Single cell capture device and method

Abstract

The application provides a single cell capturing device and method based on a glass microelectrode, comprising a simple single cell capturing device, a contamination removal scheme and a transcriptome sequencing pre-quality control scheme. The single cell capturing device comprises a power system, a micro-operation system and a microscope system. The device is simple and effective, and can be built on most experimental platforms. The contamination removal scheme effectively removes the tissue cells adhered to the outer wall of the glass microelectrode to improve the single cell sample preparation quality. The transcriptome sequencing pre-quality control scheme provides a method for detecting the contamination degree of the captured single cell. In summary, the application provides a simple single cell capturing device, and provides a unique contamination removal method and transcriptome sequencing pre-quality control scheme, and provides a new scheme for obtaining high-quality cells for downstream single cell analysis test.

Description

Technical Field

[0001] This invention belongs to the field of cell and molecular biology, and specifically relates to a single-cell capture device and method. Background Technology

[0002] In recent years, the rapid rise of single-cell omics technology has greatly promoted research progress in various fields of life sciences and medicine. The first key step in single-cell omics technology is obtaining individual cells. Currently, there are several mature technologies, including flow cytometry, manual sorting, laser microdissection, and patch-seq based on patch-clamp systems. Among them, flow cytometry relies on preparing a single-cell suspension and then further detecting and sorting cells with special labels in batches. This method has the advantages of high throughput and high reproducibility, but its disadvantage is that it is only suitable for cell samples with a high content of target cells. Manual sorting also requires the preparation of a single-cell suspension, and then the target cells are aspirated one by one under a microscope using a capillary glass tube. Compared with flow cytometry, its throughput is reduced, but its accuracy is improved. It allows for human judgment of the target, but again, this method is only suitable for cell samples with a high content of target cells. Laser microdissection technology is suitable for tissue sections. By using a laser to cut around the target cells on a two-dimensional tissue section, a single cell is captured. This technology also has the characteristics of high accuracy, but it requires a sufficient number of target cells to reduce the difficulty of sectioning, and its throughput is also relatively low. The patch-seq approach based on the patch-clamp system involves sealing target cells with high resistance and recording electrical signals, then aspirating intracellular contents to obtain the cells. This method can correlate electrical signals, neuronal morphology, and sequencing information. However, it also suffers from low throughput, and the patch-clamp technique is technically demanding and the entire system is complex, making it somewhat redundant for non-neuronal cells or cells for which electrical signals and morphology recording are not required. Furthermore, for in vivo capture of single cells, there are very few successful implementations of the patch-seq approach based solely on the patch-clamp system. A major problem with this method is the potential for introducing tissue cellular components other than the target cells, severely contaminating the already scarce single-cell samples and interfering with subsequent experiments. Although the cost of single-cell sequencing is gradually decreasing, for single-cell sequencing experiments requiring large sample volumes, the cost remains high if the proportion of low-quality cells is high.

[0003] Specifically, currently reported patch-seq single-cell capture methods are based on recording the electrical signals of neurons using a patch-clamp system, followed by applying negative pressure to aspirate the contents of individual cells. Most published patch-seq studies capture individual neurons after recording their electrical signals for high-throughput sequencing. However, sequencing analysis reveals that many cell samples contain non-target cell components. The usual practice is to remove highly contaminated samples after sequencing analysis. Although high-throughput sequencing significantly reduces costs, the large sample size of single-cell sequencing means that even a low proportion of contaminated cells can still increase research costs. Furthermore, patch-clamp-based single-cell capture systems are structurally complex, making them unsuitable for laboratories lacking such systems, and thus limiting their versatility.

[0004] In addition, some research groups study cells that are extremely rare, especially individual cells mixed in with tissues. How to capture high-quality single rare cell types remains a major challenge.

[0005] Therefore, the present invention provides a simple single-cell capture device and a method for obtaining high-quality single rare cells, while also providing a contamination removal and pre-sequencing quality control scheme, which can greatly reduce the cost of single-cell sequencing experiments. Summary of the Invention

[0006] The purpose of this invention is to provide a more versatile single-cell capture device and method, particularly for in vivo capture of single rare cell types based on glass microelectrodes. This method for in vivo capture of single rare cells improves the quality of captured single cells and reduces the cost of single-cell sequencing.

[0007] To achieve the above objectives, the present invention provides a single-cell capture device.

[0008] In one embodiment, the single-cell capture device includes a power system, a micromanipulation system, and a microscopic system. The power system includes: first and second medical syringes, a three-way valve, a rubber hose, an electrode holder, and a glass microelectrode; the micromanipulation system includes: a micromanipulation controller, a micromanipulation control center, and a micromanipulation robotic arm; the microscopic system includes: a fluorescence microscope, a CCD camera, and a computer.

[0009] The 1mL or 10mL medical syringe of the power system is connected to the first port of the three-way valve to provide power for cell aspiration. The first and second medical syringes can both be 1mL medical syringes, or the first medical syringe is a 1mL medical syringe and the second medical syringe is a 10mL medical syringe.

[0010] The second port of the three-way valve of the power system is closed, and the third port is connected to one end of the rubber hose to control the air pressure inside the power system.

[0011] The other end of the rubber hose of the power system is connected to the electrode holder, which serves as a connection.

[0012] The electrode holder of the power system is connected to the glass microelectrode and is used to fix the glass microelectrode.

[0013] The glass microelectrode of the power system is used to aspirate cell contents. The glass microelectrode is an electrode drawn by a three-step process, and the tip of the finished electrode has an opening of 2-3 μm.

[0014] The micro-operation system's micro-operation controller is connected to the micro-operation control center and is used to transmit position movement commands to the micro-operation control center.

[0015] The micro-operation control center of the micro-operation system is connected to the micro-operation robotic arm and is used to process position movement signals and output them to the micro-operation robotic arm.

[0016] The micro-manipulation system's micro-manipulation robotic arm is connected to an electrode holder to provide the power for movement and to carry glass microelectrodes to achieve micron-level displacement.

[0017] The fluorescence microscope of the microscopy system is connected to a CCD camera to magnify tissue cells. It can also excite fluorescent substances in cells or electrode tips through filters of different wavelengths and transmit the information to the CCD camera.

[0018] The CCD camera of the microscopy system is connected to a computer to convert the light signal from the fluorescence microscope into an electrical signal and transmit it to the computer.

[0019] The electronic computer of the microscopic system is used to process the electrical signals transmitted by the CCD camera, which can be converted into image information and displayed on the computer's monitor.

[0020] On the other hand, the present invention provides a method for obtaining high-quality single cells.

[0021] The protocol includes a single-cell capture protocol, a contamination removal protocol, and a pre-transcriptional sequencing quality control protocol.

[0022] The single-cell capture protocol provides a method for capturing a single rare cell using the in vivo capture device of this application.

[0023] The contamination removal solution provides a unique method for removing adhering tissue cells from the outer wall of a glass microelectrode. Specifically, the tip of the glass microelectrode, which is part of a device for capturing single rare cells in vivo, is repeatedly immersed, for example, about 10 times, in a sterile, enzyme-free container (e.g., a PCR tube, a commercially available RNase-free product, etc.) containing a reagent for lysing cells, such as an alkaline solution, preferably a 50 mM sodium hydroxide solution, to effectively remove the adhering tissue cells from the outer wall of the glass microelectrode.

[0024] The pre-transcriptional sequencing quality control protocol provides a method for detecting the proportion of non-target cells mixed in with captured cells. This method includes cDNA integrity testing and specific gene expression level testing.

[0025] The cDNA integrity detection in the pre-transcriptional sequencing quality control protocol is a detection of cDNA length and content distribution. It is not limited to using a bioanalyzer 2100 combined with a high-sensitivity DNA detection kit to detect cDNA libraries obtained by smart-seq of mRNA.

[0026] The specific gene expression level detection in the pre-transcriptional sequencing quality control protocol uses real-time quantitative PCR to detect the expression levels of target cells and non-target cells specific genes in the cDNA library. If the cells have no or very few non-target cell components, the detected proportion of non-target cell specific gene expression levels should be extremely low.

[0027] In the specific gene expression level detection of the transcriptome sequencing pre-sequencing quality control protocol, the identification of non-target cells can be achieved by analyzing the tissue cells that the target cells may encounter along the path they take when the analytical electrode contacts them.

[0028] The specific genes in the specific gene expression level detection of the aforementioned transcriptome sequencing pre-sequencing quality control protocol can be determined based on literature and database surveys.

[0029] The ratio of target cell to non-target cell specific gene expression in the specific gene expression level detection of the pre-transcriptional sequencing quality control protocol can be converted by the following formula:

[0030]

[0031] Wherein, N1, N2, and N3 represent the expression levels of target cell and non-target cell specific genes, respectively, and cq1, cq2, and cq3 represent the number of PCR cycles required for the fluorescence intensity of the corresponding gene to reach the threshold. In the specific gene expression level detection of the aforementioned transcriptome sequencing pre-sequencing quality control protocol, it is recommended to remove cells with a non-target cell specific gene ratio exceeding 5%.

[0032] Specifically, this application adopts the following technical solution:

[0033] 1. A single-cell capture device, characterized in that it comprises a power system, a micromanipulation system, and a microscopic system.

[0034] The power system includes: a first medical syringe and a second medical syringe; a three-way valve with its second port closed and its first port connected to either the first or second medical syringe as needed; a rubber hose connected to the third port of the three-way valve; an electrode holder sealed to the other end of the rubber hose; and a glass microelectrode sealed to the other end of the electrode holder.

[0035] The micro-operation system includes: a micro-operation controller connected to a micro-operation control center for transmitting position movement commands to the micro-operation control center; a micro-operation control center connected to a micro-operation robotic arm for processing position movement signals and outputting them to the micro-operation robotic arm; and a micro-operation robotic arm, one end of which is connected to an electrode holder and the other end of which is connected to the micro-operation control center to execute movement commands and carry glass microelectrodes to achieve micron-level displacement.

[0036] The microscopic system includes a fluorescence microscope, a CCD camera, and a computer. The CCD camera is fixed to a designated interface of the fluorescence microscope and connected to the computer via a data transmission line. The computer processes the electrical signals transmitted by the CCD camera, and after processing, the images observed under the fluorescence microscope can be converted into image information and displayed on the computer's monitor.

[0037] 2. A method for capturing single cells using the single-cell capture device described in item 1, characterized by comprising the following steps:

[0038] Connect the first medical syringe to the first port of the three-way valve.

[0039] The glass microelectrode is brought into the liquid surface of a solution containing single cells.

[0040] By adjusting the first medical syringe through the eyepiece of the fluorescence microscope, the solution inside the electrode flows out. Under the monitoring of the microscopic system, the glass microelectrode is controlled by a micromanipulator to adhere tightly to the cell membrane of the target single cell.

[0041] By pulling the first medical syringe propeller, a negative pressure is created inside the power system, drawing the cell contents into the glass microelectrode.

[0042] Disconnect the first medical syringe from the three-way valve and close the first port of the three-way valve.

[0043] Move the glass microelectrode to remove it from the liquid surface.

[0044] The tip of the glass microelectrode is filled with a solution containing a recombinant RNase inhibitor and a fluorescent dye, such as Alexa Fluor 594.

[0045] 3. The method according to item 2, characterized in that it further includes connecting the second medical syringe to the first port of the three-way valve, and adjusting the three-way valve to connect the first and third ports, thereby pushing the propeller of the second medical syringe to transfer the cell contents aspirated into the glass microelectrode.

[0046] 4. The method according to item 2, characterized in that it further includes repeatedly immersing the glass microelectrode tip removed from the liquid surface in item 2 with a reagent containing cell lysis, such as an alkaline solution, preferably a 50 mM sodium hydroxide solution, to remove adhering tissue.

[0047] 5. A method for obtaining high-quality target cells, characterized by comprising the following steps:

[0048] (1) Using the single-cell capture device of item 1 to capture cells, wherein the glass microelectrode in the single-cell capture device contains single-cell contents;

[0049] (2) The glass microelectrode tip in step (1) is repeatedly soaked with a reagent containing cell lysis, such as an alkaline solution, preferably a 50 mM sodium hydroxide solution;

[0050] (3) Detect the integrity of cDNA obtained by reverse transcription of the contents of a single cell aspirated into the glass microelectrode in step (2).

[0051] (4) Detect the expression levels of specific genes in target cells and non-target cells;

[0052] (5) Calculate the ratio of specific gene expression in target cells to non-target cells using the following formula;

[0053]

[0054] Where N1, N2, and N3 represent the expression levels of target-cell and non-target-cell specific genes, respectively, and cq1, cq2, and cq3 represent the number of PCR cycles required for the fluorescence intensity of the corresponding gene to reach the threshold.

[0055] (6) Based on the calculation results, remove cells whose proportion of non-target cell specific genes exceeds the set proportion.

[0056] 6. The method according to item 5, characterized in that the detection of cDNA integrity is the detection of cDNA length and content distribution.

[0057] 7. The method according to item 5, characterized in that the detection of cDNA integrity is performed using a bioanalyzer 2100 and a high-sensitivity DNA chip detection kit.

[0058] 8. The method according to item 5, characterized in that the expression levels of target cells and non-target cells specific genes are detected by real-time quantitative PCR.

[0059] 9. The method according to item 5, characterized in that the threshold for the ratio of target cell to non-target cell specific gene expression is set based on the fact that the ratio of target cell specific gene expression exceeds 95%.

[0060] definition:

[0061] Fpkm: This is an abbreviation for Fragments per kilobase of exon model per million mapped fragments, which is a normalized metric used in next-generation sequencing to measure gene expression levels.

[0062] Beneficial technical effects

[0063] This invention provides a technique for capturing single rare cells in vivo, which greatly simplifies single-cell capture systems based on patch-clamp systems. It also adds a contamination removal step and provides a lower-cost pre-transcriptional sequencing quality control scheme, which not only improves universality but also reduces research costs, allowing laboratories without patch-clamp systems to easily build a platform for capturing cells. Attached Figure Description

[0064] Figure 1 This is a structural block diagram of the device for capturing single rare cells in vivo according to the present invention.

[0065] Figure 2 This is a schematic diagram of the device for capturing single rare cells in vivo according to the present invention. Wherein: 1-1mL syringe, 2-10mL syringe, 3-three-way valve, 4-rubber tubing, 5-electrode holder, 6-glass microelectrode, 7-microcontroller, 8-microcontroller control center, 9-microcontroller robotic arm, 10-microscope objective, 11-microscope stage, 12-CCD camera, 13-computer, 14-display, 15-Mouthna cell body.

[0066] Figure 3 A flowchart illustrating how to obtain high-quality single cells using the method of this invention.

[0067] Figure 4The following are statistical graphs used in Example 2 to evaluate the effectiveness of the contamination removal and pre-sequencing quality control protocols. Graph a represents the proportion of target and non-target cell-specific gene expression in the transcriptome sequencing results of 64 single-cell samples that were not screened using the contamination removal and pre-sequencing quality control protocols. Graph b represents the proportion of target and non-target cell-specific gene expression in 106 single-cell samples detected using qPCR, where the white dashed line corresponds to the 95% proportion, and the highlighted yellow cells represent the 9 cells with higher contamination that were removed after screening. Graph c represents the proportion of target and non-target cell-specific gene expression in the transcriptome sequencing results of 97 single-cell samples that were screened using the contamination removal and pre-sequencing quality control protocols. Detailed Implementation

[0068] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0069] Unless otherwise specified, the methods used in the following examples are conventional methods, and the chemicals and raw materials used in the following examples are commercially available or prepared by known methods unless otherwise specified.

[0070] The embodiments described here are mainly aimed at the in vivo capture of rare neurons—Mauthner cells—in zebrafish juveniles. However, the present invention is actually applicable to the capture of any cells that can be seen under a microscope, such as non-neuronal cells, cells of small transparent model organisms such as nematodes and fruit flies, and in the in vivo capture of large model animals such as mice and rats. The strong penetrating power of two-photon microscopy can be used to capture cells in regions deeper than 200 μm, as well as cultured cells in vitro. If fluorescent labels are present, they are easier to identify. If fluorescent labels are not present, phase contrast microscopy can be used in conjunction with experience to identify target cells. Therefore, the single-cell capture device of the present invention is feasible, reliable, and accurate in the capture of single cells.

[0071] Example 1

[0072] like Figure 1 A structural block diagram of a device for capturing single rare cells in vivo is shown, including a power system, a micromanipulation system, and a microscopic system.

[0073] Specific examples Figure 2As shown, the power system includes a 1mL medical syringe 1 and a 10mL medical syringe 2, a three-way valve 3, a rubber tubing 4, an electrode holder 5, and a glass microelectrode 6. The glass microelectrode 6 has a short neck, a large taper angle, and a tip diameter of approximately 2-3μm. The large taper angle facilitates tissue penetration and promotes cell aspiration into the electrode. Medical syringe 1 or the 10mL medical syringe 2 is connected to the first port of the three-way valve. The second port of the three-way valve 3 is closed, and the third port is connected to the rubber tubing 4. The second port of the rubber tubing 4 is connected to one end of the electrode holder 5. The other end of the electrode holder 5 is used to hold the glass microelectrode 6. The entire power system forms a closed, interconnected system, and the release or aspiration of substances from the glass microelectrode 6 can be controlled by the injector of the medical syringe.

[0074] The micro-manipulation system includes a micro-manipulation controller 7, a micro-manipulation control center 8, and a micro-manipulation robotic arm 9. Commands are transmitted from the micro-manipulation controller 7 to the micro-manipulation control center 8, which can control the micro-manipulation robotic arm 9, which has the ability to move up and down, forward and backward, left and right, and diagonally forward and backward. The micro-manipulation robotic arm 9 is connected to the electrode holder 5, thereby enabling precise control of the movement of the glass microelectrode 6 via the micro-manipulation controller 7.

[0075] The microscopy system includes a fluorescence microscope (which comprises a microscope objective 10 and a microscope stage 11), a CCD camera 12, and a computer 13. The CCD camera 12 is fixed to a designated interface on the fluorescence microscope 10 (usually provided by the microscope) and connected to the computer 13 via a data transmission cable. The fluorescence microscope can provide fields of view at different magnifications to locate target cells and monitor the position of glass microelectrodes, while the CCD camera 12 can transmit the fluorescence microscope image to the computer screen in real time to provide feedback on the position of the glass microelectrodes.

[0076] Example 2

[0077] The mortner cells captured in the embodiments of this invention express green fluorescent protein. The experimental animal is a transgenic strain of transparent zebrafish juveniles, and there are only two of these cells in each fish. These experimental animals were donated to Saitama University in Japan, and our research group has been breeding this strain of zebrafish for a long time.

[0078] In this embodiment, we elaborate on the method of capturing single rare cells in vivo according to the present invention by combining the method of capturing mortner cells of zebrafish juveniles with green fluorescent tags.

[0079] like Figure 2As shown, we fixed transgenic zebrafish larvae in homemade agarose culture dishes and used ophthalmic scissors to cut the meninges above the hairline cells to prevent the glass microelectrodes from puncturing the meninges and causing blockage. Then, we added an isotonic solution of MS-222 (ethyl 3-aminobenzoate methanesulfonate) anesthetic at a final concentration of 133-200 mg / L to the culture dish. While maintaining anesthesia, we provided the exposed cells with an appropriate osmotic pressure (osmotic pressure varies depending on the species and tissue type; here, DMEM F12 low-glucose cell culture medium was used). The specific component ratios in this cell culture medium provided a suitable pH (7.0-7.5) and osmotic pressure (320-340 mOsmol / kg) for viable cells. Theoretically, physiological saline, PBS, or artificial cerebrospinal fluid could also be used, mainly to prevent the cells from absorbing water and bursting.

[0080] The glass microelectrode tip was filled with approximately 0.3 μL of a solution containing a recombinant RNase inhibitor and a red fluorescent dye (such as Alexa Fluor 594) (Van den Hurk, Mark, et al. "Patch-seq protocol to analyze the electrophysiology, morphology and transcriptome of whole single neurons derived from human pluripotent stem cells." Frontiers in Molecular Neuroscience 11(2018):261.) to reduce RNA degradation after cell capture. The fluorescent dye also provided feedback for the position of the glass microelectrode tip and the internal pressure of the dynamic system. Since Morthena cells are labeled with green fluorescent protein, a red fluorescent dye was used, but not limited to red; the choice could be based on the fluorescent labeling type of the target cells. Before the glass microelectrode enters the liquid, a 1mL medical syringe is connected to the first port of a three-way valve. This creates a positive pressure within the power system. After the glass microelectrode enters the liquid, the outflow rate of the red dye excited by green fluorescence inside the microelectrode is observed through the microscope eyepiece. By adjusting the medical syringe, the red dye solution flows out at a slower rate to prevent external contaminants from being drawn back into the microelectrode. The micro-manipulation controller guides the glass microelectrode into the juvenile fish brain tissue through the cut meninges. Simultaneously, the microscope stage is moved and the focus adjusted through the eyepiece to keep the glass microelectrode in the field of view. In this state, the glass microelectrode is slowly moved closer to the mortner cells. When the glass microelectrode is about to contact the mortner cells, the system switches to CCD imaging. The image previously seen through the eyepiece is transmitted to a computer monitor via the CCD camera. Using the magnification function of the CCD camera imaging software, the area where the glass microelectrode and mortner cells are about to contact is magnified. The glass microelectrode is then moved further until a tiny indentation is formed on the mortner cell membrane. At this point, by pulling the plunger of the medical syringe, a negative pressure is created inside the power system, drawing the cell contents into the glass microelectrode. The syringe is then immediately disconnected from the three-way valve to balance the internal pressure of the power system with atmospheric pressure, and the first port of the three-way valve is closed. Afterward, the glass microelectrode is moved to remove it from the brain tissue and out of the liquid.

[0081] Because glass microelectrodes may adhere to tissue cells on their outer wall as they move through brain tissue, directly transferring cells to the lysis buffer could result in these non-target cells contaminating the lysis buffer and causing significant contamination of single-cell samples. The contamination removal method of this invention uses an alkaline solution (50mM sodium hydroxide solution in this case) to repeatedly wet the electrode tip before cell transfer, effectively removing tissue cells adhering to the electrode outer wall. Specifically, manually controlling the PCR tube containing sodium hydroxide solution to repeatedly wet the electrode tip approximately 10 times effectively removes tissue cells from the electrode outer wall. Afterward, a 10mL medical syringe is connected to the first port of a three-way valve, and the three-way valve is adjusted to connect the first and third ports. By pushing the syringe plunger, the cells captured within the glass microelectrode are transferred to the lysis buffer. If necessary, the electrode tip can be gently folded into the lysis buffer.

[0082] To screen for heavily contaminated cells before conducting expensive high-throughput sequencing experiments, a pre-transcriptional sequencing quality control protocol was employed. Specifically, single cells collected in lysis buffer underwent reverse transcription and linear amplification using the Smart-seq2 strategy (Picelli, Simone, et al. "Full-length RNA-seq from single cells using Smart-seq2." Nature protocols 9.1 (2014): 171-181.). 1 μL of purified cDNA was then used to test cDNA integrity using an Agilent Bioanalyst 2100 with a high-sensitivity DNA detection chip kit. Cell samples with numerous small peaks and very low concentrations were discarded. To avoid wasting valuable cDNA libraries, 1 μL of cDNA from samples that passed the cDNA integrity test was used for a new round of linear amplification as a template for real-time quantitative PCR (qPCR). The core of the pre-transcriptional sequencing quality control protocol is to detect whether the single-cell cDNA library contains a large number of non-target cell-specific genes. Specifically, if the proportion of non-target cell-specific genes in the captured target cells is high, it indicates that the cell was contaminated with a large number of non-target cells during capture, and thus the cell is considered heavily contaminated. Conversely, if the proportion of non-target cell-specific genes is very low or undetectable, it indicates that the cell contains very few or no non-target cells. Which non-target cells are detected depends on the cellular composition of the site where the target cell is located and the cellular composition of the tissue crossed by the electrode during contact with the cell. The relative expression levels of target cell- and non-target cell-specific genes (genes expressed only in specific cell types, also known as marker genes) are detected by qPCR and calculated according to the following formula:

[0083]

[0084] Wherein, N1, N2, and N3 represent the expression levels of target cell and non-target cell specific genes, respectively, and cq1, cq2, and cq3 represent the number of PCR cycles when the fluorescence intensity of the corresponding gene reaches the threshold.

[0085] The CQ value is converted into a ratio of the relative expression levels of specific genes to filter out severely contaminated cells. The severity of contamination can be defined by setting a threshold. In this embodiment, we assess the degree of contamination by detecting the relative expression levels of specific genes in oligodendrocytes and hematopoietic cells. Oligodendrocytes are widely distributed cells in the central nervous system, and hematopoietic cells represent the circulatory system. Detection of other non-target cell types can be added here. Oligodendrocytes specifically express the mbpa gene, hematopoietic cells specifically express the hbae3 gene, and the gene specifically expressed by dermal cells is unknown, but investigations indicate that they definitely express the nefmb gene (which can be recognized by the 3A10 antibody). This gene is not expressed in either oligodendrocytes or hematopoietic cells; therefore, nefmb can be considered a heterologous gene expressed by dermal cells. The threshold is set at a relative expression rate of nefmb greater than 95%. Table 1 shows 10 cells tested (examples). The left side represents the original CQ value, and the right side represents the ratio converted to the relative expression level N using the formula. Cells 6-10 are the most severely contaminated cells. The original CQ value is just a numerical value, inversely proportional to the expression level. Since the expression levels of the same gene are not entirely identical across different cells, the CQ value only reflects the relative expression levels of different genes within the same sample. Converting it to a ratio relationship here provides a more intuitive view of the contamination level, and allows for the setting of a threshold to remove severely contaminated cells.

[0086] Table 1

[0087]

[0088] This invention also employed an external sequencing company to perform next-generation high-throughput sequencing, and the effectiveness of contamination removal and pre-sequencing quality control was experimentally tested. In 64 cells that did not undergo contamination removal and pre-sequencing quality control, a significant proportion of cells exhibited severe non-target cell contamination. In contrast, in 97 cells that underwent contamination removal and pre-sequencing quality control, almost all cells showed a very low proportion of non-target cell contamination. Table 2 shows the sequencing results of the 64 cells, with the left side representing the expression levels of target and non-target cell-specific genes, and the right side representing the relative expression levels of the three genes. Only 26.6% of the cells showed a nefmb expression rate exceeding 95%. Table 3 shows the sequencing results of the 97 cells, with the left side representing the expression levels of target and non-target cell-specific genes, and the right side representing the relative expression levels of the three genes. A staggering 92% of the cells showed a nefmb expression rate exceeding 95%, and no cells exhibited severe contamination.

[0089] Specifically, the contamination removal scheme targets contamination on the outer wall of the electrode, while pre-sequencing quality control targets the detection of total contamination, including contamination mixed into the electrode interior and contamination not completely removed from the outer wall of the electrode.

[0090] The effectiveness of the contamination removal scheme can be reflected by the results of two sequencing runs, namely Table 2 and Table 3. Table 2 shows the results without the contamination removal step, with a high proportion of non-target cell contamination (both in terms of the proportion of non-target genes in a single sample and the overall proportion of unqualified cells). Table 3 shows the results after contamination removal and pre-sequencing quality control steps, which shows a significant reduction in the proportion of non-target cell contamination.

[0091] Figure 4 Tables 2 and 3, along with a statistical chart of the contamination levels detected by qPCR, show that by comparing Figure a and Figure c, it can be seen that after adding the contamination removal and pre-sequencing quality control measures, the proportion of non-target cell-specific genes significantly decreased, which also represents a significant improvement in single-cell quality.

[0092] Table 2. Single-cell sequencing results after contamination removal and pre-sequencing quality control.

[0093]

[0094]

[0095]

[0096] Table 3 shows the single-cell sequencing results with contamination removal and pre-sequencing quality control.

[0097]

[0098]

[0099]

[0100] Therefore, this invention provides a simple device for capturing single cells and a method for obtaining high-quality single cells, including a contamination removal protocol and a pre-transcriptional sequencing quality control protocol. The unique feature of this invention is the simplification of the patch-seq device, greatly improving its versatility; it provides an unprecedented contamination removal protocol and a pre-transcriptional sequencing quality control protocol. While inheriting the "what you see is what you get" advantage of patch-seq, it provides a simpler single-cell capture device, contamination removal, and pre-transcriptional sequencing quality control protocol, improving single-cell quality and further reducing the cost of single-cell sequencing.

[0101] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for obtaining high-quality target cells, characterized in that, Includes the following steps: (1) Using a single-cell capture device to capture cells, wherein the glass microelectrode in the single-cell capture device contains single-cell contents; (2) Repeatedly wet the glass microelectrode tip from step (1) with 50 mM sodium hydroxide solution; (3) Detect the integrity of cDNA obtained by reverse transcription of the contents of a single cell aspirated into the glass microelectrode in step (2); (4) Detect the expression levels of specific genes in target cells and non-target cells; (5) Calculate the ratio of target cell to non-target cell specific gene expression using the following formula; Where N1, N2, and N3 represent the expression levels of target-cell and non-target-cell specific genes, respectively, and cq1, cq2, and cq3 represent the number of PCR cycles required for the fluorescence intensity of the corresponding gene to reach the threshold. (6) Based on the calculation results, remove cells whose proportion of non-target cell-specific genes exceeds the set proportion. The single-cell capture device includes a power system, a micromanipulation system, and a microscopic system. The power system includes: a first medical syringe and a second medical syringe; a three-way valve with its second port closed and its first port connected to either the first or second medical syringe as needed; a rubber hose connected to the third port of the three-way valve; an electrode holder sealed to the other end of the rubber hose; and a glass microelectrode sealed to the other end of the electrode holder. The micro-manipulation system includes: a micro-manipulation controller connected to a micro-manipulation control center for transmitting position movement commands to the micro-manipulation control center; a micro-manipulation control center connected to a micro-manipulation robotic arm for processing position movement signals and outputting them to the micro-manipulation robotic arm; and a micro-manipulation robotic arm, one end of which is connected to an electrode holder and the other end of which is connected to the micro-manipulation control center to execute movement commands, carrying glass microelectrodes to achieve micron-level displacement. The microscopic system includes a fluorescence microscope, a CCD camera, and a computer. The CCD camera is fixed to a designated interface of the fluorescence microscope and connected to the computer via a data transmission line. The computer processes the electrical signals transmitted by the CCD camera, and after processing, the images observed under the fluorescence microscope can be converted into image information and displayed on the computer's monitor.

2. The method according to claim 1, characterized in that, The detection of cDNA integrity involves detecting the length and content distribution of cDNA.

3. The method according to claim 1, characterized in that, cDNA integrity was detected using a Bioanalyzer 2100 and a high-sensitivity DNA chip detection kit.

4. The method according to claim 1, characterized in that, The expression levels of target-cell and non-target-cell specific genes were detected by real-time quantitative PCR.

5. The method according to claim 1, characterized in that, The threshold for the ratio of target cell to non-target cell specific gene expression is set based on the fact that the ratio of target cell specific gene expression exceeds 95%.

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