Exosome separation and extraction equipment

Abstract

The utility model discloses exosome separation and extraction equipment, which relates to the technical field of exosome separation and comprises a sample loading barrel, a compression mechanism, a filter component and a collection bottle, the sample loading barrel is provided with a sample accommodating cavity, a sample adding pipe is arranged on the side wall of the sample loading barrel, the sample adding pipe is communicated with the sample accommodating cavity, and a valve is arranged on the sample adding pipe; a lower discharge port communicated with the sample accommodating cavity is formed in the sample loading barrel; the compression mechanism comprises a driver and a compression piston; the compression piston is slidably arranged in the sample containing cavity along the axis of the sample containing cavity, and the output end of the driver is used for driving the compression piston to move; the filtering assembly comprises a filtering plate and at least two filtering membranes with different pore diameters; the filter plate is positioned in the sample accommodating cavity above the lower discharge port, and the single filter membrane is positioned above the filter plate; the collecting bottle is located below the lower discharging opening, and an upper opening of the collecting bottle is opposite to the lower discharging opening. Simple and batch exosome separation and extraction can be realized.

Description

TECHNICAL FIELD

[0001] The utility model relates to exosome separation technical field, in particular to a kind of exosome separation extraction equipment. BACKGROUND

[0002] Exosome is the extracellular vesicle of about 30-150nm in diameter size that cell secretes. Exosome is widely distributed in body fluid including blood, urine, saliva, milk after cell secretion, its outer wall is formed by lipid hollow vesicle, and vesicle contains nucleic acid, protein, amino acid, lipid etc. Exosome can be absorbed by other cells by endocytosis after cell secretion, and influence the biological function of the cell.

[0003] Global exosome research is increasingly extensive, and once become the hotspot in biological scientific research field. By artificially reconfiguring the content of exosome, exosome has specific biological benefits, and is expected to develop new disease treatment method in biological medicine field. For example, after reconfiguring certain engineering cell, exosome carrying specific nucleic acid / protein is secreted by the cell, so that exosome can be endowed with specific treatment effect. At present, some exosome products have been experimentally applied to life production, for example, exosome extracted from stem cell is injected into skin, which can play the role of skin anti-aging, and part of products has been prepared for marketing.

[0004] How to realize the concentration extraction of exosome to realize simpler batch preparation is particularly important. UTILITY MODEL CONTENT

[0005] The utility model aims at providing a kind of exosome separation extraction equipment to solve the problems existing in the prior art, realize simple and batch exosome separation extraction.

[0006] To achieve the above object, the utility model provides the following scheme:

[0007] The utility model provides a kind of exosome separation extraction equipment, including sample loading cylinder, compression mechanism, filter assembly and collection bottle;The sample loading cylinder has sample accommodating cavity, and the side wall of the sample loading cylinder is provided with sample addition tube, the sample addition tube is communicated with the sample accommodating cavity, and the valve is set on the sample addition tube;The sample loading cylinder is provided with lower discharge port communicated with the sample accommodating cavity;The compression mechanism includes driver and compression piston;The compression piston is slidably arranged in the sample accommodating cavity along the axis of the sample accommodating cavity, and the output end of the driver is used to drive the compression piston to move;The filter assembly includes filter plate and at least two different pore size filter membranes;The filter plate is located in the sample accommodating cavity above the lower discharge port, and single the filter membrane is located above the filter plate;The collection bottle is located below the lower discharge port, and the upper opening of the collection bottle is opposite to the lower discharge port.

[0008] Preferably, a pressure detector is further included, and a sensing end of the pressure detector is located in the sample accommodating cavity between the compression piston and the filter membrane.

[0009] Preferably, the filter membrane is a PCTE membrane.

[0010] Preferably, the sample loading cylinder comprises a cylinder body and an inverted conical cylinder, a first flange ring is fixedly arranged around a lower end of the cylinder body, a second flange ring is fixedly arranged around a large opening end of the inverted conical cylinder, the second flange ring is capable of being fixedly connected with the first flange ring, and an inner recess is arranged at the large opening end of the inverted conical cylinder, and the filter plate and the filter membrane are located in the inner recess.

[0011] Preferably, the first flange ring is crimped above the filter membrane.

[0012] Preferably, a support frame is further fixedly arranged on the sample loading cylinder.

[0013] Preferably, a flared tube is arranged at a top of the sample loading tube, and a large opening end of the flared tube is located above a small opening end of the flared tube.

[0014] Preferably, a plurality of one-to-one corresponding mounting holes are arranged on the first flange ring and the second flange ring respectively, a bolt is arranged in each mounting hole, and a threaded end of the bolt is threadedly connected with a nut.

[0015] Preferably, the collection bottle is made of transparent material.

[0016] Preferably, a reinforcing connecting ring is fixedly arranged close to a bottom of the support frame.

[0017] The utility model discloses relative to prior art has obtained following technical effect:

[0018] The sample accommodating cavity provides a space for placing the sample containing exosome after centrifugal treatment, so that the sample can be treated in a relatively closed and stable environment; the compression piston driven by the driver pressurizes the liquid containing exosome, so as to push the liquid containing exosome to pass through the filter membrane, thereby leaving the exosome on the filter membrane, completing the preliminary separation of the exosome, and then performing secondary separation through replacing the filter membrane with smaller aperture, and finally washing the exosome from the filter membrane; the washed liquid can be directly used for exosome freeze-drying or secondary ultracentrifugation, so as to realize simple and batch exosome separation and extraction, and improve the purity of the exosome; the sample loading tube arranged on the side facilitates the sample loading of the liquid containing exosome.

[0019] Further, the pressure detector can accurately feedback the internal pressure information, thereby providing good separation pressure conditions for the exosomes and improving the separation effect.

[0020] Further, the PCTE film (polycarbonate track-etched film) is a film material made of polycarbonate film with a specific pore size. The PCTE film has a unique manufacturing process that ensures precise material separation by increasing control over pore size and density, making it suitable for blood analysis and high-purity and general filtration. It can effectively separate the exosome liquid.

[0021] Further, when the compression piston is pressed down, the liquid containing exosomes will flow in the direction of the inverted cone under the action of pressure. Since the shape of the inverted cone is gradually reduced, it can effectively guide the filtered liquid to the center and bottom, helping to improve the flowability and collection efficiency of the filtered liquid, so that the filtered liquid can more smoothly pass through the filter assembly into the collection bottle. The first flange ring and the second flange ring are arranged to facilitate the fixed connection of the barrel body and the inverted cone. This connection method is not only stable, but also easy to disassemble and assemble.

[0022] Further, the first flange ring is pressed on the top of the filter membrane, which can firmly fix the filter membrane in its position, ensuring the stability and reliability of the filtration process. A stable filter membrane position helps to continuously and efficiently filter and separate.

[0023] Further, the presence of the support frame can provide additional support for the sample loading cylinder, ensuring that the sample loading cylinder remains stable, thereby ensuring that the filtration and other operations can be carried out smoothly, improving the precision and efficiency of exosome separation and extraction.

[0024] Further, the design of the flared tube with the large end facing up makes it easier to add samples.

[0025] Further, by threading bolts through the mounting holes corresponding to the first flange ring and the second flange ring and tightening them with nuts, the barrel body and the inverted cone can be tightly connected together. The combination of bolts and nuts can ensure that the sample loading cylinder does not separate during long-term use, ensuring the stability of the equipment structure, thereby providing a reliable hardware foundation for stable exosome separation and extraction.

[0026] Further, the transparent collection bottle allows the operator to visually observe the volume of the collected liquid.

[0027] Further, the reinforced connection ring can enhance the support strength of the support frame, and the reinforced connection ring is located near the bottom of the support frame, which can effectively reduce the center of gravity of the equipment. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0029] Figure 1 The overall structure schematic diagram of the exosome separation and extraction equipment provided by the present application is shown in the figure.

[0030] Figure 2 The internal structure schematic diagram of the exosome separation and extraction equipment provided by the present application is shown in the figure.

[0031] Figure 3 The structure explosion diagram of the filter plate, filter membrane and inverted cone type cylinder in the exosome separation and extraction equipment provided by the present application is shown in the figure.

[0032] In the figure:

[0033] 10-cylinder body; 101-first flange ring; 11-inverted cone type cylinder; 111-lower discharge port; 112-second flange ring; 113-internal recess; 12-sample adding pipe; 121-flared pipe; 122-valve; 13-top cover;

[0034] 20-compression piston; 21-rotating shaft; 22-rotating handle;

[0035] 30-filter plate; 31-filter membrane;

[0036] 40-collection bottle;

[0037] 50-support frame; 51-reinforcing connecting ring. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0039] The purpose of the present application is to provide an exosome separation and extraction equipment to solve the problems existing in the prior art, which is simple and can batch separate and extract exosomes.

[0040] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the present application will be further described in detail in combination with the drawings and specific embodiments.

[0041] Embodiment one

[0042] The embodiment provides an exosome separation and extraction device, which comprises a sample loading cylinder, a compression mechanism, a filter assembly and a collection bottle 40, as shown in the drawings. Figures 1-3 The sample loading cylinder has a sample accommodating cavity, and a sample loading pipe 12 is arranged on the side wall of the sample loading cylinder and communicates with the sample accommodating cavity. A valve 122 is arranged on the sample loading pipe 12. The sample loading cylinder has a lower discharge port 111 which communicates with the sample accommodating cavity. The compression mechanism comprises a driver and a compression piston 20. The compression piston 20 is arranged in the sample accommodating cavity and slides along the axis of the sample accommodating cavity. The output end of the driver is used to drive the compression piston 20 to move. The filter assembly comprises a filter plate 30 and at least two filter membranes 31 with different pore sizes. The filter plate 30 is arranged in the sample accommodating cavity above the lower discharge port 111, and a single filter membrane 31 is arranged above the filter plate 30. The collection bottle 40 is arranged below the lower discharge port 111, and the upper opening of the collection bottle 40 is opposite to the lower discharge port 111.

[0043] The sample accommodating cavity provides a space for placing the exosome-containing sample after centrifugal treatment, so that the sample can be treated in a relatively closed and stable environment. The driver drives the compression piston 20 to pressurize the exosome-containing liquid, so as to push the exosome-containing liquid to pass through the filter membrane 31, thereby leaving the exosomes on the filter membrane 31, completing the preliminary separation of the exosomes. The exosomes are then separated again by replacing the filter membrane 31 with a smaller pore size, and finally the exosomes are eluted from the filter membrane 31. The eluted liquid can be directly used for exosome freeze-drying or ultracentrifugation again, so as to realize simple and batch exosome separation and extraction and improve the purity of the exosomes. The sample loading pipe 12 arranged on the side facilitates the loading of the exosome-containing liquid.

[0044] In the structure of the sample loading cylinder, the sample loading pipe 12 is arranged on the side wall of the sample loading cylinder and communicates with the sample accommodating cavity.

[0045] In the optional scheme of the embodiment, preferably, the sample loading pipe 12 is arranged on the side wall of the sample loading cylinder and communicates with the sample accommodating cavity. Figure 1 and Figure 2As shown, the sample loading cylinder includes a cylinder body 10 and a reverse tapered cylinder 11; a first flange ring 101 is fixedly arranged around the lower end of the cylinder body 10; a second flange ring 112 is fixedly arranged around the large opening end of the reverse tapered cylinder 11 above; the second flange ring 112 can be fixedly connected with the first flange ring 101; the large opening end of the reverse tapered cylinder 11 is provided with an inner recess 113, and the filter plate 30 and the filter membrane 31 are located in the inner recess 113. When the compression piston 20 is pressed down, the liquid containing exosomes will flow in the direction of the reverse tapered cylinder 11 under the action of pressure. Since the shape of the reverse tapered cylinder 11 is gradually reduced, it can effectively guide the filtered liquid to converge to the center and the bottom, which helps to improve the flowability and collection efficiency of the filtered liquid, so that the filtered liquid can more smoothly pass through the filter assembly into the collection bottle 40; the arrangement of the first flange ring 101 and the second flange ring 112 facilitates the fixed connection of the cylinder body 10 and the reverse tapered cylinder 11. This connection method is not only stable, but also convenient to disassemble and assemble.

[0046] In an optional solution of the present embodiment, more preferably, as shown in Figure 1 As shown, a plurality of one-to-one corresponding mounting holes are formed in the first flange ring 101 and the second flange ring 112 respectively, a bolt is arranged in each mounting hole, and the threaded end of the bolt is threadedly connected with a nut. By threading the bolt in the corresponding mounting hole of the first flange ring 101 and the second flange ring 112 and fastening it with the nut, the cylinder body 10 and the reverse tapered cylinder 11 can be tightly connected together; the combination of the bolt and the nut can ensure that the sample loading cylinder will not separate during long-term use, ensuring the stability of the equipment structure, thereby providing a reliable hardware foundation for stable separation and extraction of exosomes.

[0047] In an optional solution of the present embodiment, more preferably, the first flange ring 101 is crimped above the filter membrane 31. Crimping the first flange ring 101 above the filter membrane 31 can firmly fix the filter membrane 31 in its position, ensuring the stability and reliability of the filtration process. A stable filter membrane 31 position helps to continuously and efficiently filter and separate.

[0048] In an optional solution of the present embodiment, more preferably, as shown in Figure 1 and Figure 2 As shown, the top of the sample adding tube 12 is provided with a flared tube 121, and the large opening end of the flared tube 121 is located above the small opening end of the flared tube 121. The upward design of the large opening end of the flared tube 121 makes it more convenient to add samples.

[0049] In an optional solution of the present embodiment, more preferably, as shown in Figure 1 and Figure 2As shown, the sample loading cylinder is also fixedly provided with a support frame 50. The presence of the support frame 50 can provide additional support for the sample loading cylinder, ensure that the sample loading cylinder remains stable, so as to ensure that the filtering and other operations can be carried out smoothly, and improve the precision and efficiency of the exosome separation and extraction.

[0050] In an optional solution of the present embodiment, more preferably, as shown in Figure 1 As shown, the support frame 50 is fixedly provided with a reinforcing connecting ring 51 near the bottom position. The reinforcing connecting ring 51 can enhance the support strength of the support frame 50, and the reinforcing connecting ring 51 is located near the bottom position of the support frame 50, which can effectively reduce the center of gravity of the equipment.

[0051] Among them, the related description of the compression mechanism is as follows:

[0052] Specifically, the compression mechanism can be an existing hydraulic device or as shown in Figure 1 As shown, it is a manual structure, at this time the compression mechanism includes a rotating handle 22 and a rotating shaft 21, one end of the rotating shaft 21 is rotatably connected with the compression piston 20, the other end of the rotating shaft 21 is fixedly connected with the rotating handle 22, at this time the compression can be realized by manual rotation.

[0053] Specifically, to achieve better compression control, an automatic control hydraulic device can be used to cooperate with a pressure detector for common control.

[0054] Specifically, the pressure value control range can be set according to the actual effect.

[0055] Specifically, the cylinder body 10 has a top cover 13, and the top cover 13 is provided with a through hole for the rotating shaft 21 to pass through.

[0056] Among them, the related description of the filtering assembly is as follows:

[0057] In an optional solution of the present embodiment, more preferably, the filtering membrane 31 is a PCTE membrane. The PCTE membrane (polycarbonate track etched membrane) is a membrane material made of polycarbonate film with a specific pore size. The PCTE membrane has a unique manufacturing process to ensure precise material separation by increasing control over pore size and density, and is very suitable for blood analysis and high-purity and general filtration; it can have a good separation effect on the liquid of the exosome.

[0058] Specifically, the pore size of the PCTE membrane can be selected according to the actual situation.

[0059] Among them, the other related description is as follows:

[0060] In the optional solution of the embodiment, preferably, a pressure detector is further included, and a sensing end of the pressure detector is located in the sample containing cavity between the compression piston 20 and the filter membrane 31. The pressure detector can accurately feedback the internal pressure information, thereby providing a good separation pressure condition for the exosomes and improving the separation effect.

[0061] Specifically, the pressure detector can be a hydraulic gauge or a pressure sensor.

[0062] In the optional solution of the embodiment, preferably, the material of the collection bottle 40 is transparent. The transparent collection bottle 40 can enable the operator to visually observe the volume of the collected liquid.

[0063] Specifically, the sample loading cylinder is made of stainless steel.

[0064] Specific extraction operation:

[0065] 1. Centrifuge 2L of cell culture supernatant for exosome extraction at 3000 rpm for 10 minutes, and remove the precipitate;

[0066] 2. Filter the liquid after centrifugation through a 220μm filter to remove impurities in the liquid, which is the liquid to be extracted;

[0067] 3. Cover the PCTE membrane with a pore size of 160nm on the filter plate 30, and connect the inverted conical cylinder 11 to the bottom of the cylinder body 10 to ensure tight connection;

[0068] 4. Stretch the rotating shaft 21 connected to the hydraulic device outward to 2L, which indicates that the sample loading cylinder can load 2L of liquid;

[0069] 5. Slowly add the liquid to be extracted into the sample loading cylinder through the sample loading tube 12, appropriately adjust the compression piston 20 so that the sample loading cylinder is full of liquid, and then close the valve 122 on the sample loading tube 12. At this time, the liquid is in contact with the PCTE membrane, but under the condition of no pressure, the liquid will not leak out quickly;

[0070] 6. Start the hydraulic device to push at 0.7MPa, and about 15 minutes later, the liquid can completely flow out through the inverted conical cylinder 11, and at this time, the collected liquid is the further separation liquid (which removes molecules larger than 160nm);

[0071] 7. Remove the inverted conical cylinder 11, remove the 160nm PCTE membrane, cover the 30nm PCTE membrane, and connect it to the bottom of the cylinder body 10 again;

[0072] 8. Adjust the hydraulic device to re-add the further separation liquid into the sample loading cylinder through the sample loading tube 12;

[0073] 9. Close the valve 122 of the sample tube 12, and start the hydraulic device to push at 1.2 MPa for about 30 minutes, and the liquid can be completely discharged from the inverted tapered cylinder 11, at this time, the liquid discharged from the inverted tapered cylinder 11 can be discarded (containing molecules less than 30 nm);

[0074] 10. Remove the inverted tapered cylinder 11, and take off the 30 nm PCTE film from the filter plate 30, and place it in a glass dish, and use 1-5 ml of PBS buffer to elute the 30-160 nm exosomes on the film;

[0075] 11. The eluted liquid can be directly used for exosome freeze-drying or re-centrifugation.

[0076] The principles and implementation modes of the specific examples applied in the utility model are described, and the above embodiment is only used for helping to understand the method and core idea of the utility model; meanwhile, for the general skilled in the art, according to the idea of the utility model, the specific implementation mode and application range will have changes. In conclusion, the content of the specification should not be understood as the limitation of the utility model.

Claims

1. An exosome separation and extraction device, characterized in that: Includes a sample carrier, a compression mechanism, a filter assembly, and a collection bottle; The sample carrier has a sample receiving cavity, and a sample addition tube is provided on the side wall of the sample carrier, which is connected to the sample receiving cavity and is equipped with a valve; the sample carrier has a lower discharge port that is connected to the sample receiving cavity. The compression mechanism includes a driver and a compression piston; the compression piston is slidably disposed within the sample receiving cavity along the axis of the sample receiving cavity, and the output end of the driver is used to drive the compression piston to move; The filtration assembly includes a filter plate and at least two filter membranes with different pore sizes; the filter plate is located in the sample receiving cavity above the lower outlet, and each filter membrane is located above the filter plate at a time. The collection bottle is located below the lower outlet, and the upper opening of the collection bottle is opposite to the lower outlet.

2. The exosome separation and extraction device according to claim 1, characterized in that: It also includes a pressure detector, the sensing end of which is located within the sample receiving cavity between the compression piston and the filter membrane.

3. The exosome separation and extraction device according to claim 1, characterized in that: The filter membrane is a PCTE membrane.

4. The exosome separation and extraction device according to claim 1, characterized in that: The sample carrier cylinder includes a cylinder body and an inverted conical cylinder; A first flange ring is fixedly installed around the lower end of the cylinder; A second flange ring is fixedly provided around the large opening at the top of the inverted conical cylinder; the second flange ring can be fixedly connected to the first flange ring. The large opening at the top of the inverted conical cylinder is provided with an inner groove, and the filter plate and the filter membrane are located within the inner groove.

5. The exosome separation and extraction device according to claim 4, characterized in that: The first flange ring is pressed onto the filter membrane.

6. The exosome separation and extraction apparatus according to claim 1, characterized in that: A support frame is also fixedly installed on the sample carrier cylinder.

7. The exosome separation and extraction apparatus according to claim 1, characterized in that: The top of the sample dispensing tube is provided with a flared tube, and the larger end of the flared tube is located above the smaller end of the flared tube.

8. The exosome separation and extraction apparatus according to claim 4, characterized in that: The first flange ring and the second flange ring are respectively provided with a plurality of one-to-one corresponding mounting holes, and a bolt is inserted into each mounting hole, and a nut is threadedly connected to the threaded end of the bolt.

9. The exosome separation and extraction apparatus according to claim 1, characterized in that: The collection bottle is made of a transparent material.

10. The exosome separation and extraction apparatus according to claim 6, characterized in that: A reinforcing connecting ring is fixedly installed near the bottom of the support frame.

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