Cryogenic elution system for extracellular vesicles

Abstract

Cryogenic elution system (10) for extracellular vesicles, comprising: an elution extraction section (20) for taking up cells and performing a separation process on the cells; a low-temperature section (30) for generating frozen crystals, which is connected to the elution extraction section (20) and configured to control a temperature in the elution extraction section (20) in order to create a low-temperature environment for the elution extraction section (20) so that a liquid in the cells forms ice crystal particles; and a pressure control section (40) connected to the elution extraction section (20) and configured to control pressure in the elution extraction section (20) so that the ice crystal particles grow to a predetermined size and move rapidly out of the cells, thus producing an isolate of extracellular vesicles.

Description

STATE OF THE ART 1. Technical field

[0001] The present invention relates to a production system for extracellular vesicles and, more particularly, to a cryogenic elution system for extracellular vesicles. 2. Description of the state of the art

[0002] Extracellular vesicles are vesicles secreted by cells, measuring 30–150 nm in size. They have a bilayer lipid structure and are rich in proteins, DNA, RNA, growth factors, and the like. They participate in cell-to-cell signaling to produce effects such as tissue repair, cell activation, cell regeneration, immune regulation, and immune system activation. Today, extracellular vesicles are widely used in biomedicine and cosmetic treatments, with natural extracellular vesicles being obtained by isolation from animal cells, plant cells, or microorganisms.

[0003] Currently, common isolation methods include ultracentrifugation, ultrafiltration, precipitation, immunoaffinity-based isolation, and size-exclusion chromatography. Ultracentrifugation does not yield high yields of extracellular vesicles and is the most time-consuming. Size-exclusion chromatography is highly reproducible but costly, requiring additional processing steps to collect the extracellular vesicles. Ultrafiltration has advantages such as low cost and speed, but tends to damage the processed extracellular vesicles. Precipitation has high processing efficiency but raises safety concerns because the extracellular vesicles may become contaminated during processing.Immunoaffinity-based isolation advantageously offers high purity and no reagent contamination, but it has disadvantages due to high antibody costs and low extracellular vesicle yield. Furthermore, structural damage to the extracellular vesicles occurs during the production process, making it ultimately less effective than expected. From the above, it is clear that an important research goal in the industry is to discover ways to stably obtain safe and effective extracellular vesicles. SUMMARY OF THE INVENTION

[0004] The main object of the present invention is to provide a cryogenic elution system for extracellular vesicles that simplifies the process of separating extracellular vesicles from cells and enables the stable and rapid isolation of structurally undamaged extracellular vesicles.

[0005] Another object of the present invention is to provide a cryogenic extracellular vesicle elution system that can process a large amount of cells, reduce cell processing costs, and thus meet the demand for extracellular vesicles in the relevant industries.

[0006] To achieve the above-mentioned objects, the present invention discloses a cryogenic elution system for extracellular vesicles, which includes, as main components, an elution-extraction section, a low-temperature section for generating frozen crystals, and a pressure-control section. The above sections may be a machine, a piece of equipment, a device, or a combination of at least two thereof. The elution-extraction section is configured to receive cells and perform a separation process on the cells. The low-temperature section for generating frozen crystals and the pressure-control section are separately coupled to the elution-extraction section and are configured to control the temperature and pressure in the elution-extraction section so that ice particles form in the cells, enabling the separation of an isolate of extracellular vesicles from the cells.

[0007] The extracellular vesicle isolate contains extracellular vesicle particles, with the particle sizes of the extracellular vesicle particles ranging from 50 to 150 nm.

[0008] In one embodiment of the present invention, the elution extraction section includes a receiving unit and a separation unit. The receiving unit is configured to receive the cells, and the separation unit is configured to separate the isolate from extracellular vesicles.

[0009] In another embodiment of the present invention, the low-temperature frozen crystal generating section includes a temperature control unit for generating a low-temperature environment for the elution extraction section so that the liquid in the cells can form the ice crystal particles.

[0010] In yet another embodiment of the present invention, the pressure control section is configured to control the pressure in the elution extraction section and thereby control the size of the grown ice crystal particles and the timing of cell damage, enabling the isolation of extracellular vesicles to be obtained.

[0011] The pressure control section includes a compressor unit, a heat exchange unit, a circulation unit, and a pressure control unit. The pressure control unit is connected to the compressor unit, the heat exchange unit, and the circulation unit and controls their operation to control the pressure in the elution extraction section.

[0012] The pressure control section allows the ice crystal particles in the cells to grow to a predetermined size. For example, the ice crystal particles can grow until their diameter is greater than or equal to 200 nm. BRIEF DESCRIPTION OF THE MULTIPLE VIEWS OF THE DRAWINGS Fig. 1 is a schematic representation of a cryogenic extracellular vesicle elution system disclosed in one embodiment of the present invention. Fig. 2 is a photograph showing the frozen crystals in the cell tissue processed with the cryogenic extracellular vesicle elution system disclosed in the present invention, wherein the solid arrows indicate extracellular vesicles and the dashed arrows indicate ice crystal particles. Fig. 3 shows a state in which the walls of the cells processed with the cryogenic extracellular vesicle elution system disclosed in the present invention are ruptured. Fig. 4 shows the result of an analysis of the particle sizes of the extracellular vesicles obtained with the cryogenic extracellular vesicle elution system disclosed in the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention discloses a cryogenic elution system for extracellular vesicles, which includes an elution-extraction section, a low-temperature section for generating frozen crystals, and a pressure control section. Through the interaction of the low-temperature section for generating frozen crystals and the pressure control section for regulating the temperature and pressure in the elution-extraction section, a temperature difference and pressure fluctuations can be generated in the elution-extraction section, causing ice crystal particles to form in the elution-extraction section and ultimately damaging the cell tissue, allowing structurally and functionally intact extracellular vesicles to be separated from the cells.

[0014] The term “ice crystal particle,” also known as “frozen crystal,” refers to a crystal that forms at low temperatures in the liquid in a cell.

[0015] The technical features of the present invention and their effects will be explained below by describing in detail a preferred embodiment of the invention with reference to the accompanying drawings.

[0016] Referring to Fig. 1, the cryogenic elution system for extracellular vesicles 10 disclosed in the present invention includes, as main components, an elution extraction section 20, a low-temperature section 30 for generating frozen crystals, and a pressure control section 40.

[0017] The elution extraction section 20 includes a receiving unit 21 and a separation unit 22. The receiving unit 21 is configured to receive cells, which may be cells obtained from an animal, a plant, or a microorganism. The separation unit 22 is connected to the receiving unit 21 and configured to separate an isolate of extracellular vesicles from the receiving unit 21.

[0018] The low-temperature frozen crystal generation section 30 is connected to the elution extraction section 20 and includes a temperature control unit 31 for receiving an external command and controlling the temperature in the elution extraction section 20 according to the command, thereby creating a low-temperature environment for the elution extraction section 20. Once the low-temperature environment for the elution extraction section 20 is created, the liquid in the cells in the receiving unit coagulates, freezes, and forms ice crystal particles.

[0019] The pressure control section 40 is connected to the elution extraction section 20 and is configured to control the pressure in the elution extraction section 20. Specifically, the pressure control section 40 includes a pressure control unit 41, a compressor unit 42, a heat exchange unit 43, and a circulation guide unit 44. The pressure control unit 41 is wirelessly or wired connected to the compressor unit 42, the heat exchange unit 43, and the circulation guide unit 44. Upon receiving an external command, the pressure control unit 41 controls the operation of the compressor unit 42, the heat exchange unit 43, and the circulation guide unit 44 according to the command to control the pressure in the elution extraction section 20, thereby allowing the ice crystal particles in the cells to grow to a predetermined size and damage the cell structure to produce an isolate of extracellular vesicles.

[0020] The cryogenic extracellular vesicle elution system 10 disclosed in the present invention, formed from the above components, may utilize the low-temperature section 30 for generating frozen crystals and the pressure control section 40 to adjust the temperature and pressure of the elution extraction section 20 so that the separation unit 22 can separate an isolate of structurally and functionally intact extracellular vesicles from the cells in the receiving unit 21.

[0021] The specific operation procedure is as follows: The low-temperature frozen crystal generation section 30 creates a low-temperature environment for the elution extraction section 20, so that the liquid in the cells in the receiving unit 21 forms ice crystal particles. With continued low-temperature conditions, the ice crystal particles continue to grow. When the size, or more specifically, the diameter, of the ice crystal particles is greater than or equal to 200 nm, the pressure control section 40 is started. Through the operation of the pressure control unit 41, the compressor unit 42, the heat exchange unit 43, and the circulation guide unit 44, the pressure in the elution extraction section 20 is changed to accelerate the damage to the cell structure and the leakage of the ice crystal particles from the inside of the cells, allowing the extracellular vesicle isolate to move out of the cells simultaneously.The isolate is then separated from extracellular vesicles by the separation unit 22.

[0022] As in Fig. 2 and Fig. As shown in Figure 3, the cryogenic elution system for extracellular vesicles 10 disclosed in the present invention proved capable of damaging the cell structure and enabling the release of ice crystal particles and extracellular vesicles from the cells, while the released extracellular vesicles remained structurally intact. Furthermore, a particle size analysis was performed on the separated isolate of extracellular vesicles, the analysis result showing that the cryogenic elution system for extracellular vesicles 10 disclosed in the invention had successfully isolated extracellular vesicles of high purity, and that, with reference to Fig.4, the particle sizes of the extracellular vesicles were in the range of 50 to 150 nm, which is advantageous for subsequent industrial applications in the production of various products.

[0023] A cryogenic elution system for extracellular vesicles includes an elution-extraction section, a low-temperature section for generating frozen crystals, and a pressure control section. The low-temperature section for generating frozen crystals and the pressure control section for regulating the temperature and pressure in the elution-extraction section work together to create a temperature difference and pressure fluctuations in the elution-extraction section, causing ice crystal particles to form in the elution-extraction section and ultimately damaging the cell tissue, allowing structurally and functionally intact extracellular vesicles to be separated from the cells.

Claims

[1] Cryogenic elution system (10) for extracellular vesicles, comprising: an elution extraction section (20) for receiving cells and performing a separation process on the cells; a low-temperature frozen crystal generating section (30) connected to the elution extraction section (20) and configured to control a temperature in the elution extraction section (20) so as to create a low-temperature environment for the elution extraction section (20) so that a liquid in the cells forms ice crystal particles; and a pressure control section (40) connected to the elution extraction section (20) and configured to control a pressure in the elution extraction section (20) so that the ice crystal particles grow to a predetermined size and move rapidly out of the cells, thus yielding an isolate of extracellular vesicles. [2] The cryogenic extracellular vesicle elution system (10) according to claim 1, wherein the low-temperature frozen crystal producing section (30) comprises a temperature control unit (31) for receiving a command and controlling the temperature in the elution extraction section (20) according to the command. [3] The cryogenic elution system (10) for extracellular vesicles according to claim 1, wherein the pressure control section (40) comprises a compressor unit (42), a heat exchange unit (43), a circulation guide unit (44), and a pressure control unit (41), the pressure control unit (41) being configured to receive a command and control the operation of the compressor unit (42), the heat exchange unit (43), and the circulation guide unit (44) according to the command, thereby controlling the pressure in the elution extraction section (20). [4] The cryogenic extracellular vesicle elution system (10) of claim 1, wherein the extracellular vesicle isolate contains extracellular vesicle particles, the extracellular vesicles having particle sizes in a range of 50 to 150 nm. [5] The cryogenic extracellular vesicle elution system (10) according to claim 1, wherein the pressure control section (40) is capable of causing the diameter of the ice crystal particles to be greater than or equal to 200 nm. [6] The cryogenic elution system (10) for extracellular vesicles according to claim 1, wherein the elution extraction section (20) comprises a receiving unit (21) for receiving the cells. [7] The cryogenic extracellular vesicle elution system (10) according to claim 6, wherein the elution extraction section (20) further comprises a separation unit (22) connected to the receiving unit (21) and configured to separate the extracellular vesicle isolate and the ice crystal particles in the receiving unit (21).

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