Elution system for frozen crystalline exosomes

Abstract

The present invention provides a system for eluting frozen crystallized exosomes, which has the effect of stably isolating exosomes. [Solution] The system includes an elution and extraction unit 20, a low-temperature freezing crystal generation unit 30, and a pressure control unit 40. By having the pressure control unit and the low-temperature freezing crystal generation unit work together and controlling the temperature and pressure of the elution and extraction unit, a temperature difference and pressure change are generated in the elution and extraction unit, ice crystals are formed within the cells, and cellular tissue is destroyed, thereby achieving the effect of stably isolating exosomes with intact structure and function from within the cells.

Description

[Technical field]

[0001] The present invention relates to a system for producing exosomes, and in particular to a system for eluting frozen crystalline exosomes. [Background technology]

[0002] Exosomes are small vesicles with a size of about 30 to 150 nanometers secreted from cells, have a lipid bilayer structure, and are rich in components such as proteins, DNA, RNA, and growth factors. They are used for intercellular information transmission, and can have effects such as tissue repair, cell activation, cell regeneration, immune regulation, and immune activation. Currently, exosomes are widely used in the fields of biomedical and cosmetic medicine, and natural exosomes are obtained by isolation from animal cells, plant cells, or microorganisms.

[0003] Currently, common isolation methods include ultracentrifugation, ultrafiltration, precipitation, immunoaffinity isolation, and size-exclusion chromatography, but the yield of exosomes by ultracentrifugation is not high and it takes the longest time. Size-exclusion chromatography has excellent reproducibility, but is expensive and requires additional processing steps to aggregate exosomes. Ultrafiltration has the advantages of low cost and high speed, but may damage exosomes during the processing process. Precipitation has high processing efficiency, but exosomes may be contaminated during the processing process, which raises safety concerns. Immunoaffinity isolation has the advantages of high purity and no reagent contamination, but also has the disadvantages of high antibody cost and low exosome yield, and damage to the structure of exosomes during the production process reduces the effectiveness of exosomes. For this reason, the focus of current research is on how to stably obtain safe and effective exosomes. Summary of the Invention [Problem to be solved by the invention]

[0004] The main objective of the present invention is to provide a system for eluting frozen crystallized exosomes, which can simplify the process of isolating exosomes from cells and can stably and quickly isolate structurally intact exosomes.

[0005] The second objective of the present invention is to provide a system for eluting frozen crystallized exosomes, which can process a large number of cells, reduce cell processing costs, and thereby supply the demand for exosomes required by industry. [Means for solving the problem]

[0006] Therefore, in order to achieve the above object, the present invention discloses a system for eluting frozen crystallized exosomes, which mainly includes an elution / extraction unit, a low-temperature freezing crystallization unit, and a pressure control unit. Each component may be a machine, equipment, device, or a combination of at least two of them. The elution / extraction unit is used to contain and isolate cells, and the low-temperature freezing crystallization unit and the pressure control unit are connected to the elution / extraction unit, respectively, and are used to form ice crystals in the cells and isolate exosomes by controlling the temperature and pressure of the elution / extraction unit.

[0007] The exosome isolate contains exosome microparticles having a particle diameter of 50 to 150 nm.

[0008] In one embodiment of the present invention, the elution and extraction section further includes a storage unit for storing cells and an isolation unit for isolating the exosome isolate.

[0009] In another embodiment of the present invention, the low-temperature freezing crystallization unit further includes a temperature control unit for making the elution extraction unit in a low-temperature environment so that the intracellular fluid can form ice crystals.

[0010] In the next embodiment of the present invention, the pressure control unit is used to control the pressure in the elution extraction unit, thereby controlling the size of the ice crystals generated and the time point of cell destruction, thereby producing the exosome isolate.

[0011] The pressure control unit further includes a compressor unit, a heat exchange unit, a circulating conduction unit, and a pressure control unit, and the pressure control unit is connected to the compressor unit, the heat exchange unit, and the circulating conduction unit, and controls their operation to achieve the purpose of controlling the pressure in the elution extraction unit.

[0012] The pressure control section can continue to generate intracellular ice crystals up to a predetermined size, for example, ice crystals with a diameter of 200 nm or more can be generated. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 is a schematic diagram of a system for eluting frozen crystalline exosomes according to one embodiment of the present invention. [Diagram 2] This is a photograph of frozen crystals in cell tissue after treatment with the frozen crystallized exosome elution system of the present invention. The solid arrow indicates an exosome (extracellular vesicle), and the dashed arrow indicates an ice crystal particle. [Diagram 3] FIG. 2 shows the state of cell wall destruction after treatment with the frozen crystallized exosome elution system of the present invention. [Figure 4] 1 is a graph showing the results of analyzing the particle size of exosomes after treatment with the frozen crystallized exosome elution system of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention discloses a system for eluting frozen crystallized exosomes, which includes an elution / extraction unit, a low-temperature freezing crystallization unit, and a pressure control unit. The pressure control unit and the low-temperature freezing crystallization unit cooperate with each other, and the temperature and pressure of the elution / extraction unit are controlled to generate a temperature difference and a pressure change in the elution / extraction unit, which forms ice crystals in cells to destroy cell tissue, and isolates cellular exosomes with intact structure and function.

[0015] The term "ice crystals", also known as cryocrystals, as used herein refers to the crystallization of intracellular fluids at low temperatures.

[0016] In order to explain the technical features and effects of the present invention, the preferred embodiments of the present invention will be described in detail below with reference to the drawings.

[0017] Referring to FIG. 1, the frozen crystallized exosome elution system 10 according to the present invention is mainly composed of an elution extraction section 20, a low-temperature frozen crystal production section 30, and a pressure control section 40.

[0018] The elution and extraction section 20 includes a storage unit 21 and an isolation unit 22. The storage unit 21 is used to receive and store cells, which may be derived from animals, plants, or microorganisms. The isolation unit 22 is connected to the storage unit 21 and is used to isolate exosomes from the storage unit 21.

[0019] The low-temperature freezing crystallization unit 30 is connected to the elution and extraction unit 20 and includes a temperature control unit 31 for receiving an external command, controlling the temperature of the elution and extraction unit 20, and making the elution and extraction unit 20 in a low-temperature environment. For example, when the elution and extraction unit 20 is in a low-temperature environment, the intracellular fluid in the storage unit 21 gathers and freezes to form ice crystals.

[0020] The pressure control unit 40 is connected to the elution and extraction unit 20 and is used to control the pressure in the elution and extraction unit 20. Specifically, the pressure control unit 40 further includes a pressure control unit 41, a compressor unit 42, a heat exchange unit 43, and a circulating conduction unit 44, and the pressure control unit 41 is connected to the compressor unit 42, the heat exchange unit 43, and the circulating conduction unit 44 in a wired or wireless manner. After the pressure control unit 41 receives an external command, it controls the operation and operation of the compressor unit 42, the heat exchange unit 43, and the circulating conduction unit 44 to change the pressure in the elution and extraction unit 20, thereby controlling the ice crystals in the cells to continue to form to a predetermined size and the time when the cell structure is destroyed, thereby producing the exosome isolate.

[0021] With the above components, the frozen crystallized exosome elution system 10 of the present invention can adjust the temperature and pressure of the elution and extraction section 20 through the low-temperature frozen crystal generation section 30 and the pressure control section 40, and the isolation unit 22 can isolate exosomes with intact structure and function from the cells in the storage unit 21.

[0022] The specific operation method is as follows: The low-temperature freezing crystallization unit 30 places the elution and extraction unit 20 in a low-temperature environment, at which time the intracellular fluid forms ice crystals, and if the low-temperature environment is maintained, the ice crystals continue to be generated, and when the size of the ice crystals reaches a diameter of 200 nm or more, the pressure control unit 40 is started, and the pressure of the elution and extraction unit 20 is changed by the operation of the pressure control unit 41, the compressor unit 42, the heat exchange unit 43, and the circulatory conduction unit, and the destruction of the cell structure and the speed of ice crystal export from the cells are also accelerated, and at the same time, the exosome isolate is also detached from the cells, and then the exosome isolate can be isolated via the isolation unit 22. [Industrial Applicability]

[0023] As shown in Figures 2 and 3, the frozen crystallized exosome elution system 10 of the present invention can reliably destroy cell structures and release ice crystals and exosomes, and the structure of the released exosomes is very complete. The isolated exosome isolate is subjected to particle size analysis, and the result is shown in Figure 4. The frozen crystallized exosome elution system 10 of the present invention can isolate high-purity exosomes with a particle size of 50 to 150 nm, which can be used in the industry to manufacture various products.

Claims

1. an elution extraction section for receiving and isolating cells; A low-temperature freezing crystallization unit connected to the elution / extraction unit for controlling the temperature of the elution / extraction unit and placing the elution / extraction unit in a low-temperature environment so that the intracellular fluid can form ice crystals; a pressure control unit connected to the elution / extraction unit, for controlling the pressure of the elution / extraction unit, and for quickly detaching the ice crystals from the cells when the ice crystals reach a predetermined size to produce an exosome isolate; A system for eluting frozen crystalline exosomes comprising:

2. The elution system for frozen crystallized exosomes according to claim 1, wherein the low-temperature frozen crystal generation unit further includes a temperature control unit for receiving an instruction and controlling the temperature in the elution extraction unit.

3. The pressure control unit further includes a compressor unit, a heat exchange unit, a circulating conduction unit, and a pressure control unit; The pressure control unit is used to receive commands and control the operation of the compressor unit, the heat exchange unit and the circulating conduction unit to control the pressure in the elution extraction unit. The system for eluting frozen crystalline exosomes according to claim 1

4. The frozen crystallized exosome elution system according to claim 1, wherein the exosome isolate contains exosome microparticles having a particle diameter of 50 to 150 nm.

5. The system for eluting frozen crystalline exosomes according to claim 1, wherein the pressure control unit can set the diameter of the ice crystals to 200 nm.

6. The elution system for frozen crystallized exosomes according to claim 1 , wherein the elution and extraction section further includes a storage unit for receiving and storing the cells.

7. The elution extraction unit is connected to the storage unit and further includes an isolation unit for isolating the exosome isolate and the ice crystals in the storage unit. The elution system for frozen crystallized exosomes according to claim 6.

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