Method for high-purity sorting and capturing of rare cells

Abstract

The invention discloses a method for high-purity sorting and capturing of rare cells. According to the method for high-purity sorting and capturing of the rare cells, the cells are spread firstly, then the positions of the target cells are searched and determined, then a probe is used for discharging a buffer solution outwards to blow away non-target cells around the target cells, and then fluid power is used for capturing the target cells without damage. Finally, the target cells can be released into a receiving container, and high-purity sorting and capturing of the rare cells are achieved. A device matched with the method is simple in structure and easy to operate, and the method has the advantages that repeated and massive dilution is not needed, the cell sample spreading speed is high, the target cell sorting purity is high, the omission rate is low, in-situ capture is achieved, and repeatability is good. The method is suitable for the fields of single cell screening and analysis, single cell culture, single cell drug screening, single molecule analysis, gene screening, sample purification, antibody screening, microbiological research and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of rare cell sorting, and in particular relates to a method for high-purity sorting and capturing of rare cells. Background Art

[0002] Cells are heterogeneous, and the study of cell groups often obscures the differences between single cells, thus limiting the development of disciplines such as biology and medicine. Focusing on the study of single cells, especially single rare cells (such as circulating tumor cells, tumor stem cells, circulating endothelial progenitor cells, hematopoietic stem cells, etc.) is not only conducive to further deepening our understanding of life, the human body, and diseases, but also conducive to the development of new drugs and the treatment of diseases. In clinical settings, identifying some special rare cells can often provide more valuable information about the patient's disease status, such as metastasis, chemotherapy efficacy, genetic disease screening, etc., but the large number of non-target cells interferes, making the development of such research relatively limited. Therefore, accurately sorting and capturing single target cells (rare cells) from a large number of non-target cells has become a key technology in single-cell research.

[0003] Currently, the main technical methods for cell sorting and capture can be summarized into the following three categories: microfluidics-based technology, cell surface marker-based sorting technology, and micromanipulation-based technology.

[0004] The microfluidics-based method has the advantages of high reaction efficiency, strong applicability, and saving reagent sample consumption. However, the high loss of disposable microfluidic chips, the complex operation of microfluidic technology, and the difficulty in separating and releasing target cells for downstream analysis have also become the main obstacles to the popularization of this method. For example, the invention application with publication number CN111718836A discloses a microfluidic chip for rare cell acquisition and single cell encapsulation, which stacks the upper flow channel layer, the filter membrane layer, and the lower flow channel layer from top to bottom, uses microporous filter membrane technology to enrich rare cells in the sample liquid, uses droplet technology to encapsulate the enriched cells, and uses dielectrophoresis technology to purify and sort the droplets.

[0005] Cell surface marker sorting technologies represented by flow cytometry usually require a relatively large amount of sample, making it difficult to process those precious and rare samples.

[0006] The equipment required for micromanipulation technology is relatively simple. Visual operation, extremely low sampling volume, and extremely simple experimental consumables can increase the complete and lossless capture of rare cells while reducing sample loss. The technology that allows capture and in situ identification is also very suitable for phenotype-related analysis and research while retaining the corresponding spatial information. For example, the invention application with publication number CN113373104A discloses a device and method for high-purity sorting of rare cells, the method comprising: (1) spreading a cell sample; (2) scanning and imaging the spread cell sample; (3) reading the position information of the target cell and the distance information between the target cell and other surrounding non-target cells according to the imaging result, and judging the obtained distance information: if the distance information does not meet the set sampling distance requirement, then sampling the area where the target cell is located according to the position information of the target cell to obtain an intermediate cell sample containing the target cell and other surrounding non-target cells, spreading the obtained intermediate cell sample again, and returning to step (2); if the distance information meets the set sampling distance requirement, directly sampling the target cell to achieve the sorting of the target cell. However, this technical solution requires repeated imaging and analysis of the distance information between the target cells and other non-target cells, and repeated sampling and re-spreading near the target cells until the target cells are far enough away from the nearby non-target cells before the target cells can be absorbed. However, the operation is relatively cumbersome and time-consuming, and it is difficult to capture a very small number of rare cells in the presence of a large number of non-target cells. Summary of the invention

[0007] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for high-purity sorting and capturing of rare cells, which has the advantages of simple device, easy operation, no need for cell sample dilution, fast cell sample spreading speed, high target cell sorting purity, low omission rate, in situ capture, repeatability, etc. The method is suitable for single cell screening and analysis, single cell culture, single cell drug screening, single molecule analysis, gene screening, sample purification, antibody screening, microbial research and other fields.

[0008] A method for high-purity sorting and capturing of rare cells, comprising the following steps: (1) The sample is spread in a spreading container, where the rare target cells in the sample are mixed among a large number of non-target cells; (2) Determine the location of the target cells in the sample, as well as the positional relationship between the target cells and the non-target cells surrounding the target cells; (3) Using a tubular probe to discharge buffer, the non-target cells around the target cells are blown away, so that the non-target cells are away from the target cells to be captured; until there are no non-target cells that affect the capture near the target cells to be captured, the target cells are captured by suction through the probe.

[0009] Specifically, a method for high-purity sorting and capture of rare cells: (1) Spreading the sample in a spreading container, where a very small number of rare target cells are mixed with a large number of non-target cells; (2) using an imaging device to image the sample spread in the container to determine the location of the target cells in the sample, as well as the positional relationship between the target cells and the surrounding non-target cells; (3) Controlling the tubular probe so that its tip is aimed directly at the target cell and inserted into the sample solution from top to bottom; in the process of the probe tip being inserted into the sample solution and moving downward, controlling the fluid drive system connected to the end of the probe to discharge the buffer through the probe tip, blowing away the non-target cells around the target cell, and moving the non-target cells away from the target cell to be captured; until there are no non-target cells that affect the capture near the target cell to be captured, and the distance between the probe tip and the target cell reaches a set range, controlling the liquid drive system to suck the target cell into the probe channel through the probe tip, thereby completing the capture operation of the target cell; (4) Control the probe so that its tip is aligned with the receiving container of the target cells, and discharge the liquid out of the probe by controlling the liquid drive system, so that the target cells in the probe channel are discharged into the receiving container through the probe tip.

[0010] Preferably, samples used for high-purity sorting and capture of rare cells include: blood, pleural effusion, urine, cerebrospinal fluid, bone marrow, lymph fluid, and the actual sample types may not be limited to the above. The target cells may be cells that account for a very small proportion of cells in the sample, such as circulating tumor cells, tumor stem cells, circulating endothelial progenitor cells, hematopoietic stem cells, etc., and the actual cell types may not be limited to the above. The target cells may be single cells or cell clusters formed by a small number of cells aggregated together.

[0011] Taking advantage of the fact that the adhesion of target cells is usually higher than that of non-target cells such as white blood cells, when the cells are blown away at an appropriate flow rate, the non-target cells are blown away, while the target cells can still remain in place due to their adhesion to the surface of the spreading container, thereby keeping the non-target cells away from the target cells to be captured, avoiding interference of non-target cells in the capture of target cells.

[0012] The present invention is aimed at the problem of sorting rare cells with strong adhesion (such as circulating tumor cells). For example, circulating tumor cells are extremely rare, usually no more than 10 per milliliter of peripheral blood, so a single-cell capture method with high recovery rate, high purity and low damage is extremely needed. The present invention uses a single directional blowing to utilize the high adhesion of the target cells to the container surface. When the buffer is discharged, only the surrounding non-target cells (low adhesion or small volume / density) are blown away, while the target cells are still firmly attached to the surface due to differences in physical properties. At the same time, the target cells are held by a probe to avoid the absorption of non-target cells during the absorption process, ensuring that only a single target cell is captured, so that the single-cell capture success rate is as high as more than 80%, while avoiding the damage to the physiological activity of cells caused by long-term strong light exposure and repeated blowing.

[0013] When the sample used for high-purity sorting and capture of rare cells is blood, it is preferred to first perform a step of red blood cell lysis before sorting and capturing the target cells. Since there are a large number of red blood cells in the blood, there is a great influence on the sorting and capture of rare target cells. The red blood cells can be first lysed by red blood cell lysis solution, and the red blood cell fragments can be removed, and the remaining white blood cells and target cells can be collected. In this way, the proportion of target cells in the remaining cells is greatly increased, and the influence of red blood cells, which are cells with more eye-catching colors, on the observation is also avoided.

[0014] The observation and identification of target cells first rely on an inverted fluorescence microscope or other imaging instrument platform to image the sample cells spread in the container. Methods for identifying target cells among a large number of non-target cells include manual naked eye recognition, machine vision recognition, artificial intelligence comprehensive analysis recognition, and other methods.

[0015] Preferably, when determining the location of the target cells, the target cells are distinguished from the non-target cells by their size and / or cell morphology. If the target cells and non-target cells can be distinguished by their natural state characteristics such as size and morphology, the next step can be performed directly without marking the target cells. Otherwise, the target cells can be distinguished from the non-target cells by adding a labeling reagent for the target cells or non-target cells. Because the number of non-target cells is large and there may be many types, generally speaking, non-target cells are less frequently labeled, and target cells are generally distinguished from non-target cells by adding a label for the target cells.

[0016] The labeling method can be used as long as it can distinguish target cells from non-target cells. Preferably, the labeling method includes: antibody labeling, nucleic acid aptamer labeling, nucleic acid probe labeling, nanoparticle labeling, isotope labeling; the reporting method of the labeling reagent includes: fluorescence spectroscopy, ultraviolet visible absorption spectroscopy, Raman spectroscopy, mass spectrometry, electrochemical method, nucleic acid PCR amplification method, etc.; the actual labeling method and reporting method may not be limited to the above.

[0017] The spreading container needs to be able to spread the cells in the sample, and its top is open so that the cells can be easily operated using a probe. Preferably, the container for spreading the sample is a cell culture dish, a multi-well plate, or a microfluidic chip or other configuration sample spreading container with an open top and a bottom surface that can contact the target cells in the sample.

[0018] The function of the probe is to blow non-target cells away from the target cells and to sort and capture the target cells, and finally release the captured cells in a selected container. As a preferred solution, the probe is a hollow tubular structure with a sampling channel that can be used for liquid or gas injection and absorption and has a certain hardness. Preferably, the probe is a capillary-shaped capture probe with a pointed tip structure, and the inner diameter of the tip of the probe should match the size of the target cell. Generally, the inner diameter of the tip of the probe is slightly larger than the diameter of the target cell. If it is a target cell cluster, the inner diameter of the tip of the probe should be slightly larger than the size of the cell cluster to be sorted and captured. Further preferably, the surface of the tip and the internal channel of the probe can be subjected to a hydrophobic surface treatment or a treatment to reduce the adsorption of the probe surface to prevent or reduce the adsorption of cells to the probe wall.

[0019] Preferably, when the probe discharges liquid to disperse non-target cells around target cells, the flow rate is 5-200 nanoliters / second and the discharge volume is 10-500 nanoliters. The probe discharges liquid in two ways: continuous discharge of liquid or intermittent pulse discharge of liquid, or the two ways are used alternately.

[0020] When performing the capture operation of target cells, the success rate of the probe capturing target cells can be improved and the probability of the probe capturing non-target interference cells can be reduced by changing the inner diameter of the probe tip, the flow rate and volume of the cells and liquids sucked into the probe, and other conditions.

[0021] Preferably, when the target cells are captured by probe suction, the distance between the probe tip and the target cells is less than 10 microns; more preferably, the distance between the probe tip and the target cells is less than 5 microns.

[0022] Preferably, when the target cells are captured by probe suction, the suction flow rate is 10-200 nanoliters / second and the suction volume is 1-20 nanoliters.

[0023] The present invention limits the blowing flow rate and the suction volume, and limits the distance between the tip of the probe and the target cells. This range can prevent the detachment of adherent cells through experimental verification (instruction manual example). For example, a flow rate greater than 200 nanoliters / second will cause excessive impact force of the buffer solution, destroying the adhesion of the target cells; a volume greater than 20 nanoliters will easily inhale non-target cells.

[0024] Preferably, when performing high-purity sorting and capturing of rare cells, a dedicated sorting and capturing device is used, and the sorting and capturing device comprises: A tubular probe used to blow away non-target cells and capture target cells by suction; The mobile stage comprises a first mobile stage and a second mobile stage; the spreading container is placed on the first mobile stage, the probe is fixed on the second mobile stage, the top of the spreading container is open to facilitate the operation of the probe, and the bottom of the spreading container can contact the target cells in the sample; A cell imaging system, used for imaging, observing and detecting the cells in the spreading container; A three-dimensional mobile control system, comprising a horizontal driving device for controlling the first mobile stage to move in a horizontal plane, and a vertical driving device for controlling the vertical movement of a probe fixed on the second mobile stage, wherein the three-dimensional mobile control system is used to control and adjust the relative positions among the probe, the cell imaging system and the spreading container; The liquid driving device is connected to the end of the probe and is used to control the flow rate and volume of liquid discharge and liquid suction when the probe performs blowing and suction operations.

[0025] The liquid driving device can apply positive pressure or negative pressure to the liquid in the probe to complete the blowing of non-target cells and the capture of target cells. The liquid driving device includes a syringe pump, a pneumatic pump, an electroosmotic pump, a gravity liquid level difference driving device, etc. connected to the end of the probe. The actual fluid driving device is not limited to the above-mentioned ones.

[0026] In the present invention, fluorescence spectroscopy, ultraviolet visible absorption spectroscopy, Raman spectroscopy, mass spectrometry, electrochemical method, nucleic acid PCR amplification method, etc. can be used for imaging and detecting sample cells, and the instruments corresponding to these methods can be used as cell imaging systems. Preferably, an inverted fluorescence microscope is used to scan the sample area to be detected and image the target cells by fluorescence.

[0027] Compared with the prior art, the advantages of the present invention are mainly: The method for high-purity sorting and capturing of rare cells of the present invention first spreads the cells, then searches and determines the location of the target cells, and then uses a probe to inject a buffer outward to blow away the non-target cells around the target cells based on the inherent physical properties of the target cells (such as adhesion, density or size), and then uses a fluid dynamic force similar to blood flow to capture the target cells without damage, and finally releases the target cells into a receiving container to achieve high-purity sorting and capture of rare cells. The device used in conjunction with the method of the present invention has a simple structure and is easy to operate. The method has the advantages of no need for repeated large-scale dilution, fast cell sample spreading speed, high target cell sorting purity, low omission rate, in-situ capture, good repeatability, etc., and is suitable for single cell screening and analysis, single cell culture, single cell drug screening, single molecule analysis, gene screening, sample purification, antibody screening, microbial research and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic diagram of the structure of the sorting and capturing device of the present invention.

[0029] Figure 2 Schematic diagram of the operation principle of high-purity sorting and capture of rare cells in the present invention.

[0030] Figure 3 Schematic diagram of the operation of high-purity sorting and capturing of rare cells in Example 3.

[0031] Figure 4 Schematic diagram of the operation of high-purity sorting and capturing of rare cells in Example 4.

[0032] Description of reference numerals: A cell imaging system 1, a liquid driving device 2, a probe 3, a first moving platform 5 for placing a spreading container 4 and a receiving container 6, a receiving container 6, a second moving platform 7 with the probe 3 fixed thereon, a sample 8, target cells 9, non-target cells 10, a target cell cluster 11, and a multi-well plate 12. DETAILED DESCRIPTION

[0033] A method for high-purity sorting and capturing of rare cells, comprising the following steps: (1) The sample is spread in a spreading container, where the rare target cells in the sample are mixed among a large number of non-target cells.

[0034] Among them, samples used for high-purity sorting and capture of rare cells include: blood, pleural effusion, urine, cerebrospinal fluid, bone marrow, lymph fluid, and the actual sample types may not be limited to the above. The target cells may be cells that account for a very small proportion of the cells in the sample, such as circulating tumor cells, tumor stem cells, circulating endothelial progenitor cells, hematopoietic stem cells, etc. The actual cell types may not be limited to the above. The target cells may be single cells or cell clusters formed by a small number of cells aggregated together.

[0035] When the sample used for high-purity sorting and capture of rare cells is blood, red blood cell lysis is performed first before sorting and capturing. Since there are a large number of red blood cells in the blood, there is a great impact on the sorting and capture of rare target cells. The red blood cells can be lysed first with red blood cell lysis solution, and red blood cell fragments can be removed, and the remaining white blood cells and target cells can be collected. In this way, the proportion of target cells in the remaining cells is greatly increased, and the influence of red blood cells, which are cells with more eye-catching colors, on observation is avoided.

[0036] The spreading container needs to be able to spread the cells in the sample, and its top is open so that the cells can be easily operated using a probe. Preferably, the sample spreading container is a cell culture dish, a multi-well plate, or a microfluidic chip or other sample spreading container with an open top and a bottom surface that can contact the target cells in the sample.

[0037] (2) Determine the location of the target cells and judge the positional relationship between the target cells and non-target cells.

[0038] The observation and identification of target cells first rely on an inverted fluorescence microscope or other imaging instrument platform to image the sample cells spread in the container. Methods for identifying target cells among a large number of non-target cells include manual naked eye recognition, machine vision recognition, artificial intelligence comprehensive analysis recognition, and other methods.

[0039] Preferably, when determining the location of the target cells, the target cells are distinguished from the non-target cells by their size and / or cell morphology. If the target cells and non-target cells can be distinguished by their natural state characteristics such as size and morphology, the next step can be performed directly without marking the target cells. Otherwise, the target cells can be distinguished from the non-target cells by adding a labeling reagent for the target cells or non-target cells. Because the number of non-target cells is large and there may be many types, generally speaking, non-target cells are less frequently labeled, and target cells are generally distinguished from non-target cells by adding a label for the target cells.

[0040] The labeling method can be used as long as it can distinguish target cells from non-target cells. Preferably, the labeling method includes: antibody labeling, nucleic acid aptamer labeling, nucleic acid probe labeling, nanoparticle labeling, isotope labeling; the reporting method of the labeling reagent includes: fluorescence spectroscopy, ultraviolet visible absorption spectroscopy, Raman spectroscopy, mass spectrometry, electrochemical method, nucleic acid PCR amplification method, etc.; the actual labeling method and reporting method may not be limited to the above.

[0041] (3) Using a tubular probe, a buffer solution is injected to disperse non-target cells around the target cell, so that the non-target cells are moved away from the target cell to be captured. When there are no non-target cells that affect the capture near the target cell to be captured, the target cell is captured by suction through the probe.

[0042] The function of the probe is to blow non-target cells away from the target cells and to sort and capture the target cells, and finally release the captured cells in a selected container. As a preferred solution, the probe is a hollow tubular structure with a sampling channel that can be used for liquid or gas injection and absorption and has a certain hardness. Preferably, the probe is a capillary-shaped capture probe with a pointed tip structure, and the inner diameter of the tip of the probe should match the size of the target cell. Generally, the inner diameter of the tip of the probe is slightly larger than the target cell. If it is a target cell cluster, the inner diameter of the tip of the probe should be slightly larger than the size of the cell cluster to be sorted and captured. Further preferably, the surface of the tip and the internal channel of the probe can be subjected to a hydrophobic surface treatment or a treatment to reduce the adsorption of the probe surface to prevent or reduce the adsorption of cells to the probe wall.

[0043] Preferably, when the probe discharges liquid to disperse non-target cells around target cells, the flow rate is 5-200 nanoliters / second and the discharge volume is 10-500 nanoliters. The probe discharges liquid in two ways: continuous discharge of liquid or intermittent pulse discharge of liquid, or the two ways are used alternately.

[0044] When performing the capture operation of target cells, the success rate of the probe capturing target cells can be improved and the probability of the probe capturing non-target interference cells can be reduced by changing the inner diameter of the probe tip, the flow rate and volume of the cells and liquids sucked into the probe, and other conditions.

[0045] Preferably, when the target cells are captured by probe suction, the distance between the probe tip and the target cells is less than 10 microns; more preferably, the distance between the probe tip and the target cells is less than 5 microns.

[0046] Preferably, when the target cells are captured by suction through the probe, the suction flow rate is 10-200 nanoliters / second and the suction volume is 1-20 nanoliters. The tubular probe is used to blow away non-target cells and to capture target cells by suction; The mobile stage comprises a first mobile stage and a second mobile stage; the spreading container is placed on the first mobile stage, the probe is fixed on the second mobile stage, the top of the spreading container is open to facilitate the operation of the probe, and the bottom of the spreading container can contact the target cells in the sample; A cell imaging system, used for imaging, observing and detecting the cells in the spreading container; A three-dimensional mobile control system, comprising a horizontal driving device for controlling the first mobile stage to move in a horizontal plane, and a vertical driving device for controlling the vertical movement of a probe fixed on the second mobile stage, wherein the three-dimensional mobile control system is used to control and adjust the relative positions among the probe, the cell imaging system and the spreading container; The liquid driving device is connected to the end of the probe and is used to control the flow rate and volume of liquid discharge and liquid suction when the probe performs blowing and suction operations.

[0047] The liquid driving device can apply positive pressure or negative pressure to the liquid in the probe to complete the blowing of non-target cells and the capture of target cells. The liquid driving device includes a syringe pump, a pneumatic pump, an electroosmotic pump, a gravity liquid level difference driving device, etc. connected to the end of the probe. The actual fluid driving device is not limited to the above-mentioned ones.

[0048] In the present invention, fluorescence spectroscopy, ultraviolet visible absorption spectroscopy, Raman spectroscopy, mass spectrometry, electrochemical method, nucleic acid PCR amplification method, etc. can be used for imaging and detecting sample cells, and the instruments corresponding to these methods can be used as cell imaging systems. Preferably, an inverted fluorescence microscope is used to scan the sample area to be detected and image the target cells by fluorescence.

[0049] The technical solution of the present invention is illustrated below in conjunction with specific embodiments.

[0050] Example 1 The structure of the sorting and capturing device in this embodiment is as follows Figures 1 to 4As shown, it includes a cell imaging system 1, a liquid driving device 2, a probe 3, a first mobile platform 5 for placing a spreading container 4 and a receiving container 6, a receiving container 6, a second mobile platform 7 fixed with the probe 3, a sample 8, a target cell 9, a non-target cell 10, a target cell cluster 11, and a multi-well plate 12. The three-dimensional mobile control system in the present invention adopts a conventional structure in the prior art, which is not shown in the figure. The three-dimensional mobile control system is used to control the horizontal movement of the first mobile platform 5 on which the spreading container 4 and the receiving container 6 are placed, and to control the vertical movement of the second mobile platform 7 on which the probe 3 is fixed. The three-dimensional mobile control system can move the spreading container 4 and the probe 3 when the position of the target cell needs to be adjusted to cooperate with the operation of imaging or probe. The three-dimensional mobile control system controls the horizontal movement of the first mobile platform 5 to drive the spreading container 4 and the receiving container 6 to move, and the three-dimensional mobile control system controls the second mobile platform 7 to drive the probe 3 to move vertically, so that the relative movement between the probe 3 and the spreading container 4 and the receiving container 6 can be realized in three-dimensional space.

[0051] In this embodiment, the probe 3 is a single cell capture probe, which is a pointed quartz capillary with a channel, and the tip diameter of the capillary matches the size of the target cell to be captured. When in use, the angle between the probe 3 and the bottom surface of the cell sample spreading container 4 is 90 degrees, that is, the probe 3 is used vertically.

[0052] In this embodiment, the spreading container 4 for spreading the cell sample is a culture dish used in conventional cell culture. The receiving container 6 for carrying the captured cells can be any container capable of carrying cells, such as a microplate, a centrifuge tube, an inner insert tube, etc. The first moving stage 5 is an xy-axis electric translation stage, and the second moving stage 7 is a z-axis electric translation stage.

[0053] Example 2

[0054] The sorting and capturing device used in this example is the same as that in Example 1.

[0055] As a specific implementation, the sample is blood from a tumor patient, the target cells are circulating tumor cells in the blood of the tumor patient, and the single cell sorting and capturing method of circulating tumor cells is as follows: (1) Collect whole blood from cancer patients into EDTA-K2 anticoagulant tubes, use red blood cell lysis buffer to lyse 1 mL of whole blood, centrifuge to remove red blood cell fragments, and collect the mixed cell pellet of white blood cells and circulating tumor cells. Use circulating tumor cell-specific aptamer W3 and Hoechst 33324 nuclear dye to specifically label the target cells in the pellet; (2) Resuspend the labeled cell sample in 1 mL of PBS and spread it evenly in the cell culture dish; (3) Place the culture dish under an inverted fluorescence microscope and use the naked eye or an artificial intelligence image recognition system to automatically determine the location of the target circulating tumor cells based on the fluorescence signal; (4) Move probe 3 to just above the target circulating tumor cells, and adjust the probe height and the flow rate and volume of the liquid pumped out by the probe. Based on the characteristic that circulating tumor cells have higher adhesion than interfering leukocytes, blow away the interfering leukocytes around the target circulating tumor cells layer by layer, and control the blowing flow rate at 5-50 nanoliters / second, and the blowing volume at 10-100 nanoliters. Try the blowing speed and volume from the minimum value. If it fails to blow away, increase the blowing flow rate and volume step by step until the probe tip completely wraps the target cell and there are no interfering cells around. Then control the probe to absorb the target cell, and control the absorption flow rate at 50-100 nanoliters / second, and the absorption volume at 2-10 nanoliters. Try the absorption flow rate and volume from the minimum value. If it fails to absorb the cell, increase the absorption flow rate and volume step by step until a single circulating tumor cell is captured. For the specific operation process, see Figure 2 ; (5) Finally, the captured target cells are injected into the inner cannula for downstream operations and analysis, such as gene and transcriptome sequencing, proteomics analysis, etc.

[0056] Example 3

[0057] The sorting and capturing device used in this example is the same as that in Example 1.

[0058] like Figure 3 As shown, as a specific embodiment, the sample is pleural effusion of a tumor patient, the target cells are rare circulating tumor cells in the pleural effusion, and the cell sorting and capturing method is as follows: (1) Collect pleural effusion from tumor patients into a 50 mL centrifuge tube and centrifuge to collect cell precipitate; (2) Resuspend the labeled cell sample in 1 mL PBS and spread it evenly in the cell culture dish; (3) placing the culture dish on a cell imaging detector platform, and utilizing the characteristic that the target cells 9, i.e., circulating tumor cells, are usually larger in diameter, to search for circulating tumor cells and determine their locations; (4) Move the probe to just above the circulating tumor cells, and adjust the probe height and the flow rate and volume of the liquid pumped out by the probe. Blow away the non-target cells 10 (such as endothelial cells, blood cells, etc.) around the circulating tumor cells layer by layer, while blowing away as you descend. The blowing flow rate is controlled at 5-50 nanoliters / second, and the blowing volume is controlled at 10-100 nanoliters. After the probe tip completely wraps around the target cell 9 and there are no non-target cells 10 around it, control the probe 3 to absorb the target cell 9 - the circulating tumor cell. The absorption flow rate is controlled at 50-100 nanoliters / second, and the absorption volume is controlled at 2-10 nanoliters. Start by trying the absorption flow rate and volume from the minimum value. If the cell cannot be absorbed, gradually increase the absorption flow rate and volume until a single target cell 9 is successfully captured; (5) Finally, the captured target cells 9 are transferred to the multi-well plate 12 for further culture to construct a tumor cell line for downstream research such as drug screening.

[0059] Example 4

[0060] The sorting and capturing device used in this example is the same as that in Example 1.

[0061] like Figure 4 As shown, as a specific embodiment, the sample is blood from a tumor patient, the target cells are circulating tumor cell clusters in the blood of the tumor patient, and the method for capturing circulating tumor cell clusters in the tumor blood is as follows: (1) Collect whole blood from cancer patients into EDTA-K2 anticoagulant tubes, lyse 1 mL of whole blood with red blood cell lysis buffer, centrifuge to remove red blood cell fragments, and collect the mixed cell pellet of white blood cells and circulating tumor cells. The epithelial marker EpCAM fluorescent antibody, the white blood cell-specific CD45 fluorescent antibody, and the Hoechst 33324 nuclear dye are used to specifically label the target cells in the pellet; (2) Resuspend the labeled cell sample in 1 mL PBS and spread it evenly in the cell culture dish; (3) The culture dish is placed under an inverted fluorescence microscope, and the location of the target cell cluster 11, i.e., the circulating tumor cell cluster, is comprehensively determined using a machine automatic recognition system based on the cell size, morphology information, and fluorescence signal under bright field; (4) Move the probe to the top of the circulating tumor cell cluster, and according to the different adhesion characteristics between the cell cluster and the suspended single cell, blow away the non-target cells 10 around the target cell cluster 11 layer by layer, and control the blowing flow rate to 5-50 nanoliters / second, and the blowing volume to 10-100 nanoliters. Try the blowing speed and volume from the minimum value, and increase the blowing flow rate and volume step by step when it is impossible to blow away, until the probe tip completely wraps the target cell cluster 11, and there are no interfering cells around, control the probe to absorb, and control the absorption flow rate to 50-100 nanoliters / second, and the absorption volume to 2-10 nanoliters. Try the absorption flow rate and volume from the minimum value, and gradually increase the absorption flow rate and volume when it is impossible to absorb cells, until the target cell cluster 11 is successfully captured; (5) Finally, the captured target cell clusters 11 are released into the multi-well plate 12 for downstream testing such as sequencing and omics analysis or experiments such as organoid culture and drug screening.

Claims

1. A method for high-purity sorting and capture of rare cells, characterized in that: The following steps are involved: (1) The sample is spread in a spreading container, where the rare target cells in the sample are mixed among a large number of non-target cells; (2) Determine the location of the target cells in the sample, as well as the positional relationship between the target cells and the non-target cells surrounding the target cells; (3) Using a tubular probe to discharge buffer to blow away non-target cells around the target cell, so that the non-target cells are away from the target cell to be captured; until there are no non-target cells that affect the capture near the target cell to be captured, the target cell is captured by suction through the probe; Based on the fact that the target cells have a higher adhesion to the container surface than the non-target cells, or the density or size of the target cells is greater than that of the non-target cells, the probe is used to discharge buffer to blow away the non-target cells near the target cells, so that the non-target cells are away from the target cells to be captured.

2. The method for high-purity separation and capture of rare cells according to claim 1, characterized in that: The source of the sample used for high-purity sorting and capture of rare cells is one of the following: blood, pleural effusion, urine, cerebrospinal fluid, bone marrow, lymph fluid.

3. The method for high-purity separation and capture of rare cells according to claim 1, characterized in that: When determining the location of target cells, the target cells are distinguished from non-target cells by size and / or cell morphology; Alternatively, target cells can be distinguished from non-target cells by adding a labeling agent to the sample to label the target cells or non-target cells.

4. The method for high-purity separation and capture of rare cells according to claim 3, characterized in that: When determining the location of target cells, target cells are distinguished from non-target cells by adding a labeling agent to the sample to label the target cells.

5. The method for high-purity separation and capture of rare cells according to claim 3 or 4, characterized in that: The labeling method is at least one of the following: antibody labeling, nucleic acid aptamer labeling, nucleic acid probe labeling, nanoparticle labeling, and isotope labeling; The reporting method of the labeling reagent is at least one of the following: fluorescence spectroscopy, ultraviolet-visible absorption spectroscopy, Raman spectroscopy, mass spectrometry, electrochemical method, and nucleic acid PCR amplification method.

6. The method for high-purity separation and capture of rare cells according to claim 1, characterized in that: The spreading container is a cell culture dish, a multi-well plate, a microfluidic chip or a sample spreading container which is open at the top to facilitate the operation of the probe and whose bottom surface can contact with the target cells in the sample.

7. The method for high-purity separation and capture of rare cells according to claim 1, characterized in that: The probe is a capillary capture probe having a pointed tip structure, and the inner diameter of the tip of the probe is slightly larger than the diameter of the target cell.

8. The method for high-purity separation and capture of rare cells according to claim 1, characterized in that: When the probe blows away non-target cells around the target cells by discharging the buffer, the flow rate is 5 to 200 nanoliters per second and the discharge volume is 10 to 500 nanoliters; When the target cell is captured by probe suction, the distance between the tip of the probe and the target cell reaches a set range for extraction, and the distance between the tip of the probe and the target cell is less than 10 microns; The distance between the tip of the probe and the target cell is less than 5 microns; When the target cells are captured by probe aspiration, the aspiration flow rate is 10-200 nL / s and the aspiration volume is 1-20 nL.

9. The method for high-purity separation and capture of rare cells according to claim 1, characterized in that: Use a dedicated sorting and capturing device, which includes: A tubular probe used to blow away non-target cells and capture target cells by suction; The mobile stage comprises a first mobile stage and a second mobile stage; the spreading container is placed on the first mobile stage, the probe is fixed on the second mobile stage, the top of the spreading container is open to facilitate the operation of the probe, and the bottom of the spreading container is in contact with the target cells in the sample; A cell imaging system, used for imaging, observing and detecting the cells in the spreading container; A three-dimensional mobile control system, comprising a horizontal drive device for controlling the movement of the first mobile stage in a horizontal plane, and a vertical drive device for controlling the vertical movement of the probe fixed on the second mobile stage, wherein the three-dimensional mobile control system is used to control and adjust the relative positions among the probe, the cell imaging system and the spreading container; The liquid driving device is connected to the end of the probe and is used to control the flow rate and volume of liquid discharge and liquid suction when the probe performs blowing and suction operations.

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