Exosome preparation system

Abstract

The invention relates to the technical field of biological extraction, in particular to an exosome preparation system which comprises a first storage tank, a second storage tank, a hollow fiber cell culture device, a tangential flow filtering device, a first peristaltic pump and a second peristaltic pump. An outlet of the second storage tank is connected with an inlet of the tangential flow filtering device through a second peristaltic pump, the ultrasonic generator and the ultrasonic transducer are additionally arranged in the air bag, and ultrasonic oscillation can stimulate cells and promote the cells to release more exosomes; a culture solution in a culture barrel is kept in a flowing state through a first peristaltic pump, nutrient elements in the culture solution can be supplemented, meanwhile, when an ultrasonic transducer drives a hollow vibration pipe to vibrate, the vibration frequencies of the two ends of the hollow vibration pipe are different, and the first peristaltic pump drives the culture solution to flow; the cells can receive oscillation stimulation of different frequencies, so that more exosomes are released, and large-scale industrial production of the exosomes is realized.

Description

Technical Field

[0001] This invention relates to the field of bio-extraction technology, and in particular to an exosome preparation system. Background Technology

[0002] Exosomes are lipid bilayer vesicles containing intracellular proteins and RNA, secreted by almost all cells. They range in diameter from 30 to 150 nm and primarily originate from multivesicles formed by the invagination of lysosomal microparticles within cells. After fusing with the cell membrane through the outer membrane of these multivesicles, they are released into the extracellular matrix. Currently, extensive research on their biological origin, composition and transport, intercellular signal transduction, and distribution in body fluids has revealed a wide variety of functions for exosomes. The function of exosomes depends on the cell type from which they originate, and they can participate in various aspects of the body's immune response, antigen presentation, cell migration, cell differentiation, and tumor invasion. Studies have shown that tumor-derived exosomes participate in the exchange of genetic information between tumor cells and basal cells, leading to the formation of numerous new blood vessels and promoting tumor growth and invasion. Exosomes also have important applications in anti-aging and skincare; therefore, the large-scale production of exosomes has become a significant factor restricting their development.

[0003] Currently, numerous companies have begun developing equipment for the mass production of exosomes. For example, a patent application with application number 2021105585609 discloses a system for the production and preparation of exosomes and a method for preparing exosomes, specifically related to the biomedical field. The method includes two parts: cell culture and exosome extraction. The system includes a first storage tank, a second storage tank, a hollow fiber cell culture device, a tangential flow filtration device, a first peristaltic pump, and a second peristaltic pump.

[0004] The aforementioned system for exosome production utilizes a large number of hollow fiber tubes in a hollow fiber device to ensure cell culture area. The culture medium delivered through the hollow fiber tubes stimulates cells to secrete exosomes, and the produced exosome solution is purified by a tangential flow filtration device to achieve exosome preparation. However, this system has certain drawbacks. First, the hollow fiber tubes lack internal support, causing the filtration speed to gradually slow down during exosome filtration. As the peristaltic pump pumps in more culture medium, the stress on the hollow fiber tubes increases, leading to tube collapse. Second, the hollow fiber cell culture device lacks methods to stimulate cells to increase exosome secretion. Currently known methods for stimulating cells to increase exosome secretion mainly include ultrasonic stimulation, pressure stimulation, electrical stimulation, compound stimulation, and culture medium stimulation.

[0005] The inventors have creatively developed an exosome preparation system by combining existing exosome preparation systems and various stimulation methods. Summary of the Invention

[0006] The purpose of this invention is to provide an exosome preparation system to solve the problems mentioned in the background art. Specific techniques...

[0007] The surgical plan is as follows:

[0008] To achieve the above and other related objectives, the present invention provides an exosome preparation system, comprising a first storage tank, a second storage tank, a hollow fiber cell culture device, a tangential flow filtration device, a first peristaltic pump, and a second peristaltic pump; the outlet of the second storage tank is connected to the inlet of the tangential flow filtration device via the second peristaltic pump, and the outlet of the tangential flow filtration device is connected to the second inlet of the second storage tank; the liquid filtered by the tangential flow filtration device flows directly out of the system; wherein,

[0009] The hollow fiber cell culture device includes a culture tank, a feed pipe on one side of the culture tank, a filtrate discharge pipe at the bottom of the culture tank, the feed pipe being connected to the outlet of a first storage tank via a first peristaltic pump, the filtrate discharge pipe being connected to the first inlet of a second storage tank, an isolation chamber on the other side of the culture tank, a discharge pipe on the isolation chamber being connected to the inlet of the first storage tank, and a plurality of hollow fiber tubes being arranged inside the culture tank near the isolation chamber, the hollow fiber tubes having a molecular weight cutoff of 20-75KD;

[0010] The tangential flow filtration device is equipped with a tangential flow filtration membrane, and the molecular weight cutoff of the tangential flow filtration membrane is 50-750KD;

[0011] A hollow vibrating tube is inserted into the hollow fiber tube. The hollow vibrating tube has multiple small holes for the inflow of culture medium. After being filtered through the hollow fiber tube, the culture medium enters the isolation chamber through the hollow vibrating tube. The end of the hollow vibrating tube penetrates the isolation chamber and is equipped with an ultrasonic transducer. The ultrasonic transducer is connected to an ultrasonic generator, which provides energy to the ultrasonic transducer.

[0012] Preferably, the top of the culture tank is provided with an inoculation port.

[0013] Preferably, the culture tank is equipped with an index detection system, including a pH detection module, a temperature detection module, and a dissolved oxygen detection module.

[0014] Preferably, the culture tank is provided with a transparent viewing window.

[0015] Preferably, a soundproof cover is also provided outside the isolation chamber.

[0016] Preferably, a rubber sealing gasket is provided at the through connection between the hollow vibrating tube and the isolation chamber.

[0017] Preferably, the ultrasonic transducer generates an ultrasonic intensity of 0.6–3.4 W / cm². 2 .

[0018] The exosome preparation system provided by this invention has the following beneficial effects:

[0019] The airbag in this invention incorporates an ultrasonic generator and an ultrasonic transducer. The ultrasonic vibrations stimulate cells, promoting the release of more exosomes. During culture, a first peristaltic pump is activated, trapping exosomes within the culture tank via a hollow fiber tube. The culture medium flows through the hollow fiber tube and through small holes into a hollow vibrating tube, then into an isolation chamber. From there, it is discharged back into a first storage tank via a discharge pipe. Nutrients are added to the first storage tank, and the first peristaltic pump keeps the culture medium flowing, ensuring replenishment of nutrients. Simultaneously, the ultrasonic transducer drives the hollow vibrating tube to vibrate at different frequencies at its two ends. The first peristaltic pump, by driving the culture medium flow, allows cells to receive different frequency vibration stimuli, resulting in the release of more exosomes and enabling large-scale industrial production of exosomes. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a schematic diagram of the structure of an exosome preparation system according to the present invention;

[0022] Figure 2 This is a schematic diagram of the hollow fiber cell culture device in this invention;

[0023] Figure 3 yes Figure 2 Enlarged view of a portion of point A in the middle. Detailed Implementation

[0024] The exosome preparation system proposed in this invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The advantages and features of this invention will become clearer from the following description. It should be noted that the drawings are all in a very simplified form and use non-precise proportions, and are only used to facilitate and clarify the illustration of the embodiments of this invention.

[0025] In the description of this invention, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0026] It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Therefore, the illustrations only show the components related to the present invention and are not drawn according to the actual number, shape and size of the components in the actual implementation. In the actual implementation, the shape, quantity and proportion of each component can be arbitrarily changed, and the layout of the components may also be more complex.

[0027] Therefore, the present invention provides an exosome preparation system, such as Figure 1-3 As shown, the system includes a first storage tank 1, a second storage tank 2, a hollow fiber cell culture device 3, a tangential flow filter 4, a first peristaltic pump 5, and a second peristaltic pump 6. The outlet of the second storage tank 2 is connected to the inlet of the tangential flow filter 4 via the second peristaltic pump 6, and the outlet of the tangential flow filter 4 is connected to the second inlet of the second storage tank 2. The liquid filtered by the tangential flow filter 4 flows directly out of the system.

[0028] The hollow fiber cell culture device 3 includes a culture tank 31. A feed pipe 32 is provided on one side of the culture tank 31, and a filtrate discharge pipe 33 is provided at the bottom of the culture tank 31. The feed pipe 32 is connected to the outlet of the first storage tank 1 via the first peristaltic pump 5, and the filtrate discharge pipe 33 is connected to the first inlet of the second storage tank 2. An isolation chamber 34 is provided on the other side of the culture tank 31. A discharge pipe 35 is provided on the isolation chamber 34 and is connected to the inlet of the first storage tank 1. A plurality of hollow fiber tubes 36 are provided inside the culture tank 31 near the isolation chamber 34. The molecular weight cutoff of the hollow fiber tubes 36 is 20-75KD.

[0029] The tangential flow filtration device 4 is equipped with a tangential flow filtration membrane, and the molecular weight cutoff of the tangential flow filtration membrane is 50-750KD;

[0030] A hollow vibrating tube 37 is inserted into the hollow fiber tube 36. The hollow vibrating tube 37 has multiple small holes for the inflow of culture medium. After being filtered by the hollow fiber tube 36, the culture medium enters the isolation chamber 34 through the hollow vibrating tube 37. The end of the hollow vibrating tube 37 penetrates the isolation chamber 34 and is equipped with an ultrasonic transducer 71. The ultrasonic transducer 71 is connected to an ultrasonic generator 7, which provides energy to the ultrasonic transducer 71.

[0031] When preparing exosomes, the culture medium is added to the first storage tank 1, the first peristaltic pump 5 is turned on, and the culture medium in the first storage tank 11 is pumped into the culture tank 31 until it is full. 10... 7 Third-generation umbilical cord mesenchymal stem cells are seeded into culture tank 31. Ultrasonic transducer 71 operates, driving hollow vibrating tube 37 to oscillate ultrasonically. The cells are cultured at 25°C, releasing exosomes as they grow. Simultaneously, the ultrasonic vibration stimulates the cells, promoting the release of even more exosomes. During culture, the first peristaltic pump 5 is activated, trapping exosomes within culture tank 31 through hollow fiber tube 36. The culture medium flows through hollow fiber tube 36 and through small holes into hollow vibrating tube 37, then into isolation chamber 34. From isolation chamber 34, the medium is discharged back into first storage tank 1 through discharge pipe 35. Nutrients are added to first storage tank 1. The first peristaltic pump 5 keeps the culture medium in culture tank 31 flowing, ensuring the replenishment of nutrients. Furthermore, when ultrasonic transducer 71 drives the hollow vibrating tube 37 to oscillate, the two ends of the tube oscillate at different frequencies. The first peristaltic pump 5 drives the culture medium flow, allowing the cells to receive different frequency vibration stimuli, thus promoting the release of more exosomes.

[0032] After the culture is completed, open the filtrate discharge tube 33 and send the culture medium containing exosomes in the culture tank 31 into the second storage tank 2. When the volume of the culture medium in the second storage tank 2 reaches 200-400mL, turn on the second peristaltic pump 6 and pump the culture medium into the tangential flow filtration device 4 to extract and purify the exosome solution in the culture medium. This step can remove small molecule impurities in the culture medium.

[0033] When the liquid volume in the second storage tank 2 is concentrated to the minimum operating volume of 50-500 mL, add 5 times the minimum operating volume of sterile PBS buffer to the second storage tank 2, and continue to pump the liquid in the second storage tank 2 into the tangential flow filtration device 4 for circulation, concentration and filtration. When the liquid volume in the second storage tank 2 is concentrated to a certain volume, collect the liquid in the second storage tank 2 to obtain the extracted and purified exosome solution.

[0034] In one embodiment of the present invention, the top of the culture tank 31 is provided with an inoculation port 311 for inoculating cells.

[0035] As one embodiment of the present invention, the culture tank 31 is equipped with an index detection system, including a pH detection module 312, a temperature detection module 313 and a dissolved oxygen detection module 314, for detecting the pH value, temperature and oxygen content of the culture medium in the culture tank 31.

[0036] Furthermore, the culture tank 31 is provided with a transparent viewing window.

[0037] As one embodiment of the present invention, a soundproof cover 38 is also provided outside the isolation chamber 34, which is used to reduce the noise generated by the ultrasonic transducer 71.

[0038] In one embodiment of the present invention, a rubber sealing gasket 39 is provided at the through connection between the hollow vibration tube 37 and the isolation chamber 34 to seal the connection between the hollow vibration tube 37 and the isolation chamber 34 and prevent the culture medium from flowing into the soundproof cover 38.

[0039] In one embodiment of the present invention, the ultrasonic transducer 71 generates an ultrasonic intensity of 0.6–3.4 W / cm². 2 .

[0040] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. An exosome preparation system, characterized in that, The system includes a first storage tank, a second storage tank, a hollow fiber cell culture device, a tangential flow filter, a first peristaltic pump, and a second peristaltic pump. The outlet of the second storage tank is connected to the inlet of the tangential flow filter via the second peristaltic pump, and the outlet of the tangential flow filter is connected to the second inlet of the second storage tank. The liquid filtered by the tangential flow filter flows directly out of the system. The hollow fiber cell culture device includes a culture tank, a feed pipe on one side of the culture tank, a filtrate discharge pipe at the bottom of the culture tank, the feed pipe being connected to the outlet of a first storage tank via a first peristaltic pump, the filtrate discharge pipe being connected to the first inlet of a second storage tank, an isolation chamber on the other side of the culture tank, a discharge pipe on the isolation chamber being connected to the inlet of the first storage tank, and a plurality of hollow fiber tubes being arranged inside the culture tank near the isolation chamber, the hollow fiber tubes having a molecular weight cutoff of 20-75KD; The tangential flow filtration device is equipped with a tangential flow filtration membrane, and the molecular weight cutoff of the tangential flow filtration membrane is 50-750KD; A hollow vibrating tube is inserted into the hollow fiber tube. The hollow vibrating tube has multiple small holes for the inflow of culture medium. After being filtered through the hollow fiber tube, the culture medium enters the isolation chamber through the hollow vibrating tube. The end of the hollow vibrating tube penetrates the isolation chamber and is equipped with an ultrasonic transducer. The ultrasonic transducer is connected to an ultrasonic generator, which provides energy to the ultrasonic transducer.

2. The exosome preparation system according to claim 1, characterized in that, The top of the culture tank is provided with an inoculation port.

3. The exosome preparation system according to claim 2, characterized in that, The culture tank is equipped with an index detection system, including a pH detection module, a temperature detection module, and a dissolved oxygen detection module.

4. The exosome preparation system according to claim 3, characterized in that, The culture tank is equipped with a transparent viewing window.

5. The exosome preparation system according to claim 1, characterized in that, The isolation chamber is also equipped with a soundproof cover.

6. The exosome preparation system according to claim 5, characterized in that, A rubber sealing gasket is provided at the through connection between the hollow vibrating tube and the isolation chamber.

7. The exosome preparation system according to claim 1, characterized in that, The ultrasonic transducer generates an ultrasonic intensity of 0.6–3.4 W / cm². 2 .

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