Separation equipment and separation method for extracting extracellular vesicles

Abstract

The invention discloses separation equipment and a separation method for extracellular vesicle extraction, and relates to the field of separation equipment.The separation equipment comprises a separation container and a centrifuge, the centrifuge can drive the separation container to rotate, the separation container comprises a lower shell, an upper shell and an ultrafiltration element, and the upper shell and the lower shell are in threaded connection; the ultrafiltration element is arranged in the upper shell and the lower shell, the separation container further comprises a sealing ring and a plugging block, the top of the ultrafiltration element is provided with an opening and abuts against the plugging block, and when the ultrafiltration element moves downwards in the upper shell and the lower shell, the bottom of the ultrafiltration element is inserted into the sealing ring, and the top of the ultrafiltration element is separated from the plugging block. Filtrate and filter residues can be collected into the lower shell and the upper shell respectively through centrifugal force, use is more convenient, meanwhile, the filter residues are discharged out of the ultrafiltration element through the centrifugal force, the discharging effect is better, the filter residues are prevented from being attached to the ultrafiltration element, the cleaning difficulty of the ultrafiltration element is reduced, and the service life of the ultrafiltration element is prolonged.

Description

Technical Field

[0001] The present invention relates to the field of separation equipment, and in particular to a separation equipment and a separation method for extracting extracellular vesicles. Background Art

[0002] Separation equipment is an important machine used in industrial production processes such as chemical, pharmaceutical, petroleum, dye, biochemical, and food to achieve chemical and physical changes such as chemical reactions and material separation, heating and cooling, liquid extraction, and gas absorption. These devices use the differences in physical properties between different substances, such as density, particle size, solubility, etc., to achieve effective separation.

[0003] Ultrafiltration membrane separation technology is a membrane separation process that uses the microporous structure of a semipermeable membrane and a certain external pressure as a driving force to achieve selective separation and recovery of substances. It can effectively retain large molecules and colloids. The main function of the selective surface layer of the ultrafiltration membrane is to form pores of a certain size and shape. Its separation mechanism mainly relies on physical screening. Ultrafiltration membranes and other membrane separations such as nanofiltration and microfiltration have a large number of applications in extracellular vesicle extraction and concentration.

[0004] Chinese patent application CN118286867A discloses an avian influenza cell vaccine ultrafiltration concentration system and its operating method, comprising: a first buffer tank, a second buffer tank, a third buffer tank, a centrifuge, and an ultrafiltration concentrator; the buffer tanks are interconnected and are respectively connected to the centrifuge and the ultrafiltration concentrator; wherein the first buffer tank is used to store virus liquid; the second buffer tank is used to store centrifuged virus liquid; the third buffer tank is used to store concentrated virus liquid, or to store centrifuged virus liquid and concentrated virus liquid; the centrifuge is used to centrifuge the virus liquid stored in the first buffer tank; the ultrafiltration concentrator is used to concentrate the virus liquid stored in the second buffer tank or the third buffer tank, and return the concentrated virus liquid to the third buffer tank through a reflux port, and is also used to output the virus liquid obtained after the concentration process through a permeate port, thereby improving work efficiency;

[0005] Chinese patent application CN101898088A discloses a centrifugal ultrafiltration device suitable for the pretreatment of trace samples, including a centrifuge tube, in which a hollow fiber membrane is provided parallel to the axis of the centrifuge tube, and the outlet end of the hollow fiber membrane is connected to the outside of the centrifuge tube. It can be widely used for the pretreatment of trace analytical samples, and can also be used for the separation, purification, and concentration of trace biological samples. Its application is simple, fast, and convenient; the treated samples have less interference and high sensitivity; and it can effectively extend the service life of the chromatographic column.

[0006] The above patents and prior art also have the following defects:

[0007] It is impossible to separate and collect the filtrate and the residue, and at the same time, centrifugation is used to move the substance to the outside of the ultrafiltration membrane to improve the filtration efficiency and speed. However, the large molecular substances will stick to and adhere to the ultrafiltration membrane under the action of centrifugal force, making it difficult to remove the residue from the ultrafiltration membrane, causing trouble in cleaning the ultrafiltration membrane and affecting the secondary use of the ultrafiltration membrane. When used in the field of biological tissue concentration and filtration, since it is necessary to collect the filtered substance, collecting the substance on the ultrafiltration membrane not only reduces the processing efficiency, but also easily causes damage to the filtered substance, resulting in a decrease in the quality of the biological sample. For example, when concentrating and separating larger molecular extracellular vesicles, using tools to scrape the extracellular vesicles on the ultrafiltration membrane can easily cause the extracellular vesicles to break, affecting the sample quality.

[0008] Therefore, the present application provides a separation device and a separation method for extracellular vesicle extraction to meet the needs. Summary of the Invention

[0009] The purpose of the present application is to provide a separation device and a separation method for extracellular vesicle extraction, which can collect the filtrate and filter residue into the lower shell and the upper shell respectively through centrifugal force, which is more convenient to use. At the same time, the filter residue is discharged from the ultrafiltration element through centrifugal force, and the discharge effect is better, preventing the filter residue from adhering to the ultrafiltration element, reducing the difficulty of cleaning the ultrafiltration element, and increasing the service life of the ultrafiltration element.

[0010] To achieve the above objectives, the present application provides the following technical solutions: a separation device, comprising a separation container and a centrifuge, wherein the centrifuge is capable of driving the separation container to rotate, the separation container comprising a lower shell, an upper shell, and an ultrafiltration element, wherein the upper shell and the lower shell are threadedly connected, and the ultrafiltration element is disposed in the upper shell and the lower shell;

[0011] The separation container further includes a sealing ring and a blocking block. The top of the ultrafiltration element is open and abuts against the blocking block. When the ultrafiltration element moves downward in the upper and lower shells, the bottom of the ultrafiltration element is inserted into the sealing ring, and the top of the ultrafiltration element is separated from the blocking block.

[0012] The centrifuge includes a downward pressure component and a rotating component. When the separation container is in a vertical state, the downward pressure component can push the ultrafiltration element downward, and the rotating component can drive the separation container to rotate, so that it can switch between a vertical state and a horizontal state.

[0013] Preferably, the separation container also includes a plurality of snap assemblies, the snap assemblies include a clamping spring and a clamping block, a connecting groove is opened on the upper shell body, one end of the clamping spring is fixedly installed on the bottom of the connecting groove, the clamping block is fixedly installed on the other end of the clamping spring, the top and bottom of the clamping block are both inclined, the ultrafiltration element includes a filter element bracket, a top ring, two clamping rings and an ultrafiltration membrane, the ultrafiltration membrane is fixedly installed around the filter element bracket, the top ring is fixedly installed on the top of the filter element bracket, and the two clamping rings are fixedly installed on the outer ring of the top ring.

[0014] Preferably, the blocking block is fixedly mounted on the inner top of the upper shell, and the separation container also includes a push rod, a push ring and a push plate. The push rod is fixedly mounted on the top of the push ring, and the push rod passes through the upper shell and extends outward. The push plate is fixedly mounted on the top of the push rod, and the blocking block is inserted in the push ring. An anti-slip groove is provided on the upper shell.

[0015] Preferably, the centrifuge includes a rotating drum, and several rotating components are arranged on the rotating drum. A rotating groove is opened on the rotating drum. The rotating component includes a placement shell with an open top, a rotating gear and a movable rack. Rotating rods are fixedly installed on both sides of the placement shell, and the rotating rods are rotatably connected to the groove wall of the rotating groove. The rotating gear is fixedly installed on the rotating rod, and the movable rack is engaged with the rotating gear.

[0016] Preferably, the centrifuge further includes a fixed shell, a drive assembly and a pushing assembly, the drum is rotatably connected to the fixed shell, the drive assembly and the pushing assembly are arranged in the fixed shell, the drive assembly can drive the drum to rotate, and the pushing assembly can push several movable racks in the rotating assemblies to move.

[0017] Preferably, the pushing assembly includes a pushing cylinder and a movable plate, the pushing cylinder is fixedly mounted on a fixed shell, the output end of the pushing cylinder is rotatably connected to the movable plate, the top of the movable rack passes through the rotating drum and is fixedly mounted on the movable plate, an avoidance groove is provided at the bottom of the rotating drum, and the pushing cylinder and the movable plate are arranged in the avoidance groove.

[0018] Preferably, a closing cover is installed on the fixed shell, and the pressing assembly includes a pressing cylinder and a pressing plate. The pressing cylinder is fixedly installed on the closing cover, and the pressing plate is fixedly installed on the output end of the pressing cylinder.

[0019] Preferably, the drive assembly includes a drive motor, a drive shaft, a driving pulley, a driven pulley, a drive belt, a drive gear and a drive inner ring gear, the drive motor is fixedly mounted in the fixed housing, the drive shaft is rotatably connected to the fixed housing, the drive inner ring gear is fixedly mounted on the rotating drum, the drive gear is fixedly mounted on the top of the drive shaft, and the drive gear is meshed with the drive inner ring gear, the driving pulley is fixedly mounted on the output end of the drive motor, the driven pulley is fixedly mounted on the drive shaft, and the drive belt is connected to the driving pulley and the driven pulley.

[0020] Preferably, a rubber block is fixedly mounted on one end of the rotating groove, the top of the rubber block is arranged in an arc shape, and a buffer pad is arranged in the placement shell.

[0021] A separation method for extracting extracellular vesicles, using the above-mentioned separation device, comprises the following steps:

[0022] The tissue fluid was separated by centrifugation, and the supernatant containing a large number of extracellular vesicles was collected;

[0023] Rotate and separate the threaded upper and lower shells to remove the ultrafiltration element;

[0024] After adding the supernatant into the ultrafiltration element, the ultrafiltration element is placed in the upper shell and the lower shell, and the upper shell and the lower shell are rotated to make them threadedly connected;

[0025] The separation container is placed vertically in the centrifuge, and the centrifuge drives the separation container to rotate. The liquid in the ultrafiltration element passes through the ultrafiltration element under the action of centrifugal force and enters the lower shell. After centrifugation for a specified time, the centrifuge stops driving the separation container to rotate and centrifuge, and the filtrate in the lower shell flows into the bottom of the lower shell under the action of gravity;

[0026] The downward pressure assembly pushes the ultrafiltration element downward, so that the ultrafiltration element is inserted into the sealing ring. The sealing ring and the ultrafiltration element seal the filtrate at the bottom of the lower shell. At the same time, the top of the ultrafiltration element is separated from the blocking block, and the top opening of the ultrafiltration element is connected to the upper shell.

[0027] The rotating assembly drives the separation container to rotate, so that the separation container rotates from a vertical state to a horizontal state, and the open end of the ultrafiltration element is located at the outer ring of the centrifuge;

[0028] The centrifuge drives the separation container to rotate and centrifuge. The extracellular vesicles in the ultrafiltration element enter the upper shell through the open end of the ultrafiltration element under the action of centrifugal force. The centrifugation stops after a specified time.

[0029] The upper shell and the lower shell are separated by rotation, and the filtrate in the lower shell and the extracellular vesicles in the upper shell are collected respectively, so as to obtain extracellular vesicles with a diameter within a set numerical range.

[0030] In summary, the technical effects and advantages of the present invention are:

[0031] 1. In the present invention, the separation container is rotated and centrifuged in a vertical state, and the filtrate and small molecular materials in the ultrafiltration element enter the lower shell under the action of centrifugal force, which can generate a certain pressure difference for the materials in the separation container through centrifugation. The materials are filtered by the ultrafiltration element under the action of the pressure difference, and the filtration time of the ultrafiltration element is short and the filtration effect is better. After that, the ultrafiltration element descends, and the filtrate in the lower shell is sealed, and the separation container is rotated to a horizontal state and rotated and centrifuged, so that the filter residue in the ultrafiltration element enters the upper shell under the action of centrifugal force, and the filtrate and filter residue can be respectively collected in the lower shell and the upper shell by centrifugal force, which is more convenient to use. At the same time, the filter residue is discharged from the ultrafiltration element by centrifugal force, and the discharge effect is better, which prevents the filter residue from adhering to the ultrafiltration element, reduces the difficulty of cleaning the ultrafiltration element, and increases the service life of the ultrafiltration element.

[0032] 2. In the present invention, the push cylinder pushes the movable plate to move vertically, and the movable plate drives the movable racks in the plurality of rotating components to move, so that the separation container rotates. The push component pushes the movable racks in the plurality of rotating components to move, so that all the separation containers rotate, which makes the operation simpler.

[0033] 3. In the present invention, when the separation container is inserted into the placement shell, the buffer pad is deformed, and the buffer pad clamps the separation container to prevent the separation container from moving, thereby improving the stability of the separation container. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 Schematic diagram of the structure of the separation container and the centrifuge in the present invention;

[0036] Figure 2 It is a schematic structural diagram of the rotary drum, drive assembly, fixed housing and closing cover in the present invention;

[0037] Figure 3 Schematic diagram of the structure of the fixed shell, separation container and drum in the present invention;

[0038] Figure 4 Schematic diagram of the structure of the driving motor, driving shaft, rotating shaft and separation container in the present invention;

[0039] Figure 5 Schematic diagram of the structure of the fixed shell and the closing cover in the present invention;

[0040] Figure 6 For the present invention Figure 5 Enlarged view of part A;

[0041] Figure 7 It is a schematic structural diagram of the rotary drum, drive shaft, drive gear and drive internal gear ring in the present invention;

[0042] Figure 8 It is a structural schematic diagram of the rotary drum, separation container and rotating assembly in the present invention;

[0043] Figure 9 Schematic diagram of the structure of the upper shell and the lower shell in the present invention;

[0044] Figure 10 Schematic diagram of the structure of the sealing ring, ultrafiltration element and push ring in the present invention;

[0045] Figure 11 For the present invention Figure 10 Enlarged view of part B;

[0046] Figure 12 It is a schematic structural diagram of the upper shell, top ring, clamping ring and filter element bracket in the present invention.

[0047] In the figure: 2, separation container; 21, lower shell; 22, upper shell; 23, ultrafiltration element; 231, filter element bracket; 232, top ring; 233, clamping ring; 24, sealing ring; 25, blocking block; 26, snap assembly; 261, clamping spring; 262, clamping block; 27, pushing rod; 28, pushing ring; 29, pushing plate; 3, centrifuge; 31, pressing assembly; 311, pressing cylinder; 312, pressing plate; 32, rotating assembly; 321, placing shell; 322, rotating gear; 323, moving rack; 33, rotating drum; 34, fixed shell; 35, driving assembly; 351, driving motor; 352, driving shaft; 353, driving gear; 354, driving inner ring; 36, pushing assembly; 361, pushing cylinder; 362, moving plate; 37, closing cover; 38, rubber block. DETAILED DESCRIPTION

[0048] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0049] Example 1: Reference Figures 1-12A separation device shown includes a separation container 2 and a centrifuge 3. The centrifuge 3 can drive the separation container 2 to rotate. The separation container 2 includes a lower shell 21, an upper shell 22, and an ultrafiltration element 23. The upper shell 22 and the lower shell 21 are threadedly connected. The ultrafiltration element 23 is disposed in the upper shell 22 and the lower shell 21.

[0050] The separation container 2 further includes a sealing ring 24 and a blocking block 25. The top of the ultrafiltration element 23 is open and abuts against the blocking block 25. When the ultrafiltration element 23 moves downward in the upper shell 22 and the lower shell 21, the bottom of the ultrafiltration element 23 is inserted into the sealing ring 24, and the top of the ultrafiltration element 23 is separated from the blocking block 25.

[0051] The centrifuge 3 includes a pressing component 31 and a rotating component 32. When the separation container 2 is in a vertical state, the pressing component 31 can push the ultrafiltration element 23 to move downward, and the rotating component 32 can drive the separation container 2 to rotate, so that it can switch between a vertical state and a horizontal state.

[0052] The threaded upper shell 22 and the lower shell 21 are rotated and separated, the ultrafiltration element 23 is taken out, and after the material to be filtered is added into the ultrafiltration element 23, the ultrafiltration element 23 is placed in the upper shell 22 and the lower shell 21, and the upper shell 22 and the lower shell 21 are rotated to make them threadedly connected, and the separation container 2 is placed vertically in the centrifuge 3. The centrifuge 3 drives the separation container 2 to rotate, and the molecules in the material to be filtered in the ultrafiltration element 23 that are smaller than the filtration diameter of the ultrafiltration element 23 pass through the ultrafiltration element 23 into the lower shell 21 under the action of centrifugal force. After centrifugation for a specified time, the centrifuge 3 stops driving the separation container 2 to rotate and centrifuge, and the filtrate in the lower shell 21 flows into the bottom of the lower shell 21 under the action of gravity, and the downward pressure component 31 pushes the ultrafiltration element 23 to move downward The upper shell 22 and the lower shell 21 are rotated and separated, and the filtrate in the lower shell 21 and the filter residue in the upper shell 22 are collected respectively.

[0053] When the separation container 2 is in a vertical state, the separation container 2 is rotated and centrifuged, and the filtrate and small molecular materials in the ultrafiltration element 23 enter the lower shell 21 under the action of centrifugal force, which can generate a certain pressure difference for the materials in the separation container 2 through centrifugation. The materials are filtered by the ultrafiltration element 23 under the action of the pressure difference. The filtration time of the ultrafiltration element 23 is short and the filtration effect is better. Thereafter, the ultrafiltration element 23 descends, and the filtrate in the lower shell 21 is sealed. The separation container 2 is rotated to a horizontal state and rotated and centrifuged, so that the filter residue in the ultrafiltration element 23 enters the upper shell 22 under the action of centrifugal force, and the filtrate and filter residue can be collected into the lower shell 21 and the upper shell 22 respectively by centrifugal force, which is more convenient to use. At the same time, the filter residue is discharged from the ultrafiltration element 23 by centrifugal force, and the discharge effect is better, which prevents the filter residue from adhering to the ultrafiltration element 23, reduces the difficulty of cleaning the ultrafiltration element 23, and increases the service life of the ultrafiltration element 23.

[0054] It should be noted that when removing the separation container 2 from the centrifuge 3, it is necessary to keep the upper shell 22 horizontal or facing downward to prevent the filter residue in the upper shell 22 from entering the ultrafiltration element 23 again under the action of gravity.

[0055] Example 2, reference Figure 1 、 Figures 9-12 , different from the above embodiment, the separation container 2 also includes a plurality of snap assemblies 26, the snap assemblies 26 include a clamping spring 261 and a clamping block 262, a connecting groove is opened on the upper shell 22, one end of the clamping spring 261 is fixedly mounted on the bottom of the connecting groove, and the clamping block 262 is fixedly mounted on the other end of the clamping spring 261, and the top and bottom of the clamping block 262 are both inclined. The ultrafiltration element 23 includes a filter element bracket 231, a top ring 232, two clamping rings 233 and an ultrafiltration membrane. The ultrafiltration membrane is fixedly mounted on the four sides of the filter element bracket 231, the top ring 232 is fixedly mounted on the top of the filter element bracket 231, and the two clamping rings 233 are fixedly mounted on the outer ring of the top ring 232.

[0056] Add the material to be filtered into the filter element bracket 231 and the ultrafiltration membrane, insert the ultrafiltration element 23 into the upper shell 22, and the clamping block 262 is pressed between the two clamping rings 233 by the clamping spring 261. At the same time, the top ring 232 is against the blocking block 25. When the ultrafiltration element 23 moves downward, the clamping ring 233 moves downward to squeeze the clamping block 262 to move horizontally. The clamping block 262 pushes the clamping spring 261 to accumulate force, so that the clamping block 262 is separated from the two clamping rings 233, and the ultrafiltration element 23 moves downward.

[0057] In addition, a support frame is provided at the bottom of the filter element bracket 231, which is not shown in the figure, to prevent the bottom of the ultrafiltration element 23 from swinging under the action of centrifugal force. At the same time, the support frame is interference fit with the lower shell 21 to provide a certain supporting force to prevent the ultrafiltration element 23 from moving downward due to gravity.

[0058] Example 3, reference Figure 1 、 Figures 9-12 , which is different from the above embodiment, is that the blocking block 25 is fixedly mounted on the inner top of the upper shell 22, and the separation container 2 also includes a push rod 27, a push ring 28 and a push plate 29. The push rod 27 is fixedly mounted on the top of the push ring 28, and the push rod 27 passes through the upper shell 22 and extends outward. The push plate 29 is fixedly mounted on the top of the push rod 27, the blocking block 25 is inserted in the push ring 28, and an anti-slip groove is provided on the upper shell 22.

[0059] When the centrifuge 3 is centrifuged in the separation container 2 in a vertical state and the separation of the internal materials is completed, the centrifuge 3 stops centrifuging, and the pressing assembly 31 squeezes the push plate 29 to move downward, and the push plate 29 drives the push rod 27 to move downward, and the push rod 27 drives the push ring 28 to move downward, and the push ring 28 drives the top ring 232 to move downward, and the top ring 232 drives the filter element bracket 231 and the ultrafiltration membrane to move downward, and the filter element bracket 231 is inserted into the sealing ring 24 to form a seal, and the push ring 28 is sealed with the upper shell 22, so that the filter residue entering the upper shell 22 will not enter the lower shell 21.

[0060] Example 4, with reference to Figures 1-9 , which is different from the above embodiment, is that the centrifuge 3 includes a rotating drum 33, a plurality of rotating assemblies 32 are arranged on the rotating drum 33, a rotating groove is opened on the rotating drum 33, the rotating assembly 32 includes a placement shell 321 with an open top, a rotating gear 322 and a movable rack 323, rotating rods are fixedly installed on both sides of the placement shell 321, the rotating rods are rotatably connected to the groove wall of the rotating groove, the rotating gear 322 is fixedly installed on the rotating rods, and the movable rack 323 is engaged with the rotating gear 322.

[0061] The separation container 2 is placed in the placement shell 321. When the separation container 2 needs to be rotated from a vertical state to a horizontal state, the movable rack 323 is pushed to move vertically. The movable rack 323 drives the rotating gear 322 to rotate. The rotating gear 322 drives the rotating rod to rotate. The rotating rod drives the placement shell 321 to rotate. The placement shell 321 drives the separation container 2 to rotate, so that the separation container 2 is rotated from a vertical state to a horizontal state.

[0062] Example 5, with reference to Figures 1-9 , which is different from the above embodiment, the centrifuge 3 further includes a fixed shell 34, a driving assembly 35 and a pushing assembly 36. The drum 33 is rotatably connected to the fixed shell 34. The driving assembly 35 and the pushing assembly 36 are arranged in the fixed shell 34. The driving assembly 35 can drive the drum 33 to rotate, and the pushing assembly 36 can push the movable racks 323 in the plurality of rotating assemblies 32 to move.

[0063] The pushing assembly 36 pushes the moving racks 323 in the plurality of rotating assemblies 32 to move, so that all the separation containers 2 rotate, making the operation simpler.

[0064] Example 6, reference Figures 1-9 , which is different from the above embodiment, is that the pushing assembly 36 includes a pushing cylinder 361 and a movable plate 362. The pushing cylinder 361 is fixedly mounted on the fixed shell 34. The output end of the pushing cylinder 361 is rotatably connected to the movable plate 362. The top of the movable rack 323 passes through the rotating drum 33 and is fixedly mounted on the movable plate 362. An avoidance groove is provided at the bottom of the rotating drum 33. The pushing cylinder 361 and the movable plate 362 are arranged in the avoidance groove.

[0065] When the drum 33 rotates, the drum 33 drives the movable plate 362 to rotate through the movable rack 323. When the separation container 2 needs to be rotated to make it horizontal, the cylinder 361 is pushed to push the movable plate 362 to move vertically. The movable plate 362 drives the movable racks 323 in the rotating components 32 to move, so that the separation container 2 rotates.

[0066] Example 7, reference Figures 1-9 , which is different from the above embodiment, the centrifuge 3 also includes a closing cover 37, the closing cover 37 is installed on the fixed shell 34, the pressing assembly 31 includes a pressing cylinder 311 and a pressing plate 312, the pressing cylinder 311 is fixedly installed on the closing cover 37, and the pressing plate 312 is fixedly installed at the output end of the pressing cylinder 311.

[0067] After the separation container 2 is placed in the placement shell 321, the closing cover 37 is closed, and the lower pressure plate 312 is located above the push plate 29. When the separation container 2 rotates centrifugally in a vertical state, the filtrate and the filter residue are separated and then the rotation stops. The lower pressure cylinder 311 pushes the lower pressure plate 312 to move downward, and the lower pressure plate 312 squeezes the push plate 29 to move downward. The push plate 29 drives the push ring 28 to move downward through the push rod 27, and the push ring 28 pushes the ultrafiltration element 23 to move downward.

[0068] Example 8, reference Figures 1-9 , which is different from the above embodiment, is that the driving assembly 35 includes a driving motor 351, a driving shaft 352, a driving pulley, a driven pulley, a driving belt, a driving gear 353 and a driving inner ring gear 354. The driving motor 351 is fixedly installed in the fixed housing 34, the driving shaft 352 is rotatably connected to the fixed housing 34, the driving inner ring gear 354 is fixedly installed on the rotating drum 33, the driving gear 353 is fixedly installed on the top of the driving shaft 352, and the driving gear 353 is meshed with the driving inner ring gear 354, the driving pulley is fixedly installed at the output end of the driving motor 351, the driven pulley is fixedly installed on the driving shaft 352, and the driving belt is connected to the driving pulley and the driven pulley.

[0069] The driving motor 351 drives the active pulley to rotate, the active pulley drives the driving belt to rotate, the driving belt causes the passive pulley to rotate, the passive pulley drives the driving shaft 352 to rotate, the driving shaft 352 drives the driving gear 353 to rotate, the driving gear 353 drives the driving inner ring gear 354 to rotate, the driving inner ring gear 354 drives the rotating drum 33 to rotate, and the rotating drum 33 drives the separation container 2 to rotate, so that the material in the separation container 2 is separated.

[0070] Example 9, reference Figures 1-9 , which is different from the above embodiment, the centrifuge 3 further includes a rubber block 38 , and the rubber block 38 is fixedly mounted on one end of the rotating groove. The top of the rubber block 38 is arc-shaped, and a buffer pad is provided in the placement shell 321 .

[0071] When the separation container 2 rotates from a vertical state to a horizontal state, the upper shell 22 rotates to one end of the rotating groove, and the rubber block 38 presses the push plate 29 to prevent the push plate 29 from moving under the centrifugal force. At the same time, the rubber block 38 has a certain deformation ability, which can enable the push plate 29 to enter the rotating groove.

[0072] When the separation container 2 is inserted into the placement shell 321 , the buffer pad is deformed, and the buffer pad clamps the separation container 2 to prevent the separation container 2 from moving, thereby improving the stability of the separation container 2 .

[0073] A separation method for extracting extracellular vesicles, using the above-mentioned separation device, comprises the following steps:

[0074] The tissue fluid was separated by centrifugation, and the supernatant containing a large number of extracellular vesicles was collected;

[0075] The upper shell 22 and the lower shell 21 that are threadedly connected are rotated and separated to remove the ultrafiltration element 23;

[0076] After adding the supernatant into the ultrafiltration element 23, the ultrafiltration element 23 is placed in the upper shell 22 and the lower shell 21, and the upper shell 22 and the lower shell 21 are rotated to make them threadedly connected;

[0077] The separation container 2 is placed vertically in the centrifuge 3. The centrifuge 3 drives the separation container 2 to rotate. Under the action of centrifugal force, the liquid in the ultrafiltration element 23 passes through the ultrafiltration element 23 and enters the lower shell 21. After centrifugation for a specified time, the centrifuge 3 stops driving the separation container 2 to rotate and centrifuge. The filtrate in the lower shell 21 flows into the bottom of the lower shell 21 under the action of gravity.

[0078] The pressing assembly 31 pushes the ultrafiltration element 23 downward, so that the ultrafiltration element 23 is inserted into the sealing ring 24. The sealing ring 24 and the ultrafiltration element 23 seal the filtrate at the bottom of the lower shell 21. At the same time, the top of the ultrafiltration element 23 is separated from the blocking block 25, and the top opening of the ultrafiltration element 23 is connected to the upper shell 22.

[0079] The rotating assembly 32 drives the separation container 2 to rotate, so that the separation container 2 rotates from a vertical state to a horizontal state, and the open end of the ultrafiltration element 23 is located at the outer ring of the centrifuge 3;

[0080] The centrifuge 3 drives the separation container 2 to rotate and centrifuge. Under the action of centrifugal force, the extracellular vesicles in the ultrafiltration element 23 pass through the open end of the ultrafiltration element 23 and enter the upper housing 22. The centrifugation stops after a specified time.

[0081] The upper shell 22 and the lower shell 21 are rotated and separated to collect the filtrate in the lower shell 21 and the extracellular vesicles in the upper shell 22 respectively.

[0082] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A separation device comprising a separation container and a centrifuge, wherein the centrifuge is capable of driving the separation container to rotate, characterized in that: The separation container comprises a lower shell, an upper shell and an ultrafiltration element, the upper shell and the lower shell are threadedly connected, and the ultrafiltration element is arranged in the upper shell and the lower shell; The separation container further includes a sealing ring and a blocking block. The top of the ultrafiltration element is open and abuts against the blocking block. When the ultrafiltration element moves downward in the upper and lower shells, the bottom of the ultrafiltration element is inserted into the sealing ring, and the top of the ultrafiltration element is separated from the blocking block. The centrifuge includes a downward pressure component and a rotating component. When the separation container is in a vertical state, the downward pressure component can push the ultrafiltration element downward, and the rotating component can drive the separation container to rotate, so that it can switch between a vertical state and a horizontal state.

2. A separation device according to claim 1, characterized in that: The separation container also includes a plurality of snap assemblies, which include a clamping spring and a clamping block. A connecting groove is provided on the upper shell body. One end of the clamping spring is fixedly mounted on the bottom of the connecting groove, and the clamping block is fixedly mounted on the other end of the clamping spring. The top and bottom of the clamping block are both inclined. The ultrafiltration element includes a filter element bracket, a top ring, two clamping rings and an ultrafiltration membrane. The ultrafiltration membrane is fixedly mounted on the four sides of the filter element bracket. The top ring is fixedly mounted on the top of the filter element bracket, and the two clamping rings are fixedly mounted on the outer ring of the top ring.

3. A separation device according to claim 2, characterized in that: The blocking block is fixedly mounted on the inner top of the upper shell. The separation container also includes a push rod, a push ring and a push plate. The push rod is fixedly mounted on the top of the push ring. The push rod passes through the upper shell and extends outward. The push plate is fixedly mounted on the top of the push rod. The blocking block is inserted in the push ring. An anti-slip groove is provided on the upper shell.

4. A separation device according to claim 1, characterized in that: The centrifuge includes a rotating drum, a plurality of rotating assemblies are arranged on the rotating drum, a rotating groove is opened on the rotating drum, and the rotating assembly includes a placement shell with an open top, a rotating gear and a movable rack. Rotating rods are fixedly installed on both sides of the placement shell, and the rotating rods are rotatably connected to the groove wall of the rotating groove. The rotating gear is fixedly installed on the rotating rod, and the movable rack is engaged with the rotating gear.

5. A separation device according to claim 4, characterized in that: The centrifuge also includes a fixed shell, a drive assembly and a pushing assembly. The drum is rotatably connected to the fixed shell. The drive assembly and the pushing assembly are arranged in the fixed shell. The drive assembly can drive the drum to rotate, and the pushing assembly can push several movable racks in the rotating assemblies to move.

6. A separation device according to claim 5, characterized in that: The pushing assembly includes a pushing cylinder and a movable plate. The pushing cylinder is fixedly mounted on a fixed shell. The output end of the pushing cylinder is rotatably connected to the movable plate. The top of the movable rack passes through the rotating drum and is fixedly mounted on the movable plate. An avoidance groove is provided at the bottom of the rotating drum. The pushing cylinder and the movable plate are arranged in the avoidance groove.

7. A separation device according to claim 5, characterized in that: A closing cover is installed on the fixed shell, and the pressing assembly includes a pressing cylinder and a pressing plate. The pressing cylinder is fixedly installed on the closing cover, and the pressing plate is fixedly installed on the output end of the pressing cylinder.

8. A separation device according to claim 5, characterized in that: The driving assembly includes a driving motor, a driving shaft, a driving pulley, a driven pulley, a driving belt, a driving gear and a driving internal gear ring. The driving motor is fixedly installed in the fixed housing, the driving shaft is rotatably connected to the fixed housing, the driving internal gear ring is fixedly installed on the rotating drum, the driving gear is fixedly installed on the top of the driving shaft, and the driving gear is meshed with the driving internal gear ring. The driving pulley is fixedly installed on the output end of the driving motor, the driven pulley is fixedly installed on the driving shaft, and the driving belt is connected to the driving pulley and the driven pulley.

9. A separation device according to claim 4, characterized in that: A rubber block is fixedly mounted on one end of the rotating groove, the top of the rubber block is arranged in an arc shape, and a buffer pad is arranged in the placement shell.

10. A separation method for extracting extracellular vesicles, using the separation device according to any one of claims 1 to 9, characterized in that: The following steps are involved: The tissue fluid was separated by centrifugation, and the supernatant containing a large number of extracellular vesicles was collected; Rotate and separate the threaded upper and lower shells to remove the ultrafiltration element; After adding the supernatant into the ultrafiltration element, the ultrafiltration element is placed in the upper shell and the lower shell, and the upper shell and the lower shell are rotated to make them threadedly connected; The separation container is placed vertically in the centrifuge, and the centrifuge drives the separation container to rotate. The liquid in the ultrafiltration element passes through the ultrafiltration element under the action of centrifugal force and enters the lower shell. After centrifugation for a specified time, the centrifuge stops driving the separation container to rotate and centrifuge, and the filtrate in the lower shell flows into the bottom of the lower shell under the action of gravity; The downward pressure assembly pushes the ultrafiltration element downward, so that the ultrafiltration element is inserted into the sealing ring. The sealing ring and the ultrafiltration element seal the filtrate at the bottom of the lower shell. At the same time, the top of the ultrafiltration element is separated from the blocking block, and the top opening of the ultrafiltration element is connected to the upper shell. The rotating assembly drives the separation container to rotate, so that the separation container rotates from a vertical state to a horizontal state, and the open end of the ultrafiltration element is located at the outer ring of the centrifuge; The centrifuge drives the separation container to rotate and centrifuge. The extracellular vesicles in the ultrafiltration element enter the upper shell through the open end of the ultrafiltration element under the action of centrifugal force. The centrifugation stops after a specified time. The upper shell and the lower shell were separated by rotation, and the filtrate in the lower shell and the extracellular vesicles in the upper shell were collected respectively.

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