A device for preparing stem cell-derived exosomes

Abstract

The utility model relates to the technical field of exosome preparation, in particular to a kind of preparation device of stem cell-derived exosome;It can realize the continuous operation of precipitation, pipetting and centrifugal separation, reduce manual operation amount, avoid supernatant waste, improve exosome preparation efficiency;Including sedimentation separation integrated device, sedimentation separation integrated device includes base, fixedly installed on the drive motor of base, centrifugal carousel fixedly installed on the output end of drive motor, sedimentation dish in the middle part of centrifugal carousel, multiple centrifugal holding mechanisms and synchronous tilt drive mechanism, multiple centrifugal holding mechanisms are evenly distributed at the periphery of centrifugal carousel, centrifugal holding mechanism includes ring frame, test tube and clamp installed on ring frame, test tube is fixedly clamped on ring frame by clamp, two sides of ring frame are fixedly installed with pivot, centrifugal carousel is equipped with multiple openings arranged corresponding with centrifugal holding mechanism, the periphery of sedimentation dish is equipped with multiple titration valves communicated with the upper portion of sedimentation dish.

Description

Technical Field

[0001] This utility model relates to the technical field of exosome preparation, and in particular to a device for preparing stem cell-derived exosomes. Background Technology

[0002] Stem cell-derived exosomes have great potential applications in drug delivery, tissue repair, and anti-aging, and are a hot topic in current biomedical research. Existing methods for preparing stem cell exosomes include mechanical extrusion, polyethylene glycol (PEG) precipitation, and serum-free culture medium optimization. Among them, the PEG precipitation method involves culturing umbilical cord mesenchymal stem cells in a serum-free environment, collecting the supernatant, and then adding 16% and 10% PEG6000 solutions in stages for centrifugation purification to obtain artificial exosomes.

[0003] However, the supernatant after precipitation needs to be manually transferred to the centrifugation equipment using pipettes and other auxiliary equipment. This manual operation is labor-intensive, and some supernatant will drip during the transfer process, resulting in waste of supernatant and affecting the preparation efficiency of exosomes. Utility Model Content

[0004] To address the shortcomings of existing technologies, this invention provides a stem cell-derived exosome preparation device that enables continuous operation of precipitation, pipetting, and centrifugation, reduces manual labor, avoids waste of supernatant, and improves the efficiency of exosome preparation.

[0005] To achieve the above objectives, this utility model provides the following technical solution: a device for preparing stem cell-derived exosomes, comprising a precipitation-separation integrated unit, the precipitation-separation integrated unit comprising a base, a drive motor fixedly mounted on the base, a centrifugal disc fixedly mounted on the output end of the drive motor, a precipitation dish located in the center of the centrifugal disc, multiple centrifugal holding mechanisms, and a synchronous tilting drive mechanism. The multiple centrifugal holding mechanisms are evenly distributed around the centrifugal disc. Each centrifugal holding mechanism includes a ring frame, test tubes, and clamps mounted on the ring frame. The test tubes are fixedly clamped to the ring frame by the clamps. Rotating shafts are fixedly mounted on both sides of the ring frame. The centrifugal disc has multiple openings corresponding to the centrifugal holding mechanisms. Bearing seats are fixedly mounted on both sides of the upper opening of the centrifugal disc. Two rotating shafts are rotatably mounted on corresponding bearing seats. Multiple titration valves communicating with the upper part of the precipitation dish are provided around the precipitation dish. The titration valves are connected to the corresponding... The centrifugal holding mechanism is located on the same radial line as the centrifugal turntable. The synchronous tilting drive mechanism includes a drive ring and multiple lifting drive components mounted on the base. The drive ring is fixedly installed at the output end of the lifting drive components, and the outer circle of the drive ring has an inclined slope. Furthermore, the opening is arranged radially along the centrifugal turntable, and the central axis of the rotating shaft is perpendicular to the radial line passing through the center of the opening on the centrifugal turntable. The bottom of the titration valve is higher than the top of the corresponding test tube. The titration valve is located on the side closer to the center of the centrifugal turntable, and the corresponding test tube is located on the side farther from the center of the centrifugal turntable, that is, the titration valve is located directly above the corresponding test tube on the side closer to the center of the centrifugal turntable. The outer diameter of the inclined slope of the drive ring gradually narrows from the bottom to the top. The lifting drive component is used to drive the drive ring to move up and down. The lifting drive component is preferably an electric cylinder, but other drive components with equivalent lifting drive effects, such as a mechanical slide, can also be used. The supernatant in the sedimentation dish is above the titration valve.

[0006] Preferably, the clamp includes two arc-shaped clamping plates and two tension springs. At least two sets of sliding rods are fixedly installed on each of the two arc-shaped clamping plates. The sliding rods are slidably installed on the ring frame, and the two arc-shaped clamping plates are elastically connected by the two tension springs. Further, after the two arc-shaped clamping plates are closed, a ring is formed in the middle that fits with the outer wall of the test tube. The two tension springs are symmetrically installed on both sides of the arc-shaped clamping plates, and the two ends of the two tension springs are fixedly connected to the two arc-shaped clamping plates respectively. The ring frame is provided with sliding holes that allow the sliding rods to pass through.

[0007] Preferably, the two arc-shaped clamps form a ring, and the outer diameter of the ring is 2-6 mm smaller than the inner diameter of the ring frame.

[0008] Preferably, it also includes a spring, with an adjusting nut screwed onto the end of the slide rod, the spring being sleeved on the slide rod, one end of the spring abutting against the outer wall of the ring frame, and the other end of the spring abutting against the adjusting nut.

[0009] Preferably, the arc-shaped clamps are provided with chamfered portions near the center of the ring frame.

[0010] Preferably, the centrifugal turntable has an annular groove in the middle that fits with the outer wall of the sedimentation dish, and the sedimentation dish is placed in the annular groove; an anti-slip pad can be attached to the top of the centrifugal turntable where it contacts the sedimentation dish to improve the friction between the centrifugal turntable and the sedimentation dish.

[0011] Preferably, it also includes an outer shell, the outer shell having an installation cavity, and the precipitation separator is installed in the installation cavity; a gap is left between the inner wall of the installation cavity and the outer wall of the test tube to allow the test tube to move freely.

[0012] Preferably, the drive motor is a speed-regulating motor; furthermore, the speed-regulating motor adopts existing technology on the market, such as a frequency conversion speed-regulating motor, a pole-changing speed-regulating motor, a voltage-regulating speed-regulating motor, etc., which will not be elaborated further here.

[0013] Compared with existing technologies, this invention provides a device for preparing stem cell-derived exosomes, which has the following beneficial effects: In this device, serum-free cultured umbilical cord mesenchymal stem cell solution is placed into a sedimentation dish. A drive motor rotates a centrifuge disc at low speed, causing the liquid in the sedimentation dish to precipitate and separate into layers under centrifugal force. The drive motor then stops, and the centrifuge disc remains stationary. After the supernatant in the sedimentation dish meets the requirements, the lifting drive is activated, causing the drive ring to move upwards. The inclined slope contacts the bottom of each test tube and pushes the bottom of each test tube away from the center of the centrifuge disc. The rotating shaft rotates around the bearing seat, moving the test tube opening towards the center of the centrifuge disc until the test tube opening is below the titration valve. The titration valve is then opened, and the supernatant is dripped into the corresponding test tube. The titration valve is closed, and 16% and 10% solutions are added to the test tubes in steps. After resetting the ring holder, the drive motor is restarted with PEG6000 solution. The centrifuge disc drives each test tube to centrifuge. After centrifugation and purification, the exosome solution is obtained. This achieves continuous operation of precipitation, pipetting and centrifugation, reducing manual operation, avoiding waste of supernatant and improving the efficiency of exosome preparation. Attached Figure Description

[0014] Figure 1 This is a three-dimensional structural schematic diagram of the present invention;

[0015] Figure 2 This is a three-dimensional structural diagram of the integrated sedimentation and separation device of this utility model;

[0016] Figure 3 This is a top view schematic diagram of the integrated sedimentation and separation device of this utility model;

[0017] Figure 4 This is the utility model Figure 2 Schematic diagram of the cross-sectional structure at point AA;

[0018] Figure 5 This is the utility model Figure 2 A magnified schematic diagram of the structure at point B in the middle;

[0019] The following are labels in the attached diagram: 1. Integrated sedimentation and separation unit; 2. Base; 3. Drive motor; 4. Centrifugal turntable; 5. Sedimentation dish; 6. Ring frame; 7. Test tube; 8. Rotating shaft; 9. Opening; 10. Bearing seat; 11. Titration valve; 12. Drive ring; 13. Lifting drive component; 14. Inclined slope; 15. Arc-shaped clamp; 16. Tension spring; 17. Slide rod; 18. Spring; 19. Adjusting nut; 20. Chamfer; 21. Ring groove; 22. Outer shell. Detailed Implementation

[0020] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0021] It should be noted that, unless otherwise specified, the embodiments and features and technical solutions in the present invention can be combined with each other.

[0022] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0023] Example 1

[0024] Please refer to Figure 2-5A device for preparing stem cell-derived exosomes includes: a precipitation and separation unit 1, which includes a base 2, a drive motor 3 fixedly mounted on the base 2, a centrifugal disc 4 fixedly mounted on the output end of the drive motor 3, a precipitation dish 5 located in the middle of the centrifugal disc 4, multiple centrifugal holding mechanisms, and a synchronous tilting drive mechanism. The multiple centrifugal holding mechanisms are evenly distributed around the centrifugal disc 4. Each centrifugal holding mechanism includes a ring frame 6, test tubes 7, and clamps mounted on the ring frame 6. The test tubes 7 are fixedly clamped to the ring frame 6 by the clamps. Rotating shafts 8 are fixedly mounted on both sides of the ring frame 6. The centrifugal disc 4 has multiple openings 9 corresponding to the centrifugal holding mechanisms. Bearing seats 10 are fixedly installed on both sides of the upper opening 9 of the centrifugal turntable 4. Two rotating shafts 8 are rotatably installed on the corresponding two bearing seats 10. Multiple titration valves 11 connected to the upper part of the sedimentation dish 5 are provided around the sedimentation dish 5. The titration valves 11 and the corresponding centrifugal holding mechanism are on the same radial line of the centrifugal turntable 4. The synchronous tilting drive mechanism includes a drive ring 12 and multiple lifting drive components 13 installed on the base 2. The drive ring 12 is fixedly installed at the output end of the lifting drive component 13. The outer circle of the drive ring 12 is provided with an inclined slope 14. Furthermore, the opening 9 is arranged radially along the centrifugal turntable 4, and the central axis of the rotating shaft 8 is perpendicular to the radial line on the centrifugal turntable 4 that passes through the center of the corresponding opening 9. The bottom of the titration valve 11 is higher than the top of the corresponding test tube 7. The titration valve 11 is closer to the center of the centrifuge turntable 4, while the corresponding test tube 7 is further away from the center of the centrifuge turntable 4. That is, the titration valve 11 is located directly above the corresponding test tube 7, close to the center of the centrifuge turntable 4. The outer diameter of the inclined slope 14 of the drive ring 12 gradually narrows from the bottom to the top. When the drive ring 12 is at its lowest position, it is no longer in contact with the test tube 7. The lifting drive 13 is preferably an electric cylinder, but other drive components with the same lifting drive effect, such as a mechanical slide, can also be used. The supernatant in the sedimentation dish 5 is above the titration valve 11. It should be noted that when the lifting drive 13 moves the drive ring 13 to its limit height... At this time, the opening of test tube 7 is exactly below the outlet of the corresponding titration valve 11 to prevent the liquid from dripping outwards and causing waste when it is added to test tube 7; the tilt angle of test tube 7 should be between 30° and 45°; when test tube 7 is separated from contact with drive ring 13 (the liquid level in test tube 7 is lower than the middle of test tube 7, and the center of gravity of test tube 7 with solution is close to the lower part of test tube 7), test tube 7 can automatically reset under the action of gravity; titration valve 11 can be a manual valve or an electric valve, and titration valve 11 is preferably an electric valve with flow rate and flow control effect to control the dripping speed and flow rate of solution; the central axis of centrifugal turntable 4 is collinear with the central axis of the output shaft of drive motor 3.

[0025] For details, please refer to Figure 2-3The centrifugal turntable 4 has an annular groove 21 in the middle that fits with the outer wall of the sedimentation dish 5, and the sedimentation dish 5 is placed in the annular groove 21. An anti-slip pad can be attached to the top of the centrifugal turntable 4 where it contacts the sedimentation dish 5 to improve the friction between the centrifugal turntable 4 and the sedimentation dish 5.

[0026] For details, please refer to Figure 1 It also includes an outer shell 22, which has an installation cavity inside, and the sedimentation separator 1 is installed in the installation cavity; a gap is left between the inner wall of the installation cavity and the outer wall of the test tube 7 to allow the test tube 7 to move freely, so as to avoid interference with the movement of the test tube 7.

[0027] The stem cell-derived exosome preparation apparatus provided in this embodiment uses an open-top precipitation dish 5. The highest liquid level in the precipitation dish 5 should be at least 5 cm below the top of the precipitation dish 5 to prevent the solution from overflowing during centrifugation. In another embodiment, to prevent overflow, a lid can be added to the top of the precipitation dish 5. To prevent liquid from splashing out of the test tube 7, the liquid in the test tube 7 should not exceed 1 / 2 of the volume of the test tube 7. A cap can also be added to the top of the test tube 7. Centrifugation can accelerate the separation efficiency of the solution, thereby further improving the preparation efficiency of stem cell-derived exosomes. The precipitation dish 5 can be movably placed on the centrifuge turntable 4 for easy addition and cleaning of the solution. The annular groove 21 can limit the precipitation dish 5 to prevent it from slipping off the centrifuge turntable 4. The outer shell 22 is equipped with a necessary control panel. The outer shell 22 can prevent the operator from contacting the rotating centrifuge turntable 4, greatly reducing the rate of mechanical injury accidents and improving operational safety.

[0028] Example 2

[0029] The apparatus for preparing stem cell-derived exosomes provided in Example 1 has been further optimized. For details, please refer to [link / reference needed]. Figure 5 The clamp includes two arc-shaped clamping plates 15 and two tension springs 16. At least two sets of sliding rods 17 are fixedly installed on each of the two arc-shaped clamping plates 15. The sliding rods 17 are slidably installed on the ring frame 6. The two arc-shaped clamping plates 15 are elastically connected by the two tension springs 16. Furthermore, after the two arc-shaped clamping plates 15 are closed, the middle part forms an annulus that fits with the outer wall of the test tube 7. The two tension springs 16 are symmetrically installed on both sides of the arc-shaped clamping plates 15, and the two ends of the two tension springs 16 are fixedly connected to the two arc-shaped clamping plates 15 respectively. The ring frame 6 is provided with sliding holes that allow the sliding rods 17 to pass through.

[0030] For details, please refer to Figure 5 Two arc-shaped clamps 15 form a ring, the outer diameter of which is 2-6 mm smaller than the inner diameter of the ring frame 6. This distance allows the test tube 7 to perform low-amplitude, high-frequency vibration, thus preventing the test tube 7 from vibrating too much.

[0031] For details, please refer to Figure 5It also includes a spring 18, an adjusting nut 19 screwed onto the end of the slide rod 17, the spring 18 is sleeved on the slide rod 17, one end of the spring 18 abuts against the outer wall of the ring frame 6, and the other end of the spring 18 abuts against the adjusting nut 19.

[0032] Specifically, drive motor 3 is a speed-regulating motor; furthermore, the speed-regulating motor adopts existing technologies on the market, such as frequency conversion speed-regulating motor, pole conversion speed-regulating motor, voltage regulation speed-regulating motor, etc., which will not be elaborated further here.

[0033] For details, please refer to Figure 5 The curved clamping plate 15 is provided with a chamfered part 20 near the center of the ring frame 6.

[0034] The stem cell-derived exosome preparation device provided in this embodiment, under the action of the tension spring 16, allows the test tube 7 to be clamped by two arc-shaped clamps 15, while the chamfered part 20 facilitates the insertion of the test tube 7 between the two arc-shaped clamps 15, improving the smoothness of the placement of the test tube 7 into the clamp; the distance between the annular body formed by the two arc-shaped clamps 15 and the inner wall of the ring frame 6 allows the annular body to move within the ring frame 6. During the rotation of the output end of the drive motor 3, the edge of the centrifugal turntable 4 vibrates due to friction and other reasons. During the rotation of the centrifugal turntable 4, low-amplitude vibration will occur due to factors such as unbalanced force (unequal liquid volume in the test tube 7), under the action of the spring 18. The vibration effect is enhanced, thereby further improving the vibration effect at test tube 7. This causes test tube 7 in ring rack 6 to vibrate at high frequency while following the centrifugal turntable 4, thus further improving the separation effect of exosomes. By tightening the adjusting nut 19, the compression of spring 18 can be adjusted, and the vibration amplitude of test tube 7 can be adaptively adjusted to meet the needs of different working conditions. The drive motor 3 is a speed-regulating motor to adjust the rotation speed of centrifugal turntable 4, thereby adjusting the centrifugal force at test tube 7. The vibration effect is generated when test tube 7 is subjected to dynamically changing centrifugal force, thereby further improving the centrifugal separation effect of exosomes.

[0035] The process of using the stem cell-derived exosome preparation device provided by this utility model is as follows: Serum-free cultured umbilical cord mesenchymal stem cell solution is placed into a sedimentation dish 5. The drive motor 3 drives the centrifuge disc 4 to rotate at low speed. Under centrifugal force, the liquid in the sedimentation dish 5 precipitates and separates into layers. Then, the drive motor 3 stops, and the centrifuge disc 4 remains stationary. After the supernatant in the sedimentation dish 5 meets the requirements, the lifting drive component 13 is activated, the drive ring 12 moves upward, and the inclined slope 14 contacts the bottom of each test tube 7, pushing the bottom of each test tube 7 away from the center of the centrifuge disc 4. The rotating shaft 8 rotates around the bearing seat 10, and the opening of the test tube 7 moves towards the center of the centrifuge disc 4 until the opening of the test tube 7 is below the titration valve 11. The titration valve 11 is opened, and the supernatant is dripped into the corresponding test tube 7. The titration valve 11 is closed, and 16% and 10% solutions are added to the test tube 7 in steps. After the PEG6000 solution and the ring holder 6 are reset, the drive motor 3 is turned on again. The centrifuge turntable 4 drives each test tube 7 to perform high-speed centrifugation and vibration. After centrifugation and purification, the exosome solution is obtained.

[0036] In this utility model, unless otherwise explicitly specified and limited, the terms "installation," "connection," "joining," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection, an electrical connection, or a connection that allows communication between them; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components, unless otherwise explicitly limited. Those skilled in the art can understand the specific meaning of the above terms in this utility model according to the specific circumstances.

Claims

1. An apparatus for preparing stem cell-derived exosomes, characterized in that, The system includes a sedimentation separator (1), which comprises a base (2), a drive motor (3) fixedly mounted on the base (2), a centrifugal disc (4) fixedly mounted on the output end of the drive motor (3), a sedimentation dish (5) located in the middle of the centrifugal disc (4), multiple centrifugal holding mechanisms, and a synchronous tilting drive mechanism. The multiple centrifugal holding mechanisms are evenly distributed around the centrifugal disc (4). Each centrifugal holding mechanism includes a ring frame (6), test tubes (7), and clamps mounted on the ring frame (6). The test tubes (7) are fixedly clamped to the ring frame (6) by the clamps. Rotating shafts (8) are fixedly mounted on both sides of the ring frame (6). The centrifugal disc (4) is provided with multiple centrifugal holding mechanisms. The opening (9) of the centrifugal container is arranged in a corresponding manner. Bearing seats (10) are fixedly installed on both sides of the upper opening (9) of the centrifugal turntable (4). The two rotating shafts (8) are respectively rotatably installed on the corresponding two bearing seats (10). Multiple titration valves (11) connected to the upper part of the sedimentation dish (5) are provided around the sedimentation dish (5). The titration valves (11) and the corresponding centrifugal container are on the same radial line of the centrifugal turntable (4). The synchronous tilting drive mechanism includes a drive ring (12) and multiple lifting drive components (13) installed on the base (2). The drive ring (12) is fixedly installed at the output end of the lifting drive component (13). The outer circle of the drive ring (12) is provided with an inclined slope (14).

2. The apparatus for preparing stem cell-derived exosomes according to claim 1, characterized in that, The clamp includes two arc-shaped clamps (15) and two tension springs (16). At least two sets of slide rods (17) are fixedly installed on each of the two arc-shaped clamps (15). The slide rods (17) are slidably installed on the ring frame (6). The two arc-shaped clamps (15) are elastically connected by two tension springs (16).

3. The apparatus for preparing stem cell-derived exosomes according to claim 2, characterized in that, The two arc-shaped clamps (15) form a ring body, and the outer diameter of the ring body is 2-6 mm smaller than the inner diameter of the ring frame (6).

4. The apparatus for preparing stem cell-derived exosomes according to claim 3, characterized in that, It also includes a spring (18), and an adjusting nut (19) is screwed onto the end of the slide rod (17). The spring (18) is sleeved on the slide rod (17), one end of the spring (18) abuts against the outer wall of the ring frame (6), and the other end of the spring (18) abuts against the adjusting nut (19).

5. The apparatus for preparing stem cell-derived exosomes according to claim 2, characterized in that, The arc-shaped clamp (15) is provided with a chamfer (20) near the center of the ring frame (6).

6. The apparatus for preparing stem cell-derived exosomes according to claim 1, characterized in that, The centrifugal turntable (4) has an annular groove (21) in the middle that fits the outer wall of the sedimentation dish (5), and the sedimentation dish (5) is placed in the annular groove (21).

7. The apparatus for preparing stem cell-derived exosomes according to claim 1, characterized in that, It also includes an outer shell (22), which has an installation cavity inside, and the precipitation separator (1) is installed in the installation cavity; a gap is left between the inner wall of the installation cavity and the outer wall of the test tube (7) to allow the test tube (7) to move freely.

8. The apparatus for preparing stem cell-derived exosomes according to claim 4, characterized in that, The drive motor (3) is a speed-regulating motor.

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