A method for high-purity sorting and capture of rare cells

Abstract

The present invention discloses a method for high-purity sorting and capturing of rare cells. The method for high-purity sorting and capturing of rare cells in the present invention first spreads the cells, then locates and determines the position of the target cells, then uses a probe to expel the buffer outward to disperse the non-target cells around the target cells, and then uses hydrodynamic force to capture the target cells without damage. Finally, the target cells can be released into a receiving container to achieve high-purity sorting and capturing of rare cells. The device used in the method of the present invention has a simple structure and is easy to operate. The method has the advantages of not requiring repeated large-scale dilution, fast spreading speed of cell samples, high sorting purity of target cells, low omission rate, in-situ capture, and good repeatability, and is applicable to fields such as single-cell screening and analysis, single-cell culture, single-cell drug screening, single-molecule analysis, gene screening, sample purification, antibody screening, and microbial research.

Description

Technical Field

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

[0002] Cells are heterogeneous. The study of population cells often masks the differences between single cells, thus restricting 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, diseases, etc., but also conducive to the development of new drugs and the treatment of diseases. In a clinical setting, the identification of 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. However, the interference of a large number of non-target cells has relatively restricted the development of such research. 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 methods, cell surface marker sorting techniques, and micromanipulation techniques.

[0004] Microfluidics-based methods have the advantages of high reaction efficiency, strong applicability, and saving reagent sample consumption. However, the high loss of disposable microfluidic chips, the complexity of microfluidic technology operation, and the difficulty of 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 the publication number CN111718836A discloses a microfluidic chip for rare cell acquisition and single-cell encapsulation, which is stacked with an upper flow channel layer, a filter membrane layer, and a lower flow channel layer from top to bottom. The microporous filter membrane technology is used to enrich rare cells in the sample liquid, the droplet technology is used to encapsulate the enriched cells, and the dielectrophoresis technology is used to purify and sort the droplets.

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

[0006] The equipment required for micromanipulation technology is relatively simple. Visualized operation, extremely low sampling volume, and minimalist experimental consumables can increase the intact capture of rare cells with reduced sample loss. The technology that allows capture and in-situ identification is also very suitable for phenotypic-related analysis research while retaining the corresponding spatial information. For example, the invention application with the publication number CN113373104A discloses a device and method for high-purity sorting of rare cells. The method includes: (1) spreading a cell sample; (2) performing scanning imaging on the spread cell sample; (3) according to the imaging result, reading the position information of the target cell and the distance information between the target cell and other non-target cells around it, and judging the obtained distance information: if the distance information does not meet the set sampling distance requirement, then according to the position information of the target cell, sample the area where it is located to obtain an intermediate cell sample containing the target cell and other non-target cells around it, re-spread the obtained intermediate cell sample, and return to step (2); if the distance information meets the set sampling distance requirement, directly sample the target cell to achieve the sorting of the target cell. However, in this technical solution, it is necessary to repeatedly image and analyze the distance information between the target cell and other non-target cells, and it is necessary to repeatedly sample near the target cell and re-spread it until the distance between the target cell and the nearby non-target cells is far enough, and then the target cell can be aspirated. However, its operation is relatively cumbersome, 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 deficiencies in the prior art, the present invention provides a method for high-purity sorting and capture of rare cells, which has the advantages of simple device, simple operation, no need for cell sample dilution, fast cell sample spreading speed, high purity of target cell sorting, low omission rate, in-situ capture, repeatability, etc., and is applicable to fields such as single-cell screening and analysis, single-cell culture, single-cell drug screening, single-molecule analysis, gene screening, sample purification, antibody screening, and microbial research.

[0008] A method for high-purity sorting and capture of rare cells includes the following steps:

[0009] (1) Spread the sample in a spreading container, and the rare target cells in the sample are mixed among a large number of non-target cells;

[0010] (2) Determine the position of the target cell in the sample and the positional relationship between the target cell and the non-target cells around the target cell;

[0011] (3) Use a tubular probe to blow away non-target cells around the target cells by discharging buffer solution, so that the non-target cells are far away from the target cells to be captured; until there are no non-target cells affecting the capture near the target cells to be captured, aspirate and capture the target cells through the probe.

[0012] Specifically, a method for high-purity sorting and capture of rare cells:

[0013] (1) Spread the sample in a spreading container, and a very small number of rare target cells contained in the sample are mixed among a large number of non-target cells;

[0014] (2) Use an imaging device to image the sample spread in the container to determine the position of the target cells in the sample and the positional relationship between the target cells and the non-target cells around them;

[0015] (3) Control the tubular probe so that its tip is aligned directly above the target cell, and insert it into the sample solution from top to bottom; during the process of the probe tip inserting into the sample solution and moving downward, control the fluid driving system connected to the end of the probe to discharge buffer solution through the probe tip to blow away the non-target cells around the target cells, so that the non-target cells are far away from the target cells to be captured; until there are no non-target cells affecting the capture near the target cells to be captured, and after the distance between the probe tip and the target cell reaches the set range, control the liquid driving system to aspirate the target cell into the probe channel through the probe tip to complete the capture operation of the target cell;

[0016] (4) Control the probe so that its tip is aligned with the receiving container of the target cell, and discharge the liquid outside the probe by controlling the liquid driving system to discharge the target cell in the probe channel into the receiving container through the probe tip.

[0017] Preferably, the samples 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 list. The target cells can be cells that account for a very small proportion of the cells in the sample. For example, 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 list. The target cells can be single cells or cell clusters formed by aggregation of a small number of cells.

[0018] Utilize the characteristic that the adhesiveness of target cells is usually higher than that of non-target cells such as white blood cells. When blowing away the cells at an appropriate flow rate, the non-target cells are blown away, while the target cells can still remain in place due to adhering to the surface of the spreading container, so that the non-target cells are far away from the target cells to be captured, avoiding the interference of non-target cells on the capture of target cells.

[0019] The present invention addresses 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. Therefore, there is an urgent need for a single-cell capture method with high recovery rate, high purity, and low damage. Through single-time directional blowing, the present invention utilizes the high adhesion of target cells to the container surface. When the buffer is drained, only the non-target cells (with lower adhesion or smaller volume / density) around are blown away, while the target cells remain firmly attached to the surface due to physical property differences. At the same time, a probe is used to hold the target cells to avoid sucking non-target cells during the aspiration process, ensuring that only a single target cell is captured. The success rate of single-cell capture is over 80%, and at the same time, the damage to the physiological activity of cells caused by long-term strong light irradiation and repeated pipetting is avoided.

[0020] When the sample for high-purity sorting and capture of rare cells is blood, preferably, erythrocyte lysis is performed first and then the sorting and capture of target cells. Since a large number of erythrocytes exist in blood, they have a great impact on the sorting and capture of rare target cells. The erythrocytes can be lysed by an erythrocyte lysis solution, and the erythrocyte fragments can be removed. The remaining white blood cells and target cells are collected. In this way, the proportion of target cells in the remaining cells is greatly increased, and moreover, the influence of erythrocytes, which have a more prominent color, on observation is avoided.

[0021] The observation and identification of target cells first rely on an inverted fluorescence microscope or other imaging instrument platforms to image the sample cells spread in the container. The methods for identifying target cells among a large number of non-target cells include manual visual identification, machine vision identification, artificial intelligence comprehensive analysis and identification, etc.

[0022] Preferably, when determining the position of target cells, they are distinguished by the size and / or cell morphology between target cells and non-target cells. If the target cells and non-target cells can be distinguished by natural state characteristics such as size and morphology, the next step can be directly carried out without labeling the target cells. Otherwise, the target cells and non-target cells can be distinguished by adding a labeling reagent specific to target cells or non-target cells. Since the number of non-target cells is large and there may be many types, generally, non-target cells are less likely to be labeled. Generally, the target cells and non-target cells are distinguished by adding a label specific to target cells.

[0023] The way of labeling only needs to be able to distinguish target cells from non-target cells. Preferably, the labeling methods include: antibody labeling, nucleic acid aptamer labeling, nucleic acid probe labeling, nanoparticle labeling, isotope labeling; the reporting methods of labeling reagents include: fluorescence spectroscopy, ultraviolet-visible absorption spectroscopy, Raman spectroscopy, mass spectrometry, electrochemistry, nucleic acid PCR amplification method, etc.; the actual labeling methods and reporting methods are not limited to the above examples.

[0024] The spreading container needs to be able to spread the cells in the sample, and its upper part is open to facilitate the operation of the probe on the cells. Preferably, the container for spreading the sample is a cell culture dish, a multi-well plate, or a microfluidic chip with an open upper part and a bottom surface that can contact the target cells in the sample or other configured sample spreading containers.

[0025] The function of the probe is to blow away non-target cells around the target cells, sort and capture the target cells, and finally release the captured cells in the selected container. As a preferred solution, the probe is a hollow tubular structure with a certain hardness that can perform liquid or gas injection and aspiration and has a sampling channel. Preferably, the probe is a capillary-shaped capture probe with a sharpened tip structure, and the inner diameter of the tip of the probe should match the size of the target cells. Generally, the inner diameter of the tip of the probe is slightly larger than the diameter of the target cells. If it is a cluster of target cells, 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 inner channel of the probe can be subjected to a hydrophobic surface treatment or a treatment to reduce the adsorption on the probe surface to prevent or reduce the adsorption of cells on the probe wall.

[0026] Preferably, when the probe blows away non-target cells around the target cells by discharging liquid, the flow rate is 5-200 nanoliters / second, and the discharged volume is 10-500 nanoliters. The way of the probe discharging liquid includes two ways: continuously discharging liquid or intermittently discharging liquid in pulses, or alternately using the two ways.

[0027] 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 interfering cells can be reduced by changing conditions such as the inner diameter size of the probe tip, the flow rate and volume of the probe inhaling cells and liquid.

[0028] Preferably, when the probe aspirates and captures target cells, 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.

[0029] Preferably, when the probe aspirates and captures target cells, the aspiration flow rate is 10-200 nanoliters / second, and the aspiration volume is 1-20 nanoliters.

[0030] The present invention defines the blowing flow rate, the suction volume, and in combination, the distance between the tip of the probe and the target cells. This range is verified by experiments to avoid the detachment of adherent cells (Examples in the specification). For example, a flow rate greater than 200 nanoliters per second will cause too large an impact force of the buffer solution and damage the adhesion of target cells; a volume greater than 20 nanoliters is likely to inhale non-target cells.

[0031] Preferably, when performing high-purity sorting and capture of rare cells, a dedicated sorting and capture device is used. The sorting and capture device includes:

[0032] A tubular probe for blowing non-target cells and aspirating and capturing target cells;

[0033] A mobile stage, including 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 spreading container is open above to facilitate probe operation, and the bottom surface of the spreading container can contact the target cells in the sample;

[0034] A cell imaging system for imaging observation and detection of the cells in the spreading container;

[0035] A three-dimensional movement control system, including a horizontal driving device for controlling the movement of the first mobile stage in the horizontal plane and a vertical driving device for controlling the vertical movement of the probe fixed on the second mobile stage. The three-dimensional movement control system is used to control and adjust the relative positions among the probe, the cell imaging system, and the spreading container;

[0036] A liquid driving device, connected to the end of the probe, for controlling the flow rate and volume of liquid discharge and liquid suction during the blowing and aspirating operations of the probe.

[0037] The liquid driving device can apply positive pressure or negative pressure to the liquid in the probe to complete the operations of blowing non-target cells and capturing target cells. The liquid driving device includes an injection 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-listed ones.

[0038] In the present invention, fluorescence spectrometry, ultraviolet-visible absorption spectrometry, Raman spectrometry, mass spectrometry, electrochemistry, nucleic acid PCR amplification method, etc. can all be used for imaging and detection of sample cells, and the instruments corresponding to these methods can all be used as the cell imaging system. Preferably, an inverted fluorescence microscope is used to image the target cells by scanning the area of the sample to be detected and relying on fluorescence.

[0039] Compared with the prior art, the main advantages of the present invention are:

[0040] The method for high-purity sorting and capturing of rare cells in the present invention first spreads the cells, then locates and determines the positions of the target cells. Based on the inherent physical properties of the target cells (such as adhesion, density or size), a probe is used to inject buffer outward to disperse the non-target cells around the target cells, and then the target cells are captured non-invasively using hydrodynamic force similar to blood flow. Finally, the target cells can be released into the receiving container to achieve high-purity sorting and capturing of rare cells. The device used in the method of the present invention has a simple structure and is easy to operate. The method has the advantages of not requiring repeated large-scale dilution, fast spreading speed of cell samples, high sorting purity of target cells, low omission rate, in-situ capture, and good repeatability, and is applicable to fields such as single-cell screening and analysis, single-cell culture, single-cell drug screening, single-molecule analysis, gene screening, sample purification, antibody screening, and microbial research. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is a schematic structural diagram of the sorting and capturing device of the present invention.

[0042] Figure 2 It is a schematic diagram of the operation principle of high-purity sorting and capturing of rare cells in the present invention.

[0043] Figure 3 It is an operation schematic diagram during high-purity sorting and capturing of rare cells in Example 3.

[0044] Figure 4 It is an operation schematic diagram during high-purity sorting and capturing of rare cells in Example 4.

[0045] Description of the reference numerals:

[0046] Cell imaging system 1, liquid driving device 2, probe 3, first moving stage 5 for placing the spreading container 4 and the receiving container 6, receiving container 6, second moving stage 7 fixed with the probe 3, sample 8, target cell 9, non-target cell 10, target cell cluster 11, multi-well plate 12. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0047] A method for high-purity sorting and capturing of rare cells includes the following steps:

[0048] (1) Spread the sample in the spreading container, and the rare target cells in the sample are mixed among a large number of non-target cells.

[0049] Among them, the samples for high-purity sorting and capture of rare cells include: blood, pleural effusion, urine, cerebrospinal fluid, bone marrow, lymph fluid. The actual sample types may not be limited to those listed above. The target cells can be cells that account for a very small proportion of the cells in the sample. For example, circulating tumor cells, tumor stem cells, circulating endothelial progenitor cells, hematopoietic stem cells, etc. The actual cell types may not be limited to those listed above. The target cells can be single cells or cell clusters formed by the aggregation of a small number of cells.

[0050] When the sample for high-purity sorting and capture of rare cells is blood, first perform a step of red blood cell lysis and then perform sorting and capture. Since a large number of red blood cells exist in blood, it has a great impact on the sorting and capture of rare target cells. The red blood cells can be lysed by a red blood cell lysis solution, and the red blood cell fragments can be removed, and the remaining white blood cells and target cells are collected. In this way, the proportion of target cells in the remaining cells is greatly increased, and moreover, the influence of red blood cells, which have a more prominent color, on observation is avoided.

[0051] The spreading container needs to be able to spread the cells in the sample, and its upper part is open to facilitate the operation of the cells with a probe. Preferably, the container for spreading the sample is a cell culture dish, a multi-well plate, or a microfluidic chip with an open upper part and a bottom surface that can contact the target cells in the sample or other configured sample spreading containers.

[0052] (2) Determine the position of the target cells and judge the positional relationship between the target cells and non-target cells.

[0053] The observation and identification of the target cells first rely on an inverted fluorescence microscope or other imaging instrument platforms to image the sample cells spread in the container. The methods for identifying target cells among a large number of non-target cells include methods such as manual visual identification, machine vision identification, and artificial intelligence comprehensive analysis and identification.

[0054] Preferably, when determining the position of the target cells, distinguish them by the size and / or cell morphology between the target cells and non-target cells. If the target cells and non-target cells can be distinguished by natural state characteristics such as size and morphology, the next step can be directly carried out without labeling the target cells. Otherwise, the target cells and non-target cells can be distinguished by adding a labeling reagent for the target cells or non-target cells. Since the number of non-target cells is large and the types may also be numerous, generally speaking, it is less common to label non-target cells. Generally, the target cells and non-target cells are distinguished by adding a label for the target cells.

[0055] The marking method only needs to be able to distinguish target cells from non-target cells. Preferably, the marking method includes: antibody marking, nucleic acid aptamer marking, nucleic acid probe marking, nanoparticle marking, isotope marking; the reporting methods of the marking reagent include: fluorescence spectroscopy, ultraviolet-visible absorption spectroscopy, Raman spectroscopy, mass spectrometry, electrochemistry, nucleic acid PCR amplification method, etc.; the actual marking method and reporting method are not limited to the above examples.

[0056] (3) Use a tubular probe to blow away non-target cells around the target cells by injecting buffer solution, so that the non-target cells are far away from the target cells to be captured. When there are no non-target cells affecting the capture near the target cells to be captured, capture the target cells by aspirating with the probe.

[0057] The function of the probe is to blow non-target cells away from the target cells, 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 certain hardness that can perform liquid or gas injection and aspiration and has a sampling channel. Preferably, the probe is a capillary-shaped capture probe with a sharpened tip structure. The inner diameter of the tip of the probe should match the size of the target cells. Generally, the inner diameter of the tip of the probe is slightly larger than the target cells. If it is a cluster of target cells, 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 inner channel of the probe can be subjected to a hydrophobic surface treatment or a treatment to reduce the adsorption on the probe surface to prevent or reduce the adsorption of cells on the probe wall.

[0058] Preferably, when the probe blows away non-target cells around the target cells by discharging liquid, the flow rate is 5-200 nanoliters / second, and the discharged volume is 10-500 nanoliters. The way the probe discharges liquid includes two ways: continuously discharging liquid or intermittently discharging liquid in pulses, or using the two ways alternately.

[0059] 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 interfering cells can be reduced by changing conditions such as the inner diameter size of the probe tip, the flow rate and volume of the probe sucking in cells and liquid.

[0060] Preferably, when capturing target cells by aspirating with the probe, 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.

[0061] Preferably, when capturing target cells by aspirating with the probe, the aspiration flow rate is 10-200 nanoliters / second, and the aspiration volume is 1-20 nanoliters. The tubular probe is used to blow away non-target cells and aspirate and capture target cells;

[0062] A mobile station, including a first mobile station and a second mobile station; the spreading container is placed on the first mobile station, the probe is fixed on the second mobile station, the spreading container is open at the top to facilitate probe operation, and the bottom surface of the spreading container can contact the target cells in the sample;

[0063] A cell imaging system for imaging observation and detection of the cells in the spreading container;

[0064] A three-dimensional movement control system, including a horizontal driving device for controlling the movement of the first mobile station in the horizontal plane and a vertical driving device for controlling the vertical movement of the probe fixed on the second mobile station. The three-dimensional movement control system is used to control and adjust the relative positions among the probe, the cell imaging system and the spreading container;

[0065] A 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.

[0066] 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 an injection pump, a pneumatic pump, an electroosmotic pump, a gravity liquid level difference driving device, etc. that are connected to the end of the probe. The actual fluid driving device is not limited to the above examples.

[0067] In the present invention, fluorescence spectrometry, ultraviolet-visible absorption spectrometry, Raman spectrometry, mass spectrometry, electrochemistry, nucleic acid PCR amplification method, etc. can all be used for imaging and detection of sample cells, and the instruments corresponding to these methods can all be used as the cell imaging system. Preferably, an inverted fluorescence microscope is used to image the target cells by scanning the area of the sample to be detected and relying on fluorescence.

[0068] The technical solution of the present invention will be illustrated by specific embodiments below.

[0069] Embodiment 1

[0070] In this embodiment, the structure of the sorting and capturing device is as Figures 1 to 4As shown, it includes a cell imaging system 1, a liquid driving device 2, a probe 3, a first moving stage 5 for placing and spreading a container 4 and a receiving container 6, the receiving container 6, a second moving stage 7 fixed with the probe 3, a sample 8, target cells 9, non-target cells 10, target cell clusters 11, and a multi-well plate 12. The three-dimensional movement control system in the present invention adopts a conventional structure in the prior art and is not shown in the figure. The three-dimensional movement control system is used to control the horizontal movement of the first moving stage 5 on which the spreading container 4 and the receiving container 6 are placed, and to control the vertical movement of the second moving stage 7 fixed with the probe 3. The three-dimensional movement control system can move the spreading container 4 and the probe 3 when the position of the target cells needs to be adjusted, in cooperation with imaging or probe operations. The three-dimensional movement control system controls the horizontal movement of the first moving stage 5 to drive the spreading container 4 and the receiving container 6 to move, and the three-dimensional movement control system controls the second moving stage 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 in three-dimensional space can be realized.

[0071] In this embodiment, the probe 3, as a single-cell capture probe, is a pulled-tip quartz capillary with a channel, and the tip diameter of the capillary is matched with the size of the target cells to be captured. When in use, the included angle between the probe 3 and the bottom surface of the cell sample spreading container 4 is 90°, that is, the probe 3 is used vertically.

[0072] In this embodiment, the spreading container 4 for cell sample spreading is a culture dish used in conventional cell culture. The receiving container 6 for carrying the captured cells can be any container capable of loading cells, such as a multi-well plate, a centrifuge tube, an inner cannula, etc. The first moving stage 5 is an x-y axis electric translation stage, and the second moving stage 7 is a z-axis electric translation stage.

[0073] Example 2

[0074] The sorting and capture device used in this embodiment is the same as that in Example 1.

[0075] As a specific implementation method, 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 capture method for circulating tumor cells is as follows:

[0076] (1) Take the whole blood of a tumor patient into an EDTA-K2 anticoagulant tube. After lysing 1 mL of whole blood with red blood cell lysate, centrifuge to remove red blood cell fragments, and collect the mixed cell precipitate of white blood cells and circulating tumor cells. Use the circulating tumor cell-specific aptamer W3 and Hoechst 33324 nuclear dye to specifically label the target cells in the precipitate;

[0077] (2) Resuspend the labeled cell sample with 1 mL of PBS and evenly spread it on a cell culture dish;

[0078] (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 position of the target circulating tumor cells based on the fluorescence signal;

[0079] (4) Move the probe 3 directly above the target circulating tumor cell, and adjust the height of the probe and the flow rate and volume of the liquid pumped out by the probe. Based on the characteristic that the adhesion of circulating tumor cells is higher than that of interfering white blood cells, layer by layer, blow away the interfering white blood cells around the target circulating tumor cells. The blowing-away flow rate is controlled at 5 - 50 nanoliters per second, and the blowing-away volume is controlled at 10 - 100 nanoliters. Start trying from the minimum values of the blowing-away speed and volume. If the cells cannot be blown away, gradually increase the blowing-away flow rate and volume until the tip of the probe completely wraps the target cell and there are no interfering cells around. Then, control the probe to aspirate the target cell. The aspiration flow rate is controlled at 50 - 100 nanoliters per second, and the aspiration volume is controlled at 2 - 10 nanoliters. Start trying from the minimum values of the aspiration flow rate and volume. If the cells cannot be aspirated, gradually increase the aspiration flow rate and volume until a single circulating tumor cell is captured. For the specific operation process, see Figure 2 ;

[0080] (5) Finally, inject the captured target cells into the inner cannula for downstream operations and analyses, such as gene and transcriptome sequencing, proteomics analysis, etc.

[0081] Example 3

[0082] The sorting and capturing device used in this example is the same as that in Example 1.

[0083] As Figure 3 shown, as a specific implementation manner, the sample is the pleural effusion of a tumor patient, and the target cells are rare circulating tumor cells in the pleural effusion. The cell sorting and capturing method is as follows:

[0084] (1) Take the pleural effusion of a tumor patient into a 50 mL centrifuge tube and centrifuge to collect the cell pellet;

[0085] (2) Resuspend the labeled cell sample with 1 mL of PBS and evenly spread it in the cell culture dish;

[0086] (3) Place the culture dish on the cell imaging detector platform, and use the characteristic that the target cell 9, namely the circulating tumor cell, usually has a larger diameter to find the circulating tumor cell and determine its location;

[0087] (4) Move the probe directly above the circulating tumor cell, and adjust the height of the probe and the flow rate and volume of the liquid pumped out by the probe. Gradually blow away the non-target cells 10 (such as endothelial cells, blood cells, etc.) around the circulating tumor cell layer by layer. Blow while descending, and control the blowing flow rate at 5 - 50 nanoliters per second and the blowing volume at 10 - 100 nanoliters. Until the tip of the probe completely wraps the target cell 9 and there are no non-target cells 10 around it, then control the probe 3 to aspirate the target cell 9 - the circulating tumor cell. Control the aspiration flow rate at 50 - 100 nanoliters per second and the aspiration volume at 2 - 10 nanoliters. Start trying from the minimum values of the aspiration flow rate and volume. If the cells cannot be aspirated, gradually increase the aspiration flow rate and volume until a single target cell 9 is successfully captured;

[0088] (5) Finally, transfer the captured target cell 9 to a multi-well plate 12 for further culture to construct a tumor cell line for downstream research such as drug screening.

[0089] Example 4

[0090] The sorting and capture device used in this example is the same as that in Example 1.

[0091] Such as Figure 4 As shown, as a specific implementation method, the sample is blood from a tumor patient, and the target cell is a circulating tumor cell cluster in the blood of the tumor patient. The capture method for the circulating tumor cell cluster in the tumor blood is as follows:

[0092] (1) Take the whole blood of a tumor patient in an EDTA-K2 anticoagulant tube. After lysing 1 mL of the whole blood with red blood cell lysate, centrifuge to remove the red blood cell debris, and collect the mixed cell pellet of white blood cells and circulating tumor cells. Specifically label the target cells in the pellet with the epithelial marker EpCAM fluorescent antibody, the white blood cell-specific CD45 fluorescent antibody, and the Hoechst 33324 nuclear dye;

[0093] (2) Resuspend the labeled cell sample with 1 mL of PBS and evenly spread it in a cell culture dish;

[0094] (3) Place the culture dish under an inverted fluorescence microscope, and use the machine automatic recognition system to comprehensively determine the position of the target cell cluster 11, that is, the circulating tumor cell cluster, according to the cell size and morphological information under bright field and the fluorescence signal;

[0095] (4) Move the probe directly above the circulating tumor cell cluster. According to the different adhesion characteristics between the cell cluster and the suspended single cells, blow away the non-target cells 10 around the target cell cluster 11 layer by layer. The blowing rate is controlled at 5 - 50 nanoliters per second, and the blowing volume is controlled within the range of 10 - 100 nanoliters. Start trying from the minimum values of the blowing speed and volume. When it is impossible to blow away the cells, gradually increase the blowing rate and volume until the probe tip completely encloses the target cell cluster 11 and there are no interfering cells around it. Then, control the probe to aspirate. The aspiration rate is controlled at 50 - 100 nanoliters per second, and the aspiration volume is controlled at 2 - 10 nanoliters. Start trying from the minimum values of the aspiration rate and volume. When it is impossible to aspirate the cells, gradually increase the aspiration rate and volume until the target cell cluster 11 is successfully captured;

[0096] (5) Finally, release the captured target cell cluster 11 into the multi-well plate 12 for downstream tests 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, It includes the following steps: (1) Spread the sample in a spreading container, where rare target cells in the sample are mixed among a large number of non-target cells; (2) Determine the positions of the target cells in the sample and the positional relationship between the target cells and the non-target cells around the target cells; (3) Use a tubular probe to blow away the non-target cells around the target cells by discharging buffer solution, so that the non-target cells move away from the target cells to be captured; until there are no non-target cells affecting the capture near the target cells to be captured, then aspirate and capture the target cells through the probe; Based on the fact that the adhesion of the target cells to the container surface is higher than that of the non-target cells, or the density or size of the target cells is greater than that of the non-target cells, use the probe to discharge buffer solution to blow away the non-target cells near the target cells, so that the non-target cells move away from the target cells to be captured.

2. The method for high-purity sorting and capture of rare cells according to claim 1, characterized in that The source of the sample 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 sorting and capturing of rare cells according to claim 1, wherein When determining the positions of the target cells, distinguish them by the size and / or cell morphology between the target cells and the non-target cells; Or, distinguish the target cells from the non-target cells by adding a labeling reagent to the sample to label the target cells or non-target cells.

4. The method for high-purity sorting and capture of rare cells according to claim 3, characterized in that, When determining the positions of the target cells, distinguish the target cells from the non-target cells by adding a labeling reagent to the sample to label the target cells.

5. The method for high-purity sorting and capturing 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 probe labeling, nanoparticle labeling, 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, electrochemistry, nucleic acid PCR amplification method.

6. The method for high-purity sorting and capturing of rare cells according to claim 1, wherein The spreading container is a cell culture dish, a multi-well plate, a microfluidic chip or a sample spreading container that is open above to facilitate the operation of the probe and whose bottom surface can contact the target cells in the sample.

7. The method for high-purity sorting and capturing of rare cells according to claim 1, characterized in that, The probe is a capillary-shaped capture probe with a sharpened tip structure, and the inner diameter of the tip of the probe is slightly larger than the diameter of the target cells.

8. The method for high-purity sorting and capturing of rare cells according to claim 1, wherein When the probe discharges buffer solution to blow away the non-target cells around the target cells, the flow rate is 5 - 200 nanoliters per second, and the discharged volume is 10 - 500 nanoliters; When aspirating and capturing the target cells through the probe, the extraction is carried out when the distance between the tip of the probe and the target cells reaches a set range, and the distance between the tip of the probe and the target cells is less than 10 micrometers; The distance between the tip of the probe and the target cells is less than 5 micrometers; When aspirating and capturing the target cells through the probe, the aspiration flow rate is 10 - 200 nanoliters per second, and the aspiration volume is 1 - 20 nanoliters.

9. The method for high-purity sorting and capture of rare cells according to claim 1, wherein Use a dedicated sorting and capture device, and the sorting and capture device includes: A tubular probe for blowing away non-target cells and aspirating and capturing target cells; A moving stage, including a first moving stage and a second moving stage; the spreading container is placed on the first moving stage, the probe is fixed on the second moving stage, the spreading container is open above to facilitate the operation of the probe, and the bottom surface of the spreading container contacts the target cells in the sample; A cell imaging system for imaging observation and detection of the cells in the spreading container; A three-dimensional movement control system includes a horizontal driving device for controlling the movement of the first mobile station in a horizontal plane, and a vertical driving device for controlling the vertical movement of a probe fixed on a second mobile station. The three-dimensional movement control system is used to control and adjust the relative positions among the probe, a cell imaging system, and a spreading container; A 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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