A method for establishing a companion tumor suspension cell line

Abstract

The application discloses a method for establishing a tumor suspension cell line, which comprises a flat adherent culture stage, a suspension enrichment stage and a suspension culture stage. The method can quickly and simply establish a corresponding tumor suspension cell line with suspension cell characteristics from a tumor cell line cultured on a flat surface, and the quantity is huge, which is convenient for preservation and expansion, so that subsequent research can be carried out. In addition, the method can be directly used as one of evaluation indexes of the long-distance migration ability of tumor cells. By using the method, all tumor cell lines cultured on a flat surface can be tested, so that whether the tumor cell line cultured on a flat surface can produce a tumor suspension cell line and the kinetic behavior difference of the tumor suspension cell line can be quickly distinguished.

Description

Technical Field

[0001] This invention belongs to the technical field of establishing cell lines, specifically relating to a method for establishing a concomitant tumor suspension cell line. Background Technology

[0002] Malignant tumors are one of the major threats to human health. The vast majority of cancer-related deaths are caused by tumor recurrence and metastasis. However, the mechanisms of tumor metastasis are not fully understood. Elucidating the mechanisms of tumor recurrence and metastasis is crucial for developing new diagnostic and therapeutic methods. Solid tumors continuously shed tumor cells, some of which enter the bloodstream and are called circulating tumor cells (CTCs). CTCs are a key medium for the hematogenous metastasis of malignant tumors. If the specific markers and biological characteristics of CTCs can be identified, it is hoped that new diagnostic methods for tumor metastasis risk can be established, and even specific drugs can be developed to reduce the incidence of tumor metastasis.

[0003] However, the number of circulating tumor cells (CTCs) is extremely small. The vast majority of cancer patients have fewer than 10 CTCs per 10 mL of peripheral blood. Yet, the same volume of peripheral blood contains tens of millions of blood cells, posing significant challenges to the extraction and study of CTCs. Furthermore, CTCs differ in function and phenotype from their parent solid tumor cells. CTCs undergo a series of changes, including epithelial-mesenchymal transition, enabling them to adapt to unanchored growth and the shear forces generated by blood flow. Moreover, studies have shown that CTC phenotypes are heterogeneous and dynamically changing, making it difficult to establish definitive, standardized CTC-specific markers. This further complicates CTC research. Additionally, for CTCs to survive in the circulatory system, they need different structural and mechanical characteristics than adherent tumor cells to withstand the shear forces of rapid blood flow. Therefore, CTCs exhibit the characteristics of suspension cells. Developing a method to obtain a large number of cells with suspension characteristics from in vitro cultured adherent tumor cell lines would be a tremendous help and breakthrough in the study of tumor metastasis and recurrence. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide a method for establishing a companion tumor suspension cell line.

[0005] To achieve the above objectives, the first technical solution of the present invention is as follows: a method for establishing a concomitant tumor suspension cell line, the method specifically including the following steps:

[0006] S1. Planar Adherent Culture Stage: During planar culture, aseptic culture flasks are used to culture tumor cell lines, with the largest surface area of ​​the aseptic culture flask facing down to maximize the area available for cell spread. The volume of culture medium used is recorded as the initial volume, and the culture flask is designated as culture flask a. The culture medium is then replaced, and the cells are passaged when they reach 80-90% confluence.

[0007] S2, Suspension enrichment stage: Take another sterile culture bottle and denote it as culture bottle b. Add all the old culture medium that has been replaced multiple times in culture bottle a to culture bottle b. Place the smaller side of culture bottle b downwards, let it stand, and then slowly aspirate the upper culture medium, retaining the lower culture medium containing the enriched concomitant tumor suspension cells.

[0008] S3, Suspension Culture Stage: Add culture medium suitable for suspension cell culture to culture flask b, so that the volume of culture medium in culture flask b is restored to the level suitable for suspension culture, and does not exceed the amount at the mouth of the culture flask. Then, place the culture flask b with the largest surface area facing down, gently shake it to disperse the cells evenly, and then carry out static suspension culture.

[0009] Preferably, in S1, the tumor cells are SK-BR-3 cells.

[0010] Preferably, in S1, the culture medium is DMEM medium containing 5-15% serum and 0.5-1.5% penicillin-dipeptide antibiotics, or α-MEM medium containing 5-15% serum and 0.5-1.5% penicillin-dipeptide antibiotics, or RPMI1640 medium containing 5-15% serum and 0.5-1.5% penicillin-dipeptide antibiotics.

[0011] Preferably, in step S1, the culture medium is changed every 2-3 days.

[0012] Preferably, in step S2, the settling time is 3-15 minutes.

[0013] Preferably, in S3, the culture medium suitable for suspension cell culture is RPMI1640 medium containing 5-15% serum and 0.5-1.5% penicillin-dextrin, or DMEM medium containing 5-15% serum and 0.5-1.5% penicillin-dextrin, or α-MEM medium containing 5-15% serum and 0.5-1.5% penicillin-dextrin.

[0014] Compared with existing technologies, the method of this invention can rapidly and easily establish corresponding companion tumor suspension cell lines with suspension cell characteristics from adherent tumor cell lines, and the quantity is huge, which is easy to preserve and expand for subsequent research. In addition, the method of this invention can be directly used as one of the evaluation indicators of tumor cell distal migration ability. By using the method of this invention to test all in vitro adherent tumor cell lines, it is possible to quickly distinguish whether the adherent tumor cell lines can produce companion tumor suspension cell lines, and the differences in the dynamics and other behaviors of the companion tumor suspension cell lines. Furthermore, for adherent tumor cells that can produce companion tumor suspension cell lines, the phenotypic and functional differences between adherent tumor cells and their corresponding suspension tumor cells can be compared, thereby providing a powerful research tool for developing new methods for the diagnosis and treatment of tumor metastasis and recurrence. Attached Figure Description

[0015] Figure 1 is a schematic diagram comparing the flow cytometry analysis of adherent cells and associated tumor suspension cells of SK-BR-3, wherein:

[0016] Figure 1A A schematic diagram of flow cytometry analysis of adherent cells of SK-BR-3.

[0017] Figure 1B This is a schematic diagram of flow cytometry analysis of suspended cells associated with tumors.

[0018] Figure 2 This is a confocal comparison diagram of adherent cells and associated tumor suspension cells of SK-BR-3. The top row of images shows a confocal comparison of adherent cells of SK-BR-3, and the bottom row shows a confocal comparison of associated tumor suspension cells.

[0019] Figure 3 The diagrams show the results of nude mouse fat pad tumor transplantation experiments using SK-BR-3 adherent cells and associated tumor suspension cells. The top row shows the results of the nude mouse fat pad tumor transplantation experiment using SK-BR-3 adherent cells, and the bottom row shows the results of the nude mouse fat pad tumor transplantation experiment using associated tumor suspension cells.

[0020] Figure 4 This image shows schematic diagrams of immunohistochemical staining (HER2) of tumor sections extracted from SK-BR-3 adherent cells and concomitant tumor suspension cells after nude mouse fat pad tumor transplantation experiments. The left and upper-middle images show the immunohistochemical staining (HER2) of tumor sections extracted from SK-BR-3 adherent cells after nude mouse fat pad tumor transplantation experiments; the right and lower-middle images show the immunohistochemical staining (HER2) of tumor sections extracted from concomitant tumor suspension cells after nude mouse fat pad tumor transplantation experiments.

[0021] Figure 5 Volcano plot of transcriptomes of adherent cells and associated tumor suspension cells of SK-BR-3;

[0022] Figure 6 A heatmap of the transcriptome of SK-BR-3 adherent cells and associated tumor suspension cells. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0024] This invention establishes a co-culture method: first, SK-BR-3 cells are cultured in an adherent state, then enriched in suspension, and finally transferred to suspension culture, successfully obtaining a suspension tumor cell line. Through in vitro cell and animal experiments, we determined that this new cell line has different phenotypic and functional characteristics from SK-BR-3. Therefore, we named this cell line the SK-BR-3 concomitant tumor suspension cell line. The co-culture method established in this invention can be used to screen more adherent tumor cell lines, with the aim of discovering more concomitant tumor suspension cell lines. This provides a powerful tool for studying the mechanisms of tumor metastasis and recurrence, as well as developing novel diagnostic and therapeutic targets for tumor metastasis.

[0025] The culture of a companion tumor suspension cell line provided in this embodiment of the invention is divided into three stages: planar adherent culture, suspension enrichment, and suspension culture. The specific establishment method includes the following steps:

[0026] S1. Planar Adherent Culture Stage: For planar culture, aseptic culture flasks are used to culture tumor cell lines, with the largest surface area of ​​the flask facing down to maximize the area available for cell spread. The culture medium used is DMEM medium containing 5-15% serum and 0.5-1.5% penicillin antibiotics, or α-MEM medium containing 5-15% serum and 0.5-1.5% penicillin antibiotics, or RPMI 1640 medium containing 5-15% serum and 0.5-1.5% penicillin antibiotics. The volume of culture medium used is recorded as the initial volume, and the culture flask is designated as culture flask a. The culture medium is changed every 2-3 days. When the cells reach 80-90% confluence, they are passaged.

[0027] S2, Suspension Enrichment Stage: Take another sterile culture flask, designated as culture flask b. Add the DMEM culture medium that has been replaced multiple times in culture flask a to culture flask b, with the smaller side of culture flask b facing down. Let it stand for 3-15 minutes, then slowly aspirate the upper layer of culture medium, retaining the lower layer containing the enriched concomitant tumor suspension cells. Steps 2 and 3 can be repeated to further increase the number of concomitant tumor suspension cells in culture flask b.

[0028] There are two key points to note at this stage. First, because the cells in the culture medium are in suspension, they will not adhere to the bottom surface; instead, they will simply settle and accumulate due to gravity. Second, not all tumor cells cultured in a planar adherent state can produce suspension tumor cells capable of surviving and proliferating in suspension. Only tumor cells cultured in a planar adherent state that can produce concomitant tumor suspension cells can be used to obtain sufficient concomitant tumor suspension cells through suspension enrichment methods for the next stage of suspension culture to achieve the goal of establishing a cell line.

[0029] S3, Suspension Culture Stage: Add RPMI 1640 medium containing 5-15% serum and 0.5-1.5% antibiotics, or DMEM medium containing 5-15% serum and 0.5-1.5% antibiotics, or α-MEM medium containing 5-15% serum and 0.5-1.5% antibiotics, to culture flask b to restore the volume of the medium to a level suitable for suspension culture, without exceeding the amount at the mouth of culture flask b. Then, place the culture flask b with the largest surface area facing down and gently shake it to disperse the cells evenly before proceeding with static suspension culture.

[0030] It is important to note that not all tumor cell lines produced from adherent culture in planar environments generate suspension cells capable of unlimited proliferation in suspension. Only concomitant tumor suspension cells with unlimited proliferation capacity in suspension can establish cell lines, such as the concomitant tumor suspension cells derived from SK-BR-3 disclosed in this invention.

[0031] The following are specific embodiments.

[0032] Example 1

[0033] The concomitant tumor suspension cell line provided in Example 1 of this invention was obtained through the following method:

[0034] S1. Planar Adherent Culture Stage: SK-BR-3 cell lines are cultured in sterile culture flasks with the largest surface area facing down to maximize cell spread. The culture medium is DMEM containing 10% serum and 1% penicillin antibiotics. The volume of culture medium used is recorded as the initial volume, and the culture flask is designated as culture flask a. The culture medium is changed every 2 days. Cells are passaged when they reach 80-90% confluence.

[0035] S2, Suspension enrichment stage: Take another sterile culture bottle and denote it as culture bottle b. Add the DMEM culture medium that has been replaced multiple times in culture bottle a to culture bottle b. Place the smaller side of culture bottle b downwards and let it stand for 10 minutes. Then slowly aspirate the upper culture medium and retain the lower culture medium containing the enriched concomitant tumor suspension cells.

[0036] S3, Suspension Culture Stage: Add RPMI 1640 medium containing 10% serum and 1% penicillin antibiotics to culture flask b to restore the volume of medium in culture flask b to a level suitable for suspension culture, without exceeding the amount at the mouth of culture flask b. Then, place the culture flask b with the largest surface area facing down and gently shake it to disperse the cells evenly before proceeding with static suspension culture.

[0037] Example 2

[0038] The concomitant tumor suspension cell line provided in Example 2 of this invention was obtained through the following method:

[0039] S1. Planar Adherent Culture Stage: SK-BR-3 cell lines are cultured in sterile culture flasks with the largest surface area facing down to maximize cell spread. The culture medium is DMEM containing 5% serum and 0.5% antibiotics. The volume of culture medium used is recorded as the initial volume, and the culture flask is designated as culture flask a. The culture medium is changed every 2 days. Cells are passaged when they reach 80-90% confluence.

[0040] S2, Suspension enrichment stage: Take another sterile culture bottle and denote it as culture bottle b. Add the DMEM culture medium that has been replaced multiple times in culture bottle a to culture bottle b. Place the smaller side of culture bottle b downwards and let it stand for 3 minutes. Then slowly aspirate the upper culture medium and retain the lower culture medium containing the enriched concomitant tumor suspension cells.

[0041] S3, Suspension Culture Stage: Add RPMI 1640 medium containing 5% serum and 0.5% antibiotics to culture flask b to restore the volume of medium in culture flask b to a level suitable for suspension culture, without exceeding the amount at the mouth of culture flask b. Then, place the culture flask b with the largest surface area facing down and gently shake it to disperse the cells evenly before proceeding with static suspension culture.

[0042] Example 3

[0043] The concomitant tumor suspension cell line provided in Example 3 of this invention was obtained through the following method:

[0044] S1. Planar Adherent Culture Stage: SK-BR-3 cell lines are cultured in sterile culture flasks with the largest surface area facing down to maximize cell spread. The culture medium is DMEM containing 15% serum and 1.5% penicillin-dextrose antibodies. The volume of culture medium used is recorded as the initial volume, and the culture flask is designated as culture flask a. The culture medium is changed every 3 days. Cells are passaged when they reach 80-90% confluence.

[0045] S2, Suspension enrichment stage: Take another sterile culture bottle and denote it as culture bottle b. Add the DMEM culture medium that has been replaced multiple times in culture bottle a to culture bottle b. Place the smaller side of culture bottle b downwards and let it stand for 15 minutes. Then slowly aspirate the upper culture medium and retain the lower culture medium containing the enriched concomitant tumor suspension cells.

[0046] S3, Suspension Culture Stage: Add RPMI 1640 medium containing 15% serum and 1.5% antibiotics to culture flask b to restore the volume of medium in culture flask b to a level suitable for suspension culture, without exceeding the amount at the mouth of culture flask b. Then, place the culture flask b with the largest surface area facing down and gently shake it to disperse the cells evenly before proceeding with static suspension culture.

[0047] Example 4

[0048] The concomitant tumor suspension cell line provided in Example 4 of this invention was obtained through the following method:

[0049] S1. Planar Adherent Culture Stage: SK-BR-3 cell lines are cultured in sterile culture flasks with the largest surface area facing down to maximize cell spread. The culture medium is α-MEM containing 10% serum and 1% penicillin antibiotics. The volume of culture medium used is recorded as the initial volume, and the culture flask is designated as culture flask a. The culture medium is changed every 2 days. Cells are passaged when they reach 80-90% confluence.

[0050] S2, Suspension enrichment stage: Take another sterile culture bottle and denote it as culture bottle b. Add the DMEM culture medium that has been replaced multiple times in culture bottle a to culture bottle b. Place the smaller side of culture bottle b downwards and let it stand for 10 minutes. Then slowly aspirate the upper culture medium and retain the lower culture medium containing the enriched concomitant tumor suspension cells.

[0051] S3, Suspension Culture Stage: Add RPMI 1640 medium containing 10% serum and 1% penicillin antibiotics to culture flask b to restore the volume of medium in culture flask b to a level suitable for suspension culture, without exceeding the amount at the mouth of culture flask b. Then, place the culture flask b with the largest surface area facing down and gently shake it to disperse the cells evenly before proceeding with static suspension culture.

[0052] Example 5

[0053] The concomitant tumor suspension cell line provided in Example 5 of this invention was obtained through the following method:

[0054] S1. Planar Adherent Culture Stage: SK-BR-3 cell lines are cultured in sterile culture flasks with the largest surface area facing down to maximize cell spread. The culture medium is α-MEM containing 5% serum and 1% penicillin antibiotics. The volume of culture medium used is recorded as the initial volume, and the culture flask is designated as culture flask a. The culture medium is changed every 2 days. Cells are passaged when they reach 80-90% confluence.

[0055] S2, Suspension enrichment stage: Take another sterile culture bottle and denote it as culture bottle b. Add the DMEM culture medium that has been replaced multiple times in culture bottle a to culture bottle b. Place the smaller side of culture bottle b downwards and let it stand for 10 minutes. Then slowly aspirate the upper culture medium and retain the lower culture medium containing the enriched concomitant tumor suspension cells.

[0056] S3, Suspension Culture Stage: Add RPMI 1640 medium containing 5% serum and 1% penicillin antibiotics to culture flask b to restore the volume of medium in culture flask b to a level suitable for suspension culture, without exceeding the amount at the mouth of culture flask b. Then, place the culture flask b with the largest surface area facing down and gently shake it to disperse the cells evenly before proceeding with static suspension culture.

[0057] Example 6

[0058] The concomitant tumor suspension cell line provided in Example 6 of this invention was obtained through the following method:

[0059] S1. Planar Adherent Culture Stage: SK-BR-3 cell lines are cultured in sterile culture flasks with the largest surface area facing down to maximize cell spread. The culture medium is α-MEM containing 15% serum and 1% penicillin antibiotics. The volume of culture medium used is recorded as the initial volume, and the culture flask is designated as culture flask a. The culture medium is changed every 2 days. Cells are passaged when they reach 80-90% confluence.

[0060] S2, Suspension enrichment stage: Take another sterile culture bottle and denote it as culture bottle b. Add the DMEM culture medium that has been replaced multiple times in culture bottle a to culture bottle b. Place the smaller side of culture bottle b downwards and let it stand for 10 minutes. Then slowly aspirate the upper culture medium and retain the lower culture medium containing the enriched concomitant tumor suspension cells.

[0061] S3, Suspension Culture Stage: Add RPMI 1640 medium containing 15% serum and 1% penicillin antibiotics to culture flask b to restore the volume of medium in culture flask b to a level suitable for suspension culture, without exceeding the amount at the mouth of culture flask b. Then, place the culture flask b with the largest surface area facing down and gently shake it to disperse the cells evenly before proceeding with static suspension culture.

[0062] Example 7

[0063] The concomitant tumor suspension cell line provided in Example 7 of this invention was obtained through the following method:

[0064] S1. Planar Adherent Culture Stage: SK-BR-3 cell lines are cultured in sterile culture flasks with the largest surface area facing down to maximize cell spread. The culture medium used is RPMI 1640 medium containing 10% serum and 1% penicillin antibiotics. The volume of culture medium used is recorded as the initial volume, and the culture flask is designated as culture flask a. The culture medium is changed every 2 days. Cells are passaged when they reach 80-90% confluence.

[0065] S2, Suspension enrichment stage: Take another sterile culture bottle and denote it as culture bottle b. Add the DMEM culture medium that has been replaced multiple times in culture bottle a to culture bottle b. Place the smaller side of culture bottle b downwards and let it stand for 10 minutes. Then slowly aspirate the upper culture medium and retain the lower culture medium containing the enriched concomitant tumor suspension cells.

[0066] S3, Suspension Culture Stage: Add DMEM medium containing 10% serum and 1% antibiotics suitable for suspension cell culture to culture flask b, so that the volume of medium in culture flask b is restored to a level suitable for suspension culture, without exceeding the amount at the mouth of culture flask b. Then, place the culture flask b with the largest surface area facing down, gently shake it to disperse the cells evenly, and then carry out static suspension culture.

[0067] Example 8

[0068] The concomitant tumor suspension cell line provided in Example 8 of this invention was obtained through the following method:

[0069] S1. Planar Adherent Culture Stage: SK-BR-3 cell lines are cultured in sterile culture flasks with the largest surface area facing down to maximize cell spread. The culture medium used is RPMI 1640 medium containing 10% serum and 1% penicillin antibiotics. The volume of culture medium used is recorded as the initial volume, and the culture flask is designated as culture flask a. The culture medium is changed every 2 days. Cells are passaged when they reach 80-90% confluence.

[0070] S2, Suspension enrichment stage: Take another sterile culture bottle and denote it as culture bottle b. Add the DMEM culture medium that has been replaced multiple times in culture bottle a to culture bottle b. Place the smaller side of culture bottle b downwards and let it stand for 10 minutes. Then slowly aspirate the upper culture medium and retain the lower culture medium containing the enriched concomitant tumor suspension cells.

[0071] S3, Suspension Culture Stage: Add α-MEM medium containing 10% serum and 1% antibiotics suitable for suspension cell culture to culture flask b, so that the volume of medium in culture flask b is restored to a level suitable for suspension culture, without exceeding the amount at the mouth of culture flask b. Then, place the culture flask b with the largest surface area facing down, gently shake it to disperse the cells evenly, and then carry out static suspension culture.

[0072] To verify the performance of the tumor suspension cell line obtained by the method of the present invention, the specific steps are shown in Figure 1-. Figure 6 The detection experiment was conducted, and the results are shown in Figure 1- Figure 6 As shown.

[0073] Through analysis Figure 1A and Figure 1B It can be seen that the adherent cells and the associated tumor suspension cells of SK-BR-3 differ in size distribution and organelle complexity;

[0074] Through analysis Figure 2 It can be seen that the adherent cells and the associated tumor suspension cells of SK-BR-3 have significantly different cell sizes and cytoskeleton distributions;

[0075] Through analysis Figure 3 It can be seen that the adherent cells and associated tumor suspension cells of SK-BR-3 formed tumors of different sizes at the same time after being implanted into mice;

[0076] Through analysis Figure 4 It can be seen that after SK-BR-3 adherent cells and associated tumor suspension cells (right, lower middle), when implanted into mice, the adherent cells form large sheets of tumor tissue, while the suspension cells form micron-sized microtumor foci.

[0077] Through analysis Figure 5 and Figure 6 It can be seen that the gene expression of SK-BR-3 adherent cells and concomitant tumor suspension cells is different (red: genes upregulated in suspension cells compared to adherent cells; blue: genes downregulated in suspension cells compared to adherent cells).

[0078] In summary, the method of this invention can rapidly and easily establish corresponding companion tumor suspension cell lines with suspension cell characteristics from adherent tumor cell lines, and the resulting numbers are large, facilitating preservation and expansion for subsequent research. Since companion tumor suspension cells share similar adaptations to survive in fluids with circulating tumor cells, they are likely to have similar origins and evolution. This method has significant advantages: First, it can be directly used as an evaluation indicator of the distal migration ability of tumor cells. Second, by testing all in vitro adherent tumor cell lines using this method, it is possible to quickly distinguish whether adherent tumor cell lines can generate companion tumor suspension cell lines, and to observe the kinetic differences in the generation of these cells. More importantly, for adherent tumor cells that can generate companion tumor suspension cell lines, the phenotypic and functional differences between adherent tumor cells and their corresponding suspension tumor cells can be compared, providing a powerful research tool for developing new methods for the diagnosis and treatment of tumor metastasis and recurrence.

[0079] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for establishing a companion tumor suspension cell line, characterized in that, The method specifically includes the following steps: S1. Planar Adherent Culture Stage: During planar culture, aseptic culture flasks are used to culture tumor cell lines, with the largest surface area of ​​the aseptic culture flask facing down to maximize the area available for cell spread. The volume of culture medium used is recorded as the initial volume, and the culture flask is designated as culture flask a. The culture medium is then replaced, and the cells are passaged when they reach 80-90% confluence. S2, Suspension enrichment stage: Take another sterile culture bottle and denote it as culture bottle b. Add all the old culture medium that has been replaced multiple times in culture bottle a to culture bottle b. Place the smaller side of culture bottle b downwards, let it stand, and then slowly aspirate the upper culture medium, retaining the lower culture medium containing the enriched concomitant tumor suspension cells. S3, Suspension Culture Stage: Add culture medium suitable for suspension cell culture to culture flask b, so that the volume of culture medium in culture flask b is restored to the level suitable for suspension culture, and does not exceed the amount at the mouth of the culture flask. Then, place the culture flask b with the largest surface area facing down, gently shake it to disperse the cells evenly, and then carry out static suspension culture.

2. The method for establishing a concomitant tumor suspension cell line according to claim 1, characterized in that, In S1, the tumor cells are SK-BR-3 cells.

3. The method for establishing a concomitant tumor suspension cell line according to claim 2, characterized in that, In S1, the culture medium is DMEM medium containing 5-15% serum and 0.5-1.5% penicillin-dextrin antibiotics, or α-MEM medium containing 5-15% serum and 0.5-1.5% penicillin-dextrin antibiotics, or RPMI1640 medium containing 5-15% serum and 0.5-1.5% penicillin-dextrin antibiotics.

4. A method for establishing a concomitant tumor suspension cell line according to any one of claims 1-3, characterized in that, In S1, the culture medium is changed every 2-3 days.

5. The method for establishing a concomitant tumor suspension cell line according to claim 1, characterized in that, In step S2, the settling time is 3-15 minutes.

6. The method for establishing a concomitant tumor suspension cell line according to claim 1, characterized in that, In S3, the suitable culture medium for suspension cell culture is RPMI1640 medium containing 5-15% serum and 0.5-1.5% penicillin-dextrin, or DMEM medium containing 5-15% serum and 0.5-1.5% penicillin-dextrin, or α-MEM medium containing 5-15% serum and 0.5-1.5% penicillin-dextrin.

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