Sorting method, sorting device and storage medium

Abstract

Provided are a sorting method, a sorting device, and a storage medium. The sorting method comprises: injecting a sample liquid into a conical centrifugal space of a centrifugal container, the sample liquid containing non-target cells and cell-microbubble complexes obtained after binding target cells to microbubbles; controlling the centrifugal container to rotate around a rotation axis, so that the cell-microbubble complexes in the sample liquid are located at a gas-liquid interface in the sample liquid close to the rotation axis, the non-target cells are located at a conical tip of the conical centrifugal space, and the rotation axis is located on the side of the centrifugal container away from the conical tip; and discharging the non-target cells from the conical tip into a first collection container to separate the non-target cells and the cell-microbubble complexes. Cell damage is avoided while separating the non-target cells and the cell-microbubble complexes, there is no need to add a procedure for removing magnetic beads, etc. The operation is simple, and high recovery rate and high purity of sorting can be ensured.

Description

Sorting method, sorting device and storage medium

[0001] The present application claims priority to the Chinese patent application No. 202411120962.0, filed on August 15, 2024, and entitled "Sorting method, sorting device and storage medium", the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application belongs to the technical field of sorting, in particular relates to a sorting method, a sorting device and a storage medium. BACKGROUND

[0003] At present, in the field of cell pharmaceuticals, it is often necessary to sort a certain specific target cell from a complex component cell solution, process and form a drug. In the prior art, the principle of antigen-antibody combination can be used to make the target cell specifically combined with magnetic beads, so as to capture the magnetic beads by magnetic force, and then complete the screening of the target cell. However, the inventors realize that the above-mentioned magnetic sorting technology has the following defects: if nanometer magnetic beads are used for magnetic sorting, a sorting column must be used, and the sorting column is a porous medium structure. The target cells are adsorbed on the sorting column, and the non-target cells flow out of the sorting column. In the above-mentioned process of magnetic sorting through the sorting column, the target cells need to be subjected to the fluid shear force inside the porous medium and the magnetic force of the magnetic field at the same time, which will cause the cells to be easily damaged or even broken. In addition, after the target cells are adsorbed on the sorting column, the target cells in the sorting column need to be eluted out. At this time, due to the existence of the porous medium, the target cells are easily hidden in the gap, which will also result in a low yield of the target cells, generally only 60%-80%. If the patient sample is fragile, the yield will be even lower. If micro-magnetic beads are used for magnetic sorting, there are strict requirements for the residual micro-magnetic beads during cell pharmaceuticals. In order to meet the requirements, a process of removing the magnetic beads needs to be further added. The above-mentioned process also has problems of low purity and inconvenient operation. SUMMARY

[0004] The present application provides a sorting method, a sorting device and a storage medium to solve the technical problems of low yield and low purity of the target cells in the prior art.

[0005] In view of the above technical problems, the present application provides a sorting method, which comprises:

[0006] Injecting a sample solution into a conical centrifugal space of a centrifugal container; the sample solution comprises non-target cells and combined cells obtained by combining target cells with microbubbles;

[0007] controlling the centrifugal container to rotate around a rotation axis, so that the combined cells in the sample liquid are located at an air-liquid interface of the sample liquid close to the rotation axis, and the non-target cells are located at a tapered tip of the tapered centrifugal space; the rotation axis is located at a side of the centrifugal container far from the tapered tip;

[0008] directing the non-target cells from the tapered tip to a first collection container to separate the non-target cells and the combined cells.

[0009] The embodiment of the present application further provides a sorting device, which comprises a centrifugal container and a controller in communication connection with the centrifugal container; the controller comprises a memory, a processor and computer readable instructions stored in the memory and executable on the processor; the processor executes the computer readable instructions to implement the sorting method.

[0010] A computer readable storage medium, which stores a computer program; the computer program is executed by a processor to implement the sorting method.

[0011] In the present application, the sorting method comprises the following steps: injecting a sample liquid into a tapered centrifugal space of a centrifugal container; the sample liquid comprises non-target cells and combined cells obtained by combining target cells with microvesicles; controlling the centrifugal container to rotate around a rotation axis, so that the combined cells in the sample liquid are located at an air-liquid interface of the sample liquid close to the rotation axis, and the non-target cells are located at a tapered tip of the tapered centrifugal space; the rotation axis is located at a side of the centrifugal container far from the tapered tip; and directing the non-target cells from the tapered tip to a first collection container to separate the non-target cells and the combined cells.

[0012] In the present application, when the sample liquid (the sample liquid includes non-target cells and combined cells obtained after the target cells are combined with microbubbles) needs to be sorted, the sample liquid is first injected into the conical centrifugal space of the centrifugal container, and then the centrifugal container is controlled to rotate around the rotation axis (the rotation axis is located on the side of the centrifugal container away from the conical tip of the conical centrifugal space), that is, the sample liquid can be spun to the side close to the conical tip of the conical centrifugal space, and the combined cells and non-target cells will be stratified. Specifically, because the combined cells in the sample liquid contain microbubbles, the combined cells will be located on the side close to the rotation axis in the sample liquid, that is, at the gas-liquid interface position of the sample liquid in the conical centrifugal space, and the non-target cells are relatively heavy because they are not combined with microbubbles, so the non-target cells will be located at the conical tip of the conical centrifugal space. At this time, the non-target cells located at the conical tip can be guided out to the first collection container, while the combined cells located at the gas-liquid interface position close to the conical bottom end are retained in the conical centrifugal space, thereby realizing the separation of non-target cells and combined cells (i.e. target cells). In the present application, the sorting column is not required, and the separation of non-target cells and combined cells (i.e. target cells) can be realized by the above sorting method, and cell damage is avoided during the separation process, and no additional process of removing magnetic beads is required. The operation is simple, and high recovery rate and high purity of sorting can be ensured.

[0013] The details of one or more embodiments of the present application are presented in the following drawings and description, and other features and advantages of the present application will become apparent from the description, drawings and claims. BRIEF DESCRIPTION OF DRAWINGS

[0014] The present application is further described below in conjunction with the drawings and examples.

[0015] FIG. 1 is a flowchart of a sorting method provided by a first embodiment of the present application.

[0016] FIG. 2 is a schematic diagram of a pipeline structure for performing the sorting method according to an embodiment of the present application.

[0017] FIG. 3 is a schematic diagram of the structure of a centrifugal container for performing the sorting method according to an embodiment of the present application.

[0018] FIG. 4 is a flowchart of a sorting method provided by a second embodiment of the present application.

[0019] FIG. 5 is a flowchart of a sorting method provided by a third embodiment of the present application.

[0020] FIG. 6 is a flowchart of a sorting method provided by a fourth embodiment of the present application.

[0021] The reference signs in the description are as follows: 1, centrifugal container; 110, conical centrifugal space; 111, conical tip; 112, conical bottom end; 120, container body; 130, first conveying pipe; 140, second conveying pipe; 2, first main pipe; 21, first control valve; 3, second main pipe; 4, gas port; 41, second control valve; 5, drive pump; 6, rotating shaft; 7, sample container; 8, sample pipe; 81, first on-off valve; 9, first collection container; 10, first collection pipe; 101, second on-off valve; 11, first cleaning container; 12, first cleaning pipe; 121, third on-off valve; 13, second cleaning container; 14, second cleaning pipe; 141, fourth on-off valve; 15, second collection container; 16, second collection pipe; 161, fifth on-off valve; 17, waste liquid container; 18, waste liquid pipe; 181, sixth on-off valve; 19, pressure sensor; 20, bubble sensor. DETAILED DESCRIPTION

[0022] In order to make the technical problems, technical solutions and beneficial effects solved in the present application clearer, the present application will be further described in detail below in combination with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and not to limit the present application.

[0023] In the description of the present application, it should be understood that the terms "longitudinal", "radial", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer" and the like indicate the orientation or positional relationship shown in the drawings, which are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the present application. In the description of the present application, unless otherwise specified, the meaning of "a plurality of" is two or more.

[0024] In the description of the present application, it should be noted that, unless otherwise specified and limited, the terms "mounting", "connection", "connection" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium; it can be the communication inside two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0025] As shown in FIGS. 1-3, an embodiment of the present application provides a sorting method, which includes the following steps S10-S30:

[0026] S10, injecting sample liquid into the conical centrifugal space 110 of the centrifugal container 1; the sample liquid includes non-target cells and combined cells obtained after the target cells are combined with microbubbles; it can be understood that the microbubbles refer to microparticles containing gas or other small density components, for example, the microbubbles can be micrometer-level phospholipid bubbles, micrometer-level glass vacuoles, micrometer-level polymer vacuoles, etc., the combined cells containing the combined microbubbles have a density less than water, and the non-target cells are relatively heavy (the density is greater than water) because they are not combined with microbubbles. In this embodiment, the sample liquid in the sample container 7 can be injected into the conical centrifugal space 110 of the centrifugal container 1 without rotating the centrifugal container 1 around the rotation axis 6.

[0027] S20, rotating the centrifugal container 1 around the rotation axis 6 to make the combined cells in the sample liquid located at the gas-liquid interface of the sample liquid close to the rotation axis 6, and the non-target cells located at the conical tip 111 of the conical centrifugal space 110; the rotation axis 6 is located at the side of the centrifugal container 1 away from the conical tip 111; because the centrifugal container 1 can rotate around the rotation axis 6, and the conical tip 111 of the conical centrifugal space 110 is located at the side of the centrifugal container 1 away from the rotation axis 6, when the centrifugal container 1 rotates around the rotation axis 6 at a high speed, the sample liquid can be thrown to the side close to the conical tip 111 of the conical centrifugal space 110, and the combined cells in the sample liquid contain microbubbles and have a density less than water, so the combined cells will be located at the side of the sample liquid close to the rotation axis 6, that is, at the gas-liquid interface position of the sample liquid in the conical centrifugal space 110, and the non-target cells are relatively heavy (the density is greater than water) because they are not combined with microbubbles, so the non-target cells will be located at the conical tip 111 of the conical centrifugal space 110, thereby realizing the stratification of the combined cells and the non-target cells under the density gradient centrifugation treatment.

[0028] The rotating shaft 6 only needs to be arranged on the side of the centrifugal container 1 away from the tapered tip 111 of the tapered centrifugal space 110, so as to realize the above-mentioned separation of the combined cells and the non-target cells. Therefore, the relative position relationship between the rotating shaft 6 and the tapered centrifugal space 110 can be set according to specific requirements. In an embodiment, the central axis of the tapered centrifugal space 110 is perpendicular to the rotating shaft 6 and located on the same plane. In this way, when the centrifugal container 1 rotates around the rotating shaft 6, the sample liquid in the tapered centrifugal space 110 can be symmetrically distributed with the central axis as the axis of symmetry, so that the distribution of the sample liquid in the tapered centrifugal space 110 is more uniform, facilitating the cell processing process. Understandably, the tapered centrifugal space 110 is not limited to a conical shape, but can also be set as a spliced shape of a cone and other shapes, or an approximate conical shape, for example, the tapered tip 111 can be set as a conical shape, and the tapered tail end can be set as other shapes, such as a cylindrical shape; the entire tapered centrifugal space 110 can also be set as a circular truncated cone, and the small-diameter end of the circular truncated cone is the tapered tip 111; the tapered centrifugal space 110 can also be set as a pyramid, and the like, which is not limited here; as long as the above-mentioned separation effect of the combined cells and the non-target cells can be achieved.

[0029] S30, the non-target cells are guided out of the tapered tip 111 to the first collection container 9, so as 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 (for example, a preset volume determined after setting according to the volume of the tapered centrifugal space 110 and through trial verification, wherein the preset volume can be set according to requirements) of sample liquid needs to be reserved in the tapered centrifugal space 110, so as to ensure that the combined cells will not flow out of the tapered centrifugal space 110. In this embodiment, the sample liquid is subjected to negative selection, that is, the non-target cells are separated from the sample liquid and collected into the first collection container 9, and the combined cells are reserved in the tapered centrifugal space 110. In this application, an optical liquid level sensor can be installed above the centrifugal container 1 to monitor the liquid level in the tapered centrifugal space 110, so as to ensure that the preset volume of sample liquid is reserved in the tapered centrifugal space 110. The volume can also be controlled by a high-precision preparation module, so as to control that the tapered centrifugal space 110 reserves the preset volume of sample liquid.

[0030] In the above embodiment of the present application, when the sample liquid (the sample liquid includes non-target cells and combined cells obtained after the target cells are combined with the microbubbles) needs to be sorted, the sample liquid is first injected into the conical centrifugal space 110 of the centrifugal container 1, and then the centrifugal container 1 is controlled to rotate around the rotation shaft 6 (the rotation shaft 6 is located on the side of the centrifugal container 1 away from the conical tip 111 of the conical centrifugal space 110), that is, the sample liquid can be spun to the side close to the conical tip 111 of the conical centrifugal space 110, and the combined cells and the non-target cells will be stratified. Specifically, because the combined cells in the sample liquid contain microbubbles, the combined cells will be located on the side close to the rotation shaft 6 in the sample liquid, that is, at the gas-liquid interface position of the sample liquid in the conical centrifugal space 110, and the non-target cells are relatively heavy because they are not combined with microbubbles, so the non-target cells will be located at the conical tip 111 of the conical centrifugal space 110; at this time, the non-target cells located at the conical tip 111 can be guided out to the first collection container 9, while the combined cells located at the gas-liquid interface position close to the conical bottom end 112 are retained in the conical centrifugal space 110, thereby realizing the separation of the non-target cells and the combined cells (i.e., the target cells). In the present application, the sorting column is not required, and the separation of the non-target cells and the combined cells (i.e., the target cells) can be realized by the above sorting method, the cells are not damaged during the separation process, and no additional process of removing the magnetic beads is required, the operation is simple, and high recovery rate and high purity of the sorting can be ensured.

[0031] In an embodiment, as shown in FIG. 2, the centrifugal container 1 includes a first delivery pipe 130 and a second delivery pipe 140 inserted into the centrifugal container 1; the conical tip 111 of the conical centrifugal space 110 is communicated with the sample container 7 through the first delivery pipe 130; and the conical bottom end 112 of the conical centrifugal space 110 is communicated with the air port 4 through the second delivery pipe 140. Understandably, the centrifugal container 1 can also include a container body 120, and the conical centrifugal space 110 is arranged 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 conical. The central axis of the conical container body 120 coincides with the central axis of the conical centrifugal space 110, and the tip of the conical container body 120 is consistent with the direction of the conical tip 111 of the conical centrifugal space 110. However, the shape of the container body 120 can also be set as other shapes according to requirements, such as cylindrical, square, etc., which are not limited herein.

[0032] Further, as shown in FIG. 1 and FIG. 2, the end of the first delivery pipe 130 away from the first main pipe 2 and the end of the first delivery pipe 130 away from the second delivery pipe 140 are both inserted into the conical centrifugal space 110 from the conical bottom end 112; that is, in this embodiment, the first delivery pipe 130 and the second delivery pipe 140 are both inserted from the conical bottom end 112, but the first delivery pipe 130 will directly insert the pipe opening into the conical tip 111 from the conical bottom end 112, while the second delivery pipe 140 will directly insert the pipe opening into the conical bottom end 112.

[0033] Further, the end of the first delivery pipe 130 away from the first main pipe 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 end 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 pipe opening is also located at the conical tip 111; the second delivery pipe 140 is inserted from the conical bottom end 112, and the insertion pipe opening is located at the conical bottom end 112.

[0034] In an embodiment, the air port 4 is also provided with an air filter. That is, in this embodiment, an air filter is installed at the air port 4 to ensure that the gas entering the second main pipe 3 from the external environment is sterile, thereby ensuring the sterile environment in the conical centrifugal space 110. Among them, the air port 4 and the centrifugal container 1 are provided with a second control valve 41, which can be provided on the second main pipe 3, or a ventilation pipe connected to the second main pipe 3, and the second control valve 41 can also be provided on the ventilation pipe, which is not limited here.

[0035] Further, step S10, that is, injecting sample liquid into the conical centrifugal space 110 of the centrifugal container 1, includes: after the conical centrifugal space 110 is connected to the external air through the second delivery pipe 140 and the air port 4, the sample liquid in the sample container 7 is injected into the conical centrifugal space 110 through the first delivery pipe 130 until the sample liquid in the conical centrifugal space 110 reaches a preset height, and the preset height is lower than the height of the pipe opening of the second delivery pipe 140 inserted into the conical bottom end 112.

[0036] That is, in this embodiment, the sample liquid in the sample container 7 is injected into the centrifugal container 1 through the first delivery pipe 130, and at this time the centrifugal container 1 needs to be connected to the outside air through the second delivery pipe 140 to balance the air pressure in the tapered centrifugal space 110. Understandably, since the sample liquid enters the tapered centrifugal space 110, the centrifugal container 1 does not rotate, so the total density of the combined cells of the target cells combined with microbubbles in the sample liquid is less than water, and the density of the non-target cells is greater than water, and under the treatment of density gradient centrifugation, the target cells will float on the liquid surface of the sample liquid, and the non-target cells will sink to the bottom of the sample liquid, and the two will be separated; at this time, in order to avoid the combined cells on the liquid surface from overflowing from the pipe opening of the second delivery pipe 140 inserted into the tapered centrifugal space 110, the height of the liquid surface of the sample liquid entering the tapered centrifugal space 110 needs to be controlled to be less than the height of the pipe opening of the second delivery pipe 140. Therefore, the sample liquid entering the centrifugal container 1 in step S10 can be set to a preset height (under the condition that the volume and shape of the tapered centrifugal space 110 are determined, the preset height corresponds to a preset volume of sample liquid, so it can also be regarded as injecting a preset volume of sample liquid. Among them, the preset height can be set according to the needs), and the sample liquid of the preset height in the tapered centrifugal space 110 needs to be lower than the pipe opening height of the second delivery pipe 140 inserted into the tapered bottom end 112, so as to ensure that the combined cells will not overflow from the second delivery pipe 140. In some embodiments, the insertion pipe opening of the second delivery pipe 140 is located above the tapered centrifugal space 110, so that the capacity of the sample liquid that can be input into the tapered centrifugal space 110 can be increased, thereby improving the capacity of the sample liquid that can be sorted at a time, and further improving the cell sorting efficiency.

[0037] In an embodiment, as shown in FIG. 2, the first delivery pipe 130 is connected to the first main pipeline 2, and the first main pipeline 2 is provided with a driving pump 5; one 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 installed on the driving pump 5, wherein the driving 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 on-off 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 on-off valve 101 for controlling the opening and closing of the first collection pipeline 10.

[0038] Further, the injecting the sample liquid in the sample container 7 into the tapered centrifugal space 110 through the first delivery pipe 130 comprises: controlling the driving pump 5 to drive the sample liquid in the sample container 7 to sequentially flow into the tapered centrifugal space 110 through the first main pipe 2 and the first delivery pipe 130. That is, in the embodiment, when the first switch valve 81 is opened and the second switch valve 101 is closed, the driving pump 5 can be controlled to drive the sample liquid in the sample container 7 to sequentially flow into the tapered centrifugal space 110 through the first main pipe 2 and the first delivery pipe 130.

[0039] The guiding the non-target cells from the tapered tip 111 to the first collection container 9 comprises: controlling the driving pump 5 to drive the sample liquid in the tapered centrifugal space 110 to sequentially flow into the first collection container 9 from the tapered tip 111 through the first delivery pipe 130 and the first main pipe 2. That is, in the embodiment, in step S20, when the first switch valve 81 and the second switch valve 101 are both closed, the centrifugal container 1 can be controlled to rotate around the rotation shaft 6, so that the combined cells in the sample liquid are located at the gas-liquid interface of the sample liquid close to the rotation shaft 6, and the non-target cells are located at the tapered tip 111 of the tapered centrifugal space 110; and the rotation shaft 6 is located at the side of the centrifugal container 1 away from the tapered tip 111. Then, after the first switch valve 81 is closed and the second switch valve 101 is opened, the driving pump 5 can be controlled to drive the sample liquid in the tapered centrifugal space 110 to sequentially flow into the first collection container 9 from the tapered tip 111 through the first delivery pipe 130 and the first main pipe 2.

[0040] In an embodiment, the first main pipe 2 is connected to a cleaning assembly; further, as shown in FIG. 4, after step S30, that is, after the non-target cells are guided from the tapered tip 111 to the first collection container 9 to separate the non-target cells and the combined cells, the method comprises:

[0041] S40, performing at least one pipe cleaning operation to flush the residual sample liquid in the pipe connected between the first collection container 9 and the tapered centrifugal space 110 into the first collection container 9 or / and the tapered centrifugal space 110 by the cleaning liquid injected by the cleaning assembly. The specific composition of the cleaning assembly can be set according to requirements. When the pipe cleaning operation is performed, the cleaning assembly can inject the cleaning liquid into the pipe (that is, the pipe connected between the first collection container 9 and the tapered centrifugal space 110) through which the non-target cells are guided to flow into the first collection container 9, so as to flush the residual sample liquid in the pipe into the first collection container 9 or / and the tapered centrifugal space 110. Understandably, the pipe cleaning operation can be set to be performed once or multiple times, and repeated multiple times, which can enhance the cleaning effect, and the purpose is to clean the residual sample liquid in the pipe completely.

[0042] In one embodiment, as shown in Fig. 2, the first main pipeline 2 is further provided with a first control valve 21 between the driving pump 5 and the centrifugal container 1; the cleaning assembly comprises a first cleaning container 11, a second cleaning container 13, a first cleaning pipeline 12 communicating between the first cleaning container 11 and the first main pipeline 2, and a second cleaning pipeline 14 communicating between the second cleaning container 13 and the first main pipeline 2; the first communication point between the first cleaning pipeline 12 and the first main pipeline 2 is located between the first control valve 21 and the driving pump 5; the second communication point between the second cleaning pipeline 14 and the first main pipeline 2 is located on the side of the driving pump 5 away from the centrifugal container 1; it is understood that the first cleaning pipeline 12 is provided with a third switch valve 121 for controlling the opening and closing of the first cleaning pipeline 12. The second cleaning pipeline 14 is provided with a fourth switch valve 141 for controlling the opening and closing of the second cleaning pipeline 14.

[0043] Further, step S40, i.e. performing at least one pipeline cleaning operation, to flush the residual sample liquid in the pipeline communicating between the first collection container 9 and the conical centrifugal space 110 into the first collection container 9 or / and the conical centrifugal space 110 by the cleaning liquid injected by the cleaning assembly, comprising:

[0044] closing the first control valve 21, and driving the cleaning liquid in the first cleaning container 11 to flow through the first cleaning pipeline 12 and the first main pipeline 2 into the first collection container 9 by the driving pump 5; in this embodiment, after closing the first control valve 21 (the third switch valve 121 and the second switch valve 101 are opened, and the remaining switch valves are closed), at this time, the cleaning liquid in the first cleaning container 11 is driven 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 by the driving pump 5, thereby cleaning the part of the first main pipeline 2 (and the first collection pipeline 10) close to the first collection container 9, and flushing the residual non-target cells in the pipeline into the first collection container 9.

[0045] The first control valve 21 is opened, and the cleaning liquid in the second cleaning container 13 is driven to flow through the second cleaning pipeline 14, the first main pipeline 2 and the first delivery pipeline 130 into the conical centrifugal space 110 by driving the pump 5. Specifically, after the first control valve 21 and the second control valve 41 are opened (the fourth switch valve 141 is opened, and the remaining switch valves are closed), the cleaning liquid in the second cleaning container 13 is driven to flow through the second cleaning pipeline 14, the first main pipeline 2 and the first delivery pipeline 130 into the conical centrifugal space 110 by driving the pump 5, and air can enter the conical centrifugal space 110 from the air port 4 through the second main pipeline 3 to balance the air pressure. In the above process, the cleaning liquid can be injected into the conical centrifugal space 110 to clean the first main pipeline 2 and the first delivery pipeline 130, and the residual sample liquid in the pipeline can be flushed into the conical centrifugal space 110. Understandably, in the above process, the cleaning liquid cannot exceed the pipe opening of the second delivery pipeline 140 inserted into the conical centrifugal space 110, so as to avoid the overflow of the combined cells from the pipe opening.

[0046] In an embodiment, as shown in FIG. 5, after step S30, that is, after the non-target cells are guided out of the conical tip to the first collection container, the method further includes:

[0047] After the centrifugal container is controlled to stop rotating around the rotation axis, a microbubble annihilation operation is performed, which includes increasing the pressure in the conical centrifugal space to a preset annihilation pressure, so as to annihilate the microbubbles of the combined cells located in the conical centrifugal space. The preset annihilation pressure can be set according to requirements, but the preset annihilation pressure must be greater than the atmospheric pressure. Specifically, in an embodiment, after the pressure in the conical centrifugal space is increased to the preset annihilation pressure, the preset annihilation pressure can be maintained for a preset time length (the preset time length can also be set according to requirements), and then it can be confirmed that the microbubbles of the combined cells in the sample liquid are all annihilated. In another embodiment, an optical liquid level sensor can also be installed above the centrifugal container 1 to monitor the liquid level in the conical centrifugal space, and then when it is monitored by the optical liquid level sensor that there is no microbubble in the liquid level, it is confirmed that the microbubbles of the combined cells in the sample liquid are all annihilated.

[0048] Further, as shown in FIG. 2, the centrifugal container 1 includes the first delivery pipeline 130 and the second delivery pipeline 140 inserted into the centrifugal container 1; the conical tip 111 of the conical centrifugal space 110 is communicated with the first main pipeline 2 through the first delivery pipeline 130; the conical bottom end 112 of the conical centrifugal space 110 is communicated with the air port 4 through the second delivery pipeline 140 and the second main pipeline 3; the first main pipeline 2 is provided with the driving pump 5; one end of the first main pipeline 2 away from the first delivery pipeline 130 is communicated with the cleaning assembly; the second main pipeline 3 is further provided with the second control valve 41; at this time, increasing the pressure in the conical centrifugal space to the preset annihilation pressure includes:

[0049] The second control valve 41 is controlled to be closed to disconnect the passage between the conical centrifugal space 110 and the gas port 4, and the cleaning liquid in the cleaning assembly is driven by the driving pump 5 to pass through the first main pipeline 2 and the first delivery pipe 130 into the conical centrifugal space 110, so as to increase the pressure in the conical centrifugal space 110 to the preset annihilation pressure.

[0050] It can be understood that the step S50 can also be performed after the pipeline cleaning operation in the step S40 is performed at least once, and the specific steps are not described here again.

[0051] In a further embodiment, after the above cleaning operation is completed, the centrifugal container 1 can be stopped rotating around the rotation shaft 6, so that the sample liquid containing the joint cells is static in the conical centrifugal space 110. Then, the first control valve 21 is controlled to be opened, the second control valve 41 is controlled to be closed (the fourth switch valve 141 is opened, and the remaining switch valves are closed), the passage between the conical centrifugal space 110 and the gas port 4 is disconnected, and the cleaning liquid in the second cleaning container 13 is driven by the driving pump 5 to pass through the first main pipeline 2 and the first delivery pipe 130 into the conical centrifugal space 110, so as to increase the pressure in the conical centrifugal space 110, and then after the pressure in the conical centrifugal space 110 is increased to the preset annihilation pressure and maintained for a preset time, it is confirmed that the microbubbles in the joint cells in the sample liquid are all annihilated. In this way, the microbubbles in the joint cells can be annihilated and removed by this embodiment, and the separation of the microbubbles and the target cells is realized.

[0052] In an embodiment, as shown in FIG. 2, the first main pipeline 2 is further provided with a pressure sensor 19; specifically, the pressure sensor 19 is located between the driving pump 5 and the centrifugal container 1. Further, in the step S50, after the cleaning liquid in the cleaning assembly is driven by the driving pump 5 to pass through the first main pipeline 2 and the first delivery pipe 130 into the conical centrifugal space 110, it further includes:

[0053] The pressure information in the first main pipeline 2 is monitored in real time by the pressure sensor 19, and the real-time pressure value is determined according to the pressure information, and 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 is 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 communicates with the conical centrifugal space 110 when the second control valve 41 controls the second main pipeline 3 to be closed, and then the real-time pressure value in the first main pipeline 2 is determined according to the pressure information. Since the first main pipeline 2 communicates with the conical centrifugal space 110, the real-time pressure value is the actual pressure in the conical centrifugal space 110, so 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 is increased to the preset annihilation pressure.

[0054] In an embodiment, as shown in Fig. 2, the centrifugal container 1 comprises a first delivery pipe 130 and a second delivery pipe 140 inserted into the centrifugal container 1; the tapered tip 111 of the tapered centrifugal space 110 is connected to the first main pipe 2 through the first delivery pipe 130; the tapered bottom end 112 of the tapered centrifugal space 110 is connected to the air port 4 through the second delivery pipe 140 and the second main pipe 3; the first main pipe 2 is provided with a driving pump 5; the end of the first main pipe 2 away from the first delivery pipe 130 is connected to the second collection container 15. Further, the first main pipe 2 is connected to the second collection container 15 through a second collection pipe 16, and the third connection point between the second collection pipe 16 and the first main pipe 2 is located on the side of the driving pump 5 away from the centrifugal container 1. Understandably, the second collection pipe 16 is provided with a fifth switch valve 161 for controlling the opening and closing of the second collection pipe 16. Further, as shown in Fig. 6, after step S50, i.e., after performing the microbubble annihilation operation, the method comprises:

[0055] S60, controlling the second control valve 41 to open to connect the tapered centrifugal space 110 and the air port 4, and reducing the pressure in the tapered centrifugal 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 can be controlled to open, and the second control valve 41 can be controlled to open (at the same time, the centrifugal container 1 is controlled to rotate at a low speed or be stationary to avoid the sample liquid from being sprayed out of the pipe opening of the second delivery pipe 140), to connect the tapered centrifugal space 110 and the air port 4, and reduce the pressure in the tapered centrifugal space 110 to be consistent with the external atmospheric pressure, to release the pressure in the tapered centrifugal space 110.

[0056] S70, controlling the centrifugal container 1 to rotate around the rotation axis 6 to make the target cells with annihilated microbubbles in the tapered centrifugal space 110 located at the tapered tip 111, and driving the target cells with annihilated microbubbles in the tapered centrifugal space 110 to be guided out to the second collection container 15 through the first delivery pipe 130 and the first main pipe 2. In this step, the centrifugal container 1 needs to be controlled to rotate around the rotation axis 6 at a high speed, at this time, since the microbubbles in the combined cells in the tapered centrifugal space 110 have been annihilated, the density of the remaining target cells with annihilated microbubbles will be greater than water, therefore, the target cells with annihilated microbubbles will be located at the tapered tip 111 under the action of the centrifugal force, at this time, the fifth switch valve 161 is controlled to open, and the remaining switch valves are controlled to close, so that the target cells with annihilated microbubbles in the tapered centrifugal space 110 can be driven to be guided out to the second collection container 15 through the first delivery pipe 130 and the first main pipe 2, and further, after sorting the non-target cells into the first collection container 9, the target cells after removing the microbubbles are also sorted into the second collection container 15.

[0057] Understandably, in the present embodiment, the sample liquid is positively selected, i.e. the non-target cells are separated from the sample liquid and collected into the first collection container 9, and the target cells are collected into the second collection container 15. In the present application, the separation of the target cells and the non-target cells is achieved without the need of a sorting column, and the cell damage is avoided.

[0058] In an embodiment, as shown in Fig. 2, the first main pipeline 2 is connected to the washing assembly; the second main pipeline 3 is connected to the waste liquid container 17 at a position away from the centrifugal container 1; the waste liquid container 17 is connected to the second main pipeline 3 through the 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 centrifugal container 1. Understandably, the waste liquid pipeline 18 is provided with a sixth switch valve 181 for controlling the opening and closing of the waste liquid pipeline 18. Further, in step S70, before the target cells with the annihilated microbubbles in the conical centrifugal space 110 are driven by the pump 5 to be guided out of the first delivery pipeline 130 and the first main pipeline 2 to the second collection container 15, the method further comprises:

[0059] The washing operation is performed, which includes driving the cleaning liquid in the cleaning assembly through the first main pipeline 2 and the first delivery pipe 130 into the conical centrifugal space 110 by driving the pump 5, so that the liquid in the conical centrifugal space 110 covers the pipe orifice of the second delivery pipe 140 inserted in the conical bottom end 112 under the centrifugal action, and then the waste liquid in the liquid except the target cells with annihilated microbubbles is extracted to the waste liquid container 17 through the second delivery pipe 140 and the second main pipeline 3. That is, in this embodiment, before the target cells are sorted into the second collection container 15, in order to reduce the volume of the final preparation, the sample liquid containing the target cells with annihilated microbubbles can also be subjected to the washing operation to remove the waste liquid in the sample liquid in the conical centrifugal space, specifically, the first control valve 21 is controlled to be opened, the second control valve 41 is controlled to be closed to disconnect the communication between the air port 4 and the second main pipeline 3, and the sixth switch valve 181 and the fourth switch valve 141 are controlled to be opened (the remaining switch valves are closed) to make the waste liquid pipeline 18 communicate with the conical centrifugal space 110 through the second main pipeline 3, and the second cleaning container 13 communicates with the first main pipeline 2 through the cleaning pipeline; then, the centrifugal container 1 is controlled to rotate around the rotation shaft 6, at this time, since the microbubbles in the combined cells in the conical centrifugal space 110 have been annihilated, the density of the remaining target cells with annihilated microbubbles will be greater than water, so the target cells with annihilated microbubbles will be located at the conical tip 111 under the centrifugal force, while the waste liquid is located at the side of the conical tail end in the conical centrifugal space 110, at this time, the cleaning liquid in the second cleaning container 13 can be driven to pass through the cleaning pipeline, the first main pipeline 2 and the first delivery pipe 130 into the conical centrifugal space 110 by driving the pump 5, so that the liquid in the conical centrifugal space 110 covers the pipe orifice of the second delivery pipe 140 inserted in the conical bottom end 112 under the centrifugal action, and then the sample liquid covering the pipe orifice of the second delivery pipe 140 is the waste liquid, so the waste liquid in the liquid except 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; and the target cells with annihilated microbubbles always located at the conical tip 111 will be left in the conical centrifugal space 110.

[0060] In some embodiments, as shown in FIG. 2, a bubble sensor 20 is arranged between the sample container 7 and the centrifugal container 1, and the sorting method further comprises: detecting, by the bubble sensor 20, whether the sample liquid in the sample container 7 is completely drained, and if not, returning to step S10 to continue injecting the sample liquid into the tapered centrifugal space 110 of the centrifugal container 1 to sort the non-target cells and the combined cells (i.e. target cells) in the sample liquid again. Further, the bubble sensor 20 is arranged between the driving pump 5 and the sample container 7. In this embodiment, when the volume of the sample liquid in the sample container 7 is too large, if the centrifugal container 1 cannot be processed at one time, the above sorting method can be repeated for multiple times for processing, at this time, it is necessary to determine whether the sample liquid in the sample container 7 is completely drained, i.e. whether the sample liquid in the sample container 7 is completely processed, by the bubble sensor 20.

[0061] It should be understood that the size of the serial number of each step in the above embodiments does not mean the order of execution, and the execution order of each process should be determined according to its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0062] The application further provides a sorting device, comprising a controller and a centrifugal container 1 connected with the controller, the controller comprising a memory, a processor and computer readable instructions stored in the memory and executable on the processor, and the processor implements the above sorting method when executing the computer readable instructions. In the sorting device of the above embodiments of the application, when it is necessary to sort sample liquid (the sample liquid comprising non-target cells and combined cells obtained after the target cells are combined with microbubbles), the sample liquid is first injected into the conical centrifugal space 110 of the centrifugal container 1, and then the centrifugal container 1 is controlled to rotate around the rotation shaft 6 (the rotation shaft 6 is located on the side of the centrifugal container 1 away from the conical tip 111 of the conical centrifugal space 110), that is, the sample liquid can be spun to the side close to the conical tip 111 of the conical centrifugal space 110, and the combined cells and the non-target cells will be stratified. Specifically, because the combined cells in the sample liquid contain microbubbles, the combined cells will be located on the side close to the rotation shaft 6 in the sample liquid, that is, at the gas-liquid interface position of the sample liquid in the conical centrifugal space 110, and the non-target cells are relatively heavy because they are not combined with microbubbles, so the non-target cells will be located at the conical tip 111 of the conical centrifugal space 110; at this time, the non-target cells located at the conical tip 111 can be guided out to the first collection container 9, while the combined cells located at the gas-liquid interface position close to the conical bottom end 112 are retained in the conical centrifugal space 110, thereby realizing the separation of the non-target cells and the combined cells (i.e. the target cells). In the application, the sorting column is not required, and the separation of the non-target cells and the combined cells (i.e. the target cells) can be realized by the above sorting method, and cell damage is avoided during the separation process, and no additional process of removing magnetic beads is required, and the operation is simple, which can ensure high recovery rate and high purity of sorting.

[0063] It can be understood that the execution functions of the controller correspond one by one to the sorting method in the above embodiments. For specific limitations of the controller, refer to the limitations of the sorting method in the above, which will not be repeated here. Each sub-module in the above controller can be realized by software, hardware and their combination in whole or in part. Each sub-module can be embedded in the processor in the computer device in hardware form or independent of the processor, or stored in the memory in the computer device in software form, so as to be called and executed by the processor to perform the corresponding operations of each sub-module.

[0064] The application further provides a computer readable storage medium, which stores a computer program, and the computer program is executed by a processor to implement the steps of the sorting method.

[0065] Those skilled in the art can understand that all or part of the processes in the above-mentioned embodiment methods can be completed by instructing relevant hardware through computer readable instructions, and the computer readable instructions can be stored in a non-volatile readable storage medium or a volatile readable storage medium. When the computer readable instructions are executed, the processes of the above-mentioned embodiments of the methods can be included. Any reference to memory, storage, database or other medium used in each embodiment provided by the present application can include non-volatile and / or volatile memory. Non-volatile memory can include read-only memory (ROM), programmable ROM (PROM), electrically programmable ROM (EPROM), electrically erasable programmable ROM (EEPROM) or flash memory. Volatile memory can include random access memory (RAM) or external cache memory. As an illustration but not limitation, RAM is available in various forms, such as static RAM (SRAM), dynamic RAM (DRAM), synchronous DRAM (SDRAM), double data rate SDRAM (DDR SDRAM), enhanced SDRAM (ESDRAM), synchronous link (Synchlink) DRAM (SLDRAM), memory bus (Rambus) direct RAM (RDRAM), direct memory bus dynamic RAM (DRDRAM), and memory bus dynamic RAM (RDRAM), etc.

[0066] Those skilled in the art can clearly understand that, for the convenience and brevity of description, only the above-mentioned division of functional units and modules is exemplified, and in actual application, the above-mentioned functions can be completed by different functional units and modules according to needs, that is, the internal structure of the system is divided into different functional units or modules to complete all or part of the above-described functions.

[0067] The above embodiments are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application, and should be included in the protection scope of the present application.

Claims

1. A method of sorting, wherein, The method comprises: injecting a sample liquid into a conical centrifugal space of a centrifugal container; the sample liquid comprises non-target cells and combined cells obtained after combining target cells with microvesicles; controlling the centrifugal container to rotate around a rotation axis, so that the combined cells in the sample liquid are located at an air-liquid interface of the sample liquid close to the rotation axis, and the non-target cells are located at a conical tip of the conical centrifugal space; the rotation axis is located at a side of the centrifugal container away from the conical tip; directing the non-target cells from the conical tip to a first collection container to separate the non-target cells and the combined cells.

2. The sorting method of claim 1, wherein, the centrifugal container comprises a first delivery pipe and a second delivery pipe inserted in the centrifugal container; a conical tip of the conical centrifugal space is communicated with a sample container through the first delivery pipe; a conical bottom end of the conical centrifugal space is communicated with an air port through the second delivery pipe; the injecting of the sample liquid into the conical centrifugal space of the centrifugal container comprises: after the conical centrifugal space is communicated with external air through the second delivery pipe and the air port, the sample liquid in the sample container is injected into the conical centrifugal space through the first delivery pipe until the sample liquid in the conical centrifugal space reaches a preset height, and the preset height is lower than a pipe opening height at which the second delivery pipe is inserted in the conical bottom end.

3. The sorting method of claim 2, wherein, the first delivery pipe is communicated with a first main pipeline, and a driving pump is arranged on the first main pipeline; an end of the first main pipeline away from the first delivery pipe is communicated with the sample container and the first collection container, and the second delivery pipe is communicated with the air port through a second main pipeline; the injecting of the sample liquid in the sample container into the conical centrifugal space through the first delivery pipe comprises: controlling the driving pump to drive the sample liquid in the sample container to be sequentially injected into the conical centrifugal space through the first main pipeline and the first delivery pipe; the directing of the non-target cells from the conical tip to the first collection container comprises: controlling the driving pump to drive the sample liquid in the conical centrifugal space to be sequentially introduced into the first collection container from the conical tip through the first delivery pipe and the first main pipeline.

4. The sorting method of claim 3, wherein, the first main pipeline is communicated with a cleaning assembly; after the directing of the non-target cells from the conical tip to the first collection container to separate the non-target cells and the combined cells, the method comprises: performing at least one pipeline cleaning operation to flush sample liquid remaining in a pipeline communicated between the first collection container and the conical centrifugal space into the first collection container or / and the conical centrifugal space by a cleaning liquid injected through the cleaning assembly.

5. The sorting method of claim 4, wherein, The first main pipeline is further provided with a first control valve between the driving pump and the centrifugal container; the cleaning assembly comprises a first cleaning container, a second cleaning container, a first cleaning pipeline communicated between the first cleaning container and the first main pipeline, and a second cleaning pipeline communicated between the second cleaning container and the first main pipeline; a first communication point between the first cleaning pipeline and the first main pipeline is located between the first control valve and the driving pump; a second communication point between the second cleaning pipeline and the first main pipeline is located on a side of the driving pump away from the centrifugal container; The execution of the at least one pipeline cleaning operation to flush the residual sample liquid in the pipeline communicated between the first collection container and the conical centrifugal space into the first collection container or / and the conical centrifugal space by the cleaning liquid injected by the cleaning assembly comprises: The first control valve is closed, and the cleaning liquid in the first cleaning container is driven by the driving 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 driving pump to flow through the second cleaning pipeline, the first main pipeline and the first conveying pipeline into the conical centrifugal space.

6. The sorting method of claim 1, wherein, After the non-target cells are guided out of the conical tip to the first collection container, further comprising: After the centrifugal container is controlled to stop rotating around the rotation axis, a microbubble annihilation operation is performed, and the microbubble annihilation operation comprises: increasing the pressure in the conical centrifugal space to a preset annihilation pressure, so that the microbubbles of the joint cells located in the conical centrifugal space are annihilated.

7. The sorting method of claim 6, wherein, The centrifugal container comprises a first conveying pipeline and a second conveying pipeline inserted in the centrifugal container; a conical tip of the conical centrifugal space is communicated with a first main pipeline through the first conveying pipeline; a conical bottom end of the conical centrifugal space is communicated with an air port through the second conveying pipeline and a second main pipeline; the first main pipeline is provided with a driving pump; an end of the first main pipeline away from the first conveying pipeline is communicated with a second collection container; The second main pipeline is provided with a second control valve; After the microbubble annihilation operation is performed, comprising: The second control valve is controlled to be opened to communicate the conical centrifugal space with the air port, so that the pressure in the conical centrifugal space is reduced to be consistent with the external atmospheric pressure; The centrifugal container is controlled to rotate around the rotation axis, so that the target cells with annihilated microbubbles in the conical centrifugal space are located at the conical tip, and the target cells with annihilated microbubbles in the conical centrifugal space are guided out of the second collection container through the first conveying pipeline and the first main pipeline by the driving pump.

8. The sorting method of claim 7, wherein, The first main pipeline is communicated with a cleaning assembly; an end of the second main pipeline away from the centrifugal container is communicated with a waste liquid container; Before the target cells with annihilated microbubbles in the conical centrifugal space are guided out of the second collection container through the first conveying pipeline and the first main pipeline by the driving pump, further comprising: The elutriation operation includes: driving the cleaning liquid in the cleaning assembly to pass through the first main pipeline and the first delivery pipe into the conical centrifugal space by the driving pump, so that the liquid in the conical centrifugal space covers the pipe orifice of the second delivery pipe inserted in the conical bottom end under the centrifugal action, and then the waste liquid except the target cells with annihilated microbubbles in the liquid is extracted to the waste liquid container through the second delivery pipe and the second main pipeline.

9. A sorting apparatus wherein, The centrifugal container comprises a controller and a centrifugal container in communication connection with the controller, the controller comprises a memory, a processor and computer readable instructions stored in the memory and executable on the processor, and the processor implements the sorting method according to any one of claims 1 to 8 when executing the computer readable instructions.

10. A computer-readable storage medium storing a computer program, wherein, The computer program is executed by the processor to implement the sorting method according to any one of claims 1 to 8.

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