Method for in-vitro study on cell molecular biology characteristics of epithelial-mesenchymal transition / mesenchymal-epithelial transition of tumor cells

Abstract

The invention discloses a method for in-vitro study of tumor cell epithelial-mesenchymal transition / interstitial-epithelial transition cell molecular biological characteristics, which comprises the following steps: in a specific flowing suspension culture environment simulating a blood flow environment, using an epithelial marker and an interstitial marker which include but are not limited to the epithelial marker and the interstitial marker, and carrying out in-vitro study on tumor cell epithelial-mesenchymal transition / interstitial-epithelial transition cell molecular biological characteristics. The cell survival rate and the change of molecular biological characteristics of the cells in the adherent culture process before flowing, after flowing and after flowing are observed, and a simple and effective circulating tumor cell transformation in-vitro research method is provided. According to the method, epithelial / mesenchymal transformation and mesenchymal / epithelial transformation characteristics of target cells are observed in in-vitro culture cells for the first time, so that the characteristics of transformation from epithelial type to mesenchymal type in an in-vitro flowing suspension culture environment are reproduced for the first time; the method is used for analyzing the selective survival of target cells in a blood flowing environment and researching the survival and diffusion mechanism and diffusion capacity of different types of tumor cells in blood, and an effective research method is provided for clinical cancer diffusion prevention.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for studying the cell molecular biological characteristics of epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET) of tumor cells in vitro. Background Art

[0002] The occurrence of cancer seriously affects human health and quality of life. The spread of cancer is the main reason for the high cancer mortality rate. A large number of studies have shown that liquid biopsy technology, especially the detection and analysis of circulating tumor cells and circulating tumor genes, can detect cancer early, improve the treatment effect and the survival rate of cancer patients. However, these liquid biopsy technologies are believed to be able to detect early cancer, so early that sometimes no symptoms are found in the clinic. So, how do doctors make appropriate clinical treatment for patients, which also poses some challenges to the clinic. For example, after the detection of circulating tumor cells, if no definite symptoms can be found in the clinic, then what kind of cells are these circulating tumor cells? Do they have the ability to spread and form fatal cancer? These are currently difficult to track and verify in the patient's body. So, as a tumor cell, can it survive after entering the blood? What kind of changes in cell molecular biological characteristics will occur? There is a special need for corresponding effective research methods to explore its biological principles.

[0003] Circulating tumor cells (CTCs) are a general term for various tumor cells present in peripheral blood. They can be released from solid tumors or metastases into the peripheral blood circulation spontaneously or due to diagnostic and treatment procedures. They are an important cause of postoperative recurrence and distant metastasis in patients with malignant tumors, and an important factor leading to death in tumor patients. They are the most direct markers for cancer in liquid biopsy. CTCs detection enriches and tests CTCs present in trace amounts in peripheral blood, and monitors the trend of changes in the number of CTCs, so as to monitor tumor dynamics in real time, evaluate treatment effects, and achieve real-time personalized treatment. Studies have found that most CTCs undergo apoptosis or are phagocytosed after entering the peripheral blood, and a few escape and develop into metastatic lesions, increasing the risk of death in patients with malignant tumors. Therefore, detecting rare CTCs from patients' peripheral blood samples has become a new research hotspot.

[0004] A large number of studies have shown that CTCs undergo epithelial to mesenchymal transition (EMT) during the process of entering the peripheral blood circulation, resulting in different phenotypes, including epithelial, mesenchymal, and mixed epithelial-mesenchymal types. Among them, mesenchymal CTCs are considered to have stronger metastatic, drug-resistant, and invasive abilities. Therefore, in clinical applications, while detecting, identifying, and counting CTCs, it is also necessary to analyze the phenotype of CTCs for a complete assessment of tumor metastasis risk, disease progression, and prognosis. The epithelial-mesenchymal transition process is generally believed to be accompanied by overexpression of mesenchymal markers (such as CSV and Twist1), while the expression of epithelial markers (such as CK and EpCAM) is reduced. Epithelial cell adhesion molecule (EpCAM) is the most commonly used epithelial CTC enrichment marker, and vimentin (CSV) can be used as a marker for enriching mesenchymal CTCs.

[0005] At present, CTCs enrichment technologies mainly include positive or negative enrichment through cell surface markers, membrane filtration based on cell size, density gradient centrifugation, etc. The most common methods in the market are mainly immune positive capture method and negative enrichment method. The positive enrichment method based on immunomagnetic beads is currently the only CTCs detection technology approved by the Food and Drug Administration (FDA) for marketing. EpCAM-expressing target cells are captured by immunomagnetic beads coated with EpCAM antibodies. The most commonly used marker in traditional positive enrichment methods is epithelial cell adhesion molecule (EpCAM), which can capture epithelial and epithelial-stromal mixed CTCs in the blood. In theory, it should not be able to capture EpCAM-negative interstitial CTCs, which may lead to missed detection of interstitial CTCs.

[0006] However, when tumor cells enter the blood from tumor tissue, their living environment changes significantly, especially from the original static environment to the blood flow environment. It has been reported that after tumor cells enter the blood, they become circulating tumor cells (CTCs), and a large number of CTCs will die within 1-2 hours. So, what kind of scientific relationship does the death of these CTCs have with blood flow? There has been no good research method and no clear conclusion. In particular, different tumors and different cells need to be studied at the experimental level to explore the survival ability of different tumor cells through blood diffusion. This difference in survival ability may be used to predict the probability of tumor spread. Summary of the invention

[0007] In order to solve the urgent need for research methods when studying the cell molecular biological characteristics of epithelial-mesenchymal transition (EMT) / mesenchymal-epithelial transition (MET) of tumor cells, the main purpose of the present invention is to provide a method for studying the cell molecular biological characteristics of epithelial-mesenchymal transition / mesenchymal-epithelial transition of tumor cells in vitro. The present invention designs a cell flow suspension culture method that simulates the blood flow environment for analyzing and comparing the cells of static adherent culture and the cells of flow culture, and the cells after flow culture are then subjected to static adherent culture and other different cell culture environments, the molecular biological characteristics of the cells change, successfully capture the change law of the molecular biological characteristics of the target cells in the flow culture environment, and show the scientific research and clinical application value of the research method. The method of the present invention is simple, easy to operate, efficient, and has good repeatability, and is successfully applied to the study of the cell biological characteristics of epithelial-mesenchymal transition (EMT) / mesenchymal-epithelial transition (MET) of tumor cells in vitro.

[0008] In order to achieve the above object, in a basic embodiment, the present invention provides a method for studying the cell molecular biological characteristics of tumor cell epithelial-mesenchymal transition (EMT) / mesenchymal-epithelial transition (MET) in vitro, comprising the following steps:

[0009] S1. Use a special cell flow culture apparatus to place cells to be cultured in flow suspension in the flow culture apparatus to achieve cell non-adherent flow suspension culture;

[0010] S2. placing the flow culture device and the cells therein into a cell culture box, setting basic parameters such as temperature and CO2 concentration, and performing flow culture on the cells;

[0011] S3. After the flow culture begins, samples are taken at regular intervals to analyze markers of epithelial and mesenchymal types and related cell molecular biology levels, including molecular biological analysis of protein levels, RNA levels, and methylation levels;

[0012] S4. The cells cultured in the flow culture were relocated to the static cell culture dish for static adherent culture;

[0013] S5. For the cells that are cultured on the wall after the above-mentioned flow culture, regular analysis of mesenchymal and epithelial types and related cell molecular biology markers is performed, including cell molecular biology analysis at the protein level, RNA level and methylation level.

[0014] Preferably, in step S1, the flow frequency can be set but not limited to 50 rpm-250 rpm.

[0015] Optionally, in step S3, epithelial and mesenchymal types and related cell molecular biology markers include but are not limited to genes EpCAM (epithelial cell adhesion molecule), CKs (cytokeratins), N-cadherin (N-cadherin), FN1 (fibronectin 1), ZEB2 (zinc finger E-box binding homologous protein 12), ZEB1 (zinc finger E-box binding homologous protein 1), Snail2 (snail family transcription repressor), Snail1 (snail family transcription repressor), NANOG (homeobox), Aldh1A1 (aldehyde dehydrogenase 1A1), Oct4 (octamer binding transcription factor), Kit (KIT proto-oncogene, receptor tyrosine kinase), Vimentin (vimentin), Twist1 (twisted family bHLH transcription factor 1).

[0016] Preferably, in step S4, the target cells in flow culture are transferred to an adherent culture dish for adherent culture, which is to transfer the cell suspension to the adherent culture dish for culture; the cell suspension includes cell culture medium and target cells and avoids any adherent cells from mixing in.

[0017] For cells that have resumed adherent culture, samples are taken at regular intervals to perform cell molecular biology analysis of cell markers including protein and RNA levels and methylation levels, including analysis at the single cell level and multi-cell level.

[0018] Compare cells cultured on adherent, flow-cultured, and recovered adherent cultures for comparative analysis of epithelial, mesenchymal, and related cell molecular biomarkers, including specific genes, or broad-spectrum analysis.

[0019] Furthermore, the cell flow culture device includes a cell culture dish portion located at the upper part, a power pump located at the lower part for controlling the flow rate, direction and frequency of the culture medium, and a conduit located in the middle part for connecting the liquid culture medium portion with the power pump to achieve flow rate and flow direction control; wherein the cell culture dish portion is added with cell culture medium and target cells to be cultured.

[0020] Through the above technical scheme, the present invention achieves several key objectives. First, an effective cell flow suspension culture environment is provided, and the heart rate can be simulated to control the flow speed. Secondly, not only can the cell molecular biological characteristics of the epithelial-mesenchymal transition (EMT) of the target cells from static adherent culture to flow culture be observed by timed sampling, but also the cell molecular biological characteristics of the mesenchymal-epithelial transition (MET) of the target cells can be observed by returning from flow culture to static adherent culture. It is an effective method for studying the effect of flow suspension culture on the molecular biological characteristics of target cells. The inventive method provides a complete process of cell molecular biological characteristic observation, which is simple, easy to implement, and efficient, and is a technical method that has been successfully developed for the first time.

[0021] Compared with the prior art, the present invention has at least the following beneficial effects:

[0022] The content of the present invention was discovered by chance. When tumor cells were cultured at different flow rates to detect their survival rates, it was unexpectedly discovered that cells under static adherent culture and flow culture showed regular changes in epithelial and mesenchymal cell markers, which is similar to the characteristics of the epithelial-mesenchymal transformation of circulating tumor cells, and can be used to repeat some phenotypic changes and principle studies of epithelial-mesenchymal cell transformation in vitro. EMT and MET are the mechanisms of tumor spread and recurrence, and tumor cells will face the problem of adapting to the flow environment from a static environment after entering the blood system from tumor tissue. How can this change be repeated in the form of an experiment to facilitate in-depth research on its principles? The lack of a suitable research method has seriously affected the research progress. This change, on the one hand, is a change in cell survival rate, and on the other hand, there are also changes in different cellular molecular biological characteristics of cells with different phenotypes (epithelial, mesenchymal and epithelial-mesenchymal mixed types), or different types of tumor cells will also produce different changes in cellular molecular biological markers. These changes have been observed using different tumor cell lines. On the one hand, studying these changes can predict the survival and diffusion capabilities of specific tumor cells in the blood. On the other hand, it is also a research method to study the principles of cell adaptation and transformation in different environments. Future research can also be expanded to the same or different tumor cells, and their different gene mutations, methylation and other changes at the multi-cell or single-cell level. These changes may directly or indirectly affect or determine the survival of cells, and thus may also determine the possibility of tumor cells spreading through the blood. Therefore, the present invention is a particularly valuable research method.

[0023] The present invention uses, including but not limited to, epithelial markers and mesenchymal markers in a specific flow suspension culture environment simulating a blood flow environment, to observe the cell survival rate and changes in its molecular biological characteristics of cells before and after the flow and in the process of adhering to the wall after the flow, and provides a scientific research method model in vitro that can repeatedly study EMT / MET transformation. It provides a simple and effective research method for the change of cell biological characteristics during the process of target cells from static adherent growth to entering the blood and then returning to the static adherent culture process from circulating cells, and a simple and feasible research method is invented for the fluid biology research of circulating tumor cells (CTC). Using this method, epithelial-mesenchymal transformation and mesenchymal-epithelial transformation of target cells were observed in in vitro cultured cells for the first time, and can be further used to analyze the selective survival of target cells by blood and study the survival and diffusion mechanism and diffusion capacity of different types of tumor cells in the blood, providing an effective research method for the prevention of clinical cancer diffusion, and providing important clinical basic information. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0025] Figure 1 A cell flow culture apparatus (Yichang Micron Silicon Valley Life Science Co., Ltd.) used in the present invention is used for flow culture of cells.

[0026] Figure 2-A This is the expression level of the protein marker under flow culture in Example 2 of the present invention.

[0027] Vim / Twist1 (vimentin and twist protein 1) is clearly expressed. BF: target cells under bright field light; DAPI: cell nucleus; CK: keratin; Merge: composite photo of each marker.

[0028] Figure 2-B This is the expression level of the protein marker under adherent culture in Example 2 of the present invention.

[0029] Vim / Twist1 (vimentin and twist protein 1) has no obvious expression. BF: target cells under bright field light; DAPI: cell nucleus; CK: keratin; Merge: composite photo of each marker.

[0030] Figure 3-A This is the expression level of RNA markers of mesenchymal cells after the cells in Example 3 of the present invention are transformed from adherent culture to flow culture.

[0031] Figure 3-B This is the expression level of the interstitial RNA marker after the cells in Example 3 of the present invention are returned to static adherent culture from flow culture.

[0032] Figure 4 The numbers of flowing cells (left figure) and static adherent culture cells (right figure) at different time points after the cells were cultured by flow in Example 4 of the present invention. DETAILED DESCRIPTION

[0033] In order to facilitate ordinary technicians in the field to understand and implement the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and implementation examples. It should be understood that the implementation examples described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0034] Figure 1A special cell flow culture device used in the embodiment of the present invention is used for flow culture of cells. The flow culture device includes a cell culture dish portion 1 located at the upper part, a power pump 3 located at the lower part for controlling the flow rate, direction and frequency of the culture medium, and a conduit 2 located in the middle part for connecting the liquid culture medium portion 1 and the power pump 3 to achieve flow rate and flow direction control; wherein the cell culture dish portion 1 is added with cell culture medium and target cells to be cultured, and a filter membrane is provided in the conduit 2 to prevent cells from flowing out.

[0035] The cultured cells and cell culture fluid are placed in the upper cell culture dish part 1, and the flow rate, direction and frequency of the culture medium are controlled by the power pump 3 at the bottom to realize the flow suspension of cells and cell culture fluid, so as to achieve the purpose of cell suspension, flow and flow culture of cells at a certain frequency.

[0036] Embodiment 1:

[0037] A method for studying the cell molecular biological characteristics of tumor cell epithelial-mesenchymal transition (EMT) / mesenchymal-epithelial transition (MET) in vitro, comprising the following steps:

[0038] 1. Use a special cell flow culture apparatus for flow culture of target cells.

[0039] In step 1, the cells to be cultured in flow suspension are placed in a flow culture apparatus to achieve non-adherent flow suspension culture. Not only is the cell non-adherent, but the flow frequency can be set but not limited to 50 rpm-250 rpm.

[0040] 2. Place the flow culture apparatus and the cells in it into a conventional cell culture incubator (such as ThermoScientific Forma CO2 incubator Class 100), set basic indicators such as temperature and CO2 concentration, and perform flow culture on the cells.

[0041] 3. After the flow culture begins, samples are taken regularly to analyze markers of epithelial and mesenchymal types and related cell molecular biological levels, including molecular biological analysis of protein levels, RNA levels, and methylation levels.

[0042] In step 3, epithelial and mesenchymal types and related cell molecular biological markers include but are not limited to genes EpCAM (epithelial cell adhesion molecule), CKs (cytokeratins), N-cadherin (N-cadherin), FN1 (fibronectin 1), ZEB2 (zinc finger E-box binding homologous protein 12), ZEB1 (zinc finger E-box binding homologous protein 1), Snail2 (snail family transcription repressor), Snail1 (snail family transcription repressor), NANOG (homeobox), Aldh1A1 (aldehyde dehydrogenase 1A1), Oct4 (octamer binding transcription factor), Kit (KIT proto-oncogene, receptor tyrosine kinase), Vimentin (vimentin), Twist1 (twist family bHLH transcription factor 1).

[0043] 4. Move some of the cells cultured in the above flow culture dish to a static conventional cell culture dish for adherent culture.

[0044] In step 4, the target cells in flow culture are transferred to an adherent culture dish for adherent culture, which is to transfer the cell suspension (cell culture medium and target cells, avoiding any adherent cells from mixing) to the adherent culture dish for culture.

[0045] 5. For the above flow cultured cells and cells cultured on the wall after flow culture, regular analysis of mesenchymal and epithelial types and related cell molecular biology markers is performed, including cell molecular biology analysis of protein level, RNA level and methylation level.

[0046] 6. Comparison of cells cultured in adherent culture, flow culture and re-adherent culture was performed to establish in vitro models of epithelial / mesenchymal transition and mesenchymal / epithelial transition for comparative analysis of epithelial, mesenchymal and related cell molecular biological markers (including designated genes, or broad-spectrum analysis).

[0047] Embodiment 2:

[0048] Changes in protein markers of target cells in flow culture

[0049] In order to simulate the changes in the molecular biological characteristics of target cells in blood flow, the changes in interstitial protein markers in flow culture and adherent culture were compared. The specific steps include:

[0050] 1. Use a special cell flow culture apparatus, add A549 cell line and DMEM cell culture medium containing 10% FBS and 1% penicillin-streptomycin for flow culture, and the culture conditions are 37°C and 5% CO2.

[0051] 2. The flow culture speed is 60 rpm.

[0052] 3. After the flow culture begins, samples are taken at regular intervals to observe the expression levels of epithelial cell protein markers keratin (CKs) and mesenchymal cell protein markers Vimentin and Twist1. Protein expression analysis is performed using the circulating tumor cell identification kit from Micron Silicon Valley Life Sciences, Inc.

[0053] 4. Observe and compare the expression levels of epithelial and mesenchymal protein markers of adherent culture (with the same medium composition as flow culture) and flow culture cells.

[0054] like Figure 2-A As shown in Figure 2, after flow culture, the expression of vimentin and twist1, which are characteristic of mesenchymal cells, increased significantly. Figure 2-B As shown, in the adherent cells cultured under conventional culture conditions of 37°C and 5% CO2, these two proteins (vimentin and twist protein 1) were not expressed or expressed at very low levels.

[0055] Embodiment 3:

[0056] Expression changes of RNA markers of target cells in flow culture

[0057] In order to simulate the changes in molecular biological characteristics of target cells in blood flow, the changes in interstitial RNA markers in flow culture and adherent culture were compared. The specific steps include:

[0058] 1. Use a special cell flow culture apparatus, add HT-29 and MDA-MB-231 cell lines and DMEM cell culture medium containing 10% FBS and 1% penicillin-streptomycin for flow culture.

[0059] 2. The flow culture speed is 80 rpm.

[0060] 3. After the start of flow culture, samples were collected 2 hours later, and the expression levels of interstitial RNA markers Vimentin, Twist1, and N-cadherin in the cells were detected by quantitative PCR.

[0061] 4. After flow culture, the HT-29 and MDA-MB-231 cell lines were re-cultured in a conventional cell culture incubator at 37°C and 5% CO2 (with the same medium composition as that of flow culture) for 24 hours.

[0062] 5. Compare the expression levels of interstitial RNA markers in the above-mentioned adherent culture (the medium composition is consistent with that of the flow culture, and the culture conditions are 37°C, 5% CO2), flow culture and cells converted back to adherent culture.

[0063] Figure 3-A The expression of RNA markers of mesenchymal cells after cells were cultured from adherent culture to flow culture. 0 hours is the result of the control experiment of adherent culture. Figure 3-A Left) and MDA-MB-231 cells ( Figure 3-A (Right) Analysis results of RNA markers extracted from cells show that the RNA expression level of the vimentin gene (Vimentin) is significantly higher than that of ordinary adherent cultured cells in a static state, indicating an increase in interstitial cell markers.

[0064] Figure 3-B is the expression level of interstitial RNA markers after cells returned from flow culture to static adherent culture. Vimentin ( Figure 3-B Left), Twist1 (Twist protein 1) ( Figure 3-B middle) and N-cadherin ( Figure 3-B Right) The RNA expression levels of the three genes increased, and then decreased after the cells were re-cultured to adherent culture, which means that the markers of mesenchymal cells decreased after flow culture and then adherent culture.

[0065] Embodiment 4:

[0066] Growth and number changes of target cells in flow culture

[0067] In order to simulate the changes in the survival rate of target cells in blood flow, the changes in the number of cells in flow culture and adherent culture were compared. The specific steps include:

[0068] 1. Use a special cell flow culture apparatus, add A549 cell line and DMEM cell culture medium containing 10% FBS and 1% penicillin-streptomycin for flow culture.

[0069] 2. The flow culture speed is 80 rpm.

[0070] 3. After the flow culture begins, take samples regularly and calculate the changes in cell number.

[0071] 4. Observe and compare the changes in the number of cells in adherent culture (consistent with the medium composition of flow culture) and flow culture cells in a conventional cell culture incubator at 37°C and 5% CO2.

[0072] like Figure 4As shown in the figure, the number of A549 cells will decrease significantly under long-term flow culture. At the beginning of the experiment, the same number of cells (1145) were placed in the control group of adherent culture and the experimental group of flow culture. After 24 hours, there were only 343 cells in the experimental group of flow culture, while the number of cells in the control group of adherent culture continued to rise. The number of surviving cells in flow culture was significantly lower than that in the control group. This shows that after the adherent cells enter the flow culture environment, some cells die or decrease, and the flow culture environment will affect the survival rate and viability of the cells.

[0073] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Although the present invention has been described in detail with reference to the above embodiments, the present invention is not limited to the above specific implementation methods. Therefore, any modification or equivalent replacement of the present invention; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the invention are included in the scope of the claims of the present invention.

Claims

1. A method for studying the cell molecular biological characteristics of epithelial-mesenchymal transition / mesenchymal-epithelial transition of tumor cells in vitro, characterized in that: The following steps are involved: S1. Use a special cell flow culture apparatus to place the cells to be cultured in flow suspension in the flow culture apparatus to achieve cell non-adherence and flow suspension culture; S2. placing the flow culture device and the cells therein into a cell culture box, setting the basic indicators of temperature and CO2 concentration, and performing flow culture on the cells; S3. After the flow culture begins, samples are taken at regular intervals to analyze markers of epithelial and mesenchymal types and related cell molecular biology levels, including molecular biological analysis of protein levels, RNA levels, and methylation levels; S4. The cells cultured in the flow culture were relocated to the static cell culture dish for static adherent culture; S5. For the cells that are cultured on the wall after the above-mentioned flow culture, regular analysis of mesenchymal and epithelial types and related cell molecular biology markers is performed, including cell molecular biology analysis at the protein level, RNA level and methylation level.

2. The method according to claim 1, characterized in that In the step S1, the flow frequency is set to 50 rpm-250 rpm.

3. The method according to claim 1, characterized in that: In step S3, the epithelial and mesenchymal types and related cell molecular biological markers include genes EpCAM, CKs, N-cadherin, FN1, ZEB2, ZEB1, Snail2, Snail1, NANCG, Aldh1A1, Oct4, Kit, Vimentin, and Twist1.

4. The method according to claim 1, characterized in that In step S4, the target cells in flow culture are transferred to an adherent culture dish for adherent culture, which is to transfer the cell suspension to the adherent culture dish for culture, wherein the cell suspension includes the cell culture medium and the target cells and avoids any adherent cells from mixing in.

5. The method according to claim 1, characterized in that In step S5, samples are taken regularly from the cells that have resumed adherent culture, and cell molecular biology analysis of cell markers including protein and RNA levels and methylation levels is performed, including analysis at the single cell level and analysis at the multi-cell level.

6. The method according to claim 1, characterized in that Also includes: Compare cells cultured on adherent, flow-cultured, and recovered adherent cultures for comparative analysis of epithelial, mesenchymal, and related cell molecular biomarkers, including specific genes, or broad-spectrum analysis.

7. The method according to claim 1, characterized in that The cell flow culture device comprises a cell culture dish portion located at the upper part, a power pump located at the lower part for controlling the flow rate, direction and frequency of the culture medium, and a conduit located in the middle part for connecting the liquid culture medium portion with the power pump to achieve flow rate and flow direction control; wherein the cell culture dish portion is added with cell culture medium and target cells to be cultured.

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