An extracellular vesicle extraction system, applications and methods of extracting extracellular vesicles

Abstract

The application discloses an extracellular vesicle extraction system, application and method for extracting extracellular vesicles, and belongs to the technical field of extracellular vesicle separation. The extracellular vesicle extraction system comprises a raw material storage tank, a deep filtration device, a continuous flow centrifuge, a pretreatment buffer tank, a first filtration device, a concentrated liquid tank, a second filtration device, a product collection tank and a waste liquid tank. The extracellular vesicle extraction system is used for extracting extracellular vesicles, and based on the ultrafiltration and microfiltration technology, the extracellular vesicles in the crushed liquid of Chinese herbal medicines or other plants are separated. Compared with the existing differential centrifugation, density gradient centrifugation, ultrafiltration, immunocapture and other technologies, the process is more suitable for large-batch, standardized and high-efficiency extraction, the product yield is high, the speed is fast, the service life of the filter membrane is greatly prolonged, the process cost is reduced, and the process has great commercial value.

Description

Technical Field

[0001] This invention belongs to the field of extracellular vesicle separation technology, specifically relating to an extracellular vesicle extraction system, its application, and a method for extracting extracellular vesicles. Background Technology

[0002] Extracellular vesicles of traditional Chinese medicine and other plants are small vesicles with a phospholipid bilayer structure secreted by living cells, ranging in diameter from 30 to 150 nm. They contain various biomolecules such as proteins, lipids, mRNA, and miRNA. The abundant miRNAs not only perform biological functions in the original system but also regulate cross-border gene expression, thereby altering the physiological or pathological functions of recipient cells and playing an important role in the organism. Studies have shown that extracellular vesicles of traditional Chinese medicine have anti-inflammatory, antiviral, anti-fibrotic, and anti-tumor effects, and can also participate in the defense response against pathogen invasion, making them a potential treatment option.

[0003] Currently, most methods for isolating and purifying extracellular vesicles include ultracentrifugation, density gradient centrifugation, and ultrafiltration. Ultracentrifugation is widely recognized as the "gold standard" for extracellular vesicle extraction, but its pretreatment process (including cell disruption and multiple low-speed centrifugations) is cumbersome, time-consuming, labor-intensive, highly dependent on manual labor, has low recovery rates, and cannot be used for large-scale production. Furthermore, the vesicles in the product exhibit inconsistent morphology and size, and high-speed centrifugation can damage the vesicle structure, affecting downstream product development. Moreover, this method requires sophisticated equipment and is unsuitable for large-scale extraction. Density gradient centrifugation can obtain high-purity extracellular vesicles, but the process is cumbersome, time-consuming, has poor reproducibility, and requires sophisticated equipment, making large-scale, standardized, and high-efficiency extraction impossible. Existing ultrafiltration methods can rapidly extract extracellular vesicles, but there is a probability of large-particle contamination, severely impacting downstream applications. The isolation and purification of extracellular vesicles has always been a focus for researchers. Obtaining high-purity extracellular vesicles in large quantities, efficiently, and in a standardized manner is crucial for subsequent research and product development, but currently, there is no extraction process that can achieve large-scale, standardized, and high-efficiency extraction. Summary of the Invention

[0004] To address the lack of a standardized and efficient method for large-scale extraction of extracellular vesicles in existing technologies, this invention provides an extracellular vesicle extraction system, its application, and a method for extracting extracellular vesicles. Based on ultrafiltration and microfiltration technologies, it separates extracellular vesicles from the lysate of traditional Chinese medicine and other plant materials. Compared with existing methods, it is more suitable for large-scale, standardized, and efficient extraction, and the product yield is high.

[0005] This invention is achieved through the following technical solution: The first aspect of the present invention provides an extracellular vesicle extraction system, which includes a raw material storage tank, a deep filtration device, a continuous flow centrifuge, a pretreatment buffer tank, a first filtration device, a concentrate tank, a second filtration device, a product collection tank, and a waste liquid tank. The feed inlet of the raw material storage tank is connected to the first peristaltic pump, the discharge outlet of the raw material storage tank is connected to the feed inlet of the deep filtration device, the discharge outlet of the deep filtration device is connected to the feed inlet of the continuous flow centrifuge, and the discharge outlet of the continuous flow centrifuge is connected to the liquid feed inlet of the pretreatment buffer tank. The outlet of the pretreatment buffer tank is connected to the inlet of the first filtration device and the waste liquid tank, respectively. The post-membrane liquid outlet of the first filtration device is connected to the inlet of the concentrate tank, and the pre-membrane liquid outlet of the first filtration device is connected to the inlet of the pretreatment buffer tank. The outlet of the liquid reducing tank is connected to the inlet of the second filtration device and the product collection tank, respectively. The post-membrane liquid outlet of the second filtration device is connected to the waste liquid tank, and the pre-membrane liquid outlet of the second filtration device is connected to the inlet of the concentrate tank.

[0006] Furthermore, the discharge port of the raw material storage tank is connected to the inlet of the deep filtration device via a second peristaltic pump, and the discharge port of the deep filtration device is connected to the inlet of the continuous flow centrifuge; the discharge port of the continuous flow centrifuge is connected to the liquid inlet of the pretreatment buffer tank via a third peristaltic pump; the discharge port of the pretreatment buffer tank is connected to the first filtration device via a fourth peristaltic pump; the discharge port of the concentrate tank is connected to the inlet of the second filtration device via a fifth peristaltic pump; the discharge port of the concentrate tank is connected to the product collection tank via a sixth peristaltic pump; and the discharge port of the pretreatment buffer tank is connected to the waste liquid tank via a seventh peristaltic pump.

[0007] Furthermore, a first switch is provided between the pretreatment buffer tank and the fourth peristaltic pump to control the flow of material to the first filtration device; a second switch is provided between the pretreatment buffer tank and the seventh peristaltic pump to control the flow of material to the waste liquid tank; a third switch is provided between the outlet of the concentrate tank and the fifth peristaltic pump to control the flow of material to the second filtration device; a fourth switch is provided between the outlet of the concentrate tank and the sixth peristaltic pump to control the flow of material to the product collection tank; a fifth switch is provided between the pre-membrane liquid outlet of the first filtration device and the inlet of the pretreatment buffer tank; a sixth switch is provided between the solid outlet of the second filtration device and the inlet of the concentrate tank; when the first, third, fifth, and sixth switches are open, the extracellular vesicle extraction system runs the purification program; when the second and fourth switches are open, the sixth peristaltic pump performs the final sample recovery, and the seventh peristaltic pump performs the liquid discharge operation.

[0008] Furthermore, the deep filtration device is filled with diatomaceous earth to clarify and filter samples entering the system, thereby extending the service life of the purification device. The deep filtration device has a thick, high-capacity diatomaceous earth coating that can be reused, saving production costs. The continuous centrifugation device uses a motor to drive a continuous flow rotor, transforming traditional intermittent production into continuous production. By controlling the rotor's centrifugal force and time through a program, it deeply removes large particles, vesicles, and impurities from the feed solution, making the extraction process simple and efficient. The first and second filtration devices are tangential flow filtration systems with filter membranes. The first filtration device is a microfiltration device with a particle size cutoff of 0.22-0.85 μm, eliminating the possibility of large protein contamination in the product of traditional tangential flow filtration. By collecting the feed solution after the membrane, large protein particles are intercepted before the membrane and ultimately discharged from the system. The second filtration device has a molecular weight cutoff of 100-300. kD's ultrafiltration device removes small molecule impurities from the feed liquid through circulating filtration, further improving product purity; different filter membranes with different rejection rates can be selected according to different types of Chinese herbal medicines or other plants.

[0009] Furthermore, the extracellular vesicle extraction system is equipped with a temperature control device. The temperature is controlled by a program, and the entire system, including the raw material storage tank, centrifuge chamber, pretreatment buffer tank, concentrate tank, product collection tank, and waste tank, is controlled by a coil. By controlling the system temperature, the activity of extracellular vesicles in the sample solution is preserved to the greatest extent. The raw material storage tank, pretreatment buffer tank, and concentrate tank are equipped with level sensors. The extracellular vesicle extraction system is equipped with a safety protection system to control the safe liquid level range of the raw material storage tank, pretreatment buffer tank, concentrate tank, product collection tank, and waste tank between 5% and 90%. In the raw material storage tank, when the liquid level is below 5%, the system controls the second peristaltic pump to stop working, subsequently controlling the continuous flow centrifuge to stop and triggering an alarm. When the liquid level is above 90%, the system controls the first peristaltic pump to stop pumping and stop feeding to prevent the liquid level in the tank from becoming too high and posing a risk of overflow.

[0010] Furthermore, pressure sensors are installed between the fourth peristaltic pump and the first filter, and between the fifth peristaltic pump and the second filter. Pressure sensors and a high-low pressure interlock control system are installed after the filter membranes in the first and second filter devices. After receiving the two pressure signals from before and after the filter membrane, the program calculates the transmembrane pressure (transmembrane pressure = (pre-membrane pressure + post-membrane pressure) / 2). The range of the transmembrane pressure is set to 0.3-1.0 bar. The program includes interlocks: a low-pressure interlock occurs when the first filter transmembrane pressure is low (<0.3 bar), and the system controls the fourth peristaltic pump speed to increase at a frequency of 10 r / min. If the pump speed reaches 80% of the maximum load, the system controls the opening of valve 21 to meet the transmembrane pressure. A high-pressure interlock occurs when the first transmembrane pressure is high (>1.0 bar), and the system simultaneously adjusts valve 21 and the pump speed to reduce the transmembrane pressure of the first filter as quickly as possible, protecting the internal membrane structure. Similarly, the second filter also has a high-low pressure interlock function.

[0011] In a second aspect, the present invention provides the application of the aforementioned extracellular vesicle extraction system in the separation and extraction of extracellular vesicles. The extracellular vesicles separated by the extracellular vesicle extraction system can be used in the preparation of medical aesthetic products (facial masks, serums, etc.), dressings to promote wound healing, sprays to relieve asthma and acute respiratory distress syndrome, and other health products and biological products related to extracellular vesicles.

[0012] In a third aspect, the present invention provides a method for extracting extracellular vesicles using an extracellular vesicle extraction system, comprising the following steps: 1) Irradiate Chinese herbal medicines or other plants with ultraviolet light. After irradiation, mix them with PBS buffer, crush them, let them stand, and filter them with gauze and 30-50μm medium-speed filter paper in turn to remove larger fragments. 2) Start the extracellular vesicle extraction system. The filtrate after filtration in step (1) enters the raw material storage tank. When the liquid in the raw material storage tank reaches a certain level, the liquid in the raw material storage tank is filtered by the deep filtration device and sent to the continuous flow centrifuge. The liquid after centrifugation by the continuous flow centrifuge enters the pretreatment buffer tank. 3) After the liquid in the pretreatment buffer tank reaches a certain level, the liquid enters the first filtration device for microfiltration. The liquid after the microfiltration membrane enters the concentrate tank, and the liquid before the membrane that does not pass through the microfiltration membrane returns to the pretreatment buffer tank. 4) After the liquid in the concentrate tank reaches a certain level, the liquid enters the second filtration device for ultrafiltration. The liquid that permeates through the membrane after ultrafiltration enters the waste liquid tank, and the liquid that does not permeate through the membrane returns to the concentrate tank. When the volume of the liquid in the concentrate tank is 1 / 10 to 1 / 20 of the feed volume of the raw material storage tank, ultrafiltration is stopped. At this time, the liquid in the concentrate tank enters the product collection tank to obtain extracellular vesicles. The processing speed of the continuous flow centrifuge is greater than the filtration speed of the first filtration device, and the filtration speed of the first filtration device is greater than the filtration speed of the second filtration device.

[0013] Furthermore, the Chinese herbal medicine or other plant mentioned in step 1) is one of ginseng, Ganoderma lucidum, Phellinus linteus, grape, and cucumber.

[0014] Furthermore, in step 2), before feeding the raw material into the storage tank, the entire system is pre-cooled to 0-5℃; in step 2), the speed of the continuous flow centrifuge is 10000-15000 g, and the residence time is 5-10 min.

[0015] The extracellular vesicle extraction system of this invention includes a transport device, a purification device, a temperature control device, a collection device, and a control system. The transport device consists of seven peristaltic pumps. The purification device includes a deep filtration device and a continuous flow centrifuge device. The purification device includes a first filtration device and a second filtration device, each equipped with a filter membrane of different pore sizes to obtain extracellular vesicles with higher purity. The temperature control device is a system composed of compressors that controls the internal temperature during operation to ensure the activity of extracellular vesicles. The collection device includes the collection of feed liquid at different stages to ensure the smooth operation of the program. The control system is a program that uniformly regulates the operation of the entire process.

[0016] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: This invention utilizes an extracellular vesicle extraction system to extract extracellular vesicles. Based on ultrafiltration and microfiltration technologies, it separates extracellular vesicles from the lysate of traditional Chinese medicine or other plants. Compared with existing technologies such as differential centrifugation, density gradient centrifugation, ultrafiltration, and immunoaffinity capture, this process is more suitable for large-scale, standardized, and efficient extraction. It also has a high product yield, fast speed, and significantly extended filter membrane lifespan, reducing process costs and possessing great commercial value. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the extracellular vesicle extraction system; Figure 2 A schematic diagram of the process for extracting extracellular vesicles; Figure 3 This is a graph showing the NTA detection results of extracellular vesicles extracted in Example 1; Figure 4 This is a transmission electron microscopy image of the extracellular vesicles extracted in Example 1; Figure 5 This is a graph showing the NTA detection results of extracellular vesicles extracted in Example 2; Figure 6 This is a transmission electron microscopy image of the extracellular vesicles extracted in Example 2; Figure 7 This is a BCA detection image of the extracellular vesicles extracted in Example 2; Figure 8 Transmission electron microscopy image of extracellular vesicles extracted in Comparative Example 1; Figure 9 The image shows the NTA detection results of extracellular vesicles extracted in Comparative Example 1. Reference numerals in the attached diagram: 1 is the raw material storage tank, 2 is the deep filtration device, 3 is the continuous flow centrifuge, 4 is the pretreatment liquid buffer tank, 5 is the first filtration device, 6 is the concentrate tank, 7 is the second filtration device, 8 is the product collection tank, 9 is the waste liquid tank, 10 is the first peristaltic pump, 11 is the second peristaltic pump, 12 is the third peristaltic pump, 13 is the fourth peristaltic pump, 14 is the fifth peristaltic pump, 15 is the sixth peristaltic pump, 16 is the seventh peristaltic pump, 17 is the first switch, 18 is the second switch, 19 is the third switch, 20 is the fourth switch, 21 is the fifth switch, and 22 is the sixth switch. Detailed Implementation

[0018] The present invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following examples are generally performed under conventional conditions or as recommended by the manufacturer.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. All reagents and materials used in this invention are readily available through conventional means, and unless otherwise specified, they shall be used in accordance with conventional methods in the art or as per the product instructions.

[0020] A schematic diagram of the extracellular vesicle extraction system in this invention is shown below. Figure 1 As shown, by Figure 1As can be seen, the extracellular vesicle extraction system includes a raw material storage tank 1, a depth filtration device 2, a continuous flow centrifuge 3, a pretreatment buffer tank 4, a first filtration device 5, a concentrate tank 6, a second filtration device 7, a product collection tank 8, and a waste liquid tank 9; the inlet of the raw material storage tank 1 is connected to a first peristaltic pump 10, the outlet of the raw material storage tank 1 is connected to the inlet of the depth filtration device 2, the outlet of the depth filtration device 2 is connected to the inlet of the continuous flow centrifuge 3, and the outlet of the continuous flow centrifuge 3 is connected to the liquid inlet of the pretreatment buffer tank 4. The outlet of the pretreatment buffer tank 4 is connected to the inlet of the first filter device 5 and the waste liquid tank 9, respectively. The post-membrane liquid outlet of the first filter device 5 is connected to the inlet of the concentrate tank 6, and the pre-membrane liquid outlet of the first filter device 5 is connected to the inlet of the pretreatment buffer tank 4. The outlet of the concentrate tank 6 is connected to the inlet of the second filter device 7 and the product collection tank 8, respectively. The post-membrane liquid outlet of the second filter device 7 is connected to the waste liquid tank 9, and the pre-membrane liquid outlet of the second filter device 7 is connected to the inlet of the concentrate tank 6.

[0021] In this invention, the outlet of the raw material storage tank 1 is connected to the inlet of the deep filtration device 2 via a second peristaltic pump 11, and the outlet of the deep filtration device 2 is connected to the inlet of the continuous flow centrifuge 3; the outlet of the continuous flow centrifuge 3 is connected to the liquid inlet of the pretreatment buffer tank 4 via a third peristaltic pump 12; the outlet of the pretreatment buffer tank 4 is connected to the first filtration device 5 via a fourth peristaltic pump 13; the outlet of the concentrate tank 6 is connected to the inlet of the second filtration device 7 via a fifth peristaltic pump 14; the outlet of the concentrate tank 6 is connected to the product collection tank 8 via a sixth peristaltic pump 15; and the outlet of the pretreatment buffer tank 4 is connected to the waste liquid tank 9 via a seventh peristaltic pump 16.

[0022] A first switch 17 is provided between the pretreatment buffer tank 4 and the fourth peristaltic pump 13 to control the flow of material to the first filter device 5; a second switch 18 is provided between the pretreatment buffer tank 4 and the seventh peristaltic pump 16 to control the flow of material to the waste liquid tank 9; a third switch 19 is provided between the outlet of the concentrate tank 6 and the fifth peristaltic pump 14 to control the flow of material to the second filter device; a fourth switch 20 is provided between the outlet of the concentrate tank 6 and the sixth peristaltic pump 15 to control the flow of material to the product collection tank 8; a fifth switch 21 is provided between the pre-membrane liquid outlet of the first filter device 5 and the inlet of the pretreatment buffer tank 4; and a sixth switch 22 is provided between the pre-membrane liquid outlet of the second filter device 7 and the inlet of the concentrate tank 6.

[0023] The deep filtration device 2 is filled with diatomaceous earth; the first filtration device 5 and the second filtration device 7 are equipped with tangential flow filtration systems with filter membranes; the first filtration device 5 is a microfiltration device that retains particles with a diameter of 0.22-0.85μm; the second filtration device 7 is an ultrafiltration device that retains molecules with a molecular weight of 100-300 kD.

[0024] The extracellular vesicle extraction system is equipped with a temperature control device; the raw material storage tank 1, the pretreatment buffer tank 4, and the concentrate tank 6 are equipped with liquid level sensors; the extracellular vesicle extraction system is equipped with a safety protection system to control the liquid level of the raw material storage tank 1, the pretreatment buffer tank 4, the concentrate tank 6, the product collection tank 8, and the waste liquid tank 9 within a safe range of 5-90%.

[0025] In this invention, pressure sensors are installed between the fourth peristaltic pump 13 and the first filter device 5, and between the fifth peristaltic pump 14 and the second filter device 7. Pressure sensors and a high-low pressure interlock control system are installed after the filter membranes in the first filter device 5 and the second filter device 7. After receiving the two pressure signals before and after the filter device, the program calculates the transmembrane pressure (transmembrane pressure = (pre-membrane pressure + post-membrane pressure) / 2). The range of the transmembrane pressure is set to 0.3-1.0 bar. The program includes interlocks, where the low-pressure interlock is as follows: when the first filter transmembrane pressure is low (<0.3 bar), the system controls the fourth peristaltic pump speed to increase at a frequency of 10 r / min. If the pump speed reaches 80% of the maximum load, the system will control the opening of valve 21 to meet the transmembrane pressure. The high-pressure interlock is as follows: when the first transmembrane pressure is high (>1.0 bar), the system will simultaneously adjust valve 21 and pump speed to reduce the transmembrane pressure of the first filter device as quickly as possible, protecting the internal membrane structure. Similarly, the second filter device also has a high-low pressure interlock function.

[0026] A flowchart illustrating the method for extracting extracellular vesicles is shown below. Figure 2 As shown, by Figure 2 As can be seen, the method for extracting extracellular vesicles includes sample washing, sample disruption, solid-liquid separation, sample injection, and product collection. The method for extracting extracellular vesicles using the extracellular vesicle extraction system of this invention is described in specific embodiments.

[0027] Example 1 Extraction of extracellular vesicles from Ganoderma lucidum: 1) Clean the fresh Ganoderma lucidum (15 kg), let it stand, and irradiate it with ultraviolet light for 30 min. Then cut the Ganoderma lucidum into coin-sized pieces, mix it with PBS buffer, crush it with a crusher, let it stand, and after standing for 2 h, filter it with gauze (4 layers) and 30-50 μm medium-speed filter paper to remove larger Ganoderma lucidum fragments, leaving 35 L of filtrate. 2) Start the extracellular vesicle extraction system and pre-cool it 30 minutes in advance. Control the system temperature to 4±1℃. Turn on the first peristaltic pump 10. The filtrate after step 1) enters the raw material storage tank 1. When the liquid in the raw material storage tank 1 is greater than 500mL, the second peristaltic pump 11 starts and puts the liquid in the raw material tank 1 into the deep filtration device 2 for filtration. The filtered liquid is sent to the continuous flow centrifuge 3. Set the speed of the continuous flow centrifuge 3 to 1000g and the residence time to 5min. The third peristaltic pump 12 sends the centrifuged liquid to the pretreatment liquid storage tank 4. When the continuous flow centrifuge 3 is emptied, the second peristaltic pump 11 starts and feeds continuously. 3) When the liquid volume of the feed solution in the pretreatment buffer tank 4 reaches 500mL, the first switch 17, the fifth switch 21 and the fourth peristaltic pump 13 are turned on, and the feed solution enters the first filter device 5 for microfiltration (the pore size of the microfiltration membrane in the first filter device is 0.22μm). The liquid after the microfiltration membrane enters the concentrate tank 6, and the liquid before the membrane that has not passed through the microfiltration membrane returns to the pretreatment buffer tank 4. After the first switch 17, the fifth switch 21 and the fourth peristaltic pump 13 are turned off, the second switch 18 and the seventh peristaltic pump 16 are turned on, and the liquid before the membrane in the pretreatment buffer tank 4 enters the waste liquid tank 9. 4) When the volume of the liquid in the concentrate tank 6 reaches 500mL, the third switch 19, the sixth switch 22 and the fifth peristaltic pump 14 are turned on, and the liquid enters the second filter device 7 (the ultrafiltration membrane in the second filter device has a pore size of 100KD) for ultrafiltration treatment. The part after the ultrafiltration membrane passes through the membrane enters the waste liquid tank 9, and the part before the membrane does not pass through the ultrafiltration membrane returns to the concentrate tank 6. When the volume of the liquid in the concentrate tank 6 is 1 / 10 of the feed volume of the raw material storage tank 1, the third switch 19, the sixth switch 22 and the fifth peristaltic pump 14 are turned off to stop ultrafiltration. At this time, the fourth switch 20 and the sixth peristaltic pump 15 are turned on, and the liquid in the concentrate tank 6 enters the product collection tank 8 to obtain extracellular vesicles (Ganoderma lucidum extracellular vesicles). The sample processing efficiency is 20L / 60min.

[0028] In this embodiment, the processing speed of the continuous flow centrifuge 3 is greater than the filtration speed of the first filter device 5, and the filtration speed of the first filter device 5 is greater than the filtration speed of the second filter device 7.

[0029] The extracellular vesicles of Ganoderma lucidum obtained in Example 1 were subjected to NTA detection, and the results are as follows: Figure 3 As shown, the particle size distribution curve is single-peaked, the concentration of extracellular vesicles in Ganoderma lucidum is 9.44E+11 particles / mL, and the average particle size is 128.7±6.8 nm; transmission electron microscopy was performed on the extracellular vesicles of Ganoderma lucidum, and the results are as follows. Figure 4 As shown, the extracted Ganoderma lucidum extracellular vesicles exhibit a typical round shape.

[0030] Example 2 Extraction of extracellular vesicles from cucumber cells: 1) Wash 45kg of fresh cucumbers, let them stand, and irradiate them with ultraviolet light for 30 minutes. Then peel the cucumbers and cut them into small pieces of about 5cm. Mix the cucumbers with PBS buffer and crush the peeled cucumbers using a crushing device. Let them stand for 2 hours. Then filter the solution through gauze (4 layers) and 30-50μm medium-speed filter paper to remove larger cucumber fragments. The filtrate is 120L. 2) Start the extracellular vesicle extraction system and pre-cool it 30 minutes in advance. Control the system temperature to 4±1℃. Turn on the first peristaltic pump 10. The filtrate after step 1) enters the raw material storage tank 1. When the liquid in the raw material storage tank 1 is greater than 500mL, the second peristaltic pump 11 starts and puts the liquid in the raw material tank 1 into the deep filtration device 2 for filtration. The filtered liquid is sent to the continuous flow centrifuge 3. Set the speed of the continuous flow centrifuge 3 to 1000g and the residence time to 5min. The third peristaltic pump 12 sends the centrifuged liquid to the pretreatment liquid storage tank 4. When the continuous flow centrifuge 3 is emptied, the second peristaltic pump 11 starts and feeds continuously. 3) When the liquid volume of the feed solution in the pretreatment buffer tank 4 reaches 500mL, the first switch 17, the fifth switch 21 and the fourth peristaltic pump 13 are turned on, and the feed solution enters the first filter device 5 for microfiltration (the pore size of the microfiltration membrane in the first filter device is 0.22μm). The liquid after the microfiltration membrane enters the concentrate tank 6, and the liquid before the membrane that has not passed through the microfiltration membrane returns to the pretreatment buffer tank 4. After the first switch 17, the fifth switch 21 and the fourth peristaltic pump 13 are turned off, the second switch 18 and the seventh peristaltic pump 16 are turned on, and the liquid before the membrane in the pretreatment buffer tank 4 enters the waste liquid tank 9. 4) When the volume of the liquid in the concentrate tank 6 reaches 500mL, the third switch 19, the sixth switch 22, and the fifth peristaltic pump 14 are turned on, and the liquid enters the second filter device 7 (the ultrafiltration membrane in the second filter device has a pore size of 100KD) for ultrafiltration treatment. The portion after the ultrafiltration membrane passes through the membrane enters the waste liquid tank 9, and the portion before the ultrafiltration membrane does not pass through the membrane returns to the concentrate tank 6. When the volume of the liquid in the concentrate tank 6 is 1 / 10 of the feed volume of the raw material storage tank 1, the third switch 19, the sixth switch 22, and the fifth peristaltic pump 14 are turned off to stop ultrafiltration. At this time, the fourth switch 20 and the sixth peristaltic pump 15 are turned on, and the liquid in the concentrate tank 6 enters the product collection tank 8 to obtain extracellular vesicles (cucumber extracellular vesicles). The sample processing efficiency is 50L / 60min.

[0031] In this embodiment, the processing speed of the continuous flow centrifuge 3 is greater than the filtration speed of the first filter device 5, and the filtration speed of the first filter device 5 is greater than the filtration speed of the second filter device 7.

[0032] The extracellular vesicles of cucumber obtained in Example 2 were subjected to NTA detection, and the results are as follows: Figure 5 As shown, the particle size distribution curve is single-peaked, the concentration of cucumber extracellular vesicles is 2.08E+11 particles / mL, and the average particle size is 145.4 ± 3.3 nm; transmission electron microscopy (TEM) analysis of cucumber extracellular vesicles yielded the following results: Figure 6 As shown, the extracted Ganoderma lucidum extracellular vesicles exhibit a typical round morphology. BCA detection results are as follows... Figure 7 As shown, the concentration of extracellular vesicle protein obtained from cucumber cells was 5.47 mg prot / mL (OD value 0.829, diluted 10 times during detection).

[0033] Comparative Example 1 Ultracentrifugation for the extraction of extracellular vesicles from Ganoderma lucidum cells 1) Clean 1 kg of fresh Ganoderma lucidum, let it stand, irradiate it with ultraviolet light for 30 min, then cut the Ganoderma lucidum into coin-sized pieces, mix it with PBS buffer, crush it with a crushing device, let it stand for 2 h. 2) Transfer the supernatant of the lysate to a centrifuge tube, centrifuge at 1000g, 4℃ for 5 min, then centrifuge at 10000g, 4℃ for 30 min, and collect the supernatant. 3) Transfer the collected supernatant to an ultracentrifuge tube and centrifuge at 100,000g, 4°C for 45 minutes. Carefully remove the supernatant, retaining the precipitate at the bottom. Add pre-chilled PBS buffer and gently mix with a pipette. Repeat the ultracentrifugation process, collect the precipitate, and carefully resuspend it in an appropriate amount of pre-chilled PBS buffer to obtain Ganoderma lucidum extracellular vesicles.

[0034] 4) Due to the cumbersome and time-consuming process, the initial sample of 200 mL of Ganoderma lucidum extract, after multiple centrifugations and concentrations, may result in an exosome suspension volume of 1-10 mL, taking approximately 6 hours. In Example 1, the initial sample of 100 L of Ganoderma lucidum extract was processed, and after purification and concentration, the final extracellular vesicle volume was between 4.5-5 L, taking approximately 4.5-5 hours. Compared to the traditional ultracentrifugation method, the extraction system described in this invention is faster, has a larger processing capacity, is easier to operate, and is more suitable for large-scale extraction processes.

[0035] Transmission electron microscopy image of the extracellular vesicles of Ganoderma lucidum prepared by the method in Comparative Example 1 is shown below. Figure 8 As shown, Figure 8As can be seen from the corresponding figure in Example 1, the extracellular vesicles extracted by both methods show a saucer-like double-layered vesicle structure under electron microscopy, with a particle size in the range of 30-150 nm and a clear structure. However, the extracellular vesicles extracted by ultracentrifugation show irregularly shaped impurities, while the extracellular vesicles extracted by the purification system of the present invention have a clean background and no impurity residue.

[0036] The NTA detection results of Ganoderma lucidum extracellular vesicles prepared by the method in Comparative Example 1 are shown in the figure below. Figure 9 As shown. Figure 9 Comparing the graphs with those in Example 1, it can be seen that the extracellular vesicles of Ganoderma lucidum extracted by the system of the present invention exhibit a single-peak particle size distribution curve, a concentration of 9.44E+11 particles / mL, and an average particle size of 128.7 ± 6.8 nm. The extracellular vesicles extracted by the method in Comparative Example 1 also exhibit a single-peak particle size distribution curve, a concentration of 1.65E+11 particles / mL, and an average particle size of 145.3 ± 2.0 nm. This comparison reveals that the extracellular vesicles obtained by the extraction method and system of the present invention, compared to those extracted by the traditional ultracentrifugation method, may contain a large number of large-particle protein particles, resulting in a larger particle size. Therefore, the extracellular vesicles obtained by the extraction system of the present invention have less large-particle protein, a more concentrated particle size distribution, and a higher concentration.

Claims

1. An extracellular vesicle extraction system, characterized in that, The extracellular vesicle extraction system includes a raw material storage tank (1), a deep filtration device (2), a continuous flow centrifuge (3), a pretreatment buffer tank (4), a first filtration device (5), a concentrate tank (6), a second filtration device (7), a product collection tank (8), and a waste liquid tank (9). The outlet of the pretreatment buffer tank (4) is connected to the inlet of the first filter device (5) and the waste liquid tank (9) respectively. The post-membrane liquid outlet of the first filter device (5) is connected to the inlet of the concentrate tank (6). The pre-membrane liquid outlet of the first filter device (5) is connected to the inlet of the pretreatment buffer tank (4). The outlet of the concentrate tank (6) is connected to the inlet of the second filter device (7) and the product collection tank (8) respectively. The liquid outlet after the membrane of the second filter device (7) is connected to the waste liquid tank (9). The liquid outlet before the membrane of the second filter device (7) is connected to the inlet of the concentrate tank (6).

2. The extracellular vesicle extraction system according to claim 1, characterized in that, The outlet of the raw material storage tank (1) is connected to the inlet of the deep filtration device (2) via the second peristaltic pump (11), and the outlet of the deep filtration device (2) is connected to the inlet of the continuous flow centrifuge (3); the outlet of the continuous flow centrifuge (3) is connected to the liquid inlet of the pretreatment buffer tank (4) via the third peristaltic pump (12); the outlet of the pretreatment buffer tank (4) is connected to the first filtration device (5) via the fourth peristaltic pump (13); the outlet of the concentrate tank (6) is connected to the inlet of the second filtration device (7) via the fifth peristaltic pump (14); the outlet of the concentrate tank (6) is connected to the product collection tank (8) via the sixth peristaltic pump (15); and the outlet of the pretreatment buffer tank (4) is connected to the waste liquid tank (9) via the seventh peristaltic pump (16).

3. The extracellular vesicle extraction system according to claim 2, characterized in that, A first switch (17) is provided between the pretreatment buffer tank (4) and the fourth peristaltic pump (13) to control the flow of material to the first filter device (5); a second switch (18) is provided between the pretreatment buffer tank (4) and the seventh peristaltic pump (16) to control the flow of material to the waste liquid tank (9); a third switch (19) is provided between the outlet of the concentrate tank (6) and the fifth peristaltic pump (14) to control the flow of material to the second filter device; a fourth switch (20) is provided between the outlet of the concentrate tank (6) and the sixth peristaltic pump (15) to control the flow of material to the product collection tank (8); a fifth switch (21) is provided between the pre-membrane liquid outlet of the first filter device (5) and the inlet of the pretreatment buffer tank (4); a sixth switch (22) is provided between the pre-membrane liquid outlet of the second filter device (7) and the inlet of the concentrate tank (6).

4. The extracellular vesicle extraction system according to claim 1, characterized in that, The deep filtration device (2) is filled with diatomaceous earth; the first filtration device (5) and the second filtration device (7) are equipped with tangential flow filtration systems with filter membranes; the first filtration device (5) is a microfiltration device that retains particles with a diameter of 0.22-0.85μm; the second filtration device (7) is an ultrafiltration device that retains molecules with a molecular weight of 100-300 kD.

5. The extracellular vesicle extraction system according to claim 1, characterized in that, The extracellular vesicle extraction system is equipped with a temperature control device; the raw material storage tank (1), the pretreatment buffer tank (4) and the concentrate tank (6) are equipped with liquid level sensors; the extracellular vesicle extraction system is equipped with a safety protection system to control the liquid level of the raw material storage tank (1), the pretreatment buffer tank (4), the concentrate tank (6), the product collection tank (8) and the waste liquid tank (9) within a safe range of 5-90%.

6. The extracellular vesicle extraction system according to claim 1, characterized in that, Pressure sensors are provided between the fourth peristaltic pump (13) and the first filter device (5), and between the fifth peristaltic pump (14) and the second filter device (7); pressure sensors and high and low pressure interlocking control systems are provided after the filter membranes in the first filter device (5) and the second filter device (7).

7. The use of the extracellular vesicle extraction system according to any one of claims 1 to 6 in the separation and extraction of extracellular vesicles.

8. A method for extracting extracellular vesicles using the extracellular vesicle extraction system according to any one of claims 1 to 6, characterized in that, Includes the following steps: 1) Irradiate Chinese herbal medicines or other plants with ultraviolet light. After irradiation, mix them with PBS buffer, crush them, let them stand, and filter them with gauze and 30-50μm medium-speed filter paper in turn. 2) Start the extracellular vesicle extraction system. The filtrate after filtration in step (1) enters the raw material storage tank (1). When the liquid in the raw material storage tank (1) reaches a certain level, the liquid in the raw material storage tank (1) is filtered by the deep filtration device (2) and sent to the continuous flow centrifuge (3). The liquid after centrifugation by the continuous flow centrifuge (3) enters the pretreatment buffer tank (4). 3) After the liquid in the pretreatment buffer tank (4) reaches a certain level, the liquid enters the first filtration device (5) for microfiltration. The liquid after the microfiltration membrane enters the concentrate tank (6), and the liquid before the membrane that has not passed through the microfiltration membrane returns to the pretreatment buffer tank (4). 4) After the liquid in the concentrate tank (6) reaches a certain level, the liquid enters the second filter device (7) for ultrafiltration. The liquid after passing through the ultrafiltration membrane enters the waste liquid tank (9), and the liquid before passing through the ultrafiltration membrane returns to the concentrate tank (6). When the volume of the liquid in the concentrate tank (6) is 1 / 10-1 / 20 of the feed volume of the raw material storage tank (1), ultrafiltration is stopped. At this time, the liquid in the concentrate tank (6) enters the product collection tank (8) to obtain extracellular vesicles. The processing speed of the continuous flow centrifuge (3) is greater than the filtration speed of the first filter device (5), and the filtration speed of the first filter device (5) is greater than the filtration speed of the second filter device (7).

9. The method for extracting extracellular vesicles according to claim 8, characterized in that, The Chinese herbal medicine or other plant mentioned in step 1) is one of ginseng, Ganoderma lucidum, Phellinus linteus, grape, or cucumber.

10. The method for extracting extracellular vesicles according to claim 8, characterized in that, Step 2) Before feeding the raw material storage tank (1), the entire system is pre-cooled to 0-5℃; in Step 2), the speed of the continuous flow centrifuge (3) is 10000-15000g and the residence time is 5-10min.

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