An extracellular vesicle continuous separation and purification device and application method

Abstract

The present invention provides a continuous extracellular vesicle separation and purification device, comprising an interconnected extracellular vesicle separation and purification device and a main control module. The extracellular vesicle separation and purification device comprises an ultrafiltration module for separating extracellular vesicles of different particle size ranges, a pressurized inlet module for providing positive pressure ultrafiltration power and positive pressure driving power for fluid flow within the ultrafiltration module, a concentration end negative pressure aspirator module for providing negative pressure suction power for fluid flow within the ultrafiltration module, a filtration end negative pressure aspirator module for providing negative pressure ultrafiltration power within the ultrafiltration module, and a pipeline temperature control module for maintaining the temperature of the liquid to be separated in the pipeline to maintain the quality of the extracellular vesicles. The device described in the present invention can continuously maintain the filtration efficiency of the ultrafiltration membrane, achieving the purpose of fully enclosed continuous flow high-throughput differential pore size series dynamic ultrafiltration separation and purification of extracellular vesicles under the conditions of positive pressure at the inlet end and negative pressure at the concentration end and filtration end.

Description

Technical Field

[0001] The present invention belongs to the technical field of extracellular vesicle separation and purification, and specifically relates to an extracellular vesicle continuous separation and purification device and an application method. Background Art

[0002] Extracellular vesicles (EVs) are small vesicles released by human, animal, plant, or microbial cells either spontaneously or in response to physical, chemical, or biological factors. They have a lipid bilayer membrane structure and range in diameter from tens of nanometers to several micrometers. They include exosomes, microvesicles, apoptotic bodies, shedding vesicles, and microparticles. EVs carry biologically active proteins, lipids, messenger RNA (mRNA), microRNA (miRNA), non-coding RNA (ncRNA), and DNA fragments. They can deliver these active biomolecules to recipient cells, thereby modulating the biological functions of target cells.

[0003] Extracellular vesicles are a new type of biotherapeutic agent and drug delivery carrier. For example, the extracellular vesicles produced by mesenchymal stem cells (MSCs) contain a variety of active biomolecules that can inhibit inflammatory responses, protect damaged tissues, promote growth and repair, and regulate immune function. Clinically, they can be used as biotherapeutic agents to inhibit inflammatory responses, reduce tissue damage, promote tissue regeneration and restore physiological functions. Extracellular vesicles can also be used as drug delivery carriers, using electroporation, chemical perforation, ultrasonic perforation, nano-microfluidics and other methods to load artificially synthesized small interfering RNA (siRNA), miRNA, chemotherapy drugs, etc. for disease treatment. It has now been verified in animal models that certain extracellular vesicles can promote angiogenesis, reduce inflammatory responses, promote nerve regeneration, reduce myocardial and cerebral ischemic damage, protect acute kidney injury and lung injury, and treat diabetes and neurodegenerative diseases.

[0004] Currently, commonly used methods for separating and purifying extracellular vesicles include ultrahigh-speed refrigerated centrifugation, rotary ultrafiltration, size exclusion chromatography, immunomagnetic capture, polymer precipitation, and membrane affinity capture. Ultrahigh-speed refrigerated centrifugation distinguishes extracellular vesicles from other components in the liquid to be separated based on their size and density, using an ultrahigh-speed refrigerated centrifuge to separate them. This method requires expensive equipment, complex procedures, is time-consuming, and has a low recovery rate. While rotary ultrafiltration can isolate extracellular vesicles of high purity and requires relatively simple equipment, the ultrafiltration membrane is prone to clogging, leading to a rapid decrease in separation and purification efficiency. Size exclusion chromatography is a screening method based on extracellular vesicle particle size, resulting in high product purity but inability to concentrate extracellular vesicles. Immunomagnetic capture isolates extracellular vesicles using antibodies that specifically bind to extracellular vesicle surface antigens. This method has high specificity, but is expensive and susceptible to differences in antigen expression, resulting in significant differences in capture efficiency for different subtypes of extracellular vesicles. The polymer precipitation method uses polymers such as polyethylene glycol (PEG) to change the microenvironment in the liquid to be separated, reducing the suspension stability of extracellular vesicles and allowing them to settle and separate. This method does not require large-scale equipment, but a large amount of polymerized impurities will settle together with the extracellular vesicles, seriously affecting the purity of the product. The above extracellular vesicle separation and purification methods have different advantages and disadvantages, but they generally have disadvantages such as complex operation, long time consumption, low yield, and poor purity, which greatly limit the further application of extracellular vesicles in the biomedical field. Therefore, it is very necessary to develop an efficient and low-cost ultra-high throughput, continuous operation, and size-classified extracellular vesicle industrial-grade separation and purification technology. Summary of the Invention

[0005] The present invention solves the shortcomings of the existing technology of separating and purifying extracellular vesicles, such as complex operation, long time consumption, low yield, poor purity, and difficulty in achieving size classification, and provides an extracellular vesicle continuous separation and purification device and an application method.

[0006] In a first aspect, the present invention provides a continuous separation and purification device for extracellular vesicles, comprising an extracellular vesicle separation and purification device and a main control module, wherein the extracellular vesicle separation and purification device is electrically connected to the main control module, and the extracellular vesicle separation and purification device comprises an ultrafilter module, a pressurized liquid inlet module, a concentration end negative pressure aspirator module, and a filtration end negative pressure aspirator module, wherein the ultrafilter module is connected to the pressurized liquid inlet module, the concentration end negative pressure aspirator module, and the filtration end negative pressure aspirator module by pipeline.

[0007] The pressurized liquid inlet module is used to provide positive pressure ultrafiltration power in the ultrafilter module and positive pressure driving power for the fluid flow in the upper chamber of the ultrafilter.

[0008] The concentrating end negative pressure suction module provides negative pressure suction power for the flow of fluid in the upper chamber of the ultrafilter module.

[0009] The negative pressure suction module at the filtering end provides negative pressure ultrafiltration power in the ultrafilter module.

[0010] The ultrafilter module is used to separate extracellular vesicles of different particle size ranges.

[0011] In some embodiments, the extracellular vesicle separation and purification device includes at least two stages of extracellular vesicle separation and purification devices connected in series, the pore size of the ultrafiltration membrane of the previous stage extracellular vesicle separation and purification device is larger than the pore size of the ultrafiltration membrane of the next stage extracellular vesicle separation and purification device, the liquid in the filter liquid bag of the previous stage extracellular vesicle separation and purification device enters the booster liquid inlet module of the adjacent next stage extracellular vesicle separation and purification device, and the concentrated liquid of each adjacent next stage is collected into the corresponding concentrated liquid bag, so that multi-stage continuous separation and purification of extracellular vesicles can be performed to obtain extracellular vesicles of different particle size ranges.

[0012] In some embodiments, the ultrafilter module includes an ultrafilter and a pipeline, the ultrafilter is provided with a liquid inlet connected to the pressurized liquid inlet module, a concentrated liquid port connected to the concentration end negative pressure suction module, and a filtered liquid port connected to the filtration end negative pressure suction module, the ultrafilter is provided with an ultrafiltration membrane, the upper chamber of the ultrafiltration membrane is above the ultrafiltration membrane, and the lower chamber of the ultrafiltration membrane is below the ultrafiltration membrane.

[0013] The ultrafilter is any one of a flat plate serpentine dynamic ultrafilter, a flat plate volute dynamic ultrafilter and a hollow fiber membrane shell and tube dynamic ultrafilter.

[0014] The upper chamber of the ultrafilter in the flat serpentine dynamic ultrafilter is a repeatedly bent serpentine chamber, one end of the serpentine chamber is connected to the liquid inlet, the other end of the serpentine chamber is connected to the concentrated liquid outlet, and the lower chamber of the ultrafiltration membrane is connected to the filtered liquid outlet.

[0015] The upper chamber of the ultrafilter in the flat spiral dynamic ultrafilter is a spiral spiral chamber from the center to the outside, the center of the spiral spiral chamber is connected to the liquid inlet, the outer end of the spiral spiral chamber is connected to the concentrated liquid outlet, and the lower chamber of the ultrafiltration membrane is connected to the filtered liquid outlet.

[0016] The hollow fiber membrane shell and tube dynamic ultrafilter includes an internal pressure hollow fiber membrane shell and tube dynamic ultrafilter and an external pressure hollow fiber membrane shell and tube dynamic ultrafilter. The internal pressure hollow fiber membrane shell and tube dynamic ultrafilter consists of a membrane tube shell, a tube sheet head and a hollow fiber membrane. The hollow fiber membrane is directly encapsulated in the membrane tube shell at both ends by two tube sheet heads. One end of the hollow fiber membrane tube cavity is collected in the front chamber cavity and connected to the liquid inlet, and the other end is collected in the rear chamber cavity and connected to the concentrated liquid port. The outer chamber cavity between the hollow fiber membrane and the membrane tube shell is connected to the filtered liquid port. ; The external pressure hollow fiber membrane shell and tube dynamic ultrafilter consists of a membrane tube shell, a tube sheet head, an end cover and a hollow fiber membrane. The hollow fiber membrane is directly encapsulated in the membrane tube shell at both ends by the tube sheet head and the end cover. One end of the outer chamber cavity between the hollow fiber membrane and the membrane tube shell is connected to the liquid inlet, and the other end of the outer chamber cavity between the hollow fiber membrane and the membrane tube shell is connected to the concentrated liquid port. One end of the hollow fiber membrane tube cavity is closed by the end cover, and the other end of the hollow fiber membrane tube cavity is collected in the filter chamber cavity and connected to the filtered liquid port.

[0017] In some embodiments, the pore size of the ultrafiltration membrane is 10 nm to 4000 nm.

[0018] In some embodiments, the booster liquid inlet module includes a booster pump, a buffer bag, a reflux liquid bag, a multi-way valve and a pipeline. The extracellular vesicle liquid bag, the buffer bag and the reflux liquid bag are collected into the multi-way valve through the pipeline. The extracellular vesicle liquid bag is an extracellular vesicle stock liquid bag or a filtered liquid bag in the extracellular vesicle separation and purification device of the previous level. The multi-way valve is connected to the booster pump through a pipeline. The booster pump is connected to the liquid inlet of the ultrafilter through a pipeline. A pressure sensor is connected in series between the booster pump and the liquid inlet of the ultrafilter. The pressure sensor is electrically connected to the main control module.

[0019] The booster pump is any one of a diaphragm pump, a peristaltic pump, a plunger pump and a gear pump, preferably a diaphragm pump or a peristaltic pump.

[0020] In some embodiments, the apparatus for continuous separation and purification of extracellular vesicles further comprises auxiliary weighing sensors corresponding to the extracellular vesicle stock solution bag, the buffer solution bag, the reflux solution bag, and the filtered solution bag, wherein the auxiliary weighing sensors are electrically connected to the main control module.

[0021] The auxiliary weighing sensor is preferably a suspended weighing sensor.

[0022] In some embodiments, the concentration end negative pressure aspirator module includes a concentration negative pressure bottle, a negative pressure aspirator, an air filter, a multi-way valve, a concentrate bag and a pipeline. The concentration negative pressure bottle is provided with a negative pressure exhaust port, a liquid inlet and a liquid discharge port. The negative pressure exhaust port is connected to the negative pressure aspirator through a pipeline. The pipeline between the negative pressure exhaust port and the negative pressure aspirator is connected in series with an air filter, a multi-way valve, an air vent, an air pressure buffer and an air pressure sensor. The liquid inlet is connected to the concentrate port of the ultrafilter and the reflux liquid bag through a pipeline and a multi-way valve. A pressure sensor is connected in series between the concentrate port of the ultrafilter and the multi-way valve. The pressure sensor is electrically connected to the main control module. The liquid discharge port is connected to the concentrate bag through a pipeline. A solenoid valve and a power pump are connected in series between the liquid discharge port and the concentrate bag.

[0023] The power pump is preferably a peristaltic pump.

[0024] In some embodiments, the concentration end negative pressure aspirator module further includes a weighing sensor, which is provided corresponding to the concentrated negative pressure bottle and the concentrated liquid bag, and is electrically connected to the main control module.

[0025] The weighing sensor corresponding to the concentrated negative pressure bottle is preferably a supporting weighing sensor.

[0026] The weighing sensor provided for the concentrate bag is preferably a suspended weighing sensor;

[0027] The concentrated negative pressure bottle is a hard bottle, which includes a bottle body and a bottle cap. A groove is provided at the bottom of the bottle body. The design of the groove facilitates the accumulation of liquid in the concentrated negative pressure bottle. The shape of the groove is preferably a cone with a wide top and a narrow bottom, or a round bottom with a wide top and a narrow bottom. The negative pressure exhaust port, liquid inlet and discharge port are arranged on the bottle cap.

[0028] In some embodiments, the filtering end negative pressure suction module includes a filtering negative pressure bottle, a negative pressure suction device, an air filter, a multi-way valve, a filtered liquid bag and a pipeline, the filtering negative pressure exhaust port is connected to the negative pressure suction device through a pipeline, the air filter, the multi-way valve, the vent, the air pressure buffer and the air pressure sensor are connected in series on the pipeline between the negative pressure exhaust port and the negative pressure suction device, the liquid inlet is connected to the filtered liquid port of the ultrafilter through a pipeline, a solenoid valve and a pressure sensor are connected in series between the liquid inlet and the filtered liquid port of the ultrafilter, the solenoid valve and the pressure sensor are electrically connected to the main control module, the liquid discharge port is connected to the filtered liquid bag through a pipeline, and a solenoid valve and a power pump are connected in series between the liquid discharge port and the filtered liquid bag.

[0029] The power pump is preferably a peristaltic pump.

[0030] In some embodiments, the filter end negative pressure aspirator module further includes a weighing sensor, which is provided corresponding to the filter negative pressure bottle and the filter liquid bag, and is electrically connected to the main control module.

[0031] The weighing sensor corresponding to the filter negative pressure bottle is preferably a supporting weighing sensor.

[0032] The weighing sensor provided for the filter liquid bag is preferably a suspended weighing sensor;

[0033] The filtering negative pressure bottle is a hard bottle, which includes a bottle body and a bottle cap. A groove is provided at the bottom of the bottle body. The design of the groove facilitates the accumulation of liquid in the filtering negative pressure bottle. The shape of the groove is preferably a cone that is wide at the top and narrow at the bottom, or a round bottom that is wide at the top and narrow at the bottom. The negative pressure exhaust port, liquid inlet and discharge port are arranged on the bottle cap.

[0034] In some embodiments, the extracellular vesicle separation and purification device further includes a pipeline temperature control module, which acts on the pipelines involved in the extracellular vesicle separation and purification device to keep the liquid in the pipelines in a desired temperature environment.

[0035] In some embodiments, the pipeline is a hose, and the pipeline temperature control module includes a winding thermostat and a cold source. The winding thermostat is connected to the cold source, and the pipeline is wound around the winding thermostat.

[0036] In some embodiments, the wrap-around thermostat is made of a metal with good thermal conductivity, and a spiral groove is provided on the surface of the wrap-around thermostat, and the pipeline is wound in the spiral groove; the cold source includes a refrigerated water circulation device and a Peltier semiconductor refrigeration plate, and the refrigerated water circulation device is a low-temperature constant-temperature cold water circulation device. A cold water circulation pipeline is provided inside the wrap-around thermostat, and the cold water circulation pipeline of the low-temperature constant-temperature cold water circulation device is connected to the cold water circulation pipeline of the wrap-around thermostat, and the wrap-around thermostat is cooled by the low-temperature constant-temperature cold water circulation device; the wrap-around thermostat corresponds to the pipeline setting, and the water temperature control range in the low-temperature constant-temperature cold water circulation device is 0 to 8°C.

[0037] The metal is preferably aluminum, copper or a metal alloy.

[0038] In some embodiments, the extracellular vesicle separation and purification device further includes an alarm module and a human-computer interaction module, and the alarm module and the human-computer interaction module are both electrically connected to the main control module.

[0039] The alarm module has visual and audible high and low pressure alarms, which are used to shut down the booster pump, the concentration end negative pressure aspirator, and the filtration end negative pressure aspirator to maintain the integrity of the ultrafiltration membrane and achieve efficient extracellular vesicle separation and purification.

[0040] In a second aspect, the present invention provides an application method based on the above-mentioned extracellular vesicle continuous separation and purification device, comprising the following steps:

[0041] S1: adding the extracellular vesicle stock solution into the extracellular vesicle stock solution bag of the first-stage extracellular vesicle separation and purification device, and the extracellular vesicle stock solution to be separated enters the ultrafilter from the liquid inlet of the ultrafilter through a booster pump;

[0042] S2: The booster inlet module provides positive pressure ultrafiltration power, and the filtration end negative pressure suction module provides negative pressure suction ultrafiltration power. Under the action of the two filtration powers of positive pressure push and negative pressure suction, the impurities in the extracellular vesicle stock solution to be separated whose particle size is larger than the pore size of the ultrafiltration membrane of the first-stage extracellular vesicle separation and purification device are retained, and flow through the concentrated liquid port of the ultrafilter through the multi-way valve, and then enter the concentrated negative pressure bottle for temporary storage. The extracellular vesicles and impurities in the extracellular vesicle stock solution to be separated whose particle size is smaller than the pore size of the ultrafiltration membrane of the first-stage extracellular vesicle separation and purification device pass through the ultrafiltration membrane, and enter the filtration negative pressure bottle for temporary storage through the filtrate port of the ultrafilter;

[0043] S3: After the filtrate obtained by the first-stage extracellular vesicle separation and purification device accumulates in the filtration negative pressure bottle to the rated capacity of the filtration negative pressure bottle, the booster pump and the negative pressure aspirator in the filtration end negative pressure aspirator module stop working, the solenoid valve of the pipeline at the liquid inlet end of the filtration negative pressure bottle is closed, the multi-way valve in the filtration end negative pressure aspirator module closes the negative pressure channel and opens the vent, and the solenoid valve of the pipeline at the liquid discharge end of the filtration negative pressure bottle is opened. Under the action of the power pump, the filtrate in the negative pressure bottle enters the filtration liquid bag;

[0044] S4: If there is a next-stage extracellular vesicle separation and purification device, the filtrate in the filter bag of the previous stage enters the ultrafilter from the liquid inlet of the ultrafilter of the next-stage extracellular vesicle separation and purification device through a booster pump;

[0045] S5: The booster inlet module provides positive pressure ultrafiltration power, and the filtration end negative pressure suction module provides negative pressure suction ultrafiltration power. Under the action of the two filtration powers of positive pressure push and negative pressure suction, the extracellular vesicles in the filtrate of the previous stage to be separated whose particle size is larger than the pore size of the ultrafiltration membrane of the next stage extracellular vesicle separation and purification device are retained, and flow through the concentrated liquid port of the ultrafilter through the multi-way valve, and then enter the concentrated negative pressure bottle for temporary storage. The extracellular vesicles and impurities in the filtrate of the previous stage to be separated whose particle size is smaller than the pore size of the ultrafiltration membrane of the next stage extracellular vesicle separation and purification device pass through the ultrafiltration membrane, and enter the filtration negative pressure bottle for temporary storage through the filtrate port of the ultrafilter;

[0046] S6: After the concentrated liquid obtained by the next-stage extracellular vesicle separation and purification device accumulates in the concentrated negative pressure bottle to the rated capacity of the concentrated negative pressure bottle, the booster pump and the negative pressure aspirator stop working, the solenoid valve of the pipeline at the liquid inlet end of the concentrated negative pressure bottle is closed, the multi-way valve in the negative pressure aspirator module at the concentration end closes the negative pressure channel and opens the vent, and the solenoid valve of the pipeline at the liquid discharge end of the concentrated negative pressure bottle is opened. Under the action of the power pump, the next-stage concentrated liquid in the negative pressure bottle enters the concentrated liquid bag; if there is still an extracellular vesicle separation and purification device at the next stage, S4-S6 are looped until all the extracellular vesicle separation and purification devices in the extracellular vesicle separation and purification device are executed.

[0047] In some embodiments, the rated capacity of the filtration negative pressure bottle refers to 0.5-0.75 times the capacity of the filtration negative pressure bottle; the rated capacity of the concentration negative pressure bottle refers to 0.5-0.75 times the capacity of the concentration negative pressure bottle.

[0048] In some embodiments, during the execution of S1-S6, the main control module monitors the pressure of the ultrafilter inlet, concentrate inlet, and filtered liquid inlet pipelines through pressure sensors, and monitors the weight of the extracellular vesicle stock solution bag, buffer bag, reflux liquid bag, filtered liquid bag, concentrated liquid bag, and the liquid in the filtration negative pressure bottle and the concentration negative pressure bottle through weighing sensors and auxiliary weighing sensors.

[0049] When the main control module senses through the pressure sensor that the pressures at the liquid inlet and concentrate port of the ultrafilter continue to rise compared to the initial state, and the pressure at the filtrate port continues to drop compared to the initial state, and senses through the data of the weighing sensor and the auxiliary weighing sensor that the weight growth rate of the liquid in the filtration negative pressure bottle continues to drop compared to the initial state, and at the same time, the weight growth rate of the liquid in the concentrate negative pressure bottle continues to increase compared to the initial state, the main control module determines that the filtration efficiency of the ultrafilter has dropped, and instructs the extracellular vesicle separation and purification device corresponding to the current filtration negative pressure bottle and the concentrate negative pressure bottle to enter the ultrafiltration membrane cleaning operation mode, that is, by increasing the positive pressure driving power of the booster pump of the booster inlet module of the extracellular vesicle separation and purification device and increasing the negative pressure suction power of the negative pressure aspirator module at the concentration end, the flow velocity of the fluid in the upper chamber of the ultrafilter is accelerated, the shear force between the fluid and the ultrafiltration membrane is increased, and the suspended particles and colloidal substances adsorbed and deposited on the surface of the ultrafiltration membrane are flushed away and taken away, thereby restoring the filtration efficiency of the ultrafiltration membrane;

[0050] The main control module senses through the pressure sensor that the pressures at the liquid inlet, concentrate port, and filtrate port of the ultrafilter have returned to their initial states, and senses through the data of the weighing sensor and the auxiliary weighing sensor that the weight growth rate of the liquid in the filtration negative pressure bottle has returned to its initial state. At the same time, the weight growth rate of the liquid in the concentrate negative pressure bottle has returned to its initial state. The main control module determines that the filtration efficiency of the ultrafilter has recovered, and then calls back the positive pressure driving power of the boosting pump of the boosting liquid inlet module and the negative pressure suction power of the negative pressure aspirator module at the concentration end, so that the corresponding extracellular vesicle separation and purification device returns to the normal operation mode of separation and purification.

[0051] The device for continuous separation and purification of extracellular vesicles described in the present invention has the following advantages:

[0052] 1. Faster processing time, enabling full-closed continuous flow high-throughput differential pore size tandem dynamic ultrafiltration separation and purification of extracellular vesicles under positive pressure at the inlet end and negative pressure at the concentration and filtration ends;

[0053] 2. Outstanding filtration power: the ultrafilter provides positive pressure ultrafiltration power at the liquid inlet end and negative pressure suction ultrafiltration power at the filtration end. Under the action of positive pressure push and negative pressure suction, the ultrafiltration power of the ultrafilter is stable, reliable and strong;

[0054] 3. Maintain high filtration efficiency for a long time. The liquid inlet end of the ultrafilter provides positive pressure driving force for the flow of fluid in the upper chamber of the ultrafilter through the booster pump. The concentrating end of the ultrafilter provides negative pressure suction force for the flow of fluid in the upper chamber of the ultrafilter. Under the action of the two fluid flow forces of positive pressure driving and negative pressure suction, the liquid in the upper chamber of the ultrafilter flows forward rapidly in the upper chamber of the ultrafilter, forming a shear force between the ultrafiltration membrane, preventing the adsorption and deposition of suspended particles and colloidal substances on the surface of the ultrafiltration membrane, overcoming the concentration polarization effect, thereby effectively reducing the attenuation rate of the ultrafiltration membrane flux and maintaining the high filtration efficiency of the ultrafiltration membrane for a long time.

[0055] 4. Ultrafiltration membrane self-cleaning function. This device has an ultrafiltration membrane cleaning operation mode. By increasing the positive pressure driving power of the booster pump of the booster inlet module of the extracellular vesicle separation and purification device and the negative pressure suction power of the negative pressure aspirator module at the concentration end, the flow velocity of the fluid in the upper chamber of the ultrafiltration chamber is accelerated, the shear force between the fluid and the ultrafiltration membrane is increased, and the suspended particles and colloidal substances adsorbed and deposited on the surface of the ultrafiltration membrane are flushed away, thereby restoring the filtration efficiency of the ultrafiltration membrane.

[0056] 5. Separation and purification of extracellular vesicles by different particle sizes: by adjusting adjacent extracellular vesicle separation and purification devices to use ultrafiltration membranes with different pore sizes for multi-stage separation and purification of extracellular vesicles, extracellular vesicles of different particle size ranges can be obtained;

[0057] 6. The concentrated liquid and the filtrate can be obtained at the same time. This device is fully functional and can obtain both the concentrated liquid and the filtrate in one step.

[0058] 7. The production capacity can be expanded directly and simply. This device can be used as a single separation unit or in parallel with multiple separation and purification units, which can increase the production of extracellular vesicle products exponentially.

[0059] 8. Pipeline temperature control function: This device can maintain the liquid to be separated in the pipeline at 0℃-8℃. The lower liquid temperature is beneficial to protecting the quality of extracellular vesicles during the separation and purification process;

[0060] 9. Fully enclosed pipeline architecture: This device completes the entire process of extracellular vesicle separation and purification in a closed pipeline. Extracellular vesicles are separated and purified under sterile conditions. It can be connected and used with extracellular vesicle stock solution production equipment, extracellular vesicle packaging and freeze-drying equipment, etc., truly achieving full-process, fully enclosed production of extracellular vesicle products;

[0061] 10. Highly automated. The microcomputer main control module of this device uses data collection from multiple pressure sensors, suspended load cells, supported load cells, air pressure sensors, temperature sensors, etc. to automatically adjust the device operation in a highly intelligent manner, maintaining efficient and safe separation and purification of extracellular vesicles. The operating data can also be automatically downloaded and plotted to form a spreadsheet of various parameters. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Schematic diagram of the framework structure of an apparatus for continuous separation and purification of extracellular vesicles in some embodiments of the present invention;

[0063] Figure 2 Schematic diagram of the structure of the device for continuous separation and purification of extracellular vesicles in Example 1;

[0064] Figure 3 This is a schematic diagram of the structure of the continuous separation and purification device for extracellular vesicles in Example 2 used in series;

[0065] Figure 4 This is a schematic diagram of the top view of the flat-plate serpentine dynamic ultrafilter, with arrows representing the direction of liquid flow;

[0066] Figure 5 This is a schematic diagram of the flat-plate scroll dynamic ultrafilter from a top view, with arrows representing the direction of liquid flow;

[0067] Figure 6 Schematic diagram of the connection structure between the winding temperature controller and the pipeline. The thin arrows represent the direction of liquid flow in the separation and purification pipeline, and the thick arrows represent the direction of liquid flow in the cold water circulation pipeline.

[0068] Figure 7This is a schematic diagram of the structure of an internal pressure hollow fiber membrane shell and tube dynamic ultrafilter, where the arrows represent the direction of liquid flow;

[0069] Figure 8 This is a schematic diagram of the structure of an external pressure hollow fiber membrane shell and tube dynamic ultrafilter, where the arrows represent the direction of liquid flow. DETAILED DESCRIPTION

[0070] Example 1

[0071] Combine Figure 1 and Figure 2 The content shown in this embodiment is a continuous separation and purification device for extracellular vesicles, including an extracellular vesicle separation and purification device and a main control module. The extracellular vesicle separation and purification device is electrically connected to the main control module. The extracellular vesicle separation and purification device includes an ultrafilter module, a pressurized liquid inlet module, a concentration end negative pressure aspirator module, a filtration end negative pressure aspirator module and a pipeline temperature control module. The ultrafilter module is connected to the pressurized liquid inlet module, the concentration end negative pressure aspirator module, and the filtration end negative pressure aspirator module through pipelines. A pipeline temperature control module is provided on the pipelines involved in the extracellular vesicle separation and purification device, wherein

[0072] The booster inlet module is used to provide positive pressure ultrafiltration power in the ultrafilter module and positive pressure driving power for the fluid flow in the upper chamber of the ultrafilter.

[0073] The negative pressure suction module at the concentration end provides negative pressure suction power for the flow of fluid in the upper chamber of the ultrafilter module.

[0074] The negative pressure suction module at the filtration end provides negative pressure ultrafiltration power in the ultrafilter module.

[0075] Ultrafilter modules are used to separate extracellular vesicles of different size ranges.

[0076] The ultrafilter module includes an ultrafilter and a pipeline. The ultrafilter is an ultrafilter with three ports, namely: a liquid inlet connected to the booster liquid inlet module, a concentrated liquid port connected to the concentration end negative pressure suction module, and a filtered liquid port connected to the filtration end negative pressure suction module.

[0077] Among them, combined Figure 5 As shown in the content, the ultrafilter uses a flat-plate scroll dynamic ultrafilter. The upper chamber of the ultrafilter in the flat-plate scroll dynamic ultrafilter is a spiral scroll chamber from the center to the outside. The center of the scroll chamber is connected to the liquid inlet, the outer end of the scroll chamber is connected to the concentrated liquid port, and the lower chamber of the ultrafiltration membrane is connected to the filtered liquid port.

[0078] The pore size of ultrafiltration membranes ranges from 10 nanometers to 4000 nanometers.

[0079] The above-mentioned ultrafiltration membrane can directly select the ultrafiltration membrane available on the market, for example: ultrafiltration membrane made of cellulose acetate, cellulose acetate ester, polyethylene, polysulfone, polyamide, aromatic polymer, nylon, polyethersulfone, hydrophilic polyvinylidene fluoride, polytetrafluoroethylene, hydrophilic polytetrafluoroethylene, mixed cellulose, hydrophilic polycarbonate, and porous alumina.

[0080] The booster inlet module includes a booster pump, an extracellular vesicle stock solution bag, a buffer bag, a reflux liquid bag, a multi-way valve 1 and a pipeline. The extracellular vesicle stock solution bag, the buffer bag and the reflux liquid bag are collected into the multi-way valve 1 through the pipeline. The multi-way valve 1 is connected to the booster pump 1 through the pipeline. The booster pump 1 is connected to the liquid inlet of the flat-plate scroll dynamic ultrafilter 1 through the pipeline.

[0081] The booster pump 1 is any one of a diaphragm pump, a peristaltic pump, a plunger pump and a gear pump.

[0082] A diaphragm pump or a peristaltic pump is preferred. The diaphragm pump is configured to separate the liquid being transported from the piston and the pump cylinder, thereby maintaining the cleanliness of the liquid during transport. The peristaltic pump is configured so that the fluid only contacts the pipeline and does not contact the pump body, thereby maintaining the cleanliness of the liquid during transport.

[0083] A pressure sensor 1 is connected in series between the booster pump 1 and the liquid inlet of the flat-plate scroll dynamic ultrafilter 1.

[0084] The buffer bag is used to hold buffer, including physiological saline, PBS buffer, Hanks Balanced Salt Solution (HBSS), etc. The buffer bag can balance the ultrafilter and pipeline before extracellular vesicle separation and purification, adjust the concentration of extracellular vesicles in the liquid to be separated during the separation and purification process, flush the upper chamber of the ultrafilter to restore filtration efficiency during the separation and purification process, and flush the pipeline after separation and purification to avoid waste of extracellular vesicles.

[0085] The functions of the reflux liquid bag are: 1. In the process of separating and purifying extracellular vesicles at each stage, the liquid to be separated can be repeatedly circulated through the ultrafilter through the reflux liquid bag, thereby increasing the separation and purification efficiency of extracellular vesicles and avoiding waste; 2. When flushing the upper chamber of the ultrafilter, under the condition that the booster pump provides the positive pressure driving force of fluid flow, the buffer solution can be repeatedly circulated through the reflux liquid bag to flush the ultrafilter, flushing away the suspended particles and colloidal substances adsorbed and deposited on the surface of the ultrafiltration membrane until the ultrafilter restores the filtration efficiency, thereby avoiding excessive waste of buffer solution.

[0086] In some specific implementations, auxiliary weighing sensors will be set corresponding to the extracellular vesicle stock solution bag, buffer solution bag, and reflux liquid bag. The auxiliary weighing sensors are electrically connected to the main control module. The auxiliary weighing sensors are preferably suspended weighing sensors, which are set above the corresponding extracellular vesicle stock solution bag, buffer solution bag, and reflux liquid bag. They are suspended weighing sensor 1, suspended weighing sensor 2, and suspended weighing sensor 3 respectively. Suspended weighing sensor 1, suspended weighing sensor 2, and suspended weighing sensor 3 are used to monitor the changes in the weight of the liquid in the extracellular vesicle stock solution bag, buffer solution bag, and reflux liquid bag, respectively.

[0087] The concentrated end negative pressure suction device module includes a concentrated negative pressure bottle 1, a weighing sensor 1, a negative pressure suction device 1, an air pressure sensor 1, an air pressure buffer 1, an air filter 1, a multi-way valve 3, a concentrated liquid bag and a pipeline. The concentrated negative pressure bottle 1 is provided with a negative pressure exhaust port, a liquid inlet and a liquid discharge port. The negative pressure exhaust port is connected to the negative pressure suction device 1 through a pipeline. The air filter 1, the multi-way valve 3, the air pressure buffer 1 and the air pressure sensor 1 are connected in series on the pipeline between the negative pressure exhaust port and the negative pressure suction device 1. The two ports on the multi-way valve 3 are connected to the air bag respectively. The filter 1 is connected to the air pressure buffer 1, and a port is provided on the multi-way valve 3 as a vent 1. The liquid inlet is connected to the concentrate port and the reflux liquid bag of the flat scroll dynamic ultrafilter 1 through a pipeline and a multi-way valve 2. The discharge port is connected to the concentrate bag through a pipeline. A solenoid valve 1 and a peristaltic pump 1 are connected in series between the discharge port and the concentrate bag. A weighing sensor 1 connected to the main control module is provided below the concentrated negative pressure bottle 1. The weighing sensor 1 is a support type weighing sensor 1, which is used to monitor the weight change of the liquid in the concentrated negative pressure bottle 1.

[0088] A pressure sensor 2 is connected in series between the multi-way valve 2 and the concentrated liquid port of the flat-plate scroll dynamic ultrafilter 1 .

[0089] In some specific implementations, a weighing sensor is provided for the corresponding concentrated liquid bag. The weighing sensor is electrically connected to the main control module. The weighing sensor is preferably a suspended weighing sensor 4, which is provided above the corresponding concentrated liquid bag to monitor the weight change of the liquid in the concentrated liquid bag.

[0090] The filter end negative pressure suction device module includes a filter negative pressure bottle 1, a weighing sensor 2, a negative pressure suction device 2, an air pressure sensor 2, an air pressure buffer 2, an air filter 2, a multi-way valve 4, a filter liquid bag and a pipeline. The filter negative pressure bottle 1 is provided with a negative pressure exhaust port, a liquid inlet and a liquid discharge port. The negative pressure exhaust port is connected to the negative pressure suction device 2 through a pipeline. The air filter 2, the multi-way valve 4, the air pressure buffer 2 and the air pressure sensor 2 are connected in series on the pipeline between the negative pressure exhaust port and the negative pressure suction device 2. The two ports on the multi-way valve 4 are connected to the filter bag respectively. The air filter 2 is connected to the air pressure buffer 2. The multi-way valve 4 is also provided with a port as a vent 2. The liquid inlet is connected to the filtered liquid port of the flat scroll dynamic ultrafilter 1 through a pipeline and an electromagnetic valve 2. The discharge port is connected to the filtered liquid bag through a pipeline. The electromagnetic valve 3 and the peristaltic pump 2 are connected in series between the discharge port and the filtered liquid bag. A weighing sensor 2 connected to the main control module is provided below the filter negative pressure bottle 1. The weighing sensor 2 is a support type weighing sensor 2 used to monitor the weight change of the liquid in the filter negative pressure bottle 1.

[0091] A pressure sensor 3 is connected in series between the solenoid valve 2 and the filtered liquid port of the flat-plate scroll dynamic ultrafilter 1 .

[0092] In some specific implementations, a weighing sensor is provided corresponding to the filter liquid bag. The weighing sensor is electrically connected to the main control module. The weighing sensor is preferably a suspended weighing sensor 5, which is provided above the corresponding filter liquid bag to monitor the weight change of the liquid in the filter liquid bag.

[0093] It should be noted that both the concentrated negative pressure bottle 1 and the filtered negative pressure bottle 1 are hard-bodied bottles, comprising a bottle body and a bottle cap, and the bottom of the bottle body is designed to facilitate the accumulation of liquid in the negative pressure bottle, such as a groove provided at the bottom of the bottle body, through which the liquid at the bottom of the negative pressure bottle is accumulated, wherein the shape of the groove can be a tapered shape with a wide top and a narrow bottom, or a round bottom with a wide top and a narrow bottom, etc. The negative pressure exhaust port, liquid inlet, and liquid discharge port are provided on the bottle cap. The concentrated negative pressure bottle 1 and the filtered negative pressure bottle 1 herein can directly use existing negative pressure bottles on the market.

[0094] The pipeline temperature control module includes a winding type thermostat and a cold source. The winding type thermostat is connected to the cold source. The pipeline adopts a hose design and the pipeline is wound on the winding type thermostat.

[0095] The winding thermostat is made of metal with good thermal conductivity, preferably aluminum, copper or metal alloy, combined with Figure 6The content shown is that the surface of the wound thermostat has spiral grooves, and the pipeline is wound around the wound thermostat along the spiral grooves. A cold water circulation pipeline is provided inside the wound thermostat. The wound thermostat is connected and fixed to the shell or other structure of the extracellular vesicle continuous separation and purification device. The cold water circulation pipeline of the wound thermostat is connected to the cold water circulation pipeline of the cold source device. The setting of the spiral grooves on the surface of the wound thermostat can not only effectively guide the wound pipeline, increase the contact area between the pipeline and the wound thermostat, and improve the heat exchange efficiency, but also can support the wound pipeline through the design of the spiral grooves, effectively reducing the chance of the pipeline wrapped on the wound thermostat slipping;

[0096] The cold source includes a refrigerated water circulation device and a Peltier semiconductor refrigeration sheet. The refrigerated water circulation device is a low-temperature constant-temperature cold water circulation device with a water temperature controlled at 0 to 8°C. The Peltier semiconductor refrigeration sheet is used to reduce the water temperature in the refrigerated water circulation so that the temperature of the circulating water is controlled within the required temperature range. The low-temperature constant-temperature cold water circulation device includes a circulation pump and a flexible conduit, which is connected to the inlet / outlet of the wrap-around thermostat through the flexible conduit. The cold water in the wrap-around thermostat circulates through the circulation pump and the flexible conduit, quickly cooling the wrap-around thermostat.

[0097] There are multiple winding temperature controllers in the extracellular vesicle separation and purification device, which are distributed in various places of the pipeline, such as before the liquid inlet of the flat-plate scroll dynamic ultrafilter 1, before the inlet of the concentrate bag, and before the inlet of the filtered liquid bag.

[0098] In addition to the above-mentioned suspended weighing sensor 1, suspended weighing sensor 2, suspended weighing sensor 3, suspended weighing sensor 4, suspended weighing sensor 5, supporting weighing sensor 1, supporting weighing sensor 2, pressure sensor 1, pressure sensor 2, pressure sensor 3, air pressure sensor 1 and air pressure sensor 2, in the specific implementation, a working environment temperature sensor is set on the extracellular vesicle separation and purification device to detect the room temperature, and a circulating water temperature sensor is set in the low-temperature constant temperature cold water circulation device of the pipeline temperature control module. These circulating water temperature sensors, working environment temperature sensors and the above-mentioned suspended weighing sensor 1, suspended weighing sensor 2, suspended weighing sensor 3, suspended weighing sensor 4, suspended weighing sensor 5, supporting weighing sensor 1, supporting weighing sensor 2, pressure sensor 1, pressure sensor 2, pressure sensor 3, air pressure sensor 1 and air pressure sensor 2 form a sensor detection module, and the sensor detection module is connected to the main control module.

[0099] A microcomputer can be directly used as the above-mentioned main control module.

[0100] In some specific embodiments, the apparatus for continuous separation and purification of extracellular vesicles further includes an alarm module and a human-computer interaction module, which are connected to the main control module. The human-computer interaction module includes an operation control panel and / or a touch screen. The alarm module can emit an audible and / or visual alarm signal. The human-computer interaction module adjusts and sets reference thresholds for various parameters and values detected by the sensor detection module. Once the data detected by the sensor detection module exceeds the reference threshold, the alarm module emits an alarm signal.

[0101] The application method of the extracellular vesicle continuous separation and purification device comprises the following steps:

[0102] S1.1: Add the extracellular vesicle stock solution to the extracellular vesicle stock solution bag of the extracellular vesicle separation and purification device. The extracellular vesicle stock solution to be separated enters the flat-plate cochlear dynamic ultrafilter 1 from the liquid inlet of the flat-plate cochlear dynamic ultrafilter 1 through the booster pump 1;

[0103] S1.2: The booster inlet module provides positive pressure ultrafiltration power, and the filtration end negative pressure suction module provides negative pressure suction ultrafiltration power. Under the action of the two filtration powers of positive pressure push and negative pressure suction, the impurities in the extracellular vesicle stock solution to be separated whose particle size is larger than the pore size of the ultrafiltration membrane of the extracellular vesicle separation and purification device are intercepted, and flow through the concentrated liquid port at the end of the spiral volute cavity of the flat-plate volute dynamic ultrafilter 1, through the multi-way valve 2, and then enter the concentrated negative pressure bottle 1 for temporary storage. The extracellular vesicles and impurities in the extracellular vesicle stock solution to be separated whose particle size is smaller than the pore size of the ultrafiltration membrane of the extracellular vesicle separation and purification device pass through the ultrafiltration membrane, and enter the filtration negative pressure bottle 1 for temporary storage through the filtrate port of the flat-plate volute dynamic ultrafilter 1;

[0104] S1.3: After the filtrate obtained by the extracellular vesicle separation and purification device accumulates to a certain amount in the filtration negative pressure bottle (the value range of the certain amount is 0.5-0.75 times the capacity of the filtration negative pressure bottle, for example, 2 / 3 times), the booster pump 1 and the negative pressure aspirator 1 stop working, the solenoid valve 3 is closed, the multi-way valve 4 closes the negative pressure channel, the vent 2 is opened, the solenoid valve 3 is opened, and under the action of the peristaltic pump 2, the filtrate in the filtration negative pressure bottle 1 enters the filtrate bag.

[0105] During the execution of S1.1-S1.3, the main control module monitors the pressure of the ultrafilter inlet, concentrate outlet, and filtrate outlet pipelines through pressure sensors 1, 2, and 3, respectively, and monitors the weight changes of the extracellular vesicle stock solution bag, buffer bag, reflux liquid bag, filtrate bag, concentrate bag, as well as the filtration negative pressure bottle and the concentration negative pressure bottle through weighing sensors and auxiliary weighing sensors.

[0106] When the main control module senses through pressure sensor 1, pressure sensor 2, and pressure sensor 3 respectively that the pressures at the liquid inlet and concentrate outlet of the ultrafilter continue to rise compared with the initial state, and the pressure at the filtrate outlet continues to drop compared with the initial state, and senses through the data changes of the weighing sensor and the auxiliary weighing sensor that the weight growth rate of the liquid in the filtration negative pressure bottle 1 continues to drop compared with the initial state, and at the same time, the weight growth rate of the liquid in the concentrate negative pressure bottle 1 continues to increase compared with the initial state, the main control module determines that the filtration efficiency of the ultrafilter has dropped, and instructs the extracellular vesicle separation and purification devices corresponding to the current filtration negative pressure bottle 1 and the concentrate negative pressure bottle 1 to enter the ultrafiltration membrane cleaning operation mode, that is, by increasing the positive pressure driving power of the booster pump 1 of the booster inlet module of the extracellular vesicle separation and purification device and increasing the negative pressure suction power of the negative pressure suction device 1 of the concentrate end negative pressure aspirator module, the flow velocity of the fluid in the spiral volute cavity is accelerated, the shear force between the fluid and the ultrafiltration membrane is increased, and the suspended particles and colloidal substances adsorbed and deposited on the surface of the ultrafiltration membrane are flushed away, thereby restoring the filtration efficiency of the ultrafiltration membrane;

[0107] The main control module senses through pressure sensor 1, pressure sensor 2, and pressure sensor 3 that the pressures at the liquid inlet, concentrate port, and filtrate port of the ultrafilter have returned to their initial states, and senses through the data of the weighing sensor and the auxiliary weighing sensor that the weight growth rate of the liquid in the filtration negative pressure bottle 1 has returned to its initial state. At the same time, the weight growth rate of the liquid in the concentrate negative pressure bottle 1 has returned to its initial state. The main control module determines that the filtration efficiency of the ultrafilter has recovered, and then calls back the positive pressure driving power of the boosting pump 1 of the boosting liquid inlet module and the negative pressure suction power of the negative pressure suction device 1 of the concentration end negative pressure aspirator module, so that the corresponding extracellular vesicle separation and purification device returns to the normal operation mode of separation and purification.

[0108] Example 2

[0109] Combine Figure 1 and Figure 3 The content shown in this embodiment proposes a continuous separation and purification device for extracellular vesicles, including two-stage extracellular vesicle separation and purification devices and a main control module connected in series. The extracellular vesicle separation and purification device is electrically connected to the main control module. The two-stage extracellular vesicle separation and purification device includes an ultrafilter module, a pressurized liquid inlet module, a concentration end negative pressure aspirator module, a filtration end negative pressure aspirator module and a pipeline temperature control module. The ultrafilter module is connected to the pressurized liquid inlet module, the concentration end negative pressure aspirator module, and the filtration end negative pressure aspirator module through pipelines. A pipeline temperature control module is provided on the pipelines involved in the extracellular vesicle separation and purification device, wherein,

[0110] The booster inlet module is used to provide positive pressure ultrafiltration power in the ultrafilter module and positive pressure driving power for the fluid flow in the upper chamber of the ultrafilter.

[0111] The negative pressure suction module at the concentration end provides negative pressure suction power for the flow of fluid in the upper chamber of the ultrafilter module.

[0112] The negative pressure suction module at the filtration end provides negative pressure ultrafiltration power in the ultrafilter module.

[0113] Ultrafilter modules are used to separate extracellular vesicles of different size ranges.

[0114] In the first-stage extracellular vesicle separation and purification device:

[0115] The ultrafilter module includes an ultrafilter and a pipeline. The ultrafilter is provided with a liquid inlet connected to the pressurized liquid inlet module, a concentrated liquid port connected to the concentrated end negative pressure suction module, and a filtered liquid port connected to the filtered end negative pressure suction module. Figure 5 The ultrafiltration device shown is a flat-plate scroll dynamic ultrafiltration device 1. A spiral volute extends from the center outward above the ultrafiltration membrane. The center of the spiral volute connects to the liquid inlet, the outer end of the spiral volute connects to the concentrate outlet, and the chamber below the ultrafiltration membrane connects to the filtrate outlet. The pore size of the ultrafiltration membrane is 150-200 nanometers.

[0116] The booster inlet module includes a booster pump, an extracellular vesicle stock solution bag, a buffer bag 1, a reflux bag 1, a multi-way valve 1, and a pipeline. The extracellular vesicle stock solution bag, the buffer bag 1, and the reflux bag 1 are collected through the pipeline to the multi-way valve 1. The multi-way valve 1 is connected to the booster pump 1 through the pipeline. The booster pump 1 is connected to the liquid inlet of the flat-plate scroll dynamic ultrafilter 1 through the pipeline. The booster pump 1 is any one of a diaphragm pump, a peristaltic pump, a plunger pump, and a gear pump.

[0117] A diaphragm pump or a peristaltic pump is preferred. The diaphragm pump is configured to separate the liquid being transported from the piston and the pump cylinder, thereby maintaining the cleanliness of the liquid during transport. The peristaltic pump is configured so that the fluid only contacts the pipeline and does not contact the pump body, thereby maintaining the cleanliness of the liquid during transport.

[0118] A pressure sensor 1 is connected in series between the booster pump 1 and the liquid inlet of the flat-plate scroll dynamic ultrafilter 1;

[0119] The buffer bag is used to hold buffer, including physiological saline, PBS buffer, Hanks Balanced Salt Solution (HBSS), etc. The buffer bag can balance the ultrafilter and pipeline before extracellular vesicle separation and purification, adjust the concentration of extracellular vesicles in the liquid to be separated during the separation and purification process, flush the spiral volute lumen to restore filtration efficiency during the separation and purification process, and flush the pipeline after separation and purification to avoid waste of extracellular vesicles.

[0120] The functions of the reflux liquid bag are: 1. During each stage of separation and purification of extracellular vesicles, the liquid to be separated can be repeatedly circulated through the ultrafilter through the reflux liquid bag, thereby increasing the efficiency of separation and purification of extracellular vesicles and avoiding waste; 2. When flushing the spiral volute lumen, under the condition that the booster pump provides positive pressure driving force for fluid flow, the buffer solution can be repeatedly circulated through the reflux liquid bag to flush the ultrafilter until the ultrafilter restores its filtration efficiency, thereby avoiding excessive waste of buffer solution.

[0121] In some specific implementations, auxiliary weighing sensors will be set corresponding to the extracellular vesicle stock solution bag, buffer solution bag, and reflux liquid bag. The auxiliary weighing sensors are electrically connected to the main control module. The auxiliary weighing sensors are preferably suspended weighing sensors, which are set above the corresponding extracellular vesicle stock solution bag, buffer solution bag, and reflux liquid bag. They are suspended weighing sensor 1, suspended weighing sensor 2, and suspended weighing sensor 3 respectively. Suspended weighing sensor 1, suspended weighing sensor 2, and suspended weighing sensor 3 are used to monitor the changes in the weight of the liquid in the extracellular vesicle stock solution bag, buffer solution bag, and reflux liquid bag, respectively.

[0122] The concentration end negative pressure suction device module includes a concentration negative pressure bottle 1, a weighing sensor 1, a negative pressure suction device 1, an air pressure sensor 1, an air pressure buffer 1, an air filter 1, a multi-way valve 3, a concentrate bag 1 and a pipeline. The concentration negative pressure bottle 1 is provided with a negative pressure exhaust port, a liquid inlet and a liquid discharge port. The negative pressure exhaust port is connected to the negative pressure suction device 1 through a pipeline. The air filter 1, the multi-way valve 3, the air pressure buffer 1 and the air pressure sensor 1 are connected in series on the pipeline between the negative pressure exhaust port and the negative pressure suction device 1. The two ports on the multi-way valve 3 are connected to the air inlet and the liquid discharge port respectively. The filter 1 is connected to the air pressure buffer 1, and a port is provided on the multi-way valve 3 as a vent 1. The liquid inlet is connected to the concentrate port and the reflux liquid bag 1 of the flat scroll dynamic ultrafilter 1 through a pipeline and a multi-way valve 2. The discharge port is connected to the concentrate bag 1 through a pipeline. A solenoid valve 1 and a peristaltic pump 1 are connected in series between the discharge port and the concentrate bag 1. A weighing sensor 1 connected to the main control module is provided below the concentrated negative pressure bottle 1. The weighing sensor 1 is a support type weighing sensor 1, which is used to monitor the weight change of the liquid in the concentrated negative pressure bottle 1.

[0123] A pressure sensor 2 is connected in series between the multi-way valve 2 and the concentrated liquid port of the flat-plate scroll dynamic ultrafilter 1 .

[0124] In some specific implementations, a weighing sensor is provided corresponding to the concentrated liquid bag 1 , and the weighing sensor is electrically connected to the main control module. The weighing sensor is preferably a suspended weighing sensor 4 , which is provided above the corresponding concentrated liquid bag 1 to monitor the weight change of the liquid in the concentrated liquid bag 1 .

[0125] The filter end negative pressure suction device module includes a filter negative pressure bottle 1, a weighing sensor 2, a negative pressure suction device 2, an air pressure sensor 2, an air pressure buffer 2, an air filter 2, a multi-way valve 4, a filter liquid bag 1 and a pipeline. The filter negative pressure bottle 1 is provided with a negative pressure exhaust port, a liquid inlet and a liquid discharge port. The negative pressure exhaust port is connected to the negative pressure suction device 2 through a pipeline. The air filter 2, the multi-way valve 4, the air pressure buffer 2 and the air pressure sensor 2 are connected in series on the pipeline between the negative pressure exhaust port and the negative pressure suction device 2. The two ports on the multi-way valve 4 are connected to the air filter 2, respectively. The air filter 2 is connected to the air pressure buffer 2, and a port is also provided on the multi-way valve 4 as a vent 2. The liquid inlet is connected to the filtered liquid port of the flat scroll dynamic ultrafilter 1 through a pipeline and an electromagnetic valve 2. The discharge port is connected to the filtered liquid bag 1 through a pipeline. The electromagnetic valve 3 and the peristaltic pump 2 are connected in series between the discharge port and the filtered liquid bag 1. A weighing sensor 2 connected to the main control module is provided below the filter negative pressure bottle 1. The weighing sensor 2 is a support type weighing sensor 2 used to monitor the weight change of the liquid in the filter negative pressure bottle 1;

[0126] A pressure sensor 3 is connected in series between the solenoid valve 2 and the filtered liquid port of the flat-plate scroll dynamic ultrafilter 1 .

[0127] In some specific implementations, a weighing sensor will be set corresponding to the filter liquid bag 1. The weighing sensor is electrically connected to the main control module. The weighing sensor is preferably a suspended weighing sensor 5, which is set above the corresponding filter liquid bag 1 to monitor the change in the weight of the liquid in the filter liquid bag 1.

[0128] In the second-stage extracellular vesicle separation and purification device:

[0129] The ultrafilter module includes an ultrafilter and a pipeline. The ultrafilter is provided with a liquid inlet connected to the pressurized liquid inlet module, a concentrated liquid port connected to the concentrated end negative pressure suction module, and a filtered liquid port connected to the filtered end negative pressure suction module. Figure 4 The ultrafilter shown is a flat-plate scroll dynamic ultrafilter 2. A spiral volute cavity extends outward from the center above the ultrafiltration membrane. The center of the spiral volute cavity connects to the liquid inlet, the outer end of the spiral volute cavity connects to the concentrate outlet, and the chamber below the ultrafiltration membrane connects to the filtrate outlet. The pore size of the ultrafiltration membrane is 30 to 70 nanometers.

[0130] The booster liquid inlet module includes a booster pump 2, a buffer bag 2, a reflux liquid bag 2, a multi-way valve 5, and a pipeline. The filter bag 1, the buffer bag 2, and the reflux liquid bag 2 in the previous extracellular vesicle separation and purification device (i.e., the first-stage extracellular vesicle separation and purification device) are collected through the pipeline to the multi-way valve 5. The multi-way valve 5 is connected to the booster pump 2 through the pipeline. The booster pump 2 is connected to the liquid inlet of the flat-plate volute dynamic ultrafilter 2 through the pipeline. The booster pump 2 is any one of a diaphragm pump, a peristaltic pump, a plunger pump, and a gear pump, preferably a diaphragm pump or a peristaltic pump. The diaphragm pump is configured to separate the liquid to be transported from the piston and the pump cylinder, thereby maintaining the cleanliness of the liquid during transport. The peristaltic pump is configured so that the fluid only contacts the pipeline and does not contact the pump body, thereby maintaining the cleanliness of the liquid during transport.

[0131] A pressure sensor 4 is connected in series between the booster pump 2 and the liquid inlet of the flat-plate scroll dynamic ultrafilter 2;

[0132] The buffer bag 2 is used to hold a buffer solution, including physiological saline, PBS buffer, Hanks Balanced Salt Solution (HBSS), etc. The buffer bag 2 can balance the ultrafilter and pipeline before extracellular vesicle separation and purification, adjust the concentration of extracellular vesicles in the liquid to be separated during the separation and purification process, flush the spiral volute lumen to restore the filtration efficiency during the separation and purification process, and flush the pipeline after separation and purification to avoid waste of extracellular vesicles.

[0133] The functions of the reflux liquid bag 2 are: 1. During each stage of separation and purification of extracellular vesicles, the liquid to be separated can be repeatedly circulated through the ultrafilter through the reflux liquid bag, thereby increasing the efficiency of extracellular vesicle separation and purification and avoiding waste; 2. When flushing the spiral volute lumen, under the condition that the booster pump provides positive pressure driving force for fluid flow, the buffer solution can be repeatedly circulated through the reflux liquid bag to flush the ultrafilter until the ultrafilter restores its filtration efficiency, thereby avoiding excessive waste of buffer solution.

[0134] In some specific implementations, auxiliary weighing sensors will be set corresponding to the buffer bag 2 and the reflux liquid bag 2. The auxiliary weighing sensors are electrically connected to the main control module. The auxiliary weighing sensors are preferably suspended weighing sensors, which are set above the corresponding buffer bag 2 and the reflux liquid bag 2. They are suspended weighing sensor 6 and suspended weighing sensor 7 respectively. The suspended weighing sensor 6 and the suspended weighing sensor 7 are used to monitor the changes in the weight of the liquid in the buffer bag 2 and the reflux liquid bag 2 respectively.

[0135] The concentration end negative pressure suction device module includes a concentration negative pressure bottle 2, a weighing sensor 3, a negative pressure suction device 3, an air pressure sensor 3, an air pressure buffer 3, an air filter 3, a multi-way valve 7, a concentrate bag 2 and a pipeline. The concentration negative pressure bottle 2 is provided with a negative pressure exhaust port, a liquid inlet and a liquid discharge port. The negative pressure exhaust port is connected to the negative pressure suction device 3 through a pipeline. The air filter 3, the multi-way valve 7, the air pressure buffer 3 and the air pressure sensor 3 are connected in series on the pipeline between the negative pressure exhaust port and the negative pressure suction device 3. The two ports on the multi-way valve 7 are connected to the air filter 3, the multi-way valve 7, the air pressure buffer 3 and the air pressure sensor 3. The filter 3 is connected to the air pressure buffer 3, and a port is also provided on the multi-way valve 7 as a vent 3. The liquid inlet is connected to the concentrate port and the reflux liquid bag of the flat scroll dynamic ultrafilter 2 through a pipeline and a multi-way valve 6. The discharge port is connected to the concentrate bag 2 through a pipeline. A solenoid valve 4 and a peristaltic pump 3 are connected in series between the discharge port and the concentrate bag 2. A weighing sensor 3 connected to the main control module is provided below the concentrated negative pressure bottle 2. The weighing sensor 3 is a support type weighing sensor 3 used to monitor the weight change of the liquid in the concentrated negative pressure bottle 2.

[0136] A pressure sensor 5 is connected in series between the multi-way valve 6 and the concentrated liquid port of the flat-plate scroll dynamic ultrafilter 2 .

[0137] In some specific implementations, a weighing sensor is provided corresponding to the concentrated liquid bag 2 , and the weighing sensor is electrically connected to the main control module. The weighing sensor is preferably a suspended weighing sensor 8 , which is provided above the corresponding concentrated liquid bag 2 to monitor the change in the weight of the liquid in the concentrated liquid bag 2 .

[0138] The filter end negative pressure suction device module includes a filter negative pressure bottle 2, a weighing sensor 4, a negative pressure suction device 4, an air pressure sensor 4, an air pressure buffer 4, an air filter 4, a multi-way valve 8, a filter liquid bag 2 and a pipeline. The filter negative pressure bottle 2 is provided with a negative pressure exhaust port, a liquid inlet and a liquid discharge port. The negative pressure exhaust port is connected to the negative pressure suction device 4 through a pipeline. The air filter 4, the multi-way valve 8, the air pressure buffer 4 and the air pressure sensor 4 are connected in series on the pipeline between the negative pressure exhaust port and the negative pressure suction device 4. The two ports on the multi-way valve 8 are connected to the air filter 4, respectively. The air filter 4 is connected to the air pressure buffer 4, and a port is also provided on the multi-way valve 8 as a vent 4. The liquid inlet is connected to the filtered liquid port of the flat scroll dynamic ultrafilter 2 through a pipeline and an electromagnetic valve 5. The discharge port is connected to the filtered liquid bag 2 through a pipeline. The electromagnetic valve 6 and the peristaltic pump 4 are connected in series between the discharge port and the filtered liquid bag 2. A weighing sensor 4 connected to the main control module is provided under the filter negative pressure bottle 2. The weighing sensor 4 is a support type weighing sensor 4, which is used to monitor the weight change of the liquid in the filter negative pressure bottle 2.

[0139] In some specific implementations, a weighing sensor is provided corresponding to the filter liquid bag 2, and the weighing sensor is electrically connected to the main control module. The weighing sensor is preferably a suspended weighing sensor 9, which is provided above the corresponding filter liquid bag 2 to monitor the weight change of the liquid in the filter liquid bag 2;

[0140] A pressure sensor 6 is connected in series between the solenoid valve 5 and the filtered liquid outlet of the flat-plate scroll dynamic ultrafilter 2 .

[0141] The above-mentioned ultrafiltration membrane can directly select the ultrafiltration membrane available on the market, for example: ultrafiltration membrane made of cellulose acetate, cellulose acetate ester, polyethylene, polysulfone, polyamide, aromatic polymer, nylon, polyethersulfone, hydrophilic polyvinylidene fluoride, polytetrafluoroethylene, hydrophilic polytetrafluoroethylene, mixed cellulose, hydrophilic polycarbonate, and porous alumina.

[0142] It should be noted that the above-mentioned concentrated negative pressure bottle 1, filtered negative pressure bottle 1, concentrated negative pressure bottle 2 and filtered negative pressure bottle 2 are all hard bottles, which include a bottle body and a bottle cap, and the bottom of the bottle body adopts a design that is conducive to the accumulation of liquid in the negative pressure bottle, such as a groove provided at the bottom of the bottle body, through which the liquid at the bottom of the negative pressure bottle is accumulated, wherein the shape of the groove can be a cone with a wide top and a narrow bottom or a round bottom with a wide top and a narrow bottom, etc. The negative pressure exhaust port, liquid inlet and liquid discharge port are provided on the bottle cap. The concentrated negative pressure bottle 1 and filtered negative pressure bottle 1 herein can directly use existing negative pressure bottles on the market.

[0143] The pipeline temperature control module in the two-stage extracellular vesicle separation and purification device includes a winding temperature controller and a cold source. The winding temperature controller is connected to the cold source. The pipeline adopts a hose design and is wound around the winding temperature controller.

[0144] The winding thermostat is made of metal with good thermal conductivity, preferably aluminum, copper or metal alloy, combined with Figure 6 The content shown is that the surface of the wound thermostat has spiral grooves, and the pipeline is wound around the wound thermostat along the spiral grooves. A cold water circulation pipeline is provided inside the wound thermostat. The wound thermostat is connected and fixed to the shell or other structure of the extracellular vesicle continuous separation and purification device. The cold water circulation pipeline of the wound thermostat is connected to the cold water circulation pipeline of the cold source device. The setting of the spiral grooves on the surface of the wound thermostat can not only effectively guide the wound pipeline, increase the contact area between the pipeline and the wound thermostat, and improve the heat exchange efficiency, but also can support the wound pipeline through the design of the spiral grooves, effectively reducing the chance of the pipeline wrapped on the wound thermostat slipping;

[0145] The cold source includes a refrigerated water circulation device and a Peltier semiconductor refrigeration sheet. The refrigerated water circulation device is a low-temperature constant-temperature cold water circulation device with a water temperature controlled at 0 to 8°C. The Peltier semiconductor refrigeration sheet is used to reduce the water temperature in the refrigerated water circulation so that the temperature of the circulating water is controlled within the required temperature range. The low-temperature constant-temperature cold water circulation device includes a circulation pump and a flexible conduit, which is connected to the inlet / outlet of the wrap-around thermostat through the flexible conduit. The cold water in the wrap-around thermostat circulates through the circulation pump and the flexible conduit, quickly cooling the wrap-around thermostat.

[0146] There are multiple winding temperature controllers in the extracellular vesicle separation and purification device, which are distributed in various places of the pipeline, such as before the ultrafilter inlet, before the concentrate bag inlet, and before the filtered liquid bag inlet.

[0147] In addition to the above-mentioned suspended weighing sensor 1-9, supporting weighing sensor 1-4, pressure sensor 1-6, and air pressure sensor 1-4, in the specific implementation, a working environment temperature sensor is set on the extracellular vesicle separation and purification device to detect the room temperature, and a circulating water temperature sensor is set in the low-temperature constant temperature cold water circulation device of the pipeline temperature control module. These circulating water temperature sensors, working environment temperature sensors and the above-mentioned suspended weighing sensor 1-9, supporting weighing sensor 1-4, pressure sensor 1-6, and air pressure sensor 1-4 are combined into a sensor detection module, and the sensor detection module is electrically connected to the main control module.

[0148] A microcomputer can be directly used as the above-mentioned main control module.

[0149] In some specific embodiments, the apparatus for continuous separation and purification of extracellular vesicles further includes an alarm module and a human-computer interaction module, which are connected to the main control module. The human-computer interaction module includes an operation control panel and / or a touch screen. The alarm module can emit an audible and / or visual alarm signal. The human-computer interaction module adjusts and sets reference thresholds for various parameters and values detected by the sensor detection module. Once the data detected by the sensor detection module exceeds the reference threshold, the alarm module emits an alarm signal.

[0150] In the two-stage extracellular vesicle separation and purification device, the particle size of the ultrafiltration membrane of the first-stage extracellular vesicle separation and purification device is larger than the pore size of the ultrafiltration membrane of the second-stage extracellular vesicle separation and purification device. The liquid in the filtered liquid bag of the first-stage extracellular vesicle separation and purification device enters the booster liquid inlet module of the adjacent second-stage extracellular vesicle separation and purification device, and the concentrated liquid of each adjacent second-stage is collected into the corresponding concentrated liquid bag, so that multi-stage continuous separation and purification of extracellular vesicles can be performed to obtain extracellular vesicles in different particle size ranges.

[0151] The application method of the extracellular vesicle separation and purification device comprises the following steps:

[0152] S1: adding the extracellular vesicle stock solution to the extracellular vesicle stock solution bag of the first-stage extracellular vesicle separation and purification device, and the extracellular vesicle stock solution to be separated enters the ultrafilter from the liquid inlet of the flat-plate scroll dynamic ultrafilter 1 through the booster pump 1. For example, the pore size of the ultrafiltration membrane of the first-stage extracellular vesicle separation and purification device is set to 150 nm;

[0153] S2: The booster inlet module provides positive pressure ultrafiltration power, and the filtration end negative pressure suction module provides negative pressure suction ultrafiltration power. Under the action of the two filtration powers of positive pressure push and negative pressure suction, the impurities in the extracellular vesicle stock solution to be separated whose particle size is larger than the pore size of the ultrafiltration membrane of the first-stage extracellular vesicle separation and purification device of 150nm are intercepted and flow through the concentrated liquid port of the ultrafilter through the multi-way valve 2, and then enter the concentrated negative pressure bottle 1 for temporary storage. The extracellular vesicles and impurities in the extracellular vesicle stock solution to be separated whose particle size is smaller than the pore size of the ultrafiltration membrane of the first-stage extracellular vesicle separation and purification device of 150nm pass through the ultrafiltration membrane and enter the filtration negative pressure bottle 1 for temporary storage through the filtrate port of the ultrafilter;

[0154] S3: After the filtrate obtained by the first-stage extracellular vesicle separation and purification device accumulates to a certain amount in the filtration negative pressure bottle 1 (the value range of the certain amount is 0.5-0.75 times the capacity of the filtration negative pressure bottle 1, for example, 2 / 3 times), the booster pump 1 and the negative pressure aspirator 1 stop working, the solenoid valve 2 is closed, the multi-way valve 4 closes the negative pressure channel, the vent 2 is opened, and the solenoid valve 3 is opened. Under the action of the peristaltic pump 2, the filtrate in the filtration negative pressure bottle 1 enters the filtration liquid bag 1;

[0155] S4: The filtrate in the filter liquid bag 1 enters the ultrafilter from the liquid inlet of the flat-plate scroll dynamic ultrafilter 2 through the booster pump 2. For example, the pore size of the ultrafiltration membrane of the second-stage extracellular vesicle separation and purification device is set to 70 nm;

[0156] S5: The booster inlet module provides positive pressure ultrafiltration power, and the filtration end negative pressure suction module provides negative pressure suction ultrafiltration power. Under the action of the two filtration powers of positive pressure push and negative pressure suction, the extracellular vesicle stock solution with a particle size larger than the 70nm pore size of the ultrafiltration membrane of the second-stage extracellular vesicle separation and purification device in the first-stage filtrate to be separated is retained, and flows through the concentrated liquid port of the flat-plate scroll dynamic ultrafilter 2 through the multi-way valve 6, and then enters the concentrated negative pressure bottle 2 for temporary storage. The extracellular vesicles and impurities with a particle size smaller than the 70nm pore size of the ultrafiltration membrane of the second-stage extracellular vesicle separation and purification device in the first-stage filtrate to be separated pass through the ultrafiltration membrane, and enter the filtration negative pressure bottle 2 for temporary storage through the filtrate port of the flat-plate scroll dynamic ultrafilter 2;

[0157] S6: After the concentrate obtained by the second-stage extracellular vesicle separation and purification device accumulates to a certain amount in the concentrated negative pressure bottle 2 (the certain amount ranges from 0.5 to 0.75 times the capacity of the concentrated negative pressure bottle 2, for example, 2 / 3 times), the booster pump 2 and the negative pressure aspirator 3 stop working, the multi-way valve 6 is closed, the multi-way valve 7 closes the negative pressure channel, the vent 3 is opened, and the solenoid valve 4 is opened. Under the action of the peristaltic pump 3, the second-level concentrate in the concentrated negative pressure bottle 2 enters the concentrated liquid bag 2, and extracellular vesicles with a particle size range of 70 nm to 150 nm are obtained;

[0158] By adjusting the pore size of the ultrafiltration membrane of adjacent extracellular vesicle separation and purification devices, multi-stage separation and purification of extracellular vesicles can be performed, thereby obtaining extracellular vesicles with different particle size ranges. For specific methods, see S1-S6.

[0159] The booster liquid inlet module provides positive pressure driving force for the flow of fluid in the spiral volute cavity, and the concentration end negative pressure suction module provides negative pressure suction force for the flow of fluid in the spiral volute cavity. Under the action of the two fluid flow forces of positive pressure driving and negative pressure suction, the extracellular vesicle stock solution flows forward rapidly in the spiral volute cavity, forming a shear force between the ultrafiltration membrane, preventing the adsorption and deposition of suspended particles and colloidal substances on the surface of the ultrafiltration membrane, overcoming the concentration polarization effect, thereby effectively reducing the attenuation rate of the ultrafiltration membrane flux and maintaining the high filtration efficiency of the ultrafiltration membrane for a long time.

[0160] During the execution of S1-S6, the main control module monitors the pressure of the liquid inlet, concentrate inlet, and filtrate inlet pipelines of the ultrafilter through pressure sensors, with pressure sensors 1-3 forming a group and pressure sensors 4-6 forming another group; and monitors the weight of the liquids in the extracellular vesicle stock solution bag, buffer bag 1, reflux liquid bag 1, filtered liquid bag 1, concentrated liquid bag 1, filtration negative pressure bottle 1, concentration negative pressure bottle 1, buffer bag 2, reflux liquid bag 2, filtered liquid bag 2, concentrated liquid bag 2, filtration negative pressure bottle 2, and concentration negative pressure bottle 2 in groups through weighing sensors and auxiliary weighing sensors, with the extracellular vesicle stock solution bag, buffer bag 1, reflux liquid bag 1, filtered liquid bag 1, concentrated liquid bag 1, filtration negative pressure bottle 1, and concentration negative pressure bottle 1 forming a group and the filtration negative pressure bottle 1, buffer bag 2, reflux liquid bag 2, filtered liquid bag 2, concentrated liquid bag 2, filtration negative pressure bottle 2, and concentration negative pressure bottle 2 forming another group;

[0161] When the main control module senses through the pressure sensor that the pressures at the liquid inlet and concentrate port of the ultrafilter continue to rise compared to the initial state, and the pressure at the filtrate port continues to drop compared to the initial state, and senses through the data changes of the weighing sensor and the auxiliary weighing sensor that the weight growth rate of the liquid in the filtration negative pressure bottle in one or more groups continues to drop compared to the initial state, and at the same time, the weight growth rate of the liquid in the concentrate negative pressure bottle continues to increase compared to the initial state, the main control module determines that the filtration efficiency of the ultrafilter has dropped, and instructs the extracellular vesicle separation and purification device corresponding to the filtration negative pressure bottle and the concentrate negative pressure bottle to enter the ultrafiltration membrane cleaning operation mode, that is, by increasing the positive pressure driving power of the booster pump of the booster inlet module of the extracellular vesicle separation and purification device and increasing the negative pressure suction power of the negative pressure aspirator module at the concentrate end, the flow velocity of the fluid in the spiral volute cavity is accelerated, the shear force between the fluid and the ultrafiltration membrane is increased, and the suspended particles and colloidal substances adsorbed and deposited on the surface of the ultrafiltration membrane are flushed away and taken away, thereby restoring the filtration efficiency of the ultrafiltration membrane;

[0162] The main control module senses through pressure sensors that the pressures at the ultrafilter's liquid inlet, concentrate port, and filtrate port have returned to their initial states. It senses through data from the weighing sensor and the auxiliary weighing sensor that the weight growth rate of the liquid in the filtration negative pressure bottle has returned to its initial state. At the same time, the weight growth rate of the liquid in the concentrate negative pressure bottle has returned to its initial state. The main control module determines that the ultrafilter's filtration efficiency has recovered, and then calls back the positive pressure driving power of the booster pump of the booster inlet module and the negative pressure suction power of the concentration end negative pressure aspirator module, allowing the corresponding extracellular vesicle separation and purification device to return to its normal separation and purification operation mode. The ultrafiltration membrane cleaning operation mode can ensure the filtration efficiency of the ultrafiltration membrane, allowing the ultrafiltration membrane to maintain high filtration efficiency for a long time.

[0163] An extracellular vesicle separation and purification device can also include multiple stages (three or more) of extracellular vesicle separation and purification devices connected in series. The structure of the extracellular vesicle separation and purification device is the same as that in Example 2. The additional extracellular vesicle separation and purification device will combine the filtered liquid bag of the previous stage with the buffer bag and reflux liquid bag of the current stage through a pipeline to a multi-way valve. The multi-way valve is connected to a booster pump via a pipeline, and the booster pump is connected to the liquid inlet of the ultrafiltration module via a pipeline. The application method of this extracellular vesicle separation and purification device is the same as that described in Example 2 and is not further described here.

[0164] Example 3

[0165] Combine Figure 4As shown in the content, the ultrafilter in this embodiment is a flat serpentine dynamic ultrafilter. The upper chamber of the ultrafilter in the flat serpentine dynamic ultrafilter is a repeatedly bent serpentine lumen. One end of the serpentine lumen is connected to the liquid inlet pipeline, the other end of the serpentine lumen is connected to the concentrate outlet pipeline, and the lower chamber of the ultrafiltration membrane is connected to the filtrate outlet pipeline. The extracellular vesicle-containing liquid to be separated enters the repeatedly bent serpentine lumen from the liquid inlet. The booster pump provides positive pressure ultrafiltration power, and the negative pressure aspirator at the filtration end provides negative pressure suction ultrafiltration power. Under the influence of the aforementioned positive pressure push and negative pressure suction, the extracellular vesicle-containing liquid to be separated, impurities with a particle size larger than the ultrafiltration membrane pore size, are retained and flow through the concentrate port of the flat-plate serpentine dynamic ultrafilter, through the multi-way valve, and then into the concentration negative pressure bottle. Extracellular vesicles and impurities with a particle size smaller than the ultrafiltration membrane pore size in the extracellular vesicle-containing liquid to be separated pass through the ultrafiltration membrane and enter the filtration negative pressure bottle through the filtrate port of the flat-plate serpentine dynamic ultrafilter. The remaining application methods of this extracellular vesicle separation and purification device are the same as those described in Examples 1 and 2, and are therefore not described in detail here.

[0166] Example 4

[0167] Combine Figure 7 As shown in the content, the ultrafilter in this embodiment is an internal pressure hollow fiber membrane shell and tube dynamic ultrafilter, which consists of a membrane tube shell, two tube sheet heads and a number of hollow fiber membranes. All hollow fiber membranes are directly encapsulated in the membrane tube shell by two tube sheet heads. One end of the hollow fiber membrane tube cavity is gathered into a front chamber cavity connected to the liquid inlet, and the other end is gathered into a rear chamber cavity connected to the concentrated liquid port. The outer chamber cavity between the hollow fiber membrane and the membrane tube shell is connected to the filtered liquid port. The extracellular vesicle-containing liquid to be separated enters the hollow fiber membrane from the liquid inlet, the booster pump provides positive pressure ultrafiltration power, and the negative pressure suction device at the filtration end provides negative pressure suction ultrafiltration power. Under the action of the two filtration powers of positive pressure push and negative pressure suction, the impurities in the extracellular vesicle-containing liquid to be separated with a particle size larger than the pore size of the hollow fiber membrane are intercepted and collected to the concentrated liquid port through the other end of the hollow fiber membrane lumen, and then enter the concentrated negative pressure bottle. The extracellular vesicles and impurities in the extracellular vesicle-containing liquid to be separated with a particle size smaller than the pore size of the hollow fiber membrane pass through the micropores of the hollow fiber membrane, enter the outer chamber cavity between the hollow fiber membrane and the membrane tube shell, and enter the filtration negative pressure bottle through the filtrate port. The rest of the application method of the extracellular vesicle separation and purification device is the same as the application method described in Example 1 and Example 2, so it will not be repeated here.

[0168] Example 5

[0169] Combine Figure 8As shown in the content, the ultrafilter in this embodiment is an external pressure hollow fiber membrane shell and tube dynamic ultrafilter, which consists of a membrane tube shell, a tube sheet head, an end cover plate and a plurality of hollow fiber membranes. All hollow fiber membranes are directly encapsulated in the membrane tube shell by a tube sheet head and an end cover plate. One end of the outer chamber cavity between the hollow fiber membrane and the membrane tube shell is connected to the liquid inlet, and the other end of the outer chamber cavity between the hollow fiber membrane and the membrane tube shell is connected to the concentrated liquid port. One end of all hollow fiber membrane tube lumens is closed by the end cover plate, and the other end of all hollow fiber membrane tube lumens is collected in the filter chamber cavity and connected to the filtered liquid port. The extracellular vesicle-containing liquid to be separated enters the outer chamber cavity between the hollow fiber membrane and the membrane tube shell from the liquid inlet, the booster pump provides positive pressure ultrafiltration power, and the negative pressure suction device at the filtration end provides negative pressure suction ultrafiltration power. Under the action of the two filtration powers of positive pressure push and negative pressure suction, the impurities in the extracellular vesicle-containing liquid to be separated with a particle size larger than the pore size of the hollow fiber membrane are intercepted and enter the concentrated negative pressure bottle through the concentrated liquid port at the other end of the outer chamber cavity between the hollow fiber membrane and the membrane tube shell. The extracellular vesicles and impurities in the extracellular vesicle-containing liquid to be separated with a particle size smaller than the pore size of the hollow fiber membrane enter the hollow fiber membrane lumen through the micropores on the hollow fiber membrane, and are collected in the filtration chamber cavity through the hollow fiber membrane lumen, and enter the filtration negative pressure bottle through the filtrate port. The rest of the application method of the extracellular vesicle separation and purification device is the same as the application method described in Example 1 and Example 2, so it will not be repeated here.

[0170] The above content is a further detailed description of the present invention in conjunction with specific implementation methods. It cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, they can make several simple deductions or substitutions without departing from the concept of the present invention, which should be regarded as falling within the scope of protection determined by the claims submitted for the present invention.

Claims

1. A device for continuous separation and purification of extracellular vesicles, characterized in that: The invention comprises an extracellular vesicle separation and purification device, a main control module and an extracellular vesicle stock solution bag. The extracellular vesicle separation and purification device is electrically connected to the main control module, and the extracellular vesicle separation and purification device is connected to the extracellular vesicle stock solution bag. The extracellular vesicle separation and purification device comprises an ultrafilter module, a pressurized liquid inlet module, a concentration end negative pressure aspirator module and a filtration end negative pressure aspirator module. The ultrafilter module is connected to the pressurized liquid inlet module, the concentration end negative pressure aspirator module and the filtration end negative pressure aspirator module by pipeline. The extracellular vesicle continuous separation and purification device comprises at least two stages of extracellular vesicle separation and purification devices connected in series, wherein The extracellular vesicle stock solution bag is used to hold the extracellular vesicle stock solution; The ultrafilter module is used to separate extracellular vesicles of different particle size ranges. The ultrafilter module includes an ultrafilter and a pipeline. The ultrafilter is provided with a liquid inlet connected to the pressurized liquid inlet module, a concentrated liquid port connected to the concentration end negative pressure aspirator module, and a filtered liquid port connected to the filtration end negative pressure aspirator module. An ultrafiltration membrane is provided in the ultrafilter. The upper part of the ultrafiltration membrane is the upper chamber of the ultrafilter, and the lower part of the ultrafiltration membrane is the lower chamber of the ultrafilter. The booster inlet module is used to provide positive pressure ultrafiltration power in the ultrafilter module and positive pressure driving power for the fluid flow in the upper chamber of the ultrafilter. The booster inlet module includes a booster pump, a buffer bag, a reflux liquid bag, a multi-way valve and a pipeline; The concentrating end negative pressure aspirator module provides negative pressure aspiration power for the flow of fluid in the upper chamber of the ultrafilter module. The concentrating end negative pressure aspirator module includes a concentrated negative pressure bottle, a negative pressure aspirator, an air filter, a multi-way valve, a concentrated liquid bag, and a pipeline. The filtration end negative pressure aspirator module provides negative pressure ultrafiltration power in the ultrafiltration module, and the filtration end negative pressure aspirator module includes a filtration negative pressure bottle, a negative pressure aspirator, an air filter, a multi-way valve, a filter liquid bag and a pipeline; The liquid in the filtered liquid bag of the extracellular vesicle separation and purification device of the previous stage enters the pressurized liquid inlet module of the extracellular vesicle separation and purification device of the adjacent next stage, and the concentrated liquid of the adjacent next stage is collected into the corresponding concentrated liquid bag, so that the multi-stage continuous separation and purification of extracellular vesicles can be carried out to obtain extracellular vesicles of different particle size ranges; The pore size of the ultrafiltration membrane of the upper-stage extracellular vesicle separation and purification device is larger than the pore size of the ultrafiltration membrane of the lower-stage extracellular vesicle separation and purification device.

2. The device for continuous separation and purification of extracellular vesicles according to claim 1, wherein: The ultrafilter is any one of a flat plate serpentine dynamic ultrafilter, a flat plate volute dynamic ultrafilter and a hollow fiber membrane shell and tube dynamic ultrafilter. The upper chamber of the ultrafilter in the flat serpentine dynamic ultrafilter is a repeatedly bent serpentine chamber, one end of the serpentine chamber is connected to the liquid inlet, the other end of the serpentine chamber is connected to the concentrated liquid outlet, and the lower chamber of the ultrafiltration membrane is connected to the filtered liquid outlet. The upper chamber of the ultrafilter in the flat spiral dynamic ultrafilter is a spiral spiral chamber from the center to the outside, the center of the spiral spiral chamber is connected to the liquid inlet, the outer end of the spiral spiral chamber is connected to the concentrated liquid outlet, and the lower chamber of the ultrafiltration membrane is connected to the filtered liquid outlet. The hollow fiber membrane shell and tube dynamic ultrafilter includes an internal pressure hollow fiber membrane shell and tube dynamic ultrafilter and an external pressure hollow fiber membrane shell and tube dynamic ultrafilter. The internal pressure hollow fiber membrane shell and tube dynamic ultrafilter consists of a membrane tube shell, a tube sheet head and a hollow fiber membrane. The hollow fiber membrane is directly encapsulated in the membrane tube shell at both ends by two tube sheet heads. One end of the tube cavity of the hollow fiber membrane is collected in the front chamber cavity and connected to the liquid inlet, and the other end is collected in the rear chamber cavity and connected to the concentrated liquid port. The outer chamber cavity between the hollow fiber membrane and the membrane tube shell is connected to the filtered liquid port. The external pressure hollow fiber membrane shell and tube dynamic ultrafilter consists of a membrane tube shell, a tube sheet head, an end cover and a hollow fiber membrane. The hollow fiber membrane is directly encapsulated in the membrane tube shell at both ends by the tube sheet head and the end cover. One end of the outer chamber cavity between the hollow fiber membrane and the membrane tube shell is connected to the liquid inlet, and the other end of the outer chamber cavity between the hollow fiber membrane and the membrane tube shell is connected to the concentrated liquid port. One end of the tube cavity of the hollow fiber membrane is closed by the end cover, and the other end of the tube cavity of the hollow fiber membrane is collected in the filter chamber cavity and connected to the filtered liquid port.

3. The device for continuous separation and purification of extracellular vesicles according to claim 2, wherein: The pore size of the ultrafiltration membrane is 10 nanometers to 4000 nanometers.

4. The device for continuous separation and purification of extracellular vesicles according to claim 1, wherein: The extracellular vesicle liquid bag in the extracellular vesicle continuous separation and purification device is an extracellular vesicle stock liquid bag or a filtered liquid bag in the extracellular vesicle separation and purification device of the previous level. The extracellular vesicle liquid bag, the buffer bag and the reflux liquid bag are collected into a multi-way valve through a pipeline. The multi-way valve is connected to the booster pump through a pipeline. The booster pump is connected to the liquid inlet of the ultrafilter through a pipeline. A pressure sensor is connected in series between the booster pump and the liquid inlet of the ultrafilter. The pressure sensor is electrically connected to the main control module. The booster pump is any one of a diaphragm pump, a peristaltic pump, a plunger pump and a gear pump.

5. The device for continuous separation and purification of extracellular vesicles according to claim 4, wherein: The booster pump is a diaphragm pump or a peristaltic pump.

6. The device for continuous separation and purification of extracellular vesicles according to claim 4, wherein: The extracellular vesicle continuous separation and purification device also includes auxiliary weighing sensors arranged corresponding to the extracellular vesicle stock solution bag, buffer solution bag, reflux solution bag, and filtered solution bag, and the auxiliary weighing sensors are electrically connected to the main control module.

7. The device for continuous separation and purification of extracellular vesicles according to claim 6, wherein: The auxiliary weighing sensor is a suspended weighing sensor.

8. The device for continuous separation and purification of extracellular vesicles according to claim 6, wherein: The concentrated negative pressure bottle is provided with a negative pressure exhaust port, a liquid inlet and a liquid discharge port. The negative pressure exhaust port on the concentrated negative pressure bottle is connected to the negative pressure suction device in the concentrated end negative pressure suction device module through a pipeline. An air filter, a multi-way valve, an air vent, an air pressure buffer and an air pressure sensor are connected in series on the pipeline between the negative pressure exhaust port on the concentrated negative pressure bottle and the negative pressure suction device in the concentrated end negative pressure suction device module. The liquid inlet on the concentrated negative pressure bottle is connected to the concentrated liquid port of the ultrafilter and the reflux liquid bag through a pipeline and a multi-way valve. A pressure sensor is connected in series between the concentrated liquid port of the ultrafilter and the multi-way valve. The pressure sensor is electrically connected to the main control module. The liquid discharge port on the concentrated negative pressure bottle is connected to the concentrated liquid bag through a pipeline. An electromagnetic valve and a power pump are connected in series between the liquid discharge port on the concentrated negative pressure bottle and the concentrated liquid bag.

9. The device for continuous separation and purification of extracellular vesicles according to claim 8, wherein: The power pump connected in series with the discharge port on the concentrated negative pressure bottle is a peristaltic pump.

10. The device for continuous separation and purification of extracellular vesicles according to claim 8, wherein: The concentration end negative pressure aspirator module further includes a weighing sensor, which is provided corresponding to the concentrated negative pressure bottle and the concentrated liquid bag, and is electrically connected to the main control module. The concentrated negative pressure bottle is a hard bottle, comprising a bottle body and a bottle cap. A groove is provided at the bottom of the bottle body of the concentrated negative pressure bottle. The design of the groove at the bottom of the bottle body of the concentrated negative pressure bottle facilitates the accumulation of liquid in the concentrated negative pressure bottle. The shape of the groove at the bottom of the bottle body of the concentrated negative pressure bottle is a cone that is wide at the top and narrow at the bottom, or a round bottom that is wide at the top and narrow at the bottom. The negative pressure exhaust port, liquid inlet and discharge port on the concentrated negative pressure bottle are arranged on the bottle cap of the concentrated negative pressure bottle.

11. The device for continuous separation and purification of extracellular vesicles according to claim 10, wherein: The weighing sensor corresponding to the concentrated negative pressure bottle is a supporting weighing sensor. The weighing sensor provided for the concentrate bag is a suspended weighing sensor.

12. The device for continuous separation and purification of extracellular vesicles according to claim 10, wherein: The filtering negative pressure bottle is provided with a negative pressure exhaust port, a liquid inlet and a discharge port. The negative pressure exhaust port on the filtering negative pressure bottle is connected to the negative pressure suction device in the filtering end negative pressure suction device module through a pipeline. An air filter, a multi-way valve, a vent, an air pressure buffer and an air pressure sensor are connected in series on the pipeline between the negative pressure exhaust port on the filtering negative pressure bottle and the negative pressure suction device in the filtering end negative pressure suction device module. The liquid inlet on the filtering negative pressure bottle is connected to the filtered liquid port of the ultrafilter through a pipeline. An electromagnetic valve and a pressure sensor are connected in series between the liquid inlet on the filtering negative pressure bottle and the filtered liquid port of the ultrafilter. The electromagnetic valve and the pressure sensor between the liquid inlet on the filtering negative pressure bottle and the filtered liquid port of the ultrafilter are electrically connected to the main control module. The discharge port on the filtering negative pressure bottle is connected to the filtered liquid bag through a pipeline. An electromagnetic valve and a power pump are connected in series between the discharge port on the filtering negative pressure bottle and the filtered liquid bag.

13. The device for continuous separation and purification of extracellular vesicles according to claim 12, wherein: The power pump connected in series with the discharge port on the filter negative pressure bottle is a peristaltic pump.

14. The device for continuous separation and purification of extracellular vesicles according to claim 12, wherein: The filter end negative pressure suction module also includes an auxiliary weighing sensor corresponding to the filter negative pressure bottle, and the auxiliary weighing sensor corresponding to the filter negative pressure bottle is electrically connected to the main control module. The filtering negative pressure bottle is a hard bottle, which includes a bottle body and a bottle cap. A groove is provided at the bottom of the bottle body of the filtering negative pressure bottle. The groove design at the bottom of the bottle body of the filtering negative pressure bottle facilitates the accumulation of liquid in the filtering negative pressure bottle. The negative pressure exhaust port, liquid inlet and liquid discharge port on the filtering negative pressure bottle are arranged on the bottle cap of the filtering negative pressure bottle.

15. The device for continuous separation and purification of extracellular vesicles according to claim 14, wherein: The auxiliary weighing sensor provided corresponding to the filter negative pressure bottle is a supporting weighing sensor.

16. The device for continuous separation and purification of extracellular vesicles according to claim 14, wherein: The shape of the groove at the bottom of the bottle body of the filtering negative pressure bottle is a cone with a wide top and a narrow bottom or a round bottom with a wide top and a narrow bottom.

17. The device for continuous separation and purification of extracellular vesicles according to claim 1, wherein: The extracellular vesicle separation and purification device also includes a pipeline temperature control module, which acts on the pipelines involved in the extracellular vesicle separation and purification device to keep the liquid in the pipelines in a required temperature environment.

18. The device for continuous separation and purification of extracellular vesicles according to claim 17, wherein: The pipeline adopts a hose, and the pipeline temperature control module includes a winding type thermostat and a cold source. The winding type thermostat is connected to the cold source, and the pipeline is wound on the winding type thermostat.

19. The device for continuous separation and purification of extracellular vesicles according to claim 18, wherein: The wound-type thermostat is made of a metal with good thermal conductivity, and has spiral grooves on its surface, in which the pipeline is wound; the cold source includes a refrigeration water circulation device and a Peltier semiconductor refrigeration plate, and the refrigeration water circulation device is a low-temperature constant-temperature cold water circulation device. A cold water circulation pipeline is provided inside the wound-type thermostat, and the cold water circulation pipeline of the low-temperature constant-temperature cold water circulation device is connected to the cold water circulation pipeline of the wound-type thermostat, and the wound-type thermostat is cooled by the low-temperature constant-temperature cold water circulation device; the wound-type thermostat corresponds to the pipeline setting, and the water temperature control range in the low-temperature constant-temperature cold water circulation device is 0 to 8°C.

20. The device for continuous separation and purification of extracellular vesicles according to claim 19, wherein: The metal is aluminum, copper or a metal alloy.

21. The device for continuous separation and purification of extracellular vesicles according to claim 1, wherein: The extracellular vesicle separation and purification device further comprises an alarm module and a human-computer interaction module, and both the alarm module and the human-computer interaction module are electrically connected to the main control module.

22. An application method of the device for continuous separation and purification of extracellular vesicles according to any one of claims 14 to 16, characterized in that: The steps include: S1: adding the extracellular vesicle stock solution to be separated into the extracellular vesicle stock solution bag, and the extracellular vesicle stock solution to be separated enters the ultrafilter from the liquid inlet of the ultrafilter through a booster pump; S2: The booster inlet module provides positive pressure ultrafiltration power, and the filtration end negative pressure suction module provides negative pressure suction ultrafiltration power. Under the action of the two filtration powers of positive pressure push and negative pressure suction, the impurities in the extracellular vesicle stock solution to be separated whose particle size is larger than the pore size of the ultrafiltration membrane of the first-stage extracellular vesicle separation and purification device are retained, and flow through the concentrated liquid port of the ultrafilter through the multi-way valve, and then enter the concentrated negative pressure bottle for temporary storage. The extracellular vesicles and impurities in the extracellular vesicle stock solution to be separated whose particle size is smaller than the pore size of the ultrafiltration membrane of the first-stage extracellular vesicle separation and purification device pass through the ultrafiltration membrane, and enter the filtration negative pressure bottle for temporary storage through the filtrate port of the ultrafilter; S3: After the filtrate obtained by the first-stage extracellular vesicle separation and purification device accumulates in the filtration negative pressure bottle to the rated capacity of the filtration negative pressure bottle, the booster pump and the negative pressure aspirator in the filtration end negative pressure aspirator module stop working, the solenoid valve of the pipeline at the liquid inlet end of the filtration negative pressure bottle is closed, the multi-way valve in the filtration end negative pressure aspirator module closes the negative pressure channel and opens the vent, and the solenoid valve of the pipeline at the discharge end of the filtration negative pressure bottle is opened. Under the action of the power pump connected in series with the discharge port on the filtration negative pressure bottle, the filtrate in the filtration negative pressure bottle enters the filtration liquid bag; S4: If there is a next-stage extracellular vesicle separation and purification device, the filtrate in the filter bag of the previous stage enters the ultrafilter from the liquid inlet of the ultrafilter of the next-stage extracellular vesicle separation and purification device through a booster pump; S5: The booster inlet module provides positive pressure ultrafiltration power, and the filtration end negative pressure suction module provides negative pressure suction ultrafiltration power. Under the action of the two filtration powers of positive pressure push and negative pressure suction, the extracellular vesicles in the filtrate of the previous stage to be separated whose particle size is larger than the pore size of the ultrafiltration membrane of the next stage extracellular vesicle separation and purification device are retained, and flow through the concentrated liquid port of the ultrafilter through the multi-way valve, and then enter the concentrated negative pressure bottle for temporary storage. The extracellular vesicles and impurities in the filtrate of the previous stage to be separated whose particle size is smaller than the pore size of the ultrafiltration membrane of the next stage extracellular vesicle separation and purification device pass through the ultrafiltration membrane, and enter the filtration negative pressure bottle for temporary storage through the filtrate port of the ultrafilter; S6: After the concentrated liquid obtained by the next-stage extracellular vesicle separation and purification device accumulates in the concentrated negative pressure bottle to the rated capacity of the concentrated negative pressure bottle, the booster pump and the negative pressure aspirator stop working, the solenoid valve of the pipeline at the liquid inlet end of the concentrated negative pressure bottle is closed, the multi-way valve in the negative pressure aspirator module at the concentration end closes the negative pressure channel and opens the vent, and the solenoid valve of the pipeline at the discharge end of the concentrated negative pressure bottle is opened. Under the action of the power pump connected in series with the discharge port on the concentrated negative pressure bottle, the next-stage concentrated liquid in the concentrated negative pressure bottle enters the concentrated liquid bag; if there is still an extracellular vesicle separation and purification device at the next stage, S4-S6 are looped until all extracellular vesicle separation and purification devices in the extracellular vesicle continuous separation and purification device are executed.

23. The application method according to claim 22, characterized in that: The rated capacity of the filtering negative pressure bottle refers to 0.5-0.75 times the capacity of the filtering negative pressure bottle; the rated capacity of the concentrating negative pressure bottle refers to 0.5-0.75 times the capacity of the concentrating negative pressure bottle.

24. The application method according to claim 22, characterized in that: During the execution of S1-S6, the main control module monitors the pressure of the ultrafilter inlet, concentrate port, and filtered liquid port pipelines through pressure sensors, monitors the weight of the liquid in the corresponding concentrated negative pressure bottle and concentrated liquid bag through weighing sensors, and monitors the weight of the liquid in the corresponding extracellular vesicle stock solution bag, buffer bag, reflux liquid bag, filtered liquid bag, and filtered negative pressure bottle through auxiliary weighing sensors. When the main control module senses through the pressure sensor that the pressures at the liquid inlet and concentrate port of the ultrafilter continue to rise compared to the initial state, and the pressure at the filtrate port continues to drop compared to the initial state, and senses through the data of the weighing sensor and the auxiliary weighing sensor that the weight growth rate of the liquid in the filtration negative pressure bottle continues to drop compared to the initial state, and at the same time, the weight growth rate of the liquid in the concentrate negative pressure bottle continues to increase compared to the initial state, the main control module determines that the filtration efficiency of the ultrafilter has dropped, and instructs the extracellular vesicle separation and purification device corresponding to the current filtration negative pressure bottle and the concentrate negative pressure bottle to enter the ultrafiltration membrane cleaning operation mode, that is, by increasing the positive pressure driving power of the booster pump of the booster inlet module of the extracellular vesicle separation and purification device and increasing the negative pressure suction power of the negative pressure aspirator module at the concentrate end, the flow velocity of the fluid in the upper chamber of the ultrafilter is accelerated, the shear force between the fluid and the ultrafiltration membrane is increased, and the suspended particles and colloidal substances adsorbed and deposited on the surface of the ultrafiltration membrane are flushed away and taken away, thereby restoring the filtration efficiency of the ultrafiltration membrane; The main control module senses through the pressure sensor that the pressures at the liquid inlet, concentrate port, and filtrate port of the ultrafilter have returned to their initial states, and senses through the data of the weighing sensor and the auxiliary weighing sensor that the weight growth rate of the liquid in the filtration negative pressure bottle has returned to its initial state. At the same time, the weight growth rate of the liquid in the concentrate negative pressure bottle has returned to its initial state. The main control module determines that the filtration efficiency of the ultrafilter has recovered, and then calls back the positive pressure driving power of the boosting pump of the boosting liquid inlet module and the negative pressure suction power of the negative pressure aspirator module at the concentration end, so that the corresponding extracellular vesicle separation and purification device returns to the normal operation mode of separation and purification.

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