Method and system for screening cell components based on flow cytometry

Abstract

The present invention relates to the field of cell biology, and specifically discloses a method and system for screening cell components based on flow cytometry. The method includes: digesting a target tissue into a cell solution, preparing a buffer solution for removing miscellaneous cells, and using the buffer solution to prepare a single-cell suspension from the solution of removing miscellaneous cells; analyzing the property specificities of target cell components and corresponding non-target cell components in the single-cell suspension, determining a fluorescent marker for the target cell components, mixing the fluorescent marker with the single-cell suspension, constructing a cell scatter plot of the labeled cell suspension, and determining the gating parameters of the cell scatter plot; calculating the cell proportion and average cell fluorescence intensity of the cell population to be screened, determining the sorting parameters of the labeled cell suspension, and using a preset flow cytometer to perform cell sorting on the cell population to be screened to obtain a target cell population. The present invention can improve the efficiency of cell component screening.

Description

Technical Field

[0001] The present invention relates to the field of cell biology, and particularly to a method and system for screening cell components based on flow cytometry. Background Art

[0002] Cell component screening refers to the process of using a series of biological experimental methods and technical means to identify, isolate, and / or study cell components with specific properties from a cell population. These cell components can be specific molecules, organelles, genetic materials, or other biological macromolecules on the cell surface or inside. By screening and identifying specific components in cells, researchers can better understand the biological behavior of cells.

[0003] Currently, cell component screening methods analyze the protein composition in cells through techniques such as mass spectrometry. Because the protein separation and identification steps in the screening process are complex, only specific cell components can be detected, while other important components are ignored, resulting in the inability to process a large number of samples and low efficiency. Summary of the Invention

[0004] The present invention provides a method and system for screening cell components based on flow cytometry, and its main purpose is to improve the efficiency of cell component screening.

[0005] To achieve the above object, a method for screening cell components based on flow cytometry provided by the present invention includes:

[0006] Obtain a target tissue containing target cell components, digest the target tissue into a cell solution, centrifuge the cell solution to obtain a cell solution with impurities removed, configure a buffer solution for the cell solution with impurities removed, and use the buffer solution to prepare the cell solution with impurities removed into a single-cell suspension;

[0007] Analyze the component type and component composition of the target cell components, and based on the component type and the component composition, determine the component chemical characteristics and component physical characteristics of the target cell components;

[0008] Based on the component chemical characteristics and the component physical characteristics, analyze the property specificity between the target cell components and the corresponding non-target cell components in the single-cell suspension. According to the property specificity, determine the fluorescent label for the target cell components, and mix the fluorescent label with the single-cell suspension to obtain a labeled cell suspension;

[0009] Use a preset flow cytometer to collect the scattered light and fluorescence signals of the labeled cell suspension. According to the scattered light and the fluorescence signals, construct a cell scatter plot of the labeled cell suspension, and determine the gating parameters of the cell scatter plot. Based on the gating parameters, determine the cell population to be screened in the labeled cell suspension;

[0010] Calculate the cell ratio and average cell fluorescence intensity of the cell population to be screened. Based on the cell ratio and the average cell fluorescence intensity, determine the sorting parameters of the labeled cell suspension. According to the sorting parameters, use a preset flow cytometer to perform cell sorting on the cell population to be screened to obtain a target cell population.

[0011] Optionally, the digesting the target tissue into a cell solution includes:

[0012] Construct a digestion environment for the target tissue; based on the digestion environment, divide the target tissue into target tissue fragments;

[0013] Transfer the target tissue fragments to a preset digestive enzyme solution to obtain a tissue-enzyme mixed solution;

[0014] Regularly check the tissue state in the tissue-enzyme mixed solution;

[0015] When the tissue state meets the preset state standard, filter the tissue-enzyme mixed solution to obtain a cell solution.

[0016] Optionally, the analyzing the component type and component composition of the target cell components includes:

[0017] Use a preset infrared spectrum to determine the macromolecular type of the target cell components;

[0018] Perform mass spectrometry analysis on the target cell components to obtain a mass spectrometry analysis result. According to the mass spectrometry result, determine the molecular weight of the target cell components;

[0019] Perform nuclear magnetic resonance on the target cell components to obtain the molecular three-dimensional structure and dynamic characteristics of the target cell components;

[0020] According to the macromolecular type, the molecular weight, the molecular three-dimensional structure, and the dynamic characteristics, determine the component type of the target cell components;

[0021] Based on the component type, perform elemental analysis on the target cell components to obtain the elemental composition of the target cell components;

[0022] Analyze the organic small molecule components of the target cell components;

[0023] According to the elemental composition and the organic small molecule components, determine the component composition of the target cell components.

[0024] Optionally, analyzing the property specificity between the target cell components and the corresponding non-target cell components in the single-cell suspension based on the chemical characteristics and physical characteristics of the components includes:

[0025] Based on the chemical characteristics and physical characteristics of the components, determining the chemical differences and physical differences between the target cell components and the non-target cell components;

[0026] According to the chemical differences and physical differences, clarifying the specific differences between the target cell components and the non-target cell components;

[0027] Quantifying the specific differences to obtain a specificity index;

[0028] According to the specificity index, constructing a specific analysis model for the target cell components and the non-target cell components;

[0029] Using the specific analysis model to predict the property specificity between the target cell components and the non-target cell components.

[0030] Optionally, determining the fluorescent marker for the target cell components according to the property specificity includes:

[0031] Obtaining the potential fluorescent markers for the target cell components and analyzing the marker characteristics of the potential fluorescent markers;

[0032] According to the marker characteristics, determining the incubation time of the fluorescent marker with the target cell components;

[0033] According to the incubation time, incubating the potential fluorescent marker with the target cell components to obtain labeled cell components;

[0034] Detecting the labeling effect of the labeled cell components, and according to the labeling effect, analyzing the marker fluorescence intensity and background noise of the potential fluorescent marker;

[0035] According to the property specificity, the marker characteristics, the marker fluorescence intensity, and the background noise, analyzing the fluorescence labeling effect of the potential fluorescent marker;

[0036] Selecting the potential fluorescent marker with the best fluorescence labeling effect as the fluorescent marker for the target cell components.

[0037] Optionally, constructing a cell scatter plot of the labeled cell suspension according to the scattered light and the fluorescence signal includes:

[0038] Performing background correction on the scattered light and the fluorescence signal to obtain corrected scattered light and corrected fluorescence signal;

[0039] Perform signal normalization on the corrected scattered light and the corrected fluorescence signal to obtain normalized scattered light and normalized fluorescence signal;

[0040] Analyze the forward scattered light and side scattered light of the normalized scattered light;

[0041] Analyze the fluorescence signal intensity of the normalized fluorescence signal, and based on the fluorescence signal intensity, determine the color coding of the normalized fluorescence signal;

[0042] Use the forward scattered light as the horizontal axis and the side scattered light as the vertical axis to establish a cell coordinate system for the labeled cell suspension;

[0043] According to the color coding, map the labeled cell suspension to the cell coordinate system to obtain a cell scatter plot of the labeled cell suspension.

[0044] Optionally, determining the gating parameters of the cell scatter plot includes:

[0045] Determine the screening requirements for the target cell components corresponding to the cell scatter plot;

[0046] Determine the signal parameters of the cell scatter plot, and analyze the signal parameter weights of the signal parameters;

[0047] According to the screening requirements, construct the gating logic of the cell scatter plot;

[0048] Based on the gating logic, combine the gating conditions of the signal parameters;

[0049] According to the gating conditions, the signal parameter weights, and the signal parameters, determine the gating parameters of the cell scatter plot.

[0050] Optionally, calculating the cell ratio and average cell fluorescence intensity of the cell population to be screened includes:

[0051] Respectively determine the specific cell number and total cell number of the cell population to be screened and the labeled cell suspension corresponding to the cell population to be screened;

[0052] Measure the cell fluorescence intensity of the cell population to be screened and the background fluorescence intensity of the cell population to be screened;

[0053] According to the specific cell number, the total cell number, the cell fluorescence intensity, and the background fluorescence intensity, calculate the cell ratio and average cell fluorescence intensity of the cell population to be screened.

[0054] Optionally, determining the sorting parameters of the labeled cell suspension based on the cell ratio and the average cell fluorescence intensity includes:

[0055] Determine the fluorescence distribution range of the labeled cell suspension according to the cell ratio and the average cell fluorescence intensity;

[0056] Determine the initial sorting parameters of the labeled cell suspension according to the fluorescence distribution range;

[0057] Based on the initial sorting parameters, perform a sorting test on the labeled cell suspension to obtain the test purity of the target cells;

[0058] When the test purity of the target cells does not meet the preset purity standard, adjust the initial sorting parameters to obtain optimized sorting parameters;

[0059] According to the optimized sorting parameters, perform a sorting test on the labeled cell suspension to obtain the optimized test purity of the cells;

[0060] When the optimized test purity of the cells does not meet the preset purity standard, return to the above step of adjusting the initial sorting parameters to obtain optimized sorting parameters;

[0061] When the optimized test purity of the cells meets the preset purity standard, use the initial sorting parameters as the sorting parameters of the labeled cell suspension.

[0062] To solve the above problems, the present invention also provides a cell component screening system based on flow cytometry. The system includes:

[0063] A single-cell suspension preparation module for obtaining a target tissue containing target cell components, digesting the target tissue into a cell solution, centrifuging the cell solution to obtain a cell solution free of impurities, preparing a buffer solution for the cell solution free of impurities, and using the buffer solution to prepare a single-cell suspension from the cell solution free of impurities;

[0064] A cell component analysis module for analyzing the component types and component compositions of the target cell components, and determining the component chemical characteristics and component physical characteristics of the target cell components based on the component types and the component compositions;

[0065] A fluorescence labeling module for analyzing the property specificities of the target cell components and the corresponding non-target cell components in the single-cell suspension based on the component chemical characteristics and the component physical characteristics, determining the fluorescence labeling agents for the target cell components according to the property specificities, and mixing the fluorescence labeling agents with the single-cell suspension to obtain a labeled cell suspension;

[0066] The fluorescence labeling detection module is used to collect the scattered light and fluorescence signals of the labeled cell suspension by using a preset flow cytometer, construct a cell scatter plot of the labeled cell suspension according to the scattered light and the fluorescence signals, determine the gating parameters of the cell scatter plot, and determine the cell population to be screened in the labeled cell suspension based on the gating parameters;

[0067] The target cell screening module is used to calculate the cell ratio and the average cell fluorescence intensity of the cell population to be screened, determine the sorting parameters of the labeled cell suspension based on the cell ratio and the average cell fluorescence intensity, and perform cell sorting on the cell population to be screened by using a preset flow cytometer according to the sorting parameters to obtain a target cell population.

[0068] In the embodiment of the present invention, preparing the cell solution after removing impurities into a single-cell suspension by using the buffer can facilitate subsequent operations such as cell counting, sorting, and culturing; optionally, in the embodiment of the present invention, detailed information about cell characteristics and functions can be obtained by analyzing the component type and component composition of the target cell component; in the embodiment of the present invention, determining the component chemical characteristics and component physical characteristics of the target cell component based on the component type and the component composition can help to discover the specificity of the target cell component and facilitate subsequent cell component screening; in the embodiment of the present invention, analyzing the property specificity between the target cell component and the corresponding non-target cell components in the single-cell suspension based on the component chemical characteristics and the component physical characteristics can ensure that the marker mainly reacts with the target component, reduce cross-reaction with non-target components, and improve the accuracy and reliability of the experiment; in the embodiment of the present invention, the scattered light and fluorescence signals of the labeled cell suspension can be collected by using a preset flow cytometer to quickly identify and screen cells with specific properties. Finally, in the embodiment of the present invention, by performing cell sorting on the cell population to be screened by using a preset flow cytometer according to the sorting parameters to obtain a target cell population, the target cells can be effectively separated, and the contamination of non-target cells can be reduced, thereby improving the purity of the target cells. Therefore, the present invention can improve the efficiency of cell component screening. Description of the Drawings

[0069] Figure 1 It is a schematic flowchart of a method for realizing cell component screening based on flow cytometry provided by an embodiment of the present invention;

[0070] Figure 2 It is a functional module diagram of a system for realizing cell component screening based on flow cytometry provided by an embodiment of the present invention;

[0071] The realization, functional characteristics, and advantages of the object of the present invention will be further described with reference to the embodiments and the accompanying drawings. Detailed Embodiments

[0072] It should be understood that the specific embodiments described herein are merely used to explain the present invention and are not intended to limit the present invention.

[0073] An embodiment of the present application provides a method for screening cell components based on flow cytometry. The execution subject of the method for screening cell components based on flow cytometry includes, but is not limited to, at least one of electronic devices such as a server, a terminal, etc. that can be configured to execute the method provided by the embodiment of the present application. In other words, the method for screening cell components based on flow cytometry can be executed by software or hardware installed on a terminal device or a server device, and the software can be a blockchain platform. The server includes, but is not limited to: a single server, a server cluster, a cloud server, or a cloud server cluster, etc. The server can be an independent server or a cloud server that provides basic cloud computing services such as cloud services, cloud databases, cloud computing, cloud functions, cloud storage, network services, cloud communications, middleware services, domain name services, security services, Content Delivery Network (CDN), and big data and artificial intelligence platforms.

[0074] Refer to Figure 1 As shown, it is a flowchart of a method for screening cell components based on flow cytometry provided by an embodiment of the present invention. In this embodiment, the method for screening cell components based on flow cytometry includes:

[0075] S1. Obtain a target tissue containing a target cell component, digest the target tissue into a cell solution, centrifuge the cell solution to obtain a cell solution with impurities removed, prepare a buffer solution for the cell solution with impurities removed, and use the buffer solution to prepare the cell solution with impurities removed into a single-cell suspension.

[0076] By obtaining a target tissue containing a target cell component in an embodiment of the present invention, it can be ensured that high-quality and high-purity cell samples can be used in subsequent biological research. Among them, the target tissue refers to a biological tissue that can extract specific types of cells or cell components.

[0077] By digesting the target tissue into a cell solution in an embodiment of the present invention, individual cells can be released from the tissue structure, enabling the cells to exist independently and facilitating subsequent operations. Among them, the cell solution refers to a liquid mixture containing single or multiple cells obtained after digesting and processing the target tissue.

[0078] As an embodiment of the present invention, the step of digesting the target tissue into a cell solution includes:

[0079] Construct a digestion environment for the target tissue; based on the digestion environment, divide the target tissue into target tissue fragments;

[0080] Transfer the target tissue fragments to a preset digestive enzyme solution to obtain a tissue-enzyme mixed solution;

[0081] Regularly check the tissue state in the tissue-enzyme mixed solution;

[0082] When the tissue state meets the preset state standard, filter the tissue-enzyme mixed solution to obtain a cell solution.

[0083] Among them, the series of conditions specifically set to effectively digest the target tissue into a cell solution, such as: temperature, pH value, gas environment, and aseptic conditions, etc. The preset digestive enzyme solution refers to a liquid mixture of a group of enzymes specially prepared to digest the target tissue and release single cells. The digestive enzyme solution includes: trypsin, collagenase, and neutral protease. The tissue-enzyme mixed solution refers to the liquid mixture formed by mixing the target tissue fragments with a solution containing digestive enzymes. The tissue state refers to the physical and chemical changes of the target tissue fragments under the action of digestive enzymes during the tissue digestion process. The preset state refers to the expected goal or standard set according to the experimental purpose and cell type during the tissue digestion process to judge when to terminate the digestion process.

[0084] Optionally, the regular check of the tissue state in the tissue-enzyme mixed solution can be carried out by using the method of microscopic observation.

[0085] Optionally, the filtration of the tissue-enzyme mixed solution can be carried out through a cell filter, such as a filter with a pore size of 40 microns.

[0086] In the embodiment of the present invention, by performing cell centrifugation on the cell solution, a cell solution with impurities removed can effectively remove most of the impurities in the solution and obtain a relatively pure cell suspension, providing a high-quality sample for subsequent cell culture or other experimental operations. Among them, the cell solution with impurities removed refers to the cell solution that has removed most of the impurities and undigested tissue fragments after centrifugation and other processing steps.

[0087] Optionally, as an embodiment of the present invention, the cell centrifugation of the cell solution can be carried out by differential centrifugation. Among them, differential centrifugation refers to separating cells and cell components with different densities through a series of centrifugation steps at different speeds.

[0088] In the embodiments of the present invention, by configuring the buffer solution of the cell impurity removal solution, an appropriate pH value and ionic strength can be provided to maintain the normal physiological state of the cells. Among them, the buffer solution refers to a liquid medium used to maintain the stability of the pH value of the solution.

[0089] In the embodiments of the present invention, by using the buffer solution to prepare the cell impurity removal solution into a single-cell suspension, subsequent operations such as cell counting, sorting, and culturing can be facilitated. Among them, the single-cell suspension refers to a cell suspension extracted from a cell mixture that contains only single cells and does not contain other cell clumps or cell debris.

[0090] Optionally, as an embodiment of the present invention, the use of the buffer solution to prepare the cell impurity removal solution into a single-cell suspension can be achieved by the method of resuspending cells. Among them, resuspending cells refers to gently pipetting the precipitated cells repeatedly with a buffer solution to make them resuspend.

[0091] S2. Analyze the component type and component composition of the target cell component, and based on the component type and the component composition, determine the chemical characteristics and physical characteristics of the target cell component.

[0092] In the embodiments of the present invention, by analyzing the component type and component composition of the target cell component, detailed information about cell characteristics and functions can be obtained. Among them, the component type refers to the types of various chemical substances and biomolecules present in the target cell component. The component composition refers to the relative content and distribution of various chemical substances and biomolecules in the target cell component.

[0093] As an embodiment of the present invention, the analysis of the component type and component composition of the target cell component includes:

[0094] Use a preset infrared spectrum to determine the macromolecular type of the target cell component;

[0095] Perform mass spectrometry analysis on the target cell component to obtain a mass spectrometry analysis result, and based on the mass spectrometry result, determine the molecular weight of the target cell component;

[0096] Perform nuclear magnetic resonance on the target cell component to obtain the three-dimensional molecular structure and dynamic characteristics of the target cell component;

[0097] Based on the macromolecular type, the molecular weight, the three-dimensional molecular structure, and the dynamic characteristics, determine the component type of the target cell component;

[0098] Based on the component type, perform elemental analysis on the target cell component to obtain the elemental composition of the target cell component;

[0099] Analyze the small organic molecule components of the target cell components;

[0100] Determine the component composition of the target cell components according to the element composition and the small organic molecule components.

[0101] Among them, the macromolecule type refers to the main biological macromolecules that make up cell components, such as proteins, nucleic acids, etc. The preset infrared spectrum refers to a set of preset infrared spectrum characteristic parameters or standard spectrograms, which are used to identify and classify biological macromolecules. The mass spectrometry analysis result refers to the data and information obtained by analyzing the target cell components with a mass spectrometer. The molecular weight refers to the mass of a molecule or compound determined by mass spectrometry or other technical means. The three-dimensional molecular structure refers to the specific arrangement and form of a molecule in three-dimensional space. The dynamic characteristics refer to the movements and changes experienced by a molecule during its life cycle, such as molecular motion, interaction, etc. The element composition refers to the types of different elements contained in the target cell components and their relative proportions. The small organic molecule components refer to the relatively small molecular weight organic compounds present in the target cell components.

[0102] Optionally, the mass spectrometry analysis of the target cell components can be performed by using MALDI-TOF for analysis, where MALDI-TOF refers to a mass spectrometry technique for analyzing biological macromolecules such as proteins, polypeptides, nucleic acids, and other large molecular organic compounds.

[0103] Optionally, based on the component type, the elemental analysis of the target cell components can be performed by X-ray fluorescence spectroscopy, where X-ray fluorescence spectroscopy is a technique for material component analysis and elemental quantitative analysis.

[0104] In the embodiments of the present invention, determining the chemical and physical characteristics of the components of the target cell components based on the component type and the component composition can help to discover the specificity of the target cell components and facilitate subsequent cell component screening. Among them, the component chemical characteristics refer to the chemical properties and chemical structure characteristics of various chemical substances in the target cell components, such as the element composition, molecular structure, functional groups, etc. of the components. The component physical characteristics refer to the physical properties of the target cell components, such as the morphology, size, structure, fluidity, etc. of the components.

[0105] Optionally, as an embodiment of the present invention, the chemical and physical characteristics of the components of the target cell components can be determined by chemical reactivity analysis and electron microscope observation respectively based on the component type and the component composition.

[0106] S3. Based on the chemical characteristics and physical characteristics of the components, analyze the property specificity between the target cell components and the corresponding non-target cell components in the single-cell suspension. According to the property specificity, determine the fluorescent marker for the target cell components, and mix the fluorescent marker with the single-cell suspension to obtain a labeled cell suspension.

[0107] In the embodiment of the present invention, by analyzing the property specificity between the target cell components and the corresponding non-target cell components in the single-cell suspension based on the chemical characteristics and physical characteristics of the components, it can be ensured that the marker mainly reacts with the target components, reducing the cross-reaction with non-target components and improving the accuracy and reliability of the experiment. Among them, the property specificity refers to the unique chemical and physical properties of the target cell components relative to the non-target components in the environment.

[0108] As an embodiment of the present invention, the analysis of the property specificity between the target cell components and the corresponding non-target cell components in the single-cell suspension based on the chemical characteristics and physical characteristics of the components includes:

[0109] Based on the chemical characteristics and physical characteristics of the components, determine the chemical differences and physical differences between the target cell components and the non-target cell components;

[0110] According to the chemical differences and the physical differences, clarify the specific differences between the target cell components and the non-target cell components;

[0111] Quantify the specific differences to obtain a specificity index;

[0112] According to the specificity index, construct a specific analysis model for the target cell components and the non-target cell components;

[0113] Use the specific analysis model to predict the property specificity between the target cell components and the non-target cell components.

[0114] Among them, the chemical difference refers to the difference in chemical properties between the target cell components and the non-target cell components. The physical difference refers to the difference in physical properties between the target cell components and the non-target cell components. The specific difference refers to the specific and significant differences in chemical properties and physical properties between the target cell components and the non-target cell components. The specificity index is a measure used to quantify the specific differences between the target cell components and the non-target cell components. The specific analysis model is a mathematical or computational model used to predict and identify target components.

[0115] Optionally, the specific differences between the target cell components and the non-target cell components determined based on the chemical differences and the physical differences can be determined by statistical analysis, such as t-test, analysis of variance, etc.

[0116] In the embodiments of the present invention, by determining the fluorescent label of the target cell component according to the property specificity, the fluorescent label can specifically bind to the target cell component without non-specific binding to the non-target cell component, thereby achieving precise identification and tracking of the target cell. Among them, the fluorescent label refers to a chemical substance or biomolecule that can be used to label the target cell component and emit fluorescence under specific conditions.

[0117] As an embodiment of the present invention, determining the fluorescent label of the target cell component according to the property specificity includes:

[0118] Obtain the potential fluorescent label of the target cell component and analyze the label characteristics of the potential fluorescent label;

[0119] According to the label characteristics, determine the incubation time of the fluorescent label with the target cell component;

[0120] According to the incubation time, incubate the potential fluorescent label with the target cell component to obtain the labeled cell component;

[0121] Detect the labeling effect of the labeled cell component, and according to the labeling effect, analyze the fluorescence intensity of the label and the background noise of the potential fluorescent label;

[0122] According to the property specificity, the label characteristics, the fluorescence intensity of the label, and the background noise, analyze the fluorescence labeling effect of the potential fluorescent label;

[0123] Select the potential fluorescent label with the best fluorescence labeling effect as the fluorescent label of the target cell component.

[0124] Among them, the potential fluorescent marker refers to a candidate fluorescent dye, fluorescent protein, fluorescent probe, or other fluorescent compound that is considered for labeling the target cell component during the process of determining the fluorescent marker for the target cell component. The marker characteristics refer to a series of specific properties of the fluorescent marker, which determine the performance and applicability of the marker in experimental applications. The incubation time refers to the time required after mixing the fluorescent marker with the target cell component and waiting for their interaction to achieve the best labeling effect. The labeled cell component refers to the cell component that can emit fluorescence by incubating the fluorescent marker with the target cell component and binding the fluorescent marker to the target cell component. The labeling effect refers to the characteristics of the fluorescence signal, such as intensity, distribution, uniformity, and background noise, exhibited after the target cell component binds to the fluorescent marker. The marker fluorescence intensity refers to the intensity of the fluorescence signal generated by the fluorescent marker under the irradiation of the excitation light. The background noise refers to the fluorescence signals generated by non-target cell components or other factors, which will interfere with the observation and analysis of the fluorescence signal of the target cell component, thereby affecting the accuracy of the experimental results.

[0125] Optionally, the analysis of the marker characteristics of the potential fluorescent marker can be performed through biocompatibility evaluation.

[0126] Optionally, the detection of the labeling effect of the labeled cell component can be performed using a fluorescence microscope.

[0127] In the embodiment of the present invention, by mixing the fluorescent marker and the single-cell suspension, a labeled cell suspension can be obtained to identify and classify different types of cells and distinguish different subpopulations or states of cells. Among them, the labeled cell suspension refers to a cell suspension in which the fluorescent marker is mixed with the single-cell suspension, and the fluorescent marker binds to the cell components in the single-cell suspension, so that the cell components can emit fluorescence.

[0128] S4. Use a preset flow cytometer to collect the scattered light and fluorescence signals of the labeled cell suspension, construct a cell scatter plot of the labeled cell suspension based on the scattered light and the fluorescence signals, and determine the gating parameters of the cell scatter plot. Based on the gating parameters, determine the cell population to be screened in the labeled cell suspension.

[0129] In the embodiment of the present invention, by using a preset flow cytometer to collect the scattered light and fluorescence signals of the labeled cell suspension, cells with specific properties can be quickly identified and screened. Among them, the scattered light refers to the non-specific light signal generated by cells detected by the flow cytometer. The fluorescence signal refers to the light signal with a specific wavelength generated by the excitation of the fluorescent marker in the flow cytometer analysis.

[0130] In an embodiment of the present invention, constructing a cell scatter plot of the labeled cell suspension based on the scattered light and the fluorescence signal can help identify different types of cells. Among them, the cell scatter plot refers to a two-dimensional or multi-dimensional distribution map of cells constructed using scattered light and fluorescence signals during the analysis by a flow cytometer.

[0131] As an embodiment of the present invention, constructing the cell scatter plot of the labeled cell suspension based on the scattered light and the fluorescence signal includes:

[0132] Performing background correction on the scattered light and the fluorescence signal to obtain corrected scattered light and corrected fluorescence signal;

[0133] Performing signal normalization on the corrected scattered light and the corrected fluorescence signal to obtain normalized scattered light and normalized fluorescence signal;

[0134] Analyzing the forward scattered light and the side scattered light of the normalized scattered light;

[0135] Analyzing the fluorescence signal intensity of the normalized fluorescence signal, and determining the color coding of the normalized fluorescence signal based on the fluorescence signal intensity;

[0136] Establishing a cell coordinate system of the labeled cell suspension with the forward scattered light as the horizontal axis and the side scattered light as the vertical axis;

[0137] Mapping the labeled cell suspension to the cell coordinate system according to the color coding to obtain the cell scatter plot of the labeled cell suspension.

[0138] Among them, the background correction refers to removing the noise caused by non-specific binding, instrument background or other irrelevant factors, so as to improve the quality of data and the accuracy of analysis. The signal normalization refers to a preprocessing step for scattered light and fluorescence signals during the analysis by a flow cytometer to eliminate the difference in signal intensity between different cells or different fluorescence channels. The forward scattered light refers to the light signal in a specific direction generated by cells detected by a flow cytometer. The side scattered light refers to the light signal generated by cells detected by a flow cytometer perpendicular to the direction of the laser beam. The fluorescence signal intensity refers to the intensity of the light signal emitted by a fluorescent marker after being excited by an excitation light during the analysis by a flow cytometer. The color coding refers to the method of using colors to represent different fluorescence channels or different cell characteristics in the data analysis software of a flow cytometer. The cell coordinate system refers to a coordinate system with forward scattered light (FSC) and side scattered light (SSC) as coordinate axes, and each cell is regarded as a point to display its position in the coordinate system.

[0139] Optionally, the signal normalization of the corrected scattered light and the corrected fluorescence signal can be obtained by a signal normalization method, where the signal normalization refers to converting the corrected scattered light and corrected fluorescence signal values into values proportional to a reference value.

[0140] In the embodiment of the present invention, by determining the gating parameters of the cell scatter plot, the noise and interference in data analysis can be reduced, and the quality and reliability of the data can be improved. Among them, the gating parameters refer to the parameters used to screen and define the cell population of interest during the data analysis of a flow cytometer.

[0141] As an embodiment of the present invention, the determination of the gating parameters of the cell scatter plot includes:

[0142] Determine the screening requirements for the target cell components corresponding to the cell scatter plot;

[0143] Determine the signal parameters of the cell scatter plot and analyze the signal parameter weights of the signal parameters;

[0144] According to the screening requirements, construct the gating logic of the cell scatter plot;

[0145] Based on the gating logic, combine the gating conditions of the signal parameters;

[0146] According to the gating conditions, the signal parameter weights, and the signal parameters, determine the gating parameters of the cell scatter plot.

[0147] Among them, the screening requirements refer to the cell characteristics or subpopulations that need to be screened for specific experimental purposes in flow cytometry analysis. The signal parameters refer to the physical quantities used to describe and distinguish cell characteristics. The signal parameter weights refer to the indicators used to reflect the relative importance or contribution degree of different signal parameters in distinguishing and identifying cell subpopulations. The gating logic refers to the logical rules used to screen and define the cell population of interest during the data analysis of a flow cytometer. The gating conditions refer to the specific conditions or thresholds used to screen and define the cell population of interest during the data analysis of a flow cytometer.

[0148] Optionally, the combination of the gating conditions of the signal parameters can be performed by data analysis software, such as FlowJo, FCS Express, Beckman Coulter Accuri Cytomation Cytobuilder, etc.

[0149] In the embodiments of the present invention, by determining the cell population to be screened of the labeled cell suspension based on the gating parameters, target cell components can be accurately identified and screened out, reducing the interference of non-target cells and improving the accuracy and efficiency of the experiment. Among them, the cell population to be screened refers to the cell subset that meets specific conditions determined through the gating analysis of a flow cytometer.

[0150] S5. Calculate the cell ratio and average cell fluorescence intensity of the cell population to be screened. Based on the cell ratio and the average cell fluorescence intensity, determine the sorting parameters of the labeled cell suspension. According to the sorting parameters, use a preset flow cytometer to perform cell sorting on the cell population to be screened to obtain a target cell population.

[0151] In the embodiments of the present invention, by calculating the cell ratio and average cell fluorescence intensity of the cell population to be screened, the purity of the target cell population can be ensured, and the efficiency of the screening process can be guaranteed. Among them, the cell ratio refers to the ratio of a specific cell subset to the entire cell population in flow cytometry analysis. The average cell fluorescence intensity refers to the average value of the fluorescence signal intensity generated by a fluorescent label under the irradiation of excitation light in flow cytometer analysis.

[0152] As an embodiment of the present invention, the calculating the cell ratio and average cell fluorescence intensity of the cell population to be screened includes:

[0153] Respectively determine the specific cell number and total cell number of the cell population to be screened and the labeled cell suspension corresponding to the cell population to be screened;

[0154] Measure the cell fluorescence intensity of the cell population to be screened and the background fluorescence intensity of the cell population to be screened;

[0155] According to the specific cell number, the total cell number, the cell fluorescence intensity, and the background fluorescence intensity, use the following formula to calculate the cell ratio and average cell fluorescence intensity of the cell population to be screened:

[0156] ;

[0157] ;

[0158] Among them, represents the cell ratio, represents the average cell fluorescence intensity, represents the specific cell number of the cell population to be screened, represents the total cell number of the labeled cell suspension, represents the th cell fluorescence intensity in the cell population to be screened, Represents the background fluorescence intensity.

[0159] Among them, the specific cell number refers to the number of labeled cells in the cell population to be screened. The total cell number refers to the total amount of labeled and unlabeled cells in the labeled cell suspension in the whole solution. The cell fluorescence intensity refers to the fluorescence signal intensity measured from a single cell. The background fluorescence intensity refers to the fluorescence signal intensity detected in the target cell or sample area without specific fluorescence labeling.

[0160] In the embodiment of the present invention, by determining the sorting parameters of the labeled cell suspension based on the cell ratio and the average cell fluorescence intensity, the sorting accuracy can be improved and the sorting speed can be optimized. Thus, the sorting efficiency is improved. Among them, the sorting parameters refer to a series of parameters used to define and optimize the sorting process during cell sorting using a flow cytometer or other cell sorting equipment.

[0161] As an embodiment of the present invention, determining the sorting parameters of the labeled cell suspension based on the cell ratio and the average cell fluorescence intensity includes:

[0162] According to the cell ratio and the average cell fluorescence intensity, use the following formula to determine the fluorescence distribution range of the labeled cell suspension:

[0163] ;

[0164] Among them, represents the fluorescence distribution range, represents the average cell fluorescence intensity, represents the specific cell number of the cell population to be screened, represents the th cell fluorescence intensity in the cell population to be screened;

[0165] According to the fluorescence distribution range, determine the initial sorting parameters of the labeled cell suspension;

[0166] Based on the initial sorting parameters, perform a sorting test on the labeled cell suspension to obtain the test purity of the target cells;

[0167] When the test purity of the target cells does not meet the preset purity standard, adjust the initial sorting parameters to obtain optimized sorting parameters;

[0168] According to the optimized sorting parameters, perform a sorting test on the labeled cell suspension to obtain the optimized test purity of the cells;

[0169] When the purity of the optimized cell test does not meet the preset purity standard, return to the above step of adjusting the initial sorting parameters to obtain the optimized sorting parameters;

[0170] When the purity of the optimized cell test meets the preset purity standard, use the initial sorting parameters as the sorting parameters for the labeled cell suspension.

[0171] Among them, the fluorescence distribution range refers to the fluctuation range of fluorescence intensity values in the cell population. The target cell test purity refers to the percentage of target cells in the collected target cell population after sorting and testing. The preset purity standard refers to the lowest acceptable level of the target cell purity preset according to the experimental purpose and requirements before the cell sorting test.

[0172] Optionally, determining the initial sorting parameters of the labeled cell suspension according to the fluorescence distribution range can be determined by combining high-throughput screening techniques, such as microfluidic chips or array sorting techniques.

[0173] In the embodiment of the present invention, by sorting the cell population to be screened using a preset flow cytometer according to the sorting parameters, a target cell population can be obtained, effectively separating the target cells and reducing the contamination of non-target cells, thereby improving the purity of the target cells. Among them, the flow cytometer refers to an instrument for cell analysis, sorting, and collection.

[0174] In the embodiment of the present invention, preparing the cell solution free of impurities into a single-cell suspension using the buffer can facilitate subsequent operations such as cell counting, sorting, and culturing; optionally, in the embodiment of the present invention, by analyzing the component type and composition of the target cell components, detailed information about the cell characteristics and functions can be obtained; in the embodiment of the present invention, by determining the chemical and physical characteristics of the target cell components based on the component type and composition, it is beneficial to discover the specificity of the target cell components and facilitate subsequent cell component screening; in the embodiment of the present invention, by analyzing the property specificity between the target cell components and the corresponding non-target cell components in the single-cell suspension based on the chemical and physical characteristics of the components, it can ensure that the marker mainly reacts with the target components, reduce cross-reaction with non-target components, and improve the accuracy and reliability of the experiment; in the embodiment of the present invention, by using a preset flow cytometer to collect the scattered light and fluorescence signals of the labeled cell suspension, cells with specific properties can be quickly identified and screened. Finally, in the embodiment of the present invention, by sorting the cell population to be screened using a preset flow cytometer according to the sorting parameters, a target cell population can be obtained, effectively separating the target cells and reducing the contamination of non-target cells, thereby improving the purity of the target cells. Therefore, the present invention can improve the efficiency of cell component screening.

[0175] As shown in Figure 2 the figure, it is a functional module diagram of a cell component screening system implemented based on flow cytometry provided by an embodiment of the present invention.

[0176] The cell component screening system 200 implemented based on flow cytometry according to the present invention can be installed in an electronic device. According to the functions achieved, the cell component screening system 200 implemented based on flow cytometry may include a single-cell suspension preparation module 201, a cell component analysis module 202, a fluorescence labeling module 203, a fluorescence labeling detection module 204, and a target cell screening module 205. The modules of the present invention may also be referred to as units, which refer to a series of computer program segments that can be executed by a processor of an electronic device and can complete fixed functions, and are stored in the memory of the electronic device.

[0177] In this embodiment, the functions of each module / unit are as follows:

[0178] The single-cell suspension preparation module 201 is used to obtain a target tissue containing target cell components, digest the target tissue into a cell solution, centrifuge the cell solution to obtain a cell solution free of impurities, prepare a buffer solution for the cell solution free of impurities, and use the buffer solution to prepare the cell solution free of impurities into a single-cell suspension;

[0179] The cell component analysis module 202 is used to analyze the component types and component compositions of the target cell components, and based on the component types and the component compositions, determine the component chemical characteristics and component physical characteristics of the target cell components;

[0180] The fluorescence labeling module 203 is used to analyze the property specificities of the target cell components and the corresponding non-target cell components in the single-cell suspension based on the component chemical characteristics and the component physical characteristics, determine the fluorescence labeling agents for the target cell components according to the property specificities, and mix the fluorescence labeling agents with the single-cell suspension to obtain a labeled cell suspension;

[0181] The fluorescence labeling detection module 204 is used to collect the scattered light and fluorescence signals of the labeled cell suspension by using a preset flow cytometer, construct a cell scatter plot of the labeled cell suspension according to the scattered light and the fluorescence signals, and determine the gating parameters of the cell scatter plot, and based on the gating parameters, determine the cell population to be screened in the labeled cell suspension;

[0182] The target cell screening module 205 is configured to calculate the cell ratio and the average cell fluorescence intensity of the cell population to be screened, determine the sorting parameters of the labeled cell suspension based on the cell ratio and the average cell fluorescence intensity, and perform cell sorting on the cell population to be screened by using a preset flow cytometer according to the sorting parameters, so as to obtain a target cell population.

[0183] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for screening cell components based on flow cytometry, characterized in that: The method comprises: Obtaining a target tissue containing target cell components, digesting the target tissue into a cell solution, centrifuging the cell solution to obtain a decontaminated cell solution, preparing a buffer for the decontaminated cell solution, and using the buffer to prepare the decontaminated cell solution into a single cell suspension; Analyzing the component type and component composition of the target cell component, and determining the component chemical characteristics and component physical characteristics of the target cell component based on the component type and the component composition; Based on the chemical characteristics of the components and the physical characteristics of the components, analyzing the property specificity of the target cell components and the non-target cell components corresponding to the single cell suspension, determining the fluorescent marker of the target cell components according to the property specificity, and mixing the fluorescent marker with the single cell suspension to obtain a labeled cell suspension; Using a preset flow cytometer to collect scattered light and fluorescence signals of the labeled cell suspension, constructing a cell scattergram of the labeled cell suspension according to the scattered light and the fluorescence signals, determining gating parameters of the cell scattergram, and determining a cell population to be screened of the labeled cell suspension based on the gating parameters; The cell ratio and average cell fluorescence intensity of the cell population to be screened are calculated, and based on the cell ratio and the average cell fluorescence intensity, the sorting parameters of the labeled cell suspension are determined. According to the sorting parameters, the cell population to be screened is sorted using a preset flow cytometer to obtain a target cell population.

2. The method for screening cell components based on flow cytometry according to claim 1, characterized in that: The step of digesting the target tissue into a cell solution comprises: constructing a digestive environment for the target tissue; and dividing the target tissue into target tissue fragments based on the digestive environment; Transferring the target tissue fragments into a preset digestive enzyme solution to obtain a tissue-enzyme mixed solution; regularly checking the status of the tissue in the tissue-enzyme mixture solution; When the tissue state meets the preset state standard, the tissue-enzyme mixed solution is filtered to obtain a cell solution.

3. The method for screening cell components based on flow cytometry according to claim 1, characterized in that: The analyzing the component type and component composition of the target cell component comprises: Determining the macromolecular type of the target cell component using a preset infrared spectrum; Performing mass spectrometry analysis on the target cell component to obtain a mass spectrometry analysis result, and determining the molecular weight of the target cell component according to the mass spectrometry result; Performing nuclear magnetic resonance on the target cell component to obtain the molecular three-dimensional structure and dynamic characteristics of the target cell component; Determining the component type of the target cell component according to the macromolecule type, the molecular weight, the molecular three-dimensional structure and the dynamic characteristics; Based on the component type, performing elemental analysis on the target cell component to obtain the elemental composition of the target cell component; analyzing the organic small molecule components of the target cell components; The composition of the target cell components is determined based on the elemental composition and the organic small molecule components.

4. The method for screening cell components based on flow cytometry according to claim 1, characterized in that: The analyzing the property specificity of the target cell component and the non-target cell component corresponding to the single cell suspension based on the chemical characteristics of the component and the physical characteristics of the component includes: Determining chemical differences and physical differences between the target cell components and the non-target cell components based on the chemical characteristics of the components and the physical characteristics of the components; According to the chemical difference and the physical difference, clarify the specific difference between the target cell component and the non-target cell component; quantifying the specificity difference to obtain a specificity index; Constructing a specific analysis model of the target cell component and the non-target cell component according to the specific index; The specificity analysis model is used to predict the property specificity of the target cell components and the non-target cell components.

5. The method for screening cell components based on flow cytometry according to claim 1, characterized in that: Determining the fluorescent marker of the target cell component according to the property specificity includes: Acquiring a potential fluorescent marker of the target cell component, and analyzing the marker characteristics of the potential fluorescent marker; Determining the incubation time of the fluorescent marker and the target cell component according to the marker characteristics; Incubating the potential fluorescent marker with the target cell component according to the incubation time to obtain a labeled cell component; Detecting the labeling effect of the labeled cell components, and analyzing the marker fluorescence intensity and background noise of the potential fluorescent marker according to the labeling effect; Analyzing the fluorescent labeling effect of the potential fluorescent label according to the property specificity, the label characteristics, the label fluorescence intensity and the background noise; The potential fluorescent marker with the best fluorescent labeling effect is selected as the fluorescent marker of the target cell component.

6. The method for screening cell components based on flow cytometry according to claim 1, characterized in that: The step of constructing a cell scattergram of the labeled cell suspension according to the scattered light and the fluorescent signal comprises: Performing background correction on the scattered light and the fluorescent signal to obtain corrected scattered light and corrected fluorescent signal; Performing signal standardization on the corrected scattered light and the corrected fluorescent signal to obtain standardized scattered light and standardized fluorescent signal; Analyzing forward scattered light and side scattered light of the standardized scattered light; analyzing the fluorescence signal intensity of the standardized fluorescence signal, and determining the color coding of the standardized fluorescence signal based on the fluorescence signal intensity; Establishing a cell coordinate system of the labeled cell suspension with the forward scattered light as the horizontal axis and the side scattered light as the vertical axis; According to the color coding, the labeled cell suspension is mapped to the cell coordinate system to obtain a cell scattergram of the labeled cell suspension.

7. The method for screening cell components based on flow cytometry according to claim 1, characterized in that: Determining the gating parameters of the cell scattergram comprises: Determining the screening requirements of the target cell components corresponding to the cell scattergram; Determining signal parameters of the cell scattergram, and analyzing signal parameter weights of the signal parameters; According to the screening requirements, constructing the gating logic of the cell scatter plot; combining gating conditions of the signal parameters based on the gating logic; The gating parameters of the cell scattergram are determined according to the gating conditions, the signal parameter weights and the signal parameters.

8. The method for screening cell components based on flow cytometry according to claim 1, characterized in that: The step of calculating the cell ratio and average cell fluorescence intensity of the cell population to be screened comprises: Respectively determining the specific cell number and the total cell number of the cell population to be screened and the labeled cell suspension corresponding to the cell population to be screened; Measuring the cell fluorescence intensity of the cell population to be screened and the background fluorescence intensity of the cell population to be screened; The cell ratio and average cell fluorescence intensity of the cell population to be screened are calculated based on the specific cell number, the total cell number, the cell fluorescence intensity and the background fluorescence intensity.

9. The method for screening cell components based on flow cytometry according to claim 1, characterized in that: Determining the sorting parameters of the labeled cell suspension based on the cell ratio and the average cell fluorescence intensity comprises: Determining the fluorescence distribution range of the labeled cell suspension according to the cell ratio and the average cell fluorescence intensity; Determining initial sorting parameters of the labeled cell suspension according to the fluorescence distribution range; Based on the initial sorting parameters, the labeled cell suspension is sorted and tested to obtain the test purity of the target cells; When the test purity of the target cells does not meet the preset purity standard, adjusting the initial sorting parameters to obtain optimized sorting parameters; According to the optimized sorting parameters, the labeled cell suspension is subjected to a sorting test to obtain an optimized cell test purity; When the optimized cell test purity does not meet the preset purity standard, returning to the above step of adjusting the initial sorting parameters to obtain optimized sorting parameters; When the optimized cell test purity meets the preset purity standard, the initial sorting parameters are used as the sorting parameters of the labeled cell suspension.

10. A cell component screening system based on flow cytometry, characterized in that: Used to perform the method for screening cell components based on flow cytometry as described in any one of claims 1 to 9, the system comprising: A single cell suspension preparation module is used to obtain a target tissue containing target cell components, digest the target tissue into a cell solution, centrifuge the cell solution to obtain a decontaminated cell solution, prepare a buffer solution for the decontaminated cell solution, and use the buffer solution to prepare a single cell suspension from the decontaminated cell solution; A cell component analysis module, used to analyze the component type and component composition of the target cell component, and determine the component chemical characteristics and component physical characteristics of the target cell component based on the component type and the component composition; A fluorescent labeling module, used for analyzing the property specificity of the target cell component and the non-target cell component corresponding to the single cell suspension based on the chemical characteristics of the component and the physical characteristics of the component, determining the fluorescent marker of the target cell component according to the property specificity, and mixing the fluorescent marker with the single cell suspension to obtain a labeled cell suspension; A fluorescent marker detection module, used to collect scattered light and fluorescent signals of the labeled cell suspension using a preset flow cytometer, construct a cell scatter plot of the labeled cell suspension according to the scattered light and the fluorescent signals, determine gating parameters of the cell scatter plot, and determine the cell population to be screened of the labeled cell suspension based on the gating parameters; The target cell screening module is used to calculate the cell ratio and average cell fluorescence intensity of the cell population to be screened, determine the sorting parameters of the labeled cell suspension based on the cell ratio and the average cell fluorescence intensity, and perform cell sorting on the cell population to be screened using a preset flow cytometer according to the sorting parameters to obtain a target cell population.

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