Sorting method, sorting equipment and storage medium

Abstract

The invention provides a sorting method, sorting equipment and a storage medium. The sorting method comprises the steps that sample liquid is injected into a conical centrifugal space of a centrifugal container; the sample liquid comprises non-target cells and combined cells obtained after target cells and microbubbles are combined; the centrifugal container is controlled to rotate around the rotating shaft, so that the combined cells in the sample liquid are located on a gas-liquid interface, close to the rotating shaft, in the sample liquid, and the non-target cells are located at the conical tip of the conical centrifugal space; the rotating shaft is positioned on one side of the centrifugal container away from the conical tip; the non-target cells are exported from the tapered tip into a first collection container to separate the non-target cells from the associated cells. According to the method, non-target cells and combined cells are separated, meanwhile, cell damage is avoided, the procedure of removing magnetic beads does not need to be added, operation is easy and convenient, and high recovery rate and high purity of separation can be ensured.

Description

Technical Field

[0001] This invention belongs to the field of sorting technology, and in particular relates to a sorting method, sorting equipment and storage medium. Background Technology

[0002] Currently, in the field of cell-based pharmaceuticals, it is often necessary to sort out specific target cells from a complex cell sap for processing and drug formulation. Existing technologies utilize the principle of antigen-antibody binding to specifically bind target cells to magnetic beads, thereby capturing the beads through magnetic force and screening the target cells. However, the aforementioned magnetic sorting technology has the following drawbacks: If nano-magnetic beads are used for magnetic sorting, a sorting column must be used. This column has a porous structure; target cells are adsorbed onto the column, while non-target cells flow out. During this magnetic sorting process, the target cells are simultaneously subjected to the fluid shear force and magnetic force within the porous medium, making them susceptible to damage or even breakage. Furthermore, after the target cells are adsorbed onto the column, elution is required. Due to the presence of the porous medium, the target cells can easily become trapped in the gaps, resulting in a low yield, typically only 60%-80%. For fragile patient samples, the yield will be even lower. If micron-sized magnetic beads are used for magnetic sorting, there are strict requirements on the residue of micron-sized magnetic beads during cell-based drug preparation. To meet these requirements, a further process of removing magnetic beads is needed. This process also has problems such as low purity and inconvenient operation. Summary of the Invention

[0003] This invention addresses the technical problems of low yield and low purity in the magnetic sorting of target cells in the prior art by providing a sorting method, sorting equipment and storage medium.

[0004] In view of the above technical problems, embodiments of the present invention provide a sorting method, including:

[0005] Inject sample solution into the conical centrifugation space of the centrifuge container; the sample solution includes non-target cells and combined cells obtained by binding target cells with microbubbles;

[0006] The centrifuge container is controlled to rotate around a rotation axis such that the conjoined cells in the sample solution are located at the gas-liquid interface in the sample solution near the rotation axis, and the non-target cells are located at the cone tip of the cone-shaped centrifuge space; the rotation axis is located on the side of the centrifuge container away from the cone tip;

[0007] The non-target cells are drawn from the conical tip into a first collection container to separate the non-target cells from the combined cells.

[0008] This invention also provides a sorting device, including a controller and a centrifuge container communicatively connected to the controller. The controller includes a memory, a processor, and computer-readable instructions stored in the memory and executable on the processor. When the processor executes the computer-readable instructions, it implements the sorting method described above.

[0009] A computer-readable storage medium storing a computer program that, when executed by a processor, implements the sorting method.

[0010] In this invention, the sorting method includes: injecting sample liquid into the conical centrifugation space of a centrifuge container; the sample liquid includes non-target cells and combined cells obtained by combining target cells with microbubbles; controlling the centrifuge container to rotate around a rotation axis such that the combined cells in the sample liquid are located at the gas-liquid interface of the sample liquid near the rotation axis, and the non-target cells are located at the conical tip of the conical centrifugation space; the rotation axis is located on the side of the centrifuge container away from the conical tip; and exporting the non-target cells from the conical tip to a first collection container to separate the non-target cells and the combined cells.

[0011] In this invention, when it is necessary to sort the sample solution (which includes non-target cells and combined cells obtained by binding target cells with microbubbles), the sample solution is first injected into the conical centrifugation space of the centrifuge container. Then, the centrifuge container is controlled to rotate around the rotation axis (located on the side of the centrifuge container away from the conical tip of the conical centrifugation space). This allows the sample solution to be swung to the side closer to the conical tip of the conical centrifugation space. The combined cells and non-target cells will then separate into layers. Specifically, since microbubbles are present in the combined cells of the sample solution, the combined cells will be located on the side of the sample solution closer to the rotation axis, that is, at the gas-liquid interface of the sample solution in the conical centrifugation space. The non-target cells, being relatively heavier due to not being bound to microbubbles, will be located at the conical tip of the conical centrifugation space. At this time, the non-target cells located at the conical tip can be exported to the first collection container, while the combined cells located at the gas-liquid interface near the bottom of the cone are retained in the conical centrifugation space, thereby achieving the separation of non-target cells and combined cells. In this invention, non-target cells and combined cells (i.e., target cells) can be separated by the above-mentioned sorting method without the need for a sorting column. Cell damage is avoided during the separation process, and there is no need to add a step to remove magnetic beads. The operation is simple and can ensure high recovery rate and high purity of the sorted cells. Attached Figure Description

[0012] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0013] Figure 1 This is a flowchart of the sorting method provided in the first embodiment of the present invention.

[0014] Figure 2 This is a schematic diagram of a pipeline structure for performing a sorting method according to an embodiment of the present invention.

[0015] Figure 3 This is a schematic diagram of the structure of a centrifuge container for performing a sorting method according to an embodiment of the present invention.

[0016] Figure 4 This is a flowchart of the sorting method provided in the second embodiment of the present invention.

[0017] Figure 5 This is a flowchart of the sorting method provided in the third embodiment of the present invention.

[0018] Figure 6 This is a flowchart of the sorting method provided in the fourth embodiment of the present invention.

[0019] The reference numerals in the accompanying drawings are as follows:

[0020] 1. Centrifuge container; 110. Conical centrifuge space; 111. Conical tip; 112. Conical bottom; 120. Container body; 130. First delivery pipe; 140. Second delivery pipe; 2. First main pipeline; 21. First control valve; 3. Second main pipeline; 4. Air inlet; 41. Second control valve; 5. Drive pump; 6. Rotating shaft; 7. Sample container; 8. Sample pipeline; 81. First switching valve; 9. First collection container; 10. First collection pipeline; 101. Second switching valve; 11. First cleaning container; 12. First cleaning pipeline; 121. Third switching valve; 13. Second cleaning container; 14. Second cleaning pipeline; 141. Fourth switching valve; 15. Second collection container; 16. Second collection pipeline; 161. Fifth switching valve; 17. Waste liquid container; 18. Waste liquid pipeline; 181. Sixth switching valve; 19. Pressure sensor; 20. Bubble sensor. Detailed Implementation

[0021] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0022] In the description of this invention, it should be understood that the terms "longitudinal," "radial," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicating orientation or positional relationships, are based on the orientation or positional relationships shown in the accompanying drawings and are only for the convenience of describing the invention and 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, and therefore should not be construed as a limitation of the invention. In the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0023] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0024] like Figures 1 to 3 As shown, an embodiment of the present invention provides a sorting method, the sorting method comprising the following steps S10-S30:

[0025] S10, inject sample solution into the conical centrifugation space 110 of centrifuge container 1; the sample solution includes non-target cells and combined cells obtained by combining target cells with microbubbles; understandably, microbubbles refer to particles containing gas or other low-density components, such as micron-sized phospholipid bubbles, micron-sized glass vacuoles, micron-sized polymer vacuoles, etc. The density of combined cells containing bound microbubbles is less than that of water, while non-target cells are relatively heavier (density greater than that of water) because they are not bound to microbubbles. In this embodiment, the sample solution in sample container 7 can be injected into the conical centrifugation space 110 of centrifuge container 1 even when centrifuge container 1 is not rotating around the rotation axis 6.

[0026] S20, control the centrifuge container 1 to rotate around the rotation axis 6, so that the conjoined cells in the sample solution are located at the gas-liquid interface of the sample solution near the rotation axis 6, and the non-target cells are located at the conical tip 111 of the conical centrifugation space 110; the rotation axis 6 is located on the side of the centrifuge container 1 away from the conical tip 111; since the centrifuge container 1 can rotate around the rotation axis 6, and the conical tip 111 of the conical centrifugation space 110 is located on the side of the centrifuge container 1 away from the rotation axis 6, therefore, when the centrifuge container 1 rotates at high speed around the rotation axis 6, it can... The sample solution is swirled to one side near the conical tip 111 of the conical centrifugation space 110. Since the combined cells in the sample solution contain microbubbles and have a density less than water, the combined cells will be located on the side of the sample solution closer to the rotation axis 6, that is, at the gas-liquid interface of the sample solution in the conical centrifugation space 110. The non-target cells, since they do not bind microbubbles, are relatively heavier (density greater than water). Therefore, the non-target cells will be located at the conical tip 111 of the conical centrifugation space 110. Thus, the stratification of combined cells and non-target cells is achieved under density gradient centrifugation.

[0027] The rotation axis 6 only needs to be positioned on the side of the centrifuge container 1 away from the conical tip 111 of the conical centrifuge space 110 to achieve the aforementioned stratification of combined cells and non-target cells. Therefore, the relative positional relationship between the rotation axis 6 and the conical centrifuge space 110 can be set according to specific needs. In one embodiment, the central axis of the conical centrifuge space 110 is perpendicular to the rotation axis 6 and lies on the same plane. This allows the sample solution in the conical centrifuge space 110 to be symmetrically distributed about the central axis when the centrifuge container 1 rotates around the rotation axis 6, resulting in a more uniform distribution of the sample solution within the conical centrifuge space 110 and facilitating the cell processing. Understandably, the conical centrifugal space 110 is not limited to a cone shape. It can also be a combination of a cone and other shapes, or an approximate cone shape. For example, the cone tip 111 can be cone-shaped, while the cone tail can be other shapes, such as a cylinder. Alternatively, the entire conical centrifugal space 110 can be shaped like a frustum, with the smaller diameter end of the frustum being the cone tip 111. The conical centrifugal space 110 can also be a pyramid, etc. There are no limitations here. As long as the above-mentioned layering effect of combined cells and non-target cells can be achieved, it is acceptable.

[0028] S30, the non-target cells are led from the conical tip 111 into the first collection container 9 to separate the non-target cells and the combined cells. Understandably, after the non-target cells enter the first collection container 9, a certain volume of sample solution (e.g., a preset volume determined by experimental verification based on the capacity of the conical centrifugation space 110, which can be set according to requirements) needs to be retained in the conical centrifugation space 110 to ensure that the combined cells do not flow out of the conical centrifugation space 110. In this embodiment, negative selection of the sample solution is achieved, that is, the non-target cells are separated from the sample solution and collected into the first collection container 9, while the combined cells are retained in the conical centrifugation space 110. In this invention, an optical liquid level sensor can be installed above the centrifugation container 1 to monitor the liquid level in the conical centrifugation space to ensure that a preset volume of sample solution is retained in the conical centrifugation space 110. Alternatively, a high-precision formulation module can be used to control the volume, thereby controlling the retention of a preset volume of sample solution in the conical centrifugation space 110.

[0029] In the above embodiments of the present invention, when it is necessary to sort the sample solution (the sample solution includes non-target cells and conjugate cells obtained by combining target cells with microbubbles), the sample solution is first injected into the conical centrifugation space 110 of the centrifugation container 1. Then, the centrifugation container 1 is controlled to rotate around the rotation axis 6 (the rotation axis 6 is located on the side of the centrifugation container 1 away from the conical tip 111 of the conical centrifugation space 110). This can cause the sample solution to be swung to the side closer to the conical tip 111 of the conical centrifugation space 110, and the conjugate cells and non-target cells will be separated into layers. Specifically, due to the conjugate cells in the sample solution... Microbubbles are present in the sample solution. Therefore, the conjoined cells will be located on the side of the sample solution closer to the rotation axis 6, that is, at the gas-liquid interface of the sample solution in the conical centrifugation space 110. Non-target cells, being relatively heavier due to the absence of microbubbles, will be located at the conical tip 111 of the conical centrifugation space 110. At this point, the non-target cells located at the conical tip 111 can be exported to the first collection container 9, while the conjoined cells located near the gas-liquid interface at the conical bottom 112 are retained in the conical centrifugation space 110, thereby achieving the separation of non-target cells and conjoined cells. In this invention, the separation of non-target cells and conjoined cells (i.e., target cells) can be achieved through the above-described sorting method without the need for a sorting column. Cell damage is avoided during the separation process, and there is no need to add a step of removing magnetic beads. The operation is simple and ensures high recovery rate and high purity of the sorted cells.

[0030] In one embodiment, such as Figure 2As shown, the centrifuge container 1 includes a first delivery tube 130 and a second delivery tube 140 inserted into the centrifuge container 1; the conical tip 111 of the conical centrifuge space 110 is connected to the sample container 7 through the first delivery tube 130; the conical bottom 112 of the conical centrifuge space 110 is connected to the air port 4 through the second delivery tube 140; understandably, the centrifuge container 1 may also include a container body 120, and the conical centrifuge space 110 is disposed on the container body 120; the shape of the container body 120 can be set according to requirements, for example, the container body 120 can also be set as conical. And the central axis of the conical container body 120 coincides with the central axis of the conical centrifuge space 110, and the tip of the conical container body 120 is aligned with the direction of the conical tip 111 of the conical centrifuge space 110. However, the shape of the container body 120 can also be set to other shapes according to requirements, such as cylindrical, square, etc., and is not limited here.

[0031] Furthermore, such as Figure 1 and Figure 2 As shown, the end of the first conveying pipe 130 away from the first main pipeline 2 and the end of the first conveying pipe 130 away from the second conveying pipe 140 are both inserted into the conical centrifugal space 110 from the conical bottom end 112; that is, in this embodiment, both the first conveying pipe 130 and the second conveying pipe 140 are inserted from the conical bottom end 112, but the first conveying pipe 130 directly inserts its opening from the conical bottom end 112 to the conical tip 111, while the second conveying pipe 140 can directly insert its opening to the conical bottom end 112.

[0032] Furthermore, the end of the first delivery pipe 130 away from the first main pipeline 2 is inserted into the conical centrifugal space 110 from the conical tip 111, and the end of the first delivery pipe 130 away from the second delivery pipe 140 is inserted into the conical centrifugal space 110 from the conical bottom 112. That is, in this embodiment, the first delivery pipe 130 is directly inserted into the conical centrifugal space 110 from the conical tip 111, and the insertion port is also located at the conical tip 111; the second delivery pipe 140 is inserted from the conical bottom 112, and the insertion port is located at the conical bottom 112.

[0033] In one embodiment, the air inlet 4 is also equipped with an air filter. That is, in this embodiment, an air filter is installed at the air inlet 4 to ensure that the gas entering the second main pipeline 3 from the external environment is sterile, thereby ensuring a sterile environment in the conical centrifuge space 110. A second control valve 41 is provided between the air inlet 4 and the centrifuge container 1. The second control valve 41 can be located on the second main pipeline 3, or it can be located on a ventilation pipe connected to the second main pipeline 3; there is no limitation on this.

[0034] Further, step S10, namely injecting sample liquid into the conical centrifugation space 110 of the centrifugation container 1, includes: after the conical centrifugation space 110 is connected to the outside air through the second delivery tube 140 and the air port 4, injecting the sample liquid in the sample container 7 into the conical centrifugation space 110 through the first delivery tube 130 until the sample liquid in the conical centrifugation space 110 reaches a preset height, the preset height being lower than the height of the second delivery tube 140 inserted into the tube opening at the bottom end 112 of the cone.

[0035] That is, in this embodiment, the sample liquid in sample container 7 is injected into centrifuge container 1 through the first delivery tube 130, and at this time, centrifuge container 1 needs to be connected to the outside air through the second delivery tube 140 to balance the air pressure in the conical centrifuge space 110. Understandably, since centrifuge container 1 does not rotate when the sample liquid enters the conical centrifuge space 110, the total density of the target cells and the combined cells after binding with microbubbles in the sample liquid is less than that of water, while the density of non-target cells is greater than that of water. Under density gradient centrifugation, the target cells will float on the surface of the sample liquid, and the non-target cells will sink to the bottom of the sample liquid, and the two will separate. At this time, in order to prevent the combined cells located on the surface of the liquid from overflowing from the opening of the second delivery tube 140 inserted into the conical centrifuge space 110, it is necessary to control the height of the sample liquid entering the conical centrifuge space 110 to be less than the height of the opening of the second delivery tube 140. Therefore, the sample liquid entering the centrifuge container 1 in step S10 can be set to a preset height (under the clamp with a defined volume and shape of the conical centrifuge space 110, the preset height corresponds to a preset volume of sample liquid, and can therefore be considered as the injection of a preset volume of sample liquid. The preset height can be set according to requirements). Thus, the sample liquid at the preset height needs to be lower than the height of the second delivery tube 140 inserted into the conical bottom end 112 within the conical centrifuge space 110 to ensure that the conjoined cells do not overflow from the second delivery tube 140. In some embodiments, the insertion port of the second delivery tube 140 is located above the conical centrifuge space 110. This increases the capacity of sample liquid that can be input into the conical centrifuge space 110, thereby increasing the capacity of sample liquid that can be sorted in a single operation, and thus improving cell sorting efficiency.

[0036] In one embodiment, such as Figure 2As shown, the first delivery pipe 130 is connected to the first main pipeline 2, and a drive pump 5 is provided on the first main pipeline 2; the end of the first main pipeline 2 away from the first delivery pipe 130 is connected to the sample container 7 and the first collection container 9, and the second delivery pipe 140 is connected to the air port 4 through the second main pipeline 3; understandably, the first main pipeline 2 is mounted on the drive pump 5, wherein the drive pump 5 can be a peristaltic pump or other pump that can drive the fluid in the pipeline, and its specific type is not limited here. Understandably, the sample container 7 can be connected to the first end of the first main pipeline 2 through the sample pipeline 8, and the sample pipeline 8 is provided with a first switching valve 81 for controlling the opening and closing of the sample pipeline 8. Further, the first collection container 9 can be connected to the first end of the first main pipeline 2 through the first collection pipeline 10, and the first collection pipeline 10 is provided with a second switching valve 101 for controlling the opening and closing of the first collection pipeline 10.

[0037] Further, the step of injecting the sample liquid in the sample container 7 into the conical centrifugation space 110 through the first delivery pipe 130 includes: controlling the drive pump 5 to drive the sample liquid in the sample container 7 to be injected into the conical centrifugation space 110 sequentially through the first main pipeline 2 and the first delivery pipe 130; that is, in this embodiment, when the first switch valve 81 is open and the second switch valve 101 is closed, the drive pump 5 can be controlled to drive the sample liquid in the sample container 7 to be injected into the conical centrifugation space 110 sequentially through the first main pipeline 2 and the first delivery pipe 130.

[0038] The step of exporting the non-target cells from the conical tip 111 to the first collection container 9 includes: controlling the drive pump 5 to drive the sample liquid in the conical centrifugation space 110 from the conical tip 111 sequentially through the first delivery tube 130 and the first main pipeline 2 into the first collection container 9. That is, in this invention, in step S20, when both the first switch valve 81 and the second switch valve 101 are closed, the centrifugation container 1 can be controlled to rotate around the rotation axis 6, so that the conjugated cells in the sample liquid are located at the gas-liquid interface in the sample liquid near the rotation axis 6, and the non-target cells are located at the conical tip 111 of the conical centrifugation space 110; the rotation axis 6 is located on the side of the centrifugation container 1 away from the conical tip 111. Furthermore, after the first switch valve 81 is closed and the second switch valve 101 is opened, the drive pump 5 can be controlled to drive the sample liquid in the conical centrifugation space 110 from the conical tip 111 sequentially through the first delivery tube 130 and the first main pipeline 2 into the first collection container 9.

[0039] In one embodiment, the first main pipeline 2 is connected to the cleaning assembly; further, as... Figure 4As shown, after step S30, that is, after the non-target cells are extracted from the conical tip 111 into the first collection container 9 to separate the non-target cells and the combined cells, the process includes:

[0040] S40, perform at least one tubing cleaning operation to flush residual sample solution in the tubing connecting the first collection container 9 and the conical centrifugation space 110 into the first collection container 9 and / or the conical centrifugation space 110 using cleaning fluid injected through the cleaning component. The specific composition of the cleaning component can be configured as needed. During the tubing cleaning operation, the cleaning component can inject cleaning fluid into the tubing that previously guided non-target cells into the first collection container 9 (i.e., the tubing between the first collection container 9 and the conical centrifugation space 110), thereby flushing residual sample solution in the tubing into the first collection container 9 and / or the conical centrifugation space 110. Understandably, the tubing cleaning operation can be configured to be performed once or multiple times as needed. Repeating the operation multiple times can enhance the cleaning effect, with the aim of thoroughly cleaning the residual sample solution in the tubing.

[0041] In one embodiment, such as Figure 2 As shown, the first main pipeline 2 is also provided with a first control valve 21 located between the drive pump 5 and the centrifuge container 1; the cleaning assembly includes a first cleaning container 11, a second cleaning container 13, a first cleaning pipeline 12 connecting the first cleaning container 11 and the first main pipeline 2, and a second cleaning pipeline 14 connecting the second cleaning container 13 and the first main pipeline 2; the first connection point between the first cleaning pipeline 12 and the first main pipeline 2 is located between the first control valve 21 and the drive pump 5; the second connection point between the second cleaning pipeline 14 and the first main pipeline 2 is located on the side of the drive pump 5 away from the centrifuge container 1; understandably, the first cleaning pipeline 12 is provided with a third switching valve 121 for controlling the on / off state of the first cleaning pipeline 12. The second cleaning pipeline 14 is provided with a fourth switching valve 141 for controlling the on / off state of the second cleaning pipeline 14.

[0042] Further, step S40, namely performing at least one tubing cleaning operation to flush residual sample solution in the tubing connecting the first collection container 9 and the conical centrifugation space 110 into the first collection container 9 and / or the conical centrifugation space 110 through the cleaning fluid injected by the cleaning assembly, includes:

[0043] When the first control valve 21 is closed, the cleaning fluid in the first cleaning container 11 is driven by the drive pump 5 to flow through the first cleaning pipeline 12 and the first main pipeline 2 into the first collection container 9. In this embodiment, after the first control valve 21 is closed (the third switch valve 121 and the second switch valve 101 are open, and the other switch valves are closed), the cleaning fluid in the first cleaning container 11 is driven by the drive pump 5 to flow through the first cleaning pipeline 12 and the first main pipeline 2 (and the first collection pipeline 10) into the first collection container 9. This allows for the cleaning of a portion of the first main pipeline 2 near the first collection container 9 (and the first collection pipeline 10), rinsing away any non-target cells remaining in the pipeline into the first collection container 9.

[0044] The first control valve 21 is opened, and the driving pump 5 drives the cleaning fluid in the second cleaning container 13 to flow through the second cleaning pipeline 14, the first main pipeline 2, and the first delivery pipe 130 into the conical centrifuge space 110. Specifically, after opening the first control valve 21 and the second control valve 41 (the fourth switch valve 141 is open, and the other switch valves are closed), the driving pump 5 drives the cleaning fluid in the second cleaning container 13 to flow through the second cleaning pipeline 14, the first main pipeline 2, and the first delivery pipe 130 into the conical centrifuge space 110, and air can enter the conical centrifuge space 110 from the air port 4 through the second main pipeline 3 to balance the air pressure. During the above process, cleaning fluid can be injected into the conical centrifuge space 110 to clean the first main pipeline 2 and the first delivery pipe 130, rinsing the residual sample liquid in the pipeline into the conical centrifuge space 110. Understandably, during the above process, the cleaning solution must not exceed the opening of the second delivery tube 140 inserted into the conical centrifuge space 110 to avoid the conical cells overflowing from the opening.

[0045] In one embodiment, such as Figure 5 As shown, after step S30, that is, after the non-target cells are extracted from the conical tip into the first collection container, the method further includes:

[0046] After the centrifuge container stops rotating around the rotation axis, a microbubble annihilation operation is performed. This operation includes increasing the pressure in the conical centrifuge space to a preset annihilation pressure, causing the microbubbles of the conjoined cells located in the conical centrifuge space to annihilate. The preset annihilation pressure can be set as needed, but it must be greater than atmospheric pressure. Specifically, in one embodiment, after increasing the pressure in the conical centrifuge space to the preset annihilation pressure, this preset annihilation pressure can be maintained for a preset duration (the preset duration can be set as needed), after which it can be confirmed that all microbubbles in the conjoined cells of the sample solution have been annihilated. In another embodiment, an optical liquid level sensor can be installed above the centrifuge container 1 to monitor the liquid level in the conical centrifuge space. When the optical liquid level sensor detects that no microbubbles remain on the liquid surface, it can be confirmed that all microbubbles in the conjoined cells of the sample solution have been annihilated.

[0047] Furthermore, such as Figure 2 As shown, the centrifuge container 1 includes a first delivery pipe 130 and a second delivery pipe 140 inserted into the centrifuge container 1; the conical tip 111 of the conical centrifuge space 110 is connected to the first main pipeline 2 through the first delivery pipe 130; the conical bottom end 112 of the conical centrifuge space 110 is connected to the air port 4 through the second delivery pipe 140 and the second main pipeline 3; a drive pump 5 is provided on the first main pipeline 2; the end of the first main pipeline 2 away from the first delivery pipe 130 is connected to a cleaning assembly; a second control valve 41 is also provided on the second main pipeline 3; at this time, increasing the pressure in the conical centrifuge space to the preset annihilation pressure includes:

[0048] The second control valve 41 is closed to disconnect the passage between the conical centrifugal space 110 and the air port 4. The cleaning fluid in the cleaning assembly is driven by the drive pump 5 to enter the conical centrifugal space 110 through the first main pipeline 2 and the first delivery pipe 130, so as to increase the pressure in the conical centrifugal space 110 to the preset annihilation pressure.

[0049] Understandably, step S50 can also be performed after at least one pipeline cleaning operation in step S40, and the specific steps will not be described in detail here.

[0050] In a further embodiment, after the above-mentioned cleaning operation is completed, the centrifuge container 1 can be stopped from rotating around the rotation axis 6, thereby allowing the sample solution containing the conical centrifuge space 110 to remain still. Then, the first control valve 21 can be opened and the second control valve 41 closed (the fourth switch valve 141 is open, and the remaining switch valves are closed) to disconnect the passage between the conical centrifuge space 110 and the air port 4. The cleaning solution in the second cleaning container 13 is driven by the drive pump 5 to enter the conical centrifuge space 110 through the first main pipeline 2 and the first delivery pipe 130, thereby increasing the pressure in the conical centrifuge space 110. After the pressure in the conical centrifuge space 110 is increased to a preset annihilation pressure and maintained for a preset time, it is confirmed that all microbubbles in the conical cells in the sample solution have been annihilated. Thus, this embodiment can annihilate and remove microbubbles in the conical cells, achieving the separation of microbubbles from the target cells.

[0051] In one embodiment, such as Figure 2 As shown, a pressure sensor 19 is also provided on the first main pipeline 2; specifically, the pressure sensor 19 is located between the drive pump 5 and the centrifuge container 1. Further, in step S50, after the cleaning fluid in the cleaning assembly is driven by the drive pump 5 through the first main pipeline 2 and the first delivery pipe 130 into the conical centrifuge space 110, the following steps are also included:

[0052] The pressure sensor 19 monitors the pressure information in the first main pipeline 2 in real time and determines the real-time pressure value based on the pressure information. When the real-time pressure value is greater than or equal to the preset annihilation pressure, it is confirmed that the pressure in the conical centrifugal space 110 has increased to the preset annihilation pressure. That is, the pressure sensor 19 is used to detect the pressure information in the first main pipeline 2, which is connected to the conical centrifugal space 110, when the second control valve 41 controls the second main pipeline 3 to close, and then determines the real-time pressure value in the first main pipeline 2 based on the pressure information. Since the first main pipeline 2 is connected to the conical centrifugal space 110, the real-time pressure value is the actual pressure in the conical centrifugal space 110. Therefore, when the real-time pressure value is greater than or equal to the preset annihilation pressure, it can be confirmed that the pressure in the conical centrifugal space 110 has increased to the preset annihilation pressure.

[0053] In one embodiment, such as Figure 2As shown, the centrifuge container 1 includes a first delivery pipe 130 and a second delivery pipe 140 inserted into the centrifuge container 1; the conical tip 111 of the conical centrifuge space 110 is connected to the first main pipeline 2 through the first delivery pipe 130; the conical bottom end 112 of the conical centrifuge space 110 is connected to the air port 4 through the second delivery pipe 140 and the second main pipeline 3; a drive pump 5 is provided on the first main pipeline 2; the end of the first main pipeline 2 away from the first delivery pipe 130 is connected to the second collection container 15. Further, the first main pipeline 2 is connected to the second collection container 15 through the second collection pipeline 16, and the third connection point between the second collection pipeline 16 and the first main pipeline 2 is located on the side of the drive pump 5 away from the centrifuge container 1. Understandably, a fifth switching valve 161 for controlling the opening and closing of the second collection pipeline 16 is provided on the second collection pipeline 16. Further, as Figure 6 As shown, after step S50, that is, after performing the microbubble annihilation operation, the following steps are included:

[0054] S60, control the second control valve 41 to open, so as to connect the conical centrifuge space 110 with the air port 4, and reduce the pressure in the conical centrifuge space 110 to be consistent with the external atmospheric pressure; in this embodiment, after confirming that the microbubbles in the combined cells have been annihilated, the first control valve 21 and the second control valve 41 can be opened (while controlling the centrifuge container 1 to rotate at a low speed or remain stationary to avoid the sample liquid from being sprayed out from the port of the second delivery tube 140), so as to connect the conical centrifuge space 110 with the air port 4, and reduce the pressure in the conical centrifuge space 110 to be consistent with the external atmospheric pressure, so as to release the pressure in the conical centrifuge space 110.

[0055] S70, control the centrifuge container 1 to rotate around the rotation axis 6 so that the target cells with annihilated microbubbles in the conical centrifuge space 110 are located at the conical tip 111, and drive the target cells with annihilated microbubbles in the conical centrifuge space 110 to be discharged to the second collection container 15 through the first delivery pipe 130 and the first main pipeline 2 by the drive pump 5. In this step, the centrifuge container 1 needs to be controlled to rotate at high speed around the rotation axis 6. At this time, since the microbubbles in the conical centrifuge space 110 have been annihilated, the density of the remaining target cells with annihilated microbubbles will be greater than that of water. Therefore, the target cells with annihilated microbubbles will be located at the tip of the cone 111 under the action of centrifugal force. At this time, the fifth switch valve 161 is opened and the other switch valves are closed. The target cells with annihilated microbubbles in the conical centrifuge space 110 can be driven by the drive pump 5 to be discharged through the first delivery pipe 130 and the first main pipeline 2 to the second collection container 15. Thus, after the non-target cells are sorted into the first collection container 9, the target cells after removing microbubbles are also sorted into the second collection container 15.

[0056] Understandably, in this embodiment, positive selection of the sample solution is achieved, that is, non-target cells are separated from the sample solution and collected into the first collection container 9, while target cells are collected into the second collection container 15. In this invention, the separation of target cells and non-target cells can be achieved without setting up a sorting column, and cell damage is avoided.

[0057] In one embodiment, such as Figure 2 As shown, the first main pipeline 2 connects to the cleaning assembly; the end of the second main pipeline 3 furthest from the centrifuge container 1 connects to the waste liquid container 17; the waste liquid container 17 connects to the second main pipeline 3 via a waste liquid pipeline 18; the fourth connection point between the waste liquid pipeline 18 and the second main pipeline 3 is located between the air port 4 and the centrifuge container 1. Understandably, a sixth switching valve 181 for controlling the opening and closing of the waste liquid pipeline 18 is provided on the waste liquid pipeline 18. Further, in step S70, before the target cells with annihilated microbubbles in the conical centrifuge space 110 are driven by the drive pump 5 to be exported to the second collection container 15 through the first delivery pipe 130 and the first main pipeline 2, the following steps are also included:

[0058] Perform a rinsing operation, which includes: driving the cleaning liquid in the cleaning assembly through the drive pump 5 into the conical centrifuge space 110 through the first main pipeline 2 and the first delivery pipe 130, so that the liquid in the conical centrifuge space 110 covers the opening of the second delivery pipe 140 inserted into the bottom end 112 of the cone under centrifugal action, and then extracting the waste liquid, except for the target cells with annihilated microbubbles, into the waste liquid container 17 through the second delivery pipe 140 and the second main pipeline 3. That is, in this embodiment, before sorting the target cells into the second collection container 15, in order to reduce the volume of the final preparation, a washing operation can be performed on the sample solution containing the annihilated microbubbles of the target cells to remove waste liquid from the sample solution in the conical centrifuge space. Specifically, the first control valve 21 is opened and the second control valve 41 is closed to disconnect the connection between the air port 4 and the second main pipeline 3. At the same time, the sixth switch valve 181 and the fourth switch valve 141 are opened (the remaining switch valves are closed) so that the waste liquid pipeline 18 is connected to the conical centrifuge space 110 through the second main pipeline 3, and the second washing container 13 is connected to the first main pipeline 2 through the washing pipeline. Afterwards, the centrifuge container 1 is controlled to rotate around the rotation axis 6. At this time, since the microbubbles in the conical centrifuge space 110 have been annihilated, the density of the remaining annihilated microbubbles of the target cells will be greater than that of water. Therefore, the annihilated microbubbles are removed from the sample solution. The target cells with annihilated microbubbles will be located at the tip 111 of the cone under centrifugal force, while the waste liquid is located on the side of the cone centrifugal space 110 near the tail end of the cone. At this time, the cleaning liquid in the second cleaning container 13 can be driven by the drive pump 5 through the cleaning pipeline, the first main pipeline 2 and the first delivery pipe 130 into the cone centrifugal space 110, so that the liquid in the cone centrifugal space 110 covers the opening of the second delivery pipe 140 inserted at the bottom end 112 of the cone under centrifugal force. Then, the sample liquid covering the opening of the second delivery pipe 140 is the waste liquid. Therefore, the waste liquid, except for the target cells with annihilated microbubbles, can be extracted to the waste liquid container 17 through the second delivery pipe 140 and the second main pipeline 3; while the target cells with annihilated microbubbles that are always located at the tip 111 of the cone will be retained in the cone centrifugal space 110.

[0059] In some embodiments, such as Figure 2As shown, a bubble sensor 20 is provided between the sample container 7 and the centrifuge container 1. The sorting method further includes: detecting whether the sample liquid in the sample container 7 has completely drained using the bubble sensor 20; if it has not drained completely, returning to step S10 to continue injecting sample liquid into the conical centrifugation space 110 of the centrifuge container 1 to sort the non-target cells and conjoint cells (i.e., target cells) in the sample liquid again. Further, the bubble sensor 20 is located between the drive pump 5 and the sample container 7. In this embodiment, if the sample liquid volume in the sample container 7 is too large and the centrifuge container 1 cannot process it all at once, the above sorting method can be repeated multiple times. In this case, it is necessary to use the bubble sensor 20 to determine whether all the sample liquid in the sample container 7 has drained completely, that is, to determine whether the sample liquid in the sample container 7 has been completely processed.

[0060] It should be understood that the sequence number of each step in the above embodiments does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present invention.

[0061] The present invention also provides a sorting device, including a controller and a centrifuge container 1 communicatively connected to the controller. The controller includes a memory, a processor, and computer-readable instructions stored in the memory and executable on the processor. When the processor executes the computer-readable instructions, it implements the above-described sorting method. In the sorting device of the above embodiment of the present invention, when it is necessary to sort a sample solution (the sample solution includes non-target cells and conjugate cells obtained by combining target cells with microvesicles), the sample solution is first injected into the conical centrifugation space 110 of the centrifuge container 1. Then, the centrifuge container 1 is controlled to rotate around a rotation axis 6 (the rotation axis 6 is located on the side of the centrifuge container 1 away from the conical tip 111 of the conical centrifugation space 110), which can swing the sample solution to the side close to the conical tip 111 of the conical centrifugation space 110. Furthermore, the conjugate cells and non-target cells will be separated into layers. Specifically, due to the conjugate cells in the sample solution... Microbubbles exist within the cells; therefore, the conjoined cells will be located on the side of the sample solution closer to the rotation axis 6, i.e., at the gas-liquid interface of the sample solution in the conical centrifugation space 110. Non-target cells, being relatively heavier due to the absence of microbubbles, will be located at the conical tip 111 of the conical centrifugation space 110. At this point, the non-target cells located at the conical tip 111 can be exported to the first collection container 9, while the conjoined cells located near the gas-liquid interface at the conical bottom 112 remain in the conical centrifugation space 110, thus achieving the separation of non-target cells and conjoined cells. In this invention, the separation of non-target cells and conjoined cells (i.e., target cells) can be achieved through the above-described sorting method without the need for a sorting column. Cell damage is avoided during the separation process, and there is no need for additional steps such as removing magnetic beads. The operation is simple and ensures high recovery rate and high purity in the sorting.

[0062] Understandably, the execution functions of this controller correspond one-to-one with the sorting methods in the above embodiments. Specific limitations of the controller can be found in the limitations of the sorting methods described above, and will not be repeated here. Each submodule in the above controller can be implemented entirely or partially through software, hardware, or a combination thereof. Each submodule can be embedded in hardware or independently of the processor in the computer device, or stored in software in the memory of the computer device, so that the processor can call and execute the operations corresponding to each submodule.

[0063] The present invention also provides a computer-readable storage medium storing a computer program that, when executed by a processor, implements the steps of the sorting method.

[0064] Those skilled in the art will understand that all or part of the processes in the methods of the above embodiments can be implemented by instructing related hardware with computer-readable instructions. These computer-readable instructions can be stored in a non-volatile readable storage medium or a volatile readable storage medium. When executed, these computer-readable instructions can include the processes of the embodiments of the above methods. Any references to memory, storage, databases, or other media used in the embodiments provided in this application can include non-volatile and / or volatile memory. Non-volatile memory may include read-only memory (ROM), programmable ROM (PROM), electrically programmable ROM (EPROM), electrically erasable programmable ROM (EEPROM), or flash memory. Volatile memory may include random access memory (RAM) or external cache memory. By way of illustration and not limitation, RAM is available in a variety of forms, such as static RAM (SRAM), dynamic RAM (DRAM), synchronous DRAM (SDRAM), dual data rate SDRAM (DDRSDRAM), enhanced SDRAM (ESDRAM), synchronous link DRAM (SLDRAM), RAMbus direct RAM (RDRAM), direct memory bus dynamic RAM (DRDRAM), and memory bus dynamic RAM (RDRAM).

[0065] Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the above-described division of functional units and modules is used as an example. In practical applications, the above functions can be assigned to different functional units and modules as needed, that is, the internal structure of the system can be divided into different functional units or modules to complete all or part of the functions described above.

[0066] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. A sorting method, characterized in that, include: Inject the sample solution into the conical centrifugation space of the centrifuge container; The sample solution includes non-target cells and combined cells obtained by combining target cells with microbubbles; The centrifuge container is controlled to rotate around a rotation axis such that the conjoined cells in the sample solution are located at the gas-liquid interface in the sample solution near the rotation axis, and the non-target cells are located at the cone tip of the cone-shaped centrifuge space; the rotation axis is located on the side of the centrifuge container away from the cone tip; The non-target cells are drawn from the conical tip into a first collection container to separate the non-target cells from the combined cells.

2. The sorting method according to claim 1, characterized in that, The centrifuge container includes a first delivery tube and a second delivery tube inserted into the centrifuge container; the conical tip of the conical centrifuge space is connected to the sample container through the first delivery tube; the conical bottom of the conical centrifuge space is connected to the air inlet through the second delivery tube. The injection of sample solution into the conical centrifugation space of the centrifuge container includes: After the conical centrifuge space is connected to the outside air through the second delivery tube and the air port, the sample liquid in the sample container is injected into the conical centrifuge space through the first delivery tube until the sample liquid in the conical centrifuge space reaches a preset height, which is lower than the height of the second delivery tube inserted into the bottom of the cone.

3. The sorting method according to claim 2, characterized in that, The first delivery pipe is connected to the first main pipeline, and a drive pump is provided on the first main pipeline; the end of the first main pipeline away from the first delivery pipe is connected to the sample container and the first collection container, and the second delivery pipe is connected to the air port through the second main pipeline. The step of injecting the sample liquid from the sample container into the conical centrifugation space through the first delivery tube includes: The drive pump is controlled to drive the sample liquid in the sample container to be injected sequentially into the conical centrifuge space through the first main pipeline and the first delivery pipe; The step of exporting the non-target cells from the conical tip into the first collection container includes: The drive pump is controlled to drive the sample liquid in the conical centrifugation space from the tip of the cone through the first delivery tube and the first main pipeline into the first collection container.

4. The sorting method according to claim 3, characterized in that, The first main pipeline is connected to the cleaning component; The step of guiding the non-target cells from the conical tip into the first collection container to separate the non-target cells and the combined cells includes: Perform at least one tubing cleaning operation to flush residual sample solution in the tubing connecting the first collection container and the conical centrifuge space into the first collection container and / or the conical centrifuge space using cleaning fluid injected through the cleaning assembly.

5. The sorting method according to claim 4, characterized in that, The first main pipeline is further provided with a first control valve located between the drive pump and the centrifuge container; the cleaning assembly includes a first cleaning container, a second cleaning container, a first cleaning pipeline connecting the first cleaning container and the first main pipeline, and a second cleaning pipeline connecting the second cleaning container and the first main pipeline; the first connection point between the first cleaning pipeline and the first main pipeline is located between the first control valve and the drive pump; the second connection point between the second cleaning pipeline and the first main pipeline is located on the side of the drive pump away from the centrifuge container; Performing at least one tubing cleaning operation to flush residual sample solution in the tubing connecting the first collection container and the conical centrifuge space into the first collection container and / or the conical centrifuge space via cleaning fluid injected through the cleaning assembly includes: The first control valve is closed, and the cleaning fluid in the first cleaning container is driven by the drive pump to flow through the first cleaning pipeline and the first main pipeline into the first collection container. The first control valve is opened, and the cleaning liquid in the second cleaning container is driven by the drive pump to flow through the second cleaning pipeline, the first main pipeline and the first delivery pipe into the conical centrifugal space.

6. The sorting method according to claim 1, characterized in that, After the non-target cells are extracted from the conical tip into the first collection container, the process further includes: After controlling the centrifuge container to stop rotating around the rotation axis, a microbubble annihilation operation is performed. The microbubble annihilation operation includes increasing the pressure in the conical centrifuge space to a preset annihilation pressure so that the microbubbles of the conjoined cells located in the conical centrifuge space are annihilated.

7. The sorting method according to claim 6, characterized in that, The centrifuge container includes a first delivery pipe and a second delivery pipe inserted into the centrifuge container; the conical tip of the conical centrifuge space is connected to a first main pipeline through the first delivery pipe; the conical bottom of the conical centrifuge space is connected to an air inlet through the second delivery pipe and the second main pipeline; a drive pump is provided on the first main pipeline; the end of the first main pipeline away from the first delivery pipe is connected to a second collection container; A second control valve is installed on the second main pipeline; After performing the microbubble annihilation operation, the following is included: The second control valve is opened to connect the conical centrifugal space with the air port, thereby reducing the pressure in the conical centrifugal space to match the external atmospheric pressure. The centrifuge container is controlled to rotate around a rotation axis so that the target cells with annihilated microbubbles in the conical centrifuge space are located at the tip of the cone, and the target cells with annihilated microbubbles in the conical centrifuge space are driven by the drive pump to be discharged to the second collection container through the first delivery tube and the first main pipeline.

8. The sorting method according to claim 7, characterized in that, The first main pipeline connects to the cleaning assembly; the end of the second main pipeline furthest from the centrifuge container connects to the waste liquid container. Before the target cells with annihilated microbubbles in the conical centrifuge space are driven by the drive pump to be exported to the second collection container through the first delivery tube and the first main pipeline, the process further includes: Perform a rinsing operation, which includes: driving the cleaning fluid in the cleaning assembly through the drive pump into the conical centrifuge space through the first main pipeline and the first delivery pipe, so that the liquid in the conical centrifuge space covers the opening of the second delivery pipe inserted at the bottom of the cone under centrifugal action, and then extracting the waste liquid, except for the target cells whose microbubbles have been annihilated, into the waste liquid container through the second delivery pipe and the second main pipeline.

9. A sorting device, characterized in that, The system includes a controller and a centrifuge container communicatively connected to the controller. The controller includes a memory, a processor, and computer-readable instructions stored in the memory and executable on the processor. When the processor executes the computer-readable instructions, it implements the sorting method as described in any one of claims 1 to 8.

10. A computer-readable storage medium storing a computer program, characterized in that, When the computer program is executed by the processor, it implements the sorting method as described in any one of claims 1 to 8.

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