A processing method and system for extracting live single cells based on deformation trend

Abstract

This invention provides a method for processing live single-cell extraction based on deformation trends, comprising the following steps: Step S1: Controlling a microfluidic chip to immerse itself in a culture medium containing adherent cells while maintaining a preset gap, controlling the injection flow rate of the injection channel and the aspiration flow rate of the aspiration channel to form a microjet acting on the cells; Step S2: Controlling an image processing module to receive and process cell morphology images on the culture substrate acquired by a microscopic imaging module, and calculating deformation parameters based on the comparison between the cell area in the current frame image and the initial cell area; Step S3: Comparing the deformation parameters with critical deformation parameters obtained through experimental calibration, and when the deformation parameters are greater than the critical deformation parameters, controlling a fluid drive module to reduce the injection flow rate and / or the aspiration flow rate; or, calculating a comprehensive damage risk index based on the deformation parameters, and when it is greater than 1, reducing the injection flow rate and / or the aspiration flow rate.

Description

Technical Field

[0001] This invention relates to the field of cell extraction technology, and in particular to a processing method and system for extracting live single cells based on deformation trends. Background Technology

[0002] Live single-cell extraction is widely used in life science research, drug screening, single-cell sequencing, and regenerative medicine. It allows for the non-destructive separation of target single cells from adherent cultured cells, playing a crucial role in maintaining cell viability, physiological function, and the accuracy of downstream analyses. Currently, extraction is mainly performed using the following methods: mechanical methods, which involve directly peeling cells using micromanipulation needles, but these are complex, experience-dependent, have low throughput, and are prone to mechanical damage; and microfluidic extraction, which uses a dual-orifice structure to generate microjets for cell separation, offering advantages such as flexible operation, integrability, and relatively less physical damage.

[0003] Currently, microfluidic extraction technology mainly operates using preset fixed flow rates or pressures. However, due to the heterogeneity in the adhesion strength, size, and mechanical properties of different cells, it is difficult to adapt to individual differences. Excessive fluid shear force can easily lead to irreversible cell deformation or even rupture, while insufficient shear force cannot effectively separate the cells. Furthermore, the current method often uses "whether cells detach" as the criterion for judgment, meaning that although the extracted cells may be morphologically intact, they may have been damaged during the detachment process. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a processing method and system for extracting live single cells based on deformation trends.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, a method for processing live single-cell extraction based on deformation trends includes the following steps:

[0007] Step S1: Control the microfluidic chip to be immersed in the culture medium containing adherent cells and maintain a preset gap, control the injection flow rate of the injection channel and the aspiration flow rate of the aspiration channel to form a microjet acting on the cells;

[0008] Step S2: The image processing module receives and analyzes the cell morphology image on the culture substrate acquired by the microscopic imaging module, and calculates the deformation parameters based on the comparison between the cell area in the current frame image and the initial cell area. ;

[0009] Step S3: The deformation parameters Compared with the critical deformation parameters obtained through experimental calibration In comparison, when the deformation parameter Greater than the At that time, the control fluid drive module reduces the injection flow rate and / or the suction flow rate; or,

[0010] According to deformation parameters Calculate the comprehensive damage risk index ,when If the value is greater than 1, reduce the injection flow rate and / or aspiration flow rate.

[0011] Optionally, in step S2, the deformation parameter include:

[0012] ;

[0013] in This represents the cell area in the current frame. This represents the initial cell area before extraction begins.

[0014] Optionally, in step S3, the comprehensive damage risk index include:

[0015] ;

[0016] ;

[0017] in For deformation rate, For deformation acceleration, The image sampling time interval, , , , These are the critical deformation parameters, critical deformation rate, critical deformation acceleration, and critical cumulative deformation, respectively, calibrated through preliminary experiments. When the value is greater than 1, reduce the injection flow rate and / or aspiration flow rate.

[0018] Optionally, the preliminary experiment is calibrated through the following steps:

[0019] (i) A stepwise increasing method was used to control the inhalation flow rate and / or injection flow rate to conduct a progressive destruction experiment on the target cell type;

[0020] (ii) During the experiment, continuously acquire cell morphology images and record the deformation parameters when irreversible cell damage occurs. Deformation rate Deformation acceleration and cumulative deformation ;

[0021] (iii) Perform repeated experiments on at least 30 cells of the same type and conduct statistical analysis on the data of each parameter, including but not limited to taking the arithmetic mean, median, or specific percentile of the sample parameters, or calculating their confidence interval by fitting a normal distribution, and use this as the critical value of the fluid handling limit of this type of cell.

[0022] Optionally, the deformation parameters in step S3 are calculated based on smoothed filtering of multiple frames of images to reduce the impact of measurement noise on the calculation.

[0023] Optionally, the control method further includes step S4: recording the separation time required from the start of the extraction process to the complete aspiration of the target single cell into the aspiration channel, and using the separation time as an indicator characterizing the adhesion strength of the single cell.

[0024] Optionally, the microfluidic component may be at least one of trypsin, a drug, or a fluorescently labeled molecule.

[0025] Secondly, a live single-cell extraction system for implementing the above-mentioned processing method includes:

[0026] The microfluidic chip has an injection channel and an intake channel, with the outlet of the injection channel and the inlet of the intake channel being adjacent to each other to form a dual-orifice structure;

[0027] A fluid drive module is connected to the injection channel and the suction channel to control the injection flow rate and the suction flow rate;

[0028] A microscopic imaging module is located below the microfluidic chip, and its optical path is aligned with the target single cell on the culture substrate.

[0029] The image processing module is communicatively connected to the microscopic imaging module and receives cell morphology images for real-time deformation analysis.

[0030] The control module is used to implement the above processing methods.

[0031] Optionally, the microfluidic chip integrates multiple extraction units, each of which has an injection channel and an aspiration channel; the fluid drive module is fluidly connected to the injection channel and aspiration channel of each extraction unit, respectively.

[0032] Optionally, the number of extraction units integrated on the microfluidic chip is 4-96, arranged in a rectangular array or a ring array.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] This invention quantifies the dynamic deformation of cells during extraction by acquiring cell morphology images in real time and calculating the deformation parameter R(t). By observing this deformation parameter, the stress state and degree of deformation of the cells can be perceived, overcoming the shortcomings of existing microfluidic extraction technologies that use preset fixed flow rates or pressures for cell separation and cannot adapt to individual cell differences. Furthermore, it compares the results with pre-calibrated critical deformation parameters obtained through experiments. or comprehensive injury risk index By comparison, when cell deformation exceeds the safety threshold, the injection flow rate and / or aspiration flow rate are reduced immediately. Instead of using "whether cells detach" as the sole endpoint, the risk of cell damage is proactively predicted and intervened. When cell deformation approaches its limit, the fluid rate is reduced in a timely manner to avoid irreversible cell deformation or even rupture due to excessive fluid shear force. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Legend:

[0037] Figure 1 This is a schematic diagram of the steps of the present invention;

[0038] Figure 2 This is a schematic diagram of the microfluidic chip of the present invention;

[0039] Figure 3 This is a front view of the invention under a microscope;

[0040] Figures 4-7 This is a diagram showing the morphological changes of cells under a microscope when exposed to microjets.

[0041] Figures 8-10 This is a schematic diagram of cells after being flushed under a microscope.

[0042] In the diagram: 1. Microfluidic chip; 11. Injection channel; 12. Suction channel. Detailed Implementation

[0043] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] It will be understood that although the terms “first,” “second,” “third,” “fourth,” etc., may be used here to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another.

[0045] Combination Figures 1-10 As shown, an embodiment of the present invention provides a method for processing live single-cell extraction based on deformation trends, comprising the following steps:

[0046] Step S1: Control the microfluidic chip 1 to be immersed in the culture medium containing adherent cells and maintain a preset gap, control the ratio between the injection flow rate of the injection channel 11 and the aspiration flow rate of the aspiration channel 12 to form a microjet acting on the cells.

[0047] The microfluidic chip 1 has an outlet of injection channel 11 and an inlet of suction channel 12, which are adjacent to each other to form a dual-orifice structure. By adjusting the ratio of injection flow rate to suction flow rate, a local microjet can be generated at the dual orifices. This microjet acts on the target single cell, providing fluid shear force to detach it from the culture substrate. Based on the working distance of the microscopic imaging system and the effective action distance of the microjet, the preset gap is typically 10-100 micrometers, determined through preliminary experiments. If the gap is too small, the chip tip may directly contact the cell, causing mechanical damage and preventing the microjet from being completely absorbed. If the gap is too large, the microjet will not generate sufficient shear force on the cell, failing to promote cell detachment.

[0048] Step S2: The image processing module receives and analyzes the cell morphology images on the culture substrate acquired by the microscopic imaging module, and calculates the deformation parameters based on the comparison between the cell area in the current frame image and the initial cell area. ;

[0049] ;

[0050] in This represents the cell area in the current frame. This represents the initial cell area before extraction begins;

[0051] The objective lens of the microscopic imaging module is aimed at the target single cell, and the camera acquires cell morphology images at a sampling frequency of approximately 30 frames per second, transmitting the images to the image processing module. The image processing module uses edge detection algorithms (such as the Canny operator or active contour model) to extract the cell contour and calculate the cell area. The deformation parameter R(t) reflects the degree of cell deformation relative to its initial state under fluid action. Its physical meaning lies in using the relative rate of change of cell area as an indicator of the cell's stress state; a larger R(t) value indicates a more severe stretching or compression of the cell.

[0052] Step S3: [The text appears to be incomplete and contains several grammatical errors. A more accurate translation would require the full context.] Compared with the critical deformation parameters obtained through experimental calibration In contrast, when Greater than At that time, the control fluid drive module reduces the injection flow rate and / or suction flow rate. Less than At that time, maintain the current flow rate. Or,

[0053] According to deformation parameters Calculate the comprehensive damage risk index ,when If the value is greater than 1, reduce the injection flow rate and / or aspiration flow rate.

[0054] ;

[0055] ;

[0056] in For deformation rate, For deformation acceleration, The image sampling time interval, , , , These are the critical deformation parameters, critical deformation rate, critical deformation acceleration, and critical cumulative deformation, respectively, calibrated through preliminary experiments.

[0057] This is the pre-determined deformation threshold for this type of cell, calibrated experimentally. The physical significance of this threshold lies in the fact that the cell membrane and cytoskeleton possess elastic deformation capabilities. When the degree of deformation exceeds a certain limit, irreversible damage will occur to the cell (such as membrane rupture or cytoskeleton breakage). When the deformation parameters monitored in real-time exceed this threshold, it indicates that the cell is approaching its elastic limit. At this point, the system reduces the injection flow rate and / or aspiration flow rate to decrease the shear force acting on the cell. The system does not use "whether the cell detaches" as a criterion; it promptly reduces the fluid rate when cell deformation approaches its limit to avoid irreversible cell deformation or even rupture due to excessive shear force.

[0058] Compared to single-parameter control that relies solely on the deformation parameter R(t), the comprehensive damage risk index... The multi-factor characteristics of cell damage coupling were considered. Deformation rate. It reflects the rate of cell deformation, and its critical value The physical significance is that, even under the same degree of deformation, rapid deformation is more likely to cause cell damage than slow deformation because the stress relaxation of the cytoskeleton takes time. Deformation acceleration. This reflects the abrupt change in fluid shear force, and its critical value. Corresponding to abrupt shocks in fluid forces; cumulative deformation This characterizes the fatigue effect of cells during prolonged extraction, and its critical value... This reflects the cumulative damage tolerance limit of cells under repeated deformation. The calculated value is... When the value is greater than 1, it indicates that the overall risk of damage has exceeded the safety threshold, and the system immediately controls the flow rate to avoid damage.

[0059] Step S4: Record the separation time required from the start of the extraction process to the complete aspiration of the target single cell into the aspiration channel 12, and use the separation time as an indicator of the adhesion strength of the single cell.

[0060] The adhesion strength of different cells, or even the same cell under different culture conditions, varies. Under the same microfluidic effect, cells with weaker adhesion have a shorter separation time, while cells with stronger adhesion have a longer separation time. By recording the separation time, the adhesion strength of the cell can be obtained, providing data for subsequent analysis.

[0061] Combination Figures 4 to 7 As shown, the continuous deformation process of the target single cell under the action of microjets can be observed: with the application of fluid shear force, the cell is gradually stretched, and its projected area is compared with the initial state ( Figure 5 As the value increases, the deformation parameter R(t) also increases. Figure 8 and Figure 9 This is a diagram of cells during the detachment process. Figure 10 The final results of extraction using the control method described in this invention are shown: the surface of the culture medium is smooth and intact, with no cell membrane fragments, cytoskeleton residues or any particulate precipitates. The target single cell has been completely aspirated into the aspiration channel, indicating that no irreversible damage occurred to the cell during the detachment process.

[0062] In some possible implementations, preliminary experiments are calibrated through the following steps:

[0063] (i) A stepwise increasing method was used to control the inhalation flow rate and / or injection flow rate to conduct a progressive destruction experiment on the target cell type;

[0064] (ii) During the experiment, continuously acquire cell morphology images and record the deformation parameters when irreversible cell damage occurs. Deformation rate Deformation acceleration and cumulative deformation ;

[0065] (iii) Perform repeated experiments on at least 30 cells of the same type and conduct statistical analysis on the data of each parameter, including but not limited to taking the arithmetic mean, median, or specific percentile of the sample parameters, or calculating the confidence interval by fitting a normal distribution, and use this as the critical value of the fluid handling limit of this type of cell.

[0066] The stepwise increasing method involves starting with a low flow rate and gradually increasing the inhalation and / or injection flow rates at fixed time intervals, causing the cells to undergo progressively more severe deformation, from slight to large-area deformation, until rupture. When irreversible damage occurs (such as membrane rupture, cell fragmentation, or irreversible deformation), various deformation parameters are recorded. The experiment is repeated on at least 30 cells of the same type, and each parameter is statistically analyzed, including but not limited to obtaining the arithmetic mean, median, or specific percentiles of the sample parameters, or using confidence intervals calculated through normal distribution fitting as critical values. This eliminates the randomness caused by individual differences. Different types of cells (such as epithelial cells, neurons, and stem cells) have different mechanical properties and require separate pre-experimental calibration.

[0067] In some possible implementations, the deformation parameter in step S3 is calculated based on smoothed and filtered multi-frame images to reduce the impact of measurement noise on the calculation. Since cell boundaries may exhibit slight jitter in microscopic images, directly calculating the area of ​​a single frame image can lead to measurement noise. Using multi-frame image smoothing filtering, such as performing a moving average of the cell area over 3-5 consecutive frames, can filter out high-frequency noise, making the curve of the deformation parameter R(t) smoother and avoiding false triggering of flow rate regulation due to instantaneous noise.

[0068] In some possible implementations, the microfluidic component is at least one of trypsin, a drug, or a fluorescently labeled molecule.

[0069] In addition to pure culture medium, the microjets can contain digestive enzymes such as trypsin to assist in cell detachment from the substrate. In this case, the system achieves non-destructive extraction of live single cells, protecting cell viability during the extraction process through real-time monitoring of deformation parameters.

[0070] The microjets can also contain the drugs to be tested, simultaneously stimulating cells during extraction. Examples of such drugs include anticancer drugs (e.g., temozolomide, paclitaxel, cisplatin, fluorouracil), antioxidants (e.g., vitamins, tea polyphenols, acetylcysteine), and other bioactive molecules (e.g., exosomes, nucleic acid drugs). In this case, the system achieves single-cell drug stimulation. Under controllable fluid shear force, drug molecules act on the target single cell through the microjets. Simultaneously, deformation parameters are used to monitor the cell's mechanical response to drug stimulation in real time, providing dynamic data at the single-cell level for drug sensitivity testing and antioxidant stress research.

[0071] The microfluidic stream can also contain fluorescently labeled molecules, such as mitochondrial fluorescent probes, cell membrane fluorescent probes, calcium ion indicators, or live-cell tracer dyes. In this case, the system achieves single-cell fluorescent labeling, completing in-situ fluorescent staining while extracting cells. The labeled cells can be used for downstream fluorescence imaging analysis, flow cytometry sorting, or single-cell sequencing, avoiding the impact of traditional post-staining processing steps on cell viability.

[0072] By integrating multiple active ingredients into a microfluidic stream, this system can not only achieve non-destructive extraction of live single cells, but also be extended to diverse applications such as single-cell drug screening, single-cell mechanical pharmacology research, single-cell fluorescent labeling and sorting, demonstrating broad compatibility and application flexibility.

[0073] The present invention also provides a live single-cell extraction system, comprising:

[0074] The microfluidic chip 1 has an injection channel 11 and an aspiration channel 12. The outlet of the injection channel 11 and the inlet of the aspiration channel 12 are arranged adjacent to each other, forming a dual-orifice structure. The microfluidic chip 1 is the actuator of the system. The dual orifice allows the fluid flowing out of the injection channel 11 and the fluid drawn in by the aspiration channel 12 to form a local microjet region between the outlet and the inlet, providing controllable fluid shear force to act on the target single cell, separating the adherent cell from the culture substrate.

[0075] A fluid drive module, connected to injection channel 11 and suction channel 12, controls the injection and suction flow rates. The fluid drive module typically consists of an injection pump, a pressure controller, or a peristaltic pump. It adjusts the flow rate and ratio of injection channel 11 and suction channel 12 according to control commands, thereby controlling the intensity and direction of the microjets. This module can adjust the flow rate in real time based on feedback signals from the image processing module.

[0076] The microscopic imaging module, located below the microfluidic chip 1, directs the optical path towards the target single cell on the culture substrate. This module typically includes an objective lens, a camera, and an illumination source, enabling continuous, high-resolution real-time imaging of the target single cell during the extraction process. This module is responsible for acquiring images of cell morphology, providing data for subsequent deformation analysis.

[0077] The image processing module communicates with the microscopic imaging module to receive cell morphology images and perform real-time deformation analysis. The image processing module processes the images acquired by the microscopic imaging module in real time, using algorithms such as edge detection, contour extraction, and area calculation to obtain deformation parameters. These parameters are then compared with pre-calibrated critical values, generating corresponding control commands that are sent to the fluid drive module.

[0078] The control module is used to implement the above processing methods.

[0079] In some possible implementations, the microfluidic chip 1 integrates multiple extraction units, each with an injection channel 11 and an aspiration channel 12. A fluid drive module is fluidly connected to the injection channel 11 and aspiration channel 12 of each extraction unit. The number of extraction units integrated on the microfluidic chip 1 is 4-96, arranged in a rectangular or ring array. By integrating multiple extraction units, simultaneous extraction of multiple target single cells can be achieved, significantly increasing extraction throughput. The injection channel 11 and aspiration channel of each extraction unit are independently connected to the fluid drive module, allowing for independent adjustment of the flow rate based on the real-time deformation of the cells in each extraction unit. The arrangement of 4-96 extraction units in a rectangular or ring array adapts to the pore positions of multi-well plates.

[0080] Furthermore, the extraction unit of the microfluidic chip can be designed with a non-perpendicular angle between the flow path body and the bottom surface. Specifically, the central axis of the flow path body forms an angle of 15 to 90 degrees with the bottom surface (i.e., the bottom view plane under the microscope).

[0081] It should be noted that the 90-degree vertical structure is only one basic embodiment among many design schemes. When using tilt angles ranging from 15 to 60 degrees, while ensuring the bottom surface remains parallel to the sample surface, it effectively reduces the obstruction of the inverted microscope's optical path by the main flow path and can form an asymmetric shear microfluidic field at the microscale, which is more conducive to the peeling and extraction of adherent cells. In terms of device manufacturing, the device of this invention is not limited to a single material. The main body of the device can be made of glass, photosensitive materials, ceramic materials, or polymer materials (such as PMMA). Preferably, it is made of polydimethylsiloxane (PDMS), whose air permeability and biocompatibility greatly improve the survival rate of live single-cell operations. Simultaneously, the cross-sectional shape of the internal channels can be designed as square, rectangular, circular, or hexagonal, etc., according to fluid dynamics requirements, all of which can achieve the objectives of this invention.

[0082] Finally, it should be noted that the above embodiments are merely specific implementations of the present invention, used to illustrate the technical solutions of the present invention, and not to limit it. The scope of protection of the present invention is not limited thereto. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any person skilled in the art can still modify or easily conceive of changes to the technical solutions described in the foregoing embodiments within the scope of the technology disclosed in the present invention, or make equivalent substitutions for some of the technical features; and these modifications, changes, or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention and should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

[0083] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for processing live single-cell extraction based on deformation trends, characterized in that, Includes the following steps: Step S1: Control the microfluidic chip to be immersed in the culture medium containing adherent cells and maintain a preset gap, control the injection flow rate of the injection channel and the aspiration flow rate of the aspiration channel to form a microjet acting on the cells; Step S2: The image processing module receives and analyzes the cell morphology image on the culture substrate acquired by the microscopic imaging module, and calculates the deformation parameters based on the comparison between the cell area in the current frame image and the initial cell area. ; Step S3: Based on deformation parameters Calculate the comprehensive damage risk index ,when If the value is greater than 1, reduce the injection flow rate and / or aspiration flow rate; In step S2, the deformation parameters include in This represents the cell area in the current frame. This represents the initial cell area before extraction begins; In step S3, the comprehensive damage risk index include: in For deformation rate, For deformation acceleration, The image sampling time interval, , , , These are the critical deformation parameters, critical deformation rate, critical deformation acceleration, and critical cumulative deformation, respectively, calibrated through preliminary experiments. The preliminary experiment was calibrated through the following steps: (i) A stepwise increasing method was used to control the inhalation flow rate and / or injection flow rate to conduct a progressive destruction experiment on the target cell type; (ii) During the experiment, continuously acquire cell morphology images and record the deformation parameters when irreversible cell damage occurs. Deformation rate Deformation acceleration and cumulative deformation ; (iii) Perform repeated experiments on at least 30 cells of the same type and conduct statistical analysis on the data of each parameter, including but not limited to taking the arithmetic mean, median, or specific percentile of the sample parameters, or calculating their confidence interval by fitting a normal distribution, and use this as the critical value of the fluid handling limit of this type of cell.

2. The method for processing live single-cell extraction based on deformation trend according to claim 1, characterized in that, The deformation parameters mentioned in step S3 are calculated based on smoothed filtering of multiple frames of images to reduce the impact of measurement noise on the calculation.

3. The method for processing live single-cell extraction based on deformation trend according to claim 1, characterized in that, The processing method further includes step S4: recording the separation time required from the start of the extraction process to the complete absorption of the target single cell into the aspiration channel, and using the separation time as an indicator of the adhesion strength of the single cell.

4. The method for processing live single-cell extraction based on deformation trend according to claim 1, characterized in that, The microfluid is composed of at least one of trypsin, a drug, or a fluorescently labeled molecule.

5. A live single-cell extraction system, characterized in that, include: The microfluidic chip has an injection channel and an intake channel, with the outlet of the injection channel and the inlet of the intake channel being adjacent to each other to form a dual-orifice structure; A fluid drive module is connected to the injection channel and the suction channel to control the injection flow rate and the suction flow rate; A microscopic imaging module is located below the microfluidic chip, and its optical path is aligned with the target single cell on the culture substrate. The image processing module is communicatively connected to the microscopic imaging module and receives cell morphology images for real-time deformation analysis. A control module for implementing the processing method according to any one of claims 1-4.

6. The live single-cell extraction system according to claim 5, characterized in that, The microfluidic chip integrates multiple extraction units, each of which has an injection channel and an aspiration channel; the fluid drive module is fluidly connected to the injection channel and aspiration channel of each extraction unit.

7. The live single-cell extraction system according to claim 6, characterized in that, The number of extraction units integrated on the microfluidic chip is 4-96, arranged in a rectangular array or a ring array.

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