Magnetic cell sorting method, device and equipment

Abstract

The invention discloses a magnetic cell sorting method, device and equipment, and the sorting method comprises the following steps: applying a magnetic field to a sorting column in a pipeline structure, and opening a control valve between a sample container and a negative sorting collection container on the pipeline structure, the cell sample in the sample container is driven by a peristaltic pump to flow towards the negative selection collection container, target cells in the cell sample are adsorbed in the sorting column, and non-target cells enter the negative selection collection container along with flowing of the cell sample; the pipeline structure is washed through a buffer solution, so that residual target cells in the pipeline structure are adsorbed in the sorting column; and opening a control valve corresponding to the positive separation collection container, eluting the separation column after the magnetic field is removed, and eluting the target cells in the separation column into the positive separation collection container through a peristaltic pump. According to the invention, the sorting efficiency is improved, the risk of target cell pollution is reduced, and the purity of the sorted target cells is ensured.

Description

Technical Field

[0001] The present invention relates to the technical field of cell sorting, and in particular to a magnetic cell sorting method, device and equipment. Background Art

[0002] In cytological research, the functional study of a specific cell, such as the detection of cytokines by ELISA analysis of cell culture supernatant, the detection of cell function by cell co-culture, etc., requires the acquisition of high-purity target cells. Therefore, efficient separation of the required target cells is a prerequisite for cell function research.

[0003] Cell sorting refers to a technique that separates a specific cell subpopulation from a mixed cell sample based on the characteristics of the cells. It is the premise and basis for biochemical and functional analysis of a specific cell.

[0004] Magnetic cell sorting: It is to incubate sample cells with immunomagnetic beads bound with antibodies. Cells expressing corresponding antigens will specifically bind to immunomagnetic particles coated with antibodies. When the mixed cell sample formed by incubation slowly passes through the magnetic field, the target cells bound with magnetic beads (i.e. positive cells) will be retained on the magnet, while non-target cells not bound with magnetic beads (i.e. negative cells) will still exist in the mixed cell sample, thereby achieving the purpose of separating and purifying cells. Magnetic sorting only puts cells in a low magnetic field, and the effect on cells can be basically ignored. The separated cells have a higher recovery rate and cell activity, and have little impact on downstream applications.

[0005] Currently, magnetic cell sorting is mainly carried out by manual sorting, and the steps are as follows:

[0006] placing the separation column in a magnetic field;

[0007] The incubated cell sample is passed through the sorting column in the magnetic field. The target cells are adsorbed on the inner wall of the sorting column under the action of the magnetic field, thereby achieving the purpose of separation;

[0008] The separation column is removed from the magnetic field, and a separation solution is introduced into the separation column to elute the target cells.

[0009] Although this sorting method has the advantages of high sensitivity, high purity, easy operation, and less stimulation to the target cells, it takes a long time to manually sort a large number of cells, which reduces the sorting efficiency. The operator needs to remain focused continuously, the labor intensity is high, and it is easy to make operational errors, which increases the possibility of cell contamination. In addition, due to differences in personnel, the purity of the sorted cells may vary greatly, which is not conducive to subsequent research. Summary of the invention

[0010] In order to overcome the shortcomings of the prior art, the present invention provides a magnetic cell sorting method, device and equipment, which shortens the sorting time, improves the sorting efficiency, is less prone to operating errors, reduces labor intensity and reduces the risk of target cell contamination, ensures the purity of the sorted target cells, and does not cause too much difference.

[0011] The technical solution adopted by the present invention to solve its technical problem is:

[0012] The first aspect of the present invention provides a magnetic cell sorting method, comprising the following steps: applying a magnetic field to a sorting column in a pipeline structure, opening a control valve located between a sample container and a negative selection collection container on the pipeline structure, and driving a cell sample in the sample container to flow toward the negative selection collection container by a peristaltic pump, wherein target cells in the cell sample are adsorbed in the sorting column, and non-target cells enter the negative selection collection container with the flow of the cell sample; flushing the pipeline structure with a buffer solution so that the target cells remaining in the pipeline structure are adsorbed in the sorting column; opening a control valve corresponding to the positive selection collection container, eluting the sorting column after the magnetic field is released, and eluting the target cells in the sorting column into the positive selection collection container by a peristaltic pump.

[0013] The second aspect of the present invention provides a magnetic cell sorting device, which uses the above-mentioned magnetic cell sorting method to perform cell sorting, including a pipeline structure, a peristaltic pump and a magnetic field application unit, the pipeline structure has a first interface, a second interface, a third interface, a fourth interface and a fifth interface, the pipeline structure is provided with a plurality of control valves, a circulation valve and a sorting column, the peristaltic pump is arranged on the pipeline structure, and the magnetic field application unit is used to apply a magnetic field to the sorting column and to release the magnetic field applied to the sorting column.

[0014] The third aspect of the present invention provides a magnetic cell sorting device, characterized in that it includes a pipeline structure, a peristaltic pump, a magnetic field application unit and a controller for executing the above-mentioned magnetic cell sorting method, the pipeline structure has a first interface, a second interface, a third interface, a fourth interface and a fifth interface, the pipeline structure is provided with a plurality of control valves, a circulation valve and a sorting column, the peristaltic pump is arranged on the pipeline structure, the magnetic field application unit is used to apply a magnetic field to the sorting column and to release the magnetic field applied to the sorting column, the peristaltic pump, the plurality of control valves, the circulation valve and the magnetic field application unit are all electrically connected to the controller.

[0015] The beneficial effects of the present invention are as follows: the magnetic cell sorting method of the present invention can automatically complete the cell sorting by controlling the control valve and the peristaltic pump action on the pipeline structure. Compared with the existing manual method, the sorting time is shortened, the sorting efficiency is improved, the operating errors are less likely to occur, the labor intensity is reduced, and the risk of cell contamination is reduced, the purity of the sorted cells is guaranteed, and there will not be too much difference, which is beneficial to subsequent research. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0017] Figure 1 It is a schematic structural diagram of a magnetic cell sorting device provided by one embodiment of the present invention;

[0018] Figure 2 yes Figure 1 A schematic structural diagram of a magnetic field applying unit of a magnetic cell sorting device is shown;

[0019] Figure 3 The present invention is based on Figure 1 The magnetic cell sorting device shown is a flowchart of a magnetic cell sorting method. DETAILED DESCRIPTION

[0020] The following will clearly and completely describe the concept, specific structure and technical effects of the present invention in combination with the embodiments and drawings, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by technicians in this field without creative work are all within the scope of protection of the present invention. In addition, all the connection / connection relationships involved in the patent do not refer to the direct connection of components, but refer to the formation of a better connection structure by adding or reducing connection accessories according to the specific implementation situation. The various technical features in the invention can be combined interchangeably without conflicting with each other.

[0021] Please refer to Figure 1 and Figure 2 A magnetic cell sorting device provided by one embodiment of the present invention includes a pipeline structure 10, a peristaltic pump 100 and a magnetic field applying unit.

[0022] The pipeline structure 10 has a first interface 11, a second interface 12, a third interface 13, a fourth interface 14, a fifth interface 15 and a sterile filter 16. The first interface 11 is used to connect with a buffer container and an eluent container, the buffer container is used to hold a buffer, the buffer container is for example a buffer storage bag, the eluent container is used to hold an eluent, the eluent container is for example an eluent storage bag, the buffer and the eluent are both existing liquids, which will not be repeated here, and the volume of the buffer container and the eluent container can be set according to actual conditions. The buffer can be a magnetic separation buffer (also known as magnetic bead separation buffer) or a nanomagnetic separation buffer for magnetic separation, the eluent can be a buffer, and can also be convenient for elution or a solution for subsequent processes, such as a culture medium, the second interface 12 is used to connect with a sample container, the sample container is used to hold a cell sample, the cell sample contains target cells combined with magnetic beads and non-target cells not combined with magnetic beads, the sample container is for example a sample bag, and the volume of the sample container can be set according to actual conditions. The third interface 13 is used to connect with the negative selection collection container 201, and the negative selection collection container 201 is used to collect non-target cells. The negative selection collection container 201 is, for example, a negative sample collection bag, and the volume of the negative selection collection container 201 can be set according to actual conditions. The fourth interface 14 is used to connect with the positive selection collection container 202, and the positive selection collection container 202 is used to collect target cells. The positive selection collection container 202 is, for example, a positive sample collection bag, and the volume of the positive selection collection container 202 can be set according to actual conditions. The fifth interface 15 is used to connect with the waste liquid container 203, and the waste liquid container 203 is used to collect buffer. The waste liquid container 203 is, for example, a waste liquid bag, and the volume of the waste liquid container 203 can be set according to actual conditions. The sterile filter 16 is used to communicate with the external environment. The sterile filter 16 is a sterile filter that can be sterilized. After passing through the sterile filter, the air can be provided to the sterilized air of the pipeline.

[0023] The first interface 11, the second interface 12, the third interface 13, the fourth interface 14 and the fifth interface 15 can all be Luer connectors, which facilitates the disassembly and assembly of the corresponding containers.

[0024] The pipeline structure 10 is provided with a plurality of control valves, a circulation valve 50 and a separation column 60. The peristaltic pump 100 is provided on the pipeline structure 10. The magnetic field applying unit is used to apply a magnetic field to the separation column 60 and to release the magnetic field applied to the separation column 60.

[0025] The pipeline structure 10 and the magnetic field applying unit are described in detail below.

[0026] Specifically, the pipeline structure 10 includes a first pipeline 21, a second pipeline 22, a third pipeline 23, a fourth pipeline 24, a fifth pipeline 25, a sixth pipeline 26, a pump pipe 27, a seventh pipeline 28, an eighth pipeline 29, a ninth pipeline 30, a tenth pipeline 31, an eleventh pipeline 32, a twelfth pipeline 33, a circulating flow pipeline 34, a thirteenth pipeline 35, a fourteenth pipeline 36 and a fifteenth pipeline 37.

[0027] The first pipeline 21, the ninth pipeline 30, and the seventh pipeline 28 are connected in sequence. The end of the first pipeline 21 away from the ninth pipeline 30 has the above-mentioned first interface 11. In this embodiment, the end of the first pipeline 21 away from the ninth pipeline 30 is connected to two branch pipelines 211, and the ends of the two branch pipelines 211 away from the first pipeline 21 respectively have a first interface 11. The second pipeline 22, the third pipeline 23, the fourth pipeline 24, and the fifth pipeline 25 are connected in sequence. The end of the second pipeline 22 away from the third pipeline 23 has the above-mentioned second interface 12. The sorting column 60 is connected between the fifth pipeline 25 and the sixth pipeline 26. The pump pipe 27 is connected between the sixth pipeline 26 and the seventh pipeline 28. The peristaltic pump 100 is arranged on the pump pipe 27. The way in which the peristaltic pump 100 is arranged on the pump pipe 27 is existing and will not be repeated here. One end of the eighth pipeline 29 is connected to the seventh pipeline 28, and the other end of the eighth pipeline 29 has the above-mentioned third interface 13. One end of the tenth pipeline 31 is connected between the first pipeline 21 and the ninth pipeline 30, and the other end of the tenth pipeline 31 is connected between the second pipeline 21 and the third pipeline 23. One end of the eleventh pipeline 32 is connected between the ninth pipeline 30 and the seventh pipeline 28, and the other end of the eleventh pipeline 32 is connected between the fourth pipeline 24 and the fifth pipeline 25. One end of the twelfth pipeline 33 is connected to the seventh pipeline 28, and the other end of the twelfth pipeline 33 has the fourth interface 14 mentioned above. One end of the circulating flow pipeline 34 is connected between the pump pipe 27 and the seventh pipeline 28, and the other end of the circulating flow pipeline 34 is connected to the fifth pipeline 25. One end of the thirteenth pipeline 35 is connected to the seventh pipeline 28, and the other end of the thirteenth pipeline 35 has the fifth interface 15 mentioned above. One end of the fourteenth pipeline 36 is connected between the fourth pipeline 24 and the fifth pipeline 25, and the other end of the fourteenth pipeline 36 is connected to one end of the fifteenth pipeline 37. The other end of the fifteenth pipeline 37 has the above-mentioned sterile filter 16, and the sterile filter 16 is used to filter the external air.

[0028] The plurality of control valves are respectively a first control valve 41, a second control valve 42, a third control valve 43, a fourth control valve 44, a fifth control valve 45, a sixth control valve 46, a seventh control valve 47, an eighth control valve 48, a ninth control valve 49, a tenth control valve 51, an eleventh control valve 52 and a twelfth control valve 53. The first control valve 41 is arranged on the first pipeline 21. The second control valve 42 is arranged on the second pipeline 22. The third control valve 43 is arranged on the third pipeline 23. The fourth control valve 44 is arranged on the fourth pipeline 24. The fifth control valve 45 is arranged on the eighth pipeline 29. The sixth control valve 46 is arranged on the seventh pipeline 28. The seventh control valve 47 is arranged on the tenth pipeline 31. The eighth control valve 48 is arranged on the eleventh pipeline 32. The ninth control valve 49 is arranged on the twelfth pipeline 33. The tenth control valve 51 is arranged on the thirteenth pipeline 35. The circulation valve 50 is arranged on the circulation flow pipeline 34. The thirteenth pipeline 35, the eighth pipeline 29 and the twelfth pipeline 33 are sequentially located between the sixth control valve 46 and the pump pipe 27. The eleventh control valve 52 is arranged on the fourteenth pipeline 36. The twelfth control valve 53 is arranged on the fifteenth pipeline 37.

[0029] The above-mentioned pipelines are connected through pipeline joints. A pinch valve 61 is respectively provided on the branch pipeline 211, the eighth pipeline 29, the twelfth pipeline 33, the thirteenth pipeline 35, and the fifteenth pipeline 37. The pinch valve 61 on the branch pipeline 211 is located between the first interface 11 and the first pipeline 21, the pinch valve 61 on the eighth pipeline 29 is located between the third interface 13 and the fifth control valve 45, the pinch valve 61 on the twelfth pipeline 33 is located between the fourth interface 14 and the ninth control valve 49, the pinch valve 61 on the thirteenth pipeline 35 is located between the fifth interface 15 and the tenth control valve 51, and the pinch valve 61 on the fifteenth pipeline 37 is located between the sterile filter 16 and the twelfth control valve 53. Before connecting the corresponding container to the interface, the corresponding pinch valve 61 needs to be closed first, and then the corresponding pinch valve 61 is opened after completion. Before disassembling the corresponding container from the interface, the corresponding pinch valve 61 also needs to be closed first to prevent air from entering the pipeline structure.

[0030] In this embodiment, a filter 231 is provided on the third pipeline 23, and the filter 231 is located between the second pipeline 22 and the third control valve 43. The filter 231 is used to filter the cell sample to filter large cells such as platelets. The fifteenth pipeline 37 is provided with a drip bucket 161, and the drip bucket 161 is located between the pinch valve 61 on the fifteenth pipeline 37 and the twelfth control valve 53.

[0031] The magnetic field applying unit includes a linear module 71, a U-shaped mounting bracket 72 and two magnets 74. The mounting bracket 72 is arranged at the top of the linear module 71. The linear module 71 is an existing structure and is not described here. The open end of the mounting bracket 72 faces the sorting column 60. The two magnets 74 are respectively magnetically attracted to the inner walls on both sides of the mounting bracket 72 and the two are arranged opposite to each other. The two magnets 74 are respectively close to the open ends of the mounting bracket 70. Two outer covers 73 ( Figure 3 Only the outer cover 73 on the right is shown in the figure. The two outer covers 73 are respectively arranged on the inner walls on both sides of the mounting bracket 72. The two outer covers 73 are provided to protect the two magnets 74. The linear module 71 is used to drive the mounting bracket 72 to move in the direction close to or away from the sorting column 60, thereby driving the two magnets 74 to move in the direction close to or away from the sorting column 60. When the two magnets 74 move to a predetermined position in the direction close to the sorting column 60, the sorting column 60 is located between the two magnets 74 and there is a gap between the two magnets 74. At this time, the two magnets 74 can generate a magnetic force around the sorting column 60, so that the magnetic field is applied to the sorting column 60. When the two magnets 74 move to a predetermined position in the direction away from the sorting column 60, the sorting column 60 is located on one side of the two magnets 74, so that no magnetic force is generated around the sorting column 60, so that the magnetic field applied to the sorting column 60 is released.

[0032] Please refer to Figure 3 The present invention also provides a magnetic cell sorting method, comprising the following steps:

[0033] S1. Perfusion: Connect the buffer container containing buffer, the sample container containing cell samples, the negative selection collection container 201, the positive selection collection container 202, and the waste liquid container 203 to the first interface 11, the second interface 12, the third interface 13, the fourth interface 14, and the fifth interface 15 of the pipeline structure 10 respectively.

[0034] Open the control valve located between the buffer solution container and the waste liquid container 203 on the pipeline structure 10 and close the remaining control valves, start the peristaltic pump 100, and make the peristaltic pump 100 rotate in the clockwise direction. The peristaltic pump 100 drives the buffer solution in the buffer solution container to flow toward the waste liquid container 203 to fill the pipeline structure 10 and discharge the bubbles in the separation column 60.

[0035] In the above step S1 , the control valves opened are the first control valve 41 , the third control valve 43 , the fourth control valve 44 , the seventh control valve 47 , the circulation valve 50 , the eighth control valve 48 and the tenth control valve 51 . The peristaltic pump 100 drives the buffer solution in the buffer solution container to flow along the first pipeline 21, the ninth pipeline 30, the tenth pipeline 31, the eleventh pipeline 32, the third pipeline 23, the fourth pipeline 24, the fifth pipeline 25, the circulating flow pipeline 34, the sorting column 60, the sixth pipeline 26, the pump tube 27, the seventh pipeline 28, and the thirteenth pipeline 35, so that the bubbles in the sorting column 60 can be discharged through the buffer solution, and the first pipeline 21, the ninth pipeline 30, the tenth pipeline 31, the eleventh pipeline 32, the third pipeline 23, the fourth pipeline 24, the fifth pipeline 25, the circulating flow pipeline 34, the sorting column 60, the sixth pipeline 26, the pump tube 27 and the seventh pipeline 28 can be perfused with buffer solution to achieve the purpose of cleaning the pipelines.

[0036] The rotation speed of the peristaltic pump 100 is 0-500 ml / min (milliliters / minute), preferably 100 ml / min.

[0037] S2. Injection: Apply a magnetic field to the sorting column 60 in the pipeline structure 10, open the control valve located between the sample container and the negative selection collection container 201 on the pipeline structure 10, and drive the cell sample in the sample container to flow toward the negative selection collection container 201 through the peristaltic pump 100, wherein the target cells in the cell sample are adsorbed in the sorting column 60, and the non-target cells enter the negative selection collection container 201 along with the flow of the cell sample.

[0038] It can be understood that a magnetic field is applied to the sorting column 60 in the pipeline structure 10 by the magnetic field application unit, the control valve located between the sample container and the negative selection collection container 201 on the pipeline structure 10 is opened and the remaining control valves are closed, the peristaltic pump 100 is started, and the peristaltic pump 100 is rotated in a clockwise direction, and the cell sample in the sample container is driven by the peristaltic pump 100 to flow toward the negative selection collection container 201, wherein when the cell sample passes through the sorting column 60, the target cells in the cell sample are adsorbed on the inner wall of the sorting column 60, and the non-target cells in the cell sample enter the negative selection collection container 201 along with the flow of the cell sample.

[0039] In the above step S2, the opened control valves are the second control valve 42, the third control valve 43, the fourth control valve 44 and the fifth control valve 45. The peristaltic pump 100 drives the cell sample in the sample container to flow along the second pipeline 22, the third pipeline 23, the fourth pipeline 24, the fifth pipeline 25, the sorting column 60, the sixth pipeline 26, the pump tube 27, the seventh pipeline 28 and the eighth pipeline 29 of the pipeline structure 10.

[0040] The rotation speed of the peristaltic pump 100 is 5-500 ml / min, preferably 10 ml / min.

[0041] S3, rinsing: flushing the pipeline structure 10 with a buffer solution so that the target cells remaining in the pipeline structure 10 are adsorbed in the separation column 60.

[0042] The specific steps include:

[0043] S31. Confirm that the sorting column 60 is in a state of applying a magnetic field, open the control valve located between the buffer container and the sample container on the pipeline structure 10 and close the remaining control valves, start the peristaltic pump 100, and make the peristaltic pump 100 rotate in a clockwise direction. The peristaltic pump 100 drives the buffer solution in the buffer container to flow toward the sample container to resuspend the target cells remaining in the sample container.

[0044] In the above step S31, the opened control valves are the first control valve 41, the sixth control valve 46, the fourth control valve 44, the third control valve 43 and the second control valve 42. The peristaltic pump 100 drives the buffer in the buffer container to flow along the first pipeline 21, the ninth pipeline 30, the seventh pipeline 28, the pump tube 27, the sixth pipeline 26, the sorting column 60, the fifth pipeline 25, the fourth pipeline 24, the third pipeline 23 and the second pipeline 22 of the pipeline structure 100.

[0045] The rotation speed of the peristaltic pump 100 is 5-100 ml / min, preferably 10 ml / min.

[0046] S32, open the control valve located between the sample container and the negative selection collection container 201 on the pipeline structure 10 and close the remaining control valves, start the peristaltic pump 100, and make the peristaltic pump 100 rotate in the clockwise direction, and drive the buffer solution in the sample container and the target cells remaining in the pipeline structure 10 to flow toward the negative selection collection container 201 through the peristaltic pump 100, wherein the sorting column 60 absorbs the target cells flowing through, and the buffer solution continues to flow and enters the negative selection collection container 201.

[0047] In the above step S32, the control valves opened are the second control valve 42, the third control valve 43, the fourth control valve 44 and the fifth control valve 45. The peristaltic pump 100 drives the buffer in the sample container and the target cells remaining in the second pipeline 22, the third pipeline 23, the fourth pipeline 24 and the fifth pipeline 25 to flow along the second pipeline 22, the third pipeline 23, the fourth pipeline 24, the fifth pipeline 25, the sorting column 60, the sixth pipeline 26, the pump tube 27, the seventh pipeline 28 and the eighth pipeline 29 of the pipeline structure 10, and fills the sixth pipeline 26 and the pump tube 27 with the buffer.

[0048] The rotation speed of the peristaltic pump 100 is 5-100 ml / min, preferably 10 ml / min.

[0049] Through the above-mentioned step S3, it is possible to avoid target cells remaining in the sample container and the pipeline structure 10, and to avoid wasting target cells.

[0050] S4, internal circulation cleaning, internal circulation cleaning includes:

[0051] S41. Release the magnetic field applied to the sorting column 60 through the magnetic field applying unit, open the circulation valve 50 on the pipeline structure 10 and close the remaining control valves, and drive the buffer solution in the circulation pipeline in the pipeline structure 10 to circulate through the peristaltic pump 100. When the buffer solution passes through the sorting column 60, the target cells in the sorting column 60 enter the buffer solution under the flushing of the buffer solution, and then flow with the buffer solution.

[0052] S42 . After the circulation flow, a magnetic field is applied to the separation column 60 by the magnetic field application unit, and the buffer solution in the circulation pipeline is driven to continue to flow by the peristaltic pump 100 , wherein the target cells in the circulation pipeline are re-adsorbed in the separation column 60 .

[0053] S43, confirm that the sorting column 60 is in a state of applying a magnetic field, open the control valve between the sorting column 60 and the negative separation collection container 201 and close the circulation valve 50 and the remaining control valves, and drive the buffer solution in the pipeline structure 10 through the peristaltic pump 100 to flush the sorting column 60.

[0054] The above step S41 specifically includes:

[0055] S411 , the two magnets 74 of the magnetic field applying unit are moved away from the sorting column 60 to release the magnetic field of the sorting column 60 .

[0056] S412, open the circulation valve 50 on the pipeline structure 10 and close the other control valves, start the peristaltic pump 100, and make the peristaltic pump 100 rotate in the clockwise direction, and the buffer solution in the circulation pipeline in the pipeline structure 10 is positively driven by the peristaltic pump 100 to circulate clockwise.

[0057] S413, reversely drive the buffer solution in the circulation pipeline in the pipeline structure 10 to circulate counterclockwise through the peristaltic pump 100.

[0058] Repeat the above steps S411-S413 twice, three times, etc., so that the buffer solution circulates clockwise and counterclockwise for multiple times. The specific number of times can be set according to actual conditions. By circulating multiple times, non-target cells stuck in the gaps between target cells can be washed out, thereby improving the purity of target cells.

[0059] In the above steps, the fifth pipeline 25, the separation column 60, the sixth pipeline 26, the pump tube 27 and the circulating flow pipeline 34 constitute the circulation pipeline of the pipeline structure 10. The rotation speed of the peristaltic pump 100 is 5-500 ml / min, preferably 200 ml / min.

[0060] In the above step S42, the peristaltic pump 100 rotates in a clockwise direction, and the buffer solution in the circulation pipeline circulates in a clockwise direction. The rotation speed of the peristaltic pump 100 is 5-100 ml / min, preferably 10 ml / min. In the above step S43, the peristaltic pump 100 rotates in a clockwise direction, and the rotation speed of the peristaltic pump is 5-100 ml / min, preferably 10 ml / min. The opened control valve is the fifth control valve 45. The buffer solution in the sixth pipeline 26 and the pump tube 27 of the pipeline structure 10 is driven by the peristaltic pump 100 to enter the negative selection collection container 201 along the fifth pipeline 25, the sorting column 60, the sixth pipeline 26, the pump tube 27, the seventh pipeline 28, and the eighth pipeline 29 of the pipeline structure 10, so as to collect the buffer solution through the negative selection collection container 201.

[0061] Through the internal circulation cleaning step, the magnetic field applied to the sorting column 60 is first released, and the peristaltic pump 100 is used to drive the buffer to circulate clockwise and counterclockwise, so that the buffer can be used to flush the target cells on the inner wall of the sorting column 60 in a positive and reverse direction, thereby cleaning out the non-target cells stuck in the gaps between the target cells, improving the purity of the target cells, and the purity of the target cells can reach 97%-99%. After that, the magnetic field is re-applied to the sorting column 60, and the peristaltic pump 100 is used to drive the buffer to circulate clockwise, so that the flushed target cells can be re-adsorbed on the inner wall of the sorting column 60, which is convenient for subsequent elution of the target cells.

[0062] The magnetic field applied to the sorting column 60 is released, the circulation valve 50 on the pipeline structure 10 is opened, and the buffer solution in the circulation pipeline of the pipeline structure 10 is driven to circulate by the peristaltic pump 100, and the process also includes: by controlling the opening and closing of the air valve, the fifteenth pipeline 37 is connected or disconnected with the external environment, so that the pressure in the pipeline can be released during the circulation of the buffer solution. Among them, the air valve is a control valve corresponding to the pipeline connected to the air through the sterile filter 16, and the air valve is the twelfth control valve 53, which connects the fifteenth pipeline 37 with the external environment through the sterile filter 16.

[0063] S5, elution: open the control valve corresponding to the positive selection collection container 202 and close the other control valves, elute the sorting column 60 after the magnetic field is released, and elute the target cells in the sorting column 60 into the positive selection collection container 202 through the peristaltic pump 100.

[0064] In the above step S5, in one embodiment, the control valve located between the buffer container and the positive selection collection container 202 on the pipeline structure 10 is opened, the peristaltic pump 100 is started, and the peristaltic pump 100 is rotated in a clockwise direction. The buffer in the buffer container is driven by the peristaltic pump 100 to flow toward the positive selection collection container 202. When the buffer passes through the sorting column 60, the buffer can elute the target cells in the sorting column 60, and then enter the positive selection collection container 202 with the flow of the buffer.

[0065] In the above step S5, in another implementation, it can be switched to eluent, that is, the buffer solution container is first removed from the first interface 11, and the eluent container containing eluent is connected to the first interface 11. The magnetic field applied to the sorting column 60 is released by the magnetic field application unit, and the control valve located between the eluent container and the positive selection collection container 202 on the pipeline structure 10 is opened, and the peristaltic pump 100 is started to rotate in a clockwise direction. The eluent in the eluent container is driven by the peristaltic pump 100 to flow toward the positive selection collection container 202, wherein when the eluent passes through the sorting column 60, the eluent can elute the target cells in the sorting column 60, and then enter the positive selection collection container 202 with the flow of the eluent.

[0066] It is understandable that step S5 can be eluted by a buffer solution or an eluent, and elution by a buffer solution does not require switching.

[0067] In the above step S5, the control valves opened are the first control valve 41, the eighth control valve 48 and the ninth control valve 49. The peristaltic pump 100 drives the eluent in the eluent container to flow along the first pipeline 21, the eleventh pipeline 32, the fifth pipeline 25, the separation column 60, the sixth pipeline 26, the pump tube 27, the seventh pipeline 28 and the twelfth pipeline 33.

[0068] The rotation speed of the peristaltic pump 100 is 50-500 ml / min, preferably 100 ml / min.

[0069] The magnetic cell sorting method of the present invention can automatically complete the steps of sampling, rinsing and elution by controlling the control valve and the peristaltic pump 100 on the pipeline structure 10, thereby automatically completing the cell sorting. Compared with the existing manual method, the sorting time is shortened, the sorting efficiency is improved, the operating errors are less likely to occur, the labor intensity is reduced, and the risk of cell contamination is reduced, thereby ensuring the purity of the sorted cells and preventing too much difference, which is beneficial to subsequent research.

[0070] The present invention also provides a magnetic cell sorting device, comprising the above-mentioned pipeline structure 10, a peristaltic pump 100, a magnetic field applying unit and a controller, wherein the peristaltic pump 100, the linear module 71 of the magnetic field applying unit, the multiple control valves of the pipeline structure 100 and the circulation valve 50 are all electrically connected to the controller. The controller is used to execute the above-mentioned magnetic cell sorting method.

[0071] The above is a specific description of the preferred implementation of the present invention, but the invention is not limited to the embodiments. Those skilled in the art can make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A magnetic cell sorting method, characterized in that: The following steps are involved: Apply a magnetic field to the sorting column in the pipeline structure, open the control valve between the sample container and the negative selection collection container on the pipeline structure, and drive the cell sample in the sample container to flow toward the negative selection collection container through the peristaltic pump, wherein the target cells in the cell sample are adsorbed in the sorting column, and the non-target cells enter the negative selection collection container along with the flow of the cell sample; The pipeline structure is flushed with a buffer solution so that the target cells remaining in the pipeline structure are adsorbed in the separation column; The control valve corresponding to the positive selection collection container is opened to elute the separation column after the magnetic field is released, and the target cells in the separation column are eluted into the positive selection collection container by a peristaltic pump.

2. The magnetic cell sorting method according to claim 1, characterized in that: After flushing the pipeline structure with a buffer solution so that the target cells remaining in the pipeline structure are adsorbed in the sorting column, and before opening the control valve corresponding to the positive selection collection container, the method further includes: The magnetic field applied to the separation column is released, the circulation valve on the pipeline structure is opened, and the buffer solution in the circulation pipeline of the pipeline structure is driven to circulate by the peristaltic pump; After the circulation flow, a magnetic field is applied to the separation column, and the buffer solution in the circulation line is driven to continue to flow by a peristaltic pump, wherein the target cells in the circulation line are re-adsorbed in the separation column; Confirm that the sorting column is in a state of applying a magnetic field, open the control valve between the sorting column and the negative separation collection container and close the circulation valve, and drive the buffer solution in the pipeline structure through the peristaltic pump to flush the sorting column.

3. The magnetic cell sorting method according to claim 2, characterized in that: The method of releasing the magnetic field applied to the separation column, opening the circulation valve on the pipeline structure, and driving the buffer solution in the circulation pipeline of the pipeline structure to circulate by a peristaltic pump includes: By moving the magnetic field applying unit away from the separation column, the magnetic field of the separation column is released; Open the circulation valve on the pipeline structure, and use the peristaltic pump to positively drive the buffer solution in the circulation pipeline of the pipeline structure to circulate clockwise; The peristaltic pump drives the buffer solution in the circulation pipeline in the pipeline structure in a reverse direction to circulate counterclockwise.

4. The magnetic cell sorting method according to claim 1, characterized in that: The step of flushing the pipeline structure with a buffer so that the target cells remaining in the pipeline structure are adsorbed in the separation column comprises: Confirm that the separation column is in a state of applying a magnetic field, open the control valve located between the buffer container and the sample container on the pipeline structure, and drive the buffer in the buffer container to flow to the sample container through the peristaltic pump to resuspend the target cells remaining in the sample container; The control valve located between the sample container and the negative selection collection container on the pipeline structure is opened, and the buffer solution in the sample container and the target cells remaining in the pipeline structure are driven by the peristaltic pump to flow toward the negative selection collection container, wherein the sorting column adsorbs the target cells flowing through.

5. The magnetic cell sorting method according to claim 1, characterized in that: The method of applying a magnetic field to the sorting column in the pipeline structure and opening the control valve located between the sample container and the negative selection collection container on the pipeline structure includes: connecting a buffer container containing a buffer solution, a sample container containing a cell sample, a negative selection collection container, a positive selection collection container, and a waste liquid container to the first interface, the second interface, the third interface, the fourth interface, and the fifth interface of the pipeline structure respectively.

6. The magnetic cell sorting method according to claim 2, characterized in that: The process of releasing the magnetic field applied to the separation column, opening the circulation valve on the pipeline structure and controlling the control valve corresponding to the internal circulation cleaning, and driving the buffer solution in the circulation pipeline in the pipeline structure to circulate by the peristaltic pump includes: By controlling the opening and closing of the air valve, the pressure in the tube is released during the circulation of the buffer solution; wherein the air valve is a control valve corresponding to the pipeline connected to the air through the sterile filter.

7. The magnetic cell sorting method according to claim 5, characterized in that: After the buffer container containing buffer, the sample container containing cell samples, the negative selection collection container, the positive selection collection container, and the waste liquid container are respectively connected to the first interface, the second interface, the third interface, the fourth interface, and the fifth interface of the pipeline structure, the process includes: opening a control valve located between the buffer container and the waste liquid container on the pipeline structure, and driving the buffer in the buffer container to flow toward the waste liquid container through a peristaltic pump to perfuse the pipeline structure and discharge bubbles in the sorting column.

8. A magnetic cell sorting device, which uses the magnetic cell sorting method according to any one of claims 1 to 7 to sort cells, characterized in that: It includes a pipeline structure, a peristaltic pump and a magnetic field applying unit. The pipeline structure has a first interface, a second interface, a third interface, a fourth interface and a fifth interface. The pipeline structure is provided with a plurality of control valves, a circulation valve and a sorting column. The peristaltic pump is arranged on the pipeline structure. The magnetic field applying unit is used for applying a magnetic field to the sorting column and for releasing the magnetic field applied to the sorting column.

9. The magnetic cell sorting device according to claim 8, characterized in that: The pipeline structure includes a first pipeline, a second pipeline, a third pipeline, a fourth pipeline, a fifth pipeline, a sixth pipeline, a pump pipe, a seventh pipeline, an eighth pipeline, a ninth pipeline, a tenth pipeline, an eleventh pipeline, a twelfth pipeline, a circulating flow pipeline and a thirteenth pipeline. The first pipeline, the ninth pipeline and the seventh pipeline are connected in sequence, and the end of the first pipeline away from the ninth pipeline has the first interface. The second pipeline, the third pipeline, the fourth pipeline and the fifth pipeline are connected in sequence, and the end of the second pipeline away from the third pipeline has the second interface. The sorting column is connected between the fifth pipeline and the sixth pipeline, the pump pipe is connected between the sixth pipeline and the seventh pipeline, the peristaltic pump is arranged on the pump pipe, and one end of the eighth pipeline is connected to the seventh pipeline. The eighth pipeline is connected, the other end of the eighth pipeline has the third interface, one end of the tenth pipeline is connected between the first pipeline and the ninth pipeline, the other end of the tenth pipeline is connected between the second pipeline and the third pipeline, one end of the eleventh pipeline is connected between the ninth pipeline and the seventh pipeline, the other end of the eleventh pipeline is connected between the fourth pipeline and the fifth pipeline, one end of the twelfth pipeline is connected to the seventh pipeline, the other end of the twelfth pipeline has the fourth interface, one end of the circulating flow pipeline is connected between the pump pipe and the seventh pipeline, the other end of the circulating flow pipeline is connected to the fifth pipeline, one end of the thirteenth pipeline is connected to the seventh pipeline, and the other end of the thirteenth pipeline has the fifth interface; The multiple control valves are respectively a first control valve, a second control valve, a third control valve, a fourth control valve, a fifth control valve, a sixth control valve, a seventh control valve, an eighth control valve, a ninth control valve and a tenth control valve. The first control valve is arranged on the first pipeline, the second control valve is arranged on the second pipeline, the third control valve is arranged on the third pipeline, the fourth control valve is arranged on the fourth pipeline, the fifth control valve is arranged on the eighth pipeline, the sixth control valve is arranged on the seventh pipeline, the seventh control valve is arranged on the tenth pipeline, the eighth control valve is arranged on the eleventh pipeline, the ninth control valve is arranged on the twelfth pipeline, the tenth control valve is arranged on the thirteenth pipeline, the circulation valve is arranged on the circulating flow pipeline, and the thirteenth pipeline, the eighth pipeline and the twelfth pipeline are located respectively between the sixth control valve and the pump pipe.

10. A magnetic cell sorting system, characterized in that: It includes a pipeline structure, a peristaltic pump, a magnetic field applying unit and a controller for executing the magnetic cell sorting method as described in any one of claims 1 to 7, the pipeline structure has a first interface, a second interface, a third interface, a fourth interface and a fifth interface, the pipeline structure is provided with a plurality of control valves, a circulation valve and a sorting column, the peristaltic pump is arranged on the pipeline structure, the magnetic field applying unit is used to apply a magnetic field to the sorting column and to release the magnetic field applied to the sorting column, and the peristaltic pump, the plurality of control valves, the circulation valve and the magnetic field applying unit are all electrically connected to the controller.

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