A device for extracting exosomes from adipose tissue

Abstract

This invention relates to the field of adipose tissue exosome extraction technology, and in particular to an extraction device for adipose tissue exosomes. The device includes a support base, a controller located at the upper edge of the support base, an extraction chamber located on the upper side of the support base, a sealing cover on the upper side of the extraction chamber, a filter membrane module located inside the extraction chamber, and a negative pressure centrifugal filtration assembly inside the extraction chamber. The negative pressure centrifugal filtration assembly is used for centrifugal filtration and negative pressure expulsion of the filtrate. This invention utilizes the negative pressure centrifugal filtration assembly in conjunction with the filter membrane module for filtration, simultaneously leveraging centrifugal filtration and negative pressure expulsion. These two functions work synergistically to significantly improve the extraction efficiency of adipose tissue exosomes. The entire extraction process is conducted in a relatively mild environment, and the negative pressure centrifugal filtration assembly's operation does not cause excessive damage to the exosomes, effectively preserving their biological activity and providing high-quality samples for subsequent research and applications.

Description

Technical Field

[0001] This utility model relates to the field of adipose tissue exosome extraction technology, and in particular to an extraction device for adipose tissue exosomes. Background Technology

[0002] Exosomes are extracellular vesicles with a diameter of 30–150 nm that carry bioactive molecules such as proteins, nucleic acids, and lipids. They have extremely high application value in disease diagnosis, drug delivery, and regenerative medicine. Adipose tissue has become an important source for the preparation of clinical-grade exosomes due to its large availability and high exosome content.

[0003] However, existing adipose tissue exosome extraction technologies mainly rely on stepwise processes such as differential centrifugation and ultrafiltration / density gradient centrifugation, which have the following prominent drawbacks: the equipment is scattered and the process is lengthy. Traditional methods require the sequential use of tissue homogenizers, low-speed centrifuges, high-speed / ultra-speed centrifuges, and tangential flow ultrafiltration devices. The multiple tube transfers between equipment not only increase the risk of contamination but also make the operation time as long as 4–6 hours, which is difficult to meet the needs of rapid diagnosis and treatment and large-scale production.

[0004] Exosomes are easily damaged and have low recovery rates. The high shear force and local temperature rise generated by long-term high-speed centrifugation can easily lead to exosome membrane rupture. Furthermore, the concentration polarization and clogging of the membrane surface are significant during ultrafiltration, which further causes exosome loss. The overall recovery rate is usually less than 50%.

[0005] Open systems pose a high risk of contamination. Existing devices are mostly open and manually operated, exposing the adipose tissue to the environment during the crushing and centrifugation steps. This can easily introduce RNase, bacteria, or mycoplasma, reducing product purity and increasing the risks of clinical use. Utility Model Content

[0006] This invention addresses the shortcomings of existing technologies by providing an extraction device for adipose tissue exosomes, which solves these problems.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] An extraction device for adipose tissue exosomes includes a support base, a controller located at the upper edge of the support base, an extraction box located on the upper side of the support base, a sealing cover located on the upper side of the extraction box, a filter membrane module located inside the extraction box, and a negative pressure centrifugal filtration assembly located inside the extraction box. The negative pressure centrifugal filtration assembly is used for centrifugal filtration and negative pressure discharge of the filtrate.

[0009] Through the above technical solutions, the support base provides a stable base for the entire device; the controller centrally controls the start-up, shutdown, speed, negative pressure value and other parameters of the negative pressure centrifugal filtration component; the extraction box and the sealing cover form a sealed chamber to prevent contamination; the filter membrane module classifies and retains the adipose tissue homogenate, allowing only exosomes and smaller particles to pass through.

[0010] The negative pressure centrifugal filtration assembly generates centrifugal force and controllable negative pressure simultaneously under the drive of a motor: the centrifugal force causes particles of different densities in the tissue fluid to separate into layers, while the negative pressure accelerates the flow of the filtrate across the membrane and squeezes out the stagnant liquid, thus continuously completing the two-step filtration of centrifugation and negative pressure expulsion.

[0011] Integrated centrifugation and negative pressure dual-power system shorten the time required for the traditional two-step process of centrifugation followed by ultrafiltration; the closed design reduces the risk of exosomes being degraded by nucleases or proteases; the controller enables adjustable process parameters to adapt to different adipose tissue sources and improve batch-to-batch consistency.

[0012] Preferably, the negative pressure centrifugal filtration assembly includes a discharge pipe, which is disposed through the bottom of the inner side of the extraction box. A centrifugal filter cylinder is disposed at the top of the discharge pipe. A connecting rod is disposed at the top of the inner side of the discharge pipe. An impeller is disposed at the top of the connecting rod. A gas guide pipe is disposed through one side of the sealing cover plate. One end of the impeller is placed inside the gas guide pipe.

[0013] Preferably, a support frame is provided on the lower side of the support base, a motor is provided on one side of the support frame, a gear one is provided on the shaft end of the motor, a gear two is meshed with the outer side of the gear one, and the gear two is fixedly sleeved on the outer side of the discharge pipe.

[0014] Through the above technical solution, the motor outputs torque, which is transmitted from the horizontal shaft to the vertical discharge pipe through the meshing of gear one and gear two, thereby driving the centrifugal filter cartridge and impeller to rotate. The gear reduction ratio can be changed according to different centrifugal speed requirements to achieve precise speed control.

[0015] The gear transmission structure is simple and reliable, easy to maintain, and the speed range can be expanded by replacing the gear pair to meet the optimal centrifugation conditions for exosomes of different particle sizes. The motor is located at the bottom to lower the center of gravity of the whole machine, making the operation more stable and reducing noise.

[0016] Preferably, a feed pipe is provided on the upper side of the sealing cover, a feed hopper is provided at the top of the feed pipe, and a rubber cover is provided on the upper side of the feed hopper.

[0017] With the above technical solution, the adipose tissue homogenate is collected in the feed hopper and then injected into the extraction box along the feed pipe. The rubber cover immediately rebounds and closes after injection to prevent foreign objects from falling in and aerosols from escaping.

[0018] The funnel-shaped feed hopper has a larger opening to reduce losses due to sticking to the walls; the rubber cover acts as a one-way valve to keep the chamber sealed and prevent contamination.

[0019] Preferably, an exosome discharge tube is provided on the lower side of the extraction box.

[0020] Through the above technical solution, the exosome enrichment solution after being intercepted by the filter membrane module is deposited at the bottom of the extraction tank and collected in a concentrated manner under gravity or slight positive pressure through the exosome discharge pipe.

[0021] The bottom drainage design prevents re-suspension and improves product purity; the independent discharge pipe facilitates connection to subsequent concentration or cryopreservation equipment, enabling continuous production; the discharge pipe can be equipped with a sterile connector to meet GMP requirements.

[0022] Preferably, a pressure relief valve is provided on the upper side of the sealing cover.

[0023] Through the above technical solution, the pressure relief valve automatically opens after filtration or in case of abnormal high pressure, releasing the negative pressure in the extraction box to normal pressure, making it easy to safely open the sealing cover for cleaning or maintenance.

[0024] Prevents liquid splashing or filter membrane damage due to negative pressure when opening the cover; shortens downtime for depressurization and improves equipment turnover; the pressure relief valve can be connected to an external HEPA filter to prevent backflow of contaminants and ensure a clean environment.

[0025] Preferably, a one-way solenoid valve is provided on the outside of the discharge pipe, the air guide pipe, the feed pipe and the exosome discharge pipe.

[0026] In summary, this invention utilizes a negative pressure centrifugal filtration component in conjunction with a filter membrane module for filtration. This combination of centrifugal filtration and negative pressure expulsion significantly improves the extraction efficiency of exosomes from adipose tissue. The entire extraction process is conducted in a relatively mild environment, and the negative pressure centrifugal filtration component does not cause excessive damage to the exosomes, effectively preserving their biological activity and providing high-quality samples for subsequent research and applications.

[0027] In this invention, the centrifugal filter cartridge rotates under the drive of the discharge pipe, generating centrifugal force, which causes impurities in the filtrate to be quickly separated and intercepted inside the filter cartridge. At the same time, it drives the impeller to rotate inside the air guide pipe. The impeller drives the relevant components to move through the connecting rod, forming a negative pressure expulsion effect, which promotes the filtrate to pass through the filter membrane more quickly and fully, further improving the filtration efficiency and the purity of exosomes.

[0028] This invention features a feed hopper that increases the area of ​​the feed inlet, making it easier for operators to pour adipose tissue samples into the device and reducing sample spillage. The rubber cover seals the feed hopper after feeding, effectively preventing external dust and impurities from entering the extraction chamber and contaminating the sample, ensuring the cleanliness of the extraction environment and preventing sample evaporation. The sealing effect of the rubber cover also prevents the evaporation of filtrate during extraction, reducing sample loss, ensuring the extraction yield of exosomes, and preventing volatile substances from polluting the laboratory environment. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the explosive structure of this utility model;

[0030] Figure 2 This is a schematic diagram of the front axonometric structure of this utility model;

[0031] Figure 3 This is a front-section axial side view of the present invention.

[0032] In the diagram: 1. Support base; 2. Extraction box; 3. Sealing cover; 4. Filter membrane module; 5. Discharge pipe; 6. Centrifugal filter cartridge; 7. Connecting strip; 8. Connecting rod; 9. Impeller; 10. Air guide pipe; 11. Support frame; 12. Motor; 13. Gear 1; 14. Gear 2; 15. Feed pipe; 16. Feed hopper; 17. Rubber cover; 18. Exosome discharge pipe; 19. Pressure relief valve; 20. Controller. Detailed Implementation

[0033] The technical solutions of the present utility model will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present utility model. Obviously, the described embodiments are only some embodiments of the present utility model, and not all embodiments.

[0034] like Figures 1-3 As shown, an extraction device for adipose tissue exosomes includes a support base 1. An extraction box 2 is fixed to the upper surface of the support base 1 by bolts or welding to ensure a stable center of gravity. Mechanical calculations show that the bottom area of ​​the support base is ≥0.15 m². 2 At that time, the shaking amplitude of the device under centrifugation at 3000 rpm was < ±0.5 mm.

[0035] The controller 20 is embedded in the edge of the support base 1 and is isolated from the support base by a waterproof sealing ring, with a protection level of IP54.

[0036] The discharge pipe 5 passes through the bottom of the extraction box 2 via a bearing, model 6204-2RS, ensuring smooth rotation and a friction coefficient of <0.01.

[0037] The centrifugal filter cartridge 6 is fixed to the top of the discharge pipe 5 by threaded connection or keyway. The filter cartridge wall has micropores with a diameter of 0.22μm, made of polyethersulfone, with a porosity of 75%.

[0038] Impeller 9 is welded to the inner top of discharge pipe 5 via connecting rod 8. The blade angle of impeller 9 is 45° to ensure maximum airflow drive efficiency.

[0039] Motor 12, model 57HS76-4004A, has a rated power of 400W and is fixed to one side of support frame 11 via a flange. Gear 13 is connected to the shaft of motor 12 by a flat key.

[0040] Gear 2 14 with module m=2 and number of teeth z=45 is heat-fitted onto the outside of discharge pipe 5, forming a 3:1 reduction ratio with gear 1 13 with module m=2 and number of teeth z=15, ensuring that the centrifugal filter cartridge speed reaches 3000-5000 rpm.

[0041] In this embodiment, the sample pretreatment involves digesting the adipose tissue with collagenase, centrifuging it at 300g for 10 minutes to remove cell debris, and collecting the supernatant.

[0042] Add PBS buffer (pH=7.4) to the supernatant and adjust the sample volume to 50-100mL.

[0043] Start the device and set the parameters: turn on controller 20, set the centrifugal speed to 3500 rpm, the negative pressure value to -0.06 MPa, and the running time to 30 minutes.

[0044] The pretreated sample is added through the feed hopper 16, and the rubber cover 17 automatically rebounds and seals after feeding, with a rebound time of <0.5 seconds.

[0045] During the centrifugal filtration and negative pressure expulsion process, motor 12 drives gear 13 to transmit gear 14, which in turn drives discharge pipe 5, causing centrifugal filter cartridge 6 to rotate and generate centrifugal force. Exosomes in the sample are discharged to the outside of the filter cartridge due to the density difference of 1.13-1.19 g / mL.

[0046] The discharge pipe 5 drives the connecting strip 7 and the connecting rod 8 to rotate. The connecting rod 8 drives the impeller 9 to rotate inside the air guide pipe 10, so that a vacuum negative pressure environment is formed inside the extraction box 2, so that the centrifuged sample can be evenly filtered through the filter membrane module 4 for secondary filtration.

[0047] The filter module 4 adopts a three-layer structure: the upper layer is a 10μm polypropylene fiber membrane to intercept cell debris, the middle layer is a 0.45μm mixed cellulose membrane to intercept microorganisms, and the lower layer is a 0.1μm polycarbonate membrane to enrich exosomes.

[0048] Exosomes were collected and enriched, then flowed into a collection bottle through an exosome discharge tube 18. The inner diameter of the connecting tube was 6 mm, and the flow rate was controlled at 5-8 mL / min.

[0049] Impurities are discharged through discharge pipe 5. A one-way solenoid valve, model ZCG-15, is installed at the outlet of the discharge pipe and is opened by controller 20 at regular intervals (opening for 5 seconds every 10 minutes).

[0050] The pressure protection mechanism relief valve, model 19 (A41H-16C), is set to open at -0.08MPa. When the negative pressure inside extraction tank 2 exceeds the threshold, the valve automatically opens to relieve pressure, with a response time of <0.1 seconds.

[0051] The controller 20 monitors the pressure sensor data (model MPX5100DP) in real time. If the pressure is abnormal, such as a leak causing a pressure rise, it will automatically shut down and trigger an alarm.

[0052] Loosen the buckle of the sealing cover 3 using a quick-release buckle, model: S18-22, and remove the filter membrane module 4.

[0053] After replacing the filter membrane, the inside of the extraction chamber 2 is disinfected by irradiating it with a UV lamp at a wavelength of 254nm and a power of 15W for 15 minutes.

[0054] Extraction box 2 and sealing cover 3 are made of 316L stainless steel, with surface electrolytic polishing treatment, roughness Ra≤0.4μm, corrosion resistant and easy to clean.

[0055] Centrifuge filter cartridge 6 is made of polyetheretherketone (PEEK) material with a density of 1.3 g / cm³. 3 It has high strength and good biocompatibility, and complies with ISO10993-5.

[0056] A silicone rubber O-ring with a Shore hardness of 70±5 is used between the sealing cover plate 3 and the extraction box 2 to ensure airtightness and a leakage rate of <0.01mL / min.

[0057] The feed pipe 15, air guide pipe 10 and other interfaces are sealed with clamps (model: DIN32676), which are easy to disassemble and provide a reliable seal.

[0058] One-way solenoid valves are installed on the outside of the discharge pipe 5, the air guide pipe 10, the feed pipe 15, and the exosome discharge pipe 18.

[0059] The above description is only a preferred embodiment of the present utility model, but the protection scope of the present utility model is not limited thereto. Any equivalent substitutions or changes made by those skilled in the art within the technical scope disclosed in the present utility model, based on the technical solution and the inventive concept of the present utility model, should be included within the protection scope of the present utility model.

Claims

1. A device for extracting adipose tissue exosomes, comprising a support seat (1), the upper side edge position of which is provided with a controller (20), characterized in that, An extraction box (2) is provided on the upper side of the support base (1), a sealing cover plate (3) is provided on the upper side of the extraction box (2), a filter membrane module (4) is provided on the inner side of the extraction box (2), and a negative pressure centrifugal filtration assembly is provided inside the extraction box (2). The negative pressure centrifugal filtration assembly is used to centrifuge and filter the filtrate and to discharge it under negative pressure.

2. The apparatus for extracting exosomes from adipose tissue according to claim 1, characterized in that, The negative pressure centrifugal filtration assembly includes a discharge pipe (5), which is installed through the bottom of the extraction box (2). A centrifugal filter cylinder (6) is installed at the top of the discharge pipe (5). A connecting rod (8) is installed at the top of the inner side of the discharge pipe (5). An impeller (9) is installed at the top of the connecting rod (8). A gas guide pipe (10) is connected through one side of the sealing cover plate (3). One end of the impeller (9) is placed inside the gas guide pipe (10).

3. The apparatus for extracting exosomes from adipose tissue according to claim 2, characterized in that, A support frame (11) is provided on the lower side of the support base (1), and a motor (12) is provided on one side of the support frame (11). A gear one (13) is provided on the shaft end of the motor (12), and a gear two (14) is meshed on the outer side of the gear one (13). The gear two (14) is fixedly sleeved on the outer side of the discharge pipe (5).

4. The apparatus for extracting exosomes from adipose tissue according to claim 1, characterized in that, The upper side of the sealing cover plate (3) is provided with a feed pipe (15), the top of the feed pipe (15) is provided with a feed hopper (16), and the upper side of the feed hopper (16) is provided with a rubber cover plate (17).

5. The apparatus for extracting exosomes from adipose tissue according to claim 1, characterized in that, An exosome discharge tube (18) is provided on the lower side of the extraction box (2).

6. The apparatus for extracting exosomes from adipose tissue according to claim 1, characterized in that, A pressure relief valve (19) is provided on the upper side of the sealing cover plate (3).

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