A device and method for extracting exosomes

Abstract

The application discloses a device and a method for extracting exosomes, and the device comprises a first centrifugal mechanism composed of a first separation cabin and a second separation cabin which are axially communicated, and a second centrifugal mechanism composed of a third separation cabin and a fourth separation cabin which are axially communicated; the first separation cabin is arranged above the second separation cabin, and the third separation cabin is arranged above the fourth separation cabin; the first centrifugal mechanism is arranged in the third separation cabin of the second centrifugal mechanism; and a valve mechanism which can be opened and closed is arranged between the first separation cabin and the second separation cabin, between the third separation cabin and the fourth separation cabin and between the first separation cabin and the third separation cabin; and a separation filter membrane is arranged on the third separation cabin and the fourth separation cabin. The exosomes can be extracted without liquid external transfer.

Description

Technical Field

[0001] This invention belongs to the field of exosome extraction technology, and particularly relates to an apparatus and method for extracting exosomes. Background Technology

[0002] Exosomes (EVs) are small extracellular vesicle structures that play a vital role in cell biology. They can transmit information between cells both inside and outside the body, carrying various types of biomolecules, including proteins, RNA, and DNA. Exosomes are considered important biomolecular delivery mediators in cell communication, tissue repair, immune surveillance, and cancer metastasis.

[0003] Existing methods for exosome extraction generally employ ultracentrifugation, which involves centrifuging cell culture medium or tissue homogenate (containing exosomes) and then gradually increasing the centrifugation speed and time to separate vesicle structures of different sizes, thereby separating the precipitate containing exosomes.

[0004] Chinese patent CN202021640398.2 discloses an exosome extraction device, including an outer tube mechanism, an inner tube mechanism that is snapped into the inner side of the outer tube mechanism, a first extraction mechanism that is snapped into the inner side of the inner tube mechanism, and a second extraction mechanism that is snapped into the inner side of the first extraction mechanism. The extraction device inserts the inner tube into the first inner groove, drives the first extraction tube into the second inner groove, and drives the second extraction tube into the first extraction groove. Cell culture supernatant is stored in the second extraction groove, filtered by a second filter in the second extraction groove, then filtered by a first filter in the first inner groove, and then flows into the second inner groove. Impurities are filtered into the first inner groove and discharged by a third filter, and the separation and extraction are performed sequentially.

[0005] The extraction device uses a single centrifugation followed by filtration to extract exosomes, thus improving purity. However, extracting exosomes from small batches of samples such as whole blood requires multiple centrifugations. For example, extracting exosomes from whole blood requires three centrifugations. Therefore, the exosome extraction device disclosed in the aforementioned patent is not suitable for experiments requiring multiple centrifugations to extract exosomes. Summary of the Invention

[0006] The purpose of this invention is to provide an apparatus and method for extracting exosomes from whole blood, which can achieve multiple centrifugations without external liquid transfer or additional centrifugation equipment, thereby extracting an appropriate amount of exosomes from small batches of samples such as whole blood.

[0007] To solve the aforementioned technical problems, the present invention specifically adopts the following technical solution: an apparatus for extracting exosomes, comprising a first centrifuge mechanism consisting of a first separation chamber and a second separation chamber connected axially, wherein the volume of the first separation chamber is larger than the volume of the second separation chamber, and a second centrifuge mechanism consisting of a third separation chamber and a fourth separation chamber connected axially; the first separation chamber is disposed above the second separation chamber, and the third separation chamber is disposed above the fourth separation chamber; the first centrifuge mechanism is placed in the third separation chamber of the second centrifuge mechanism; and openable and closable valve mechanisms are provided between the first separation chamber and the second separation chamber, between the third separation chamber and the fourth separation chamber, and between the first separation chamber and the third separation chamber; separation filter membranes are provided on the third separation chamber and the fourth separation chamber;

[0008] The first separation chamber has a first through hole on its side wall, and the third separation chamber has a corresponding second through hole on its side wall. The first through hole and the second through hole are coaxial. The piston, which serves as a valve mechanism between the first and third separation chambers, can pass through both the first and second through holes to block the communication channel between the first and third separation chambers, thereby allowing the first centrifugation operation to be performed in both chambers; or it can pass through only the second through hole to connect the first and third separation chambers, so that after the first centrifugation, the upper liquid in the first separation chamber enters the third separation chamber through the first through hole.

[0009] The top of the first separation chamber is provided with a cylindrical plug that serves as a valve mechanism; the cylindrical plug is used to close the communication channel between the first separation chamber and the second separation chamber.

[0010] As an improvement, the bottom of the first separation chamber is connected to the second separation chamber via a narrowed channel I to form a connecting channel between the first and second separation chambers.

[0011] As an improvement, the piston is marked with graduation lines, so that when the graduation lines are flush with the outer wall of the third separation chamber, the piston closes the second through hole while opening the first through hole, so that the first separation chamber and the third separation chamber are connected, thereby allowing the upper liquid in the first separation chamber after the first centrifugation to enter the third separation chamber for a second centrifugation.

[0012] As an improvement, it also includes a cover for sealing on the third separation chamber, with a cylindrical plug serving as a valve mechanism disposed on the cover; and the cylindrical plug can extend through the top wall of the first separation chamber into the narrowing channel I to close the narrowing channel I.

[0013] As an improvement, the top wall of the first separation chamber has a hole for a cylindrical plug to pass through, and the inner wall of the hole has an annular groove, in which a Gladley sealing ring is installed.

[0014] As an improvement, the outer wall of the third separation chamber is provided with one-way teeth, and the cover body cooperates with the one-way teeth so that the cover body can only drive the cylindrical plug to move in the direction of approaching the narrowing channel I.

[0015] As an improvement, the end of the cylindrical plug is a tapered section, and a sealing groove is opened at the root of the tapered section; the reduced diameter channel I is a tapered cavity that mates with the tapered section, and a sealing annular protrusion that mates with the sealing groove is provided at its opening.

[0016] As an improvement, the bottom of the third separation chamber is connected to the fourth separation chamber via a reduced-diameter channel II; the valve mechanism provided on the reduced-diameter channel II includes a valve body, and the valve body is provided with a valve core that is threadedly engaged with it.

[0017] As an improvement, a puncture area is provided at the bottom of the fourth separation chamber, and the puncture area has the same puncture holes as the interior of the fourth separation chamber. The puncture holes are sealed with a membrane, and a pinhole marker is provided on the membrane corresponding to the position of the puncture hole. Since the pinholes at the bottom of the fourth separation chamber are very small and difficult to identify with the naked eye, a sealing membrane is provided over the puncture holes, and a pinhole marker is provided on the membrane, thereby avoiding the need to search for the pinholes.

[0018] The present invention also provides a method for extracting exosomes, applied to the above-mentioned apparatus for extracting exosomes, comprising:

[0019] The piston that simultaneously closes the first through hole on the side wall of the first separation chamber and the second through hole on the side wall of the third separation chamber is opened, and the sample to be separated (such as whole blood or other samples that require multiple centrifugations) is injected into the first centrifugation mechanism through the second through hole of the third separation chamber and the first through hole of the first separation chamber using a syringe. At this time, the first separation chamber and the second separation chamber are connected, while the third separation chamber is blocked from the fourth separation chamber.

[0020] After the piston seals the first and second through holes to block the communication channel between the first and third separation chambers, the first centrifugation is performed. After the first centrifugation, the narrowed channel I between the first and second separation chambers is sealed by the plunger, thus blocking the communication channel between the first and second separation chambers. The waste liquid (e.g., lower layer liquid, or lower layer liquid and a very small amount of upper layer liquid) in the second separation chamber and the upper layer liquid in the first separation chamber are obtained respectively.

[0021] Open the first through hole on the first separation chamber, but keep the second through hole closed, so that the first separation chamber and the third separation chamber are connected. Pour the liquid in the first separation chamber into the third separation chamber through the first through hole for a second centrifugation.

[0022] After the second centrifugation is completed, open the valve mechanism between the third and fourth separation chambers, put the lower layer liquid obtained from the second centrifugation in the third separation chamber into the fourth separation chamber, and then close the third and fourth separation chambers again.

[0023] The liquid in the fourth separation chamber was centrifuged for the third time. After the third centrifugation, the lower layer of liquid was extracted through the puncture hole at the bottom of the fourth separation chamber using a syringe to obtain exosomes.

[0024] In other embodiments, PRP liquid can also be obtained by using a syringe to extract the lower layer liquid that has not undergone a third centrifugation through a puncture hole at the bottom of the fourth separation chamber.

[0025] The advantages of this invention are as follows: For multiple centrifugations, where the required liquid position differs after each centrifugation (e.g., the upper layer needs to be extracted after the first centrifugation, while the lower layer is needed after the second centrifugation), multiple centrifuge devices are typically required. For example, a corresponding centrifuge device is prepared for each centrifugation, and after each centrifugation, the liquid that needs to be centrifuged again after the previous centrifugation is transferred to a new centrifuge device under aseptic conditions for further centrifugation. In addition, for applications involving the extraction of trace amounts of exosomes where it is desirable to minimize space usage, using a medical institution's laboratory is not only cumbersome, requiring multiple centrifuge devices and occupying a large space, but also involves multiple transfers, making the operation tedious and redundant. Furthermore, for the extraction of trace amounts of exosomes (e.g., extracting exosomes from 10ml-50ml of whole blood), preparing multiple centrifuge devices and complicated operating steps is very inefficient.

[0026] In view of this, and taking into account: 1) multiple centrifugations; 2) different liquid locations after each centrifugation; 3) micro-volume exosome extraction; and 4) small space requirement, this invention proposes an exosome extraction device that is simpler to operate and more cost-effective. It is suitable for applications requiring small-batch exosome extraction and where space is a constraint. Its core lies in designing a nested centrifugation mechanism, and by changing the opening and closing of the channels between the nested centrifugation mechanisms, ensuring direct transfer of liquid at different locations within a sealed environment without exposure to the external environment. Specifically, the nested arrangement of the first and second centrifugation mechanisms significantly reduces the overall size of the device. Furthermore, the design of the corresponding channels in the first and second centrifugation mechanisms enables transfer of liquid at different locations after centrifugation within a sealed environment, making it suitable for small-batch exosome extraction.

[0027] Typically, to obtain the liquid in the first separation chamber, it is sufficient to design the first and second separation chambers to be staggered, and to design an opening at the bottom of the first separation chamber. However, in practice, it has been found that the staggered design leads to a significant difference in centrifugal force between the two separation chambers, thus affecting the centrifugation effect. Therefore, in this invention, the axes or center lines of the first and second separation chambers are kept highly aligned, and an opening and corresponding valve mechanism are provided on the side wall of the first separation chamber. Furthermore, this opening also serves as a sample injection port, thus eliminating the need to create a separate injection port at the top or other locations of the first separation chamber, further simplifying the structure and reducing the complexity of the device manufacturing process.

[0028] In this invention, the first centrifugation mechanism is used for the first centrifugation, and the second centrifugation mechanism is used for the second and third centrifugations. After the first centrifugation, a valve mechanism between the first and second separation chambers is used to separate the upper and lower liquids. Specifically, since the volume of the first separation chamber is larger than that of the second separation chamber (and the volume of the second separation chamber is greater than or equal to the volume of the lower liquid obtained after the first separation of the whole blood sample), the separation between the upper and lower liquids is automatically achieved while blocking the flow. (In practical operation, after the first centrifugation, due to the design of the volume ratio of the two separation chambers, a clear non-boundary line is formed between the second separation chamber and channel I, that is, the upper liquid is in channel I. Therefore, when the plug in the valve mechanism cooperates with channel I, it will automatically squeeze the liquid in channel I into the first separation chamber, thereby achieving automatic separation and isolation between the upper and lower liquids.) This makes it easy to pour the upper liquid in the first separation chamber into the third separation chamber without the need for additional transfer equipment, and also avoids the mixing of lower liquid during pouring. Normally, the volume of the second separation chamber is equal to the volume of the lower layer liquid (the volume of the lower layer liquid obtained after the first centrifugation of a whole blood sample of a predetermined volume, estimated based on experience; for example, the volume of the lower layer liquid obtained after the first centrifugation of a 50ml whole blood sample is 42%-50% of 50ml). Thus, once the first and second separation chambers are blocked, as mentioned above, the upper and lower layers of liquid can be automatically separated and isolated. However, in practical applications, some unexpected situations may occur: after the first centrifugation of a whole blood sample of a specified volume, the volume of the lower layer liquid is slightly smaller than the volume of the second separation chamber, resulting in a small amount of upper layer liquid remaining in the second separation chamber after the first centrifugation. However, this amount does not affect the final extraction of exosomes. Therefore, by using a valve mechanism to block the first and second separation chambers, it can be completely ensured that no lower layer liquid remains in the first separation chamber, thus guaranteeing the smooth progress of exosome extraction.

[0029] After a second centrifugation in the third separation chamber, the lower layer liquid can be transferred into the fourth separation chamber via a valve mechanism between the third and fourth separation chambers, thus preventing the upper layer liquid from mixing in. Specifically, since the extraction is done in small batches, the amount of upper and lower layer liquid in the third separation chamber is very small. Furthermore, the diameter of the narrowed channel II between the third and fourth separation chambers is very small (typically a few millimeters), and the entire device is made of transparent material. Therefore, the amount of lower layer liquid flowing into the fourth separation chamber from the third separation chamber can be observed and controlled through this channel II, thereby achieving the separation of the upper and lower layer liquids.

[0030] After a third centrifugation in the fourth separation chamber, the lower layer of liquid is extracted through a puncture hole at the bottom, ensuring that the entire centrifugation and extraction process is carried out in a closed space, reducing the possibility of contamination.

[0031] In addition, the present invention selects the upper liquid after the first centrifugation and the lower liquid after the second and third centrifugation, which fully meets the process requirements for whole blood exosome extraction. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0033] Figure 1 This is a schematic diagram of the structure of an exosome extraction device according to an exemplary embodiment of the present invention;

[0034] Figure 2 for Figure 1 Enlarged view of point A in the exosome extraction device shown;

[0035] Figure 3 for Figure 1 Enlarged view of point B in the exosome extraction device shown;

[0036] Figure 4 for Figure 1 Enlarged view of point C in the exosome extraction device shown;

[0037] Figure 5 for Figure 1 Enlarged view of point D in the exosome extraction device shown;

[0038] Figure 6 for Figure 1Enlarged view of point E in the exosome extraction device shown;

[0039] Figure 7 for Figure 1 Enlarged view of point F in the exosome extraction device shown;

[0040] Figure 8A , 8B This is a schematic diagram of the first centrifugation in an exemplary embodiment of the exosome extraction method of the present invention;

[0041] Figure 9A , 9B This is a schematic diagram of the second centrifugation in an exemplary embodiment of the exosome extraction method of the present invention;

[0042] Figure 10 This is a schematic diagram of the third centrifugation in an exemplary embodiment of the exosome extraction method of the present invention;

[0043] Figure 11 This is a schematic diagram to show the process of not performing a third centrifugation.

[0044] The markings in the diagram are: 1 First separation chamber, 2 Second separation chamber, 3 Third separation chamber, 4 Fourth separation chamber, 5 Reduction channel I, 6 Reduction channel II, 7 Cover, 8 Plug, 9 Piston, 10 Valve body, 11 Glyd seal ring; 31 Separation filter membrane I, 32 One-way tooth, 41 Separation filter membrane II, 42 Puncture area, 51 Sealing ring, 81 Conical section, 82 Sealing groove, 101 Valve core. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0046] In this document, suffixes such as "module," "part," or "unit" used to denote elements are used only for the purpose of illustrative purposes and have no specific meaning in themselves. Therefore, "module," "part," or "unit" may be used interchangeably.

[0047] In this document, the terms "upper," "lower," "inner," "outer," "front," "rear," "one end," and "the other end," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the present invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0048] In this document, unless otherwise explicitly specified and limited, the terms "installed," "equipped with," "connected," etc., should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, a direct connection, or an indirect connection through an intermediate medium; it can be a connection within two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0049] In this document, "and / or" includes any and all combinations of one or more of the listed related items. "Multiple" in this document means two or more, i.e., it includes two, three, four, five, etc.

[0050] The whole blood mentioned in this invention refers to an unprocessed or unseparated blood sample collected from a person or animal. It contains all blood cells (red blood cells, white blood cells, and platelets) as well as plasma. Therefore, the process of extracting exosomes from whole blood involves multiple centrifugations and even multiple filtrations. This invention is designed for the process of extracting exosomes from small batches of samples such as whole blood, which require multiple centrifugations.

[0051] Specifically, such as Figure 1 As shown, the present invention provides an apparatus for extracting exosomes, comprising a first centrifuge mechanism consisting of a first separation chamber 1 and a second separation chamber 2 connected axially, and a second centrifuge mechanism consisting of a third separation chamber 3 and a fourth separation chamber 4 connected axially; the first separation chamber 1 is disposed above the second separation chamber 2, and the third separation chamber 3 is disposed above the fourth separation chamber 4; the first centrifuge mechanism is placed in the third separation chamber 3 of the second centrifuge mechanism; and openable and closable valve mechanisms are provided between the first separation chamber 1 and the second separation chamber 2, between the third separation chamber 3 and the fourth separation chamber 4, and between the first separation chamber 1 and the third separation chamber 3; separation filter membrane I31 and separation filter membrane II41 are respectively disposed on the third separation chamber 3 and the fourth separation chamber 4.

[0052] In this invention, the separation chambers are preferably cylindrical in shape, with the first separation chamber 1, the second separation chamber 2, and the fourth separation chamber 4 all having flat bottoms. The flat bottom of the first separation chamber 1 facilitates the pouring of the upper liquid. The bottom of the third separation chamber 3 has a certain taper to facilitate liquid concentration. Of course, in some other embodiments, the separation chambers may not be cylindrical, or the first separation chamber 1, the second separation chamber 2, and the fourth separation chamber 4 may not be designed with flat bottoms.

[0053] In this invention, the first separation chamber 1, the second separation chamber 2, the third separation chamber 3, and the fourth separation chamber 4 are located on the same axis. The first separation chamber 1 is positioned above the second separation chamber 2, and the third separation chamber 3 is positioned above the fourth separation chamber 4. The first separation chamber 1 has a passageway leading to the third separation chamber 3. The purpose of this passageway is to collect the liquid in the first separation chamber 1 after the first centrifugation for subsequent processes. The second separation chamber 2 is used to store waste liquid (including the lower layer liquid after the first centrifugation, or the lower layer liquid after the first centrifugation and a small amount of upper layer liquid).

[0054] The third separation chamber 3 is equipped with a separation filter membrane 31, which is used to perform a filtration during the second centrifugation.

[0055] The fourth separation chamber 4 is also equipped with a separation filter membrane 31, which is used to perform a second filtration during the third centrifugation.

[0056] like Figure 2 As shown, the bottom of the first separation chamber 1 is connected to the second separation chamber 2 via a narrowed channel I5, and the volume of the first separation chamber 1 is larger than the volume of the second separation chamber 2; the first separation chamber 1 has a first through hole on its side wall, and the third separation chamber 3 has a corresponding second through hole on its side wall; the piston 9, which serves as a valve mechanism, passes through the first through hole and the second through hole to seal the first through hole and the second through hole.

[0057] The purpose of setting the volume of the first separation chamber 1 to be larger than that of the second separation chamber 2 is that, generally, nearly half or less of the lower layer liquid after the first centrifugation is unnecessary. Therefore, by setting the volume ratio, it can be ensured that the liquid in the first separation chamber 1 after the first centrifugation is all usable liquid. The function of the narrowed-diameter channel I5 is that, since its diameter at the end near the first separation chamber is larger than its diameter at the end near the second separation chamber, when it cooperates with the piston, the piston's squeezing action can guide the upper layer liquid originally in the narrowed-diameter channel I5 into the first separation chamber.

[0058] In this embodiment, a piston acts as a valve mechanism to open or close the passage, i.e., the first through hole, between the first and third separation chambers. The second through hole on the side wall of the third separation chamber is mainly used for injecting whole blood into the first separation chamber initially using a syringe.

[0059] like Figure 5 As shown, the first and second through holes can be closed by a piston 9. The piston 9 can be moved to disengage from the first through hole and remain inside the second through hole. To facilitate this purpose, the piston 9 is marked with graduation lines, so that when the graduation lines are flush with the outer wall of the third separation chamber 3, the piston 9 closes the second through hole while opening the first through hole.

[0060] The purpose of designing the passageway between the first separation chamber 1 and the second separation chamber 2 as a narrow-diameter channel I5 is to facilitate sealing. This invention also includes a cover 7 disposed on the third separation chamber 3, within which a cylindrical plug 8 serving as a valve mechanism is installed. The cylindrical plug 8 extends through the top wall of the first separation chamber 1 into the narrow-diameter channel I5 to seal it. The end of the cylindrical plug 8 is a tapered section 81, and a sealing groove 82 is formed at the root of the tapered section 81. The narrow-diameter channel I5 is a tapered cavity that mates with the tapered section 81, and its opening is provided with a sealing ring 51 that mates with the sealing groove 82. During sealing, the tapered section 81 is inserted into the narrow-diameter channel I5, and the sealing ring 51 performs a secondary seal with the embedded sealing groove 82, ensuring a sealing effect.

[0061] like Figure 3 As shown, to prevent the plunger 8 from loosening (especially during subsequent centrifugation), the outer wall of the third separation chamber 3 in this embodiment is provided with one-way teeth 32. The cover 7 cooperates with the one-way teeth 32 so that the cover 7 can only drive the plunger 8 to move towards the narrowing channel I5. This one-way movement of the cover 7 and the third sealing chamber 3 prevents the plunger 8 from disengaging from the narrowing channel I5. Of course, other means can also be used to achieve the above effect, such as threading the cover 7 and the third separation chamber 3.

[0062] like Figure 4 As shown, to achieve a seal between the plunger 8 and the first separation chamber 1, a hole for the plunger 8 to pass through is provided on the top wall of the first separation chamber 1. An annular groove is provided on the inner wall of the hole, and a Gladley sealing ring 11 is provided in the annular groove. After the plunger 8 is inserted into the reduced-diameter channel I5, the Gladley sealing ring 11 fills the gap between the top wall of the first separation chamber 1 and the plunger 8, thus achieving a sealing effect.

[0063] like Figure 6As shown in this embodiment, the bottom of the third separation chamber 3 is also connected to the fourth separation chamber 4 via a narrowing channel II6, the purpose of which will not be elaborated further. The valve mechanism on the narrowing channel II6 includes a valve body 10, on which a valve core 101 is threadedly fitted. Rotation of the valve core 101 allows it to move axially within the valve body 10, thereby opening or closing the narrowing channel II6. The size of the passage can be adjusted according to the amount of rotation, making the acquisition of the lower layer liquid in the third separation chamber more precise. Similarly, graduations can be provided on the valve core 101 to indicate the size of the opening passage.

[0064] In addition, such as Figure 7 As shown, to facilitate the acquisition of the bottom liquid in the fourth separation chamber 4, a puncture area 42 is provided at the bottom of the fourth separation chamber 4. The puncture area 42 has puncture holes identical to those inside the fourth separation chamber 4; the puncture holes are sealed with a membrane. The puncture area can be marked on the membrane, and puncture hole markers are provided corresponding to the positions of the puncture holes, thereby facilitating the location of the puncture holes.

[0065] In addition, the present invention also provides a method for extracting exosomes, applied to the above-mentioned apparatus for extracting exosomes, comprising:

[0066] like Figure 8A As shown, a syringe is used to inject small batches of samples, such as whole blood, into the first centrifuge mechanism through the second through hole of the third separation chamber and the first through hole of the first separation chamber. At this time, the first separation chamber and the second separation chamber are connected, while the third separation chamber and the fourth separation chamber are blocked.

[0067] In the initial state, the plunger disengages from the narrowed channel I, connecting the first and second separation chambers. The valve core is fully screwed into the valve body, blocking the third and fourth separation chambers. Additionally, the piston disengages from the first and second through holes, opening both. At this point, a small batch (e.g., 10ml-50ml, with different sizes of devices for each 10ml-50ml batch) of whole blood can be drawn using a syringe for sample extraction. The whole blood sample is then injected into the first centrifugation mechanism (including the first and second separation chambers) through the second and first through holes.

[0068] After sealing the first and second through holes with a piston, the first centrifugation is performed. After the first centrifugation is completed, the narrowed channel I between the first and second separation chambers is sealed by a cylindrical plug. Figure 8B The image shows the situation after the first centrifugation. At this point, the second separation chamber contains waste liquid, so the first and second separation chambers are blocked by a plunger.

[0069] like Figure 9AAs shown, the first through hole on the first separation chamber is opened, and the liquid in the first separation chamber is poured into the third separation chamber through the first through hole for the first filtration (the third separation chamber is equipped with a separation filter membrane, so filtration is achieved as the liquid enters) and then centrifuged a second time.

[0070] As the liquid in the first separation chamber enters the third separation chamber, a portion of the liquid is filtered out. The second centrifugation is performed on the remaining liquid after filtration.

[0071] like Figure 9B As shown, after the second centrifugation is completed, the valve mechanism between the third and fourth separation chambers is opened, the lower liquid is put into the fourth separation chamber, and then the third and fourth separation chambers are blocked again.

[0072] After the second filtration (the fourth separation chamber is equipped with a separation filter membrane, which filters the liquid as it enters), the liquid in the fourth separation chamber is centrifuged for the third time. After the third centrifugation, the lower layer of liquid is extracted through the puncture hole at the bottom of the fourth separation chamber using a syringe to obtain exosomes.

[0073] like Figure 10 As shown, after the lower layer of liquid in the third separation chamber enters the fourth separation chamber, it is first filtered, and then the filtered liquid undergoes a third centrifugation. After the third centrifugation, the lower layer of liquid is the exosome, which can then be extracted using a syringe through the puncture port.

[0074] like Figure 11 As shown, if a third centrifugation is not performed, the lower layer of liquid in the fourth separation chamber is a PRP layer, which can also be extracted using a syringe through the puncture hole.

[0075] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0076] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A device for extracting exosomes, characterized in that: The system includes a first centrifuge mechanism consisting of a first separation chamber and a second separation chamber connected axially, the first separation chamber having a larger volume than the second separation chamber; and a second centrifuge mechanism consisting of a third separation chamber and a fourth separation chamber connected axially. The first separation chamber is positioned above the second separation chamber, and the third separation chamber is positioned above the fourth separation chamber. The first centrifuge mechanism is located within the third separation chamber of the second centrifuge mechanism. Openable and closable valve mechanisms are provided between the first and second separation chambers, between the third and fourth separation chambers, and between the first and third separation chambers. Separation filter membranes are provided on the third and fourth separation chambers. The first separation chamber has a first through hole on its side wall, and the third separation chamber has a corresponding second through hole on its side wall. The first through hole and the second through hole are coaxial. A piston, which serves as a valve mechanism between the first and third separation chambers, can simultaneously penetrate the first through hole and the second through hole to block the communication channel between the first and third separation chambers, thereby allowing the first centrifugation operation to be performed in the first and second separation chambers; or it can only penetrate the second through hole to connect the first and third separation chambers, thereby allowing the upper liquid in the first separation chamber to enter the third separation chamber through the first through hole after the first centrifugation. The top of the first separation chamber is provided with a cylindrical plug, which serves as a valve mechanism; the cylindrical plug is used to close the communication channel between the first separation chamber and the second separation chamber.

2. The device for extracting exosomes according to claim 1, characterized in that: The bottom of the first separation chamber is connected to the second separation chamber via a narrowed channel I to form a connecting channel between the first separation chamber and the second separation chamber.

3. The device for extracting exosomes according to claim 2, characterized in that: The piston is marked with graduation lines, so that when the graduation lines are flush with the outer wall of the third separation chamber, the piston closes the second through hole while opening the first through hole, thereby connecting the first separation chamber and the third separation chamber.

4. The device for extracting exosomes according to claim 2, characterized in that: It also includes a cover disposed on the third separation chamber, the top of the cylindrical plug serving as a valve mechanism being fixed to the cover; and the cylindrical plug can penetrate the top wall of the first separation chamber and extend into the narrowing channel I to close the narrowing channel I.

5. The apparatus for extracting exosomes according to claim 4, characterized in that: The outer wall of the third separation chamber is provided with one-way teeth, and the cover body cooperates with the one-way teeth so that the cover body can only move in the direction of driving the cylindrical plug towards the narrowing channel I.

6. The apparatus for extracting exosomes according to claim 4, characterized in that: The end of the cylindrical plug is a tapered section, and a sealing groove is opened at the root of the tapered section; the reduced diameter channel I is a tapered cavity that mates with the tapered section, and a sealing annular protrusion that mates with the sealing groove is provided at its opening.

7. The apparatus for extracting exosomes according to claim 4, characterized in that: The top wall of the first separation chamber has a hole through which the cylindrical plug passes, and the inner wall of the hole has an annular groove, in which a Gladley sealing ring is installed.

8. The apparatus for extracting exosomes according to claim 1, characterized in that: The bottom of the third separation chamber is connected to the fourth separation chamber via a reduced-diameter channel II; the valve mechanism provided on the reduced-diameter channel II includes a valve body, and the valve body is provided with a valve core that is threadedly engaged with it.

9. The apparatus for extracting exosomes according to claim 1, characterized in that: The bottom of the fourth separation chamber is provided with a puncture area, and the puncture area has a puncture hole that communicates with the interior of the fourth separation chamber; the puncture hole is sealed with a membrane, and a pinhole mark is provided on the membrane corresponding to the position of the puncture hole.

10. A method for extracting exosomes, applied to the exosome extraction apparatus according to any one of claims 1 to 9, characterized in that... include: The piston that simultaneously closes the first through hole on the side wall of the first separation chamber and the second through hole on the side wall of the third separation chamber is opened, and the sample to be separated is injected into the first centrifuge mechanism through the second through hole of the third separation chamber and the first through hole of the first separation chamber using a syringe. At this time, the first separation chamber and the second separation chamber are connected, and the third separation chamber is blocked from the fourth separation chamber. After sealing the first and second through holes with a piston, the first centrifugation is performed. After the first centrifugation is completed, the narrowed channel I between the first and second separation chambers is sealed by a plunger, thereby obtaining the waste liquid in the second separation chamber and the upper liquid in the first separation chamber. Open the first through hole on the first separation chamber, but keep the second through hole closed, so that the first separation chamber and the third separation chamber are connected, and pour the upper liquid obtained from the first centrifugation in the first separation chamber into the third separation chamber through the first through hole for a second centrifugation; After the second centrifugation is completed, the valve mechanism between the third and fourth separation chambers is opened to allow the lower layer liquid obtained from the second centrifugation in the third separation chamber to be placed into the fourth separation chamber. Then the third and fourth separation chambers are closed again. The liquid in the fourth separation chamber is centrifuged for the third time. After the third centrifugation, the lower layer of liquid obtained after the third centrifugation is extracted through the puncture hole at the bottom of the fourth separation chamber using a syringe to obtain exosomes; or, the lower layer of liquid that has not undergone the third centrifugation is extracted through the puncture hole at the bottom of the fourth separation chamber using a syringe to obtain PRP liquid.

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