Method and system for screening cell components based on flow cytometry

Abstract

The invention relates to the field of cytobiology, and particularly discloses a method and a system for screening cell components based on flow cytometry, the method comprises the following steps: digesting a target tissue into a cell solution, preparing a buffer solution of an impurity-removed cell solution, and preparing the impurity-removed cell solution into a single-cell suspension by using the buffer solution; analyzing the property specificity of the target cell component and a non-target cell component corresponding to the single-cell suspension, determining a fluorescent marker of the target cell component, mixing the fluorescent marker and the single-cell suspension, constructing a cell scatter diagram of the marked cell suspension, and determining gating parameters of the cell scatter diagram; calculating the cell proportion and the average cell fluorescence intensity of the to-be-screened cell population, determining sorting parameters of the labeled cell suspension, and performing cell sorting on the to-be-screened cell population by using a preset flow cytometer to obtain a target cell population. The cell component screening efficiency can be improved.

Description

Technical Field

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

[0002] Cell component screening refers to the process of identifying, isolating and / or studying cell components with specific properties from a cell population using a series of biological experimental methods and technical means. 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, the method for screening cell components is to analyze the protein composition in cells through mass spectrometry and other technologies. Due to the complex protein separation and identification steps in the screening process, this method can only detect specific cell components and ignores other important components, 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, the main purpose of which is to improve the efficiency of cell component screening.

[0005] To achieve the above object, the present invention provides a method for screening cell components based on flow cytometry, comprising: 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.

[0006] Optionally, 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.

[0007] Optionally, 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.

[0008] Optionally, 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.

[0009] Optionally, 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 latent 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.

[0010] Optionally, 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 the forward scattered light and the 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.

[0011] Optionally, 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.

[0012] Optionally, the 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.

[0013] Optionally, 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 purity of the optimized cell test does not meet the preset purity standard, returning to the above step of adjusting the initial sorting parameters to obtain the 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.

[0014] In order to solve the above problems, the present invention also provides a system for screening cell components based on flow cytometry, 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.

[0015] The embodiment of the present invention uses the buffer to prepare the decontaminated cell solution into a single cell suspension, which can facilitate subsequent operations such as cell counting, sorting, and culturing; optionally, the embodiment of the present invention can obtain detailed information about cell characteristics and functions by analyzing the component type and component composition of the target cell component; the embodiment of the present invention determines the component chemical characteristics and component physical characteristics of the target cell component based on the component type and the component composition, which can be beneficial to discovering the specificity of the target cell component and facilitate subsequent cell component screening; the embodiment of the present invention analyzes the target cell based on the component chemical characteristics and the component physical characteristics. The specificity of the properties of the cell components and the non-target cell components corresponding to the single cell suspension can ensure that the marker mainly reacts with the target component, reduce cross-reactions with non-target components, and improve the accuracy and reliability of the experiment; the embodiment of the present invention can quickly identify and screen out cells with specific properties by using a preset flow cytometer to collect the scattered light and fluorescent signals of the labeled cell suspension. Finally, the embodiment of the present invention uses a preset flow cytometer to perform cell sorting on the cell population to be screened according to the sorting parameters to obtain a target cell population that can effectively separate the target cells, reduce the contamination of non-target cells, and thus improve the purity of the target cells. Therefore, the present invention can improve the efficiency of cell component screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A schematic diagram of a flow chart of a method for screening cell components based on flow cytometry provided in one embodiment of the present invention; Figure 2 A functional module diagram of a cell component screening system based on flow cytometry provided in one embodiment of the present invention; The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0017] It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0018] The 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 the electronic devices that can be configured to perform the method provided by the embodiment of the present application, such as a server, a terminal, etc. 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 it can be 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 distribution networks (Content Delivery Network, CDN), and big data and artificial intelligence platforms.

[0019] Reference Figure 1 FIG. 1 is a flow chart of a method for screening cell components based on flow cytometry according to an embodiment of the present invention. In this embodiment, the method for screening cell components based on flow cytometry includes: S1. Obtain a target tissue containing target cell components, digest the target tissue into a cell solution, perform cell centrifugation on the cell solution to obtain a decontaminated cell solution, prepare a buffer for the decontaminated cell solution, and use the buffer to prepare the decontaminated cell solution into a single cell suspension.

[0020] The embodiment of the present invention can ensure that high-quality, high-purity cell samples can be used in subsequent biological research by obtaining target tissues containing target cell components. The target tissue refers to a biological tissue from which specific types of cells or cell components can be extracted.

[0021] In the embodiment of the present invention, the target tissue can be digested into a cell solution to release individual cells from the tissue structure, so that the cells can exist independently, which is convenient for subsequent operations. The cell solution refers to a liquid mixture containing a single or multiple cells obtained after the target tissue is digested.

[0022] As an embodiment of the present invention, 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.

[0023] Among them, the series of conditions specially set for effectively digesting the target tissue into a cell solution, such as: temperature, pH value, gas environment and sterile conditions. The preset digestive enzyme solution refers to a liquid mixture of a group of enzymes specially formulated to digest the target tissue and release single cells, and 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, which is used to determine when to terminate the digestion process.

[0024] Optionally, the periodic inspection of the tissue status in the tissue-enzyme mixed solution may be performed by observation using a microscope.

[0025] Optionally, the tissue-enzyme mixed solution may be filtered through a cell filter, for example, a filter with a pore size of 40 microns.

[0026] In the embodiment of the present invention, by centrifuging the cell solution, a decontaminated cell solution is obtained, which can effectively remove most of the impurities in the solution, obtain a relatively pure cell suspension, and provide a high-quality sample for subsequent cell culture or other experimental operations. The decontaminated cell solution refers to a cell solution from which most of the impurities and undigested tissue fragments have been removed after centrifugation and other processing steps.

[0027] Optionally, as an embodiment of the present invention, the cell solution may be centrifuged by differential centrifugation, wherein the differential centrifugation refers to separating cells and cell components of different densities through a series of centrifugation steps at different speeds.

[0028] The embodiment of the present invention can provide a suitable pH value and ionic strength by configuring the buffer of the decontaminated cell solution to maintain the normal physiological state of the cells. The buffer refers to a liquid medium used to maintain a stable pH value of the solution.

[0029] In the embodiment of the present invention, the decontaminated cell solution is prepared into a single cell suspension by using the buffer solution, which can facilitate subsequent operations such as cell counting, sorting, and culturing. The single cell suspension refers to a cell suspension extracted from a cell mixture that contains only a single cell and does not contain other cell aggregates or cell fragments.

[0030] Optionally, as an embodiment of the present invention, the preparation of the decontaminated cell solution into a single cell suspension using the buffer solution may be performed by a method of resuspending cells, wherein the resuspending cells refers to gently and repeatedly blowing the precipitated cells with a buffer solution multiple times to resuspend them.

[0031] S2. 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.

[0032] The embodiment of the present invention can obtain detailed information about cell characteristics and functions by analyzing the component type and component composition of the target cell component. 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.

[0033] As an embodiment of the present invention, the analyzing the component type and component composition of the target cell component includes: 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.

[0034] Among them, the macromolecule type refers to the main biological macromolecules that constitute the cell components, such as proteins, nucleic acids, etc. The preset infrared spectrum refers to a set of pre-set infrared spectrum characteristic parameters or standard spectra, which are used to identify and classify biological macromolecules. The mass spectrometry analysis results refer to the data and information obtained after analyzing the target cell components by 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 morphology of molecules in three-dimensional space. The dynamic characteristics refer to the movement and changes that molecules undergo during their life cycle, such as molecular movement, interaction, etc. The elemental composition refers to the types of different elements contained in the target cell components and their relative proportions. The small organic molecule component refers to an organic compound with a relatively small molecular mass present in the target cell components.

[0035] Optionally, the mass spectrometry analysis of the target cell components can be performed by using MALDI-TOF, wherein the MALDI-TOF refers to a mass spectrometry technology used to analyze biological macromolecules, such as proteins, peptides, nucleic acids and other macromolecular organic compounds.

[0036] Optionally, the elemental analysis of the target cell components based on the component type can be performed by X-ray fluorescence spectroscopy, wherein X-ray fluorescence spectroscopy is a technology used for material composition analysis and elemental quantitative analysis.

[0037] The embodiment of the present invention can be helpful in discovering the specificity of the target cell component by determining the component chemical characteristics and component physical characteristics of the target cell component based on the component type and the component composition, and facilitates 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 component, such as the elemental composition, molecular structure, functional group, etc. of the component. The component physical characteristics refer to the physical properties of the target cell component, such as the shape, size, structure, fluidity, etc. of the component.

[0038] Optionally, as an embodiment of the present invention, the component chemical characteristics and component physical characteristics of the target cell components based on the component type and the component composition can be determined by chemical reactivity analysis and electron microscopy observation, respectively.

[0039] S3. Based on the chemical characteristics of the components and the physical characteristics of the components, the property specificity of the target cell components and the non-target cell components corresponding to the single-cell suspension is analyzed, and according to the property specificity, the fluorescent marker of the target cell component is determined, and the fluorescent marker and the single-cell suspension are mixed to obtain a labeled cell suspension.

[0040] The embodiment of the present invention can ensure that the marker mainly reacts with the target component, reduces cross-reaction with non-target components, and improves the accuracy and reliability of the experiment by analyzing the property specificity of the target cell component and the corresponding non-target cell component of the single cell suspension based on the chemical characteristics of the component and the physical characteristics of the component. The property specificity refers to the unique chemical and physical properties of the target cell component relative to the non-target components in the environment.

[0041] As an embodiment of the present invention, 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.

[0042] Wherein, 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 and physical properties between the target cell components and the non-target cell components. The specific index refers to the metric used to quantify the specific difference between the target cell components and the non-target cell components. The specific analysis model refers to a mathematical or computational model used to predict and identify the target component.

[0043] Optionally, the specific differences between the target cell components and the non-target cell components based on the chemical differences and the physical differences can be determined by statistical analysis, such as t-test, analysis of variance, etc.

[0044] The embodiment of the present invention determines the fluorescent marker of the target cell component according to the property specificity so that the fluorescent marker can be specifically bound to the target cell component without non-specific binding with non-target cell components, thereby achieving accurate identification and tracking of the target cell. Wherein, the fluorescent marker refers to a chemical substance or biological molecule that can be used to mark the target cell component so that it emits fluorescence under specific conditions.

[0045] As an embodiment of the present invention, the step of 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 latent 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.

[0046] Among them, the potential fluorescent marker refers to a candidate fluorescent dye, fluorescent protein, fluorescent probe or other fluorescent compound considered for marking the target cell component in the process of determining the fluorescent marker of the target cell component. The marker property refers to a series of specific properties possessed by the fluorescent marker, which determine the performance and applicability of the marker in experimental applications. The incubation time refers to the time required for the fluorescent marker and the target cell component to interact with each other to achieve the best labeling effect after the fluorescent marker is mixed. The labeled cell component refers to the labeled cell component that is incubated with the fluorescent marker and the target cell component to make the fluorescent marker and the target cell component combine, so that the target cell component can emit fluorescence. The labeling effect refers to the intensity, distribution, uniformity and background noise of the fluorescent signal exhibited by the target cell component after the fluorescent marker is combined. The marker fluorescence intensity refers to the intensity of the fluorescent signal generated by the fluorescent marker under the irradiation of the excitation light. The background noise refers to the fluorescent signal generated by non-target cell components or other factors, which will interfere with the observation and analysis of the fluorescent signal of the target cell component, thereby affecting the accuracy of the experimental results.

[0047] Optionally, said analyzing the marker properties of said potential fluorescent markers may be analyzed by biocompatibility assessment.

[0048] Optionally, the detecting of the labeling effect of the labeled cell components can be detected by fluorescence microscopy.

[0049] In the embodiment of the present invention, the fluorescent marker and the single cell suspension are mixed to obtain a labeled cell suspension that can identify and classify different types of cells and distinguish cells in different subpopulations or states. The labeled cell suspension refers to a cell suspension that is obtained by mixing the fluorescent marker with the single cell suspension so that the fluorescent marker is combined with the cell components in the single cell suspension, thereby enabling the cell components to emit fluorescence.

[0050] S4. Use a preset flow cytometer to collect 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, determine the 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.

[0051] The embodiment of the present invention can quickly identify and screen out cells with specific properties by using a preset flow cytometer to collect scattered light and fluorescent signals of the labeled cell suspension. The scattered light refers to the non-specific light signal generated by the cell detected by the flow cytometer. The fluorescent signal refers to the light signal of a specific wavelength generated by the excitation of the fluorescent marker in the flow cytometer analysis.

[0052] The embodiment of the present invention can help identify different types of cells by constructing a cell scattergram of the labeled cell suspension based on the scattered light and the fluorescent signal. The cell scattergram refers to a two-dimensional or multi-dimensional distribution diagram of cells constructed using scattered light and fluorescent signals during flow cytometer analysis.

[0053] As an embodiment of the present invention, constructing a cell scattergram of the labeled cell suspension according to the scattered light and the fluorescent signal includes: 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 the forward scattered light and the 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.

[0054] Among them, the background correction refers to the removal of 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 standardization refers to a pre-processing step of scattered light and fluorescent signal in order to eliminate the difference in signal intensity between different cells or different fluorescent channels during flow cytometer analysis. The forward scattered light refers to the light signal in a specific direction generated by the cell detected by the flow cytometer. The side scattered light refers to the light signal perpendicular to the direction of the laser beam generated by the cell detected by the flow cytometer. The fluorescence signal intensity refers to the intensity of the light signal emitted by the fluorescent marker after being excited by the excitation light in the flow cytometer analysis. The color coding refers to the method used in the flow cytometer data analysis software to represent the color of different fluorescent channels or different cell characteristics. The cell coordinate system refers to the forward scattered light (FSC) and side scattered light (SSC) as the coordinate axes, and each cell is regarded as a point to display its position in the coordinate system.

[0055] Optionally, the signal standardization of the corrected scattered light and the corrected fluorescence signal can be achieved by a signal normalization method, wherein the signal normalization refers to converting the corrected scattered light and the corrected fluorescence signal values ​​into values ​​proportional to a reference value.

[0056] The embodiment of the present invention can reduce noise and interference in data analysis and improve the quality and reliability of data by determining the gating parameters of the cell scattergram. Wherein, the gating parameters refer to the parameters used to screen and define the cell population of interest during the flow cytometer data analysis process.

[0057] As an embodiment of the present invention, determining the gating parameters of the cell scattergram includes: 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.

[0058] Wherein, the screening requirement refers to the cell characteristics or subpopulations that need to be screened for specific experimental purposes in flow cytometric analysis. The signal parameter refers to the physical quantity used to describe and distinguish cell characteristics. The signal parameter weight refers to an indicator used to reflect the relative importance or contribution 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 flow cytometer data analysis process. The gating condition refers to the specific conditions or thresholds used to screen and define the cell population of interest during the flow cytometer data analysis process.

[0059] Optionally, the gating conditions of the combination of the signal parameters can be combined by data analysis software, such as FlowJo, FCS Express, Beckman Coulter Accuri Cytomation Cytobuilder, etc.

[0060] The embodiment of the present invention can accurately identify and screen out target cell components by determining the cell population to be screened of the labeled cell suspension based on the gating parameters, reduce interference from non-target cells, and improve the accuracy and efficiency of the experiment. Wherein, the cell population to be screened refers to the cell subpopulation that meets specific conditions determined by gating analysis of a flow cytometer.

[0061] S5. 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.

[0062] The embodiment of the present invention can ensure the purity of the target cell population and the efficiency of the screening process by calculating the cell ratio and the average cell fluorescence intensity of the cell population to be screened. Wherein, the cell ratio refers to the ratio of a specific cell subpopulation to the entire cell population in flow cytometric analysis. The average cell fluorescence intensity refers to the mean value of the intensity of the fluorescent signal generated by the fluorescent marker under the irradiation of the excitation light in the flow cytometric analysis.

[0063] As an embodiment of the present invention, the calculating of the cell ratio and the average cell fluorescence intensity of the cell population to be screened includes: 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; According to the specific cell number, the total cell number, the cell fluorescence intensity and the background fluorescence intensity, the cell ratio and the average cell fluorescence intensity of the cell population to be screened are calculated using the following formula: ; ; in, Represents the cell ratio, represents the average cell fluorescence intensity, Indicates the specific number of cells in the cell population to be screened, Indicates the total number of cells in the labeled cell suspension. Indicates the first The fluorescence intensity of each cell, represents the background fluorescence intensity.

[0064] Wherein, 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 cells and unlabeled cells in the labeled cell suspension in the entire solution. The cell fluorescence intensity refers to the measured fluorescence signal intensity from a single cell. The background fluorescence intensity refers to the fluorescence signal intensity detected in the target cell or sample area without a specific fluorescent marker.

[0065] The embodiment of the present invention can improve the accuracy of sorting and optimize the sorting speed by determining the sorting parameters of the labeled cell suspension based on the cell ratio and the average cell fluorescence intensity, thereby improving the efficiency of sorting. 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.

[0066] 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: According to the cell ratio and the average cell fluorescence intensity, the fluorescence distribution range of the labeled cell suspension is determined using the following formula: ; in, represents the fluorescence distribution range, represents the average cell fluorescence intensity, Indicates the specific number of cells in the cell population to be screened, Indicates the first Fluorescence intensity of each cell; 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 purity of the optimized cell test does not meet the preset purity standard, returning to the above step of adjusting the initial sorting parameters to obtain the 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.

[0067] The fluorescence distribution range refers to the fluctuation range of the fluorescence intensity value in the cell population. The target cell test purity refers to the percentage of target cells in the target cell population collected after the sorting test. The preset purity standard refers to the minimum acceptable level of target cell purity pre-set according to the experimental purpose and requirements before the cell sorting test.

[0068] Optionally, the initial sorting parameters of the labeled cell suspension determined according to the fluorescence distribution range can be determined by combining high-throughput screening technology, such as microfluidic chip or array sorting technology.

[0069] The embodiment of the present invention performs cell sorting on the cell population to be screened according to the sorting parameters using a preset flow cytometer to obtain a target cell population, which can effectively separate target cells and reduce contamination from non-target cells, thereby improving the purity of target cells. The flow cytometer refers to an instrument used for cell analysis, sorting and collection.

[0070] The embodiment of the present invention uses the buffer to prepare the decontaminated cell solution into a single cell suspension, which can facilitate subsequent operations such as cell counting, sorting, and culturing; optionally, the embodiment of the present invention can obtain detailed information about cell characteristics and functions by analyzing the component type and component composition of the target cell component; the embodiment of the present invention determines the component chemical characteristics and component physical characteristics of the target cell component based on the component type and the component composition, which can be beneficial to discovering the specificity of the target cell component and facilitate subsequent cell component screening; the embodiment of the present invention analyzes the target cell based on the component chemical characteristics and the component physical characteristics. The specificity of the properties of the cell components and the non-target cell components corresponding to the single cell suspension can ensure that the marker mainly reacts with the target component, reduce cross-reactions with non-target components, and improve the accuracy and reliability of the experiment; the embodiment of the present invention can quickly identify and screen out cells with specific properties by using a preset flow cytometer to collect the scattered light and fluorescent signals of the labeled cell suspension. Finally, the embodiment of the present invention uses a preset flow cytometer to perform cell sorting on the cell population to be screened according to the sorting parameters to obtain a target cell population that can effectively separate the target cells, reduce the contamination of non-target cells, and thus improve the purity of the target cells. Therefore, the present invention can improve the efficiency of cell component screening.

[0071] like Figure 2 , which is a functional module diagram of a cell component screening system based on flow cytometry provided by one embodiment of the present invention.

[0072] The cell component screening system 200 based on flow cytometry of the present invention can be installed in an electronic device. According to the functions implemented, the cell component screening system 200 based on flow cytometry can include a single cell suspension preparation module 201, a cell component analysis module 202, a fluorescent labeling module 203, a fluorescent labeling detection module 204 and a target cell screening module 205. The module of the present invention can also be referred to as a unit, which refers to a series of computer program segments that can be executed by an electronic device processor and can complete fixed functions, which are stored in the memory of the electronic device.

[0073] In this embodiment, the functions of each module / unit are as follows: 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 decontaminated cell solution, prepare a buffer for the decontaminated cell solution, and use the buffer to prepare the decontaminated cell solution into a single cell suspension; The cell component analysis module 202 is 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; The fluorescent labeling module 203 is used to analyze 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, determine the fluorescent marker of the target cell component according to the property specificity, and mix the fluorescent marker with the single cell suspension to obtain a labeled cell suspension; The fluorescent marker detection module 204 is used to collect scattered light and fluorescent signals of the labeled cell suspension using a preset flow cytometer, construct a cell scattergram of the labeled cell suspension according to the scattered light and the fluorescent signals, and determine the gating parameters of the cell scattergram, and determine the cell population to be screened of the labeled cell suspension based on the gating parameters; The target cell screening module 205 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.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution 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 latent 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 the forward scattered light and the 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 purity of the optimized cell test does not meet the preset purity standard, returning to the above step of adjusting the initial sorting parameters to obtain the 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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