Extracellular vesicle preserving fluid containing histidine buffering agent and preparation

Abstract

The invention provides a preserving fluid for preserving adipose tissue-derived stem cell extracellular vesicles and a formula of a corresponding preparation, and particularly, the formula comprises sodium chloride, cane sugar, polysorbate 80, histidine and histidine hydrochloride. According to the formula, the storage stability of the extracellular vesicles secreted by adipose tissue can be improved, substances in the extracellular vesicles are prevented from being degraded, convenience is provided for clinical application of the extracellular vesicles, and good market prospects are achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an extracellular vesicle preservation solution and preparation using histidine and histidine hydrochloride as buffers, which can maintain the stability of extracellular vesicle properties under low-temperature storage conditions. Background Art

[0002] Extracellular vesicles are phospholipid bilayer membrane structures secreted by tissues with diameters ranging from 30 to 200 nm, which act as vesicles for cell communication. Different from larger apoptotic bodies, etc., extracellular vesicles are structures actively secreted by cells through various pathways and contain proteins, DNA, RNA (including mRNA, miRNA, and ncRNA, etc.). These substances are received by recipient cells and regulate the signal pathways of recipient cells. CD63, CD81, CD9, etc. are membrane proteins specifically present on extracellular vesicles. At the same time, extracellular vesicles also contain intracellular proteins such as hSP70, TSG101, and ALIX. miRNA is an important and effective substance in extracellular vesicles. Extracellular vesicles of adipose mesenchymal stem cells are rich in hsa-miR-21-5p, hsa-miR-146a-5p, hsa-miR-24-3p, has-let-7a-5p, etc. These microRNAs can inhibit the inflammatory response by inhibiting the NF-κB pathway and play an anti-inflammatory role.

[0003] Isolated and purified 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 isolated and purified extracellular vesicles 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 RNA will also degrade. The stability of extracellular vesicles can be improved by adding cryoprotectants and providing a buffer system. At the same time, attention needs to be paid to maintaining the osmotic pressure of the solution.

[0004] Histidine and histidine hydrochloride are widely used in the formulation of protein drugs and play a role in regulating the pH of the preparation. The specific chemical formula is as Figure 1 shown. L-histidine, as an essential amino acid in the human body, the nitrogen atom in its imidazole ring can buffer the pH of the solution by binding or releasing protons, and at the same time can chelate with metal ions to play an antioxidant role. L-histidine hydrochloride has good biocompatibility, has a certain protective effect on proteins, and can protect protein products from oxidative degradation during long-term storage as an antioxidant.

[0005] Sucrose is a disaccharide structure in which glucose is linked to fructose. It binds to proteins by replacing the hydroxyl groups of water on the protein surface with its own hydroxyl groups, thereby stabilizing the proteins. It is a non-permeating sugar-based cryoprotectant and is widely used in various antibody formulations. It is a cost-effective cryoprotectant. Polysorbate 80 is a non-ionic surfactant and emulsifier. It is an amber oily liquid, soluble in water, and is often used as an emulsifier in foods. It is also frequently used in antibody and liposome formulations to reduce surface tension, reduce aggregation, and protect proteins and liposomes from surface denaturation.

[0006] The object of the present invention is to solve the storage problem of extracellular vesicle products. Summary of the Invention

[0007] The present invention provides a preservation solution formulation for improving the stability of extracellular vesicles during cryopreservation.

[0008] 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 5 - 8, preferably 5.5 - 7.7, more preferably 6 - 7, and most preferably 6.3 - 6.4.

[0009] In a preferred embodiment, the buffer comprises a phosphate buffer system, a Tris (tris(hydroxymethyl)aminomethane) buffer system, an organic acid buffer system, and / or an amino acid buffer system. In a preferred embodiment, the buffer comprises an amino acid buffer system. In a preferred embodiment, the buffer comprises a histidine buffer system, such as histidine and / or histidine hydrochloride, preferably L-histidine and / or L-histidine monohydrochloride monohydrate. In some specific embodiments, the buffer comprises histidine and / or histidine monohydrochloride, wherein the histidine concentration is 1 - 30 mM, and / or the histidine hydrochloride concentration is 1 - 10 mM.

[0010] In a preferred embodiment, the non-permeating cryoprotectant comprises a sugar, preferably a non-reducing sugar, more preferably a 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.

[0011] 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.

[0012] 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.

[0013] 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, histidine and / or histidine monohydrochloride. In some embodiments, the aqueous composition consists of extracellular vesicles, sodium chloride, sucrose, polysorbate 80, histidine and / or histidine monohydrochloride and water. In a more specific embodiment, the sucrose concentration is 10-100 mM; the polysorbate-80 concentration is 0.1-2 mg / mL; the sodium chloride concentration is 50-150 mM; the histidine concentration is 1-30 mM, and the histidine hydrochloride concentration is 1-10 mM.

[0014] More preferably, the sodium chloride concentration is 110-120 mM; the histidine concentration is 1-20 mM; the histidine hydrochloride concentration is 2-10 mM.

[0015] Most preferably, the sodium chloride concentration is 115-120 mM; the histidine concentration is 10-20 mM; the histidine hydrochloride concentration is 5-10 mM.

[0016] 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; the buffer, histidine and histidine hydrochloride, are in a ratio of 1:1.3, histidine at a final concentration of 3 - 5 mM, preferably 3.5 - 4.5 mM, more preferably 3.8 - 4.2 mM, most preferably 4 mM, and histidine hydrochloride at a final concentration of 3.9 - 6.5 mM, preferably 4.5 - 5.9 mM, more preferably 4.9 - 5.5 mM, most preferably 5.2 mM; sodium chloride, at a final concentration of 121.0 - 122.0 mM, preferably 121.5 mM or 121.8 mM, and the above components are dissolved / resuspended in pure water.

[0017] 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; histidine, at a final concentration of 19 - 21 mM, preferably 19.5 - 20.5 mM, more preferably 19.8 - 20.2 mM, most preferably 20 mM; sodium chloride, at a final concentration of 118 - 120 mM, preferably 119 mM, and the above components are dissolved / resuspended in pure water.

[0018] 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; the buffer, histidine and histidine - hydrochloride, are in a ratio of 2:1, histidine at a final concentration of 12 - 14 mM, preferably 12.5 - 13.5 mM, more preferably 12.8 - 13.2 mM, most preferably 13 mM, and histidine hydrochloride at a final concentration of 6.0 - 7.0 mM, preferably 6.2 - 6.8 mM, more preferably 6.4 - 6.6 mM, most preferably 6.5 mM; sodium chloride, at a final concentration of 115 - 117 mM, preferably 116 mM, and the above components are dissolved / resuspended in pure water.

[0019] In one embodiment of the present invention, the aqueous composition comprises extracellular vesicles and further comprises or essentially consists of the following components: sucrose, with a final concentration of 40 - 60 mM, preferably 45 - 55 mM, more preferably 48 - 52 mM, and 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, and most preferably 0.2 mg / mL; the buffer agent is a mixture of histidine and histidine hydrochloride in a ratio of 10:1, with a final concentration of histidine being 19 - 21 mM, preferably 19.5 - 20.5 mM, more preferably 19.8 - 20.2 mM, and most preferably 20 mM, and a final concentration of histidine hydrochloride being 1.9 - 2.1 mM, preferably 2 mM; sodium chloride, with a final concentration of 116 - 118 mM, preferably 117 mM. The above components are dissolved / suspended in pure water.

[0020] 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, and more preferably about 300 mOsmol / kg.

[0021] 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.

[0022] 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, such as 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).

[0023] In some specific embodiments of the present invention, the extracellular vesicles are characterized by having a particle size of 30-200 nm, as measured by NTA. In some more specific embodiments, the extracellular vesicles maintain said 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).

[0024] 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%.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] In a preferred embodiment, the buffer comprises a phosphate buffer system, a Tris (tris(hydroxymethyl)aminomethane) buffer system, an organic acid buffer system, and / or an amino acid buffer system. In a preferred embodiment, the buffer comprises an amino acid buffer system. In a preferred embodiment, the buffer comprises a histidine buffer system, such as histidine and / or histidine hydrochloride, preferably, L-histidine and / or L-histidine monohydrochloride monohydrate. In some specific embodiments, the buffer comprises histidine and / or histidine monohydrochloride.

[0030] In a preferred embodiment, the non-permeating cryoprotectant comprises sugars, preferably non-reducing sugars, more preferably non-reducing disaccharides. 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.

[0031] 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.

[0032] In a preferred embodiment, the osmotic pressure regulator comprises sodium chloride.

[0033] In one embodiment of the present invention, there is provided a preservation solution for cryopreserving extracellular vesicles, comprising the following components: sodium chloride, sucrose, polysorbate 80, histidine and / or histidine hydrochloride. In a more specific embodiment, the molar concentration ratio of each component is: sodium chloride: sucrose: polysorbate 80: histidine: histidine hydrochloride = 50-150:10-100:0.07-1.53:1-30:1-10, and the molar concentration of histidine is higher than 1 mM. More preferably, the molar concentration ratio of the sodium chloride is 110-120; the molar concentration ratio of the histidine is 1-20; the molar concentration ratio of the histidine hydrochloride is 2-10. Most preferably, the molar concentration ratio of the sodium chloride is 115-120; the molar concentration ratio of the histidine is 10-20; the molar concentration ratio of the histidine hydrochloride is 5-10.

[0034] Another aspect of the present invention provides a method for preparing the preservation solution, which is prepared by dissolving the buffer, non-permeating cryoprotectant, surfactant and osmotic pressure regulator separately / collectively / sequentially in water.

[0035] In some embodiments, the preservation solution is prepared by separately dissolving the buffer, non-permeating cryoprotectant, surfactant and osmotic pressure regulator in water, and mixing the solutions evenly.

[0036] In some embodiments, the preservation solution is prepared by collectively / dissolving the buffer, non-permeating cryoprotectant, surfactant and osmotic pressure regulator in water in a certain order, and mixing evenly.

[0037] In a specific embodiment, the preservation solution is prepared according to the following steps:

[0038] In the first step, prepare the equipment and pure water used during preparation, place them in a laminar flow hood, and sterilize before use.

[0039] In the second step, prepare the 100 mM histidine mother liquor: Weigh 1.55 g of histidine, 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.

[0040] In the third step, prepare the 100 mM histidine hydrochloride mother liquor: Weigh 2.10 g of histidine hydrochloride, 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.

[0041] In the fourth step, prepare the 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.

[0042] In the fifth step, prepare the 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.

[0043] In the sixth step, calculate the amount of each mother liquor and pure water required according to the component ratio and the concentration multiple of the preservation solution to be prepared (for example, 2×, 5× or 10×), accurately measure and mix them evenly for preparation. Optionally, store it at -90 - -70°C afterwards.

[0044] 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.

[0045] Another aspect of the present invention provides a method for preparing the aqueous composition of the present invention, comprising the following steps:

[0046] Uniformly mix the extracellular vesicle stock solution and the preservation solution of the present invention.

[0047] In some embodiments, the volume ratio of mixing 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 for mixing is 1:10.

[0048] More preferably, optionally, the extracellular vesicle stock solution is prepared by resuspending the obtained extracellular vesicles with sodium chloride injection (e.g., medicinal 0.9% (w / v) sodium chloride solution), compound electrolyte injection (e.g., Plasmalyte A (medicinal 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, 0.30‰ (w / v) magnesium chloride)), and / or PBS and other isotonic solutions.

[0049] More preferably, the particle concentration of the extracellular vesicle stock solution is 1 - 100×10 10 / mL.

[0050] Most preferably, the extracellular vesicle stock solution is resuspended with medicinal 0.9% (w / v) sodium chloride solution.

[0051] In an exemplary embodiment, the particle concentration of the extracellular vesicle stock solution is 1 - 10×10 10 / mL.

[0052] 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 comprises the following steps:

[0053] a) Culturing adipose mesenchymal stem cells;

[0054] b) Adding cell-free extracellular vesicle culture medium for culturing;

[0055] c) Separating and purifying extracellular vesicles from the culture medium.

[0056] In an exemplary embodiment, the specific steps of the cell culture are as follows:

[0057] 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%, with the culture time being 2 - 3 days;

[0058] 2) Removing the upper layer of the culture medium and washing the cells with PBS;

[0059] 3) Adding cell-free extracellular vesicle culture medium again and culturing in a 37°C 5% CO2 incubator;

[0060] 4) Collecting the conditioned medium after 48 hours.

[0061] Preferably, the cell-free extracellular vesicle culture medium is prepared by ultracentrifugation of a complete medium.

[0062] Preferably, the step of separating and purifying extracellular vesicles is as follows:

[0063] 1) Take the conditioned medium, perform centrifugation at 3000 - 10000 g to remove cell debris and apoptotic bodies;

[0064] 2) Add an equal volume of extracellular vesicle isolation reagent, let it stand, and then centrifuge to obtain a precipitate containing extracellular vesicles;

[0065] 3) Add PBS, perform ultracentrifugation at 3000 - 120000 g, and resuspend with sodium chloride injection solution to obtain the separated extracellular vesicle stock solution.

[0066] 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 of extracellular vesicles after cryopreservation, such as concentration changes, particle size changes, surface / intracellular marker (e.g., protein marker, miRNA) changes.

[0067] 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.

[0068] Preferably, the degradation of extracellular vesicles includes a decrease in particle concentration, degradation of protein markers, and degradation of miRNA in extracellular vesicles.

[0069] Preferably, the particle concentration is detected by nano particle tracking analysis (NTA).

[0070] Preferably, the degradation of the protein marker is detected by Western method, and CD63 is selected as the protein marker.

[0071] Preferably, the degradation of miRNA is detected by qPCR method, and hsa-miR-21-5p and / or hsa-miR-146a-5p are selected as miRNA in extracellular vesicles.

[0072] The present invention provides an aqueous composition suitable for preserving extracellular vesicles, which can ensure that extracellular vesicles do not degrade severely after long-term preservation (e.g., cryopreservation (e.g., -80 - -20 °C) preservation) and can meet clinical applications. It contains sucrose, polysorbate 80, sodium chloride, histidine and histidine hydrochloride. Among them, sucrose and polysorbate 80 are used as cryoprotectants, histidine and histidine hydrochloride are used as pH buffers, and sodium chloride is added to maintain the osmotic pressure of the solution, so as to stabilize extracellular vesicles under preservation (e.g., cryopreservation) conditions and extend the product shelf life.

[0073] 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 - -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:

[0074] (i) As measured by nanoparticle tracking analysis technology (NTA), the concentration of extracellular vesicle particles in the dissolved state shows no significant change compared to the concentration immediately after formulation, 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%;

[0075] (ii) As detected by Western blot for the expression of extracellular vesicle protein markers (e.g., CD63), there is no significant change compared to immediately after formulation, for example, the change in the expression of the protein marker (e.g., 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%;

[0076] (iii) As detected by ELISA / Western blot for the expression of extracellular vesicle protein inclusions, there is no significant change compared to immediately after formulation, for example, the change in the expression of the 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%;

[0077] (iv) As detected by fluorescence quantitative PCR (qPCR) for the expression of extracellular vesicle nucleic acid inclusions (e.g., hsa-miR-21-5p and / or hsa-miR-146a-5p), there is no significant change compared to immediately after formulation, for example, the change in the content of the nucleic acid inclusions (e.g., hsa-miR-21-5p and / or hsa-miR-146a-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%.

[0078] In a preferred embodiment of the present invention, the storage solution 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 storage at low temperature (e.g., ultra-low temperature), and the particles are not easily aggregated and / or degraded during storage.

[0079] In a preferred embodiment of the present invention, when formulating the aqueous composition of the present invention, all components meet the standards for human medication, and at this time, the aqueous composition of the present invention has the safety suitable for direct administration.

[0080] In one aspect, the present invention provides a delivery device comprising the storage liquid 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 storage liquid preparation of the present invention, for example, for intravenous, subcutaneous, intradermal or intramuscular injection, intravenous infusion.

[0081] 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 storage liquid preparation of the present invention, and the delivery is implemented, for example, by using a delivery device of a pre-filled syringe. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 It is a schematic diagram of the chemical formulas of histidine and histidine hydrochloride. The molecular formula of L-histidine is: C6H9N3O2, and the molecular weight is 155.15; the molecular formula of L-histidine monohydrochloride monohydrate is: C6H9N3O2·HCl·H2O, and the molecular weight is 209.63.

[0083] Figure 2 It is a graph showing the changes of extracellular vesicles after being stored at 2-8 °C for 2 weeks in Experimental Example 1 and Control Example 1.

[0084] Among them, (A) is the ratio change of particle concentration compared to the 0 point; (B) is the ratio change of median particle size compared to the 0 point.

[0085] Figure 3 It is a graph showing the changes of extracellular vesicles after being stored at -25 - -15 °C for 2 months in Experimental Examples 1-4. Among them, (A) is the ratio change of particle concentration compared to the 0 point; (B) is the ratio change of median particle size compared to the 0 point.

[0086] Figure 4 It is a graph showing the changes of extracellular vesicle microRNA after being stored at -25 - -15 °C for 2 months in Experimental Examples 1-4. Among them, (A) is the 2 of hsa-miR-21-5p -△CT ; (B) is the 2 of hsa-miR-146a-5p -△CT .

[0087] Figure 5 It is a graph showing the relative content change of extracellular vesicle CD63 after being stored at -25 - -15 °C for 2 months in Experimental Examples 1-4. Among them, (A) is the representative band of western detection of CD63; (B) is the ratio of the gray value of CD63 stored at -25 - -15 °C for 6 months to the 0 point.

[0088] Figure 6 Graph showing the changes in extracellular vesicles after storage of Experimental Examples 1-4 at -90 to -70 °C for 6 months. Among them, (A) shows the change in the ratio of particle concentration compared to the 0 point; (B) shows the change in the ratio of median particle size compared to the 0 point.

[0089] Figure 7 Graph showing the changes in extracellular vesicle microRNA after storage of Experimental Examples 1-4 at -90 to -70 °C for 6 months. Among them, (A) shows 2 of hsa-miR-21-5p -△CT ; (B) shows 2 of hsa-miR-146a-5p -△CT .

[0090] Figure 8 Graph showing the relative content change of extracellular vesicle CD63 after storage of Experimental Examples 1-4 at -90 to -70 °C for 6 months. Among them, (A) shows the representative band of CD63 detected by western blot; (B) shows the ratio of the gray value of CD63 stored at -90 to -70 °C for 6 months to the 0 point. Detailed implementation manner

[0091] 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 pertains. 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..

[0092] 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.

[0093] 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. Alternatively, it is directly equivalently expressed as, for example, mg / mL.

[0094] A "separated" substance has been altered by human effort from its natural state. If a "separated" component or substance occurs in nature, then it has been altered or removed from its original environment, or both. For example, the extracellular vesicles mentioned in the present invention are substantially separated when present in a suspension or storage solution, unless otherwise specified.

[0095] 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, which, when combined with the active ingredient and / or one or more additional inactive ingredients, directly or indirectly makes the formulation more suitable for therapeutic administration to a human or non-human animal.

[0096] The extracellular vesicle formulation of the present invention can be an aqueous composition, such as an aqueous solution or suspension, such as a solid, such as a lyophilized product.

[0097] 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 (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 quite significantly, and even between EVs from the same source, the size distribution may be relatively dispersed, but 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 presenting as 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, CD9 on the surface, and contain cytokines, growth factors, signaling lipids, mRNA, microRNA, and siRNA substances derived from the parent cell. Additionally, the term should also be understood to refer to extracellular vesicle mimics, 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 、1011 、10 12 、10 13 、10 14 、10 15 、10 16 、10 17 EV particles per unit volume (e.g., mL). In some embodiments of the present invention, the EVs in the formulation can be multiple. 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 delivery vectors 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 storm.

[0098] 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 carrying abundant 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 various activities of target cells through different pathways. MSC exosomes are involved in physiological and pathological processes such as body 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 data, 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., and these factors 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.

[0099] 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 exert activity, 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.

[0100] "Particle size (Diameter)" and "Median Diameter" refer to the intensity volume diameter corresponding to the extracellular vesicle particle preparation / storage solution, which can be measured by conventional measurement methods in the art.

[0101] In another preferred embodiment of the present invention, the extracellular vesicles comprise a lipid bilayer membrane structure.

[0102] In another preferred embodiment of the present invention, the median particle size of the vesicles is between 30 - 200 nm.

[0103] 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.

[0104] In another preferred embodiment of the present invention, the soluble active cytokines are soluble active cytokines produced by the somatic cells of the human body.

[0105] In another preferred embodiment of the present invention, the soluble active cytokines include active cytokines produced by paracrine of somatic cells of the human body cultured in a natural state, active cytokines expressed and produced by somatic cells of the human body modified with foreign genes, or a combination thereof.

[0106] 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.

[0107] In another preferred embodiment of the present invention, the median particle size of the extracellular vesicles of the cell membrane is 30 - 200 nm.

[0108] 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:

[0109] (a) Cells obtained by directly isolating and purifying from human tissues;

[0110] (b) Cells obtained by directly separating and purifying from human tissues and then minimally manipulating them in a GMP laboratory to proliferate these cells;

[0111] (c) Cells obtained by specific gene modification, specific gene editing, specific gene transduction, or introduction of specific microRNA (miRNA) in a GMP laboratory;

[0112] (d) Cells obtained by pre - treating under special culture conditions in a GMP laboratory.

[0113] In another preferred embodiment of the present invention, the cells for producing the extracellular vesicles of the cell membrane include primary cells and passage cells with 1 - 10 passages.

[0114] In another preferred embodiment of the present invention, the cells for producing the extracellular vesicles of the cell membrane include cells without genetic manipulation and cells with genetic manipulation.

[0115] 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.

[0116] In another preferred embodiment of the present invention, the cells for producing the extracellular vesicles of the cell membrane are pre - treated cells.

[0117] 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.

[0118] 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 passed ethical review and filing, signed an informed consent form, and meet the criteria for establishing a cell bank and enrolling in the adipose stem (progenitor) cell product after strict laboratory examinations, adipose tissue is obtained by abdominal wall skin puncture liposuction or abdominal surgery, placed in a cell preservation solution for storage, transported at low temperature to a qualified GMP laboratory, and after separation, purification, and proliferation treatments, the obtained adipose mesenchymal progenitor cells, namely the working cell bank cells (intermediate products).

[0119] 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%, and the percentage of cells positive for CD34 / CD45 and HLA-DR being less than 2%. They also meet the detection standards for antibiotic residues and serum and serum substitute residues, and are passage P1-P6 adipose mesenchymal stem (progenitor) cells.

[0120] In another preferred embodiment of the present invention, the adipose mesenchymal stem (progenitor) cells are passage P3-P4 adipose mesenchymal stem (progenitor) cells.

[0121] 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).

[0122] 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).

[0123] Components of the extracellular vesicle preparation

[0124] The extracellular vesicle preparation of the present invention may contain various components, such as one or more acids, salts, and / or sugars.

[0125] In some preferred embodiments of the present invention, the extracellular vesicle preparation does not require the addition of protein components to enhance stability.

[0126] The extracellular vesicle preparation described herein has 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 a pH value selected from the pH range of 5.5 to about 7.7, such as a pH value selected from the pH range of 5.5 - 5.6, a pH value selected from the pH range of 5.6 - 5.7, a pH value selected from the pH range of 5.7 - 5.8, a pH value selected from the pH range of 5.8 - 5.9, a pH value selected from the pH range of 5.9 - 6.0, a pH value selected from the pH range of 6.0 - 6.1, a pH value selected from the pH range of 6.1 - 6.2, a pH value selected from the pH range of 6.2 - 6.3, a pH value selected from the pH range of 6.3 - 6.4, a pH value selected from the pH range of 6.4 - 6.5, a pH value selected from the pH range of 6.5 - 6.6, a pH value selected from the pH range of 6.6 - 6.7, a pH value selected from the pH range of 6.7 - 6.8, a pH value selected from the pH range of 6.8 - 6.9, a pH value selected from the pH range of 6.9 - 7.0, a pH value selected from the pH range of 7.0 - 7.1, a pH value selected from the pH range of 7.1 - 7.2, a pH value selected from the pH range of 7.2 - 7.3, a pH value selected from the pH range of 7.3 - 7.4, a pH value selected from the pH range of 7.4 - 7.5, a pH value selected from the pH range of 7.5 - 7.6, a pH value selected from the pH range of 7.6 - 7.7, or a combined range selected from one or more of the foregoing ranges. For example, the pH value is 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 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 between 6.0 - 6.5. In some embodiments, the pH of the extracellular vesicle preparation is between 6.3 - 6.4. In some embodiments, the pH of the extracellular vesicle preparation is 6.34. In some embodiments, the pH of the extracellular vesicle preparation is between 7.3 - 7.8. In some embodiments, the pH of the extracellular vesicle preparation is 7.62.

[0127] 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, a "histidine buffer / solution / system" refers to a mixture containing histidine and its histidine hydrochloride. The ionizable functional groups of histidine, the carboxyl group and the amino group, enable it to undergo corresponding ionization reactions according to the pH change of the solution, so that when a small amount of acid or base is added to the solution, the histidine buffer will counteract the sudden change in pH value.

[0128] In an embodiment of the present invention, the buffer can be selected from phosphate buffer systems, Tris (tris(hydroxymethyl)aminomethane) buffer systems, organic acid buffer systems, and amino acid buffer systems. And, naturally, various buffers can be used alone or in combination. In a preferred embodiment, the buffer is selected from histidine and histidine hydrochloride, and their usual hydrates, etc. The appropriate concentration of these buffers varies, but generally the optimal range is 1-100 mM, although lower and higher concentrations and various sub-ranges may be useful.

[0129] The preservation solution according to the present invention further comprises a cryoprotectant comprising 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 can 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 can be in the range of 1-200 mM, preferably 1-100 mM.

[0130] 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 can be ionic or non-ionic. Exemplary non-ionic surfactants that can 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 can 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; poloxamer, 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.

[0131] The amount of surfactant contained in the formulation 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. For example, the formulation 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.

[0132] 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 2 weeks, preferably at least 1 month, more preferably at least 2 months, and most preferably at least 3 months. The stability is manifested, for example, as the ratio of the concentration of extracellular vesicle particles in the dissolved state to the concentration at the time of just formulation exceeding 70%, exceeding 80%, exceeding 85%, or exceeding 90%, 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 formulation, 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 reduction of vesicle surface markers (e.g., CD63) in quantitative / semi-quantitative detection being less than 30%, less than 20%, less than 15%, or less than 10% compared to that at the time of just formulation, 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 reduction of vesicle content substances (e.g., miRNA substances, such as hsa-miR-21-5p and / or hsa-miR-146a-5p) in quantitative / semi-quantitative detection being less than 30%, less than 20%, less than 15%, or less than 10% compared to that at the time of just formulation, preferably less than 5%, more preferably less than 3%, more preferably less than 2%, and more preferably less than 1%.

[0133] 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--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 the dissolved state compared to the concentration at the time of just formulation exceeds 80%, exceeds 85%, or exceeds 90%, preferably exceeds 95%, and more preferably exceeds 98%.

[0134] 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.

[0135] Use of the formulation

[0136] 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 the preparation of a drug. The drug is particularly suitable in the form of dosage forms such as injections, nebulized inhalants, topical skin / mucosal medications, eye drops, nasal drops, etc.

[0137] 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.

[0138] 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.

[0139] In some embodiments, the disease is a tumor.

[0140] Through extensive and in-depth research and a large number of screenings, the inventors have developed for the first time a cryopreservation solution for the preservation of extracellular vesicles under freezing conditions. Specifically, the inventors have screened out a cryopreservation solution formulation that can effectively protect the properties of extracellular vesicles from changing under freezing conditions. Experiments show that the extracellular vesicles preserved in the cryopreservation solution can maintain their properties basically unchanged after 6 months. The present invention has been completed on this basis.

[0141] The present invention relates to a method for stably storing extracellular vesicles, comprising the steps of: introducing isolated extracellular vesicles into a preservation solution described herein and storing at a suitable temperature. Suitable temperatures for storage include sub-zero temperatures, although temperatures above 0 °C are also contemplated, and it works well for short- 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), even more preferably below -50 °C (preferably about -70 °C to -90 °C, such as -80 °C).

[0142] Exemplary embodiments

[0143] This application may include the following exemplary embodiments:

[0144] 1. An aqueous composition comprising extracellular vesicles, further comprising a buffer, a non-permeating cryoprotectant, a surfactant, and an osmotic pressure regulator.

[0145] 2. The aqueous composition according to embodiment 1, wherein the aqueous composition has a pH value of about 5 - 8, preferably 5.5 - 7.7, more preferably 6 - 7, and most preferably 6.3 - 6.4.

[0146] 3. The aqueous composition according to embodiment 1, wherein the pH buffer comprises histidine and / or histidine hydrochloride, wherein the histidine concentration is 1 - 30 mM, and / or the histidine hydrochloride concentration is 1 - 10 mM.

[0147] 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, most preferably sucrose and / or trehalose, wherein the concentration of the sugar is 10 - 100 mM.

[0148] 5. The aqueous composition according to embodiment 1, wherein the surfactant comprises polysorbate-80 and / or polysorbate-20, wherein the surfactant concentration is 0.1 - 2 mg / mL.

[0149] 6. The aqueous composition according to embodiment 1, wherein the osmotic pressure regulator comprises sodium chloride, and its concentration is 50 - 150 mM.

[0150] 7. An aqueous composition comprising extracellular vesicles, further comprising the following components: sodium chloride, sucrose, polysorbate 80, and histidine and / or histidine hydrochloride.

[0151] 8. The aqueous composition according to embodiment 7, characterized in that the sodium chloride concentration is 50-150 mM; the sucrose concentration is 10-100 mM; the polysorbate 80 concentration is 0.1-2 mg / mL; the histidine concentration is 1-30 mM.

[0152] 9. The aqueous composition according to embodiment 7, characterized in that the sodium chloride concentration is 50-150 mM; the sucrose concentration is 10-100 mM; the polysorbate 80 concentration is 0.1-2 mg / mL; the histidine hydrochloride concentration is 1-10 mM.

[0153] 10. The aqueous composition according to embodiment 7, characterized in that the sodium chloride concentration is 50-150 mM; the sucrose concentration is 10-100 mM; the polysorbate 80 concentration is 0.1-2 mg / mL; the histidine concentration is 1-30 mM; the histidine hydrochloride concentration is 1-10 mM.

[0154] 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.

[0155] 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.

[0156] 13. The aqueous composition according to embodiment 11, wherein the weight osmotic concentration of the aqueous composition is about 300 mOsmol / kg.

[0157] 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.

[0158] 15. 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.

[0159] 16. The aqueous composition according to embodiment 15, wherein the cells are natural cells or genetically modified cells.

[0160] 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).

[0161] 2. 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.

[0162] 3. 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.

[0163] 4. 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.

[0164] 5. 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 40% after being placed for 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, or 6 months.

[0165] 6. The aqueous composition according to any one of embodiments 1-21, wherein the extracellular vesicles maintain the content of hsa-miR-21-5p and / or hsa-miR-146a-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.

[0166] 7. The aqueous composition according to any one of embodiments 1-22, which is a liquid.

[0167] 8. The aqueous composition according to any one of embodiments 1-22, which is a solid, preferably a freeze-dried powder.

[0168] 9. 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.

[0169] 10. 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 injection, aerosol inhalation, topical skin / mucosal application, eye drops, nasal drops, etc.

[0170] 11. The use according to embodiment 26, wherein the drug is used for preventing and / or treating inflammation or injury.

[0171] 12. The use according to embodiment 26, wherein the drug is used for preventing and / or treating tumors.

[0172] 29. A pharmaceutical composition comprising the aqueous composition of any one of embodiments 1-24.

[0173] 30. A kit comprising the aqueous composition of any one of embodiments 1-24.

[0174] 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.

[0175] 32. The preservation solution according to embodiment 31, characterized in that the preservation solution consists of a buffer, a non-permeating cryoprotectant, a surfactant and an osmotic pressure regulator dissolved in water.

[0176] 33. The preservation solution according to embodiment 31, characterized in that the pH buffer comprises histidine and / or histidine hydrochloride.

[0177] 34. The preservation solution according to embodiment 31, characterized in that 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.

[0178] 35. The preservation solution according to embodiment 31, characterized in that the surfactant comprises polysorbate-80 and / or polysorbate-20.

[0179] 36. The preservation solution according to embodiment 31, characterized in that the osmotic pressure regulator comprises sodium chloride.

[0180] 37. The preservation solution according to embodiment 31, comprising the following components: sodium chloride, sucrose, polysorbate 80, and histidine and / or histidine hydrochloride.

[0181] 38. The preservation solution according to embodiment 37, characterized in that the molar concentration ratio of each component is: sodium chloride: sucrose: polysorbate 80: histidine = 50-150:10-100:0.07-1.53:1-30, and the histidine concentration is higher than 1 mM.

[0182] 39. The preservation solution according to embodiment 37, characterized in that the molar concentration ratio of each component is: sodium chloride: sucrose: polysorbate 80: histidine hydrochloride = 50-150:10-100:0.07-1.53:1-10, and the histidine hydrochloride concentration is higher than 0.1 mM.

[0183] 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: histidine: histidine hydrochloride = 50 - 150: 10 - 100: 0.07 - 1.53: 1 - 30: 1 - 10, and the histidine concentration is higher than 1 mM.

[0184] 41. Use of the preservation solution according to Embodiments 31 - 40, characterized in that it is used for preparing an aqueous composition as described in Embodiments 1 - 23.

[0185] 42. A method for separating and preserving extracellular vesicles, characterized by comprising the steps of:

[0186] 1) Culturing and washing the cells;

[0187] 2) Performing conditioned culture with a cell - free extracellular vesicle - containing complete medium and separating the extracellular vesicles;

[0188] 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 according to any one of Embodiments 31 - 40 and an appropriate amount of water to obtain an aqueous composition according to any one of Embodiments 1 - 24;

[0189] And optionally

[0190] 4) Storing the aqueous composition at - 90 - 45 °C, for example, 37 °C, 20 - 25 °C, 2 - 8 °C, - 25 - - 15 °C, or - 90 - - 70 °C.

[0191] 43. The method for separating 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.

[0192] 44. The method according to Embodiment 42 or 43, wherein the extracellular vesicles are natural.

[0193] 45. The method according to Embodiment 42 or 43, wherein the extracellular vesicles are genetically engineered and / or are produced by genetically engineered cells.

[0194] Examples

[0195] Through the following examples, the present disclosure provides to those skilled in the art a description of how to use, manufacture, and evaluate the compositions and methods described herein, and is intended solely as an example of the present invention and not intended to limit the scope of the present invention.

[0196] Example 1

[0197] This embodiment provides a method for the isolation, extraction and identification of adipose mesenchymal stem cell extracellular vesicles.

[0198] Isolation and extraction of adipose mesenchymal stem cell extracellular vesicles

[0199] Place the 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 inoculate the cells into a HYPERFlask culture flask at a density of 2×10 7 and culture at 37 °C under 5% CO2 concentration.

[0200] Generally, the adipose mesenchymal stem cells are cultured for 3 - 4 days until the cell confluence reaches 80%, and then the culture medium is discarded. Wash the cells once with 50 mL of PBS, and then add cell-free extracellular vesicle (ultracentrifugation to remove extracellular vesicles) complete medium at a dosage of 500 mL / bottle, and continue to culture at 37 °C under 5% CO2 concentration for 48 h.

[0201] 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.

[0202] The next day, centrifuge the incubated mixture at 3000 - 120000 g at 4 °C. After centrifugation, discard the supernatant and retain the precipitate. Resuspend the precipitate with sodium chloride injection and aliquot.

[0203] Identification of adipose mesenchymal stem cell extracellular vesicles

[0204] Use nanoparticle tracking analysis technology (NTA) to measure the particle size and concentration of extracellular vesicles: Detect the samples using NTA, and repeat the detection three times for each sample. The results are shown in Table 1:

[0205] Table 1 Particle size and concentration of extracellular vesicles

[0206]

[0207] Western blot to detect protein markers: Take the aliquoted and stored extracellular vesicles, add 1 / 5 volume of 6×SDS-PAGE loading buffer, let it stand at room temperature for 5 minutes, then heat at 95 °C for 10 minutes to denature the protein, load the sample onto the SDS-PAGE precast gel for gel electrophoresis and membrane transfer, incubate with the antibody, add the chromogenic solution for color development, and use ImageJ to perform gray-scale analysis on the bands to evaluate the content of the corresponding markers.

[0208] Table 2 Antibodies Used for Protein Biomarker Detection

[0209] antibody brand article number primary antibody CD63 invitrogen 10628D secondary antibody Anti-Mouse IgG, HRP-linked Antibody CST 7076S

[0210] Fluorescence 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 GeneExpression assays as primers to detect the expression levels of relevant miRNAs by fluorescence 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 method to calculate the fold change of the corresponding sample compared to the 0-point, and analyze the experimental data.

[0211] Table 3 Fluorescence Quantitative PCR Primers and Probes

[0212]

[0213] The concentration, particle size, and characteristics of the extracellular vesicles obtained by the method for isolating and extracting adipose mesenchymal stem cell extracellular vesicles in this example are consistent with the literature reports, that is, the obtained adipose mesenchymal stem cell extracellular vesicles can be applied to subsequent experiments.

[0214] Example Two

[0215] This example provides a method for preparing the extracellular vesicle preservation solution described above. The method includes the following steps:

[0216] 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 min before use.

[0217] Second step, preparation of 100 mM histidine mother liquor: Weigh 1.55 g of histidine, dissolve it in 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] Third step, preparation of 100 mM histidine hydrochloride mother liquor: Weigh 2.10 g of histidine hydrochloride, dissolve it in pure water and make up the volume to 100 mL. After filtering with a 0.22 μm filter, store it at 2 - 8 °C.

[0219] 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. After filtering with a 0.22 μm filter, store it at 2 - 8 °C.

[0220] Step 5, Preparation of 400 mM sodium chloride stock solution: Weigh 1.17 g of sodium chloride, dissolve it with pure water and make up the volume to 50 mL. After filtration through a 0.22 μm filter, store it at 2 - 8 °C.

[0221] Step 6, Prepare the extracellular vesicles prepared in Example 1 according to different cryopreservation fluid formulations and store them at -90 - -70 °C, including:

[0222] Experimental Example 1: The final concentration of sucrose is 50 mM; the final concentration of polysorbate 80 is 0.2 mg / mL; the ratio of the pH buffer histidine and histidine hydrochloride is 1:1.3, the final concentration of histidine is 4 mM, and the final concentration of histidine hydrochloride is 5.2 mM; the final concentration of the isotonic regulator sodium chloride is 121.8 mM, the solvent is pure water; the final concentration of extracellular vesicles is 2×10 10 / mL.

[0223] Experimental Example 2: 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 histidine is 20 mM; the final concentration of the isotonic regulator sodium chloride is 119 mM, the solvent is pure water; the final concentration of extracellular vesicles is 2×10 10 / mL.

[0224] 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 the pH buffer histidine and histidine hydrochloride is 2:1, the final concentration of histidine is 13 mM, and the final concentration of histidine hydrochloride is 6.5 mM; the final concentration of the isotonic regulator sodium chloride is 116 mM, the solvent is pure water; the final concentration of extracellular vesicles is 2×10 10 / mL.

[0225] 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 the pH buffer histidine and histidine hydrochloride is 10:1, the final concentration of histidine is 20 mM, and the final concentration of histidine hydrochloride is 2 mM; the final concentration of the isotonic regulator sodium chloride is 117 mM, the solvent is pure water; the final concentration of extracellular vesicles is 2×10 10 / mL.

[0226] Step 7, To verify the effect of the extracellular vesicle preservation solution, a commonly used solution for preserving extracellular vesicles, sodium chloride injection, is selected as a comparison.

[0227] Control Example 1: Dilute the extracellular vesicles to 2×10 10 / mL with sodium chloride injection.

[0228] Step 8, Detect the pH and osmotic pressure of Experimental Examples 1 - 4 and Control Example 1, and the results are shown in Table 4.

[0229] Table 4 Measured pH and Osmotic Pressure Values of Experimental Examples 1-4 and Control Example

[0230] number pH osmotic pressure (mOsmol / kg) Experimental Example 1 5.92 299.31 Experimental Example 2 7.62 299.56 Experimental Example 3 6.34 299.48 Experimental Example 4 7.01 299.49 Control Example 6.08 298.73

[0231] Step 9: Detect the particle size and concentration of extracellular vesicles of Experimental Examples 1-4 and Control Example 1, and the expression contents of CD63, hsa-miR-21-5p, and has-miR-146a-5p are used as the sample contents at the 0 point.

[0232] Example 3

[0233] This example compares the preservation effects of extracellular vesicles of Experimental Example 1 and Control Example 1.

[0234] Storage conditions: 2-8°C, time: 2 weeks.

[0235] After Experimental Example 1 and Control Example 1 were stored at 2-8°C for 2 weeks, the concentration and median particle size of extracellular vesicles were detected and analyzed by nanoparticle tracking analysis technology (NTA), as shown in Table 5. Ratio analysis was performed with the 0-point results, and the analysis results are as Figure 2 shown. The cryopreservation solution of Experimental Example 1 has a significantly higher protective effect on particle concentration than Control Example 1. The two groups have no obvious effect on particle size, and the particle size is intact, that is, the stability of its particle size distribution can be maintained.

[0236] Table 5 Concentration and Median Particle Size of Extracellular Vesicles of Experimental Example 1 and Control Example at the 0 Point and after Storage at 2-8°C for 2 Weeks

[0237]

[0238] Therefore, the cryopreservation solution with histidine and histidine hydrochloride (ratio 1:1.3) as the buffer can improve the preservation effect of extracellular vesicles, maintain the stability of extracellular vesicles, avoid changes in particle size and concentration of extracellular vesicles, and its preservation effect is better than that of the commonly used solvent sodium chloride injection.

[0239] Example 4

[0240] This example compares the preservation effects of extracellular vesicles of cryopreservation solutions containing different ratios of histidine and histidine hydrochloride. Storage conditions: -25--15°C, time: 2 months.

[0241] The particle size and concentration of extracellular vesicles of Experimental Examples 1-4 were detected and analyzed by nanoparticle tracking analysis technology (NTA), and the results are shown in Table 6. Ratio analysis was performed with the 0-point results, and the analysis shows that the ratio of the particle concentration of Experimental Example 2 to the 0 point is significantly lower than that of Experimental Example 1, while there is no obvious difference between Experimental Examples 3, 4 and Experimental Example 1. As Figure 3As shown, there was no significant difference in the median particle size of the extracellular vesicles in Experimental Examples 1-4 after storage at -25 to -15°C for 2 months. The slight increase in the values of each index may be due to measurement errors, and they actually remained stable.

[0242] Table 6 Particle size and concentration of extracellular vesicles in Experimental Examples 1-4 after storage at -25 to -15°C for 2 months

[0243]

[0244] Fluorescence quantitative PCR (qPCR) was used to detect the expression of the nucleic acid inclusions hsa-miR-21-5p and hsa-miR-146a-5p in extracellular vesicles. The Ct values of the extracellular vesicles stored at -25 to -15°C for 2 months and the Ct values at time zero were used for result analysis according to the 2 -△CT method. As Figure 4 shown, the relative contents of hsa-miR-21-5p in Experimental Examples 2-4 were all significantly higher than that in Experimental Example 1, and the relative content in Experimental Example 3 was the highest; the relative contents of hsa-miR-146-a-5p in Experimental Examples 3 and 4 were significantly higher than that in Experimental Example 1, and there was no significant difference between Experimental Example 2 and Experimental Example 1.

[0245] Western blotting was used to detect the expression of the extracellular vesicle protein marker CD63, and the gray value of CD63 in the extracellular vesicles stored at -25 to -15°C for 2 months was used for ratio analysis with the value at time zero. As Figure 5 shown, the relative contents of CD63 in Experimental Examples 2 and 3 were slightly higher than that in Experimental Example 1, and there was no significant difference among the groups.

[0246] Example Five

[0247] In this example, the preservation effects of extracellular vesicles in cryopreservation solutions containing different ratios of histidine and histidine hydrochloride were compared. Storage conditions: -90 to -70°C, time: 6 months.

[0248] The particle size and concentration of the extracellular vesicles in Experimental Examples 1-4 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 time zero. As Figure 6 shown, after storage at -90 to -70°C for 6 months, there was no significant difference in the ratio of particle concentration to the value at time zero among the groups, but the median particle sizes of Experimental Examples 2 and 4 were significantly larger than that of Experimental Example 1, and there was no significant difference in the median particle size between Experimental Example 3 and Experimental Example 1. The slight increase in the values of each index may be due to measurement errors, and they actually remained stable.

[0249] Table 7 Particle size and concentration of extracellular vesicles in Experimental Examples 1-4 after storage at -90 to -70°C for 6 months

[0250]

[0251] The expression of extracellular vesicle nucleic acid inclusions hsa-miR-21-5p and hsa-miR-146a-5p was detected by fluorescence quantitative PCR (qPCR). The Ct value of extracellular vesicles stored at -90 - -70°C for 6 months was compared with the Ct value at 0 point according to the 2 -△CT method for result analysis. As Figure 7 shown, the relative contents of hsa-miR-21-5p and hsa-miR-146a-5p in Experimental Example 2 were significantly higher than those in Experimental Example 1, and the results of Experimental Example 3 were significantly higher than those of Experimental Example 2.

[0252] The expression of extracellular vesicle protein marker CD63 was detected by Western blotting. The gray value of CD63 in extracellular vesicles stored at -90 - -70°C for 6 months was analyzed by ratio with the value at 0 point. As Figure 8 shown, the relative content of CD63 in Experimental Example 2 was slightly lower than that in Experimental Example 1, and there was no significant difference between Experimental Examples 3, 4 and Experimental Example 1.

[0253] Therefore, fresh extracellular vesicles can be stored at a lower temperature using a cryopreservation solution with histidine and histidine hydrochloride as buffers, which can improve the preservation effect, maintain the stability of extracellular vesicles, avoid changes in the particle size and concentration of extracellular vesicles, and reduce the degradation of extracellular vesicle inclusions.

[0254] 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, 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 about 5 - 8, preferably a pH value of 5.5 - 7.7, more preferably a pH value of 6 - 7, and most preferably a pH value of 6.3 - 6.

4.

3. The aqueous composition according to claim 1, characterized in that, The pH buffer comprises histidine and / or histidine hydrochloride, wherein the concentration of histidine is 1 - 30 mM, and / or the concentration of histidine hydrochloride is 1 - 10 mM.

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

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, a topical skin / mucosal medication, an eye drop, a nasal drop, 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 preparing 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 with a complete medium without extracellular vesicles and separating 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 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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