Method for in-situ separation of cochlea related cells based on patch clamp system

Abstract

The invention discloses a method for separating cochlea related cells in situ based on a patch clamp system. The method comprises the following steps: contacting an electrode with a target single cell under a microscope, and carrying out in-situ grabbing on the target single cell through negative pressure adsorption; the target single cell and surrounding cells are kept in a connection state before in-situ grabbing; before the electrode approaches the target single cell, positive pressure is continuously applied, and when the electrode is in contact with the surface of the target single cell, the positive pressure is instantly stopped and switched to negative pressure. According to the method provided by the invention, high-quality cochlea related cells can be obtained in situ for deep sequencing analysis, and the method can be used for basic research and accurate diagnosis and treatment of clinical hearing impairment, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for in-situ isolation of cochlear-related cells based on a patch-clamp system. More specifically, it provides a method for stably isolating and obtaining single cochlear hair cells with precise spatial location information, which can be used for single-cell RNA sequencing (SRNA-seq). The method provided by this invention has good scalability and is applicable to single-cell transcriptomics studies of various cell types within the cochlea, other sensory receptor cells, and rare tissues. Background Technology

[0002] Single-cell RNA sequencing technology can acquire transcriptome information at the single-cell scale, providing a single-cell perspective and greater information depth for research on auditory system development, functional analysis, and disease mechanisms, and offering important evidence for target discovery and drug development. The cochlea, as an auditory receptor, possesses a delicate microstructure and highly specialized cell types (such as inner and outer hair cells, various supporting cells, and spiral ganglion neurons), which play crucial roles in auditory signal transduction and frequency encoding. However, the cochlea is located deep within the petrous part of the temporal bone, surrounded by a hard, dense bony structure; its internal cells are few in number, tightly connected to surrounding cells, and highly sensitive to mechanical and chemical stimuli. Separation processes can easily lead to cell death or induce transcriptome bias.

[0003] Currently, no research reports the ability to perform transcriptome sequencing on single cells with clearly defined spatial locations within cochlear tissue. Existing techniques typically involve enzymatic digestion combined with mechanical pipetting to obtain single-cell suspensions or tissue clumps, followed by flow cytometry or glass microelectrode aspiration to acquire the target cells. These methods are technically challenging: cochlear hair cells are sensitive and fragile, easily affected by mechanical and chemical stimuli during separation, leading to cell stress, quality degradation, and even death. Furthermore, this strategy often struggles to precisely locate single cells at specific spatial locations, usually only separating a certain cell type or a large group of cells, inevitably resulting in the loss of the cells' original precise spatial location information and increasing the risk of cell type misidentification in subsequent data analysis. For regenerated or genetically modified cells, accurate identification and differentiation from mixed cell populations may be difficult. Therefore, developing a new method that targets the anatomical characteristics of the cochlea and enables the acquisition of high-quality single cells in situ is of urgent practical significance for basic auditory research and the precise diagnosis and treatment of hearing impairment in clinical practice. Attached Figure Description

[0004] Figure 1 To accurately separate non-target cells and locate target outer hair cells using differential interferometry imaging.

[0005] Figure 2To accurately separate non-target cells and locate target inner hair cells using differential interferometry imaging.

[0006] Figure 3 Pearson correlation coefficient analysis for cochlear outer hair cells.

[0007] Figure 4 Pearson correlation coefficient analysis for cochlear inner hair cells.

[0008] Figure 5 A dimensionality reduction visualization of UMAP.

[0009] Figure 6 This shows the expression of the outer hair cell marker gene Slc26a5 between two cell groups.

[0010] Figure 7 This shows the expression of the inner hair cell marker gene Slc17a8 between two cell groups.

[0011] Figure 8 This represents the distribution of mitochondrial gene content in cells.

[0012] Figure 9 The stress score of the obtained cells.

[0013] Figure 10 The expression levels of stress genes in the cells were obtained. Summary of the Invention

[0014] This invention aims to address the following problems existing in the prior art: (1) how to accurately obtain single cells of a specific target location and type in highly heterogeneous cell groups (such as the cochlea); (2) how to obtain high-quality single cells in fragile tissues; and (3) how to minimize RNA degradation of target cells and effectively prevent contamination by RNA from non-target cells (such as surrounding supporting cells) during the acquisition of target single cells, thereby ensuring the acquisition of high-quality, representative target cell RNA for deep sequencing analysis. To solve the above problems, this invention provides a method for in situ separation of cochlear-related cells based on a patch-clamp system. The method provided by this invention can accurately obtain target single cells of a specific target location and type, and is particularly suitable for obtaining high-quality single cells from fragile tissues for deep sequencing analysis, while effectively preventing contamination by RNA from non-target cells.

[0015] To address the above problems, the present invention provides the following technical solution.

[0016] The first aspect of the present invention provides a method for in-situ separation of target single cells based on a patch clamp system, the method comprising contacting the target single cell with an electrode under a microscope and grasping the target single cell in situ by negative pressure adsorption;

[0017] The target single cell, as described above, is kept connected to its surrounding cells during in-situ grasping.

[0018] Before the electrode approaches the target single cell, a positive pressure is continuously applied. When the electrode contacts the surface of the target single cell, the positive pressure is instantly stopped and switched to a negative pressure.

[0019] In this invention, "in situ capture" refers to the precise acquisition of a single or specific cell directly from its original location without damaging the tissue or microenvironment.

[0020] In some embodiments of the present invention, the target single cell is a cell on the organ of Corti in the cochlea.

[0021] In some preferred embodiments of the present invention, the cells on the organ of Corti of the cochlea are cochlear hair cells, inner marginal cells, interdental cells, or regenerating hair cells.

[0022] In this invention, "regenerated hair cells" refer to hair cells that are transformed from supporting cells through a specific mechanism. The ultimate goal is to replace damaged hair cells to restore auditory function. They can be distinguished from surrounding cells by fluorescent labeling and / or morphological characteristics.

[0023] In some embodiments of the present invention, the cochlear hair cells are inner hair cells and / or outer hair cells.

[0024] In some embodiments of the present invention, the target single cell is derived from mice. .

[0025] In some embodiments of the present invention, the pressure of the negative pressure adsorption is 0.01-0.05 MPa.

[0026] In some preferred embodiments of the present invention, the pressure of the negative pressure adsorption is 0.01-0.02 MPa.

[0027] In some embodiments of the present invention, the positive pressure is 0.01-0.05 MPa.

[0028] In some preferred embodiments of the present invention, the positive pressure is 0.01-0.02 MPa.

[0029] In some specific embodiments of the present invention, the positive pressure is 0.01 MPa.

[0030] In this invention, the range of positive pressure continuously applied before the electrode approaches the target single cell is consistent with the range of negative pressure used for in-situ grasping.

[0031] In some embodiments of the present invention, the electrode is a borosilicate glass electrode or a glass electrode.

[0032] In some embodiments of the present invention, the tip diameter of the electrode is 1-5 μm.

[0033] In some preferred embodiments of the present invention, the tip diameter of the electrode is 1-4 μm.

[0034] In some specific embodiments of the present invention, when the target single cell is an inner hair cell or a regenerated hair cell, the tip diameter of the electrode is 2-4 μm.

[0035] In some specific embodiments of the present invention, when the target single cell is an outer hair cell, the tip diameter of the electrode is 2-3 μm.

[0036] In some specific embodiments of the present invention, when the target single cell is an inner edge cell or an interdental cell, the tip diameter of the electrode is 1-2 μm.

[0037] In some embodiments of the present invention, the in-situ grasping step does not include an enzymatic hydrolysis step.

[0038] In this invention, when the target single cell is an outer hair cell, it can optionally undergo slight enzymatic hydrolysis. After slight enzymatic hydrolysis, the outer hair cell is separated from the surrounding supporting cells, and generally, positive pressure blowing / blowing is not required.

[0039] In some embodiments of the present invention, when the target single cell is an outer hair cell, the in-situ grasping includes a slight enzymatic hydrolysis step; after the slight enzymatic hydrolysis, the target single cell and its surrounding hair cells remain connected.

[0040] In some preferred embodiments of the present invention, the slight enzymatic hydrolysis comprises contacting the organ of Corti in the cochlea with an enzyme-containing composition comprising type IV collagenase and CELL DISSOC MEDIUM derived from Thermo / Life / Invitrogen.

[0041] In some preferred embodiments of the present invention, the content of type IV collagenase is 3-8 mg per 1 mL of CELL DISSOCMEDIUM in the enzyme-containing composition.

[0042] In some further preferred embodiments of the present invention, the content of type IV collagenase is 4-6 mg.

[0043] In some preferred embodiments of the present invention, the enzyme-containing composition further comprises deoxyribonuclease I.

[0044] In some further preferred embodiments of the present invention, the content of deoxyribonuclease I is 0.5-2 U per 1 mL of CELLDISSOC MEDIUM contained in the enzyme-containing composition.

[0045] In some preferred embodiments of the present invention, the enzyme-containing composition further comprises CaCl2.

[0046] In some further preferred embodiments of the present invention, the content of CaCl2 in each 1 mL of CELLDISSOC MEDIUM contained in the enzyme-containing composition is 5-10 mM.

[0047] In some embodiments of the present invention, the enzyme-containing composition is provided in the form of a premixed lyophilized powder or a ready-to-use liquid.

[0048] In some embodiments of the present invention, the conditions for the slight enzymatic hydrolysis meet one or more of the following: the time for the slight enzymatic hydrolysis is 5-20 minutes; the temperature for the slight enzymatic hydrolysis is 32-38°C; and the oscillation speed for the slight enzymatic hydrolysis is 150-300 rpm.

[0049] In some preferred embodiments of the present invention, the conditions for the slight enzymatic hydrolysis satisfy one or more of the following: the time for the slight enzymatic hydrolysis is 8-15 minutes; the temperature for the slight enzymatic hydrolysis is 35-36°C; and the oscillation speed for the slight enzymatic hydrolysis is 180-220 rpm.

[0050] In some embodiments of the present invention, the in-situ grasping process further includes a step of separating non-target cells.

[0051] In some preferred embodiments of the present invention, the separation is performed using electrodes as defined in the method described in the first aspect.

[0052] In some preferred embodiments of the present invention, the separation is positive pressure blow-off and / or negative pressure peel-off of non-target cells.

[0053] In some preferred embodiments of the present invention, the pressure of the positive pressure blow-off and / or negative pressure stripping is 0.01-0.1 MPa.

[0054] In some further preferred embodiments of the present invention, the pressure of the positive pressure blow-off and / or negative pressure stripping is 0.01-0.04 MPa.

[0055] In some preferred embodiments of the present invention, the separation is performed under a microscope.

[0056] In some specific embodiments of the present invention, when the target single cell is an inner hair cell, an undigested outer hair cell, or an endogenous cell, the pressure of the positive pressure blow-off is 0.02-0.04 MPa.

[0057] In some specific embodiments of the present invention, when the target single cell is an interdental cell, the pressure of the positive pressure blow-off is 0.001-0.01 MPa.

[0058] In some specific embodiments of the present invention, when the target single cell is a regenerated hair cell, the pressure of the positive pressure blow-off is 0.001-0.02 MPa.

[0059] In some embodiments of the present invention, the in-situ grasping and / or the slight enzymatic hydrolysis are further included in a pretreatment step; the pretreatment is to remove the bone layer and peel off the organ of Corti of the cochlea after the cochlea is removed.

[0060] In some preferred embodiments of the present invention, the bone layer is removed under the following conditions: at a temperature of 0-4°C; and / or, in a liquid.

[0061] In some preferred embodiments of the present invention, the liquid is PBS buffer.

[0062] A second aspect of the present invention provides a single-cell sequencing method, the sequencing method comprising:

[0063] S1: Obtain the single cell as described in the first aspect;

[0064] S2: Sequencing.

[0065] In some embodiments of the present invention, S2 involves extracting RNA from the single cell, constructing a library, and performing high-throughput sequencing to obtain transcriptome data.

[0066] In some preferred embodiments of the present invention, the high-throughput sequencing is based on any of the following platforms: Illumina NovaSeq 6000, Illumina HiSeq 4000, Illumina NextSeq 2000, BGI MGISEQ-2000, and Element AVITI.

[0067] A third aspect of the present invention provides a method for data analysis after single-cell sequencing, the method comprising:

[0068] (a) Obtain a single cell using the method described in the first aspect, and record the spatial coordinates of the single cell in the original tissue;

[0069] (b) Obtain transcriptome data from the single cells obtained in (a);

[0070] (c) Correlate the spatial coordinates of the transcriptome data (a) from (b) to generate an expression matrix that includes spatial locations;

[0071] (d) Based on the expression matrix of step (c), the functional state of the single cell in the microenvironment is obtained using graph neural networks or spatial transcriptomics algorithms;

[0072] (e) Output the result of step (d).

[0073] In some embodiments of the present invention, the transcriptome data is obtained using the sequencing method described in the second aspect.

[0074] This invention transforms the technical bottlenecks of traditional methods into technical advantages. Existing patents and literature typically obtain individual hair cells by disrupting the tight junction complex between hair cells and surrounding supporting cells using various methods. However, this junction is extremely robust and difficult to disrupt, and hair cells themselves are fragile and easily damaged, resulting in high operational challenges and low success rates. The method provided by this invention does not disrupt this tight junction, but cleverly utilizes its stability to anchor the hair cell, thereby achieving efficient and low-damage acquisition of individual hair cells.

[0075] This invention innovatively integrates electrophysiological techniques with molecular biology methods to obtain high-quality single-cell samples and data characterization while ensuring cell activity and function.

[0076] The technical solution is to use a micromanipulation system to precisely locate the target single cell in the cochlear tissue (when the target single cell is an outer hair cell, it can be selected after gentle and rapid enzymatic hydrolysis) and achieve in situ non-destructive capture.

[0077] Compared with existing technologies (such as flow cytometry sorting after enzymatic tissue digestion or electrode aspiration), the present invention has the following significant advantages and beneficial effects:

[0078] 1. In-situ, efficient acquisition of single cells from cochlear tissue. This invention successfully solves the problem of directly and efficiently acquiring single cells in situ within cochlear tissue using glass microelectrodes. Figure 1 and Figure 2 This process addresses the technical challenges of subsequent RNA sequencing (RNA-seq). It has low dependence on enzyme digestion, the steps are easily standardized and highly reproducible, and it can reliably obtain high-quality single-cell samples for subsequent analysis.

[0079] 2. Expanding the application of this method in multiple scenarios in cochlear research. The single cells obtained using this method retain the original spatial location information of the target cells, greatly facilitating in-depth research on gene expression differences among different cell individuals within the same cell type (i.e., intercellular heterogeneity). Figures 3-7 This method can also be used for unknown cell types with well-defined spatial locations but not yet fully characterized, providing unprecedented single-cell resolution for revealing cellular functional diversity and regulatory mechanisms. Simultaneously, this method can also be used to obtain cells with specific fluorescent tags, aiding in drug development and gene therapy.

[0080] 3. Integrating molecular information and morphological characteristics: During the capture of target cells, in situ microscopic imaging can be used to simultaneously capture the three-dimensional morphology of cells, the biophysical properties of cell membranes, and the connections between cells in complex organs. This is beneficial for constructing a multi-dimensional map of "spatial location-morphological characteristics-transcriptome information", which empowers the analysis of developmental, functional and disease mechanisms and drives the precise design of regeneration and gene therapy.

[0081] 4. Captures high-quality target cells: This technology simplifies the tissue digestion process, eliminating mechanical pipetting, and achieves gentle and efficient single-cell acquisition. This method significantly reduces cell damage and stress levels, ensuring the acquisition of highly viable single cells. Figures 8-10 It is particularly well-suited for complex transcriptome analysis needs, providing a highly reliable data foundation for subsequent research.

[0082] 5. This method has significant scalability. It has been extended to other cell types within the cochlea; by adjusting parameters such as electrode diameter and suction force, it can be adapted to different cell types and states, demonstrating good universality and expansion potential. It can be widely applied to single-cell transcriptomics research on other types of receptor cells and various rare tissues, providing a powerful tool to overcome sample scarcity limitations and deeply analyze these key cell types.

[0083] The present invention uses a common pressure gauge with a range of 0 ~ 0.1 MPa (gauge pressure) and an accuracy class of 2.5.

[0084] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0085] The reagents and raw materials used in this invention are all commercially available.

[0086] The significant advantages of this invention are: the method provided can obtain high-quality cochlear-related cells in situ, and the obtained single cells can be further used for deep sequencing analysis. The method provided by this invention can be used for basic research and precise diagnosis and treatment of hearing impairment in clinical practice, and has promising application prospects. Detailed Implementation

[0087] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0088] Example 1: Obtaining a single cochlear cell

[0089] The methods for obtaining single cochlear cells are shown in Table 1.

[0090] Table 1. Methods for obtaining single cochlear cells

[0091]

[0092] The operating steps are as follows:

[0093] 1. Cochlear tissue pretreatment

[0094] Remove the cochlea from mice (P5-P15), remove the bone layer in pre-cooled (4℃) PBS, and quickly dissect the organ of Corti in the cochlea.

[0095] As shown in Table 1, the organ of Corti of the cochlea was placed on a round coverslip and gently pressed with an insect needle fixed at one end to stabilize the tissue. If the target cells required enzymatic digestion, they were quickly transferred to 100 μL of preheated (37°C) enzymatic digestion solution and incubated at 37°C and 200 rpm for 8-10 min with shaking. The digestion was then terminated by perfusion with room temperature cochlear extracorporeal fluid.

[0096] The above-mentioned enzymatic hydrolysate is ACCUMAX® CELL DISSOC MEDIUM (Thermo / Life / Invitrogen, #00-4666-56) containing 0.5 mg / mL, 1 mg / mL, 5 mg / mL or 10 mg / mL Collagenase IV (Sigma-Aldrich, #C5138), 1 U / mL DNase I (Thermo Scientific, #EN0521), and 7.5 mMCaCl2.

[0097] The cochlear extracorporeal fluid composition is as follows: 144 mM NaCl, 0.7 mM NaH2PO4, 5.8 mM KCl, 1.3 mM CaCl2, 0.9 mM MgCl2, 10 mM HEPES and 5.6 mM D-glucose (300-302 mOsm, pH = 7.40, 24℃), and all raw materials are from Sigma.

[0098] When the concentration of Collagenase IV (Sigma-Aldrich, #C5138) in the enzymatic hydrolysate was 0.5 mg / mL or 1 mg / mL, no significant changes were observed in the morphological observation of the organ of Corti in the cochlea, and the cells still maintained a tight junction structure; however, a high concentration of 10 mg / mL led to a significant deterioration in the cell state.

[0099] 2. Target cell localization and capture

[0100] The digested or undigested organ of Corti was placed on a micromanipulation stage (integrated with an upright microscope imaging system and micromanipulator), and continuously perfused with room temperature, oxygenated cochlear extracorporeal fluid. A borosilicate glass electrode (BF 100-50-10 (SUTTER INSTRUMENT), drawn using a P-97 Micropipette Puller (SUTTER INSTRUMENT), with a tip diameter of 1-3 μm, was used. 1-2 μL of PBS solution filtered through a 0.22 μm filter was perfused into the electrode. The following procedures were performed:

[0101] (1) Spatial localization: Refer to the anatomical atlas of the organ of Corti in the cochlea, identify the target cells (such as the outer hair cells in the third row of the apical gyrus) under a 60× objective lens and differential interference optical path; or use the fusion of fluorescent labeling signals for precise localization.

[0102] (2) Fine separation: According to the target cell type, as recorded in Table 1, apply the corresponding positive pressure with a borosilicate glass electrode of the corresponding tip diameter to gently and repeatedly detach the target cell body, or use negative pressure to selectively absorb non-target cells to achieve physical separation between the target cell and the surrounding cells.

[0103] (3) Single cell capture (absorption): The electrode contacts the target cell membrane (the membrane body around the cell nucleus), and the positive pressure (0.01 MPa) is released instantaneously and switched to negative pressure. The negative pressure and positive pressure range are consistent, and the cell is slowly aspirated into the electrode cavity. The electrode position and suction force are dynamically adjusted to keep the electrode tracking the cell body at all times, ensuring good sealing between the electrode and the cell.

[0104] Pressure control can be achieved by suction and blowing through a soft tube connected to the electrode (a common practice in electrophysiology experiments) or by applying pressure with a syringe. The negative pressure can be increased appropriately when aspirating cell nuclei or improving cell entry efficiency. A microscopic imaging system can record the entire procedure, facilitating subsequent review to assess the integrity of cell aspiration and the presence of exogenous / nearby cell contamination, thus aiding in determining whether to proceed with the more costly and lengthy sequencing experiments.

[0105] Specifically, the operational procedures for (2) fine separation and (3) single-cell capture of cochlear hair cells are as follows:

[0106] Positive pressure blowing: During the blowing process, the focal plane of the microscope is dynamically adjusted to clearly identify the elongated and easily overlooked supporting cells; the blowing continues until the boundary between the hair cells and the adjacent supporting cells is completely clear and physical separation is achieved.

[0107] Aspiration: Begin aspiration from the side furthest from the tight junctions, initially drawing in a portion of the cell body. Then, slightly move the electrode towards the tight junctions, ensuring the electrode remains directly aligned with the cell and reducing the pull on the cell. Continue aspiration, repeating the process. Significant resistance will be encountered when aspirating to the cell nucleus; the negative pressure can be increased appropriately. Once most of the cell has entered the electrode, slowly withdraw it. The cellular portion within the electrode gradually separates from the remaining portion until complete separation, at which point the cell membrane automatically seals. Ultimately, the electrode should contain most or all of the cell, while the tight junction region and surrounding supporting cells remain in place.

[0108] The procedure for (2) fine separation and (3) single-cell capture of cochlear outer hair cells is as follows:

[0109] After weak enzymatic hydrolysis, the cell bodies of the outer hair cells are freed, and the tight junctions on the epidermal plates remain intact, allowing for direct aspiration using a glass electrode without the need for blowing. If the enzymatic hydrolysis step is skipped, the procedure for inner hair cells is the same: positive pressure is applied to each outer hair cell individually until it can be easily aspirated. The aspiration steps are the same as for inner hair cells.

[0110] For other cochlear cells (inner marginal cells, interdental cells, and regenerating hair cells), the blowing and suction steps are the same as those for the inner hair cells, as described in Table 1.

[0111] 3. RNA Integrity Assurance System

[0112] Pre-operation treatment: UV sterilization of the operating environment for 30 min, and dry heat sterilization of the glass electrode at 200℃ for 2 h; dry heat sterilization is not only for sterilization, but also to remove RNase and prevent RNA degradation.

[0113] Protective measures during operation: Operators should wear sterile masks, clean gowns, and powder-free gloves;

[0114] Pressure barrier: A positive pressure of 0.01 MPa is continuously applied before the electrode contacts the cell to form a fluid protective layer;

[0115] Rapid single-cell collection: After cell capture, the electrode was quickly attached to a silicone tube and then inserted into a 0.2 mL PCR tube pre-loaded with lysis buffer (containing 20 U / μL RNase inhibitor). After confirming under a stereomicroscope that the electrode tip was immersed in the solution, positive pressure was applied to expel the electrode contents. The positive pressure was then quickly removed, and the electrode was rapidly withdrawn to prevent the introduction of air bubbles at the liquid-phase junction. The sample was placed on ice, and reverse transcription was initiated within 30 min.

[0116] Example 2

[0117] 1. Differential Interference Imaging

[0118] like Figure 1 and Figure 2As shown, this invention enables the direct acquisition of specific cochlear hair cells with precise anatomical location information within the cochlear tissue microenvironment. After enzymatic hydrolysis, the outermost layer of the three outer hair cells (dashed line) is in a suspended state with no supporting cells around it; the tight junctions at the top of this layer remain intact (arrow), and the hair cells are preserved in situ. Figure 1 After enzymatic hydrolysis and blowing, the two inner hair cells remained in situ but were in a free state (dashed line). The inner hair cells on the right side of these two cells remained tightly arranged (arrow). The inner and outer hair cells were in good condition, exhibiting a full three-dimensional morphology. Figure 2 ).

[0119] 2. High cell resolution and reproducible verification

[0120] Table 2 and Figures 3-7 To verify the high cell resolution and reproducibility of the obtained single-cell data.

[0121] 2.1 Housekeeper Genetic Testing

[0122] The captured cells were subjected to mouse housekeeping gene detection (for mouse housekeeping genes, please refer to the database HRT Atlas v1.0: redefining human and mouse housekeeping genes and candidate reference transcripts by mining massive RNA-seq datasets; database information can be found on the webpage housekeeping.unicamp.br).

[0123] As shown in Table 2, the high proportion of captured cell housekeeping genes demonstrates that the cochlear hair cell capture protocol (outer hair cells OHC and inner hair cells IHC) has extremely high mRNA capture efficiency and library construction quality with very low technical noise.

[0124] Table 2. Proportion of housekeeping genes in single cells

[0125]

[0126] 2.2 Pearson Correlation Analysis

[0127] In the single-cell sequencing data analysis workflow described in this invention, to quantitatively assess the similarity of gene expression patterns among individual cells of a specific cell category (e.g., outer hair cells or inner hair cells) within the experimental group, thereby verifying the homogeneity of the population at the transcriptome level, Pearson correlation coefficient analysis is performed using R language (version 4.4.2). The specific implementation method is as follows: First, the expression matrix of the target cell category is extracted from the single-cell sequencing data and exported, where rows represent cells and columns represent genes. The expression matrix is ​​then standardized (e.g., logarithmic transformation based on CPM) and high-quality genes (e.g., only genes in the list of highly variable genes are retained) to construct a core data frame for correlation analysis. Subsequently, the cor() function is used in the R environment, with the parameter set to method = The "pearson" function transposes the data frame and calculates the Pearson correlation coefficient between each pair of cells across all selected gene expression vectors, resulting in a symmetric inter-cell similarity matrix. To further assess the statistical significance of this correlation, the `cor.test()` function can be used to perform hypothesis testing on randomly sampled cell pairs or specific comparison groups to obtain p-values ​​and confidence intervals. Finally, the results are presented through visualization (e.g., using the `corrplot` package to create a correlation heatmap). If the inter-cell correlation coefficient is significantly close to 1, it indicates that the treated cell population has high transcriptomic consistency, providing crucial quantitative evidence for the core conclusions of this invention.

[0128] like Figure 3 and Figure 4 The Pearson correlation coefficient analysis of the cochlear outer hair cells and inner hair cells shown in the figure demonstrates that the high correlation of the same type of cells directly reflects the high fidelity and reproducibility of this technique.

[0129] 2.3 UMAP Dimensionality Reduction Visualization Analysis

[0130] In the single-cell sequencing data analysis described in this invention, to visualize and verify the clustering similarity of cells of the same type and the distribution differences of cells of different types, UMAP (Uniform Manifold Approximation and Projection) dimensionality reduction analysis is performed using R language. The specific method is as follows: First, based on the single-cell gene expression matrix after quality control and standardization, the NormalizeData() and FindVariableFeatures() functions in the Seurat package are used for data standardization and screening of highly variable genes. Then, the ScaleData() function is used for centering and scaling to eliminate technical bias. Next, using the highly variable genes obtained from the screening as input features, the RunUMAP() function in the Seurat package is used for dimensionality reduction calculation. The key parameters are set as: n_neighbors = 5, min_dist = 0.3; Finally, the coordinates of all cells in the two dimensions of UMAP1 and UMAP2 are generated. The coordinate data is visualized as a UMAP scatter plot using the ggplot2 package, and the points are color-coded according to the category labels in the cell metadata. If cells of the same cell type significantly cluster in the UMAP space to form independent clusters, while cell clusters of different cell types are clearly separated, it indicates that the cell type has high consistency in transcriptomic characteristics and is clearly distinguishable from other cell types. This provides intuitive dimensionality reduction visualization evidence for the accuracy of cell classification and the specificity of treatment effects in this invention.

[0131] like Figure 5 The UMAP dimensionality reduction visualization shows that different cell types (such as external hair cells (OHC) and internal hair cells (IHC)) form well-defined and separated independent clusters in two-dimensional space, proving that the above method can effectively capture cell-specific gene expression profiles and significantly distinguish heterogeneous cell populations.

[0132] 2.4 Detection of cell marker gene expression

[0133] Two cell populations (n=8) were formed from single cochlear outer hair cells (n=4) and single cochlear inner hair cells obtained by the method in Example 1, and single-cell sequencing data were performed. The expression of the outer hair cell marker gene Slc26a5 and the inner hair cell marker gene Slc17a8 in the two cell populations are as follows: Figure 6 and 7 As shown.

[0134] It is evident that the above method has high cell resolution and reproducibility.

[0135] 2.5 Analysis of the distribution of mitochondrial gene content in cells

[0136] Figure 8 The distribution of mitochondrial gene content in cells is shown. It is evident that all captured cells obtained using the above method are below the quality threshold (dashed line), indicating a low cell damage rate.

[0137] 2.6 Analysis of Cellular Stress Level

[0138] In the single-cell sequencing data analysis described in this invention, to quantitatively assess the biological response of different cell samples under stress, R language is used to calculate and statistically analyze cell stress scores. The specific implementation method is as follows: First, based on a predefined mouse stress gene set ("Hspa1a", "Hspa1b", "Hsp90aa1", "Hspd1", "Dnajb1", "Hsph1", "Fos", "Jun", "Atf3", "Ddit3", ... The dataset was filtered to include a list of available stress genes ("Xbp1"). Then, the AddModuleScore() function from the Seurat package was used to calculate a comprehensive stress score (stress_score1) for each individual cell, based on the expression levels of these stress genes. This score was obtained by calculating the average expression level of the feature gene set and correcting for background gene expression levels. To compare the differences in stress levels among different samples, the VlnPlot() function was used to plot a violin plot of the stress score distribution by sample group. The plot clearly shows the distribution range, median, and dispersion of the stress scores for each experimental group. This analysis directly reveals the differences in stress states among different cell populations, providing an important quantitative indicator for assessing the impact of external stimuli on cellular physiological states, and confirming that the experimental treatment in this invention can significantly regulate the stress response level of cells.

[0139] Figure 9 and Figure 10 The stress score and the expression levels of cellular stress genes (Dnajb1, Xbp1, Fos, Hsp90aa1, Ddit3, Hsph1, and Jun) are shown, respectively. It is evident that the cell population stress score obtained using the method of this invention is low. Figure 9 And the expression level of cellular stress genes is low. Figure 10 This demonstrates that the above method can yield a large number of high-quality, low-stress intact cells.

[0140] like Figures 8-10 As shown, the obtained single cells exhibited low stress levels and high cell quality.

[0141] In summary, the above method achieves the acquisition of complete single cells, which can be directly used for subsequent RNA sequencing (RNA-seq). The obtained single-cell RNA data completely preserves the original state information of the target cells, greatly facilitating in-depth research on gene expression differences (i.e., intercellular heterogeneity) among different cell types within the same cell type.

Claims

1. A method for in-situ separation of target single cells based on a patch-clamp system, characterized in that, The method includes contacting the target single cell with an electrode under a microscope and grasping the target single cell in situ by negative pressure adsorption. The target single cell, as described above, is kept connected to its surrounding cells during in-situ grasping. Before the electrode approaches the target single cell, a positive pressure is continuously applied. When the electrode contacts the surface of the target single cell, the positive pressure is instantly stopped and switched to a negative pressure.

2. The method as described in claim 1, characterized in that, The target single cell is a cell on the organ of Corti in the cochlea, such as a cochlear hair cell, inner marginal cell, interdental cell, or regenerating hair cell. Preferably, the cochlear hair cells are inner hair cells and / or outer hair cells; and / or, the target single cell is derived from mice. .

3. The method as described in claim 1 or 2, characterized in that, The negative pressure adsorption pressure is 0.01-0.05 MPa; and / or, The positive pressure is 0.01-0.05 MPa, preferably 0.01-0.02 MPa; and / or, The electrode is a borosilicate glass electrode or a glass electrode; and / or, The tip diameter of the electrode is 1-5 μm; preferably, the tip diameter of the electrode is 1-4 μm.

4. The method according to any one of claims 1-3, characterized in that, The in-situ grasping process does not include an enzymatic digestion step.

5. The method according to any one of claims 1-3, characterized in that, When the target single cell is an outer hair cell, the in situ grasping process includes a slight enzymatic hydrolysis step; after the slight enzymatic hydrolysis, the target single cell still maintains its connection with the surrounding cells; Preferably, the slight enzymatic hydrolysis includes contacting the organ of Corti in the cochlea with an enzyme-containing composition comprising type IV collagenase and CELL DISSOC MEDIUM, wherein the CELL DISSOC MEDIUM is derived from Thermo / Life / Invitrogen. More preferably, for every 1 mL of CELL DISSOC MEDIUM contained in the enzyme-containing composition, the content of type IV collagenase is 3-8 mg, preferably 4-6 mg; and / or, The enzyme-containing composition further comprises deoxyribonuclease I; preferably, the content of deoxyribonuclease I is 0.5-2 U per 1 mL of CELL DISSOC MEDIUM in the enzyme-containing composition; and / or, The enzyme-containing composition further comprises CaCl2; preferably, the CaCl2 content is 5-10 mM for every 1 mL of CELL DISSOCMEDIUM contained in the enzyme-containing composition; and / or, The enzyme-containing composition is provided in the form of a premixed lyophilized powder or a ready-to-use liquid.

6. The method as described in claim 5, characterized in that, The conditions for the slight enzymatic hydrolysis meet one or more of the following: The time for slight enzymatic hydrolysis is 5-20 minutes; preferably, the time for slight enzymatic hydrolysis is 8-15 minutes. The temperature for the mild enzymatic hydrolysis is 32-38°C; preferably, the temperature for the mild enzymatic hydrolysis is 35-36°C; and, The oscillation speed for the slight enzymatic hydrolysis is 150-300 rpm; preferably, the oscillation speed for the slight enzymatic hydrolysis is 180-220 rpm.

7. The method according to any one of claims 1-6, characterized in that, The in-situ grasping process also includes a step of separating non-target cells; Preferably, the separation is performed using an electrode as defined in the method of claim 3; and / or, The separation is achieved by positive pressure blowing away and / or negative pressure peeling away non-target cells, wherein the pressure of the positive pressure blowing away and / or negative pressure peeling away is preferably 0.001-0.1 MPa, more preferably 0.001-0.04 MPa; and / or, The separation was performed under a microscope.

8. The method according to any one of claims 1-7, characterized in that, The in-situ grasping and / or the slight enzymatic hydrolysis are further preceded by a pretreatment step; the pretreatment involves removing the bone layer and peeling off the organ of Corti of the cochlea after the cochlea is removed. Preferably, the bone layer is removed under the following conditions: a temperature of 0-4°C; and / or, the bone layer is removed in a liquid, such as PBS buffer.

9. A single-cell sequencing method, characterized in that, The sequencing method includes: S1: Obtain the single cell by the method according to any one of claims 1-8; S2: Sequencing; Preferably, S2 involves extracting RNA from the single cell, constructing a library, and performing high-throughput sequencing to obtain transcriptome data; the high-throughput sequencing is preferably based on any of the following platforms: Illumina NovaSeq 6000, Illumina HiSeq4000, Illumina NextSeq 2000, BGI MGISEQ-2000, and Element AVITI.

10. A method for data analysis after single-cell sequencing, characterized in that, The method includes: (a) Obtaining a single cell by the method of any one of claims 1-8, and recording the spatial coordinates of the single cell in the original tissue; (b) Obtain the transcriptome data of the single cell obtained in (a); preferably obtained by the sequencing method described in claim 9; (c) Correlate the spatial coordinates of the transcriptome data (a) from (b) to generate an expression matrix that includes spatial locations; (d) Based on the expression matrix of step (c), the functional state of the single cell in the microenvironment is obtained using graph neural networks or spatial transcriptomics algorithms; (e) Output the result of step (d).

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