A primary isolation device for screening extracellular vesicles in stem cell supernatant

Abstract

The utility model relates to the technical field of extracellular vesicle extraction, and discloses a primary separation device for screening extracellular vesicles in stem cell supernatant, which comprises a workbench, a filtering and concentrating mechanism, a gentle filtering mechanism and a centrifugal mechanism. The filtering and concentrating mechanism is arranged on the top of the workbench and is used for rough filtering the supernatant. The gentle filtering mechanism is arranged on the top of the workbench and is used for constant low-pressure filtering of the medium treated by the filtering and concentrating mechanism. The utility model can rough filter the supernatant treated by centrifugation, remove macromolecular impurities in the supernatant, concentrate extracellular vesicles, and then through the overall design of the gentle filtering mechanism, gentle filtering can be carried out under constant low pressure to form preliminarily enriched extracellular vesicle liquid, reduce vesicle rupture, and ensure the accuracy of subsequent functional research results.

Description

Technical Field

[0001] This utility model relates to the field of extracellular vesicle extraction technology, specifically to a primary separation device for screening extracellular vesicles in stem cell supernatant. Background Technology

[0002] Stem cell supernatant is the liquid component secreted into the culture medium by stem cells during in vitro culture. It contains active substances such as extracellular vesicles, growth factors, cytokines, and metabolites. These components have potential therapeutic effects such as tissue repair and immune regulation, and are of great value in regenerative medicine and disease treatment research.

[0003] Extracellular vesicles (EVs) in stem cell supernatants are nanoscale vesicles secreted by cells, carrying bioactive molecules such as proteins and nucleic acids. They have functions such as mediating intercellular communication, promoting tissue repair, and regulating immunity. The purpose of screening EVs is to remove impurities and obtain high-purity samples for medical applications such as disease diagnostic biomarker research, targeted drug delivery system development, or anti-aging therapy. Standardized extraction technology is one of the key challenges in current research.

[0004] When using traditional separation devices to screen extracellular vesicles, the probability of vesicle rupture is relatively high. Rupture releases key molecules such as proteins and nucleic acids carried within the vesicle, which may distort the results of subsequent functional studies. Utility Model Content

[0005] The purpose of this invention is to provide a preliminary separation device for screening extracellular vesicles in stem cell supernatant, thereby solving the problem that the probability of vesicle rupture is relatively high during the extraction of extracellular vesicles in the prior art.

[0006] This utility model provides the following technical solution: a primary separation device for screening extracellular vesicles in stem cell supernatant, comprising:

[0007] Workbench;

[0008] A filtration and concentration mechanism is installed on the top of the workbench and is used to coarsely filter the supernatant.

[0009] A mild filtration mechanism is located on top of the workbench and is used to perform constant low-pressure filtration on the medium processed by the filtration and concentration mechanism.

[0010] The mild filtration mechanism includes a limiting foot and a vacuum pump. The limiting foot is fixedly installed on the top of the workbench. A collection cup is movably inserted into the inner side of the limiting foot. A movable cover is movably inserted into the top of the collection cup. The vacuum pump is fixedly installed on the top of the workbench. The working end of the vacuum pump is fixedly connected to the top of the movable cover.

[0011] As a preferred embodiment of the above technical solution, a filter cup is movably inserted into the top of the movable cover, a porous plate is fixedly installed on the inner wall of the filter cup, and a microporous membrane is movably connected to the top of the porous plate.

[0012] As a preferred embodiment of the above technical solution, the mild filtration mechanism further includes a support foot, which is fixedly installed on the top of the workbench. A connecting seat one is fixedly installed on the top of the support foot, a limiting rod is fixedly installed on the top of the connecting seat one, and a connecting seat two is fixedly installed on the top of the limiting rod. A lead screw is rotatably connected between adjacent sides of the connecting seat one and the connecting seat two. A rotating handle is rotatably connected to the top of the connecting seat two, and the bottom of the rotating handle is fixedly connected to the top of the lead screw. A lifting seat is slidably connected to the outer wall of the limiting rod and the lead screw, and the lifting seat is fixedly installed on the outer wall of the movable cover.

[0013] As a preferred embodiment of the above technical solution, the filtration and concentration mechanism includes a column, which is fixedly installed on the top of the workbench. A rotating sleeve is rotatably connected to the outer wall of the column, and a positioning bolt is threadedly connected to the outer wall of the rotating sleeve. The threaded end of the positioning bolt movably abuts against the outer wall of the column. A connecting frame is fixedly installed on the top of the rotating sleeve, and a concentration cylinder is fixedly installed on the inner wall of the connecting frame.

[0014] As a preferred embodiment of the above technical solution, a sealing slide plate is detachably connected to the inner wall of the concentration cylinder, and a counterweight is fixedly installed on the top of the sealing slide plate.

[0015] As a preferred embodiment of the above technical solution, a concentration filter element is detachably connected to the bottom of the concentration cylinder.

[0016] Compared with the prior art, the beneficial effects of this utility model are:

[0017] This invention, through the overall design of the filtration and concentration mechanism, can perform coarse filtration on the supernatant after centrifugation to remove large molecular impurities from the supernatant, while concentrating extracellular vesicles. Then, through the overall design of the gentle filtration mechanism, it can perform gentle filtration under constant low pressure to form a preliminarily enriched extracellular vesicle fluid, while reducing vesicle rupture and ensuring the accuracy of subsequent functional research results. Attached Figure Description

[0018] Figure 1 This is a perspective view of the present utility model;

[0019] Figure 2 This is a schematic diagram of the filtration and concentration mechanism of this utility model;

[0020] Figure 3 This is a schematic diagram of the bottom structure of the concentration cylinder of this utility model;

[0021] Figure 4 This is a schematic diagram of the structure of the mild filtration mechanism of this utility model;

[0022] Figure 5 This is a schematic diagram of the structure of the collecting cup and the movable lid of this utility model;

[0023] Figure 6 This is a cross-sectional structural diagram of the filter cup of this utility model.

[0024] In the diagram: 1. Workbench; 2. Filtration and concentration mechanism; 21. Column; 22. Rotating sleeve; 23. Positioning bolt; 24. Connecting frame; 25. Concentration cylinder; 251. Concentration filter element; 26. Sealing slide plate; 27. Counterweight; 3. Mild filtration mechanism; 31. Limiting foot; 32. Collection cup; 33. Movable cover; 34. Vacuum pump; 35. Filter cup; 351. Perforated plate; 352. Microporous membrane; 36. Support foot; 361. Connecting seat one; 362. Limiting rod; 363. Connecting seat two; 364. Lead screw; 365. Rotary handle; 366. Lifting seat. Detailed Implementation

[0025] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention.

[0026] like Figures 1-6 As shown, this utility model provides a technical solution: a primary separation device for screening extracellular vesicles in stem cell supernatant, comprising:

[0027] Workbench 1;

[0028] The filtration and concentration mechanism 2 is located on the top of the workbench 1 and is used to coarsely filter the supernatant.

[0029] The mild filtration mechanism 3 is located on the top of the workbench 1 and is used to perform constant low-pressure filtration on the medium processed by the filtration and concentration mechanism 2.

[0030] The mild filtration mechanism 3 includes a limiting foot 31 and a vacuum pump 34. The limiting foot 31 is fixedly installed on the top of the workbench 1. A collection cup 32 is movably inserted into the inner side of the limiting foot 31, and a movable cover 33 is movably inserted into the top of the collection cup 32. The vacuum pump 34 is fixedly installed on the top of the workbench 1, and the working end of the vacuum pump 34 is fixedly connected to the top of the movable cover 33. The working end of the vacuum pump 34 communicates with the cavity formed by the collection cup 32 and the movable cover 33. The vacuum pump 34 is a vacuum pump with a flow control knob, and the working pressure is adjusted to 0.05 MPa. This design ensures mild filtration under constant low pressure, reducing vesicle rupture. It is worth noting that the vacuum pump 34 in this solution is a device that can be purchased commercially by those skilled in the art. The device has not been structurally modified in this paper. Therefore, those skilled in the art are familiar with its working principle based on their professional knowledge and can apply it skillfully. Therefore, this paper will not elaborate on it further.

[0031] As one implementation method in this embodiment, such as Figure 6 As shown, a filter cup 35 is movably inserted into the top of the movable cover 33. A porous plate 351 is fixedly installed on the inner wall of the filter cup 35. A microporous membrane 352 is movably connected to the top of the porous plate 351. The microporous membrane 352 has a size of 0.15μm. In use, the filter cup 35 is inserted into the top of the movable cover 33. The medium processed by the filtration and concentration mechanism 2 flows into the inner cavity of the filter cup 35. At this time, it is filtered by the microporous membrane 352 under constant low pressure to form a preliminarily enriched extracellular vesicle fluid, which is stored in the collection cup 32.

[0032] As one implementation method in this embodiment, such as Figure 4 As shown, the mild filtration mechanism 3 also includes a support leg 36, which is fixedly installed on the top of the workbench 1. A connecting seat 361 is fixedly installed on the top of the support leg 36. A limit rod 362 is fixedly installed on the top of the connecting seat 361. A connecting seat 363 is fixedly installed on the top of the limit rod 362. A lead screw 364 is rotatably connected between adjacent sides of the connecting seat 361 and the connecting seat 363. A handle 365 is rotatably connected to the top of the connecting seat 363. The bottom of the handle 365 is connected to the lead screw 364. The top of the rod 364 is fixedly connected, and the lifting seat 366 is slidably connected to the outer wall of the limiting rod 362 and the lead screw 364. The lifting seat 366 is fixedly installed on the outer wall of the movable cover 33. After the collecting cup 32 is inserted into the inner side of the limiting foot 31, the manual rotation of the handle 365 can drive the lead screw 364 to rotate, causing the lifting seat 366 to slide downward on the outer wall of the limiting rod 362, and simultaneously driving the movable cover 33 to be inserted into the top of the collecting cup 32, while fixing the collecting cup 32 and the movable cover 33 as a whole.

[0033] As one implementation method in this embodiment, such as Figure 2As shown, the filtration and concentration mechanism 2 includes a column 21, which is fixedly installed on the top of the workbench 1. A rotating sleeve 22 is rotatably connected to the outer wall of the column 21. A positioning bolt 23 is threadedly connected to the outer wall of the rotating sleeve 22. The threaded end of the positioning bolt 23 is in movable contact with the outer wall of the column 21. A connecting frame 24 is fixedly installed on the top of the rotating sleeve 22. A concentration cylinder 25 is fixedly installed on the inner wall of the connecting frame 24. In the initial state, the positioning bolt 23 is in a tight state. Loosening the positioning bolt 23 allows the concentration cylinder 25 to be rotated as a whole, removing the concentration cylinder 25 from above the mild filtration mechanism 3, thus avoiding any restriction on the concentration cylinder 25 when operating the mild filtration mechanism 3.

[0034] As one implementation method in this embodiment, such as Figure 2 As shown, a sealing slide plate 26 is detachably connected to the inner wall of the concentration cylinder 25. A counterweight block 27 is fixedly installed on the top of the sealing slide plate 26. After the supernatant is added into the inner cavity of the concentration cylinder 25, the sealing slide plate 26 can be inserted from the top of the concentration cylinder 25. Through the combined gravity of the sealing slide plate 26 and the counterweight block 27, pressure can be applied to the supernatant, thereby increasing the speed of coarse filtration of the supernatant.

[0035] As one implementation method in this embodiment, such as Figure 3 As shown, a concentration filter element 251 is detachably connected to the bottom of the concentration cylinder 25. The concentration filter element 251 is used to coarsely filter the supernatant inside the concentration cylinder 25. The concentration filter element 251 is an Amicon filter element.

[0036] Working principle: In use, the collection cup 32 is inserted into the inner side of the limiting foot 31. Manually rotating the handle 365 drives the lead screw 364 to rotate, causing the lifting seat 366 to slide downward on the outer wall of the limiting rod 362. Simultaneously, the movable cover 33 is inserted into the top of the collection cup 32, thus fixing the collection cup 32 and the movable cover 33 together. Then, the filter cup 35 is inserted into the top of the movable cover 33. The stem cell culture supernatant is poured into the centrifuge tube, and the sample is centrifuged at high speed to promote the sedimentation of large particles and clear stratification. The supernatant is then added to the inner cavity of the concentration cylinder 25. The sealing slide plate 26 is inserted from the top of the concentration cylinder 25. At this time, the concentration filter element 251 performs coarse filtration on the supernatant inside the concentration cylinder 25. The coarsely filtered medium enters the filter cup 35. Then, the vacuum pump 34 is controlled to work, and the working pressure is adjusted to 0.05. The extracellular vesicle fluid is filtered through a microporous membrane 352 at a constant low pressure (MPa) to form a preliminarily enriched extracellular vesicle fluid, which is then stored in a collection cup 32, thus achieving the function of extracting extracellular vesicles.

[0037] The above embodiments are only used to illustrate the technical solution of this utility model, and are not intended to limit it.

Claims

1. A primary separation device for screening extracellular vesicles in stem cell supernatant, characterized in that, include: Workbench (1); A filtration and concentration mechanism (2) is provided on the top of the workbench (1) and is used to coarsely filter the supernatant. A mild filtration mechanism (3) is provided on the top of the workbench (1) and is used to perform constant low-pressure filtration on the medium processed by the filtration and concentration mechanism (2). The mild filtration mechanism (3) includes a limiting foot (31) and a vacuum pump (34). The limiting foot (31) is fixedly installed on the top of the workbench (1). A collection cup (32) is movably inserted into the inner side of the limiting foot (31). A movable cover (33) is movably inserted into the top of the collection cup (32). The vacuum pump (34) is fixedly installed on the top of the workbench (1). The working end of the vacuum pump (34) is fixedly connected to the top of the movable cover (33).

2. The primary separation device for screening extracellular vesicles in stem cell supernatant according to claim 1, characterized in that: A filter cup (35) is movably inserted into the top of the movable cover (33), and a porous plate (351) is fixedly installed on the inner wall of the filter cup (35). A microporous membrane (352) is movably connected to the top of the porous plate (351).

3. The primary separation device for screening extracellular vesicles in stem cell supernatant according to claim 2, characterized in that: The mild filtration mechanism (3) also includes a support foot (36), which is fixedly installed on the top of the workbench (1). A connecting seat one (361) is fixedly installed on the top of the support foot (36). A limit rod (362) is fixedly installed on the top of the connecting seat one (361). A connecting seat two (363) is fixedly installed on the top of the limit rod (362). A lead screw (364) is rotatably connected between adjacent sides of the connecting seat one (361) and the connecting seat two (363). A rotating handle (365) is rotatably connected to the top of the connecting seat two (363). The bottom of the rotating handle (365) is fixedly connected to the top of the lead screw (364). A lifting seat (366) is slidably connected to the outer wall of the limit rod (362) and the lead screw (364). The lifting seat (366) is fixedly installed on the outer wall of the movable cover (33).

4. The primary separation device for screening extracellular vesicles in stem cell supernatant according to claim 1, characterized in that: The filtration and concentration mechanism (2) includes a column (21), which is fixedly installed on the top of the workbench (1). A rotating sleeve (22) is rotatably connected to the outer wall of the column (21). A positioning bolt (23) is threadedly connected to the outer wall of the rotating sleeve (22). The threaded end of the positioning bolt (23) is in movable contact with the outer wall of the column (21). A connecting frame (24) is fixedly installed on the top of the rotating sleeve (22). A concentration cylinder (25) is fixedly installed on the inner wall of the connecting frame (24).

5. The primary separation device for screening extracellular vesicles in stem cell supernatant according to claim 4, characterized in that: The inner wall of the concentration cylinder (25) is detachably connected to a sealing slide plate (26), and a counterweight block (27) is fixedly installed on the top of the sealing slide plate (26).

6. The primary separation device for screening extracellular vesicles in stem cell supernatant according to claim 5, characterized in that: The bottom of the concentration cylinder (25) is detachably connected to a concentration filter element (251).

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