicts a further embodiment of the characterization method. In the depicted embodiment, the method comprises exposing the reconstituted vesicle 20 to an active compound 30.

[0150] Fig. 2B schematically depicts a further embodiment of the characterization method. In the depicted embodiment the opening 140 is provided by a solid state nanopore.

[0151] Fig. 3 schematically depicts an embodiment of the system 200 for combining extracellular vesicles 10 from a biological sample 15 into a reconstituted vesicle 20. In the depicted embodiment the system 200 comprises a sample receiver 210, an isolation element 215, a support 120, a drying element 225, a liquid supply 230 configured to host an aqueous reconstitution liquid 130, an electric field generator 250, and a control system 300. The sample receiver 205 is configured to receive a biological sample 15 and to provide the biological sample 15 to the isolation element 215. The control system 300 may have an operational mode comprising an isolation stage, a drying stage, a reconstitution stage, and a separation stage. The isolation stage may comprise the isolation element 215 isolating the extracellular vesicles 10 from the biological sample 15 to provide a prepared sample 20, wherein the prepared sample 20 comprises a concentration of extracellular vesicles 10 selected from the range of 0.02 - 3 mg / mL. The drying stage may comprise (a) the drying element 225 receiving at least part of the prepared sample 15 from the isolation element 215 and providing a droplet 125 of the prepared sample 25 on the support 120 , and (b) the drying element 225 drying the droplet 125 to provide a multilayer 135 of dried extracellular vesicles 10 on the support 120. The reconstitution stage may comprise (a) the liquid supply 230 providing the aqueous reconstitution liquid 130 to the support 120, wherein the reconstitution liquid 130 comprises < 600 mM of ions, and (b) the electric field generator 250 exposing the multilayer 135 to an AC electric field, wherein the AC electric field has a field strength selected from the range of 20 - 1500 V / m at a frequency selected from the range of 5 - 20 Hz, thereby providing the reconstituted vesicle 20. The reconstituted vesicle 20 may especially have an equivalent spherical diameter selected from the range of 2 - 50 pm, such as from the range of 2 - 30 pm. The separation stage may comprise the electric field generator 250 exposing the multilayer 135 to a second AC electric field to separate the reconstituted vesicle 20 from the support 120, the second AC electric field especially having a second field strength selected from the range of 1600 - 3000 V / m at a second frequency selected from the range of 2 - 8 Hz.

[0152] In the depicted embodiment, the isolation element 215 may comprise an affinity purification element 216, wherein the affinity purification element 216 is configured to selectively isolate a first type 11 of extracellular vesicles 10 from the biological sample 15. The first type 11 of extracellular vesicles 10 may especially correspond to a first cell type or a first disease type, such as correspond to tumor cells, i.e., the first type 11 of extracellular vesicles 10 may comprise extracellular vesicles originating from tumor cells.

[0153] In the depicted embodiment, the system 200 further comprises a measurement element 240. The measurement element 240 may be configured to receive the reconstituted vesicle 20, such as during the separation stage. The measurement element 240 may be configured to arrange the reconstituted vesicle 20 between a first electrolyte 141 and a second electrolyte 142. In the depicted embodiment, the measurement element 240 may comprise a chip comprising a plurality of (individually addressable) pores, wherein the pores separate the first electrolyte 141 and the second electrolyte 142. The measurement element 240 may be configured to impose a voltage through the reconstituted vesicle and to measure a resulting current and / or to impose a current through the reconstituted vesicle and to measure a resulting voltage. In particular, the measurement stage may comprise the measurement element (a) controlling an absolute voltage through the reconstituted vesicle 20 in the range of 1 - 100 mV, recording a resulting current, and providing a related current signal to the control system 300; and / or (b) controlling a current through the reconstituted vesicle 20 in the range of 1 pA - 10 pA, recording a resulting voltage change, and providing a related voltage signal to the control system 300. The analysis stage may comprise the control system 300 determining a parameter of the reconstituted vesicle 20 based on the (related) current signal and / or the (related) voltage signal. The depicted system 200 further comprises outlets 261, 262 for providing (intermediate) products. In particular, the system 200 comprises a first outlet 261 and a second outlet 261, wherein the first outlet 261 is configured to provide (a second part of) the prepared sample 115, and wherein the second outlet 262 is configured to provide the reconstituted vesicle 20.

[0154] Experiments

[0155] Unless specified otherwise, the experiments described hereinafter were performed using the following materials and methods.

[0156] Cell line - SH-EP2 cells (also called SHEP2 cells and Tet2 cells) were obtained from Cellosaurus. SH-EP2 cells are known under Research Resource Identifier (RRID) CVCL HF70..

[0157] Isolating EVs using gold standard ultracentrifugation - EVs were isolated from a cell line in serum-free medium (DMEM) using the gold standard ultracentrifugation method. Specifically: SH-EP2 cells were seeded (5000 cells / cm2) in a T182.5 flask (25 mL) and grown for 3 days in Dulbecco's Modified Eagle Medium (DMEM) High Glucose supplemented with 1 mM Sodium pyruvate. Specifically, the DMEM High Glucose (4.5 g / 1), with L-Glutamine medium (Cat-No: DMEM-HA) was obtained from Capricorn Scientific. Afterwards, the culture media were centrifuged at 300g for 10 minutes and at 2000g for 20 min to spin down large / heavy particles, e.g., cells. Subsequently, the supernatant was subjected to ultracentrifugation (UC) at 110,000g for 2:15 h at 4 °C with a fixed angle MLA-50 rotor to form UC pellets containing the EVs. The UC pellets containing the EVs were resuspended in 20% phosphate buffered saline (PBS) obtained from Sigma-Aldrich (Cat-no:P5368) and was adjusted to pH 7.2 with a IM HC1 solution. The resuspended EVs were used directly in subsequent steps of the method or were stored at -80°C for later use.

[0158] Isolating EVs from cell lines using affinity purification - As a proof of concept, EVs from tumor cells were marked with a synthetic epitope that can be targeted using affinity purification. The SHEP cells were metabolically labelled on their proteins or on their glycoproteins by feeding them the synthetic amino acid L-azidohomoalanine (AHA), or N- azidoacetylmannosamine-tetraacylated (ManNAz), respectively. Next, a light-cleavable linkerbiotin was bound to proteins on the EV surface using click chemistry with AHA or ManNAz. Streptavidin functionalized agarose beads were used affinity purification of biotinylated EVs.

[0159] Cell culturing and metabolic labeling - SH-EP2 cells were maintained in high- glucose DMEM (Capricorn) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich), 2 mM L-glutamine (Lonza), 1 mM sodium pyruvate (Gibco), 1% penicillin- streptomycin (Lonza), and 1% MEM non-essential amino acids solution (Gibco) at 37 °C in a humified incubator with 5% CO2. For metabolic incorporation of L-azidohomoalanine (AHA; Sigma-Aldrich), cells were washed with warm Hank’s Balanced Salt Solution (HBSS) and incubated with methionine-free DMEM (Gibco) with 1 mM sodium pyruvate. For metabolic incorporation of N-azidoacetylmannosamine-tetraacylated (ManNAz; Sigma-Aldrich), high- glucose DMEM (Capricorn) with 1 mM sodium pyruvate was added.

[0160] Obtaining marked EVs - SH-EP2 cells were seeded (5000 cells / cm2) in T 182.5 flasks and cultured for three days to reach 75%-80% confluency. Prior to incubation with EV- depleted FBS, the cells were washed twice with warm HBSS. Fresh media with EV-depleted FBS and the respective azide-substrate (for click chemistry) were added and cells were incubated until harvest. After addition of a complete EDTA-free protease inhibitor cocktail (Roche), conditioned media were centrifuged at 300g for 10 min to discard cell contamination and at 2000g for 20 min to discard cell debris and large apoptotic bodies. EVs were pelleted by ultracentrifugation at 110,000g for 2:15 h at 4 °C (fixed angle MLA-50 rotor, k-factor 118) and resuspended in cold PBS (pH 7.2).

[0161] Biotinylation of azide-labeled EVs - Free thiols were blocked by incubation with 5 mM maleimide for 2 h at room temperature. The samples were centrifuged at 3000g for 10 min to pellet and discard any formed aggregates. The azide-labeled EVs were biotinylated by addition of the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) components in the following order: 25 pM PC biotin alkyne, 750 pM 2-(4-((bis((l-(tert-butyl)-lH-l,2,3-triazol- 4-yl)methyl)amino)methyl)-lH-l,2,3-triazol-l-yl)acetic acid (BTTAA) ligand and 150 pM CuSO4 (added as a premixed solution), 3.75 mM aminoguanidine hydrochloride, and 3.75 mM sodium ascorbate. Following overnight incubation at room temperature in the dark, samples were centrifuged at 3000g for 10 min to pellet and discard any formed aggregates. Samples were concentrated to ~1 mL in Amicon Ultra-4 ultrafiltration units (10 kDa MWCO, Millipore) and buffer-exchanged to PBS (pH 7.2) using three rounds of dilution and filtration.

[0162] Affinity purification and UV-elution - For each replicate, 150 pL Pierce high capacity NeutrAvidin agarose (Thermo Fisher Scientific) was prewashed with PBS (pH 7.2) three times and was subsequently incubated with the biotinylated sample at 4 °C overnight on an end-over-end rotator in a Protein LoBind® tube (Eppendorf). The agarose was pelleted by centrifugation at 300g for 2 min and the unbound fraction was collected. The agarose was then washed in a wash step by adding PBS, inverting the tube 10 times, and centrifuging at 300g for 2 min, and removing the supernatant. The wash step was repeated five times. After the final wash, the agarose was resuspended in PBS and transferred to clear side macro cuvettes (UV grade PMMA, Kartell). EVs specifically resuspended for subsequent drying and reconstitution into a reconstituted vesicles were resuspended in a 20% PBS elution buffer. Proteins and EVs were eluted from the agarose by irradiation with UV light (365 nm) for 2 h using a 6 W Spectroline Model ENF-260C lamp at a distance of 4 cm while the agarose was continuously stirred by a magnetic micro stirring bar. Agarose was spun down and the eluted fraction, i.e. purified EVs, was collected.

[0163] Cryo-TEM analysis - for transmission electron cryomicroscopy (cryo-TEM) analysis of enriched EVs, three sample aliquots of 3 pL were applied to a glow-discharged (40 s, 10 mA) Quantifoil R 2 / 1 holey-carbon 200 mesh gold TEM grid (Electron Microscopy Sciences) and blotted manually for 3 s. Excess liquid was drained with a filter paper. Another 3 pL aliquot was blotted on the grid and plunge-frozen into liquid ethane with a Vitrobot Mark IV (room temperature, 100% humidity, force -10, blot time of 6 s; FEI). Vitrified samples were stored in liquid nitrogen until imaging in a TALOS F200C-G2 transmission electron microscope (Thermo Fisher Scientific) at an accelerating voltage of 200 kV. Images were recorded with 10 s exposure times by a Falcon 4i direct electron detector (Thermo Fisher Scientific) at a defocus range of 1 to 6 pm, with an electron dose of 0.65 Q-lklls or 4.5 Q-lklls at a nominal magnification of 36,000* (0.397 nm pixel size) or 73,000* (0.194 nm pixel size), respectively.

[0164] Preparation of reconstituted vesicles - the EVs in 20% PBS (see above) were used to obtain a thin lipid film on the surface of conductive glass slides (ITO). Droplets of 2 pl of a prepared sample comprising EVs were applied to the ITO glass conducting surface. The ITO glass was placed in a vacuum desiccator and a tissue paper wetted in saturated NaCl was placed in an open petri dish in the vacuum desiccator, followed by overnight dehydration in the vacuum desiccator at room temperature. After the EV lipid films were obtained, an electroformation chamber was prepared in NanionVecicle Prep Pro (VPP) (Nanion Technologies GmbH, Munich, Germany). The ITO-coated glass with EV film was placed in the VPP chamber. A rubber O-ring on its lower side was placed on the ITO- glass circulating the edges of the dried EV lipid film. The chamber was filled with 200 mM sucrose solution and a second ITO-coated glass was placed on top with its conductive side facing downwards. An AC electric field was applied for 4 hours across the cell unit with stepwise increases from 100 - 1100 V / m at 12 kHz frequency with 20 min steps to form the reconstituted vesicles. At the end, in order to separate the reconstituted vesicles, the frequency was lowered to 4 Hz and the field strength was raised to 2000 V / m for 30 min. Finally, the top ITO-glass was carefully tilted and a suspension comprising reconstituted vesicles wass taken out with a large-tip pipette (1000 ml pipette) into an Eppendorf tube. The reconstituted vesicles were directly used or were stored up to a week at 4 °C for later use.

[0165] Patch clamp analysis of reconstituted vesicles - The electrophysiology experiments were carried out using the Port-a-Patch (Nani on Technologies GmbH, Munich, Germany). The following buffers were used: an internal potassium solution (KF 110): 10 mM KC1, 10 mM NaCl, 110 mM K-fluoride, 10 mM EGTA and 10 mM Hepes / KOH (pH 7.2); an internal sodium solution (CsFl 10): 10 mM CsCl, 10 mM NaCl, 110 mM Cs-fluoride, 10 mM EGTA and 10 mM Hepes / KOH (pH 7.2); and an external standard solution: 140 mM NaCl, 4 mM KC1, 1 mM MgCF 1 mM, 2 mM CaCh, 5 mM D-Glucose monohydrate, 10 mM Hepes / NaOH( pH 7.4). NPC-1 patch clamp chips, 1-2 Mohm, with 5 pl of buffer on the internal and 10 pl of buffer on the external side were used. After adjustment of voltage offset, 10 pl of a suspension containing reconstituted vesicles was added to the chip. Bilayers with several GOhm resistance were formed by applying a negative pressure of -10 mbar to -50 mbar. 30 pl of potassium or sodium external buffer were added to the external side. Ion channels can be activated by different mechanisms: a) temporary changes in the membrane potential, b) temporary changes in membrane tension, and c) the presence of a ligand. Here, first, a voltageprotocol was applied to determine the presence of voltage-gated (or voltage activated) sodium or potassium conducting channels. Channels were activated by a series of 300 ms depolarizing test potentials from -90 to 90 mV (in 10 mV steps) and resulting potassium and sodium currents were recorded. Mechanosensitive ion channels were activated by applying stepwise suction to the patch membrane in the range of 0 to 160 mbar, in 20 mbar steps at membrane potentials from -90 to + 90 mV , such as described in BARTHMES et al., Studying mechanosensitive ion channels with an automated patch clamp, European Biophysics Journal, 2014, Vol. 43, pages 97-104, which is hereby herein incorporated by reference. The patch data was analyzed using the Clampfit software (pClamp, Axon Instruments, USA). The procedures were performed as described in the manual of the Port-a-Patch from Nanion.

[0166] Data analysis - During patch clamp experiments, voltage- or tension-activated ion channels and receptors open a pore by changing their conformation and allow the passage of ions; the resulting ionic current was recorded. The recorded ionic currents were analyzed to identify single channels properties according to the procedures described in Chapter 9 of the fifth edition (2021) of The Axon Guide, Electrophysiology and Biophysics Laboratory Techniques, from Molecular Devices. In particular, the following properties were used to identify individual channel types: i) the unitary channel conductance (amount of ionic current at an applied voltage), ii) open channel dwell time, i.e., how long the channels stay open, iii) current-voltage relation, iv) ion selectivity, v) ionic response to known ion channel-specific toxins or drugs in the form of blocked ionic current, and vi) ionic response to known ion channel-specific activators.

[0167] A biological sample 15 was obtained from children tumor ‘neuroblastoma’ SHEP cell lines. The biological sample 15 was subjected to the isolation stage of the method of the method of the invention to isolate extracellular vesicles 10. Specifically, affinity purification using neutravidin affinity tags was used. Neutravidin may have a strong affinity for biotin, which may be found on the surface of EVs and / or associated with glycoproteins. Specifically, neutravidin was attached to agarose beads, which beads were exposed to the biological sample. The EVs 10 were released from the agarose beads by cleaving biotin using ultraviolet light having a wavelength in the range of 200 - 325 nm..

[0168] Fig. 4A schematically depicts the extracellular vesicles 10 that were isolated from the children tumor ‘neuroblastoma’ SHEP cell lines using affinity purification. The scale bar indicates a size of 100 nm. The depicted EVs 10 may thus have equivalent spherical diameters approximately in the range of 50 - 250 nm.

[0169] A prepared sample 115 comprising the extracellular vesicles 10 of Fig. 4A was subjected to the drying stage, the reconstitution stage and the separation stage of the method of the invention to provide reconstituted vesicles 20.

[0170] Fig. 4B schematically depicts the reconstituted vesicles 20 obtained from the EVs 10 shown in Fig. 4A. The scale bar indicates a size of 20 pm. The depicted reconstituted vesicles 20 may thus have equivalent spherical diameters approximately in the range of 10 - 35 pm.

[0171] Hence, the method of the invention facilitates providing reconstituted vesicle having an equivalent spherical diameter substantially larger than the equivalent spherical diameter of the extracellular vesicles 10 from which the reconstituted vesicle 20 is formed.

[0172] Fig. 4C and Fig. 4D schematically depict current measurements obtained by imposing a voltage through a reconstituted vesicle of Fig. 4B. Both figures depict the current I against measurement time T, wherein the axis bars indicate 0.5 nA for current I and 50 ms for measurement time T.

[0173] For the currents depicted in Fig. 4C, a voltage of ... was imposed through the reconstituted vesicle. By imposing this voltage, potassium channels may be activated. A stepwise increase in the current I may indicate the opening of one or more potassium channels, with the size of the step corresponding to the number of channels that have opened. For the currents depicted in Fig. 4D, a voltage of ... was imposed through the reconstituted vesicle. By imposing this voltage, sodium channels may be activated. A negative peak in the recorded current may indicate the opening of one or more sodium channels, with the size of the peak corresponding to the number of channels that opened.

[0174] As may be observed from Fig. 4C and Fig. 4D, different types of ion channels may be separately activated through the imposed voltage (or current). Further, the different types of ion channels, when activated, provide substantially different current patterns. These differences facilitate distinguishing between different ion channels that may be present in the reconstituted vesicle. Further, as the magnitude of the current changes depends on the number of ion channels that have opened, these current data may further be indicative of the number of ion channels present in the reconstituted vesicle.

[0175] The term “plurality” refers to two or more. Furthermore, the terms “a plurality of’ and “a number of’ may be used interchangeably.

[0176] The terms “substantially” or “essentially” herein, and similar terms, will be understood by the person skilled in the art. The terms “substantially” or “essentially” may also include embodiments with “entirely”, “completely”, “all”, etc. Hence, in embodiments the adjective substantially or essentially may also be removed. Where applicable, the term “substantially” or the term “essentially” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. Moreover, the terms ’’about” and “approximately” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. For numerical values it is to be understood that the terms “substantially”, “essentially”, “about”, and “approximately” may also relate to the range of 90% - 110%, such as 95%-105%, especially 99%-101% of the values(s) it refers to.

[0177] The term “comprise” also includes embodiments wherein the term “comprises” means “consists of’.

[0178] The term “and / or” especially relates to one or more of the items mentioned before and after “and / or”. For instance, a phrase “item 1 and / or item 2” and similar phrases may relate to one or more of item 1 and item 2. The term "comprising" may in an embodiment refer to "consisting of' but may in another embodiment also refer to "containing at least the defined species and optionally one or more other species".

[0179] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0180] The devices, apparatus, or systems may herein amongst others be described during operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation, or devices, apparatus, or systems in operation.

[0181] The term “further embodiment” and similar terms may refer to an embodiment comprising the features of the previously discussed embodiment, but may also refer to an alternative embodiment.

[0182] It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims.

[0183] In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim.

[0184] Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, “include”, “including”, “contain”, “containing” and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

[0185] The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.

[0186] The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In a device claim, or an apparatus claim, or a system claim, enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

[0187] The invention also provides a control system that may control the device, apparatus, or system, or that may execute the herein described method or process. Yet further, the invention also provides a computer program product, when running on a computer which is functionally coupled to or comprised by the device, apparatus, or system, controls one or more controllable elements of such device, apparatus, or system.

[0188] The invention further applies to a device, apparatus, or system comprising one or more of the characterizing features described in the description and / or shown in the attached drawings. The invention further pertains to a method or process comprising one or more of the characterizing features described in the description and / or shown in the attached drawings. Moreover, if a method or an embodiment of the method is described being executed in a device, apparatus, or system, it will be understood that the device, apparatus, or system is suitable for or configured for (executing) the method or the embodiment of the method, respectively.

[0189] The various aspects discussed in this patent can be combined in order to provide additional advantages. Further, the person skilled in the art will understand that embodiments can be combined, and that also more than two embodiments can be combined. Furthermore, some of the features can form the basis for one or more divisional applications.

Claims

CLAIMS:

1. A method for combining extracellular vesicles (10) from a biological sample (15) into a reconstituted vesicle (20), the method comprising: an isolation stage comprising isolating the extracellular vesicles (10) from the biological sample (15) to provide a prepared sample (115) comprising the extracellular vesicles; a drying stage comprising (a) providing a droplet (125) of the prepared sample (115) on a support (120) wherein the support (120) is electrically conductive (121), and (b) drying the droplet (125) to provide a multilayer (135) of dried extracellular vesicles (10); a reconstitution stage comprising (a) exposing the multilayer (135) to an aqueous reconstitution liquid (130), wherein the reconstitution liquid (130) comprises < 600 mM of ions, and (b) exposing the multilayer (135) to an AC electric field having a field strength selected from the range of 20-1500 V / m and a frequency selected from the range of 5 - 20 Hz, thereby providing the reconstituted vesicle (20), wherein the reconstituted vesicle (20) has a equivalent spherical diameter (DR) selected from the range of 2 - 50 pm; and a separation stage comprising removing the reconstituted vesicle (20) from the support (120).

2. The method according to claim 1, wherein the reconstitution stage comprises exposing the multilayer to the AC electric field during a reconstitution duration, wherein the electric field strength is selected from the range of 20 - 1100 V / m, and wherein the frequency of the AC electric field is selected from the range of 5 - 10 Hz, wherein the reconstitution duration is selected from the range of 60 - 200 min.

3. The method according to claim 2, wherein the separation stage comprises exposing the reconstituted vesicle (20) to a second AC electric field during a separation duration, wherein the second AC electric field has a second field strength selected from the range of 1600 - 3000 V / m and a second frequency selected from the range of 2 - 8 Hz, wherein the separation stage has a separation duration selected from the range of 10 - 60 min.

4. The method according to any one of the preceding claims 2-3, wherein the reconstitution stage comprises ramping up the field strength and / or the frequency over time.

5. The method according to any one of the preceding claims, wherein the biological sample (15) is a bodily fluid selected from the group comprising a blood sample, a urine sample, a saliva sample, a sputum sample, a stool sample, a lymph fluid sample, a pancreatic juice sample, a tear sample, an ascitic fluid sample, a pleural fluid sample, a thoracic fluid sample, a breast milk sample, and a cerebrospinal fluid sample.

6. The method according to any one of the preceding claims, wherein at least part of the multilayer (135) of dried extracellular vesicles comprises n layers (136) of dried extracellular vesicles (10), wherein n > 10.

7. The method according to any one of the preceding claims, wherein the drying stage comprises drying the droplet (125) using vacuum drying.

8. The method according to any one of the preceding claims, wherein the reconstitution liquid (130) comprises 200 - 300 mM of sugars, wherein the reconstitution liquid (130) comprises < 10 mM of the ions, and wherein the reconstitution liquid has a pH selected from the range of 5.5 - 8.

9. The method according to any one of the preceding claims, wherein the drying stage comprising providing a dried synthetic lipid layer (35) on the support (120), and subsequently providing the droplet (125) of the prepared sample (115) on the synthetic lipid layer (35).

10. The method according to any one of the preceding claims, wherein the extracellular vesicles (10) are of a first type (11), and wherein the isolation stage comprises selectively isolating the extracellular vesicles (10) of the first type (11) from the biological sample (15) using affinity purification, wherein the prepared sample (115) comprises the extracellular vesicles (10) of the first type (11).

11. The method according to claim 10, wherein the first type (11) of extracellular vesicles (10) corresponds to tumor cells12. A characterization method for determining a parameter of a reconstituted vesicle (20) , the characterization method comprising:a preparation stage comprising (I) providing the reconstituted vesicle (20) using the method of any one of the preceding claims, and (II) arranging the reconstituted vesicle (20) between a first electrolyte (141) and a second electrolyte (142) ; a measurement stage comprising: controlling an absolute voltage through the reconstituted vesicle (20) in the range of 1 - 100 mV, recording a resulting current, and providing a related current signal; and / or controlling a current through the reconstituted vesicle (20) in the range of 1 pA - 10 pA, recording a resulting voltage change, and providing a related voltage signal; and an analysis stage comprising determining the parameter based on the current signal and / or the voltage signal.

13. The characterization method according to claim 13, wherein the parameter comprises an ion channel parameter selected from the group comprising ion channel identity and ion channel abundance , and wherein the analysis stage comprises determining the ion channel parameter.

14. The characterization method according to any one of the preceding claims 12- 13, wherein the measurement stage comprises exposing the reconstituted vesicle to an active compound (30).

15. A system (200) for combining extracellular vesicles (10) from a biological sample (15) into a reconstituted vesicle (20), the system (200) comprising a sample receiver (210), an isolation element (215), a support (120), a drying element (225), a liquid supply (230) configured to host an aqueous reconstitution liquid (130), an electric field generator (250), and a control system (300), wherein the sample receiver (205) is configured to receive a biological sample (15) and to provide the biological sample (15) to the isolation element (215), wherein the control system (300) has an operational mode comprising: an isolation stage comprising the isolation element (215) isolating the extracellular vesicles (10) from the biological sample (15) to provide a prepared sample (20) comprising the extracellular vesicles (10); a drying stage comprising (a) the drying element (225) receiving at least part of the prepared sample (15) from the isolation element (215) and providing a droplet (125) of theprepared sample (25) on the support (120) , and (b) the drying element (225) drying the droplet (125) to provide a multilayer (135) of dried extracellular vesicles (10) on the support (120); a reconstitution stage comprising (a) the liquid supply (230) providing the aqueous reconstitution liquid (130) to the support (120), wherein the reconstitution liquid (130) comprises < 600 mM of ions, and (b) the electric field generator (250) exposing the multilayer (135) to an AC electric field, the AC electric field having a field strength selected from the range of 20 - 1500 V / m at a frequency selected from the range of 5 - 20 Hz, thereby providing the reconstituted vesicle (20), wherein the reconstituted vesicle (20) has an equivalent spherical diameter (DR) selected from the range of 2 - 50 pm; and a separation stage comprising the electric field generator (250) exposing the multilayer (135) to a second AC electric field to separate the reconstituted vesicle (20) from the support (120), the second AC electric field having a second field strength selected from the range of 1600 - 3000 V / m at a second frequency selected from the range of 2 - 8 Hz.

16. The system (200) according to claim 15, wherein the isolation element (215) comprises an affinity purification element (216), wherein the affinity purification element (216) is configured to selectively isolate a first type (11) of extracellular vesicles (10) from the biological sample (15), wherein the first type (11) of extracellular vesicles (10) corresponds to a first cell type or a first disease type.

17. The system (200) according to any one of the preceding claims 15-16, wherein the system (200) further comprises a measurement element (240), wherein the measurement element (240) is configured to receive the reconstituted vesicle (20) during the separation stage, wherein the measurement element (240) is configured to arrange the reconstituted vesicle (20) between a first electrolyte (141) and a second electrolyte (142) , and wherein the operational mode further comprises: a measurement stage comprising (i) the measurement element (240) controlling an absolute voltage through the reconstituted vesicle (20) in the range of 1 - 100 mV, recording a resulting current, and providing a related current signal to the control system (300); and / or (ii) the measurement element (200) controlling a current through the reconstituted vesicle (20) in the range of 1 pA - 10 pA, recording a resulting voltage change, and providing a related voltage signal to the control system (300); and an analysis stage comprising the control system (300) determining a parameter of the reconstituted vesicle (20) based on the current signal and / or the voltage signal.

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