A separation and extraction device for stem cell exosomes

Abstract

The application provides a separation and extraction device for stem cell exosomes, which comprises a shell, a partition plate arranged in the middle of the shell, a driving motor mounted at the lower part of the shell, a driving gear arranged upwardly through the partition plate, a plurality of transmission gears rotatably connected to the upper end of the partition plate and engaged with the driving gear, and a centrifugal driving pipe rotatably connected to the upper end of the transmission gear; further comprising a centrifugal sleeve pipe matched with the centrifugal driving pipe, a mesh filter ring arranged at the upper end of the centrifugal sleeve pipe, an annular ultrafiltration membrane arranged at one side of the mesh filter ring, an exosome retention pipe with a porous outer wall detachably connected in the centrifugal sleeve pipe, an inner ultrafiltration membrane arranged in the exosome retention pipe, a large protein retention pipe with a porous outer wall detachably connected in the exosome retention pipe, an inner ultrafiltration membrane arranged in the large protein retention pipe, and a sealing cover detachably connected to the upper end of the centrifugal sleeve pipe. The application can effectively improve the purity of exosomes, prevent the filter membrane from being blocked, and improve the separation efficiency.

Description

Technical Field

[0001] This invention belongs to the field of cell separation and extraction, and relates to a device for separating and extracting stem cell exosomes. Background Technology

[0002] In the field of stem cell research, exosomes serve as an important intercellular communication medium, making their isolation and extraction techniques particularly crucial. Currently, the main methods for extracting stem cell exosomes include ultracentrifugation, density gradient centrifugation, immunoaffinity assays, and ultrafiltration. Each of these methods has its advantages and disadvantages, but all aim to efficiently and purely isolate exosomes from complex biological samples.

[0003] Ultrafiltration has attracted considerable attention due to its simplicity and low cost. This method separates exosomes based on their size by selecting a suitable ultrafiltration membrane. However, despite its many advantages, the problem of large protein particles remaining after separation leads to insufficient purity of the extracted exosomes, affecting subsequent research applications. Furthermore, the ultrafiltration membrane is prone to clogging during the process, affecting separation efficiency, and exosomes attached to the membrane are difficult to recover completely, further reducing extraction efficiency. Therefore, although ultrafiltration has advantages in some aspects, it is not the optimal choice for the separation and extraction of stem cell exosomes. Summary of the Invention

[0004] The purpose of this invention is to provide a device for separating and extracting stem cell exosomes, which can effectively improve the purity of stem cell exosomes separated and extracted by ultrafiltration, prevent filter membrane clogging, improve separation efficiency, and make exosomes easier to recover.

[0005] To solve the above-mentioned technical problems, the present invention provides a device for separating and extracting stem cell exosomes, including a shell with an open top, a cover hinged to the open end of the shell, a partition plate provided in the middle of the shell, a drive motor installed inside the shell below the partition plate, a drive gear provided through the power output axis of the drive motor passing upward through the partition plate, and a plurality of circumferentially distributed transmission gears meshing with the drive gears rotatably connected to the upper end of the partition plate, and a centrifugal drive tube with an open top connected to the upper end of each transmission gear;

[0006] It also includes a centrifugal outer tube that cooperates with the centrifugal drive tube. A mesh filter ring is disposed inwardly at the upper end of the centrifugal outer tube, and an annular ultrafiltration membrane is disposed on one side of the mesh filter ring. An exosome indwelling tube with an open upper end is detachably connected inside the centrifugal outer tube. The outer diameter of the exosome indwelling tube is equal to the inner diameter of the mesh filter ring. The connection between the exosome indwelling tube and the mesh filter ring is damped. Multiple intermediate filtration holes are formed on the outer periphery of the exosome indwelling tube inside the centrifugal outer tube. An intermediate-ultrafiltration membrane is disposed inside the exosome indwelling tube. The indwelling tube is detachably connected to a large protein indwelling tube with an open top. A sealing ring with the same outer diameter as the inner diameter of the exosome indwelling tube is provided on the outer side of the upper part of the large protein indwelling tube. The sealing ring is damped when connected to the inner wall of the exosome indwelling tube. The large protein indwelling tube has multiple internal filtration pores on the outer wall inside the exosome indwelling tube. An internal ultrafiltration membrane is provided inside the large protein indwelling tube. The pore size of the annular ultrafiltration membrane is 1-5 nm, the pore size of the intermediate ultrafiltration membrane is 30-40 nm, and the pore size of the internal ultrafiltration membrane is 100-150 nm.

[0007] The upper end of the centrifuge outer tube is detachably connected to a sealing cap for covering the upper end of the exosome indwelling tube and the large protein indwelling tube. The inner wall of each centrifuge drive tube has multiple limiting slots extending downward from its opening end. The outer wall of the centrifuge outer tube is provided with limiting inserts corresponding to the limiting slots. A pressure plate is detachably connected inside the shell. The pressure plate is rotatably connected to multiple rotating shafts corresponding to the centrifuge drive tubes. The upper end of each rotating shaft is provided with a limiting piece, and the lower end of each rotating shaft is provided with a pressure cap that cooperates with the sealing cap.

[0008] By adopting the above technical solution, firstly, supernatant is obtained from stem cell culture, and the supernatant is added to a large protein indwelling tube. An appropriate amount of PBS is added to the large protein indwelling tube as a buffer, and an appropriate amount of PBS is added to the centrifuge outer tube. The large protein indwelling tube is inserted into the exosome indwelling tube, the exosome indwelling tube is inserted into the centrifuge outer tube, the sealing cap is closed, the centrifuge outer tube is aligned with the limiting slot and inserted into the centrifuge drive tube, the pressure plate is closed, and the pressure cap on the pressure plate presses against the sealing cap. The drive motor is started to perform low-speed centrifugation.

[0009] During centrifugation, due to centrifugal force, liquid, exosomes, and small protein particles pass through the inner ultrafiltration membrane into the exosome indwelling tube, while large protein particles remain in the large protein indwelling tube. Liquid and small protein particles pass through the intermediate ultrafiltration membrane into the outer centrifuge tube, while exosomes remain in the exosome indwelling tube. Under centrifugal force, the liquid rises and passes through the circular ultrafiltration membrane, filtering small protein particles into the outer centrifuge tube. The liquid passing through the circular ultrafiltration membrane continues to rise and flow back into the large protein indwelling tube, allowing the PBS liquid to continuously circulate among the large protein indwelling tube, exosome indwelling tube, and outer centrifuge tube. This prevents excessive residues from adhering to the filter membranes and affecting the separation effect. After separation, the exosome indwelling tube is removed from the outer centrifuge tube, and the large protein indwelling tube is removed from the exosome indwelling tube, allowing the exosomes to be recovered from the exosome indwelling tube.

[0010] The present invention is further configured such that the inner wall of the centrifugal outer tube is provided with a plurality of spiral drainage strips distributed in a circular pattern, and the spiral direction of each spiral drainage strip is the same as the direction of the centrifugal outer tube during centrifugal rotation.

[0011] The present invention is further configured such that a sealing ring is provided on the inner ring of the mesh filter ring toward the bottom of the centrifuge outer tube, and the inner diameter of the sealing ring is equal to the outer diameter of the exosome indwelling tube.

[0012] The present invention is further configured such that the sealing cap is fitted with the centrifugal outer tube, and the lower edge of the sealing cap is provided with a plurality of slots that engage with the limiting inserts one by one.

[0013] The present invention is further configured such that a support plate is provided above the partition plate of the housing, and the support plate has a plurality of support holes corresponding one-to-one with the centrifugal drive tubes, and a bearing corresponding to the centrifugal drive tube is provided in each support hole.

[0014] The present invention is further configured such that a connecting sleeve with internal threads is provided in the middle of the support plate, and a threaded connecting shaft that is threadedly connected to the connecting sleeve is rotatably connected in the middle of the pressure plate, and a knob is provided at the upper end of the threaded connecting shaft.

[0015] The present invention is further configured such that the inner wall of the housing is provided with a plurality of vertically arranged limiting strips, and the edge of the pressure plate is provided with a plurality of limiting notches corresponding one to one of the limiting strips. After the limiting strips and the limiting notches are connected one to one, the pressure cover and the sealing cover are connected one to one.

[0016] The present invention is further configured such that a handle is provided at the upper end of the pressure cap.

[0017] The present invention is further configured such that a rubber pad is provided at the lower end of each pressure cap.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] Firstly, this invention combines ultrafiltration with centrifugation, allowing exosomes to quickly permeate through the ultrafiltration membrane under low-speed centrifugation, thus achieving faster exosome separation and extraction and improving separation efficiency.

[0020] Secondly, this invention filters large protein particles into a large protein indwelling tube through an inner ultrafiltration membrane, retains exosomes in an exosome indwelling tube through a middle ultrafiltration membrane, and filters small protein particles into a centrifuge outer tube, effectively reducing impurities in exosomes and improving the purity of exosomes.

[0021] Thirdly, in the centrifugation process, the present invention adds a buffer solution. Under centrifugation, the buffer solution can continuously circulate in the large protein indwelling tube, exosome indwelling tube and centrifuge outer tube, preventing excessive residues from adhering to the filter membrane and affecting the separation effect. At the same time, the exosomes are diluted by the buffer solution, rather than being attached to the filter membrane in large quantities, making exosome recovery easier and with a higher recovery rate. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the overall structure of the present invention;

[0023] Figure 2 This is a schematic diagram used to demonstrate the removal of the pressure plate inside the housing;

[0024] The third is a partial sectional view used to show the internal structure of the shell;

[0025] Figure 4 Used to demonstrate the connection of centrifuge outer tube, exosome indwelling tube, large protein indwelling tube and sealing cap;

[0026] Figure 5 It is a partial cross-sectional view used to show the internal structure of centrifuge outer tube, exosome indwelling tube and large protein indwelling tube;

[0027] Figure 6 The pressure cap used to display the bottom of the pressure plate.

[0028] The components include: 1. Shell; 11. Cover; 12. Divider plate; 13. Support plate; 14. Bearing; 15. Connecting sleeve; 16. Limiting strip; 2. Drive motor; 21. Drive gear; 3. Transmission gear; 31. Centrifuge drive tube; 32. Limiting slot; 4. Centrifuge outer sleeve; 41. Spiral drainage strip; 42. Mesh filter ring; 43. Annular ultrafiltration membrane; 44. Sealing ring; 45. Limiting insert; 5. Exosome indwelling tube; 51. Middle filter hole; 52. Middle ultrafiltration membrane; 6. Large protein indwelling tube; 61. Sealing connecting ring; 62. Inner filter hole; 63. Inner ultrafiltration membrane; 7. Sealing cap; 71. Bayonet; 8. Pressure plate; 81. Limiting piece; 82. Pressure cap; 83. Rubber pad; 84. Threaded connecting shaft; 85. Knob; 86. Limiting notch; 87. Handle. Detailed Implementation

[0029] The following detailed description, in conjunction with the accompanying drawings and specific embodiments, provides a more detailed account of the apparatus for separating and extracting stem cell exosomes proposed in this invention. The advantages and features of this invention will become clearer from the following description. It should be noted that the accompanying drawings are all in a very simplified form and use non-precise proportions, and are only used to facilitate and clarify the illustration of the embodiments of this invention. The same or similar reference numerals in the drawings represent the same or similar parts.

[0030] Example, refer to Figure 1-6 A device for separating and extracting stem cell exosomes includes a shell 1 with an open top. A cover 11 is hinged to the open end of the shell 1. A partition plate 12 is provided in the middle of the shell 1. A drive motor 2 is installed inside the shell 1 below the partition plate 12. A drive gear 21 is provided through the power output axis of the drive motor 2 and passes upward through the partition plate 12. Twelve transmission gears 3 are rotatably connected to the upper end of the partition plate 12, which are circumferentially distributed and mesh with the drive gears 21. A centrifugal drive tube 31 with an open top is rotatably connected to the upper end of each transmission gear 3. A support plate 13 is provided above the partition plate 12 of the shell 1. The support plate 13 has twelve support holes (not shown) corresponding to the centrifugal drive tubes 31. A bearing 14 corresponding to the centrifugal drive tube 31 is provided in each support hole.

[0031] It also includes a centrifugal outer tube 4 that cooperates with the centrifugal drive tube 31. The inner wall of the centrifugal outer tube 4 is provided with four circumferentially distributed spiral guide strips 41. During centrifugation, the spiral guide strips 41 can further drive the liquid to rotate upward. The spiral direction of each spiral guide strip 41 is the same as the direction of centrifugal rotation of the centrifugal outer tube 4. A mesh filter ring 42 is provided inward at the upper end of the centrifugal outer tube 4. An annular ultrafiltration membrane 43 is provided inside the mesh filter ring 42. A sealing collar 44 is provided on the inner ring of the mesh filter ring 42 towards the bottom of the centrifugal outer tube 4.

[0032] An exosome indwelling tube 5 with an open upper end is detachably connected inside the centrifuge outer tube 4. The inner diameter of the sealing collar 44 is equal to the outer diameter of the exosome indwelling tube 5. The connection between the exosome indwelling tube 5 and the sealing collar 44 is damped. The upper end of the exosome indwelling tube 5 extends out of the centrifuge outer tube 4. The exosome indwelling tube 5 has multiple intermediate filtration holes 51 on its outer periphery inside the centrifuge outer tube 4. A layer of intermediate ultrafiltration membrane 52 is provided inside the exosome indwelling tube 5. A large protein indwelling tube 6 with an open top is detachably connected to the exosome indwelling tube 5. The upper end of the large protein indwelling tube 6 extends out of the exosome indwelling tube 5. A sealing ring 61 with the same outer diameter as the inner diameter of the exosome indwelling tube 5 is provided on the outer side of the upper part of the large protein indwelling tube 6. The sealing ring 61 is damped when connected to the inner wall of the exosome indwelling tube 5. The large protein indwelling tube 6 has multiple internal filtration pores 62 on its outer wall inside the exosome indwelling tube 5. An internal ultrafiltration membrane 63 is provided inside the large protein indwelling tube 6. The pore size of the annular ultrafiltration membrane 43 is 1-5 nm, allowing only buffer solution to pass through. The pore size of the intermediate ultrafiltration membrane 52 is 30-40 nm, allowing only buffer solution and particles smaller than exosomes to pass through. The pore size of the internal ultrafiltration membrane 63 is 100-150 nm, allowing only buffer solution, exosomes, and particles smaller than exosomes to pass through.

[0033] Each centrifuge drive tube 31 has multiple limiting slots 32 extending downwards from its opening end on its inner wall. The outer wall of the centrifuge outer tube 4 is provided with limiting inserts 45 corresponding to the limiting slots 32, allowing the centrifuge outer tube 4 to rotate with the centrifuge drive tube 31. The upper end of the centrifuge outer tube 4 is detachably connected to a sealing cap 7 for covering the upper end of the exosome indwelling tube 5 and the large protein indwelling tube 6. The sealing cap 7 covers the centrifuge outer tube 4, and the lower edge of the sealing cap 7 has multiple locking slots 71 that engage with the limiting inserts 45, allowing the sealing cap 7 to rotate with the centrifuge drive tube 31 and returning the upstream buffer solution to the large protein indwelling tube 6. Inside the housing 1, there is a detachable pressure plate 8. The pressure plate 8 is rotatably connected to twelve rotating shafts that correspond one-to-one with the centrifugal drive tube 31 (figure omitted). Each rotating shaft has a limiting plate 81 at its upper end and a pressure cap 82 that cooperates with the sealing cap 7 at its lower end. Each pressure cap 82 has a rubber pad 83 at its lower end to press the sealing cap 7 and make it rotate stably.

[0034] A connecting sleeve 15 with internal threads is provided upward in the middle of the support plate 13. A threaded connecting shaft 84, which is threaded to the connecting sleeve 15, is rotatably connected to the middle of the pressure plate 8. A knob 85 is provided at the upper end of the threaded connecting shaft 84. Three vertically arranged limiting strips 16 are provided on the inner wall of the housing 1. Three limiting notches 86, which correspond one-to-one with the limiting strips 16, are opened on the edge of the pressure plate 8. After the limiting strips 16 and the limiting notches 86 are connected one-to-one, the pressure cover 82 and the sealing cover 7 are connected one-to-one. A handle 87 is provided at the upper end of the pressure cover 82.

[0035] Instructions for use: First, obtain the supernatant from the stem cell culture, add the supernatant to the large protein indwelling tube 6, and add an appropriate amount of PBS as a buffer to the large protein indwelling tube 6. At the same time, add an appropriate amount of PBS to the centrifuge outer tube 4. Insert the large protein indwelling tube 6 into the exosome indwelling tube 5, insert the exosome indwelling tube 5 into the centrifuge outer tube 4, and cover it with the sealing cap 7. Align the centrifuge outer tube 4 with the limiting slot 32 and insert it into the centrifuge drive tube 31. Cover it with the pressure plate 8, so that the pressure cap 82 on the pressure plate 8 presses down on the sealing cap 7. Start the drive motor 2 to perform low-speed centrifugation.

[0036] During centrifugation, due to centrifugal force, liquid, exosomes, and small protein particles pass through the inner ultrafiltration membrane 63 into the exosome indwelling tube 5, while large protein particles remain in the large protein indwelling tube 6. Liquid and small protein particles pass through the intermediate ultrafiltration membrane 52 into the centrifuge outer tube 4, while exosomes remain in the exosome indwelling tube 5. Under centrifugal force, the liquid rises and passes through the annular ultrafiltration membrane 43, filtering small protein particles into the centrifuge outer tube 4. The liquid passing through the annular ultrafiltration membrane 43 continues to rise and flow back into the large protein indwelling tube 6, allowing the PBS liquid to continuously circulate in the large protein indwelling tube 6, exosome indwelling tube 5, and centrifuge outer tube 4, preventing excessive residues from adhering to the filter membranes and affecting the separation effect. After separation, the exosome indwelling tube 5 is removed from the centrifuge outer tube 4, and the large protein indwelling tube 6 is removed from the exosome indwelling tube 5, allowing the exosomes to be recovered from the exosome indwelling tube 5.

[0037] It should be noted that the various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0038] The above description is merely a description of preferred embodiments of the present invention and is not intended to limit the scope of the present invention in any way. Any changes or modifications made by those skilled in the art based on the above disclosure shall fall within the protection scope of the claims.

Claims

1. A device for separating and extracting stem cell exosomes, comprising a shell (1) with an opening at the upper end, characterized in that, The shell (1) has a cover (11) hinged to the open end. A partition plate (12) is provided in the middle of the shell (1). A drive motor (2) is installed in the shell (1) below the partition plate (12). The drive motor (2) has a drive gear (21) that passes through the partition plate (12) axially. A plurality of transmission gears (3) that are circumferentially distributed and mesh with the drive gear (21) are rotatably connected to the upper end of the partition plate (12). Each transmission gear (3) has a centrifugal drive tube (31) with an open upper end that is rotatably connected to its upper end. It also includes a centrifugal outer tube (4) that cooperates with the centrifugal drive tube (31). A mesh filter ring (42) is provided inward at the upper end of the centrifugal outer tube (4). An annular ultrafiltration membrane (43) is provided on one side of the mesh filter ring (42). An exosome indwelling tube (5) with an open upper end is detachably connected inside the centrifugal outer tube (4). The outer diameter of the exosome indwelling tube (5) is equal to the inner diameter of the mesh filter ring (42). The connection between the exosome indwelling tube (5) and the mesh filter ring (42) is damped. The exosome indwelling tube (5) has multiple intermediate filter holes (51) on the outer periphery inside the centrifugal outer tube (4). An intermediate ultrafiltration membrane (52) is provided inside the exosome indwelling tube (5). The exosome indwelling tube (5) is detachably connected to a large protein indwelling tube (6) with an open top. A sealing connection ring (61) with the same outer diameter as the inner diameter of the exosome indwelling tube (5) is provided on the outer side of the upper part of the large protein indwelling tube (6). The sealing connection ring (61) is damped to the inner wall of the exosome indwelling tube (5). The large protein indwelling tube (6) has multiple internal filter holes (62) on the outer wall inside the exosome indwelling tube (5). An internal ultrafiltration membrane (63) is provided inside the large protein indwelling tube (6). The pore size of the annular ultrafiltration membrane (43) is 1-5 nm, the pore size of the intermediate ultrafiltration membrane (52) is 30-40 nm, and the pore size of the internal ultrafiltration membrane (63) is 100-150 nm. The upper end of the centrifuge outer tube (4) is detachably connected to a sealing cap (7) for covering the upper end of the exosome indwelling tube (5) and the large protein indwelling tube (6). The inner wall of each centrifuge drive tube (31) has multiple limiting slots (32) extending downward from its opening end. The outer wall of the centrifuge outer tube (4) is provided with limiting inserts (45) corresponding to the limiting slots (32). The housing (1) is detachably connected to a pressure plate (8). The pressure plate (8) is rotatably connected to multiple rotating shafts corresponding to the centrifuge drive tubes (31). The upper end of each rotating shaft is provided with a limiting piece (81), and the lower end of each rotating shaft is provided with a pressure cap (82) that cooperates with the sealing cap (7).

2. The device for separating and extracting stem cell exosomes according to claim 1, characterized in that, The inner wall of the centrifugal outer tube (4) is provided with multiple spiral guide strips (41) arranged in a circular pattern. The spiral direction of each spiral guide strip (41) is the same as the direction of the centrifugal outer tube (4) during centrifugal rotation.

3. The device for separating and extracting stem cell exosomes according to claim 1, characterized in that, The inner ring of the mesh filter ring (42) is provided with a sealing ring (44) at the bottom of the centrifuge outer tube (4), and the inner diameter of the sealing ring (44) is equal to the outer diameter of the exosome indwelling tube (5).

4. The apparatus for separating and extracting stem cell exosomes according to claim 1, characterized in that, The sealing cap (7) is fitted over the centrifugal outer tube (4), and the lower edge of the sealing cap (7) is provided with a plurality of slots (71) that engage with the limiting insert (45) one by one.

5. The apparatus for separating and extracting stem cell exosomes according to claim 1, characterized in that, The housing (1) is provided with a support plate (13) above the partition plate (12). The support plate (13) has a plurality of support holes corresponding to the centrifugal drive tube (31). Each support hole is provided with a bearing (14) corresponding to the centrifugal drive tube (31).

6. The apparatus for separating and extracting stem cell exosomes according to claim 5, characterized in that, The support plate (13) has a connecting sleeve (15) with internal threads in the middle and is rotatably connected to the middle of the pressure plate (8) and a threaded connecting shaft (84) that is threaded to the connecting sleeve (15). A knob (85) is provided at the upper end of the threaded connecting shaft (84).

7. A device for separating and extracting stem cell exosomes according to any one of claims 1 or 5, characterized in that, The inner wall of the housing (1) is provided with a number of vertically arranged limiting strips (16), and the edge of the pressure plate (8) is provided with a number of limiting notches (86) corresponding to the limiting strips (16). After the limiting strips (16) and the limiting notches (86) are connected one by one, the pressure cover (82) and the sealing cover (7) are connected one by one.

8. The apparatus for separating and extracting stem cell exosomes according to claim 7, characterized in that, The upper end of the pressure cap (82) is provided with a handle (87).

9. The apparatus for separating and extracting stem cell exosomes according to claim 7, characterized in that, Each pressure cap (82) has a rubber pad (83) at its lower end.

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