Preparation method capable of controlling temperature of tissue and dissociating tissue into single cells and application

Abstract

The invention discloses a temperature-controllable preparation method for tissue temperature dissociation into single cells and application, the temperature-controllable preparation method for tissue temperature dissociation into single cells comprises the following steps: S1, providing a tissue sample, and pre-treating the tissue sample to obtain a sample block; s2, mixing the sample blocks with a compound digestive enzyme, and filtering to obtain a single-cell suspension after digestion treatment is completed; wherein the compound digestive enzyme is prepared from collagenase, hyaluronidase and papain; and selectively, S3, carrying out red removing treatment. The invention not only provides an improved preparation method of the single-cell suspension, but also has higher efficiency on the premise of ensuring the yield and activity of single cells, and provides a new strategy and tool for cancer research and treatment by particularly enriching CSCs.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a preparation method and application of temperature-controllable tissue gentle dissociation into single cells. Background Art

[0002] In biomedical research and clinical treatment, the preparation of single-cell suspension from animal tissue is a basic and critical technology. Single-cell suspension is not only used for cell counting and cell viability assessment, but is also widely used in cell signaling research, cell culture, flow cytometry analysis, and high-throughput drug screening. However, traditional methods mainly include mechanical methods and enzymatic methods, but there are some limitations. For example, mechanical methods may cause cell damage, and existing enzymatic methods have problems such as difficulty in controlling enzyme activity, long processing time, complex operation, and high cost.

[0003] The main disadvantages of the existing technology include cell damage, low efficiency, complex operation, difficulty in standardization and high cost. Mechanical methods are prone to damage to cells due to the large physical shear force. Although the enzymatic method is relatively mild, the activity of the enzyme is difficult to accurately control, and the sensitivity of tissues from different sources to enzymes varies greatly, resulting in difficulty in standardizing digestion conditions and low cell yield and activity. In addition, the multi-step operation process increases the complexity of the experiment and takes a long time.

[0004] Therefore, technicians in this field are committed to developing an improved method for preparing single-cell suspensions, which can reduce cell damage, improve preparation efficiency, simplify the operation process, standardize conditions, and reduce experimental costs. Summary of the invention

[0005] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present application is to propose a preparation method and application of temperature-controllable tissue gentle dissociation into single cells, which not only improves the efficiency and quality of the experiment, but also helps to reduce costs, expand the scope of application, and enhance the potential for scientific research and clinical applications.

[0006] To achieve the above objectives, in a first aspect, the present application provides a method for preparing a temperature-controllable tissue by mildly dissociating it into single cells, which comprises the following steps:

[0007] S1. providing a tissue sample, and pre-processing the tissue sample to obtain a sample block;

[0008] S2, mixing the sample block with a composite digestive enzyme, and filtering to obtain a single cell suspension after the digestion treatment is completed; wherein the composite digestive enzyme includes collagenase, hyaluronidase and papain;

[0009] and selectively,

[0010] S3, perform red removal treatment.

[0011] In step S2, the digestion treatment is performed in a single cell suspension instrument;

[0012] In step S2, the sample block is mixed with a composite digestive enzyme, and after the digestion treatment is completed, filtering to obtain a single cell suspension comprises:

[0013] adding the sample block and complex digestive enzyme into a test tube;

[0014] placing the test tube in the single cell suspension instrument and performing digestion treatment after preheating;

[0015] After the digestion treatment is completed, the single cell suspension in the test tube is filtered out through a cell sieve, and a digestion stop solution is added to the single cell suspension.

[0016] During the digestion process, the digestion temperature is 36.5-37.5°C, mechanical stirring is performed at a rotation speed of 40-60RPM, and regular sampling is performed every 5-10 minutes to observe the cell separation.

[0017] In one or more feasible embodiments, in step S1, providing a tissue sample and pre-treating the tissue sample to obtain sample fragments includes:

[0018] Obtain tissue samples from living animals and transfer them into culture medium;

[0019] washing the tissue sample to remove red blood cells and useless tissue;

[0020] The cleaned tissue sample is cut into small pieces and washed with a buffer solution to remove debris generated during the process, thereby obtaining the sample piece.

[0021] In one or more feasible embodiments, the culture medium is a culture solution at 4-6° C. or a sterile culture dish containing PBS, and the culture medium does not contain serum.

[0022] In one or more feasible embodiments, in step S3, adding red blood cell lysis solution to perform red blood cell removal treatment includes:

[0023] Add red blood cell lysis solution to the single cell suspension, centrifuge to remove lysed red blood cells, add PBS to resuspend, then wash by centrifugation and resuspend in culture medium.

[0024] In one or more feasible embodiments, the complex digestive enzymes include:

[0025] Collagenase: 1-2 mg / mL

[0026] Hyaluronidase: 0.05-0.2U / mL

[0027] Papain: 0.2-0.5 mg / mL.

[0028] In one or more feasible embodiments, the complex digestive enzyme further comprises:

[0029] Elastase: 0.02-0.1 U / mL

[0030] Chondroitinase ABC: 0.01-0.1U / mL.

[0031] In a second aspect, the present application provides a single-cell sequencing method, which performs sequencing after preparing the single-cell suspension by the preparation method of the temperature-controllable tissue gentle dissociation into single cells as described in the first aspect.

[0032] In a third aspect, the present application provides an application of the method for preparing temperature-controllable tissue gentle dissociation into single cells according to the first aspect in the preparation of cells for tumor detection and treatment.

[0033] The method provided by this application has the following technical effects:

[0034] Improved cell viability: The optimized enzyme combination and mild digestion conditions reduce physical and chemical damage to all cell types, including cancer stem cells (CSCs), and improve the overall viability and yield of cells in single cell suspensions.

[0035] Specific enrichment of CSCs: The temperature-controlled tissue gentle dissociation into single-cell preparation method of the present application can specifically enrich CSCs. The enriched CSCs can be used for rapid screening and development of drugs targeting cancer stem cells on the one hand, and on the other hand, it can also help develop new diagnostic markers and detection methods, which may have an important impact on the early detection and treatment of cancer, thereby accelerating the discovery and development process of new drugs.

[0036] Improve experimental efficiency: Simplified operating procedures and shortened preparation time improve the overall efficiency of the experiment and speed up the output of scientific research results.

[0037] Improve experimental reproducibility: Standardized digestion conditions ensure high consistency of experimental results between different batches or different operators.

[0038] Promote automation and large-scale production: The simplified and standardized operation process is suitable for the implementation of automated equipment, which will help to achieve automation and large-scale preparation of single-cell suspensions in the future.

[0039] In summary, the present invention not only provides an improved method for preparing single-cell suspensions, which has high efficiency while ensuring the single-cell yield and activity, but also provides new strategies and tools for cancer research and treatment by specifically enriching CSCs.

[0040] The concept, specific structure and technical effects of the present application will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is a flow chart of a preferred embodiment of the method for preparing temperature-controlled tissue gentle dissociation into single cells of the present application;

[0042] Figure 2 This is a graph showing the quality inspection results of the cell suspension after initial dissociation in the kidney 1 experimental group shown in an embodiment of the present application;

[0043] Figure 3 This is a graph showing the quality inspection results of the cell suspension after initial dissociation in the kidney 2 experimental group shown in an embodiment of the present application;

[0044] Figure 4 This is a graph showing the quality inspection results of the cell suspension after initial dissociation in the kidney 3 experimental group shown in an embodiment of the present application;

[0045] Figure 5 This is a diagram of the quality inspection results of the final cell suspension of the kidney 1 experimental group shown in an embodiment of the present application;

[0046] Figure 6 This is a diagram of the quality inspection results of the final cell suspension of the kidney 2 experimental group shown in an embodiment of the present application;

[0047] Figure 7 This is a diagram of the quality inspection results of the final cell suspension of the kidney 3 experimental group shown in an embodiment of the present application. DETAILED DESCRIPTION

[0048] The following describes the embodiments of the present application through specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the contents disclosed in this specification. The present application can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that the following embodiments and features in the embodiments can be combined with each other without conflict.

[0049] Some exemplary embodiments of the present application are described for the purpose of explanation, and it should be understood that the present application may be implemented in other ways not specifically shown in the drawings.

[0050] Figure 1The present invention shows a method for preparing a temperature-controlled tissue by gently dissociating it into single cells. Figure 1 As shown, the method comprises the following steps:

[0051] S1. providing a tissue sample, and pre-processing the tissue sample to obtain a sample block;

[0052] S2, mixing the sample block with a composite digestive enzyme, and filtering to obtain a single cell suspension after the digestion treatment is completed; wherein the composite digestive enzyme includes collagenase, hyaluronidase and papain;

[0053] and selectively,

[0054] S3, perform red removal treatment.

[0055] Wherein, step S1 comprises:

[0056] Obtain tissue samples from living animals and transfer them into culture medium;

[0057] washing the tissue sample to remove red blood cells and useless tissue;

[0058] The cleaned tissue sample is cut into small pieces, and washed with a buffer solution to remove debris generated during the process, thereby obtaining the sample piece.

[0059] As an example but not a limitation, the culture medium is a sterile culture dish of 4-6°C culture medium or PBS, and the culture medium does not contain serum. Specifically, after obtaining the tissue sample, quickly transfer it to a sterile culture dish filled with 4°C serum-free culture medium or PBS, and place the culture dish on ice. When experiments are needed, the tissue sample can be washed several times with PBS containing 1% double antibody to remove red blood cells as much as possible, and then cut away useless tissues such as fat and necrotic tissue in the sample. Then use scissors to cut the tissue into 1-2mm 3 The sample blocks were cut into pieces of about 50 mm in size and washed once with 4°C D-Hank's solution to remove as much tissue fragments as possible during the tissue cutting process.

[0060] Wherein, step S2 needs to perform the digestion process in a single cell suspension instrument, which includes:

[0061] adding the sample block and complex digestive enzyme into a test tube;

[0062] placing the test tube in the single cell suspension instrument and performing digestion treatment after preheating;

[0063] After the digestion treatment is completed, the single cell suspension in the test tube is filtered out through a cell sieve, and a digestion stop solution is added to the single cell suspension.

[0064] During the digestion process, the digestion temperature is 36.5-37.5°C, mechanical stirring is performed at a speed of 40-60RPM, and regular sampling is performed every 5-10 minutes to observe the cell separation. Specifically, after the single-cell preparation reagent is completely thawed, add the sample block and the composite digestion enzyme to the single-cell preparation test tube, the liquid level is higher than the test tube mouth, the test tube cover is horizontally closed and placed in the single-cell suspension instrument, and the built-in program is selected to run. The process of operating the single-cell suspension instrument for digestion is as follows:

[0065] First, place the prepared test tube into the preheating module and preheat for 3-5 minutes (the time difference between the samples can be ignored);

[0066] Place the preheated sample in the digestion module, close the instrument cover, select the program and click Start (select the corresponding program according to the ease of sample processing);

[0067] After the digestion process is completed, take out the test tube and transfer the sample in the test tube to the cell tube through the cell sieve.

[0068] In order to ensure that the overall digestion temperature of the test tube can be stably maintained at 36.5-37.5℃, the single-cell suspension instrument is equipped with three infrared temperature sensors for real-time temperature measurement of the front, middle and back positions of the test tube. At the same time, the single-cell suspension instrument also has an amplitude adjustment mechanism for adjusting the reciprocating amplitude of the test tube. When the temperature difference between the front, middle and back of the tested test tube is less than 0.3℃ and both are within the range of 36.5-37.5℃, the maximum amplitude is maintained for reciprocating motion, the maximum amplitude is 150mm, and the reciprocating oscillation frequency is 40 times / minute; when the temperature difference between the front, middle and back of the tested test tube is greater than 0.3℃ and less than 0.5℃, the center amplitude is maintained for reciprocating motion, the center amplitude is 100mm, and the reciprocating oscillation frequency is 50 times / minute; when the temperature difference between the front, middle and back of the tested test tube is greater than 0.5℃, the minimum amplitude is maintained for reciprocating motion, the minimum amplitude is 50mm, and the reciprocating oscillation frequency is 60 times / minute.

[0069] When processing certain tissue types (erythrocyte-rich tissues such as liver, spleen, and bone marrow), because erythrocytes greatly outnumber other cell types and their presence may interfere with flow cytometry analysis, cell counting, and other cell biology experiments, removal of erythrocytes is critical to improve the accuracy and clarity of subsequent experiments.

[0070] Wherein, in step S3, the step of adding red blood cell lysis solution to perform red blood cell removal treatment comprises:

[0071] Add red blood cell lysis solution to the single cell suspension, centrifuge to remove lysed red blood cells, add PBS to resuspend, then centrifuge to wash and resuspend in culture medium. Specifically:

[0072] Use red blood cell lysis buffer (also called red blood cell lysis buffer or lysate), which usually contains a low pH saline solution that can penetrate the red blood cell membrane, causing an imbalance of osmotic pressure inside and outside the cell, thereby rupturing the red blood cells. Add the lysate to the test tube containing the single cell suspension in a certain proportion (such as 1 or 10 times the cell volume), mix gently, and incubate at room temperature for 5-10 minutes. Centrifuge the lysed cell suspension at a low speed (about 300g) for 5 minutes to precipitate cell debris and ruptured red blood cells. After centrifugation, carefully remove the supernatant, taking care not to aspirate the precipitated cells. Then, resuspend the cell pellet with phosphate buffered saline (PBS) or appropriate culture medium. Resuspend the cells again with PBS or culture medium, then centrifuge at the same low speed, and repeat the steps of removing the supernatant and resuspending at least once to ensure that the residual lysate and red blood cell fragments are completely removed.

[0073] Optionally, the present application uses a specific enzyme combination, including collagenase, hyaluronidase and papain. The use of this composite digestive enzyme can optimize the efficiency of tissue decomposition to more effectively decompose the extracellular matrix, thereby releasing more single cells and reducing damage to the cells.

[0074] Optionally, the components of the above-mentioned complex digestive enzyme are as follows:

[0075] Collagenase: 1-2 mg / mL

[0076] Hyaluronidase: 0.05-0.2U / mL

[0077] Papain: 0.2-0.5 mg / mL

[0078] When performing flow cytometry analysis on the digested cell suspension, the inventors found that when the following composite digestive enzymes are used, they have a higher affinity for tumor stem cells (CSCs) in tumor tissues and can specifically separate tumor stem cells, especially CD133+ cell populations, thereby achieving enrichment of CSCs in the conventional single cell suspension preparation process:

[0079] Collagenase: 1-2 mg / mL

[0080] Hyaluronidase: 0.05-0.2U / mL

[0081] Papain: 0.2-0.5 mg / mL

[0082] Elastase: 0.02-0.1 U / mL

[0083] Chondroitinase ABC: 0.01-0.1U / mL

[0084] The present application will be further described in detail below with reference to specific embodiments.

[0085] It should be noted that in the following examples, the following reagents / articles used were purchased from the market:

[0086] General purpose collagenase (C5894, CV1), collagenase (C1889, CV2), hyaluronidase (H4272, Hya), papain (Papain), elastase (Elastase), chondroitinase ABC (Chondroitinase ABC), culture medium (gibco), stop solution (RPMI-1640 complete culture medium).

[0087] In the following examples, liver, kidney and intestinal tissues of mice were collected from Slake Jingda Animal Company and stored in cell preservation medium at 4°C.

[0088] Embodiment 1

[0089] In this embodiment, the kidneys of the same mouse were used as experimental materials, and three groups were set up respectively:

[0090] 1. Use the tissue dissociation solution prepared with the composite digestive enzyme in this example and use a single cell suspension instrument to perform tissue dissociation to prepare a primary cell suspension;

[0091] 2. Use universal tissue dissociation solution and single cell suspension instrument to prepare primary cell suspension. The universal tissue dissociation solution was purchased from Shanghai Jingxin and contains CV1.

[0092] 3. Use the tissue dissociation solution containing the self-prepared complex digestive enzyme in this example to manually dissociate the tissue to prepare the primary cell suspension.

[0093] Tissue acquisition and cleaning

[0094] 1. Take 300 mg of liver, kidney and intestinal tissue from the same mouse, transfer the samples quickly to a sterile culture dish filled with 4°C serum-free culture medium or PBS, and place the culture dish on ice.

[0095] 2. Take a mung bean or soybean-sized tissue according to the experimental requirements. Wash the above tissues three times with PBS containing 1% double antibody, and cut away fat, necrotic tissue and other useless tissues in the tissue blocks.

[0096] Enzymatic digestion, mechanical separation

[0097] 1. Use scissors to cut the tissue into 1-2 mm pieces 3 The small pieces on the left and right were washed once with 4°C D-Hank's solution to remove as much tissue fragments as possible during the tissue cutting process.

[0098] 2. After the single cell preparation reagent is completely thawed, add 300 mg of tissue and compound digestive enzymes to the single cell preparation test tubes respectively. The liquid level is higher than the test tube mouth. The test tube caps are moved horizontally and tightly closed (try to avoid bubbles). Put them into the single cell suspension instrument and select the built-in program to run (select the corresponding program according to the ease of sample processing, select program 1 for visceral tissue, program 2 for lesion fibrosis tissue, and program for skin, gastrointestinal and other tissues).

[0099] 3. After digestion is completed, shake the sample tube appropriately, observe the turbidity and color, and filter out the single cell suspension with a cell sieve, then add the digestion stop solution to finally obtain the cell suspension.

[0100] In this embodiment, the composite digestive enzyme used is:

[0101] CV1: 1mg / mL

[0102] CV2: 2mg / mL

[0103] Hya: 0.08U / mL

[0104] Papain: 0.3 mg / mL.

[0105] In the third group of experimental samples, after instrument dissociation, there were still large pieces of tissue visible to the naked eye in the tissue dissociation solution, indicating that the samples were not completely dissociated; after manual dissociation, there were no large pieces of tissue in the tissue dissociation solution, indicating that the samples were basically digested completely. The steps for manual tissue dissociation are as follows:

[0106] First, place the sample in the preheating area and set the preheating temperature to 37.5°C. Keep the sample heated to 37.5°C for 5 minutes. Then, set the temperature of the digestion area to 37.5°C and transfer the sample from the preheating area to the digestion area. Next, use mechanical tools such as blades, glass rods or cell scrapers and other physical forces to chop or scrape the tissue. In this embodiment, the shear speed is set to 1 rpm, and digestion is performed according to a specific reciprocating operation mode, that is, after running 1 / 2 turn, pause for 0.3 seconds, and then continue to run 1 / 2 turn. This process requires 20 minutes of reciprocating operation.

[0107] Figures 2 to 4 Three groups of cell quality inspection result diagrams of this embodiment are shown respectively. At the same time, the quality inspection results of the initial cell suspensions of the three groups of experimental samples are shown in Table 1. It can be seen from Table 1 that the cell suspensions obtained by the composite digestive enzyme treatment of the present application have higher cell activity.

[0108] Table 1

[0109]

[0110] Embodiment 2

[0111] This example performs red lysis treatment on the basis of Example 1, and after the treatment, the cells are washed twice and then resuspended for detection.

[0112] Figures 5 to 7 The three groups of cell quality inspection results of this embodiment are shown in Figure 2. At the same time, the final cell suspension quality inspection results of the three groups of experimental samples are shown in Table 2. It can be seen from Table 2 that the cell suspension obtained by the composite digestive enzyme treatment of the present application has a high cell activity. On the other hand, compared with Table 1, it can be seen that after red lysis treatment and washing, the cell concentration has changed: the cell concentration of kidney 1 and kidney 2 has increased significantly, which is 7.77×10 5 cell / mL and 5.88×10 5 cell / mL, while the cell concentration of kidney 3 decreased to 1.02×10 6 The cell activity remained at a high level, with a slight decrease in the cell activity of kidney 1 and kidney 2, which were 93.55% and 83.71%, respectively, while the cell activity of kidney 3 was 91.8%.

[0113] Table 2

[0114]

[0115] In combination with Example 1 and Example 2, it can be seen that using the composite digestive enzyme of the present application, cells can be effectively released from kidney tissues of different weights through a single cell suspension preparation instrument. At the same time, despite dissociation and red lysis treatment, cell activity is still maintained at a high level, which indicates that the method used causes less damage to the cells.

[0116] Embodiment 3

[0117] The inventors found that when using collagenase, hyaluronidase, papain, elastase and chondroitinase ABC as a composite digestive enzyme, the prepared single cell suspension can specifically enrich CSCs.

[0118] In this example, the liver of the same tumor model mouse was used as the experimental material, and three groups were set up respectively:

[0119] 1. Use the tissue dissociation solution prepared with the composite digestive enzyme in this example and use a single cell suspension instrument to perform tissue dissociation to prepare a primary cell suspension;

[0120] 2. Use universal tissue dissociation solution and single cell suspension instrument to prepare primary cell suspension. The universal tissue dissociation solution was purchased from Shanghai Jingxin and contains CV1.

[0121] 3. Use the tissue dissociation solution of Example 1 and a single cell suspension instrument to perform tissue dissociation to prepare a primary cell suspension.

[0122] The steps of this embodiment are as follows:

[0123] Tissue dissociation: Mechanically cut the tissue into 2 mm 3 Use sterile surgical scissors to manipulate the small pieces.

[0124] Enzyme digestion: Under sterile conditions, transfer the tissue pieces into a digestion solution containing complex digestive enzymes. The digestion solution in this example consists of the following enzymes:

[0125] CV1: 1mg / mL

[0126] CV2: 2mg / mL

[0127] Hya:0.1U / mL

[0128] Papain: 0.3 mg / mL

[0129] Elastase: 0.05U / mL

[0130] Chondroitinase ABC: 0.05U / mL

[0131] Digestion process: The digestion solution containing the tissue was placed in a single cell suspension instrument and incubated at 37°C with gentle mechanical agitation during the digestion process. The initial digestion time was 20 minutes.

[0132] Real-time monitoring: Take samples every 5 minutes and use a microscope to observe the cell separation and assess whether the digestion time needs to be extended.

[0133] Stop digestion: After digestion is completed, mix the digestion solution with pre-cooled stop solution (DMEM medium containing 10% FBS) to stop the activity of the enzyme.

[0134] Filtration: Filter the digested cell suspension through a 70 μm cell sieve to remove undigested tissue debris.

[0135] Lysis treatment: Add 4 times the cell volume of lysate to the test tube containing the single cell suspension, mix gently, and incubate at room temperature for 5 minutes. Centrifuge the filtered cell suspension at 300g for 5 minutes, remove the supernatant, resuspend the cell pellet with PBS, and centrifuge again to wash the cells and remove residual enzymes.

[0136] Cell counting and activity detection: Trypan blue staining exclusion method was used to detect cell activity of cell suspension, and flow cytometry was used to analyze the proportion of CD133+CSCs.

[0137] The quality inspection results of the three groups of experimental samples in this embodiment are shown in Table 3.

[0138] Table 3

[0139]

[0140] As shown in Table 3, the composite digestive enzyme of the present embodiment significantly increases the proportion of CD133+CSCs. Preliminary judgment shows that chondroitin sulfate is an abundant component in many tumor ECMs and is closely related to the invasion and metastasis of tumor cells. Chondroitin sulfate enzyme ABC can specifically degrade chondroitin sulfate, which may help reduce the adhesion and diffusion of CSCs, thereby promoting their separation from tumor tissue. Papain and elastase have a synergistic effect on those ECM components that have been partially degraded by collagenase and hyaluronidase, which may lead to further disintegration of the ECM structure, providing more pathways for the release of CSCs.

[0141] This example uses a specific enzyme combination and optimized enzymatic hydrolysis conditions to effectively improve the enrichment efficiency of CD133+CSCs while maintaining cell activity, thereby providing high-quality cell materials for subsequent tumor detection and treatment applications.

[0142] The above embodiments are merely illustrative of the principles and effects of the present application and are not intended to limit the present application. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed in the present application shall still be covered by the claims of the present application.

Claims

1. A method for preparing a temperature-controlled tissue by gently dissociating it into single cells, characterized in that: The following steps are involved: S1. providing a tissue sample, and pre-processing the tissue sample to obtain a sample block; S2, mixing the sample block with a composite digestive enzyme, and filtering to obtain a single cell suspension after the digestion treatment is completed; wherein the composite digestive enzyme includes collagenase, hyaluronidase and papain; and selectively, S3, performing red removal treatment; In step S2, the digestion treatment is performed in a single cell suspension instrument; In step S2, the sample block is mixed with a composite digestive enzyme, and after the digestion treatment is completed, filtering to obtain a single cell suspension comprises: adding the sample block and complex digestive enzyme into a test tube; placing the test tube in the single cell suspension instrument and performing digestion treatment after preheating; After the digestion treatment is completed, the single cell suspension in the test tube is filtered out through a cell sieve, and a digestion stop solution is added to the single cell suspension; During the digestion process, the digestion temperature is 36.5-37.5°C, mechanical stirring is performed at a rotation speed of 40-60RPM, and regular sampling is performed every 5-10 minutes to observe the cell separation.

2. The method for preparing temperature-controllable tissue gentle dissociation into single cells as claimed in claim 1, characterized in that: In step S1, providing a tissue sample and pre-treating the tissue sample to obtain sample fragments comprises: Obtain tissue samples from living animals and transfer them into culture medium; washing the tissue sample to remove red blood cells and useless tissue; The cleaned tissue sample is cut into small pieces and washed with a buffer solution to remove debris generated during the process, thereby obtaining the sample piece.

3. The method for preparing temperature-controllable tissue gentle dissociation into single cells as claimed in claim 2, characterized in that: The culture medium is a culture solution at 4-6° C. or a sterile culture dish containing PBS, and the culture medium does not contain serum.

4. The method for preparing temperature-controllable tissue gentle dissociation into single cells as claimed in claim 1, characterized in that: In step S3, the step of adding red blood cell lysis solution to perform red blood cell removal treatment comprises: Add red blood cell lysis solution to the single cell suspension, centrifuge to remove lysed red blood cells, add PBS to resuspend, then wash by centrifugation and resuspend in culture medium.

5. The method for preparing temperature-controllable tissue gentle dissociation into single cells as claimed in claim 1, characterized in that: The complex digestive enzymes include: Collagenase: 1-2 mg / mL Hyaluronidase: 0.05-0.2U / mL Papain: 0.2-0.5 mg / mL.

6. The method for preparing temperature-controllable tissue gentle dissociation into single cells according to any one of claims 1 to 5, characterized in that: The complex digestive enzyme also includes: Elastase: 0.02-0.1 U / mL Chondroitinase ABC: 0.01-0.1U / mL.

7. A single-cell sequencing method, characterized in that: The single-cell sequencing method is to prepare the single-cell suspension according to the method for preparing temperature-controlled tissue gentle dissociation into single cells as described in any one of claims 1 to 6, and then perform sequencing.

8. Use of the method for preparing temperature-controllable tissue gentle dissociation into single cells according to any one of claims 1 to 6 in preparing cells for tumor detection and treatment.

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