Extracellular vesicle preserving fluid taking citric acid and sodium citrate as buffering agents and preparation

Abstract

The invention provides an extracellular vesicle preserving fluid taking citric acid and sodium citrate as buffering agents and a formula of a corresponding preparation. The formula comprises sodium chloride, cane sugar, polysorbate 80, citric acid and sodium citrate. The formula can improve the storage stability of the extracellular vesicles and protect substances in the extracellular vesicles from being degraded, and is suitable for long-term storage and application of the extracellular vesicles.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an extracellular vesicle preservation solution using citric acid and sodium citrate as buffers and corresponding preparations. Background Art

[0002] As a new treatment method, cell therapy mainly achieves the treatment effect by transplanting or injecting normal or bioengineered human cells into patients. Cell therapy can be divided into two categories: immune cell therapy and stem cell therapy according to the types of cells used for treatment.

[0003] Immune cells are an important part of the human immune system, including human T cells, NK cells, B cells, DC (dendritic cell) cells, etc. Currently, the relatively mature one is CAR-T (Chimeric antigen receptor T cell) therapy. Immune cells can secrete and uptake extracellular vesicles, thereby playing an immunomodulatory role between innate immunity and adaptive immunity. Therefore, extracellular vesicles secreted by immune cells can be applied to cancer diagnosis and immunotherapy, and may be used for vaccination and chemotherapy drug transportation. The extracellular vesicles released by NK cells carry proteins and microRNAs with antitumor effects and can be used in the field of tumor treatment, and are expected to become a promising new anti-cancer method.

[0004] With stem cells as the research focus, stem cells can be divided into embryonic stem cells (ESCs) and adult stem cells (ASCs) according to their developmental state. ESCs and induced pluripotent stem cells (iPSCs) can maintain an undifferentiated state or differentiate into most of the pluripotent stem cells in the human body. ASCs are pluripotent stem cells, including hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and neural stem cells (NSCs). These cells are committed to further differentiation into cells of specific organs or systems, such as HSCs differentiate into cells of the blood and immune system, and NSCs differentiate into cells of the brain and nervous system. MSCs have a wide range of sources, such as adipose MSCs (AD-MSCs), bone marrow MSCs (BM-MSCs), umbilical cord MSCs (UC-MSCs), fetal dermal MSCs (FD-MSCs), endometrium and menstrual blood (eMSC / MenSC), etc. A large number of studies have shown that in addition to relying on self-proliferation and differentiation, most of the functions of mesenchymal stem cells are to interact with other cells through secreted proteins and extracellular vesicles. Among them, adipose-derived mesenchymal stem cells are easy to obtain, have no ethical issues, can proliferate in vitro, and have good prospects for medical applications.

[0005] Extracellular vesicles are nanoscale vesicles secreted by cells, with a maximum diameter of 2000nm, mostly 30-200nm. The formation process is: the cell membrane invaginates into endosomes, and the endosomal membrane invaginates multiple times to form multivesicular bodies and late endosomes, and then fuses with the cell membrane and is released outside the cell. Extracellular vesicles have a double-layer lipid membrane structure, with four transmembrane proteins such as CD63, CD81, and CD9 highly expressed on the surface. The interior contains cytokines, growth factors, signaling lipids, mRNA, microRNA, and siRNA derived from mother cells. As endogenously produced vesicles, EVs have various advantages such as non-toxicity, low immunogenicity, and higher stability. Using EVs instead of cell therapy may reduce the risks of whole-cell therapy, such as uncontrolled cell division or cytokine storms.

[0006] After isolation and purification, extracellular vesicles generally need to be stored at -90 to -70 °C, and repeated freezing and thawing should be avoided. Some studies have shown that if extracellular vesicles after isolation and purification are stored at 2-8 °C and -25 to -15 °C for a long time, the number of extracellular vesicle particles will decrease, and proteins and RNAs will also degrade, which hinders the development of extracellular vesicles as a cell-free therapy method in terms of usage scenarios and shelf life. At ultra-low temperatures, methods to increase the stability of extracellular vesicles include adding cryoprotectants and providing a buffer system, while attention needs to be paid to maintaining the osmotic pressure of the solution.

[0007] Citric acid and sodium citrate are widely used in the formulation of protein drugs to regulate the pH of the formulation. The specific chemical formula is as Figure 1 shown. Citric acid has biocompatibility, is biodegradable, is easy to obtain and has a low cost, and is applied to the design and construction of new delivery systems such as polymers, hydrogels, and nanoparticles (NPs). It has the functions of preventing NPs aggregation, acting as a co-surfactant, and maintaining structural stability in the delivery system of NPs. As a weak acid and strong base salt, sodium citrate has good pH regulation and buffering capabilities. In addition, sodium citrate has good abilities to complex metal ions and resist redeposition. Sucrose is a commonly used non-permeating cryoprotectant with a relatively high cost performance. It has many hydroxyl groups in its structure, so it can provide a stabilizing effect by replacing the hydrogen bonds between proteins and water, and maintain the uniform distribution of ions by preventing the formation of ice crystals and reducing damage to biological structures. As a surfactant, polysorbate 80 can interact with proteins to reduce aggregation and maintain the structural stability of proteins.

[0008] The purpose of the present invention is to solve the storage problem of extracellular vesicle products. Summary of the Invention

[0009] The present invention provides a formulation for improving the stability of extracellular vesicles stored under freezing conditions.

[0010] In a first aspect of the present invention, there is provided an aqueous composition comprising extracellular vesicles, which further comprises or essentially comprises a buffer, a non-permeating cryoprotectant, a surfactant, and an osmotic pressure regulator. In some embodiments, the aqueous composition consists of extracellular vesicles, a buffer, a non-permeating cryoprotectant, a surfactant, an osmotic pressure regulator, and water. In some embodiments, the aqueous composition has a pH value of about 4-9, preferably about 5-8, more preferably about 6-8.

[0011] In a preferred embodiment, the buffer comprises a carbonate buffer system, a phosphate buffer system, a citrate buffer system, a Tris (tris(hydroxymethyl)aminomethane) buffer system, and / or an acetate buffer system, etc. In a preferred embodiment, the buffer comprises a citrate buffer system, such as citric acid and / or sodium citrate, preferably citric acid monohydrate and / or sodium citrate dihydrate. In some specific embodiments, the buffer comprises citric acid and / or sodium citrate, wherein the concentration of citric acid is 0-10 mM; and / or the concentration of sodium citrate is 1-50 mM.

[0012] In a preferred embodiment, the non-permeating cryoprotectant comprises sugar, preferably non-reducing sugar, more preferably non-reducing disaccharide. In a preferred embodiment, the non-permeating cryoprotectant comprises sucrose. In a preferred embodiment, the non-permeating cryoprotectant comprises trehalose. In a preferred embodiment, the non-permeating cryoprotectant comprises sucrose and trehalose. In some specific embodiments, the non-permeating cryoprotectant comprises sucrose at a concentration of 10-100 mM.

[0013] In a preferred embodiment, the surfactant comprises polysorbate, such as polysorbate-20, polysorbate-80, polysorbate-60, polysorbate-40; and / or pluronic, etc. In a preferred embodiment, the surfactant comprises polysorbate-80. In another preferred embodiment, the surfactant comprises polysorbate-20. In some specific embodiments, the surfactant comprises polysorbate-80 at a concentration of 0.1-2 mg / mL.

[0014] In a preferred embodiment, the osmotic pressure regulator comprises sodium chloride. In some specific embodiments, the osmotic pressure regulator comprises sodium chloride at a concentration of 50-150 mM.

[0015] In one embodiment of the present invention, there is provided an aqueous composition for cryopreserving extracellular vesicles, which comprises extracellular vesicles and further comprises or substantially comprises the following components: sodium chloride, sucrose, polysorbate 80, citric acid and / or sodium citrate. In some embodiments, the aqueous composition consists of extracellular vesicles, sodium chloride, sucrose, polysorbate 80, citric acid and / or sodium citrate and water. In a more specific embodiment, the concentration of sucrose is 10-100 mM; the concentration of polysorbate-80 is 0.1-2 mg / mL; the concentration of sodium chloride is 50-150 mM; the concentration of citric acid is 0-10 mM; the concentration of sodium citrate is 1-50 mM.

[0016] More preferably, the sodium chloride concentration is 90 - 120 mM; the citric acid concentration is 0 - 5 mM; the sodium citrate concentration is 5 - 30 mM.

[0017] Most preferably, the sodium chloride concentration is 110 - 120 mM; the citric acid concentration is 0.1 - 1 mM; the sodium citrate concentration is 5 - 10 mM.

[0018] In one embodiment of the present invention, the aqueous composition contains extracellular vesicles and further contains or essentially contains the following components: sucrose, with a final concentration of 40 - 60 mM, preferably 45 - 55 mM, more preferably 48 - 52 mM, most preferably 50 mM; polysorbate 80, with a final concentration of 0.1 - 0.3 mg / mL, preferably 0.15 - 0.25 mg / mL, more preferably 0.18 - 0.22 mg / mL, most preferably 0.2 mg / mL; sodium citrate, with a final concentration of 10 - 30 mM, preferably 15 - 25 mM, more preferably 18 - 22 mM, most preferably 20 mM; sodium chloride, with a final concentration of 98 - 100 mM, preferably 99 mM. The above components are dissolved / suspended in pure water.

[0019] In one embodiment of the present invention, the aqueous composition contains extracellular vesicles and further contains or essentially contains the following components: sucrose, with a final concentration of 40 - 60 mM, preferably 45 - 55 mM, more preferably 48 - 52 mM, most preferably 50 mM; polysorbate 80, with a final concentration of 0.1 - 0.3 mg / mL, preferably 0.15 - 0.25 mg / mL, more preferably 0.18 - 0.22 mg / mL, most preferably 0.2 mg / mL; citric acid and sodium citrate, with a ratio of 1:15, the final concentration of citric acid is 0.4 - 0.6 mM, preferably 0.45 - 0.55 mM, more preferably 0.5 mM, and the final concentration of sodium citrate is 6.0 - 9.0 mM, preferably 6.7 - 8.3 mM, more preferably 7.5 mM; sodium chloride, with a final concentration of 117.0 - 118.0 mM, preferably 117.5 mM. The above components are dissolved / suspended in pure water.

[0020] In one embodiment of the present invention, the aqueous composition comprises extracellular vesicles and further comprises or consists essentially of the following components: sucrose at a final concentration of 40 - 60 mM, preferably 45 - 55 mM, more preferably 48 - 52 mM, most preferably 50 mM; polysorbate 80 at a final concentration of 0.1 - 0.3 mg / mL, preferably 0.15 - 0.25 mg / mL, more preferably 0.18 - 0.22 mg / mL, most preferably 0.2 mg / mL; citric acid and sodium citrate in a ratio of 1:15 - 20, such as 1:15, 1:16, 1:17, 1:18, 1:19 or 1:20, with the final concentration of citric acid being 0.5 - 2.0 mM, preferably 0.7 - 1.5 mM, more preferably 0.9 - 1.2 mM, most preferably 1 mM, and the final concentration of sodium citrate being 7.5 - 40 mM, preferably 10 - 40 mM, more preferably 15 - 30 mM, most preferably 15 - 20 mM; sodium chloride at a final concentration of 98 - 107 mM, preferably 98.5 mM or 106 mM. The above components are dissolved / suspended in pure water.

[0021] In one embodiment of the present invention, the aqueous composition comprises extracellular vesicles and further comprises or consists essentially of the following components: sucrose at a final concentration of 40 - 60 mM, preferably 45 - 55 mM, more preferably 48 - 52 mM, most preferably 50 mM; polysorbate 80 at a final concentration of 0.1 - 0.3 mg / mL, preferably 0.15 - 0.25 mg / mL, more preferably 0.18 - 0.22 mg / mL, most preferably 0.2 mg / mL; citric acid and sodium citrate in a ratio of 1:20, with the final concentration of citric acid being 0.4 - 0.6 mM, preferably 0.45 - 0.55 mM, more preferably 0.5 mM, and the final concentration of sodium citrate being 8.0 - 12.0 mM, preferably 9.0 - 11.0 mM, more preferably 10 mM; sodium chloride at a final concentration of 113.0 - 114.0 mM, preferably 113.5 mM or 113.75 mM. The above components are dissolved / suspended in pure water.

[0022] In some embodiments of the present invention, the weight osmotic concentration of the aqueous composition is about 270 mOsmol / kg to about 330 mOsmol / kg, preferably 290 mOsmol / kg to about 310 mOsmol / kg, more preferably about 300 mOsmol / kg.

[0023] In some embodiments of the present invention, the aqueous composition contains extracellular vesicles, and the particle concentration thereof is 1 - 100×10 9 / mL. For example, about 1×10 9 / mL, about 10×10 9 / mL or about 50×10 9 / mL.

[0024] In some specific embodiments of the present invention, the extracellular vesicles are derived from cells selected from the group consisting of immune cells, hematopoietic stem cells, neural stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, and umbilical cord mesenchymal stem cells. In some more specific embodiments, the cells are natural cells or genetically modified cells, for example, natural immune cells or genetically modified immune cells, natural stem cells or genetically modified stem cells. In some more specific embodiments, the cells are mesenchymal stem cells, such as adipose mesenchymal stem cells. In some more specific embodiments, the cells are chimeric antigen receptor-modified T cells (CAR-T) or chimeric antigen receptor-modified NK cells (CAR-NK).

[0025] In some specific embodiments of the present invention, the extracellular vesicles are characterized by a median particle size of 30 - 200 nm, as measured by NTA. In some more specific embodiments, the extracellular vesicles maintain or substantially maintain the particle size within the temperature range of -90°C to 45°C, for example, within the temperature range of about 37°C, within the temperature range of 20 - 25°C, within the temperature range of 2 - 8°C (e.g., 4°C), within the temperature range of -25 - -15°C (e.g., -20°C), or within the temperature range of -90 - -70°C (e.g., -80°C).

[0026] In some specific embodiments of the present invention, the extracellular vesicles are stable after being placed for 6 months within the temperature range of -90°C to 45°C, for example, showing a concentration decrease of no more than 30%.

[0027] In some embodiments, the aqueous composition of the present invention is a liquid. In some embodiments, the aqueous composition of the present invention is a solid, such as a powder, such as a lyophilized powder.

[0028] In some embodiments, the aqueous composition of the present invention is a pharmaceutical composition, for example, which further comprises other pharmaceutically acceptable carriers, diluents, and / or excipients.

[0029] In a corresponding aspect of the present invention, there is provided a preservation solution for cryopreserving extracellular vesicles, which comprises a buffer, a non-permeating cryoprotectant, a surfactant, and an osmotic pressure regulator, preferably dissolved in water. The preservation solution, for example, can be used to prepare the aqueous composition of the present invention.

[0030] In some preferred embodiments, there is provided a preservation solution for cryopreserving extracellular vesicles, which consists of a buffer, a non-permeating cryoprotectant, a surfactant, and an osmotic pressure regulator dissolved in water.

[0031] In a preferred embodiment, the buffer comprises a carbonate buffer system, a phosphate buffer system, a citrate buffer system, a Tris (tris(hydroxymethyl)aminomethane) buffer system, and / or an acetate buffer system, etc. In a preferred embodiment, the buffer comprises a citrate buffer system, such as citric acid and / or sodium citrate, preferably, citric acid monohydrate and / or sodium citrate dihydrate. In some specific embodiments, the buffer comprises citric acid and / or sodium citrate.

[0032] In a preferred embodiment, the non-permeating cryoprotectant comprises sugar, preferably non-reducing sugar, more preferably non-reducing disaccharide. In a preferred embodiment, the non-permeating cryoprotectant comprises sucrose. In a preferred embodiment, the non-permeating cryoprotectant comprises trehalose. In a preferred embodiment, the non-permeating cryoprotectant comprises sucrose and trehalose.

[0033] In a preferred embodiment, the surfactant comprises polysorbate, such as polysorbate-20, polysorbate-80, polysorbate-60, or polysorbate-40; and / or pluronic, etc. In a preferred embodiment, the surfactant comprises polysorbate-80. In another preferred embodiment, the surfactant comprises polysorbate-20.

[0034] In a preferred embodiment, the osmotic pressure regulator comprises sodium chloride.

[0035] In one embodiment of the present invention, a preservation solution for cryopreserving extracellular vesicles is provided, which comprises the following components: sodium chloride, sucrose, polysorbate 80, citric acid and / or sodium citrate. In a more specific embodiment, the molar concentration ratio of each component in the preservation solution is: sodium chloride:sucrose:polysorbate 80:sodium citrate:citric acid = 50 - 150:10 - 100:0.07 - 1.53:1 - 50:0 - 10, and the molar concentration of sodium citrate is higher than 1 mM. More preferably, the molar concentration ratio of sodium chloride is 90 - 120, the molar concentration ratio of citric acid is 0 - 5, and the molar concentration ratio of sodium citrate is 5 - 30. Most preferably, the molar concentration ratio of sodium chloride is 110 - 120; the molar concentration ratio of citric acid is 0.1 - 1; the molar concentration ratio of sodium citrate is 5 - 10.

[0036] In another aspect of the present invention, a preparation method of the preservation solution of the present invention is provided, which is prepared by dissolving the buffer, the non-permeating cryoprotectant, the surfactant and the osmotic pressure regulator in water respectively / together / sequentially.

[0037] In some embodiments, the preservation solution is prepared by dissolving the buffer, non-permeating cryoprotectant, surfactant, and osmotic pressure regulator in water respectively, and then mixing the solutions evenly.

[0038] In some embodiments, the preservation solution is prepared by dissolving the buffer, non-permeating cryoprotectant, surfactant, and osmotic pressure regulator in water together / sequentially in a certain order, and then mixing evenly.

[0039] In a specific embodiment, the preservation solution is prepared according to the following steps:

[0040] First step, prepare the equipment, pure water, etc. used for preparation, place them in a laminar flow hood, and sterilize before use.

[0041] Second step, preparation of 100 mM citric acid mother liquor: Weigh 2.10 g of citric acid, dissolve it in pure water and make up the volume to 100 mL, filter it through a 0.22 μm filter, and store it at 2 - 8 °C.

[0042] Third step, preparation of 100 mM sodium citrate mother liquor: Weigh 2.94 g of sodium citrate, dissolve it in pure water and make up the volume to 100 mL, filter it through a 0.22 μm filter, and store it at 2 - 8 °C.

[0043] Fourth step, preparation of 200 mM sucrose mother liquor: Weigh 3.42 g of sucrose, dissolve it in pure water and make up the volume to 50 mL, filter it through a 0.22 μm filter, and store it at 2 - 8 °C.

[0044] Fifth step, preparation of 400 mM sodium chloride mother liquor: Weigh 1.17 g of sodium chloride, dissolve it in pure water and make up the volume to 50 mL, filter it through a 0.22 μm filter, and store it at 2 - 8 °C.

[0045] Sixth step, according to the proportion of each component and the concentration multiple (e.g., 2×, 5× or 10×) of the required preservation solution, calculate the amount of each mother liquor and pure water needed, accurately measure and mix evenly for preparation. Optionally, store it at -90 - -70 °C afterwards.

[0046] Another aspect of the present invention provides the use of the preservation solution, which is used to preserve extracellular vesicle products at -90 - 45 °C, for example, for preparing the aqueous composition of the present invention.

[0047] Another aspect of the present invention provides a preparation method of the aqueous composition of the present invention, which comprises the following steps:

[0048] Uniformly mix the extracellular vesicle stock solution and the preservation solution of the present invention.

[0049] In some embodiments, the volume ratio of the mixture is between 1:3 and 1:30. For example, the volume ratio is 1:5, 1:10, 1:15, or 1:20. Preferably, the volume ratio of the two mixtures is 1:10.

[0050] More preferably, optionally, the extracellular vesicle stock solution is prepared by resuspending the obtained extracellular vesicles with sodium chloride injection (e.g., pharmaceutical 0.9% (w / v) sodium chloride solution), compound electrolyte injection (e.g., Plasmalyte A (a pharmaceutical solution containing 5.26‰ (w / v) sodium chloride, 5.02‰ (w / v) sodium gluconate, 3.68‰ (w / v) sodium acetate, 0.37‰ (w / v) potassium chloride, and 0.30‰ (w / v) magnesium chloride)), and / or PBS and other isotonic solutions.

[0051] More preferably, the particle concentration of the extracellular vesicle stock solution is 1 - 100×10 10 / mL.

[0052] Most preferably, the extracellular vesicle stock solution is resuspended with pharmaceutical 0.9% (w / v) sodium chloride solution.

[0053] In an exemplary embodiment, the particle concentration of the extracellular vesicle stock solution is 1 - 10×10 10 / mL.

[0054] In an exemplary embodiment, the extracellular vesicles are secreted by adipose mesenchymal stem cells. In a more specific exemplary embodiment, the method for separating the extracellular vesicles includes the following steps:

[0055] a) Culturing adipose mesenchymal stem cells;

[0056] b) Adding cell-free extracellular vesicle culture medium for culturing;

[0057] c) Separating and purifying extracellular vesicles from the culture medium.

[0058] In an exemplary embodiment, the specific steps of the cell culture are as follows:

[0059] 1) Resuscitating cryopreserved adipose mesenchymal stem cells and inoculating them into a cell culture flask at a density of 1.2×10 4 / cm 2 and culturing until the cell confluence reaches 80% - 90%, and the culture time is 2 - 3 days;

[0060] 2) Removing the upper layer of the culture medium and washing the cells with PBS;

[0061] 3) Adding cell-free extracellular vesicle culture medium again and culturing in a 37°C, 5% CO2 incubator;

[0062] 4) Collect the conditioned medium after 48 hours.

[0063] Preferably, the acellular extracellular vesicle culture medium is prepared by ultracentrifugation of a complete medium.

[0064] Preferably, the steps for isolating and purifying extracellular vesicles are as follows:

[0065] 1) Take the conditioned medium and perform centrifugation at 3000 - 10000g to discard cell debris and apoptotic bodies;

[0066] 2) Add an equal volume of an extracellular vesicle isolation reagent, let it stand, and then centrifuge to obtain a precipitate containing extracellular vesicles;

[0067] 3) Add PBS, perform ultracentrifugation at 3000 - 120000g, and resuspend with sodium chloride injection solution to obtain a separated extracellular vesicle stock solution.

[0068] Preferably, after mixing the separated extracellular vesicle stock solution with the preservation solution, store it at 2 - 8°C, -25 - -15°C, and -90 - -70°C, and optionally detect the changes in extracellular vesicles after cryopreservation, such as changes in concentration, particle size, and surface / intracellular markers (e.g., protein markers, miRNAs).

[0069] Another aspect of the present invention provides a method for detecting the protective ability of an extracellular vesicle preservation solution, including detecting the degradation of extracellular vesicles.

[0070] Preferably, the degradation of extracellular vesicles includes a decrease in particle concentration, degradation of protein markers, and degradation of miRNAs in extracellular vesicles.

[0071] Preferably, the particle concentration is detected by Nano Tracking Analysis (NTA).

[0072] Preferably, the degradation of the protein marker is detected by Western Blot, and the protein marker can be CD63.

[0073] Preferably, the degradation of miRNAs is detected by qPCR, and the miRNA in extracellular vesicles can be hsa-miR-21-5p.

[0074] The present invention provides an aqueous composition suitable for cryopreserving extracellular vesicles, which can ensure that extracellular vesicles are not significantly degraded after long-term storage (for example, cryopreservation (for example, at -80 to -20 °C)). It contains sucrose, polysorbate 80, sodium chloride, citric acid and sodium citrate. Among them, sucrose and polysorbate 80 are used as cryoprotectants, citric acid and sodium citrate are used as pH buffers, and sodium chloride is added to maintain the osmotic pressure of the solution, so as to protect and stabilize extracellular vesicles under storage (for example, cryopreservation) conditions and extend the product shelf life.

[0075] In some embodiments, the aqueous composition is stable after storage, for example, after storage at 2-8 °C for at least 1 week, 2 weeks or preferably 1 month, or for example, after storage at -25 °C to -15 °C for at least 1 month or 2 months, or for example, after storage at -90 °C to -70 °C for at least 6 months or 12 months, and preferably has one or more of the following characteristics:

[0076] (i) Measured by nanoparticle tracking analysis technology (NTA), the concentration and / or particle size of extracellular vesicle particles in the dissolved state show no obvious change compared with the concentration at the time of just preparation. For example, the decrease in concentration is less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%;

[0077] (ii) Detecting the expression of extracellular vesicle protein markers (for example, CD63) by Western blot shows no obvious change compared with the time of just preparation. For example, the change in the expression of the protein marker (for example, CD63) is less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%;

[0078] (iii) Detecting the expression of extracellular vesicle protein inclusions by ELISA / Western blot shows no obvious change compared with the time of just preparation. For example, the change in the expression of protein inclusions is less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%;

[0079] (iv) The content of extracellular vesicle nucleic acid inclusions (e.g., hsa-miR-21-5p) is detected by fluorescence quantitative PCR (qPCR), and there is no significant change compared with that at the time of just preparation. For example, the change in the content of nucleic acid inclusions (e.g., hsa-miR-21-5p) is less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%.

[0080] In a preferred embodiment of the present invention, the aqueous composition of the present invention has a high extracellular vesicle concentration, and at the same time it has high physical and chemical stability suitable for long-term preservation at low temperature (e.g., ultra-low temperature), and the particles are not easily aggregated and / or degraded during storage.

[0081] In a preferred embodiment of the present invention, when the aqueous composition of the present invention is formulated, all components meet the human drug use standards, and at this time the aqueous composition of the present invention has the safety suitable for direct administration.

[0082] In one aspect, the present invention provides a delivery device comprising the aqueous composition preparation of the present invention. In one embodiment, the delivery device of the present invention is provided in the form of a pre-filled syringe containing the aqueous composition preparation of the present invention, for example, for intravenous, subcutaneous, intradermal or intramuscular injection, intravenous infusion.

[0083] In yet another aspect, the present invention provides a method for administering extracellular vesicles to a subject, such as a mammal, such as a human, comprising the step of administering to the subject the aqueous composition preparation of the present invention, and the delivery is implemented, for example, by using a delivery device of a pre-filled syringe. Description of the Drawings

[0084] Figure 1 It is a schematic diagram of the chemical formulas of citric acid and sodium citrate. The molecular formula of citric acid monohydrate is: HOC(COOH)(CH2COOH)2·H2O, molecular weight: 210.14; the molecular formula of sodium citrate dihydrate is: HOC(COONa)(CH2COONa)2·2H2O, molecular weight: 294.10;

[0085] Figure 2 It is a graph showing the change in the concentration of extracellular vesicle particles of Experimental Example 1 and the control example stored at -25 to -15 °C for 3 months;

[0086] Figure 3 It is the change of extracellular vesicles after Experimental Example 1-5 were stored at 2-8 °C for 7 days. Among them, (A) is the ratio change of the particle concentration compared with the 0 point; (B) is the median particle size.

[0087] Figure 4Changes in extracellular vesicles after storage of Experimental Examples 1-5 at -90 to -70 °C for 6 months. Among them, (A) is the ratio change of particle concentration compared to the 0 point; (B) is the median particle size.

[0088] Figure 5 Quantification diagram of CD63 protein expression in extracellular vesicles after storage of Experimental Examples 1-5 at -90 to -70 °C for 6 months. Among them, (A) is the representative band detected by Western blot for CD63; (B) is the ratio of the gray value of CD63 stored at -90 to -70 °C for 6 months to the 0 point.

[0089] Figure 6 Quantification diagram of the expression of hsa-miR-21-5p in extracellular vesicles after storage of Experimental Examples 1-5 at -90 to -70 °C for 6 months. Detailed implementation manners

[0090] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, when used in connection with a specific recited numerical value or range of values, the term "about" will cause the numerical value with which it is associated to vary by ±10%. For example, as used herein, the expression "about 100" includes 90 and 110 and all values therebetween (such as 90.5, 95, 101, 105, 109.95... etc.). For ratios, the term "about" is used to define each number of the given ratio. For example, a ratio of about 1:1 means a ratio of 0.9 - 1.1:0.9 - 1.1.

[0091] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are now described. All patents, applications, and non-patent publications mentioned in this specification are incorporated herein by reference in their entirety.

[0092] As used herein, the term "weight percentage by volume" or "% w / v" means the percentage weight (in grams) of a single component relative to the total volume of the mixture containing that component. For example, 500 mg of a component in a total volume of 8 mL is 6.25% w / v, and 500 mg of a component in a total volume of 5 mL is 10% w / v. Or, equivalently expressed directly, for example, as mg / mL.

[0093] A "separated" substance has been changed by human effort from its natural state. If a "separated" component or substance occurs in nature, then it has been changed or removed from its original environment, or both. For example, the extracellular vesicles mentioned in the present invention are substantially separated unless otherwise stated when present in a suspension or aqueous composition.

[0094] As used herein, a formulation refers to a combination of at least one active ingredient (such as extracellular vesicles) and at least one inactive ingredient that, when combined with the active ingredient and / or one or more additional inactive ingredients, directly or indirectly renders the formulation more suitable for therapeutic administration to a human or non-human animal.

[0095] The extracellular vesicle formulation of the present invention can be an aqueous composition, such as an aqueous solution or suspension, or a solid, such as a lyophilized product.

[0096] The term "extracellular vesicle (EV)" refers to any type of vesicle that can be obtained from any form of cell, such as exosomes (e.g., any vesicle derived from the endolysosomal pathway, microvesicles, any vesicle shed from the plasma membrane of a cell), apoptotic bodies (e.g., obtainable from apoptotic cells), microparticles (which can be derived from, for example, platelets), ectosomes (which can be derived from, for example, neutrophils and monocytes in serum), etc., where the main types are exosomes, microvesicles, and apoptotic bodies. The size of EVs can vary considerably, and even among EVs from the same source, the size distribution may be relatively dispersed. However, EVs generally have a hydrodynamic particle size in the nanometer range in a liquid / suspension, i.e., a particle size less than 2000 nm, and most EVs have a particle size of 30 - 200 nm, thus representing the average particle size and / or median particle size of the vesicle population. EVs can be derived from any cell type in vivo, in vitro, and ex vivo. EVs have a bilayer lipid membrane structure, are rich in tetraspanin proteins such as CD63, CD81, and CD9 on the surface, and contain cytokines, growth factors, signaling lipids, mRNA, microRNA, and siRNA substances derived from the parent cell. Additionally, the term also encompasses extracellular vesicle mimics, cell membrane-based vesicles obtained by, for example, membrane extrusion, sonication, or other techniques. It will be apparent to those skilled in the art that the technical solutions of the present invention generally relate to a population of EVs, among which EVs can be present in various concentration ranges and concentrations, such as 10 5 、10 6 、10 7 、10 8 、10 9 、10 10 、10 11 、10 12 、10 13 、10 14 、10 15 、10 16 、10 17EV particles per unit volume (e.g., mL). In some embodiments of the present invention, there can be multiple types of EVs in the formulation. The EVs of the present invention can carry various types of payloads (e.g., drugs), and / or can contain specific molecules such as mRNA, shRNA, miRNA, proteins, peptides, etc., for example, genetically engineered to contain specific molecules such as mRNA, shRNA, miRNA, proteins (e.g., antibodies), peptides, etc. The genetic engineering can also be intended to enhance their activity as a delivery vehicle for exogenous drug payloads. As endogenously produced vesicles, EVs have various advantages such as being non-toxic, having low immunogenicity, and higher stability. Using EVs to replace cell therapy may reduce the risks of whole-cell therapy, such as uncontrolled cell division or cytokine storms.

[0097] The term exosome refers to a subclass of EVs derived from endosomes, which are a major component of the paracrine secretions of various cell types, including mesenchymal stem cells (MSCs). As carriers of rich payloads, their functions are mainly exerted by continuously transporting microRNAs (miRNAs) and proteins. More than 150 miRNAs and more than 850 unique proteins have been identified in MSC-derived exosomes, which can alter the various activities of target cells through different pathways. MSC exosomes are involved in physiological and pathological processes such as organism development, epigenetic regulation, immune regulation (miR-155 and miR-146), tumorigenesis and tumor progression (miR-23b, miR-451, miR-223, miR-24, miR-125b, miR-31, miR-214, and miR-122). According to ExoCarta, more than 900 proteins have been collected from MSC exosomes. Some studies have shown that MSC exosomes carry some cytokines and growth factors, such as TGFβ1, interleukin-6 (IL-6), IL-10, and hepatocyte growth factor (HGF), etc., which have been proven to contribute to immune regulation. Vascular endothelial growth factor (VEGF), extracellular matrix metalloproteinase inducer (EMMPRIN), and MMP-9 have all been reported in MSC exosomes, and these three proteins play important roles in stimulating angiogenesis, which may be the basis for the tissue repair effect of exosomes.

[0098] EVs contain and carry a variety of proteins, lipids, and RNAs. It is generally believed in the art that after reaching the target region / cells, the proteins and / or RNAs in EVs act as effector molecules either outside or inside the cells. Therefore, if EVs are expected to be active, the protein and / or RNA load in or on their surface should be substantially intact, undegraded or denatured. This is also the theoretical basis for judging the stability of EVs after storage by measuring the content changes of proteins and / or RNAs.

[0099] "Particle size (Diameter)" and "Median Diameter" refer to the intensity volume diameter corresponding to the extracellular vesicle particle preparation / hydrous composition, which can be measured by conventional measurement methods in the art.

[0100] In another preferred embodiment of the present invention, the extracellular vesicles comprise a lipid bilayer membrane structure.

[0101] In another preferred embodiment of the present invention, the median particle size of the extracellular vesicles is between 30 - 200 nm.

[0102] In another preferred embodiment of the present invention, the vesicles contain different types of microRNAs (miRNAs), small RNAs (sRNAs), non-coding DNA fragments, transfer RNAs (t-RNAs), soluble cytokines, growth factors, and other proteins with specific functions.

[0103] In another preferred embodiment of the present invention, the soluble active cytokines are the soluble active cytokines produced by the somatic cells of a human.

[0104] In another preferred embodiment of the present invention, the soluble active cytokines include the active cytokines produced by the paracrine of somatic cells of a human cultured in a natural state, the active cytokines expressed and produced by somatic cells of a human modified with exogenous genes, or a combination thereof.

[0105] In another preferred embodiment of the present invention, the extracellular vesicles of the cell membrane specifically express the following proteins: CD9, CD63, CD81, and TSG101; and do not express or substantially do not express the protein CANX.

[0106] In another preferred embodiment of the present invention, the median particle size of the extracellular vesicles of the cell membrane is 30 - 200 nm.

[0107] In another preferred embodiment of the present invention, the cells used for producing the extracellular vesicles of the cell membrane include cells from the following sources:

[0108] (a) Cells obtained by directly separating and purifying from human tissues;

[0109] (b) Cells obtained by directly separating and purifying from human tissues and then minimally manipulating them in a GMP laboratory to proliferate these cells;

[0110] (c) Cells obtained by specific gene modification, specific gene editing, specific gene transduction, or introduction of specific microRNA (miRNA) in a GMP laboratory;

[0111] (d) Cells obtained by pre - treating under special culture conditions in a GMP laboratory.

[0112] In another preferred embodiment of the present invention, the cells used for producing the extracellular vesicles of the cell membrane include primary cells and sub - cultured cells with a sub - culture passage number of 1 - 10 times.

[0113] In another preferred embodiment of the present invention, the cells used for producing the extracellular vesicles of the cell membrane include cells without genetic manipulation and cells with genetic manipulation.

[0114] In another preferred embodiment of the present invention, the genetic manipulation includes gene editing, gene introduction, gene down - regulation (knock - down), gene knockout (knock - out), or a combination thereof.

[0115] In another preferred embodiment of the present invention, the cells used for producing the extracellular vesicles of the cell membrane are pre - treated cells.

[0116] In another preferred embodiment of the present invention, the somatic cells are adipose - derived mesenchymal stem (progenitor) cells, placenta - amnion - derived mesenchymal stem cells, or a combination thereof.

[0117] In another preferred embodiment of the present invention, the adipose - derived mesenchymal stem (progenitor) cells are obtained as follows: For healthy male or female volunteers who have undergone ethical review and filing, signed an informed consent form, and passed strict laboratory tests to meet the criteria for establishing a cell bank and enrolling in the adipose stem (progenitor) cell product, adipose tissue is obtained by abdominal wall skin puncture liposuction or abdominal wall surgery, placed in a cell preservation solution for storage, transported at low temperature to a qualified GMP laboratory, and after separation, purification, and proliferation treatment, the adipose - derived mesenchymal progenitor cells, that is, the working cell bank cells (intermediate products), are obtained.

[0118] In another preferred embodiment of the present invention, the mesenchymal stem (progenitor) cells derived from human adipose tissue are produced in a GMP production laboratory according to the production process of intermediate products, meeting the negative standards of various etiological detections, with the percentage of cells positive for specific surface markers such as CD73, CD90, and CD105 being approximately 98%, the percentage of cells positive for CD34 / CD45 and HLA-DR being less than 2%, meeting the detection standards for antibiotic residues and serum and serum substitute residues, and being passage P1-P6 adipose mesenchymal stem (progenitor) cells.

[0119] In another preferred embodiment of the present invention, the adipose mesenchymal stem (progenitor) cells are passage P3-P4 adipose mesenchymal stem (progenitor) cells.

[0120] In another preferred embodiment of the present invention, the somatic cells are mesenchymal stem (progenitor) cells derived from adipose tissue stored under cryogenic conditions of -196°C to -80°C (preferably, -196°C to -135°C) for 0-36 months (preferably 0-24 months or 0.5-24 months).

[0121] In another preferred embodiment of the present invention, the cells are chimeric antigen receptor-modified T cells (CAR-T) or chimeric antigen receptor-modified NK cells (CAR-NK).

[0122] Components of the extracellular vesicle preparation

[0123] The extracellular vesicle preparation of the present invention may comprise various components, such as one or more acids, salts, and / or sugars.

[0124] In some preferred embodiments of the present invention, the extracellular vesicle preparation does not require the addition of protein components to enhance stability.

[0125] The extracellular vesicle preparations described herein have a pH value that is favorable for stability, and the pH value is selected from the pH range of about 5.0 to about 8.0, such as the pH range selected from 5.0 - 5.5, the pH range selected from 5.5 - 6.0, the pH range selected from 6.0 - 6.1, the pH range selected from 6.1 - 6.2, the pH range selected from 6.2 - 6.3, the pH range selected from 6.3 - 6.4, the pH range selected from 6.4 - 6.5, the pH range selected from 6.5 - 6.6, the pH range selected from 6.6 - 6.7, the pH range selected from 6.7 - 6.8, the pH range selected from 6.8 - 6.9, the pH range selected from 6.9 - 7.0, the pH range selected from 7.0 - 7.1, the pH range selected from 7.1 - 7.2, the pH range selected from 7.2 - 7.3, the pH range selected from 7.3 - 7.4, the pH range selected from 7.4 - 7.5, the pH range selected from 7.5 - 7.6, the pH range selected from 7.6 - 7.7, the pH range selected from 7.7 - 7.8, the pH range selected from 7.8 - 7.9, the pH range selected from 7.9 - 8.0, or a combined range selected from one or more of the foregoing ranges. For example, the pH value is 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0. In some embodiments, the pH of the extracellular vesicle preparation is 6.0 - 6.5. In some embodiments, the pH of the extracellular vesicle preparation is 6.0 - 6.3. In some embodiments, the pH of the extracellular vesicle preparation is 6.1 - 6.2. In some embodiments, the pH of the extracellular vesicle preparation is 6.14.

[0126] As used herein, the term "buffer" refers to a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. For example, as used herein, "citrate buffer / solution / system" refers to a mixture containing sodium citrate and its conjugate acid, citric acid. Due to the establishment of a chemical equilibrium between the weak acid and its conjugate base, when a small amount of acid or base is added to the solution, the solution containing the buffer resists sudden changes in pH value.

[0127] In an embodiment of the present invention, the buffer can be selected from a carbonate buffer system, a phosphate buffer system, a citrate buffer system, Tris (tris(hydroxymethyl)aminomethane) buffer, or an acetate buffer, etc. And, naturally, various buffers can be used alone or in combination. In a preferred embodiment, the buffer is selected from citric acid and its usual sodium salts, and usual hydrates, etc. The suitable concentrations of these buffers vary, but generally the optimal range is 1 - 100 mM, although lower and higher concentrations and various sub-ranges may be useful.

[0128] The cryoprotectant of the preservation solution according to the present invention further comprises at least one sugar. The cryoprotectant comprising at least one sugar has a stabilizing function and is important for the short-term and long-term stability of extracellular vesicles, especially the stability at ultra-low temperatures. The sugar may be a monosaccharide, disaccharide, trisaccharide, oligosaccharide, polysaccharide, sugar alcohol and / or any combination thereof. In a preferred embodiment, the sugar is a non-reducing sugar, more preferably a non-reducing disaccharide, such as sucrose and / or trehalose. The concentration of the sugar may be in the range of 1-200 mM, preferably 1-100 mM.

[0129] The pharmaceutical preparation of the present invention may further comprise one or more surfactants, the type and amount of which are sufficient to render the extracellular vesicles stable at -90°C to 8°C. As used herein, the term "surfactant" means a substance that reduces the surface tension of the fluid in which the substance is dissolved and / or reduces the interfacial tension between oil and water. The surfactant may be ionic or non-ionic. Exemplary non-ionic surfactants that may be included in the preparation of the present invention include, for example, alkyl poly(ethylene oxide), alkyl polyglucosides (such as octyl glucoside and decyl maltoside), fatty alcohols such as cetyl alcohol and oleyl alcohol, coconut amide MEA, coconut amide DEA and coconut amide TEA. Specific non-ionic surfactants that may be included in the preparation of the present invention include, for example, polysorbates, such as polysorbate 20, polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81 and polysorbate 85; poloxamers, such as poloxamer 188 (also known as Pluronic F68), poloxamer 407; polyethylene-polypropylene glycol; or polyethylene glycol (PEG). Polysorbate 20 is also known as TWEEN 20, sorbitan monolaurate and polyoxyethylene sorbitan monolaurate. In some embodiments, the surfactant is polysorbate 80 or polysorbate 20, preferably polysorbate 80.

[0130] The amount of surfactant contained in the formulations of the present invention can vary depending on the specific properties desired for the formulation and the particular circumstances and purposes for which the formulation is intended to be used. In certain embodiments, the formulation may contain from about 0.01% to about 1% surfactant; from about 0.01% to about 0.5% surfactant; from about 0.05% to about 0.15%; from about 0.08% to about 0.12%; or from about 0.09% to about 0.11% surfactant. By way of example, the formulations of the present invention may comprise about 0.01%; about 0.02%; about 0.03%; about 0.04%; about 0.05%; about 0.06%; about 0.07%; about 0.08%; about 0.09%; about 0.10%; about 0.11%; about 0.12%; about 0.13%; about 0.14%; about 0.15%; about 0.16%; about 0.17%; about 0.18%; about 0.19%; about 0.20%; about 0.21%; about 0.22%; about 0.23%; about 0.24%; about 0.25%; about 0.26%; about 0.27%; about 0.28%; about 0.29%; or about 0.30% surfactant (such as polysorbate 80). In some embodiments, the formulation contains about 0.1% surfactant (such as polysorbate 80). Each of the above percentages corresponds to a weight / volume percentage (w / v). In some cases, the formulation contains 0.1 - 2 mg / mL of polysorbate 80.

[0131] In some embodiments, the aqueous composition of the present invention can be stably stored for at least 24 months. In some embodiments, the aqueous composition of the present invention is stable at any temperature between -90°C and 45°C. For example, it is stable at about 37°C, at 20 - 25°C, at 2 - 8°C (e.g., 4°C), at -25 - -15°C (e.g., -20°C), or at -90 - -70°C (e.g., -80°C). In some embodiments, the aqueous composition of the present invention remains stable at about -90°C to -70°C for at least 3 months, preferably at least 12 months, more preferably at least 24 months, and most preferably at least 36 months. In some embodiments, the aqueous composition of the present invention remains stable at about -25°C to -15°C for at least 3 months, preferably at least 6 months, more preferably at least 12 months, and most preferably at least 24 months. In one embodiment, the aqueous composition of the present invention remains stable at about 2 - 8°C for at least 1 week, preferably at least 3 weeks, more preferably at least 1 month, and most preferably at least 2 months. - The stability is manifested, for example, as the concentration ratio of extracellular vesicle particles in the dissolved state exceeding 70%, exceeding 80%, exceeding 85%, or exceeding 90% compared to the concentration at the time of just being formulated, preferably exceeding 95%, more preferably exceeding 98%. The stability is manifested, for example, as the change in the particle size of extracellular vesicle particles in the dissolved state being less than 30%, less than 20%, less than 15%, or less than 10% compared to that at the time of just being formulated, preferably less than 5%, more preferably less than 3%, more preferably less than 2%, and more preferably less than 1%. The stability is manifested, for example, as the amount of vesicle surface markers (e.g., CD63) decreasing by less than 30%, less than 20%, less than 15%, or less than 10% in quantitative / semi-quantitative detection compared to that at the time of just being formulated, preferably less than 5%, more preferably less than 3%, more preferably less than 2%, and more preferably less than 1%. The stability is manifested, for example, as the amount of vesicle internal substances (e.g., miRNA substances, e.g., hsa-miR-21-5p) decreasing by less than 30%, less than 20%, less than 15%, or less than 10% in quantitative / semi-quantitative detection compared to that at the time of just being formulated, preferably less than 5%, more preferably less than 3%, more preferably less than 2%, and more preferably less than 1%.

[0132] In one embodiment, the stability of the formulation after storage can be indicated by detecting changes in the appearance, visible foreign matters, particle content, marker content, and / or content of the aqueous composition. In one embodiment, the stability of the aqueous composition of the present invention can be detected in a forced experiment under high-temperature stress, for example, after storage at 2-8°C for at least 1 week, 2 weeks, or preferably 1 month, or for example, after storage at -25 to -15°C for at least 1 month or 2 months, or in a long-term experiment, for example, after storage at -90°C to -70°C for at least 6 months or 12 months. The stability is manifested, for example, in that the concentration ratio of extracellular vesicle particles in a dissolved state to the concentration at the time of just being formulated exceeds 80%, exceeds 85%, or exceeds 90%, preferably exceeds 95%, and more preferably exceeds 98%.

[0133] In some embodiments, the aqueous composition has high dispersibility, that is, when placed at 0-25°C for 6-24 hours, it is colorless and transparent, and there are no visible flocs and precipitates to the naked eye.

[0134] Use of the formulation

[0135] The aqueous composition containing extracellular vesicles of the present invention can be formulated to meet the requirements for direct administration. Therefore, in some specific embodiments, the aqueous composition is a pharmaceutical composition. Thus, the present invention also provides the use of the aqueous composition for preparing a drug. The drug is particularly suitable for dosage forms such as injections, nebulized inhalants, topical skin / mucosal medications, eye drops, nose drops, etc.

[0136] In some embodiments, the drug is used to treat diseases that will benefit from the administration of extracellular vesicles. In some embodiments, the diseases are inflammation and / or injury.

[0137] In some embodiments, the inflammation is selected from the group consisting of viral infectious inflammation, bacterial infectious inflammation, fungal infectious inflammation, autoimmune reactive inflammation, or a combination thereof, such as neurodegenerative diseases or neurological autoimmune diseases. In some embodiments, the injury is selected from the group consisting of ischemic injury, hypoxic injury, chemical injury, physical injury, or a combination thereof, such as acute / chronic lung injury, acute / chronic joint injury.

[0138] In some embodiments, the disease is a tumor.

[0139] After extensive and in-depth research and a large number of screenings, the present inventor has developed for the first time a cryopreservation solution for extracellular vesicle preservation under freezing conditions. Specifically, the present inventor has screened out a cryopreservation solution formulation using citric acid and sodium citrate as pH buffers, which can effectively protect the properties of extracellular vesicles from changing under freezing conditions. Experiments show that extracellular vesicles preserved in the said cryopreservation solution can maintain their properties basically unchanged after 6 months. On this basis, the present invention has been completed.

[0140] The present invention relates to a method for stably storing extracellular vesicles, which includes the steps of introducing the isolated extracellular vesicles into the preservation solution described herein and storing them at a suitable temperature. Suitable temperatures for storage include sub-zero temperatures, although temperatures above 0°C are also considered, and it works well for short-term and medium-term storage of extracellular vesicles with maintained activity, especially if the temperature is 2-8°C, preferably below 5°C. For long-term storage, it is most preferred to store the extracellular vesicle-containing composition at sub-zero temperatures, such as below -15°C (preferably about -20°C), and even more preferably below -50°C (preferably about -70°C to -90°C, such as -80°C).

[0141] Exemplary embodiments

[0142] This application may include the following exemplary embodiments:

[0143] 1. An aqueous composition comprising extracellular vesicles, further comprising a buffer, a non-permeating cryoprotectant, a surfactant, and an osmotic pressure regulator.

[0144] 2. The aqueous composition according to embodiment 1, wherein the aqueous composition has a pH value of 4-9, preferably 5-8, and more preferably 6-8.

[0145] 3. The aqueous composition according to embodiment 1, wherein the pH buffer comprises citric acid and / or sodium citrate, with the citric acid concentration being 0-10 mM; and / or the sodium citrate concentration being 1-50 mM.

[0146] 4. The aqueous composition according to embodiment 1, wherein the non-permeating cryoprotectant comprises a sugar, preferably a non-reducing sugar, more preferably a non-reducing disaccharide, and most preferably sucrose and / or trehalose, with the concentration of the sugar being 10-100 mM.

[0147] 5. The aqueous composition according to embodiment 1, wherein the surfactant comprises polysorbate-80 and / or polysorbate-20, with the surfactant concentration being 0.1-2 mg / mL.

[0148] 6. The aqueous composition according to embodiment 1, characterized in that the osmotic pressure regulator comprises sodium chloride at a concentration of 50-150 mM.

[0149] 7. An aqueous composition comprising extracellular vesicles, further comprising the following components: sodium chloride, sucrose, polysorbate 80, and citric acid and / or sodium citrate.

[0150] 8. The aqueous composition according to embodiment 7, characterized in that the concentration of sodium chloride is 50-150 mM; the concentration of sucrose is 10-100 mM; the concentration of polysorbate 80 is 0.1-2 mg / mL; the concentration of sodium citrate is 1-50 mM.

[0151] 9. The aqueous composition according to embodiment 7, characterized in that the concentration of sodium chloride is 50-150 mM; the concentration of sucrose is 10-100 mM; the concentration of polysorbate 80 is 0.1-2 mg / mL; the concentration of citric acid is 0.1-10 mM.

[0152] 10. The aqueous composition according to embodiment 7, characterized in that the concentration of sodium chloride is 50-150 mM; the concentration of sucrose is 10-100 mM; the concentration of polysorbate 80 is 0.1-2 mg / mL; the concentration of citric acid is 0.1-10 mM, and the concentration of sodium citrate is 1-50 mM.

[0153] 11. The aqueous composition according to any one of embodiments 1-10, wherein the weight osmotic concentration of the aqueous composition is about 270 mOsmol / kg to about 330 mOsmol / kg, more preferably about 280-320 mOsmol / kg.

[0154] 12. The aqueous composition according to embodiment 11, wherein the weight osmotic concentration of the aqueous composition is about 290 mOsmol / kg to about 310 mOsmol / kg.

[0155] 13. The aqueous composition according to embodiment 11, wherein the weight osmotic concentration of the aqueous composition is about 300 mOsmol / kg.

[0156] 14. The aqueous composition according to any one of embodiments 1-13, characterized in that the particle concentration of the extracellular vesicles is 1-100×10 9 / mL.

[0157] The aqueous composition according to any one of embodiments 1-14, characterized in that the extracellular vesicles are derived from cells selected from the group consisting of immune cells, hematopoietic stem cells, neural stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, and umbilical cord mesenchymal stem cells.

[0158] 16. The aqueous composition according to embodiment 15, wherein the cells are natural cells or genetically modified cells.

[0159] 17. The aqueous composition according to embodiment 15, wherein the immune cells are chimeric antigen receptor-modified T cells (CAR-T) or chimeric antigen receptor-modified NK cells (CAR-NK).

[0160] 18. The aqueous composition according to any one of embodiments 1-17, wherein the extracellular vesicles are characterized by having a median particle size of 30-200 nm, as measured by NTA.

[0161] 19. The aqueous composition according to embodiment 18, wherein the extracellular vesicles maintain the particle size in the temperature range of -90°C to 45°C.

[0162] 20. The aqueous composition according to any one of embodiments 1-19, wherein the extracellular vesicles maintain the concentration in the temperature range of -90°C to 45°C. For example, the concentration does not decrease by more than 30% after being placed for 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, or 6 months.

[0163] 21. The aqueous composition according to any one of embodiments 1-20, wherein the extracellular vesicles maintain the expression of surface CD63 in the temperature range of -90°C to 45°C. For example, the expression level does not decrease by more than 30% after being placed for 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, or 6 months.

[0164] 22. The aqueous composition according to any one of embodiments 1-21, wherein the extracellular vesicles maintain the content of hsa-miR-21-5p in the vesicles in the temperature range of -90°C to 45°C. For example, the content does not decrease by more than 30% after being placed for 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, or 6 months.

[0165] 23. The aqueous composition according to any one of embodiments 1-22 is a liquid.

[0166] 24. The aqueous composition according to any one of embodiments 1-22 is a solid, preferably a lyophilized powder.

[0167] 25. The aqueous composition according to any one of Embodiments 1-24, characterized in that the aqueous composition is a pharmaceutical composition, which further comprises other pharmaceutically acceptable carriers, diluents and / or excipients.

[0168] 26. Use of the aqueous composition according to any one of Embodiments 1-25 in the preparation of a drug, the drug being in dosage forms such as injections, nebulized inhalants, topical skin / mucosal medications, eye drops, nasal drops, etc.

[0169] 27. The use according to Embodiment 26, wherein the drug is used for preventing and / or treating inflammation and / or injury.

[0170] 28. The use according to Embodiment 26, wherein the drug is used for preventing and / or treating tumors.

[0171] 29. A pharmaceutical composition comprising the aqueous composition according to any one of Embodiments 1-24.

[0172] 30. A kit comprising the aqueous composition according to any one of Embodiments 1-24.

[0173] 31. A formulated preservation solution for preserving extracellular vesicles, characterized in that it comprises a buffer, a non-permeating cryoprotectant, a surfactant and an osmotic pressure regulator.

[0174] 32. The preservation solution according to Embodiment 31, characterized in that the preservation solution is composed of a buffer, a non-permeating cryoprotectant, a surfactant and an osmotic pressure regulator dissolved in water.

[0175] 33. The preservation solution according to Embodiment 31, characterized in that the pH buffer comprises citric acid and / or sodium citrate.

[0176] 34. The preservation solution according to Embodiment 31, characterized in that the non-permeating cryoprotectant comprises sugars, preferably non-reducing sugars, more preferably non-reducing disaccharides, most preferably sucrose and / or trehalose.

[0177] 35. The preservation solution according to Embodiment 31, characterized in that the surfactant comprises polysorbate-80 and / or polysorbate-20.

[0178] 36. The preservation solution according to Embodiment 31, characterized in that the osmotic pressure regulator comprises sodium chloride.

[0179] 37. The preservation solution according to Embodiment 31, which comprises the following components: sodium chloride, sucrose, polysorbate 80, and citric acid and / or sodium citrate.

[0180] 38. The preservation solution as described in embodiment 37, characterized in that the molar concentration ratio of each component is: sodium chloride: sucrose: polysorbate 80: sodium citrate = 50 - 150: 10 - 100: 0.07 - 1.53: 1 - 50, and the concentration of sodium citrate is higher than 1 mM.

[0181] 39. The preservation solution as described in embodiment 37, characterized in that the molar concentration ratio of each component is: sodium chloride: sucrose: polysorbate 80: citric acid = 50 - 150: 10 - 100: 0.07 - 1.53: 0.1 - 10, and the concentration of citric acid is higher than 0.1 mM.

[0182] 40. The preservation solution as described in embodiment 37, characterized in that the molar concentration ratio of each component is: sodium chloride: sucrose: polysorbate 80: sodium citrate: citric acid = 50 - 150: 10 - 100: 0.07 - 1.53: 1 - 50: 0.1 - 10, and the concentration of sodium citrate is higher than 1 mM.

[0183] 41. Use of the preservation solution of embodiments 31 - 40, characterized in that it is used for preparing the aqueous composition as described in embodiments 1 - 23.

[0184] 42. A method for isolating and preserving extracellular vesicles, characterized by comprising the steps of:

[0185] 1) Culturing and washing the cells;

[0186] 2) Conditionally culturing and isolating extracellular vesicles with a cell - free - extracellular - vesicle complete medium;

[0187] 3) Adding the extracellular vesicles obtained in step 2) to i) an aqueous solution containing a buffer, a non - permeable cryoprotectant, a surfactant, and an osmotic pressure regulator, or ii) the preservation solution of any one of embodiments 31 - 40 and an appropriate amount of water to obtain an aqueous composition of any one of embodiments 1 - 24;

[0188] and optionally

[0189] 4) Storing the aqueous composition at - 90°C - 45°C, for example, 37°C, 20 - 25°C, 2 - 8°C, - 25 - - 15°C, or - 90 - - 70°C.

[0190] 43. The method for isolating and preserving extracellular vesicles as described in embodiment 42, characterized in that the cells are immune cells, hematopoietic stem cells, neural stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, etc.

[0191] 44. The method as described in embodiment 42 or 43, wherein the extracellular vesicles are natural.

[0192] 45. The method according to embodiment 42 or 43, wherein the extracellular vesicles are genetically engineered and / or are produced by genetically engineered cells.

[0193] Example

[0194] The following examples provide a description to those skilled in the art on how to use, manufacture and evaluate the compositions and methods described herein, and are intended to be purely examples of the present invention and are not intended to limit the scope of the present invention.

[0195] Example 1

[0196] This example provides a method for the isolation, extraction and identification of adipose mesenchymal stem cell extracellular vesicles.

[0197] Isolation and extraction of adipose mesenchymal stem cell extracellular vesicles

[0198] Place adipose mesenchymal stem cells in a 37 °C water bath for resuscitation. After the cells are thawed, immediately add the cell suspension to 10 mL of pre-prepared complete medium, and centrifuge at 300 g for 5 min at room temperature. Discard the supernatant, resuspend the cells with complete medium, and seed the cells at 2×10 7 Cells into one HYPERFlask culture flask (Conring), and culture at 37 °C and 5% CO2 concentration.

[0199] Generally, adipose mesenchymal stem cells are cultured for 3-4 days. When the cells reach 80% confluence, discard the culture medium. Wash the cells once with 50 mL of PBS, then add cell-free (ultracentrifuged to remove extracellular vesicles) complete medium, with an addition amount of about 500 mL / bottle, and continue to culture at 37 °C and 5% CO2 concentration for 48 h.

[0200] Centrifuge the collected conditioned medium at 3000-10000 g at 4 °C, collect the supernatant, add the prepared extracellular vesicle precipitation solution in a volume ratio of 1:1, and incubate overnight at 4 °C.

[0201] The next day, centrifuge the incubated mixture at 3000 g - 120000 g at 4 °C. After centrifugation, discard the supernatant and retain the precipitate. Resuspend the precipitate with sodium chloride injection solution and aliquot.

[0202] Identification of adipose mesenchymal stem cell extracellular vesicles

[0203] Use nanoparticle tracking analysis technology (NTA) to measure the particle size and concentration of extracellular vesicles: Use NTA to detect the samples, and each sample is detected three times repeatedly. The results are shown in Table 1:

[0204] Table 1 Particle size and concentration of extracellular vesicles

[0205]

[0206] Protein marker detection by Western blot: Take the extracellular vesicles stored after aliquoting, add 1 / 5 volume of 6×SDS PAGE loading buffer, place at room temperature for 5 minutes, then heat at 95°C for 10 minutes to denature the protein. Load the sample onto a precast SDS-PAGE gel for gel electrophoresis and membrane transfer. After incubating with the antibody, add the chromogenic solution for color development. Use ImageJ to perform gray-scale analysis on the bands to evaluate the content of the corresponding markers.

[0207] Table 2 Antibodies used for protein marker detection

[0208] antibody brand article number primary antibody CD63 invitrogen 10628D secondary antibody Anti-Mouse IgG, HRP-linked Antibody CST 7076S

[0209] Fluorescent quantitative PCR (qPCR) detection: Extract RNA using the RNeasy Mini Kit, and reverse transcribe 10 ng of total RNA using the TaqMan TM Advanced miRNA cDNA Synthesis Kit. According to the experimental requirements, use the probes in Gene Expression assays as primers to detect the expression levels of related miRNAs by fluorescent quantitative PCR. Each sample is repeated 3 times. After qPCR, record the Ct value. Using the Ct value of the 0-point sample as a reference, use the 2 -△CT -ΔΔCt method to calculate the fold change of the corresponding sample compared to the 0-point, and analyze the experimental data.

[0210] Table 3 Primers and probes for fluorescent quantitative PCR

[0211]

[0212] The concentration, particle size, and characteristics of the extracellular vesicles obtained by the method for isolating and extracting extracellular vesicles of adipose mesenchymal stem cells in this example are consistent with the literature reports, that is, the obtained extracellular vesicles of adipose mesenchymal stem cells can be applied to subsequent experiments.

[0213] Example 2

[0214] This example provides a method for preparing the extracellular vesicle preservation solution described above. The method includes the following steps:

[0215] First step, prepare centrifuge tubes, beakers, 0.22 μm filters, pure water, etc. used during configuration. The configuration is carried out in a laminar flow hood, and the laminar flow hood is sterilized with ultraviolet light for 30 minutes before use.

[0216] Step 2, Preparation of 100 mM citric acid mother liquor: Weigh 2.10 g of citric acid, dissolve it with pure water and make up the volume to 100 mL. After filtering with a 0.22 μm filter, store it at 2 - 8°C.

[0217] Step 3, Preparation of 100 mM sodium citrate mother liquor: Weigh 2.94 g of sodium citrate, dissolve it with pure water and make up the volume to 100 mL. After filtering with a 0.22 μm filter, store it at 2 - 8°C.

[0218] Step 4, Preparation of 200 mM sucrose mother liquor: Weigh 3.42 g of sucrose, dissolve it with pure water and make up the volume to 50 mL. After filtering with a 0.22 μm filter, store it at 2 - 8°C.

[0219] Step 5, Preparation of 400 mM sodium chloride mother liquor: Weigh 1.17 g of sodium chloride, dissolve it with pure water and make up the volume to 50 mL. After filtering with a 0.22 μm filter, store it at 2 - 8°C.

[0220] Step 6, Prepare the extracellular vesicles prepared in Example 1 according to different preservation solution formulas and store them at -90 - -70°C, including:

[0221] Experimental Example 1: The final concentration of sucrose is 50 mM; the final concentration of polysorbate 80 is 0.2 mg / mL; the final concentration of the pH buffer sodium citrate is 20 mM; the final concentration of the isotonic regulator sodium chloride is 99 mM, and the solvent is pure water; the final concentration of extracellular vesicles is 2×10 10 / mL.

[0222] Experimental Example 2: The final concentration of sucrose is 50 mM; the final concentration of polysorbate 80 is 0.2 mg / mL; the ratio of citric acid to sodium citrate in the pH buffer is 1:15, the final concentration of citric acid is 0.5 mM, and the final concentration of sodium citrate is 7.5 mM; the final concentration of the isotonic regulator sodium chloride is 117.5 mM, and the solvent is pure water; the final concentration of extracellular vesicles is 2×10 10 / mL.

[0223] Experimental Example 3: The final concentration of sucrose is 50 mM; the final concentration of polysorbate 80 is 0.2 mg / mL; the ratio of citric acid to sodium citrate in the pH buffer is 1:15, the final concentration of citric acid is 1 mM, and the final concentration of sodium citrate is 15 mM; the final concentration of the isotonic regulator sodium chloride is 106 mM, and the solvent is pure water; the final concentration of extracellular vesicles is 2×10 10 / mL.

[0224] Experimental Example 4: The final concentration of sucrose is 50 mM; the final concentration of polysorbate 80 is 0.2 mg / mL; the ratio of citric acid and sodium citrate as pH buffer is 1:20, the final concentration of citric acid is 0.5 mM, and the final concentration of sodium citrate is 10 mM; the final concentration of sodium chloride as isotonic regulator is 113.75 mM, and the solvent is pure water; the final concentration of extracellular vesicles is 2×10 10 / mL.

[0225] Experimental Example 5: The final concentration of sucrose is 50 mM; the final concentration of polysorbate 80 is 0.2 mg / mL; the ratio of citric acid and sodium citrate as pH buffer is 1:20, the final concentration of citric acid is 1 mM, and the final concentration of sodium citrate is 20 mM; the final concentration of sodium chloride as isotonic regulator is 98.5 mM, and the solvent is pure water; the final concentration of extracellular vesicles is 2×10 10 / mL.

[0226] In the seventh step, in order to verify the effect of the extracellular vesicle preservation solution, sodium chloride injection, a commonly used solvent for preserving extracellular vesicles, was selected as the control.

[0227] Control Example: The extracellular vesicles were diluted to 2×10 10 / mL with sodium chloride injection.

[0228] In the eighth step, the pH and osmotic pressure of Experimental Examples 1-5 and Control Example 1 were measured, and the results are shown in Table 4.

[0229] Table 4 Measured values of pH and osmotic pressure of Experimental Examples 1-5 and the Control Example

[0230] number pH osmotic pressure (mOsmol / kg) Experimental Example 1 7.85 299.89 Experimental Example 2 6.14 300.81 Experimental Example 3 6.16 301.23 Experimental Example 4 6.29 300.81 Experimental Example 5 6.30 300.54 Control Example 6.08 299.08

[0231] In the ninth step, the particle size and concentration of extracellular vesicles, and the expression levels of CD63 and hsa-miR-21-5p in Experimental Examples 1-5 and the Control Example were detected and used as the 0 point.

[0232] Example 3

[0233] In this example, the preservation effects of extracellular vesicles in Experimental Example 1 and the Control Example were compared.

[0234] Storage conditions: -25--15°C, time: 3 months.

[0235] The particle size and concentration of extracellular vesicles in Experimental Example 1 and the Control Example were detected and analyzed by nanoparticle tracking analysis technology (NTA), and the results are shown in Table 5. Ratio analysis was performed with the 0 point results, as Figure 2 shown. The results indicate that the preservation effect of Experimental Example 1 is significantly higher than that of the Control Example, and the concentration of extracellular vesicles in Experimental Example 1 shows no obvious change after being stored at -25--15°C for 3 months.

[0236] Table 5 Particle size and concentration of extracellular vesicles in Experimental Example 1 and Control Example at 0 hour and after storage at -25 to -15°C for 3 months

[0237]

[0238] Therefore, the effect of storing fresh extracellular vesicles with sodium citrate under low-temperature conditions is better than that with sodium chloride injection, which can maintain the stability of extracellular vesicles and avoid changes in the concentration of extracellular vesicles.

[0239] Example 4

[0240] This example compares the effects of the extracellular vesicle preservation solutions of Experimental Examples 1-5 after storage at 2-8°C for 1 week.

[0241] The particle size and concentration of the extracellular vesicles in Experimental Examples 1-5 were detected and analyzed by nanoparticle tracking analysis technology (NTA), and the results are shown in Table 6. Ratio analysis was performed with the results at 0 hour. As Figure 3 (A) shows, the results indicate that the particle concentration ratio of Experimental Example 2 after storage at 2-8°C for 1 week is significantly higher than that of Experimental Example 1 (the slight increase in the concentration value may be due to measurement error and is actually stable), and the concentration ratios of the extracellular vesicles in Examples 2-4 are slightly higher than that of Experimental Example 1. The results of the particle size are as Figure 3 (B) shows, and there is no obvious difference among Experimental Examples 1-5.

[0242] Table 6 Particle size and concentration of extracellular vesicles in Experimental Examples 1-5 after storage at 2-8°C for 1 week

[0243]

[0244] Therefore, using a preservation solution with citric acid and sodium citrate as buffers for extracellular vesicles can improve the preservation effect under lower-temperature conditions, maintain the stability of extracellular vesicles, and avoid changes in the particle size, concentration, and activity of extracellular vesicles.

[0245] Example 5

[0246] This example compares the effects of the extracellular vesicle preservation solutions of Experimental Examples 1-5 after storage at -90 to -70°C for 6 months.

[0247] The particle size and concentration of the extracellular vesicles in Experimental Examples 1-5 were detected and analyzed by nanoparticle tracking analysis technology (NTA), and the results are shown in Table 7. Ratio analysis was performed with the results at 0 hour. As Figure 4 (A) shows, the results indicate that the particle concentration ratios of Experimental Examples 2, 3, and 5 after storage at -90 to -70°C for 6 months are significantly higher than that of Experimental Example 1. There is no obvious difference in the particle size among Experimental Examples 1-5 after storage at -90 to -70°C for 6 months, and the results are as Figure 4 (B) shows.

[0248] Table 7 Particle size and concentration of extracellular vesicles after 6 months of storage at -90 to -70°C in Experimental Examples 1-5

[0249]

[0250] The expression of the extracellular vesicle protein marker CD63 was detected by Western blot, and the gray value of CD63 in extracellular vesicles stored at -90 to -70°C for 6 months was analyzed by ratio with the 0 time point. As Figure 5 shown, the relative content of CD63 in Experimental Examples 2-5 after 6 months of storage at -90 to -70°C was higher than that in Experimental Example 1.

[0251] The expression of the extracellular vesicle nucleic acid inclusion hsa-miR-21-5p was detected by fluorescence quantitative PCR (qPCR), and the Ct value of extracellular vesicles stored at -90 to -70°C for 6 months and the Ct value at the 0 time point were analyzed according to the 2 -△CT method. As Figure 6 shown, the relative content of hsa-miR-21-5p in Experimental Example 2 after 6 months of storage at -90 to -70°C was significantly higher than that of other groups.

[0252] Therefore, when fresh extracellular vesicles are stored under low temperature conditions using a preservation solution with citric acid and sodium citrate as buffers, the preservation effect can be improved, the stability of extracellular vesicles can be maintained, changes in the concentration and particle size of extracellular vesicles can be avoided, and at the same time, the degradation of extracellular vesicle inclusions can be reduced.

[0253] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An aqueous composition comprising extracellular vesicles, and further comprising a buffer, a non-permeating cryoprotectant, a surfactant, and an osmotic pressure regulator.

2. The aqueous composition according to claim 1, characterized in that, The aqueous composition has a pH value of 4 - 9, preferably 5 - 8, more preferably 6 - 8.

3. The aqueous composition according to claim 1, characterized in that, The pH buffer comprises citric acid and / or sodium citrate, wherein the concentration of citric acid is 0 - 10 mM; and / or the concentration of sodium citrate is 1 - 50 mM.

4. An aqueous composition comprising extracellular vesicles, and further comprising the following components: sodium chloride, sucrose, polysorbate 80, and citric acid and / or sodium citrate.

5. Use of the aqueous composition according to any one of claims 1 - 4 in the preparation of a drug, the drug being in dosage forms such as an injection, an aerosol inhalant, topical skin / mucosa medication, eye drops, nasal drops, etc.

6. A pharmaceutical composition comprising the aqueous composition according to any one of claims 1 - 4.

7. A kit comprising the aqueous composition according to any one of claims 1 - 4.

8. A preservation solution formulated for preserving extracellular vesicles, characterized in that, Comprising a buffer, a non-permeating cryoprotectant, a surfactant, and an osmotic pressure regulator.

9. Use of the preservation solution according to claim 8, characterized in that, For the preparation of the aqueous composition as claimed in claims 1 - 4.

10. A method for isolating and preserving extracellular vesicles, characterized in that, Comprising the steps of: 1) Culturing and washing the cells; 2) Conditioned culturing and isolating extracellular vesicles with a complete medium without extracellular vesicles; 3) Adding the extracellular vesicles obtained in step 2) to i) an aqueous solution comprising a buffer, a non-permeating cryoprotectant, a surfactant, and an osmotic pressure regulator, or ii) the preservation solution of claim 8 and an appropriate amount of water to obtain the aqueous composition according to any one of claims 1 - 4; and optionally 4) Storing the aqueous composition at -90°C to 45°C, for example, 37°C, 20 to 25°C, 2 to 8°C, -25 to -15°C, or -90 to -70°C.

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