A tangential flow membrane separation apparatus and method for exosome isolation

Abstract

The application relates to an exosome separation device, in particular to a tangential flow membrane separation device and method for exosome separation. The device comprises a first filter device and a second filter device, and a primary filtrate bin is arranged between the first filter device and the second filter device, and the primary filtrate bin is communicated with the first filter device and the second filter device through pipelines; the first filter device and the second filter device are structured as follows: a raw liquid bin, an outlet of the raw liquid bin is communicated with an inlet of a tangential flow filtration module through a first pipeline, a first outlet of the tangential flow filtration module is communicated with a second pipeline, a second outlet of the tangential flow filtration module is communicated with a third pipeline, the second pipeline is communicated with a backflow pipeline and a waste liquid pipeline through a first electromagnetic valve, the third pipeline is connected with a cleaning pipeline and a collection pipeline through a second electromagnetic valve, and the cleaning pipeline is communicated with a cleaning liquid bin. The device combines the cleaning and filtering processes in one pipeline, reduces the cost, and makes the pipeline use more reasonable by controlling the power-on and power-off of the electromagnetic valves.

Description

Technical Field

[0001] This invention relates to an exosome separation device, and more particularly to a tangential flow membrane separation device and method for exosome separation. Background Technology

[0002] Exosomes are lipid bilayer microvesicles with diameters ranging from 30 to 150 nm. They are released from cells into the extracellular matrix via exocytosis and participate extensively in normal physiological and pathological processes. In recent years, with the advancement of biomedical research, exosomes have been shown to carry bioactive molecules such as proteins, nucleic acids, and lipids from their source cells, serving as key mediators of intercellular communication. Particularly in the field of traditional Chinese medicine, exosome-like nanoparticles derived from Chinese medicinal herbs have shown great clinical application potential in targeted drug delivery systems and adjuvant disease treatment due to their low immunogenicity, good biocompatibility, and natural pharmacological activity. Among them, exosomes secreted by high-value Chinese medicinal herbs such as ginseng have been shown to be rich in unique pharmacologically active substances, demonstrating extremely high application prospects in areas such as tissue repair and anti-inflammation.

[0003] Exosomes are a primary medium for intercellular communication and have shown great clinical potential in disease diagnosis and targeted drug delivery. However, achieving efficient and high-purity large-scale separation of exosomes has been a major technical challenge limiting their clinical translation. Traditional ultracentrifugation methods suffer from time consumption and low recovery rates. Membrane separation technology, due to its ease of scale-up and operation, has become a key focus of engineering research to address this issue. However, current membrane separation equipment often encounters severe membrane fouling and flux decline during actual operation. Inappropriate pore size matching or a coarse flow field design can damage the structure of exosomes under high shear forces and result in excessive residual proteins. Therefore, it is necessary to comprehensively develop and optimize the equipment configuration and cascade filtration parameters. Membrane rinsing is required during separation, but the rinsing pipelines need to be set up separately. Only by setting up separate pipelines can the high instantaneous flow rate required for backwashing of ultrafiltration external pressure membranes be avoided, as sharing pipelines can lead to insufficient pressure and uneven backwashing.

[0004] Existing equipment suffers from physical shear force damage: Exosomes are relatively fragile, and pressure pulsations generated by the pumping system and fluid shear forces within the pipelines, if poorly designed, can directly damage the structural integrity of the vesicles. Membrane fouling and flux decline: When processing high-concentration traditional Chinese medicine separation solutions or cell culture media, membrane pores are easily clogged by proteins and lipids, causing a rapid decrease in filtration efficiency, thus requiring additional cleaning devices. Low equipment integration: Existing commercial separation systems are typically large and expensive.

[0005] Therefore, the main challenge encountered in the clinical translation of exosomes is achieving high purity and large-scale separation. This is because exosome particles are very small and have low density. These properties are similar to some protein complexes. Therefore, developing a physical separation device and method that can accurately separate nanoscale vesicles with industrial scale-up potential and precise sieving is not only a pressing need in the field of bioengineering, but also promotes the modernization of research on exosomes from traditional Chinese medicine. Summary of the Invention

[0006] The present invention aims to solve the above-mentioned technical problems by providing a tangential flow membrane separation device and method for exosome separation, with the goal of achieving pollution-free process, high separation purity, high separation yield, and simple structure.

[0007] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution: A tangential flow membrane separation device for exosome separation includes a first filtration device and a second filtration device. A primary filtrate chamber is provided between the first and second filtration devices, and the primary filtrate chamber is connected to the first and second filtration devices respectively through pipelines. The first and second filtration devices are structured as follows: a raw material tank, the outlet of which is connected to the inlet of the tangential flow filtration module through a first pipeline; a first outlet of the tangential flow filtration module is connected to a second pipeline; a second outlet of the tangential flow filtration module is connected to a third pipeline; the second pipeline is connected to a return pipeline and a waste liquid pipeline through a first solenoid valve; the third pipeline is connected to a cleaning pipeline and a collection pipeline through a second solenoid valve; and the cleaning pipeline is connected to a cleaning liquid tank.

[0008] Furthermore, a magnetic pump is installed on the first pipeline. A manual valve is installed on the first pipeline between the magnetic pump and the raw liquid silo. A third solenoid valve is installed on the first pipeline between the magnetic pump and the tangential flow filter module. The third solenoid valve is also connected to the drain pipeline. The magnetic pump is driven by an electric motor. A flow valve is installed on the second pipeline to regulate the flow rate of the liquid in the second pipeline and control the pressure.

[0009] Furthermore, a gear pump is installed on the cleaning pipeline between the cleaning fluid tank and the second solenoid valve, and the gear pump is driven by an electric motor.

[0010] Furthermore, the tangential flow filtration module is equipped with three sets of filtration chambers, each containing a tubular alumina ceramic membrane, which enables tangential flow filtration as the liquid moves from top to bottom.

[0011] Furthermore, the tangential flow filtration module in the first filtration device is equipped with a 200nm alumina ceramic membrane, and the tangential flow filtration module in the second filtration device is equipped with a 30nm alumina ceramic membrane.

[0012] Furthermore, the magnetic pump of the second filtration device is replaced by a peristaltic pump.

[0013] Furthermore, the collection pipe of the first filtration device is connected to the inlet of the primary filtrate chamber, and the outlet of the primary filtrate chamber is connected to the inlet of the raw material chamber of the second filtration device; the collection pipe of the second filtration device is connected to the inlet of the waste liquid chamber.

[0014] Furthermore, the filtrate exiting from the bottom of the tangential flow filtration module of the first and second filtration devices is returned to the raw material silo for recycling filtration via a return pipe.

[0015] Furthermore, the first, second, and third solenoid valves are three-way solenoid valves. When the first solenoid valve is energized, the second pipe connects to the return pipe. When the second solenoid valve is energized, the third pipe connects to the collection pipe. When the third solenoid valve is energized, the first pipe sends the raw liquid to the tangential flow filtration module.

[0016] A separation method using a tangential flow membrane separation device for exosome separation includes the following steps: S1. Break the cell walls of the raw materials, centrifuge to remove large fibers and cell debris, and take the supernatant; S2. The supernatant is filtered in the first filtration device: the supernatant is sent to the raw material tank of the first filtration device, the gear pump is turned off, the first solenoid valve, the second solenoid valve and the third solenoid valve are energized, and the magnetic suction pump is turned on to send the liquid to the tangential flow filtration module. The liquid moves from top to bottom within the tangential flow filtration module. During this movement, the liquid undergoes tangential flow filtration. The liquid flows out from the bottom of the tangential flow filtration module and passes through the second pipe, the first solenoid valve, and the return pipe to return to the raw material tank of the first filtration device for circulation filtration. Liquid smaller than 200nm flows out from the side wall of the tangential flow filtration module and passes through the third pipe, the second solenoid valve, and the collection pipe before entering the primary filtrate tank. The filtrate in the primary filtrate tank is the exosome liquid smaller than 200nm obtained by tangential flow filtration through a 200nm alumina ceramic membrane. S3. The liquid in the primary filtrate tank is sent to the second filtration device for filtration: In the raw material tank of the second filtration device, the gear pump is turned off, the first solenoid valve, the second solenoid valve and the third solenoid valve are energized, and the magnetic suction pump is turned on to send the liquid to the tangential flow filtration module. The liquid moves from top to bottom within the tangential flow filtration module, undergoing tangential flow filtration during its movement. The liquid flows out from the bottom of the tangential flow filtration module, passing sequentially through the second pipe, the first solenoid valve, and the return pipe back to the raw material tank of the second filtration device for further circulation filtration. Liquid smaller than 30nm flows out from the side wall of the tangential flow filtration module, passing sequentially through the third pipe, the second solenoid valve, and the collection pipe before entering the waste liquid tank. The filtrate in the waste liquid tank is the liquid smaller than 30nm obtained through tangential flow filtration using a 30nm alumina ceramic membrane. Circulation filtration in the second filtration device reduces the liquid volume in the raw material tank of the second filtration device, allowing the liquid smaller than 30nm to be filtered and discharged, while the exosome liquid (30-200nm) remains in the raw material tank of the second filtration device. S4. When the tangential flow filter modules in the first and second filter devices need to be flushed, turn off the magnetic pump and turn on the gear pump to de-energize the first and second solenoid valves. At this time, the cleaning liquid in the cleaning tank flows into the tangential flow filter module through the gear pump, the second solenoid valve and the third pipe to back-flush the alumina ceramic membrane. After the flushing liquid passes through the alumina ceramic membrane, it is discharged from the bottom of the tangential flow filter module and discharged from the waste liquid pipe through the second pipe and the first solenoid valve. S5. When the liquid in the raw material tank of the first filter device needs to be discharged, the third solenoid valve of the first filter device is de-energized and discharged from the drain pipe of the first filter device. S6. When the 30-200nm exosome liquid in the raw material tank of the second filter needs to be discharged, the third solenoid valve of the second filter is de-energized and discharged from the drain pipe of the second filter. When the membrane flux of the tangential flow filtration module (3) of the first and second filtration devices mentioned above decreases by 20%, reverse flushing is performed.

[0017] Advantages and effects of the present invention: This invention combines the cleaning and filtration processes into a single pipeline, reducing costs, and makes the use of the pipeline more efficient by controlling the energization and de-energization of the solenoid valve.

[0018] By setting up two sets of filtration devices, two independently operating filtration systems were established. Each filtration device is equipped with a magnetic pump and a gear pump for main circulation and pressure compensation, thereby achieving complete decoupling of the power system.

[0019] This invention optimizes the process flow, employing a two-stage, step-by-step approach: a first filtration device for coarse filtration to remove impurities, and a second filtration device for concentration and purification. Separation and cleaning are achieved by controlling the energization and de-energization of the solenoid valve.

[0020] After filtration by this invention, the exosomes of traditional Chinese medicine can be rapidly separated, thereby ensuring a recovery rate of over 90%. Moreover, the separation process does not significantly damage the vesicle morphology. This invention incorporates hypochlorous acid cleaning during the washing process, which rapidly degrades the proteins attached to the membrane surface under strong oxidizing action, thereby achieving the goal of high-efficiency chemical regeneration of the membrane flux. Attached Figure Description

[0021] Figure 1 This is a connection diagram of the first filtration device of the present invention.

[0022] Figure 2 This is a connection diagram of the second filtration device of the present invention.

[0023] Figure 3 This is the particle size distribution spectrum of the finished product.

[0024] Figure 4 This is an image of ginseng exosomes under a transmission electron microscope.

[0025] In the diagram, 1. Raw material tank; 2. First pipeline; 3. Tangential flow filtration module; 4. Second pipeline; 5. Third pipeline; 6. Cleaning pipeline; 7. Cleaning liquid tank; 8. Magnetic pump; 9. Gear pump; 10. Finished product tank; 11. Return pipeline; 12. Collection pipeline; 13. Waste liquid pipeline; 14. Flow valve; 15. First solenoid valve; 16. Second solenoid valve; 17. Third solenoid valve; 18. Drain pipeline; 19. Waste liquid tank; 20. Primary filtrate tank; 21. Manual valve. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1

[0027] like Figure 1 and 2As shown, a tangential flow membrane separation device for exosome separation includes a first filtration device and a second filtration device. A primary filtrate chamber 20 is provided between the first and second filtration devices, and the primary filtrate chamber 20 is connected to both the first and second filtration devices via pipes. The first and second filtration devices are structured as follows: a raw material tank 1, the outlet of which is connected to the inlet of a tangential flow filtration module 3 via a first pipe 2. The tangential flow filtration module 3 in the first filtration device is equipped with a 200nm alumina ceramic membrane, and the tangential flow filtration module 3 in the second filtration device is equipped with a 30nm alumina ceramic membrane. This allows the collection of exosome liquid in the 30-200nm range to be achieved in two steps. In this embodiment, the tangential flow filtration module 3 has three sets of filtration chambers, each equipped with a tubular alumina ceramic membrane, so that tangential flow filtration is formed as the liquid moves from top to bottom.

[0028] The first outlet of the tangential flow filter module 3 is connected to the second pipe 4, and the second outlet of the tangential flow filter module 3 is connected to the third pipe 5. The first outlet of the tangential flow filter module 3 is located at the bottom of the tangential flow filter module 3, and the second outlet of the tangential flow filter module 3 is located on the side wall of the tangential flow filter module 3. This results in the filtered liquid and the unfiltered liquid flowing out from different outlets. The second pipe 4 is connected to the return pipe 11 and the waste liquid pipe 13 through the first solenoid valve 15, which is connected to the differential relay KD2. The third pipe 5 is connected to the cleaning pipe 6 and the collection pipe 12 through the second solenoid valve 16, which is connected to the differential relay KD1. The cleaning pipe 6 is connected to the cleaning liquid tank 7.

[0029] To power the first and second filtration devices, a magnetic pump 8 is installed on the first pipe 2. A manual valve 21 is installed on the first pipe 2 between the magnetic pump 8 and the raw material tank 1. A third solenoid valve 17 is installed on the first pipe 2 between the magnetic pump 8 and the tangential flow filtration module 3. The third solenoid valve 17 is connected to a time relay KT1 and is connected to an empty pipe 18. The magnetic pump 8 is driven by an electric motor. A gear pump 9 is installed on the cleaning pipe 6 between the cleaning liquid tank 7 and the second solenoid valve 16, and the gear pump 9 is driven by an electric motor. Thus, when liquid filtration is required, the magnetic pump 8 is turned on and the gear pump 9 is turned off; when backwashing of the tangential flow filtration module 3 is required, the magnetic pump 8 is turned off and the gear pump 9 is turned on. The magnetic pump is used to reduce mechanical damage to exosomes. The gear pump is used for cleaning, can instantly generate greater cleaning pressure, and can control intermittent backwashing, resulting in higher efficiency, lower cost, and higher reliability.

[0030] In order to control the pressure in the pipeline, a flow valve 14 is installed on the second pipeline 4 to regulate the flow rate of the liquid in the second pipeline 4 and control the pressure.

[0031] To address the issue of pressure differences during the two filtration processes, this embodiment performs the two filtrations separately. Therefore, the collection pipe 12 of the first filter device is connected to the inlet of the primary filtrate chamber 20, and the outlet of the primary filtrate chamber 20 is connected to the inlet of the raw material chamber 1 of the second filter device. The collection pipe 12 of the second filter device is connected to the inlet of the waste liquid chamber 19. Thus, the filtrate exiting from the bottom of the tangential flow filtration module 3 of the first and second filters returns to the raw material chamber 1 for recirculation filtration via the return pipe 11. However, the liquid entering the primary filtrate chamber 20 from the first filter device is the primary filtrate, while the 30-200nm exosome liquid remains in the raw material chamber 1 after continuous recirculation filtration in the second filter device, and finally enters the finished product chamber 10 via the drain pipe 18.

[0032] When the first and second filtration devices are filtering, the first solenoid valve 15, the second solenoid valve 16 and the third solenoid valve 17 are three-way solenoid valves. After the first solenoid valve 15 is energized, the second pipe 4 is connected to the return pipe 11. After the second solenoid valve 16 is energized, the third pipe 5 is connected to the collection pipe 12. After the third solenoid valve 17 is energized, the first pipe 2 sends the raw liquid to the tangential flow filtration module 3.

[0033] When using the above-mentioned equipment for separation, 5-year-old ginseng roots were used as the source of plant-derived exosome-like nanoparticles (ELNs). The medicinal materials were purchased from Jilin City, Jilin Province. The alumina ceramic membrane was an inorganic alumina ceramic membrane with a porosity of 30% for the support and 40% for the membrane layer.

[0034] Includes the following steps: S1. Break the cell walls of the raw materials and centrifuge them continuously for 30 minutes at a relative centrifugal force of 3000 times the gravitational acceleration to remove large fibers and cell debris. Take the supernatant and filter it through a 5μm security filter.

[0035] S2. The supernatant is filtered in the first filtration device: The supernatant is sent to the raw material tank 1 of the first filtration device, the gear pump 9 is turned off, the first solenoid valve 15, the second solenoid valve 16 and the third solenoid valve 17 are energized, and the magnetic suction pump 8 is turned on to send the liquid to the tangential flow filtration module 3.

[0036] The liquid moves from top to bottom within the tangential flow filtration module 3, undergoing tangential flow filtration during its movement. The liquid flows out from the bottom of the tangential flow filtration module 3, passing sequentially through the second pipe 4, the first solenoid valve 15, and the return pipe 11 back to the raw material tank 1 of the first filtration device for circulation filtration. Liquid smaller than 200nm flows out from the side wall of the tangential flow filtration module 3, passing sequentially through the third pipe 5, the second solenoid valve 16, and the collection pipe 12 before entering the primary filtrate tank 20. The filtrate in the primary filtrate tank 20 is the exosome clear liquid smaller than 200nm obtained through tangential flow filtration using a 200nm alumina ceramic membrane. The operating parameters of the first filtration device are: flow rate: 150ml / min, temperature: 4-18℃, pressure: 0.12-0.14Mpa. Backwashing is required when the membrane flux of the tangential flow filtration module 3 decreases by 20%. This ensures the working efficiency of the tangential flow filtration module 3.

[0037] S3. The liquid in the primary filtrate tank 20 is sent to the second filtration device for filtration: In the raw liquid tank 1 of the second filtration device, the gear pump 9 is turned off, the first solenoid valve 15, the second solenoid valve 16 and the third solenoid valve 17 are energized, and the magnetic suction pump 8 is turned on to send the liquid to the tangential flow filtration module 3.

[0038] Liquid flows downwards within the tangential flow filtration module 3, undergoing tangential flow filtration during its movement. The liquid flows out from the bottom of the module 3, passing sequentially through the second pipe 4, the first solenoid valve 15, and the return pipe 11 back to the raw material tank 1 of the second filtration device for further circulation filtration. Liquid smaller than 30nm flows out from the side wall of the module 3, passing sequentially through the third pipe 5, the second solenoid valve 16, and the collection pipe 12 before entering the waste liquid tank 19. The filtrate in the waste liquid tank 19 is the liquid smaller than 30nm obtained through tangential flow filtration using a 30nm alumina ceramic membrane. Circulation filtration in the second filtration device reduces the liquid volume in the raw material tank 1, allowing small molecule impurities smaller than 30nm to pass through the membrane and be discharged. Exosomes are efficiently concentrated in the loop, with the final 30-200nm exosome liquid remaining in the raw material tank 1 of the second filtration device. The operating parameters of the second filtration device are: flow rate: 90-150ml / min, temperature: 4-18℃, pressure: 0.1-0.12. MPa; Backwashing is required when the membrane flux of the tangential flow filter module 3 decreases by 20%. This ensures the working efficiency of the tangential flow filter module 3.

[0039] S4. When the tangential flow filter module 3 in the first and second filter devices needs to be rinsed, turn off the magnetic pump 8 and turn on the gear pump 9 to de-energize the first solenoid valve 15 and the second solenoid valve 16. At this time, the cleaning liquid in the cleaning tank 7 flows into the tangential flow filter module 3 through the gear pump 9, the second solenoid valve 16 and the third pipe 5 to backwash the alumina ceramic membrane. After entering the alumina ceramic membrane, the rinsing liquid is discharged from the bottom of the tangential flow filter module 3 and discharged from the waste liquid pipe 13 through the second pipe 4 and the first solenoid valve 15. The temperature during rinsing is 18-25 degrees Celsius, the flow rate is 300-500 ml / min, and 3L of RO water is used each time for 3 consecutive times. The forward flow follows the filtration process with 3L of RO water. Disinfection: use 200ppm sodium hypochlorite, pressure 0.12-0.15Mpa, duration 30min, and then rinse again with RO water.

[0040] S5. When the liquid in the raw material tank 1 of the first filter device needs to be discharged, the third solenoid valve 17 of the first filter device is de-energized and discharged from the drain pipe 18 of the first filter device.

[0041] S6. When the 30-200nm exosome liquid in the raw material tank 1 of the second filter device needs to be discharged, the third solenoid valve 17 of the second filter device is de-energized and discharged from the drain pipe 18 of the second filter device into the finished product tank 10. Example 2

[0042] In this embodiment, the magnetic pump in the second filtration device is replaced by a peristaltic pump. When the exosome liquid is sent to the tangential flow filtration module 3, the concentration of exosomes increases during the concentration process. Using a peristaltic pump can reduce damage to the exosomes. Other aspects are the same as in Embodiment 1.

[0043] The following tests were performed on the filtered exosome fluid at a density of 30-200 nm. 100 μL of the concentrated sample from the finished product container was taken.

[0044] The ginseng exosome products collected by the second filtration device were detected using nanoparticle tracking analysis (NTA) technology (ZetaView). To ensure detection accuracy, the collected concentrate was diluted 10-fold with ultra-clean PBS buffer before injection analysis.

[0045] like Figure 3The NTA detection results show that the median particle size (X50) of the extracted product is 109.2 nm, and the average particle size is 120.4 nm (standard deviation 49.1 nm). The particle size distribution spectrum shows an excellent single-peak distribution, with a core peak at 105.3 nm, where the particle count is as high as 100%. Based on the range of three standard deviations, the particles are mainly distributed within the upper limit of 267.7 nm (120.4 nm ± 147.3 nm). Furthermore, logarithmic coordinate analysis confirms that although approximately 90% of the particles are concentrated below 176.2 nm (X90 = 176.2 nm), the peak tail does extend smoothly to the right to around 500 nm due to the aggregation of proteins and exosomes after separation, forming larger particles. However, most impurities larger than 200 nm are retained by the 200 nm membrane. This distribution characteristic not only accurately reflects the detection results, but also closely matches the classic size and natural morphological characteristics of plant-derived exosome-like nanoparticles (TCM-ELNs).

[0046] The extremely high initial particle concentration and precise particle size distribution directly confirm the effectiveness of this invention. On one hand, most of the detected particle sizes were controlled within the 3σ distribution upper limit of 267.7 nm, indicating that the 200 nm alumina ceramic membrane in the first filtration device effectively achieved the initial filtration and retention of large molecular impurities and coarse cell debris; on the other hand, the particle size distribution was as high as 5.1 × 10⁻⁶. 8 The final concentration of particles / mL indicates that the 30nm alumina ceramic membrane on the second filtration device, driven by a stable feed from a magnetic pump, not only avoids vesicle rupture caused by mechanical shearing, but also achieves extremely high retention and concentration of the target exosomes.

[0047] To visually assess the morphology and structural integrity of the separated products, the ginseng exosome concentrate finally collected by the second filtration device was treated with traditional phosphotungstic acid negative staining and then observed under a transmission electron microscope (TEM).

[0048] like Figure 4 As shown, at a 100 nm scale reference, numerous clearly visible nanovesicle particles are visible in the field of view. Due to the drying and dehydration effect during traditional negative staining sample preparation, these ginseng-derived exosomes do not exhibit perfect spherical shapes, but rather a classic "teacup" or unilaterally concave saucer-like morphology. The black arrows in the figure clearly indicate the deeper contours of the vesicle edges, representing the typical lipid bilayer structure of exosomes. Furthermore, the particle diameters observed in the field of view are generally distributed within the 50-150 nm range, which highly matches the particle size distribution results of previous NTA detection, confirming that the separated substances conform to the standard physical characteristics of ginseng exosome-like nanoparticles (G-ELNs).

[0049] TEM results show that almost all vesicles maintained intact "teacup-shaped" edges, and no obvious fragments of vesicle rupture caused by mechanical stretching were found in the background. This crucial microscopic morphological evidence strongly demonstrates the scientific merit and superiority of the device's dynamic decoupling design: this non-tangential, large-circulation, low-shear transport mode successfully avoids physical tearing of sensitive biofilms caused by high-velocity water flow, thereby maximally protecting the fragile vesicle structure of ginseng exosomes.

[0050] In order to preserve the physical integrity of ginseng exosomes to the greatest extent, the parameters of the second filtration device of this invention are determined as follows: transmembrane pressure 0.1-0.12 MPa, circulation flow rate 90-150 ml / min (5-9 L / h), and material temperature 4-18℃.

[0051] (1) Optimization of transmembrane pressure: When the operating pressure is stably controlled at 0.1-0.12 MPa, not only can sufficient mass transfer driving force be provided to maintain high membrane flux, but also the concentration polarization exacerbation or the rapid thickening of the membrane gel layer caused by excessive pressure can be effectively avoided. In addition, the mild pressure environment of 0.1-0.12 MPa effectively prevents leakage of highly flexible exosome nanoparticles due to excessive compression, ensuring a very high target recovery rate.

[0052] (2) Flow rate and velocity control: The feed flow rate is set to 90-150 ml / min (i.e., 5-9 L / h), which is highly matched with the internal flow channel structure of the 30 nm alumina ceramic membrane module. The moderate cross-flow velocity can generate reasonable micro-fluid shear force on the membrane surface, dynamically flushing away small molecule impurities that attempt to deposit on the membrane surface, thereby significantly slowing down the rate of membrane flux decay. At this flow rate, the system operates smoothly, avoiding physical tearing of sensitive vesicle structures by severe mechanical turbulence.

[0053] (3) Low-temperature environment protection: The material temperature is strictly controlled within the low-temperature range of 4-18℃ throughout the filtration process. The low-temperature environment is crucial for maintaining the biological activity and morphological integrity of natural ginseng exosomes (TCM-ELNs), which can effectively inhibit the degradation of plant endogenous enzymes and the tendency of vesicles to accumulate heat, thereby ensuring the natural quality of the extracted products.

[0054] During the operation of the second filtration device, a large amount of free herbal plant proteins can pass through the 30 nm alumina ceramic membrane without hindrance and be discharged with the clear permeate. More importantly, because the first and second filtration devices are completely decoupled in terms of physical pressure, the feed of the second filtration device strictly relies on the constant flow delivery of the magnetic pump, completely eliminating fluid pulses that may be transmitted from the upstream coarse filtration stage. This extremely stable transmembrane pressure difference (TMP) effectively delays the concentration polarization and adsorption of impurity proteins on the ceramic membrane surface and within the pores, maximizing the physical sieving function of the 30 nm membrane and ultimately achieving high-purity enrichment of ginseng-derived exosomes.

Claims

1. A tangential flow membrane separation device for exosome separation, characterized in that... The system includes a first filter device and a second filter device. A primary filtrate chamber (20) is provided between the first filter device and the second filter device. The primary filtrate chamber (20) is connected to the first filter device and the second filter device through pipes. The first filter device and the second filter device are structured as follows: a raw material tank (1), the outlet of the raw material tank (1) is connected to the inlet of the tangential flow filter module (3) through the first pipe (2), the first outlet of the tangential flow filter module (3) is connected to the second pipe (4), the second outlet of the tangential flow filter module (3) is connected to the third pipe (5), the second pipe (4) is connected to the return pipe (11) and the waste liquid pipe (13) through the first solenoid valve (15), the third pipe (5) is connected to the cleaning pipe (6) and the collection pipe (12) through the second solenoid valve (16), and the cleaning pipe (6) is connected to the cleaning liquid tank (7).

2. The tangential flow membrane separation device for exosome separation according to claim 1, characterized in that... The first pipe (2) is equipped with a magnetic pump (8), and a manual valve is provided on the first pipe (2) between the magnetic pump (8) and the raw liquid silo (1). A third solenoid valve (17) is provided on the first pipe (2) between the magnetic pump (8) and the tangential flow filter module (3). The third solenoid valve (17) is also connected to the drain pipe (18). The magnetic pump (8) is driven by an electric motor. The second pipe (4) is equipped with a flow valve (14). The flow valve (14) is used to adjust the flow rate of the liquid on the second pipe (4) and control the pressure.

3. The tangential flow membrane separation device for exosome separation according to claim 1, characterized in that... A gear pump (9) is installed on the cleaning pipe (6) between the cleaning liquid tank (7) and the second solenoid valve (16), and the gear pump (9) is driven by an electric motor.

4. A tangential flow membrane separation device for exosome separation according to claim 1, characterized in that... The tangential flow filtration module (3) is equipped with 3 sets of filtration chambers, each of which is equipped with a tubular alumina ceramic membrane, so that tangential flow filtration is formed when the liquid moves from top to bottom.

5. A tangential flow membrane separation device for exosome separation according to claim 4, characterized in that... The tangential flow filter module (3) in the first filter device is equipped with a 200nm alumina ceramic membrane, and the tangential flow filter module (3) in the second filter device is equipped with a 30nm alumina ceramic membrane.

6. A tangential flow membrane separation device for exosome separation according to claim 2, characterized in that, The magnetic pump in the second filtration device is replaced by a peristaltic pump.

7. A tangential flow membrane separation device for exosome separation according to claim 4, characterized in that... The collection pipe (12) of the first filter device is connected to the inlet of the primary filtrate tank (20), and the outlet of the primary filtrate tank (20) is connected to the inlet of the raw material tank (1) of the second filter device; the collection pipe (12) of the second filter device is connected to the inlet of the waste liquid tank (19).

8. A tangential flow membrane separation device for exosome separation according to claim 5 or 7, characterized in that... The filtrate from the bottom of the tangential flow filtration module (3) of the first and second filtration devices returns to the raw material silo (1) through the return pipe (11) for circulation filtration.

9. A tangential flow membrane separation device for exosome separation according to claim 1, characterized in that... The first solenoid valve (15), the second solenoid valve (16) and the third solenoid valve (17) are three-way solenoid valves. After the first solenoid valve (15) is energized, the second pipe (4) is connected to the return pipe (11). After the second solenoid valve (16) is energized, the third pipe (5) is connected to the collection pipe (12). After the third solenoid valve (17) is energized, the first pipe (2) sends the raw liquid to the tangential flow filtration module (3).

10. A separation method for a tangential flow membrane separation device for exosome separation according to any one of claims 1-9, characterized in that... Includes the following steps: S1. Break the cell walls of the raw materials, centrifuge to remove large fibers and cell debris, and take the supernatant; S2. The supernatant is filtered in the first filter device: The supernatant is sent to the raw material silo (1) of the first filter device, the gear pump (9) is turned off, the first solenoid valve (15), the second solenoid valve (16) and the third solenoid valve (17) are energized, and the magnetic suction pump (8) is turned on to send the liquid to the tangential flow filter module (3). The liquid moves from top to bottom in the tangential flow filtration module (3). During the movement, the liquid undergoes tangential flow filtration. The liquid flows out from the bottom of the tangential flow filtration module (3) and passes through the second pipe (4), the first solenoid valve (15) and the return pipe (11) to return to the raw liquid tank (1) of the first filtration device for circulation filtration. Liquid smaller than 200nm flows out from the side wall of the tangential flow filtration module (3) and passes through the third pipe (5), the second solenoid valve (16) and the collection pipe (12) before entering the primary filtrate tank (20). The filtrate in the primary filtrate tank (20) is the exosome liquid smaller than 200nm obtained by tangential flow filtration through a 200nm alumina ceramic membrane. S3. The liquid in the primary filtrate tank (20) is sent to the second filtration device for filtration: In the raw liquid tank (1) of the filtrate to be sent to the second filtration device, the gear pump (9) is turned off, the first solenoid valve (15), the second solenoid valve (16) and the third solenoid valve (17) are energized, and the magnetic suction pump (8) is turned on to send the liquid to the tangential flow filtration module (3). The liquid moves from top to bottom in the tangential flow filtration module (3). During the movement, the liquid undergoes tangential flow filtration. The liquid flows out from the bottom of the tangential flow filtration module (3) and passes through the second pipe (4), the first solenoid valve (15) and the return pipe (11) to return to the raw liquid tank (1) of the second filter device for circulation filtration. Liquid smaller than 30nm flows out from the side wall of the tangential flow filtration module (3) and passes through the third pipe (5), the second solenoid valve (16) and the collection pipe (12) before entering the waste liquid tank (19). The filtrate in the waste liquid tank (19) is the liquid smaller than 30nm obtained by tangential flow filtration through a 30nm alumina ceramic membrane. After circulation filtration in the second filter device, the liquid volume in the raw liquid tank (1) of the second filter device is reduced. Liquid smaller than 30nm is discharged after filtration, while exosome liquid of 30-200nm remains in the raw liquid tank (1) of the second filter device. S4. When the tangential flow filter module (3) in the first filter device and the second filter device needs to be flushed, turn off the magnetic pump (8) and turn on the gear pump (9) to de-energize the first solenoid valve (15) and the second solenoid valve (16). At this time, the cleaning liquid in the cleaning liquid tank (7) flows into the tangential flow filter module (3) through the gear pump (9), the second solenoid valve (16) and the third pipe (5) to back-flush the alumina ceramic membrane. After the flushing liquid passes through the alumina ceramic membrane, it is discharged from the bottom of the tangential flow filter module (3) and discharged from the waste liquid pipe (13) through the second pipe (4) and the first solenoid valve (15). S5. When the liquid in the raw material tank (1) of the first filter device needs to be discharged, the third solenoid valve (17) of the first filter device is de-energized and discharged from the drain pipe (18) of the first filter device. S6. When the 30-200nm exosome liquid in the raw material tank (1) of the second filter device needs to be discharged, the third solenoid valve (17) of the second filter device is de-energized and discharged from the drain pipe (18) of the second filter device. When the throughput of the tangential flow filtration module (3) of the first and second filtration devices mentioned above decreases by 20%, backwashing is performed.

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