CN114874884A

CN114874884A - Extracellular vesicle continuous separation and purification device and application method

Info

Publication number: CN114874884A
Application number: CN202210665723.8A
Authority: CN
Inventors: 何向锋; 梁高峰; 施文; 何守贵
Original assignee: Jiangsu Sailana Biomedical Co ltd
Current assignee: Jiangsu Sailana Biomedical Co ltd
Priority date: 2022-06-13
Filing date: 2022-06-13
Publication date: 2022-08-09

Abstract

The invention provides an extracellular vesicle continuous separation and purification device which comprises an extracellular vesicle separation and purification device and a main control module which are connected with each other, wherein the extracellular vesicle separation and purification device comprises an ultrafilter module for separating extracellular vesicles with different particle size ranges, a pressurizing liquid inlet module for providing positive pressure ultrafiltration power and fluid flowing positive pressure pushing power in the ultrafilter module, a concentration end negative pressure aspirator module for providing fluid flowing negative pressure suction power in the ultrafilter module, a filtration end negative pressure aspirator module for providing negative pressure ultrafiltration power in the ultrafilter module and a pipeline temperature control module for maintaining the temperature of liquid to be separated in a pipeline and keeping the quality of the extracellular vesicles. The device described in the invention can continuously maintain the filtration efficiency of the ultrafiltration membrane, and realizes the purpose of carrying out totally-enclosed continuous flow high-flux difference aperture series dynamic ultrafiltration separation and purification of extracellular vesicles under the conditions of positive pressure at the liquid inlet end, negative pressure at the concentration end and negative pressure at the filtration end.

Description

Extracellular vesicle continuous separation and purification device and application method

Technical Field

The invention belongs to the technical field of extracellular vesicle separation and purification, and particularly relates to an extracellular vesicle continuous separation and purification device and an application method thereof.

Background

The Extracellular vesicles (Extracellular vesicles) are small vesicles obtained by the automatic secretion release of cells of human, animals, plants or microorganisms or the release of the cells after the cells are influenced by physical, chemical and biological factors, have a lipid bilayer membrane structure, have diameters ranging from tens of nanometers to several micrometers, and include exosomes (exosomes), microvesicles (microviscles), Apoptotic bodies (Apoptotic bodies), Shedding vesicles (Shedding vesicles), microparticles (microparticles) and the like. The extracellular vesicles carry biologically active proteins, lipids, messenger rna (mrna), micro rna (mirna), non-coding rna (ncrna), DNA fragments, etc., and are capable of transferring these active biomolecules to recipient cells, thereby modulating the biological function of target cells.

Extracellular vesicles are a new class of biotherapeutic agents and drug delivery vehicles. For example, 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 modulate immune function, and can be clinically used as biotherapeutic agents to inhibit inflammatory responses, reduce tissue damage, promote tissue regeneration, and restore physiological function. The extracellular vesicles can also be used as drug delivery carriers, and artificially synthesized small interfering RNA (siRNA), miRNA, chemotherapeutic drugs and the like are loaded in the extracellular vesicles by utilizing methods such as electroporation, chemical perforation, ultrasonic perforation, nano microfluidic technology and the like, so that the extracellular vesicles can be used for treating diseases. It has now been demonstrated in animal models that certain extracellular vesicles can promote angiogenesis, reduce inflammatory responses, promote nerve regeneration, reduce myocardial and cerebral ischemic injury, protect against acute kidney and lung injury, and treat diabetes and neurodegenerative diseases.

Currently, common methods for separating and purifying extracellular vesicles include ultra-high-speed refrigerated centrifugation, rotary ultrafiltration, size exclusion chromatography, immunomagnetic bead capture, polymer precipitation, and membrane affinity capture. The ultra-high-speed freezing and centrifuging method is based on the difference of the sizes and densities of extracellular vesicles and other components in a liquid to be separated, and the extracellular vesicles and the other components are separated by means of an ultra-high-speed freezing and centrifuging machine. Although the rotary ultrafiltration method can separate and obtain extracellular vesicles with high purity and has relatively simple requirements on equipment, the ultrafiltration membrane is easy to block, so that the separation and purification efficiency is rapidly reduced. Size exclusion chromatography is a screening method based on the size of the extracellular vesicle particle size, and the product has high purity, but cannot realize the concentration of the extracellular vesicle. The immunomagnetic bead capture method is used for separating extracellular vesicles by means of specific binding of antibodies and extracellular vesicle surface antigens, has high specificity, is expensive, is easily influenced by differences of antigen expression quantity, and has great difference of capture efficiency of the extracellular vesicles of different subtypes. The polymer precipitation method is to utilize polymers such as polyethylene glycol (PEG) and the like, reduce the suspension stability of extracellular vesicles by changing the microenvironment in a liquid to be separated so as to enable the extracellular vesicles to be precipitated and separated, and the method does not need large-scale equipment, but can cause a large amount of polymerized heteroprotein to be precipitated together with the extracellular vesicles so as to seriously affect the purity of products. The above extracellular vesicle separation and purification methods have different advantages and disadvantages, but generally have the defects of complex operation, long time consumption, low yield, poor purity and the like, and greatly limit the further application of the extracellular vesicles in the field of biomedicine. Therefore, it is necessary to develop a high-efficiency, low-cost, ultra-high-throughput, continuous-operation, and size-scalable separation and purification technique for extracellular vesicles on an industrial scale.

Disclosure of Invention

The invention solves the defects of complex operation, long time consumption, low yield, poor purity and difficulty in realizing size classification in the prior art for separating and purifying the extracellular vesicles, and provides the extracellular vesicle continuous separation and purification device and the application method thereof.

In a first aspect, the invention provides an extracellular vesicle continuous separation and purification device, which comprises an extracellular vesicle separation and purification device and a main control module, wherein the extracellular vesicle separation and purification device is electrically connected with the main control module, the extracellular vesicle separation and purification device comprises an ultrafilter module, a pressurizing liquid inlet module, a concentration end negative pressure aspirator module and a filtration end negative pressure aspirator module, the ultrafilter module is connected with the pressurizing liquid inlet module, the concentration end negative pressure aspirator module and the filtration end negative pressure aspirator module through pipelines,

wherein the pressurizing liquid inlet module is used for providing positive pressure ultrafiltration power in the ultrafilter module and fluid flowing positive pressure pushing power in an upper chamber cavity of the ultrafilter,

the concentration end negative pressure suction device module provides the fluid flow negative pressure suction power in the upper chamber of the ultrafilter module,

the filtration end negative pressure suction apparatus module provides negative pressure ultrafiltration power in the ultrafilter module,

the ultrafilter module is used for separating extracellular vesicles with different particle size ranges.

In some embodiments, the extracellular vesicle separation and purification device at least comprises two stages of extracellular vesicle separation and purification devices connected in series in sequence, the pore diameter of the ultrafiltration membrane of the previous stage of extracellular vesicle separation and purification device is larger than that of the ultrafiltration membrane of the next stage of extracellular vesicle separation and purification device, liquid in the filtrate bag of the previous stage of extracellular vesicle separation and purification device enters the pressurizing liquid inlet module of the next stage of extracellular vesicle separation and purification device, and concentrated liquid of each next stage is collected into the corresponding concentrated liquid bag, so that multi-stage continuous separation and purification of extracellular vesicles can be performed, and extracellular vesicles with different particle size ranges can be obtained.

In some embodiments, the ultrafilter module comprises an ultrafilter and a pipeline, the ultrafilter is provided with a liquid inlet connected with the pressurizing liquid inlet module, a concentrated liquid port connected with the concentrated end negative pressure aspirator module and a filtered liquid port connected with the filtered end negative pressure aspirator module, an ultrafiltration membrane is arranged in the ultrafilter, an upper chamber of the ultrafilter is arranged above the ultrafiltration membrane, a lower chamber of the ultrafilter is arranged below the ultrafiltration membrane,

the ultrafilter is any one of a flat coiled pipe dynamic ultrafilter, a flat scroll pipe dynamic ultrafilter and a hollow fiber membrane shell pipe type dynamic ultrafilter,

the upper chamber of the ultrafilter in the flat-plate coiled pipe dynamic ultrafilter is a coiled pipe chamber which is repeatedly bent, one end of the coiled pipe chamber is connected with the liquid inlet, the other end of the coiled pipe chamber is connected with the concentrated solution port, the lower chamber of the ultrafilter is connected with the filtered solution port,

the upper chamber of the ultrafilter in the flat spiral pipe dynamic ultrafilter is a spiral pipe cavity from the center to the outside, the center of the spiral pipe cavity is connected with the liquid inlet, the peripheral tail end of the spiral pipe cavity is connected with the concentrated liquid port, the lower chamber of the ultrafilter is connected with the filtered liquid port,

the hollow fiber membrane shell-tube type dynamic ultrafilter comprises an internal pressure type hollow fiber membrane shell-tube type dynamic ultrafilter and an external pressure type hollow fiber membrane shell-tube type dynamic ultrafilter, wherein the internal pressure type hollow fiber membrane shell-tube type dynamic ultrafilter consists of a membrane tube shell, tube plate end sockets and hollow fiber membranes, the hollow fiber membranes are directly encapsulated in the membrane tube shell at two ends by 2 tube plate end sockets, one end of a hollow fiber membrane tube cavity is converged to a front chamber cavity and connected with a liquid inlet, the other end of the hollow fiber membrane tube cavity is converged to a rear chamber cavity and connected with a concentrated liquid port, and an outer chamber between the hollow fiber membranes and the membrane tube shell is connected with a filtered liquid port; the external pressure type hollow fiber membrane shell tube type dynamic ultrafilter is composed of a membrane tube shell, a tube plate end socket, an end cover plate and a hollow fiber membrane, wherein the hollow fiber membrane is composed of the tube plate end socket and the end cover plate which are directly encapsulated at two ends in the membrane tube shell, the hollow fiber membrane is connected with one end of an outer chamber between the membrane tube shells and a liquid inlet, the hollow fiber membrane is connected with the other end of the outer chamber between the membrane tube shells and a concentrated liquid port, one end of a hollow fiber membrane tube cavity is sealed by the end cover plate, and the other end of the hollow fiber membrane tube cavity is collected into a filter chamber and connected with a filtered liquid port.

In some embodiments, the ultrafiltration membrane has a pore size of 10 nm to 4000 nm.

In some embodiments, the pressurizing liquid inlet module comprises a pressurizing pump, a buffer liquid bag, a backflow liquid bag, a multi-way valve and a pipeline, the cell outer vesicle liquid bag, the buffer liquid bag and the backflow liquid bag collect the multi-way valve through the pipeline, the cell outer vesicle liquid bag is a cell outer vesicle raw liquid bag or a filtering liquid bag in a cell outer vesicle separation and purification device of the previous stage, the multi-way valve is connected with the pressurizing pump through the pipeline, the pressurizing pump is connected with a liquid inlet of the ultrafilter through the pipeline, a pressure sensor is connected in series between the pressurizing pump and the liquid inlet of the ultrafilter, and the pressure sensor is electrically connected with the main control module,

the booster pump is any one of a diaphragm pump, a peristaltic pump, a plunger pump and a gear pump, and preferably the diaphragm pump or the peristaltic pump.

In some embodiments, the extracellular vesicle continuous separation and purification device further comprises auxiliary weighing sensors arranged corresponding to the extracellular vesicle stock solution bag, the buffer solution bag, the reflux solution bag and the filtration solution bag, the auxiliary weighing sensors are electrically connected with the main control module,

the auxiliary weighing sensor is preferably a suspension type weighing sensor.

In some embodiments, the concentration end negative pressure aspirator module comprises a concentration negative pressure bottle, a negative pressure aspirator, an air filter, a multi-way valve, a concentrate bag and a pipeline, wherein a negative pressure exhaust port, a liquid inlet and a liquid outlet are arranged on the concentration negative pressure bottle, the negative pressure exhaust port is connected with the negative pressure aspirator through the pipeline, the air filter, the multi-way valve, a vent, an air buffer and an air pressure sensor are connected in series on the pipeline between the negative pressure exhaust port and the negative pressure aspirator, the liquid inlet is connected with the concentrate port of the ultrafilter and the reflux bag through the pipeline and the 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 with the main control module, the liquid outlet is connected with the concentrate bag through the pipeline, and an electromagnetic valve and a power pump are connected in series between the liquid outlet and the concentrate bag,

the power pump is preferably a peristaltic pump.

In some embodiments, the concentration end negative pressure suction apparatus module further comprises a weighing sensor, the weighing sensor is arranged corresponding to the concentration negative pressure bottle and the concentration liquid bag, the weighing sensor is electrically connected with the main control module,

the weighing sensor arranged corresponding to the concentration negative pressure bottle is preferably a supporting weighing sensor,

the suspension type weighing sensor is preferably selected as the weighing sensor arranged corresponding to the concentrated solution bag;

the concentrated negative pressure bottle is a hard bottle, the concentrated negative pressure bottle comprises a bottle body and a bottle cap, a groove is formed in the bottom of the bottle body, liquid in the concentrated negative pressure bottle can be conveniently gathered through the groove, the shape of the groove is preferably a cone with the upper part wide and the lower part narrow or a round bottom with the upper part wide and the lower part narrow, and the negative pressure air outlet, the liquid inlet and the liquid outlet are formed in the bottle cap.

In some embodiments, the filtration end negative pressure aspirator module comprises a filtration negative pressure bottle, a negative pressure aspirator, an air filter, a multi-way valve, a filtration liquid bag and a pipeline, the filtration negative pressure exhaust port is connected with the negative pressure aspirator through the pipeline, the air filter, the 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 and the negative pressure aspirator, the liquid inlet is connected with a filtration liquid port of the ultrafilter through the pipeline, an electromagnetic valve and a pressure sensor are connected in series between the liquid inlet and the filtration liquid port of the ultrafilter, the electromagnetic valve and the pressure sensor are electrically connected with the main control module, the liquid discharge port is connected with the filtration liquid bag through the pipeline, and an electromagnetic valve and a power pump are connected in series between the liquid discharge port and the filtration liquid bag,

the power pump is preferably a peristaltic pump.

In some embodiments, the filter end negative pressure suction apparatus module further comprises a weighing sensor, the weighing sensor is arranged corresponding to the filter negative pressure bottle and the filter liquid bag, the weighing sensor is electrically connected with the main control module,

the weighing sensor arranged corresponding to the filtering negative pressure bottle is preferably a supporting weighing sensor,

the suspension type weighing sensor is preferably selected as the weighing sensor arranged corresponding to the filtering liquid bag;

the filtering negative pressure bottle is a hard bottle and comprises a bottle body and a bottle cap, a groove is formed in the bottom of the bottle body, liquid in the filtering negative pressure bottle can be conveniently collected by the aid of the groove, the shape of the groove is preferably a cone with the upper part wide and the lower part narrow or a round bottom with the upper part wide and the lower part narrow, and the negative pressure air outlet, the liquid inlet and the liquid outlet are formed in the bottle cap.

In some embodiments, the extracellular vesicle separation and purification device further comprises a pipeline temperature control module, wherein the pipeline temperature control module acts on a pipeline involved in the extracellular vesicle separation and purification device, so that the liquid in the pipeline is in a required temperature environment.

In some embodiments, the pipeline adopts a hose, the pipeline temperature control module comprises a winding type temperature controller and a cold source device, the winding type temperature controller is connected with the cold source device, and the pipeline is wound on the winding type temperature controller.

In some embodiments, the wound thermostat is made of a metal with good heat conductivity, a spiral groove is formed on the surface of the wound thermostat, and the pipeline is wound in the spiral groove; the cold source device comprises a refrigeration water circulating device and a Peltier semiconductor refrigeration sheet, the refrigeration water circulating device is a low-temperature constant-temperature cold water circulating device, a cold water circulating pipeline is arranged in the winding type temperature controller, the cold water circulating pipeline of the low-temperature constant-temperature cold water circulating device is communicated with the cold water circulating pipeline of the winding type temperature controller, and the winding type temperature controller is cooled through the low-temperature constant-temperature cold water circulating device; the winding temperature controller is arranged corresponding to the pipeline, the water temperature control range in the low-temperature constant-temperature cold water circulating device is 0-8 ℃,

the metal is preferably metallic aluminum, metallic copper or a metal alloy.

In some embodiments, the extracellular vesicle separation and purification device further comprises an alarm module and a human-computer interaction module, both of which are electrically connected with the main control module,

the alarm module is provided with an audio-visual high-low pressure alarm and is used for closing the booster pump, the concentration end negative pressure suction apparatus and the filtering end negative pressure suction apparatus so as to maintain the integrity of the ultrafiltration membrane and achieve high-efficiency extracellular vesicle separation and purification.

In a second aspect, the invention provides an application method of the device for continuously separating and purifying extracellular vesicles, which comprises the following steps:

s1: adding the extracellular vesicle stock solution into an extracellular vesicle stock solution bag of a first-stage extracellular vesicle separation and purification device, and enabling the extracellular vesicle stock solution to be separated to enter an ultrafilter from a liquid inlet of the ultrafilter through a booster pump;

s2: the pressurized liquid inlet module provides positive pressure ultrafiltration power, the filtration end negative pressure aspirator module provides negative pressure suction ultrafiltration power, impurities with the particle size larger than the ultrafiltration membrane pore size of the first-stage extracellular vesicle separation and purification device in the extracellular vesicle stock solution to be separated are intercepted under the action of the positive pressure pushing and negative pressure suction filtration power, flow through a multi-way valve through a concentrated solution port of the ultrafilter and then enter the concentrated negative pressure bottle for temporary storage, and extracellular vesicles with the particle size smaller than the ultrafiltration membrane pore size of the first-stage extracellular vesicle separation and purification device in the extracellular vesicle stock solution to be separated pass through the ultrafiltration membrane and enter the filtration negative pressure bottle for temporary storage through a filtration solution port of the ultrafilter;

s3: after the filtrate obtained by the first-stage extracellular vesicle separation and purification device is accumulated in the filtering negative pressure bottle to reach the rated capacity of the filtering negative pressure bottle, a booster pump and a negative pressure aspirator in a filtering end negative pressure aspirator module stop working, an electromagnetic valve of a pipeline at the liquid inlet end of the filtering negative pressure bottle is closed, a multi-way valve in the filtering end negative pressure aspirator module closes a negative pressure channel and opens an air vent, the electromagnetic valve of the pipeline at the liquid outlet end of the filtering negative pressure bottle is opened, and the filtrate in the negative pressure bottle enters a filtering liquid bag under the action of a power pump;

s4: if the next-stage extracellular vesicle separation and purification device exists, the filtrate in the filtrate 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 the booster pump;

s5: the pressurized liquid inlet module provides positive pressure ultrafiltration power, the filtration end negative pressure aspirator module provides negative pressure suction ultrafiltration power, the filtrate to be separated from the upper stage is under the action of the positive pressure pushing and negative pressure suction filtration power, extracellular vesicles of which the particle size is larger than the pore size of the ultrafiltration membrane of the next stage extracellular vesicle separation and purification device in the filtrate to be separated from the upper stage are intercepted and pass through a multi-way valve through a concentrated liquid port of the ultrafilter and then enter the concentrated negative pressure bottle for temporary storage, and the extracellular vesicles of which the particle size is smaller than the pore size of the ultrafiltration membrane of the next stage extracellular vesicle separation and purification device in the filtrate to be separated from the upper stage pass through the ultrafiltration membrane and enter the filtration negative pressure bottle for temporary storage through a filtrate port of the ultrafilter;

s6: after the concentrated solution obtained by the next-stage extracellular vesicle separation and purification device is accumulated in the concentrated negative pressure bottle to reach the rated capacity of the concentrated negative pressure bottle, the booster pump and the negative pressure aspirator stop working, the electromagnetic valve of the pipeline at the liquid inlet end of the concentrated negative pressure bottle is closed, the multi-way valve in the module of the concentrated negative pressure aspirator closes the negative pressure channel and opens the air vent, the electromagnetic valve of the pipeline at the liquid outlet end of the concentrated negative pressure bottle is opened, and the next-stage concentrated solution in the negative pressure bottle enters the concentrated solution bag under the action of the power pump; and if the next extracellular vesicle separating and purifying device is still present, circulating S4-S6 until all extracellular vesicle separating and purifying devices in the extracellular vesicle separating and purifying devices are completed.

In some embodiments, the rated capacity of the filtration negative pressure bottle refers to 0.5 to 0.75 times the capacity in the filtration negative pressure bottle; the rated capacity of the concentrated negative pressure bottle is 0.5-0.75 times of the capacity in the concentrated negative pressure bottle.

In some embodiments, during execution of S1-S6, the main control module monitors the pressure of the liquid inlet, the concentrated liquid inlet and the filtered liquid inlet of the ultrafilter through pressure sensors, monitors the weight of the liquid in the extracellular vesicle stock solution bag, the buffer solution bag, the reflux solution bag, the filtered liquid bag, the concentrated liquid bag and the filtering negative pressure bottle and the concentrating negative pressure bottle through weighing sensors and auxiliary weighing sensors,

when the main control module senses that the pressure of a liquid inlet and a concentrated liquid inlet of the ultrafilter continuously rises compared with the initial state through the pressure sensor, the pressure of the filtered liquid inlet continuously falls compared with the initial state, senses that the increase of the weight of liquid in the filtering negative pressure bottle continuously falls compared with the initial state through the data of the weighing sensor and the auxiliary weighing sensor, and simultaneously, the increase of the weight of liquid in the concentrating negative pressure bottle continuously increases compared with the initial state, the main control module judges that the filtering efficiency of the ultrafilter falls, and commands the extracellular vesicle separation and purification devices corresponding to the current filtering negative pressure bottle and the concentrating negative pressure bottle to enter an ultrafiltration membrane cleaning operation mode, namely, the flow speed of fluid in an upper chamber of the ultrafilter is accelerated together by increasing the positive pressure pushing power of a pressurizing liquid inlet module of the extracellular vesicle separation and purification device and increasing the negative pressure suction power of a negative pressure device module at a concentrating end, increasing the shearing force between the ultrafiltration membrane and the membrane, flushing away suspended particles and colloidal substances adsorbed and deposited on the surface of the ultrafiltration membrane, and recovering the filtration efficiency of the ultrafiltration membrane;

the main control module senses the initial state of pressure recovery of a liquid inlet, a concentrated liquid port and a filtered liquid port of the ultrafilter through the pressure sensor, senses the initial state of liquid weight acceleration recovery in the filtered negative pressure bottle through the data of the weighing sensor and the auxiliary weighing sensor, simultaneously, the initial state of liquid weight acceleration recovery in the concentrated negative pressure bottle, judges that the filtering efficiency of the ultrafilter is recovered, and then, the booster liquid inlet module booster pump is adjusted back to the positive pressure pushing power and the concentration end negative pressure suction device module is adjusted back to the negative pressure suction power, so that the corresponding extracellular vesicle separation and purification device is recovered to a separation and purification normal operation mode.

The extracellular vesicle continuous separation and purification device provided by the invention has the following advantages:

1. the treatment time is faster, and the totally-enclosed continuous flow high-flux difference aperture series dynamic ultrafiltration separation and purification of extracellular vesicles can be carried out under the conditions of positive pressure at the liquid inlet end, negative pressure at the concentration end and negative pressure at the filtration end;

2. the liquid inlet end of the ultrafilter provides positive pressure ultrafiltration power, the filtering end of the ultrafilter provides negative pressure suction ultrafiltration power, and the ultrafiltration power of the ultrafilter is stable, reliable and strong under the action of positive pressure pushing and negative pressure suction ultrafiltration power;

3. the high filtration efficiency is kept for a long time, the liquid inlet end of the ultrafilter provides positive pressure pushing power for the fluid flowing in the upper chamber cavity of the ultrafilter through a booster pump, the concentration end of the ultrafilter provides negative pressure suction power for the fluid flowing in the upper chamber cavity of the ultrafilter, and the liquid in the upper chamber cavity of the ultrafilter rapidly flows forwards in the upper chamber cavity of the ultrafilter under the action of the positive pressure pushing power and the negative pressure suction power to form shearing force with the ultrafiltration membrane, so that the adsorption and deposition of suspended particles and colloidal substances on the surface of the ultrafiltration membrane are prevented, the concentration polarization effect is overcome, the attenuation rate of the ultrafiltration membrane flux is effectively reduced, and the high filtration efficiency of the ultrafiltration membrane is kept for a long time;

4. the device has an ultrafiltration membrane cleaning operation mode, and increases positive pressure pushing power of a booster pump of a pressurized liquid inlet module of the extracellular vesicle separation and purification device and negative pressure suction power of a negative pressure suction module of a concentration end by increasing the pressure of the booster pump, so that the flow speed of fluid in an upper chamber of the ultrafiltration membrane is increased, the shearing force between the fluid and the ultrafiltration membrane is increased, suspended particles and colloidal substances adsorbed and deposited on the surface of the ultrafiltration membrane are flushed away, and the filtration efficiency of the ultrafiltration membrane is recovered;

5. separating and purifying the extracellular vesicles according to different particle sizes, and adjusting adjacent extracellular vesicle separation and purification devices to perform multi-stage separation and purification on the extracellular vesicles by using ultrafiltration membranes with different pore sizes so as to obtain the extracellular vesicles with different particle size ranges;

6. obtain concentrate and filtrate simultaneously, this device is multiple functional, both can obtain the concentrate through the one-step method, can obtain the filtrate again.

7. The device can directly and simply enlarge the capacity on a large scale, can be independently applied by one set of separation unit, can also be applied by a plurality of separation and purification units in parallel, and can multiply increase the yield of extracellular vesicle products on a large scale;

8. the device has the function of controlling the temperature of the pipeline, can keep the liquid to be separated in the pipeline at 0-8 ℃, and the lower liquid temperature is beneficial to protecting the quality of extracellular vesicles in the separation and purification process;

9. the whole process and the whole closed pipeline structure are adopted, the whole process of separating and purifying the extracellular vesicles is completed in the closed pipeline, the extracellular vesicles are separated and purified under the whole process sterile condition, and the device can be communicated with extracellular vesicle stock solution production equipment, extracellular vesicle split-packaging freeze-drying equipment and the like for use, so that the whole process and the whole closed production of extracellular vesicle products are really realized;

10. the high automation, the microcomputer host system of this device can highly intelligent automatic regulating apparatus operation with the help of the data acquisition of a plurality of pressure sensor, suspension type weighing sensor, bearing formula weighing sensor, baroceptor, temperature sensor etc. keep high-efficient, the outer vesicle of safe separation purification cell to can download and draw the spreadsheet that forms each parameter to operation data is automatic.

Drawings

Fig. 1 is a schematic diagram of a framework structure of an extracellular vesicle continuous separation and purification apparatus according to some embodiments of the present invention;

FIG. 2 is a schematic view showing the structure of the device for continuous separation and purification of extracellular vesicles according to example 1;

FIG. 3 is a schematic view of the serial connection structure of the extracellular vesicle continuous separation and purification device in example 2;

FIG. 4 is a schematic diagram of a top view of a flat coiled dynamic ultrafilter, with arrows indicating the flow direction;

FIG. 5 is a schematic diagram of a top view of a dynamic ultrafilter with flat spiral tubes, wherein the arrows indicate the flow direction;

FIG. 6 is a schematic view of the connection structure between the wound thermostat and the pipeline, wherein the thin arrows represent the direction of the liquid flow in the separation and purification pipeline, and the thick arrows represent the direction of the liquid flow in the cold water circulation pipeline;

FIG. 7 is a schematic structural view of a shell-and-tube dynamic ultrafilter of an internal pressure hollow fiber membrane, wherein arrows represent the flow direction;

FIG. 8 is a schematic structural view of a shell-and-tube type dynamic ultrafilter of an external pressure type hollow fiber membrane, wherein arrows represent the flow direction.

Detailed Description

Example 1

With reference to fig. 1 and fig. 2, the present embodiment provides an extracellular vesicle continuous separation and purification apparatus, which includes an extracellular vesicle separation and purification apparatus and a main control module, the extracellular vesicle separation and purification apparatus is electrically connected to the main control module, the extracellular vesicle separation and purification apparatus includes an ultrafilter module, a pressurizing 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 pressurizing liquid inlet module, the concentration end negative pressure aspirator module and the filtration end negative pressure aspirator module through pipelines, the pipeline temperature control module is disposed on a pipeline involved in the extracellular vesicle separation and purification apparatus, wherein the pipeline temperature control module is disposed on the pipeline involved in the extracellular vesicle separation and purification apparatus, and the device is further configured to control the temperature of the pipeline

The pressurizing liquid inlet module is used for providing positive pressure ultrafiltration power in the ultrafilter module and positive pressure pushing power for fluid flowing in an upper chamber of the ultrafilter,

the concentration end negative pressure suction module provides the fluid flow negative pressure suction power in the upper chamber of the ultrafilter module,

the negative pressure suction device module at the filtering end provides negative pressure ultrafiltration power in the ultrafilter module,

The ultrafilter module comprises an ultrafilter and a pipeline, the ultrafilter is an ultrafilter with three ports, the three ports are respectively a liquid inlet connected with the pressurizing liquid inlet module, a concentrated liquid port connected with the concentrated end negative pressure suction device module and a filtered liquid port connected with the filtered end negative pressure suction device module,

the ultrafilter is a flat scroll dynamic ultrafilter, the upper chamber of the ultrafilter in the flat scroll dynamic ultrafilter is a spiral scroll chamber from the center to the outside, the center of the scroll chamber is connected with the liquid inlet, the peripheral tail end of the scroll chamber is connected with the concentrated liquid port, and the lower chamber of the ultrafilter is connected with the filtered liquid port.

The aperture of the ultrafiltration membrane is 10-4000 nm.

The ultrafiltration membrane can be directly selected from the existing ultrafiltration membranes on the market, such as: the material is an ultrafiltration membrane made of cellulose acetate, cellulose acetate esters, polyethylene, polysulfone, polyamide, aromatic polymer, nylon, polyethersulfone, hydrophilic polyvinylidene fluoride, polytetrafluoroethylene, hydrophilic polytetrafluoroethylene, mixed cellulose, hydrophilic polycarbonate and porous alumina.

The pressurizing liquid inlet module comprises a pressurizing pump, an extracellular vesicle stock solution bag, a buffer solution bag, a reflux solution bag, a multi-way valve 1 and a pipeline, wherein the extracellular vesicle stock solution bag, the buffer solution bag and the reflux solution bag are converged into the multi-way valve 1 through the pipeline, the multi-way valve 1 is connected with the pressurizing pump 1 through the pipeline, the pressurizing pump 1 is connected with a liquid inlet of the flat spiral pipe dynamic ultrafilter 1 through the pipeline, and the pressurizing liquid inlet module comprises a pressurizing pump, an extracellular vesicle stock solution bag, a buffer solution bag, a reflux solution bag, a multi-way valve 1 and a pipeline

The booster pump 1 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 is adopted, the conveyed liquid is separated from the plunger and the pump cylinder through the arrangement of the diaphragm pump, and the cleanliness of the liquid in the conveying process is kept; through the arrangement of the peristaltic pump, the fluid only contacts the pipeline and does not contact the pump body, so that the cleanliness of the liquid in the conveying process is kept;

a pressure sensor 1 is connected in series between the booster pump 1 and the liquid inlet of the flat worm pipe dynamic ultrafilter 1,

the buffer bag is used for containing buffer Solution, the buffer Solution comprises normal saline, PBS buffer Solution, Hanks Balanced Salt Solution (HBSS) and the like, the buffer bag can balance the ultrafilter and the pipeline before the separation and purification of the extracellular vesicles, adjust the concentration of the extracellular vesicles in the liquid to be separated in the separation and purification process, flush the upper chamber of the ultrafilter in the separation and purification process to recover the filtration efficiency, flush the pipeline after the separation and purification process, and avoid the waste of the extracellular vesicles;

the function of the backflow liquid bag is as follows: 1. in each stage of the process of separating and purifying the extracellular vesicles, liquid to be separated can repeatedly and circularly pass through the ultrafilter through the reflux liquid bag for multiple times, so that the separation and purification efficiency of the extracellular vesicles is improved, and waste is avoided; 2. when washing the ultrafilter upper chamber, under the condition that the booster pump provides fluid flow malleation promotion power, buffer solution can be through the circulation flushing ultrafilter that returns the liquid bag repeatedly, will adsorb and take away with the suspended particles and the colloidal substance that deposit on milipore filter surface, resumes filtration efficiency until the ultrafilter, avoids extravagant too much buffer solution.

In some specific implementation manners, the cell outer vesicle stock solution bag can be corresponded, the buffer solution bag and the backflow solution bag are provided with auxiliary weighing sensors, the auxiliary weighing sensors are electrically connected with the main control module, the suspension type weighing sensors are preferably selected as the auxiliary weighing sensors, the auxiliary weighing sensors are arranged above the corresponding cell outer vesicle stock solution bag, the buffer solution bag and the backflow solution bag, the suspension type weighing sensors 1, the suspension type weighing sensors 2 and the suspension type weighing sensors 3 are respectively arranged, the suspension type weighing sensors 1, the suspension type weighing sensors 2 and the suspension type weighing sensors 3 are respectively used for monitoring the weight change conditions of the liquid in the cell outer vesicle stock solution bag, the buffer solution bag and the backflow solution bag.

The concentration end negative pressure aspirator module comprises a concentration negative pressure bottle 1, a weighing sensor 1, a negative pressure aspirator 1, an air pressure sensor 1, an air pressure buffer 1, an air filter 1, a multi-way valve 3, a concentrated solution bag and a pipeline, wherein a negative pressure exhaust port, a liquid inlet and a liquid outlet are arranged on the concentration negative pressure bottle 1, the negative pressure exhaust port is connected with the negative pressure aspirator 1 through the 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 aspirator 1, two ports on the multi-way valve 3 are respectively connected with the air filter 1 and the air pressure buffer 1, a port is also arranged on the multi-way valve 3 as a vent 1, the liquid inlet is connected with the concentrated solution port and the reflux solution bag of the flat spiral dynamic ultrafilter 1 through the pipeline and the multi-way valve 2, the concentrated solution bag is connected with the peristaltic solution bag through the pipeline, and an electromagnetic valve 1 and the liquid outlet 1 are connected in series between the concentrated solution bag, a weighing sensor 1 connected with the main control module is arranged below the concentration negative pressure bottle 1, the weighing sensor 1 is a supporting weighing sensor 1 and is used for monitoring the weight change condition of the liquid in the concentration negative pressure bottle 1,

a pressure sensor 2 is connected in series between the multi-way valve 2 and the concentrated solution port of the flat spiral pipe dynamic ultrafilter 1.

In some specific implementation manners, a weighing sensor is arranged corresponding to the concentrated liquid bag, the weighing sensor is electrically connected with the main control module, and the weighing sensor is preferably a suspension type weighing sensor 4 arranged above the corresponding concentrated liquid bag and used for monitoring the weight change condition of the liquid in the concentrated liquid bag.

The filtering end negative pressure aspirator module comprises a filtering negative pressure bottle 1, a weighing sensor 2, a negative pressure aspirator 2, an air pressure sensor 2, an air pressure buffer 2, an air filter 2, a multi-way valve 4, a filtering liquid bag and a pipeline, wherein a negative pressure exhaust port, a liquid inlet and a liquid outlet are arranged on the filtering negative pressure bottle 1, the negative pressure exhaust port is connected with the negative pressure aspirator 2 through the 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 aspirator 2, two ports on the multi-way valve 4 are respectively connected with the air filter 2 and the air pressure buffer 2, a port is also arranged on the multi-way valve 4 as a vent 2, the liquid inlet is connected with the filtering liquid port of the flat spiral pipe dynamic ultra-filter 1 through the pipeline and the electromagnetic valve 3 and a pump 2 between the liquid outlet and the filtering liquid bag through the pipeline and the peristaltic pump 2, a weighing sensor 2 connected with the main control module is arranged below the filtering negative pressure bottle 1, the weighing sensor 2 is a supporting weighing sensor 2 and is used for monitoring the weight change condition of the liquid in the filtering negative pressure bottle 1,

a pressure sensor 3 is connected in series between the electromagnetic valve 2 and the filtering liquid port of the flat worm pipe dynamic ultrafilter 1.

In some specific implementation manners, a weighing sensor is arranged corresponding to the filtering liquid bag and electrically connected with the main control module, and a suspension type weighing sensor 5 is preferably arranged above the corresponding filtering liquid bag and used for monitoring the weight change condition of the liquid in the filtering liquid bag.

It should be noted that, the concentration negative pressure bottle 1 and the filtration negative pressure bottle 1 are both hard bottles, each hard bottle includes a bottle body and a bottle cap, and the bottom of the bottle body adopts a design favorable for liquid accumulation in the negative pressure bottle, for example, a groove is arranged at the bottom of the bottle body, the liquid accumulation at the bottom of the negative pressure bottle is realized through the groove, the shape of the groove can be a cone shape with a wide upper part and a narrow lower part or a round bottom with a wide upper part and a narrow lower part, and the negative pressure exhaust port, the liquid inlet and the liquid outlet are arranged on the bottle cap. The concentration negative pressure bottle 1 and the filtering negative pressure bottle 1 can directly select the existing negative pressure bottles on the market.

The pipeline temperature control module comprises a winding type temperature controller and a cold source device, the winding type temperature controller is connected with the cold source device, the pipeline is designed by a hose, and the pipeline is wound on the winding type temperature controller.

The wound temperature controller is made of metal with good heat conduction, preferably metal aluminum, metal copper or metal alloy, and combines the content shown in fig. 6, the surface of the wound temperature controller is provided with a spiral groove, a pipeline is wound on the wound temperature controller along the spiral groove, a cold water circulation pipeline is arranged in the wound temperature controller, the wound temperature controller is fixedly connected with a shell or other structures of the extracellular vesicle continuous separation and purification device, the cold water circulation pipeline of the wound temperature controller is communicated with the cold water circulation pipeline of the cold source device, the arrangement of the spiral groove on the surface of the wound temperature controller can effectively guide the wound pipeline, so that the contact area between the pipeline and the wound temperature controller is increased, the heat exchange efficiency is improved, the wound pipeline can also be supported by the design of the spiral groove, and the probability of pipeline slipping on the wound temperature controller is effectively reduced;

the cold source device comprises a refrigeration water circulating device and a Peltier semiconductor refrigerating sheet, the refrigeration water circulating device is a low-temperature constant-temperature refrigeration water circulating device, the water temperature is controlled to be 0-8 ℃, the Peltier semiconductor refrigerating sheet is used for reducing the water temperature in the refrigeration water circulation, so that the temperature of the water in the circulation is controlled to be within a required temperature range value, the low-temperature constant-temperature refrigeration water circulating device comprises a circulating pump and a soft guide pipe, the low-temperature constant-temperature refrigeration water circulating device is connected with a water inlet/outlet of the winding type temperature controller through the soft guide pipe, the cold water in the winding type temperature controller circularly flows through the circulating pump and the soft guide pipe, and the winding type temperature controller is quickly cooled;

the device for separating and purifying the extracellular vesicles is provided with a plurality of winding temperature controllers which are distributed at each position of the pipeline, such as before the liquid inlet of the flat spiral dynamic ultrafilter 1, before the inlet of the concentrated solution bag, before the inlet of the filtered solution bag and the like.

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, supported weighing sensor 1, supported weighing sensor 2, pressure sensor 1, pressure sensor 2, pressure sensor 3, pressure sensor 1 and pressure sensor 2, in the specific implementation, a working environment temperature sensor is arranged on the extracellular vesicle separation and purification device to detect the room temperature, a circulating water temperature sensor is arranged on the low-temperature constant-temperature cold water circulation device of the pipeline temperature control module, and the circulating water temperature sensor, the working environment temperature sensor, the suspended weighing sensor 1, suspended weighing sensor 2, suspended weighing sensor 3, suspended weighing sensor 4, suspended weighing sensor 5, supported weighing sensor 1, pressure sensor 2, and the above-mentioned suspended weighing sensor 1, suspended weighing sensor 3, suspended weighing sensor 4, suspended weighing sensor 5, suspended weighing sensor 1, pressure sensor 2, The bearing type weighing sensor 2, the pressure sensor 1, the pressure sensor 2, the pressure sensor 3, the air pressure sensor 1 and the air pressure sensor 2 form a sensor detection module, and the sensor detection module is connected with the main control module.

The microcomputer can be directly used as the main control module.

In some specific embodiments, the extracellular vesicle continuous separation and purification device further comprises an alarm module and a human-computer interaction module, and the alarm module and the human-computer interaction module are connected with the main control module. The man-machine interaction module comprises an operation control panel and/or a touch screen. The alarm module may emit an audible and/or visual alarm signal. The human-computer interaction module is used for adjusting and setting reference thresholds of various parameters and values detected by the sensor detection module, and the alarm module sends out an alarm signal once data detected by the sensor detection module exceeds the reference thresholds.

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

s1.1: adding the extracellular vesicle stock solution into an extracellular vesicle stock solution bag of an extracellular vesicle separation and purification device, and allowing the extracellular vesicle stock solution to be separated to enter a flat spiral pipe dynamic ultrafilter 1 from a liquid inlet of the flat spiral pipe dynamic ultrafilter 1 through a booster pump 1;

s1.2: the pressurized liquid inlet module provides positive pressure ultrafiltration power, the filtration end negative pressure aspirator module provides negative pressure suction ultrafiltration power, impurities in the extracellular vesicle stock solution to be separated, which have the particle size larger than the ultrafiltration membrane pore size of the extracellular vesicle separation and purification device, are intercepted under the action of the positive pressure pushing and negative pressure suction filtration power, flow through the multi-way valve 2 through a concentrated liquid port at the tail end of the spiral tube cavity of the flat spiral tube dynamic ultrafilter 1, then enter the concentrated negative pressure bottle 1 for temporary storage, and extracellular vesicles and impurities in the extracellular vesicle stock solution to be separated, which have the particle size smaller than the ultrafiltration membrane pore size of the extracellular vesicle separation and purification device, pass through the ultrafiltration membrane and enter the filtration negative pressure bottle 1 through a filtration liquid port of the flat spiral tube dynamic ultrafilter 1 for temporary storage;

s1.3: after the filtrate obtained by the extracellular vesicle separation and purification device is accumulated to a certain amount (the value range of a certain amount is 0.5-0.75 times of the capacity in the filtration negative pressure bottle, for example 2/3 times) in the filtration negative pressure bottle, the booster pump 1 and the negative pressure aspirator 1 stop working, the electromagnetic valve 3 is closed, the multi-way valve 4 closes the negative pressure channel, opens the vent 2, opens the electromagnetic valve 3, and the filtrate in the filtration negative pressure bottle 1 enters the filtrate bag under the action of the peristaltic pump 2.

S1.1-S1.3, the main control module monitors the pressure of the liquid inlet, the concentrated liquid port and the filtered liquid port of the ultrafilter respectively through the pressure sensor 1, the pressure sensor 2 and the pressure sensor 3, monitors the change conditions of the liquid weight in the extracellular vesicle stock solution bag, the buffer solution bag, the reflux solution bag, the filtered liquid bag, the concentrated liquid bag and the filtering negative pressure bottle and the concentrated negative pressure bottle through the weighing sensor and the auxiliary weighing sensor,

when the main control module senses that the pressure of a liquid inlet and a concentrated liquid inlet of the ultrafilter continuously rises compared with the initial state and the pressure of the filtered liquid inlet continuously falls compared with the initial state through the pressure sensor 1, the pressure sensor 2 and the pressure sensor 3, senses that the weight increasing rate of liquid in the filtering negative pressure bottle 1 continuously falls compared with the initial state through the data change of the weighing sensor and the auxiliary weighing sensor, and simultaneously the weight increasing rate of liquid in the concentrating negative pressure bottle 1 continuously increases compared with the initial state, the main control module judges that the filtering efficiency of the ultrafilter falls, and commands the extracellular vesicle separation and purification devices corresponding to the current filtering negative pressure bottle 1 and the concentrating negative pressure bottle 1 to enter an ultrafiltration membrane cleaning operation mode, namely, the extracellular vesicle separation and purification devices are boosted to push the power of a liquid inlet module 1 by increasing the positive pressure of the extracellular vesicle separation and purification devices and the negative pressure suction power of a negative pressure suction device module 1 at a concentrating end is increased, the flow speed of the fluid in the spiral volute cavity is increased, the shearing force between the fluid and the ultrafiltration membrane is increased, suspended particles and colloidal substances adsorbed and deposited on the surface of the ultrafiltration membrane are washed away, and the filtration efficiency of the ultrafiltration membrane is recovered;

the main control module respectively senses a liquid inlet, a concentrated liquid inlet and a pressure recovery initial state of the filtered liquid inlet through the pressure sensor 1, the pressure sensor 2 and the pressure sensor 3, senses the weight acceleration recovery initial state of liquid in the filtered negative pressure bottle 1 through the data of the weighing sensor and the auxiliary weighing sensor, simultaneously, the weight acceleration recovery initial state of liquid in the concentrated negative pressure bottle 1, the main control module judges that the filtering efficiency of the ultrafilter is recovered, and then the booster liquid inlet module booster pump 1 is adjusted back to push the power and the concentrated end negative pressure suction device module 1 is adjusted back to suck the power under negative pressure, so that the corresponding extracellular vesicle separation and purification device is recovered to a normal state operation mode of separation and purification.

Example 2

With reference to fig. 1 and fig. 3, the present embodiment provides an extracellular vesicle continuous separation and purification apparatus, which includes two stages of extracellular vesicle separation and purification apparatuses and a main control module connected in series in sequence, the extracellular vesicle separation and purification apparatuses are electrically connected to the main control module, the two stages of extracellular vesicle separation and purification apparatuses include an ultrafilter module, a pressurizing liquid inlet module, a concentration end negative pressure extractor module, a filtration end negative pressure extractor module and a pipeline temperature control module, the ultrafilter module is connected to the pressurizing liquid inlet module, the concentration end negative pressure extractor module and the filtration end negative pressure extractor module through pipelines, the pipeline temperature control module is disposed on a pipeline related to the extracellular vesicle separation and purification apparatus, wherein,

In the first stage extracellular vesicle separation and purification device:

the ultrafilter module comprises an ultrafilter and a pipeline, wherein the ultrafilter is provided with a liquid inlet connected with the pressurizing liquid inlet module, a concentrated liquid port connected with the concentrated end negative pressure suction device module and a filtered liquid port connected with the filtered end negative pressure suction device module, the ultrafilter adopts a flat spiral pipe dynamic ultrafilter 1 by combining the contents shown in figure 5, a spiral pipe cavity from the center to the outside is arranged above an ultrafiltration membrane in the ultrafilter, the center of the spiral pipe cavity is connected with the liquid inlet, the peripheral tail end of the spiral pipe cavity is connected with the concentrated liquid port, and the lower chamber of the ultrafiltration membrane is connected with the filtered liquid port. The pore diameter of the ultrafiltration membrane is 150 nm-200 nm.

The pressurizing liquid inlet module comprises a pressurizing pump, an extracellular vesicle stock solution bag, a buffer solution bag 1, a backflow solution bag 1, a multi-way valve 1 and a pipeline, the extracellular vesicle stock solution bag, the buffer solution bag 1 and the backflow solution bag 1 are converged to the multi-way valve 1 through the pipeline, the multi-way valve 1 is connected with the pressurizing pump 1 through the pipeline, and the pressurizing pump 1 is connected with a liquid inlet of the flat worm dynamic ultrafilter 1 through the pipeline. Wherein the booster pump 1 is any one of a diaphragm pump, a peristaltic pump, a plunger pump and a gear pump,

a pressure sensor 1 is connected in series between the booster pump 1 and the liquid inlet of the flat worm pipe dynamic ultrafilter 1;

the buffer Solution bag is used for containing buffer Solution, the buffer Solution comprises normal saline, PBS buffer Solution, Hanks Balanced Salt Solution (HBSS) and the like, the buffer Solution bag is arranged to balance an ultrafilter and a pipeline before the separation and purification of the extracellular vesicles, the concentration of the extracellular vesicles in the liquid to be separated is adjusted in the separation and purification process, the spiral volute lumen is flushed in the separation and purification process to recover the filtration efficiency, and the pipeline is flushed after the separation and purification process, so that the waste of the extracellular vesicles is avoided;

the function of the backflow liquid bag is as follows: 1. in each stage of the process of separating and purifying the extracellular vesicles, liquid to be separated can repeatedly and circularly pass through the ultrafilter through the reflux liquid bag for multiple times, so that the efficiency of separating and purifying the extracellular vesicles is increased, and waste is avoided; 2. when washing spiral volute lumen, under the condition that the booster pump provides fluid flow malleation promotion power, buffer solution can be through the circulation flushing ultrafilter of backward flow liquid bag relapse until the ultrafilter resumes filtration efficiency, avoids extravagant too much buffer solution.

The concentration end negative pressure aspirator module comprises a concentration negative pressure bottle 1, a weighing sensor 1, a negative pressure aspirator 1, an air pressure sensor 1, an air pressure buffer 1, an air filter 1, a multi-way valve 3, a concentrated solution bag 1 and a pipeline, wherein a negative pressure exhaust port, a liquid inlet and a liquid outlet are arranged on the concentration negative pressure bottle 1, the negative pressure exhaust port is connected with the negative pressure aspirator 1 through the 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 aspirator 1, two ports on the multi-way valve 3 are respectively connected with the air filter 1 and the air pressure buffer 1, a port is also arranged on the multi-way valve 3 as a vent 1, the liquid inlet is connected with the concentrated solution port and the reflux solution bag 1 of the flat spiral dynamic ultrafilter 1 through the pipeline and the multi-way valve 2, and the liquid outlet is connected with the concentrated solution bag 1 through the pipeline, an electromagnetic valve 1 and a peristaltic pump 1 are connected in series between the liquid outlet and the concentrated solution bag 1, a weighing sensor 1 connected with a main control module is arranged below the concentrated negative pressure bottle 1, the weighing sensor 1 is a supporting weighing sensor 1 and is used for monitoring the weight change condition of the liquid in the concentrated negative pressure bottle 1,

In some specific implementation manners, a weighing sensor is arranged corresponding to the concentrated solution bag 1, the weighing sensor is electrically connected with the main control module, and the weighing sensor is preferably a suspension type weighing sensor 4 arranged above the corresponding concentrated solution bag 1 and used for monitoring the weight change condition of the liquid in the concentrated solution bag 1.

The filtering end negative pressure aspirator module comprises a filtering negative pressure bottle 1, a weighing sensor 2, a negative pressure aspirator 2, an air pressure sensor 2, an air pressure buffer 2, an air filter 2, a multi-way valve 4, a filtering liquid bag 1 and a pipeline, wherein a negative pressure exhaust port, a liquid inlet and a liquid outlet are arranged on the filtering negative pressure bottle 1, the negative pressure exhaust port is connected with the negative pressure aspirator 2 through the 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 aspirator 2, two ports on the multi-way valve 4 are respectively connected with the air filter 2 and the air pressure buffer 2, a port is also arranged on the multi-way valve 4 as an air vent 2, the liquid inlet is connected with a filtering liquid port of a flat spiral pipe dynamic ultra-filter 1 through the pipeline and an electromagnetic valve 2, the liquid outlet is in peristaltic connection with the filtering liquid bag 1 through the pipeline, the electromagnetic valve 3 and the liquid pump 2 are connected in series between the liquid outlet and the filtering liquid bag 1, a weighing sensor 2 connected with the main control module is arranged below the filtering negative pressure bottle 1, and the weighing sensor 2 is a bearing type weighing sensor 2 and is used for monitoring the weight change condition of liquid in the filtering negative pressure bottle 1;

In some specific implementation manners, a weighing sensor is arranged corresponding to the filtering liquid bag 1 and is electrically connected with the main control module, and a suspension type weighing sensor 5 is preferably arranged above the corresponding filtering liquid bag 1 and is used for monitoring the weight change condition of the liquid in the filtering liquid bag 1.

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

the ultrafilter module comprises an ultrafilter and a pipeline, wherein the ultrafilter is provided with a liquid inlet connected with the pressurizing liquid inlet module, a concentrated solution port connected with the concentrated end negative pressure suction device module and a filtered solution port connected with the filtered end negative pressure suction device module, the ultrafilter is a flat spiral pipe dynamic ultrafilter 2 according to the content shown in figure 4, a spiral pipe cavity from the center to the outside is arranged above an ultrafiltration membrane in the flat spiral pipe dynamic ultrafilter 2, the center of the spiral pipe cavity is connected with the liquid inlet, the peripheral tail end of the spiral pipe cavity is connected with the concentrated solution port, and the lower ultrafiltration membrane chamber cavity is connected with the filtered solution port. The aperture of the ultrafiltration membrane is 30-70 nm.

The pressurizing liquid inlet module comprises a pressurizing pump 2, a buffer liquid bag 2, a backflow liquid bag 2, a multi-way valve 5 and a pipeline, wherein a filter bag 1, the buffer liquid bag 2 and the backflow liquid bag 2 in the upper-stage extracellular vesicle separation and purification device (namely, the first-stage extracellular vesicle separation and purification device) are converged to the multi-way valve 5 through the pipeline, the multi-way valve 5 is connected with the pressurizing pump 2 through the pipeline, and the pressurizing pump 2 is connected with a liquid inlet of the flat scroll 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 the diaphragm pump or the peristaltic pump, and separates the conveyed liquid from the plunger and the pump cylinder through the arrangement of the diaphragm pump, so that the cleanliness of the liquid in the conveying process is kept; through the arrangement of the peristaltic pump, the fluid only contacts the pipeline and does not contact the pump body, so that the cleanliness of the liquid in the conveying process is kept;

a pressure sensor 4 is connected in series between the booster pump 2 and the liquid inlet of the flat worm pipe dynamic ultrafilter 2;

the buffer Solution bag 2 is used for containing buffer Solution, the buffer Solution comprises normal saline, PBS buffer Solution, Hanks Balanced Salt Solution (HBSS) and the like, the buffer Solution bag 2 is arranged to balance an ultrafilter and a pipeline before the separation and purification of extracellular vesicles, adjust the concentration of the extracellular vesicles in the liquid to be separated in the separation and purification process, flush a spiral volute lumen in the separation and purification process to recover the filtration efficiency, flush the pipeline after the separation and purification process, and avoid the waste of the extracellular vesicles;

the function of the reflux liquid bag 2 is as follows: 1. in each stage of the process of separating and purifying the extracellular vesicles, liquid to be separated can repeatedly and circularly pass through the ultrafilter through the reflux liquid bag for multiple times, so that the separation and purification efficiency of the extracellular vesicles is improved, and waste is avoided; 2. when washing spiral volute lumen, under the condition that the booster pump provides fluid flow malleation promotion power, buffer solution can be through the circulation flushing ultrafilter of backward flow liquid bag relapse until the ultrafilter resumes filtration efficiency, avoids extravagant too much buffer solution.

In some concrete implementation modes, can correspond buffer bag 2 and return fluid bag 2 and set up supplementary weighing sensor, supplementary weighing sensor is connected with the host system electricity, the preferred suspension type weighing sensor of supplementary weighing sensor, set up in the top of buffer bag 2 and return fluid bag 2 that correspond, be suspension type weighing sensor 6 and suspension type weighing sensor 7 respectively, suspension type weighing sensor 6 and suspension type weighing sensor 7 are used for monitoring the liquid weight situation of changing in buffer bag 2 and the return fluid bag 2 respectively.

The concentration end negative pressure aspirator module comprises a concentration negative pressure bottle 2, a weighing sensor 3, a negative pressure aspirator 3, an air pressure sensor 3, an air pressure buffer 3, an air filter 3, a multi-way valve 7, a concentrated solution bag 2 and a pipeline, wherein a negative pressure exhaust port, a liquid inlet and a liquid outlet are arranged on the concentration negative pressure bottle 2, the negative pressure exhaust port is connected with the negative pressure aspirator 3 through the 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 aspirator 3, two ports on the multi-way valve 7 are respectively connected with the air filter 3 and the air pressure buffer 3, a port is also arranged on the multi-way valve 7 as a vent 3, the liquid inlet is connected with the concentrated solution port and the reflux solution bag of the flat spiral dynamic ultrafilter 2 through the pipeline and the multi-way valve 6, and the liquid outlet is connected with the concentrated solution bag 2 through the pipeline, an electromagnetic valve 4 and a peristaltic pump 3 are connected in series between the liquid outlet and the concentrated solution bag 2, a weighing sensor 3 connected with a main control module is arranged below the concentrated negative pressure bottle 2, the weighing sensor 3 is a supporting weighing sensor 3 and is used for monitoring the weight change condition of the liquid in the concentrated negative pressure bottle 2,

a pressure sensor 5 is connected in series between the multi-way valve 6 and the concentrated solution port of the flat spiral pipe dynamic ultrafilter 2.

In some specific implementation manners, a weighing sensor is arranged corresponding to the concentrated liquid bag 2 and electrically connected with the main control module, and a suspension type weighing sensor 8 is preferably arranged above the corresponding concentrated liquid bag 2 and used for monitoring the weight change condition of the liquid in the concentrated liquid bag 2.

The filtering end negative pressure aspirator module comprises a filtering negative pressure bottle 2, a weighing sensor 4, a negative pressure aspirator 4, an air pressure sensor 4, an air pressure buffer 4, an air filter 4, a multi-way valve 8, a filtering liquid bag 2 and a pipeline, wherein a negative pressure exhaust port, a liquid inlet and a liquid outlet are arranged on the filtering negative pressure bottle 2, the negative pressure exhaust port is connected with the negative pressure aspirator 4 through the 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 aspirator 4, two ports on the multi-way valve 8 are respectively connected with the air filter 4 and the air pressure buffer 4, a port is also arranged on the multi-way valve 8 as a vent 4, the liquid inlet is connected with a filtering liquid port of the flat spiral pipe dynamic ultrafilter 2 through the pipeline and an electromagnetic valve 5, the liquid outlet is connected with the filtering liquid bag 2 through the pipeline in a peristaltic manner, the electromagnetic valve 6 and the pump 4 are connected in series between the liquid outlet and the filtering liquid bag 2, the weighing sensor 4 connected with the main control module is arranged below the filtering negative pressure bottle 2, and the weighing sensor 4 selects the bearing type weighing sensor 4 for monitoring the weight change condition of the liquid in the filtering negative pressure bottle 2.

In some specific implementation manners, a weighing sensor is arranged corresponding to the filtering liquid bag 2, the weighing sensor is electrically connected with the main control module, and the weighing sensor is preferably a suspension type weighing sensor 9 which is arranged above the corresponding filtering liquid bag 2 and used for monitoring the weight change condition of the liquid in the filtering liquid bag 2;

a pressure sensor 6 is connected in series between the electromagnetic valve 5 and the filtering liquid port of the flat worm pipe dynamic ultrafilter 2.

It should be noted that the above-mentioned concentrated negative pressure bottle 1, the filtration negative pressure bottle 1, the concentrated negative pressure bottle 2 and the filtration negative pressure bottle 2 are all hard bottles, each hard bottle includes a bottle body and a bottle cap, and the bottom of the bottle body adopts a design beneficial to the collection of liquid in the negative pressure bottle, for example, a groove is arranged at the bottom of the bottle body, the liquid at the bottom of the negative pressure bottle is collected by the groove, wherein the shape of the groove can be a cone shape with a wide top and a narrow bottom or a round bottom with a wide top and a narrow bottom, and the negative pressure exhaust port, the liquid inlet and the liquid outlet are arranged on the bottle cap. The concentration negative pressure bottle 1 and the filtering negative pressure bottle 1 can directly select the existing negative pressure bottles on the market.

The pipeline temperature control module in the two-stage extracellular vesicle separation and purification device comprises a winding type temperature controller and a cold source device, wherein the winding type temperature controller is connected with the cold source device, the pipeline adopts a hose design, and the pipeline is wound on the winding type temperature controller.

the device for separating and purifying extracellular vesicles is provided with a plurality of winding temperature controllers which are distributed at each position of the pipeline, such as before the liquid inlet of the ultrafilter, before the inlet of the concentrated solution bag, before the inlet of the filtering solution bag and the like.

In addition to the suspended weighing sensors 1-9, the supported weighing sensors 1-4, the pressure sensors 1-6 and the air pressure sensors 1-4, in the specific implementation, a working environment temperature sensor is arranged on the extracellular vesicle separation and purification device to detect the room temperature, a circulating water temperature sensor is arranged on a low-temperature constant-temperature cold water circulating device of the pipeline temperature control module, the circulating water temperature sensor, the working environment temperature sensor, the suspended weighing sensors 1-9, the supported weighing sensors 1-4, the pressure sensors 1-6 and the air pressure sensors 1-4 form a sensor detection module, and the sensor detection module is electrically connected with the main control module.

The microcomputer can be directly used as the main control module.

The particle size of the ultrafiltration membrane of the previous-stage extracellular vesicle separation and purification device in the two-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, liquid in the filtrate bag of the previous-stage extracellular vesicle separation and purification device enters the pressurizing liquid inlet module of the adjacent next-stage extracellular vesicle separation and purification device, and concentrated liquid of each adjacent next stage is collected into the corresponding concentrated liquid bag, so that the multistage continuous separation and purification of extracellular vesicles can be carried out, and the extracellular vesicles with different particle size ranges are obtained.

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

s1: adding the extracellular vesicle stock solution into an extracellular vesicle stock solution bag of a first-stage extracellular vesicle separation and purification device, and allowing the extracellular vesicle stock solution to be separated to enter an ultrafilter from a liquid inlet of a flat spiral tube dynamic ultrafilter 1 through a booster pump 1, wherein the liquid inlet comprises: setting the aperture of an ultrafiltration membrane of the first-stage extracellular vesicle separation and purification device to be 150 nm;

s2: the pressurized liquid inlet module provides positive pressure ultrafiltration power, the filtration end negative pressure aspirator module provides negative pressure suction ultrafiltration power, impurities with the particle size larger than the 150nm pore size of the ultrafiltration membrane of the first-stage extracellular vesicle separation and purification device in the extracellular vesicle stock solution to be separated are intercepted under the action of the positive pressure pushing and negative pressure suction filtration power, flow through the multi-way valve 2 through a concentrated solution port of the ultrafilter and then enter the concentrated negative pressure bottle 1 for temporary storage, extracellular vesicles with the particle size smaller than the 150nm pore size of the ultrafiltration membrane of the first-stage extracellular vesicle separation and purification device in the extracellular vesicle stock solution to be separated pass through the ultrafiltration membrane and enter the filtration negative pressure bottle 1 for temporary storage through a filtration solution port of the ultrafilter;

s3: after the filtrate obtained by the first-stage extracellular vesicle separation and purification device is accumulated to a certain amount (the value range of the certain amount is 0.5-0.75 times of the capacity of the filtration negative pressure bottle 1, such as 2/3 times) in the filtration negative pressure bottle 1, the booster pump 1 and the negative pressure aspirator 1 stop working, the electromagnetic valve 2 is closed, the multi-way valve 4 closes the negative pressure channel, opens the vent 2, opens the electromagnetic valve 3, and the filtrate in the filtration negative pressure bottle 1 enters the filtration liquid bag 1 under the action of the peristaltic pump 2;

s4: the filtrate in the filtrate bag 1 enters the ultra-filter from the liquid inlet of the flat worm pipe dynamic ultra-filter 2 through the booster pump 2, such as: setting the aperture of an ultrafiltration membrane of the second-stage extracellular vesicle separation and purification device to be 70 nm;

s5: the pressurized liquid inlet module provides positive pressure ultrafiltration power, the filtration end negative pressure aspirator module provides negative pressure suction ultrafiltration power, extracellular vesicle stock solution is under the action of the positive pressure pushing and negative pressure suction filtration power, extracellular vesicles with the particle size larger than the 70nm pore size of an ultrafiltration membrane of a second-stage extracellular vesicle separation and purification device in a first-stage filtrate to be separated are intercepted, flow through a multi-way valve 6 through a concentrated solution port of a flat spiral dynamic ultrafilter 2 and then enter a concentrated negative pressure bottle 2 for temporary storage, extracellular vesicles and impurities with the 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 a filtration negative pressure bottle 2 for temporary storage through a filtrate port of the flat spiral dynamic ultrafilter 2;

s6: after the concentrated solution obtained by the second-stage extracellular vesicle separation and purification device is accumulated in the concentrated negative pressure bottle 2 to a certain amount (the value range of the certain amount is 0.5-0.75 times of the volume in the concentrated negative pressure bottle 2, such as 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, opens the vent 3, and opens the electromagnetic valve 4, and under the action of the peristaltic pump 3, the second-stage concentrated solution in the concentrated negative pressure bottle 2 enters the concentrated solution bag 2 to obtain extracellular vesicles with the particle size range of 70-150 nm;

the multi-stage separation and purification of the extracellular vesicles can be carried out by adjusting the pore size of the ultrafiltration membrane of the adjacent extracellular vesicle separation and purification devices, so that the extracellular vesicles with different particle size ranges can be obtained, and the specific method is shown in S1-S6.

The pressurized liquid inlet module provides positive pressure pushing power for fluid flowing in the spiral volute tube cavity, the concentration end negative pressure aspirator module provides negative pressure suction power for fluid flowing in the spiral volute tube cavity, and extracellular vesicle stock solution flows forward rapidly in the spiral volute tube cavity under the action of the positive pressure pushing power and the negative pressure suction power to form shearing force with an ultrafiltration membrane, so that adsorption and deposition of suspended particles and colloidal substances on the surface of the ultrafiltration membrane are prevented, concentration polarization effect is overcome, attenuation rate of flux of the ultrafiltration membrane is effectively reduced, and high filtration efficiency of the ultrafiltration membrane is maintained for a long time.

In the execution process of S1-S6, the main control module monitors the pipeline pressure of a liquid inlet, a concentrated liquid port and a filtered liquid port of the ultrafilter through pressure sensors, wherein the pressure sensors 1-3 are one group, and the pressure sensors 4-6 are the other group; the method comprises the following steps of monitoring the weight of liquid in an extracellular vesicle stock solution bag, a buffer solution bag 1, a reflux solution bag 1, a filter solution bag 1, a concentrate bag 1, a filter negative pressure bottle 1, a concentrate negative pressure bottle 1, a buffer solution bag 2, a reflux solution bag 2, a filter solution bag 2, a concentrate bag 2, a filter negative pressure bottle 2 and a concentrate negative pressure bottle 2 in groups through a weighing sensor and an auxiliary weighing sensor, wherein the extracellular vesicle stock solution bag, the buffer solution bag 1, the reflux solution bag 1, the filter solution bag 1, the concentrate bag 1, the filter negative pressure bottle 1 and the concentrate negative pressure bottle 1 form one group, and the filter negative pressure bottle 1, the buffer solution bag 2, the reflux solution bag 2, the filter solution bag 2, the concentrate bag 2, the filter negative pressure bottle 2 and the concentrate bottle 2 form another group;

when the main control module senses that the pressure of a liquid inlet and a concentrated liquid inlet of the ultrafilter continuously rises compared with the initial state through the pressure sensor, the pressure of the filtered liquid inlet continuously falls compared with the initial state, senses that the weight acceleration rate of liquid in one or more groups of filtering negative pressure bottles continuously falls compared with the initial state through the data change of the weighing sensor and the auxiliary weighing sensor, simultaneously the weight acceleration rate of the liquid in the concentrated negative pressure bottles continuously increases compared with the initial state, the main control module judges that the filtering efficiency of the ultrafilter falls, commands the extracellular vesicle separation and purification devices corresponding to the filtering negative pressure bottles and the concentrated negative pressure bottles to enter an ultrafiltration membrane cleaning operation mode, namely, the extracellular vesicle separation and purification devices boost the liquid inlet module booster pump to boost the power and boost the negative pressure suction power of the concentrated end negative pressure suction device module through increasing the positive pressure of the extracellular vesicle separation and purification devices, and jointly accelerates the flow speed of fluid in the spiral volute, increasing the shearing force between the ultrafiltration membrane and the membrane, flushing away suspended particles and colloidal substances adsorbed and deposited on the surface of the ultrafiltration membrane, and recovering the filtration efficiency of the ultrafiltration membrane;

the main control module senses the initial state of pressure recovery of a liquid inlet, a concentrated liquid port and a filtered liquid port of the ultrafilter through the pressure sensor, senses the initial state of liquid weight acceleration recovery in the filtered negative pressure bottle through the data of the weighing sensor and the auxiliary weighing sensor, simultaneously, the initial state of liquid weight acceleration recovery in the concentrated negative pressure bottle, judges that the filtering efficiency of the ultrafilter is recovered, and then, the booster liquid inlet module booster pump is adjusted back to the positive pressure pushing power and the concentration end negative pressure suction device module is adjusted back to the negative pressure suction power, so that the corresponding extracellular vesicle separation and purification device is recovered to a separation and purification normal operation mode. The ultrafiltration membrane cleaning operation mode can ensure the filtration efficiency of the ultrafiltration membrane, so that the ultrafiltration membrane can keep high filtration efficiency for a long time.

The utility model provides an extracellular vesicle separation purification device also can include the multistage (be greater than and equal to tertiary) extracellular vesicle separation purification device that connects gradually in series, extracellular vesicle separation purification device's structure is the same with the structure in embodiment 2, the extracellular vesicle separation purification device that increases can assemble the filtrate bag of last level and buffer solution bag, the backward flow liquid bag of this level to the multidirectional valve through the pipeline, the multidirectional valve passes through the pipeline and is connected with the booster pump, the booster pump passes through the pipeline and meets with the inlet of ultrafilter module. The application method of the extracellular vesicle separation and purification device is the same as that described in embodiment 2, and therefore, the detailed description thereof is omitted.

Example 3

With reference to fig. 4, the ultrafilter in this embodiment is a flat coiled dynamic ultrafilter, an upper chamber of the ultrafilter in the flat coiled dynamic ultrafilter is a repeatedly bent coiled lumen, one end of the coiled lumen is connected to a fluid inlet pipeline, the other end of the coiled lumen is connected to a concentrated fluid port pipeline, a lower chamber of the ultrafilter is connected to a filtered fluid port pipeline, the fluid to be separated from the extracellular vesicles enters the repeatedly bent coiled lumen from the fluid inlet, a booster pump provides positive pressure ultrafiltration power, a negative pressure suction unit provides negative pressure suction ultrafiltration power, the fluid to be separated from the extracellular vesicles is pushed by the positive pressure and the negative pressure suction, impurities with a particle size larger than the pore size of the ultrafilter are retained, and flow through a multi-way valve through a concentrated fluid port of the flat coiled dynamic ultrafilter, and then enter a concentrated negative pressure bottle, and the extracellular vesicles and impurities with the particle size smaller than the aperture of the ultrafiltration membrane in the extracellular vesicle-containing liquid to be separated pass through the ultrafiltration membrane and enter the negative pressure filtering bottle through a filtrate port of the flat coiled tube dynamic ultrafilter. The rest of the application method of the extracellular vesicle separation and purification device is the same as the application method described in embodiment 1 and embodiment 2, and therefore, the description thereof is omitted.

Example 4

With reference to fig. 7, the ultrafilter in this embodiment is an internal pressure type hollow fiber membrane shell-and-tube dynamic ultrafilter, and is composed of a membrane tube shell, 2 tube sheet end sockets, and a plurality of hollow fiber membranes, all the hollow fiber membranes are directly encapsulated in the membrane tube shell by the 2 tube sheet end sockets, one end of the hollow fiber membrane tube cavity is converged into a front chamber cavity connected to the liquid inlet, the other end is converged into a rear chamber cavity connected to the concentrated liquid port, and an outer chamber cavity between the hollow fiber membrane and the membrane tube shell is connected to the filtered liquid port. The liquid containing extracellular vesicles to be separated enters the hollow fiber membrane from the liquid inlet, the booster pump provides positive pressure ultrafiltration power, the negative pressure suction device at the filtering end provides negative pressure suction ultrafiltration power, under the action of the positive pressure pushing and negative pressure suction filtration power, impurities with the particle size larger than the pore size of the hollow fiber membrane in the liquid containing extracellular vesicles to be separated are intercepted and collected to the concentrated liquid port through the other end of the hollow fiber membrane tube cavity, then the liquid enters the concentrated negative pressure bottle, extracellular vesicles with the particle size smaller than the pore size of the hollow fiber membrane in the liquid containing extracellular vesicles to be separated pass through the micropores of the hollow fiber membrane, enter an outer chamber between the hollow fiber membrane and the membrane tube shell, and enter the negative pressure filtering bottle through the filtered liquid port. The rest of the application method of the extracellular vesicle separation and purification device is the same as the application method described in embodiment 1 and embodiment 2, and therefore, the description thereof is omitted.

Example 5

With reference to fig. 8, the ultrafilter in this embodiment is an external pressure type hollow fiber membrane shell-and-tube dynamic ultrafilter, which is composed of a membrane tube shell, 1 tube sheet end enclosure, 1 end cover plate and a plurality of hollow fiber membranes, all the hollow fiber membranes are directly encapsulated in the membrane tube shell by the 1 tube sheet end enclosure and the 1 end cover plate, one end of an outer chamber between the hollow fiber membranes and the membrane tube shell is connected to a liquid inlet, the other end of the outer chamber between the hollow fiber membranes and the membrane tube shell is connected to a concentrated liquid port, one end of each hollow fiber membrane chamber is sealed by the end cover plate, and the other end of each hollow fiber membrane chamber is collected to a filtration chamber and connected to a filtration liquid port. The liquid containing the extracellular vesicles to be separated enters an outer chamber between the hollow fiber membrane and the membrane tube shell from the liquid inlet, the booster pump provides positive pressure ultrafiltration power, the negative pressure suction device at the filtration end provides negative pressure suction ultrafiltration power, the liquid containing the extracellular vesicles to be separated is under the action of the positive pressure pushing and negative pressure suction filtration power, impurities with the particle size larger than the pore size of the hollow fiber membrane in the liquid containing the extracellular vesicles to be separated are intercepted and enter a concentrated negative pressure bottle through a concentrated liquid port at the other end of the outer chamber between the hollow fiber membrane and the membrane tube shell, and extracellular vesicles and impurities with the particle size smaller than the pore size of the hollow fiber membrane in the liquid containing the extracellular vesicles to be separated enter a lumen of the hollow fiber membrane through micropores in the hollow fiber membrane, are collected to the filtration chamber through the lumen of the hollow fiber membrane, and enter the filtration negative pressure bottle through a filtration liquid port. The rest of the application method of the extracellular vesicle separation and purification device is the same as the application method described in embodiment 1 and embodiment 2, and therefore, the description thereof is omitted.

While the invention has been described in further detail in connection with specific embodiments thereof, it will be understood that the invention is not limited thereto, and that various other modifications and substitutions may be made by those skilled in the art without departing from the spirit of the invention, which should be considered to be within the scope of the invention as defined by the appended claims.

Claims

1. An extracellular vesicle continuous separation and purification device is characterized by comprising an extracellular vesicle separation and purification device and a main control module, wherein the extracellular vesicle separation and purification device is electrically connected with the main control module, the extracellular vesicle separation and purification device comprises an ultrafilter module, a pressurizing liquid inlet module, a concentration end negative pressure aspirator module and a filtration end negative pressure aspirator module, the ultrafilter module is connected with the pressurizing liquid inlet module, the concentration end negative pressure aspirator module and the filtration end negative pressure aspirator module through pipelines,

2. The continuous separation and purification device for extracellular vesicles according to claim 1, wherein the continuous separation and purification device for extracellular vesicles comprises at least two stages of extracellular vesicle separation and purification devices connected in series in sequence, the pore size of the ultrafiltration membrane of the previous stage of extracellular vesicle separation and purification device is larger than that of the ultrafiltration membrane of the next stage of extracellular vesicle separation and purification device, liquid in the filtrate bag of the previous stage of extracellular vesicle separation and purification device enters the pressurized liquid inlet module of the next stage of extracellular vesicle separation and purification device, and concentrated liquid of each next stage is collected into the corresponding concentrated liquid bag, so that multi-stage continuous separation and purification of extracellular vesicles can be performed, and extracellular vesicles with different particle size ranges can be obtained.

3. The continuous extracellular vesicle separation and purification device according to claim 1 or 2, wherein the ultrafilter module comprises an ultrafilter and a pipeline, the ultrafilter is provided with a liquid inlet connected with the pressurizing liquid inlet module, a concentrated liquid port connected with the concentrated-end vacuum extractor module and a filtered liquid port connected with the filtered-end vacuum extractor module, the ultrafilter is provided with an ultrafiltration membrane, an upper chamber of the ultrafilter is arranged above the ultrafiltration membrane, a lower chamber of the ultrafilter is arranged below the ultrafiltration membrane,

4. The continuous extracellular vesicle separation and purification apparatus according to claim 3, wherein the pore size of the ultrafiltration membrane is 10 nm to 4000 nm.

5. The continuous extracellular vesicle separation and purification device according to claim 1 or 2, wherein the pressurized liquid inlet module comprises a pressurizing pump, a buffer liquid bag, a reflux liquid bag, a multi-way valve and a pipeline, the extracellular vesicle liquid bag, the buffer liquid bag and the reflux liquid bag are collected to the multi-way valve through the pipeline, the extracellular vesicle liquid bag is an extracellular vesicle raw liquid bag or a filtration liquid bag in the previous stage of extracellular vesicle separation and purification device, the multi-way valve is connected with the pressurizing pump through the pipeline, the pressurizing pump is connected with a liquid inlet of the ultrafilter through the pipeline, a pressure sensor is connected in series between the pressurizing pump and the liquid inlet of the ultrafilter, and the pressure sensor is electrically connected with the main control module,

6. The continuous extracellular vesicle separation and purification device according to claim 5, further comprising an auxiliary weighing sensor corresponding to the extracellular vesicle stock solution bag, the buffer solution bag, the reflux solution bag and the filtration solution bag, wherein the auxiliary weighing sensor is electrically connected to the main control module,

the auxiliary weighing sensor is preferably a suspension type weighing sensor.

7. The continuous extracellular vesicle separation and purification device according to claim 1 or 2, wherein the concentrated end negative pressure aspirator module comprises a concentrated negative pressure bottle, a negative pressure aspirator, an air filter, a multi-way valve, a concentrated solution bag and a pipeline, the concentrated negative pressure bottle is provided with a negative pressure exhaust port, a liquid inlet and a liquid outlet, the negative pressure exhaust port is connected with the negative pressure aspirator through the pipeline, the pipeline between the negative pressure exhaust port and the negative pressure aspirator is connected with the air filter, the multi-way valve, an air vent, an air pressure buffer and an air pressure sensor in series, the liquid inlet is connected with the concentrated solution port of the ultrafilter and the reflux solution bag through the pipeline and the multi-way valve, the concentrated solution port of the ultrafilter and the multi-way valve are connected with a pressure sensor in series, the pressure sensor is electrically connected with the master control module, and the liquid outlet is connected with the concentrated solution bag through the pipeline, an electromagnetic valve and a power pump are connected in series between the liquid outlet and the concentrated solution bag,

the power pump is preferably a peristaltic pump.

8. The continuous extracellular vesicle separation and purification device according to claim 7, wherein the concentration-end negative pressure extractor module further comprises a weighing sensor, the weighing sensor is arranged corresponding to the concentration negative pressure bottle and the concentration liquid bag, the weighing sensor is electrically connected with the main control module,

9. The continuous extracellular vesicle separation and purification device according to claim 1 or 2, wherein the filtration-end vacuum extractor module comprises a filtration vacuum bottle, a vacuum extractor, an air filter, a multi-way valve, a filtration fluid bag and a pipeline, the filtration vacuum exhaust port is connected with the vacuum extractor through a pipeline, the air filter, the multi-way valve, a vent, a pneumatic buffer and a pneumatic sensor are connected in series on the pipeline between the vacuum exhaust port and the vacuum extractor, the fluid inlet is connected with the filtration fluid port of the ultrafilter through a pipeline, a solenoid valve and a pressure sensor are connected in series between the fluid inlet and the filtration fluid port of the ultrafilter, the solenoid valve and the pressure sensor are electrically connected with the main control module, the fluid outlet is connected with the filtration fluid bag through a pipeline, and a solenoid valve and a power pump are connected in series between the fluid outlet and the filtration fluid bag,

the power pump is preferably a peristaltic pump.

10. The extracellular vesicle continuous separation and purification device according to claim 9, wherein the filtration-end negative pressure aspirator module further comprises a weighing sensor, the weighing sensor is arranged corresponding to the filtration negative pressure bottle and the filtration liquid bag, the weighing sensor is electrically connected with the main control module,

11. The continuous extracellular vesicle separation and purification device according to claim 1 or 2, wherein the extracellular vesicle separation and purification device further comprises a pipeline temperature control module, and the pipeline temperature control module acts on a pipeline involved in the extracellular vesicle separation and purification device, so that a liquid in the pipeline is in a desired temperature environment.

12. The continuous extracellular vesicle separation and purification device according to claim 11, wherein the pipeline is a flexible tube, the pipeline temperature control module comprises a winding temperature controller and a cold source device, the winding temperature controller is connected to the cold source device, and the pipeline is wound on the winding temperature controller.

13. The continuous extracellular vesicle separation and purification device according to claim 12, wherein the winding thermostat is made of a metal with good heat conductivity, the winding thermostat has a spiral groove on its surface, and the pipeline is wound in the spiral groove; the cold source device comprises a refrigeration water circulating device and a Peltier semiconductor refrigeration sheet, the refrigeration water circulating device is a low-temperature constant-temperature cold water circulating device, a cold water circulating pipeline is arranged in the winding type temperature controller, the cold water circulating pipeline of the low-temperature constant-temperature cold water circulating device is communicated with the cold water circulating pipeline of the winding type temperature controller, and the winding type temperature controller is cooled through the low-temperature constant-temperature cold water circulating device; the winding temperature controller is arranged corresponding to the pipeline, the water temperature control range in the low-temperature constant-temperature cold water circulating device is 0-8 ℃,

the metal is preferably metallic aluminum, metallic copper or a metal alloy.

14. The continuous separation and purification device for extracellular vesicles according to claim 1 or 2, wherein the continuous separation and purification device for extracellular vesicles further comprises an alarm module and a human-computer interaction module, and the alarm module and the human-computer interaction module are both electrically connected with the main control module.

15. The method for using the extracellular vesicle continuous separation and purification device according to any one of claims 1 to 14, comprising the steps of:

16. The method of use according to claim 15, wherein the rated capacity of the filtration negative pressure bottle is 0.5-0.75 times the capacity of the filtration negative pressure bottle; the rated capacity of the concentrated negative pressure bottle is 0.5-0.75 times of the capacity in the concentrated negative pressure bottle.

17. The method of application according to claim 15,

in the execution process of S1-S6, the main control module monitors the pipeline pressure of the liquid inlet, the concentrated liquid inlet and the filtered liquid outlet of the ultrafilter through pressure sensors, monitors the weight of liquid in the extracellular vesicle stock solution bag, the buffer solution bag, the reflux solution bag, the filtered liquid bag, the concentrated liquid bag, the filtering negative pressure bottle and the concentrating negative pressure bottle through weighing sensors and auxiliary weighing sensors,