Preparation method and application of liver tumor tissue single cell suspension

Abstract

The application provides a preparation method and application of a liver tumor tissue single-cell suspension, and the preparation method comprises the following steps: step 1, pretreating liver tumor tissue; step 2, step-by-step enzymolysis; the pretreated liver tumor tissue in step 1 is subjected to step-by-step enzymolysis by using an enzyme solution, and the enzyme solution in each step is collected after filtration to obtain a cell suspension; step 3, gradient centrifugation; the cell suspension is subjected to gradient centrifugation by using a cell separation solution to separate immune cells and liver parenchymal cells; step 4, red blood cell lysis; the immune cells obtained in step 3 are subjected to red blood cell lysis by using a red blood cell lysis solution, and centrifugal washing is carried out to obtain an immune cell precipitate; step 5, collecting the liver parenchymal cells in step 3 and the immune cell precipitate in step 4, washing the liver parenchymal cells and the immune cell precipitate by using a cell washing solution, centrifuging to obtain a cell precipitate, resuspending the cell precipitate by using a cell resuspension solution, and filtering to obtain the liver tumor tissue single-cell suspension.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and particularly relates to a method for dissociating liver tumor tissue, in particular a method for preparing liver tumor tissue single-cell suspension and application thereof. BACKGROUND

[0002] Liver is one of the common sites of tumors, and benign tumors are less common, and most of them are primary malignant tumors, among which metastatic tumors are more common. Primary tumors can occur in hepatocyte cords, bile duct epithelium, blood vessels or other mesoderm tissue sites, and can occur in adults and children. Early liver malignant tumor patients are mainly treated by surgical resection, liver transplantation and ablation methods, and the treatment methods for advanced patients mainly include transcatheter hepatic arterial chemoembolization and targeted drug therapy. Although the existing therapies have made some progress, the efficacy is still limited, so studying the heterogeneity of liver malignant tumors will help to analyze the evolution of the disease and find new treatment targets, and provide new ideas and schemes for clinical treatment.

[0003] Single-cell sequencing technology is increasingly applied to analyze the interaction relationship of complex cell types in the tumor microenvironment to elucidate the occurrence and development of the disease and the dynamic evolution process in the microenvironment. It can detect the heterogeneity of cells and microenvironments at single-cell resolution, analyze the types, proportions and states of tumor cells, immune cells and other members in the microenvironment through multi-dimensional single-cell sequencing data such as single-cell genomics, transcriptomics, epigenomics, proteomics and metabolomics, and elucidate the potential mechanisms of tumor biological behavior, which has very wide application prospects in promoting tumor lineage diagnosis, targeted therapy and prognosis prediction. However, single-cell sequencing requires a large number of single cells with high activity (more than 80% of the survival rate) from the target tissue, and it is expected to obtain cell types as close as possible to the types and proportions in the in vivo tissue. However, the current commonly used liver tumor dissociation method obtains a single-cell suspension with limited total cell number and low survival rate, and causes the loss of some fragile cells, especially hepatocytes. In actual research, hepatocytes play an important role in the evolution of liver tumors, so maintaining a cell proportion close to the real in vivo environment is very important for studying disease targets and guiding clinical drug use.

[0004] Since most of the current methods for obtaining liver tumor tissue cells by separating fresh tissues are not suitable for single-cell analysis experiments, therefore, developing an efficient, high-cell-viability, single-cell experiment method for preparing liver tumor single-cell suspension is conducive to improving the quality of single-cell experiment results and accelerating the application of single-cell related experiments in liver tumor research. SUMMARY

[0005] In order to overcome the defects of low activity and insufficient cell types of single cell suspension in the prior art, the application provides a preparation method of liver tumor tissue single cell suspension.

[0006] The preparation method of liver tumor tissue single cell suspension provided by the application comprises the following steps:

[0007] Step 1, pretreatment: the liver tumor tissue is pretreated (the liver tumor tissue is washed and trimmed to obtain pretreated tissue);

[0008] Step 2, step-by-step enzymolysis: the pretreated liver tumor tissue in step 1 is subjected to step-by-step enzymolysis by using an enzyme solution, and the enzyme solution in each step is collected after filtration to obtain a cell suspension (a certain amount of enzyme solution is used to enzymolyze and incubate the pretreated tumor tissue obtained in step 1 for a certain time, the supernatant of the enzyme solution is collected by filtering through a cell filter screen to obtain a cell suspension; the remaining precipitate of the enzyme solution can be continuously added with a certain amount of enzyme solution for repeated operation for multiple enzymolysis);

[0009] Step 3, gradient centrifugation: the cell suspension is subjected to gradient centrifugation by using a cell separation solution to separate immune cells and liver parenchymal cells (a certain amount of density gradient separation solution and PBS are uniformly mixed to form cell separation solutions with different concentrations. The immune cells and liver parenchymal cells are separated by gradient centrifugation by using the cell separation solution, washed and centrifuged under certain conditions to obtain cell precipitate);

[0010] Step 4, red blood cell lysis: the immune cells obtained in step 3 are subjected to red blood cell lysis by using a red blood cell lysis solution, and the immune cell precipitate is obtained after washing and centrifugation (the immune cell precipitate obtained in step 3 is subjected to red blood cell lysis by using a certain amount of red blood cell lysis solution, washed and centrifuged under certain conditions to obtain cell precipitate);

[0011] Step 5, collection: the liver parenchymal cells in step 3 and the immune cell precipitate in step 4 are washed by using a cell washing solution, centrifuged to obtain cell precipitate, resuspended by using a cell resuspension solution and filtered to obtain the liver tumor tissue single cell suspension (the cell precipitate is washed by using a certain amount of cell washing solution and centrifuged under certain conditions, the cells are resuspended by using a certain amount of cell resuspension solution and filtered to obtain the liver tumor tissue single cell suspension).

[0012] The step 1 specifically comprises: the liver tumor tissue is washed with pre-cooled PBS solution, and the tissue block is trimmed, and the tissue with complete structure is selected to avoid blood clots.

[0013] Preferably, step 1 specifically involves rinsing the liver tumor tissue with PBS solution pre-cooled at 4°C 2-3 times, and trimming the tissue blocks with sterilized scissors, selecting structurally intact tissues and avoiding blood clots.

[0014] In step 2, the enzyme solution is a type II collagenase with a mass concentration of 0.1% to 0.5% and an enzyme activity unit of not less than 125 CDU / mg.

[0015] Step 2 includes the following steps:

[0016] Step 2.1: At 4-8℃, place the tissue obtained in Step 1 into 5-10 mL of enzymatic hydrolysis solution. In the enzymatic hydrolysis solution, physically cut the tumor tissue into 1-2 mm pieces using scissors. 3 The sample was initially enzymatically hydrolyzed at 37°C in a water bath shaker for 5–10 min. After incubation, the sample was filtered through a 70–100 μm membrane, and the filtrate was collected and temporarily stored at 4–8°C.

[0017] Step 2.2: Add 5-10 mL of the enzyme solution to the precipitate for a second enzymatic hydrolysis, and incubate in a 37°C water bath shaker for 5-10 min. After incubation, filter and collect the second enzymatic hydrolysis filtrate, and mix it with the preliminary enzymatic hydrolysis filtrate collected in Step 2.1 to obtain the cell suspension.

[0018] It can undergo 2-4 enzyme digestion processes.

[0019] The density gradient separation solution described in step 3 is Percoll cell separation solution.

[0020] Step 3 specifically involves: centrifuging the cell suspension obtained in step 2 at 4-8℃ and 300-400g, resuspending the precipitate in 2-3 mL of pre-cooled PBS to prepare a cell dispersion; sequentially adding 3 mL of Percoll separation solution 3, 3 mL of Percoll separation solution 2, and 2-3 mL of cell dispersion to a centrifuge tube for gradient centrifugation; the gradient centrifugation conditions are 400g, 4℃, acceleration / deceleration set to level 4, for 20-30 min; after centrifugation, the bottom layer consists of immune cells and red blood cells, and the middle layer between Percoll separation solution 3 and Percoll separation solution 2 is the hepatocyte layer; the immune cells, red blood cells, and hepatocyte layer are separated and collected.

[0021] The Percoll separation solution 2 is prepared as follows: Percoll and 10x PBS solution are mixed at a volume ratio of 9:1 to obtain Percoll separation solution 1; Percoll separation solution 1 and 10x PBS solution are mixed at a volume ratio of 3-4:7 to obtain Percoll separation solution 2.

[0022] The Percoll separation solution 3 is prepared by mixing Percoll and 10x PBS solution at a volume ratio of 9:1 to obtain Percoll separation solution 1; and mixing Percoll separation solution 1 and 10x PBS solution at a volume ratio of 1:1 to obtain Percoll separation solution 2.

[0023] Step 4 specifically includes the following steps:

[0024] Add the red blood cells collected in step 3 to red blood cell lysis buffer and lyse at room temperature for 3-5 minutes; after red blood cell lysis, add cell washing buffer, centrifuge at 400g for 5-10 minutes, and collect the immune cells.

[0025] The preparation methods for Percoll separation solution 2 and Percoll separation solution 3 are as follows: first, mix 9 volumes of Percoll with 1 volume of 10x PBS solution to obtain Percoll separation solution 1; then, mix 3 volumes of Percoll separation solution 1 with 7 volumes of PBS solution to obtain Percoll separation solution 2; and finally, mix 5 volumes of Percoll separation solution 1 with 5 volumes of PBS solution to obtain Percoll separation solution 3.

[0026] The amount of erythrocyte lysis buffer in step 4 is 1–3 mL, used only for the precipitation of immune cells. The erythrocyte lysis conditions are room temperature lysis for 3 minutes. The washing and centrifugation process involves washing the cell suspension after lysing the erythrocytes with 5–10 mL of pre-cooled PBS and centrifuging at 500 g for 5–10 minutes to enrich the immune cells.

[0027] The cell washing solution and cell resuspension solution used in step 5 are DEME medium solution containing 10% FBS or PBS solution containing 0.1% BSA. The volume of the cell washing solution is 5-15 mL, and the centrifugation conditions are 400 g, 4°C, and centrifugation for 5-10 min.

[0028] Further, the amount of cell resuspension in step 5 is 0.5–5 mL, used to resuspend the tissue single-cell suspension. The filtration conditions are a 30 μm filter membrane to remove double cells and other impurities.

[0029] The present invention also provides a single-cell suspension of liver tumor tissue prepared by the preparation method described above.

[0030] This invention also provides an application of a single-cell suspension of liver tumor tissue for single-cell sequencing, cell sorting, and research on tumor mechanisms.

[0031] A method for preparing a single-cell suspension of liver tumor tissue is carried out under aseptic conditions. Except for the enzymatic incubation at 37°C, all other steps are performed at 4°C or on ice.

[0032] The liver tumor single-cell suspension prepared by this invention has a high yield and high cell activity of each cell type, and the proportion of each cell type is closer to the actual proportion in vivo, which is beneficial for carrying out downstream single-cell sequencing and other experiments.

[0033] The present invention has the following beneficial effects:

[0034] This invention provides, for the first time, a method for preparing a single-cell suspension of liver tumor tissue for single-cell sequencing. It has the following beneficial effects:

[0035] (1) This method reduces the time that single cells exist in the enzyme solution and takes advantage of the difference in floating density of different types of cells to first separate the liver parenchymal cells that are less tolerant in the red blood cell lysis step by gradient centrifugation, while only performing red blood cell lysis on other cell components such as immune cells, thereby improving cell activity.

[0036] (2) The single-cell suspension obtained by this method can restore the proportion of various cell types in the body to a greater extent, which is helpful for subsequent single-cell related experiments.

[0037] (3) This method has a high cell yield, so the required amount of ex vivo tissue can be lower, making it suitable for a wider range of samples, such as puncture samples.

[0038] (4) This method does not require the use of flow cytometry or other kits to sort or enrich live cells, thus saving costs and reducing the complexity and time of operation.

[0039] (5) The method provided by the present invention can flexibly select the number of multi-step dissociations according to the pyrolysis results, and does not require highly specialized equipment, reagents or skills, making it easy to operate and implement, and has good practical application value. Attached Figure Description

[0040] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0041] Figure 1 This is a schematic diagram of the cell state of the liver parenchymal cell layer and the immune cell layer obtained by gradient centrifugation in Embodiment 1 of the present invention, wherein A is the liver parenchymal cell layer; B is the immune cell layer;

[0042] Figure 2 This is a schematic diagram of the concentration counting and viability detection of a single-cell suspension obtained in Example 1 of the present invention; wherein A shows the composition of the single-cell suspension; B shows the count of live cells in the same field of view; and C shows the count of dead cells in the same field of view.

[0043] Figure 3 It is a quality control image of a single-cell library;

[0044] Figure 4 It is a two-dimensional display diagram of cell clusters;

[0045] Figure 5 This is a schematic diagram of the viability detection and counting of single-cell suspensions obtained in Comparative Example 1 of the present invention; wherein, A shows the composition of the single-cell suspension, B shows the dead cells in the single-cell suspension under the same field of view; and C shows the total cell count.

[0046] Figure 6 This is a schematic diagram of the viability detection and counting of the single-cell suspension obtained in Comparative Example 2 of the present invention; Figure A shows the composition of the single-cell suspension, where cell adhesion is relatively severe; Figure B shows the dead cells in the single-cell suspension under the same field of view; Figure C shows the total number of cells. Detailed Implementation

[0047] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0048] This invention proposes a method for preparing single-cell suspensions of liver tumor tissue for single-cell-related experiments. The resulting single-cell suspensions exhibit high yield, good activity, and a comprehensive range of cell types. The steps in the embodiments and comparative examples of this invention include reagent preparation, tissue enzymatic digestion, erythrocyte lysis, cell washing, and single-cell suspension quality control. Tissue enzymatic digestion includes two methods: one-step dissociation and stepwise dissociation. Erythrocyte lysis includes two methods: direct erythrocyte lysis and erythrocyte lysis of only a subset of cells after gradient centrifugation. Example 1 and Comparative Examples 1-2 are provided below, using a pediatric malignant liver tumor, specifically a hepatoblastoma sample. Example 1 uses a stepwise dissociation method followed by gradient centrifugation and erythrocyte lysis; Comparative Example 1 uses a one-step dissociation method combined with direct erythrocyte lysis; and Comparative Example 2 uses a one-step dissociation method with other enzymes combined with direct erythrocyte lysis. By comparing the technical effects of the comparative examples with those of Example 1, it is shown that the method for preparing single-cell suspensions of hepatoblastoma tissue for single-cell related experiments provided by the present invention can obtain single-cell suspensions with high cell yield, high cell activity and a large number of viable liver parenchymal cells, which is beneficial for carrying out downstream single-cell related experiments.

[0049] Example 1

[0050] The implementation steps are as follows:

[0051] (I) Reagent Preparation

[0052] 1. Tissue dissociation solution: Dissolve 0.1g of type II collagenase (Sigma, C6885) in 50mL of DMEM medium (Gibco, 10313021);

[0053] 2. Density gradient separation buffer: Mix 9 mL of Percoll (Solarbio, 65455-52-9) and 1 mL of 10x PBS (Gibco, 70011044) solution to obtain Percoll separation buffer 1; mix 3 mL of Percoll separation buffer 1 and 7 mL of PBS (Gibco, 10010023) solution to obtain 10 mL of Percoll separation buffer 2; mix 5 mL of Percoll separation buffer 1 and 5 mL of PBS solution to obtain 10 mL of Percoll separation buffer 3.

[0054] 3. Cell washing solution: Dissolve 5 mL of FBS (Gibco, 26140079) in 45 mL of DMEM medium;

[0055] 4. Cell resuspension: Dissolve 50 mg of BSA (Gibco, 11020039) powder in 50 mL of PBS.

[0056] (II) Organizational Dissolution

[0057] 1. Rinse the liver tumor tissue with 10-20 mL of cell flushing solution, remove the blood clots, remove the cell flushing solution, and replenish with fresh cell flushing solution;

[0058] 2. Select a structurally intact tissue, add 5 mL of tissue dissociation solution, and use scissors to cut the tissue into 1 mm pieces. 3 size;

[0059] Incubate in a 3.37℃ water bath shaker for 8 minutes to digest and dissociate the tissue;

[0060] 4. Pass the supernatant of the dissociated tissue through a 70 μm filter membrane, add 5 mL of cell washing buffer to the filtrate, and store on ice;

[0061] 5. Collect the remaining incompletely dissociated tissue on the filter screen from the previous step, add 5 mL of tissue dissociation solution and lyse for 10 min;

[0062] 6. After digestion, filter through a 70μm filter membrane, and store the filtrate together with the filtrate from step 4 for later use;

[0063] 7. Centrifuge the filtrate collected in step 6 (i.e., 300g of digestion solution) at 4°C for 6 minutes;

[0064] 8. After centrifugation, remove the supernatant and resuspend the precipitate in 10 mL of cell washing buffer;

[0065] 9.400g, centrifuged at 4℃ for 10min;

[0066] 10. After centrifugation, resuspend the above precipitate in 3 mL of cell resuspension to obtain a cell suspension.

[0067] (III) Red blood cell lysis and cell washing

[0068] 1.4 Centrifuge the cell suspension obtained in step (II) at -8℃ and 300-400g, and resuspend the precipitate in 2-3 mL of pre-cooled PBS to prepare a cell dispersion.

[0069] 2. Add 3 mL of Percoll separation solution 3, 3 mL of Percoll separation solution 2, and 3 mL of cell dispersion to a 15 mL centrifuge tube in sequence to form a gradient centrifugation system;

[0070] 3. Select centrifuge speed setting 4, and centrifuge the gradient centrifugation system at 400g and 4℃ for 20 minutes.

[0071] 4. The bottom layer of the gradient centrifugation system after centrifugation consists of immune cells and red blood cells, requiring red blood cell lysis. The middle layer between Percoll separation solution 3 and Percoll separation solution 2 consists of liver parenchymal cells, which do not require red blood cell lysis.

[0072] 5. Resuspend the basal immune cells (red blood cells) in 1 mL of erythrocyte lysis buffer (Gibco ACK Lysing Buffer, A1049201) and lyse at room temperature for 3 min;

[0073] 6. After erythrocyte lysis, add 9 mL of cell washing buffer, centrifuge at 400g for 10 min to enrich immune cells;

[0074] 7. After resuspending the immune cells, mix them with the liver parenchymal cells obtained in step 3, add cell washing solution to make up the volume to 15 mL, and centrifuge again at 400g for 10 min to enrich the cells.

[0075] 8. Resuspend the cell pellet in 1 mL of cell resuspension solution and filter through a 30 μm filter membrane to obtain a single-cell suspension.

[0076] (iv) Cell suspension quality inspection

[0077] 10 μL of the liver parenchymal cell suspension after Percoll gradient centrifugation (step 3 in step (III)) and 10 μL of the immune cell resuspension after erythrocyte lysis (step 7 in step (III)) were stained with 10 μL of trypan blue, and the cell state was examined using a fluorescence microscope. The results are as follows: Figure 1 As shown, Figure 1 A represents the liver parenchymal cell layer, where the overall cell diameter is relatively large. Figure 1B represents the immune cell layer, with cells generally having a smaller diameter. Another 10 μL of the final single-cell suspension was stained with 10 μL of AOPI (Nexclome, CS-0106), and cell viability and concentration were analyzed using a Countstar Altair intelligent cell analyzer. The automatic counting results are shown below. Figure 2 As shown, Figure 2 A shows the composition of a single-cell suspension, where the larger cells are generally liver parenchymal cells. Figure 2 B shows the count of live cells in the same field of view. Figure 2 C shows the count of dead cells in the same field of view. Total cell concentration: 1.13 × 10⁻⁶ 6 / mL, of which the concentration of live cells is 1.09×10 6 / mL, accounting for 96.5%, dead cell concentration 3.97×10 4 / mL, accounting for 3.5%.

[0078] (V) Library construction, quality control and sequencing

[0079] This invention is based on the 10x Genomics platform. The cell suspension was loaded into a Chromium Chip G chip and placed in a Chromium Controller for oil droplet encapsulation. Library construction was then performed using the Chromium Next GEM Single Cell30 Kit v3.1. Quality control was performed after library construction. Figure 3 The fragments were concentrated between 300-700 bp. Sequencing was performed using the Illumina sequencing platform.

[0080] (vi) Data Analysis

[0081] Raw sequencing data were used, and FastQC software was employed for data quality assessment. Cell-barcodes and UMIs (Unique Molecular Identifiers) were extracted. RNA sequences were aligned with the reference genome. Based on the unique alignment results of the RNA sequences and the UMI sequences, UMIs were corrected to remove PCR duplications in the sequencing process. After identifying valid cells, a Gene-barcode matrix was generated to record the gene expression values ​​of different cells. Cell ranger software was used to cluster cells based on gene expression levels. Expression data were normalized, and UMAP dimensionality reduction analysis was performed for visualization. Cell pairs that are close to each other have more similar gene expression profiles, resulting in the final cell clustering results. Sequencing quality control data and cell count results are shown in Table 1. To further analyze sample heterogeneity, cells were clustered based on gene expression levels, and the results are shown in Table 1. Figure 4The single-cell suspension prepared by the method of this invention can be conveniently, quickly, and efficiently applied to single-cell sequencing detection. The above results all indicate that the single-cell suspension prepared by this invention is of high quality and high activity, and has high application value.

[0082] Table 1 Sequencing quality control data and cell count results

[0083]

[0084] Comparative Example 1

[0085] The implementation steps are as follows:

[0086] (I) Reagent Preparation

[0087] 1. Tissue dissociation solution: Dissolve 0.1g of type II collagenase (Sigma, C6885) in 50mL of DMEM medium (Gibco, 10313021);

[0088] 2. Cell washing solution: Dissolve 5 mL of FBS (Gibco, 26140079) in 45 mL of DMEM medium.

[0089] 3. Cell resuspension: Dissolve 50 mg of BSA (Gibco, 11020039) powder in 50 mL of PBS (Gibco, 10010023).

[0090] (II) Organizational Dissolution

[0091] 1. Rinse the tissue with 10-20 mL of cell flushing solution, remove the blood clots, remove the cell flushing solution, and replenish with fresh cell flushing solution;

[0092] 2. Select a structurally intact tissue, add 5 mL of tissue dissociation solution, and use scissors to cut the tissue into 1 mm pieces. 3 size;

[0093] Incubate in a 3.37℃ water bath shaker for 20 min to lyse the tissue;

[0094] 4. Filter all tissue lysates through a 70 μm filter membrane, add 5 mL of cell washing buffer to wash the filter membrane, and store on ice;

[0095] 5. Collect 300g of all filtrate and centrifuge at 4℃ for 6 minutes;

[0096] 6. After centrifugation, remove the supernatant and resuspend in 10 mL of cell washing buffer;

[0097] 7.400g, centrifuged at 4℃ for 10min;

[0098] 8. After centrifugation, resuspend the cell pellet in 3 mL of cell resuspension solution.

[0099] (III) Red blood cell lysis and cell washing

[0100] 1. Resuspend the cell pellet in 1 mL of erythrocyte lysis buffer (Gibco ACK Lysing Buffer, A1049201) and lyse at room temperature for 3 min;

[0101] 2. After erythrocyte lysis, add 9 mL of cell washing buffer, centrifuge at 400 g for 10 min to enrich the cells;

[0102] 3. After resuspending the cells, wash them again with cell washing buffer to bring the volume to 15 mL, then centrifuge at 400g for 10 min to enrich the cells.

[0103] 4. Resuspend the cell pellet in 1 mL of cell resuspension solution and filter through a 30 μm filter membrane to obtain a single-cell suspension.

[0104] (iv) Cell suspension quality inspection

[0105] 10 μL of single-cell suspension was stained with 10 μL of DAPI, and the cell viability was examined using a fluorescence microscope. The results are as follows: Figure 5 As shown, Figure 5 A shows the composition of a single-cell suspension. Figure 5 B shows the status of dead cells in the single-cell suspension under the same field of view, appearing bright white. Another 10 μL of single-cell suspension was stained with trypan blue, and cell viability and concentration were determined using a Countess II FL Automated Cell Counter. The automated counting results are as follows... Figure 5 As shown in C, the total cell concentration was 4.69 × 10⁻⁶. 5 / mL, with a live cell concentration of 2.70×10⁶. 5 / mL, accounting for 57.6%, dead cell concentration 1.99×10 5 / mL, accounting for 42.4%.

[0106] Comparative Example 2

[0107] The implementation steps are as follows:

[0108] (I) Reagent Preparation

[0109] 1. Tissue dissociation solution: Dissolve 0.1g of type IV collagenase (Sigma, C5138) in 50mL of DMEM medium (Gibco, 10313021);

[0110] 2. Density gradient separation buffer: Mix 9 mL of Percoll (Solarbio, 65455-52-9) and 1 mL of 10x PBS (Gibco, 70011044) solution to obtain Percoll separation buffer 1; mix 3 mL of Percoll separation buffer 1 and 7 mL of PBS (Gibco, 10010023) solution to obtain 10 mL of Percoll separation buffer 2; mix 5 mL of Percoll separation buffer 1 and 5 mL of PBS solution to obtain 10 mL of Percoll separation buffer 3.

[0111] 3. Cell washing solution: Dissolve 5 mL of FBS (Gibco, 26140079) in 45 mL of DMEM medium.

[0112] 4. Cell resuspension: Dissolve 50 mg of BSA (Gibco, 11020039) powder in 50 mL of PBS (Gibco, 10010023).

[0113] (II) Organizational Dissolution

[0114] 1. Rinse the tissue with 10-20 mL of cell flushing solution, remove the blood clots, remove the cell flushing solution, and replenish with fresh cell flushing solution;

[0115] 2. Select a structurally intact tissue, add 5 mL of tissue dissociation solution, and use scissors to cut the tissue into 1 mm pieces. 3 size;

[0116] Incubate in a 3.37℃ water bath shaker for 20 min to lyse the tissue;

[0117] 4. Pass the supernatant of the dissociated tissue through a 70 μm filter membrane, add 5 mL of cell washing buffer to the filtrate, and store on ice;

[0118] 5. Collect the remaining incompletely dissociated tissue on the filter screen from the previous step, add 5 mL of tissue lysis buffer and lyse for 10 min;

[0119] 6. After digestion, filter through a 70μm filter membrane, and store the filtrate together with the filtrate from step 4 for later use;

[0120] 7. Centrifuge the filtrate collected in step 6 (i.e., 300g of digestion solution) at 4°C for 6 minutes;

[0121] 8. After centrifugation, remove the supernatant and resuspend the precipitate in 10 mL of cell washing buffer;

[0122] 9.400g, centrifuged at 4℃ for 10min;

[0123] 10. After centrifugation, resuspend the above precipitate in 3 mL of cell resuspension.

[0124] (III) Red blood cell lysis and cell washing

[0125] 1. Add 3 mL of 50% Percoll, 3 mL of 30% Percoll, and 3 mL of the cell suspension from step 2 to a 15 mL centrifuge tube in sequence to form a gradient centrifugation system;

[0126] 2. Select centrifuge speed setting 4, and centrifuge the gradient centrifugation system at 400g and 4℃ for 20 minutes.

[0127] 3. The bottom layer of the gradient centrifugation system after centrifugation consists of immune cells and red blood cells, requiring red blood cell lysis. The layer between the 50% and 30% Percoll layers is a layer of liver parenchymal cells, which does not require red blood cell lysis.

[0128] 4. Resuspend the basal immune cells (red blood cells) in 1 mL of erythrocyte lysis buffer (Gibco ACK Lysing Buffer, A1049201) and lyse at room temperature for 3 min;

[0129] 5. After erythrocyte lysis, add 9 mL of cell washing buffer, centrifuge at 400g for 10 min to enrich immune cells;

[0130] 6. After resuspending the immune cells, mix them with the liver parenchymal cells obtained in step 3, add cell washing solution to make up the volume to 15 mL, and centrifuge again at 400g for 10 min to enrich the cells.

[0131] 7. Resuspend the cell pellet in 1 mL of cell resuspension solution and filter through a 30 μm filter membrane to obtain a single-cell suspension.

[0132] (iv) Cell suspension quality inspection

[0133] 10 μL of single-cell suspension was stained with 10 μL of DAPI, and the cell viability was examined using a fluorescence microscope. The results are as follows: Figure 6 As shown, Figure 6 A shows the composition of a single-cell suspension, indicating significant cell adhesion. Figure 6 B shows the status of dead cells in the single-cell suspension under the same field of view, appearing bright white. Another 10 μL of single-cell suspension was stained with trypan blue, and cell viability and concentration were determined using a Countess II FL Automated Cell Counter. The automated counting results are as follows... Figure 6 As shown in C, the total cell concentration was 6.69 × 10⁻⁶. 5 / mL, with a live cell concentration of 4.46×10⁻⁶. 5 / mL, accounting for 66.7%, dead cell concentration 2.23×10 5 / mL, accounting for 33.3%.

[0134] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for preparing a single cell suspension of liver tumor tissue, characterized by, The method comprises the following steps: Step 1: pretreating liver tumor tissue; Step 2: step-by-step enzymolysis of the pretreated liver tumor tissue by using an enzyme solution, collecting the enzymolysis solution of each step after filtration to obtain a cell suspension; the enzyme solution is a type II collagenase with a mass concentration of 0.1-0.5% and an enzyme activity unit of not less than 125 CDU / mg; Step 3: centrifuging the cell suspension obtained in step 2 under the condition of 4-8 ℃ and 300-400 g, resuspending the precipitate in 2-3 mL of pre-cooled PBS to prepare a cell dispersion; sequentially adding 3 mL of Percoll separation solution 3, 3 mL of Percoll separation solution 2 and 2-3 mL of the cell dispersion into a centrifuge tube to perform gradient centrifugation; the gradient centrifugation is performed under the condition of 400 g, 4 ℃ and 20-30 min; after centrifugation, the bottom layer is immune cell red blood cells, and the liver parenchymal cell layer is between the Percoll separation solution 3 and the Percoll separation solution 2; the immune cell red blood cells and the liver parenchymal cell layer are separated and collected; Step 4: performing red blood cell lysis on the immune cells obtained in step 3 by using a red blood cell lysis solution, and centrifuging and washing to obtain an immune cell precipitate; Step 5: collecting the liver parenchymal cells in step 3 and the immune cell precipitate in step 4, washing by using a cell washing solution, centrifuging to obtain a cell precipitate, resuspending the cell precipitate by using a cell resuspension solution and filtering to obtain the liver tumor tissue single-cell suspension; The step 2 comprises the following steps: At 4-8℃, place the tissue obtained in step 1 into 5-10 mL of enzyme solution, and use scissors to cut the tumor tissue into 1-2 mm pieces in the enzyme solution. 3 The sample was initially enzymatically hydrolyzed at 37°C in a water bath shaker for 5-10 minutes. After incubation, the sample was filtered through a 70-100 μm filter membrane, and the filtrate from the initial enzymatic hydrolysis was collected. the precipitate is continuously added with 5-10 mL of the enzyme solution to perform secondary enzymolysis, and incubated in a 37 ℃ water bath shaker for 5-10 min; after the incubation, the secondary enzymolysis filtrate is collected and mixed with the primary enzymolysis filtrate to obtain the cell suspension; The preparation method of the Percoll separation solution 2 is as follows: mixing Percoll cell separation solution and 10x PBS solution at a volume ratio of 9:1 to obtain Percoll separation solution 1; mixing the Percoll separation solution 1 and 10x PBS solution at a volume ratio of 3-4:7 to obtain the Percoll separation solution 2; The preparation method of the Percoll separation solution 3 is as follows: mixing Percoll cell separation solution and 10x PBS solution at a volume ratio of 9:1 to obtain Percoll separation solution 1; mixing the Percoll separation solution 1 and 10x PBS solution at a volume ratio of 1:1 to obtain the Percoll separation solution 3.

2. The production method according to claim 1, characterized by, The step 1 is specifically as follows: washing the liver tumor tissue with pre-cooled PBS solution, trimming the tissue block, and selecting a tissue with complete structure to avoid blood clots.

3. The preparation method according to claim 1, characterized in that, Step 4 specifically comprises the following steps: adding the immune cell red blood cells collected in step 3 into a red blood cell lysis solution to lyse for 3-5 min at room temperature; after the red blood cell lysis, adding a cell washing solution, centrifuging at 400 g for 5-10 min, and collecting the immune cells.

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