Method for establishing a primary cell culture system of endometrial cancer

Abstract

This invention relates to the field of biological cell technology and discloses a method for establishing a primary cell culture system for endometrial cancer. The method includes: obtaining surgically removed specimens of clinical endometrial cancer, chopping them, washing them with erythrocyte lysis buffer, and digesting them with collagenase type IV; collecting the digestion solution, centrifuging it, resuspending the cells in complete culture medium containing fetal bovine serum and antibiotics, and performing primary culture until the cell confluence reaches 90%; subsequently, performing passage culture, digesting the cells with trypsin digestion solution containing EDTA and phenol red, and adding an equal volume of complete culture medium for neutralization, resuspending the cells, transferring them to new culture flasks for continued culture until passage to the tenth generation or higher, ultimately obtaining a stable human endometrial cancer cell line HEC2507. The cell line established by this invention has stable characteristics and can be directly applied to important research fields such as cell biology, molecular biology, and anti-tumor drug screening.

Description

Technical Field

[0001] This invention belongs to the field of biological cell technology, specifically relating to a method for establishing a primary cell culture system for endometrial cancer. Background Technology

[0002] Endometrial cancer is one of the most common malignant tumors of the female reproductive system, with an increasing incidence rate year by year. It exhibits significant molecular subtyping and clinical heterogeneity, and its clinical treatment still faces enormous challenges. In basic research and drug development for endometrial cancer, cell lines play an irreplaceable role as an important tool in exploring tumorigenesis mechanisms, drug screening, and the development of treatment strategies.

[0003] Currently, several endometrial cancer cell lines have been established and applied both domestically and internationally, including Ishikawa and HEC-1-A. However, traditional endometrial cancer cell lines widely used in scientific research have a series of inherent and significant limitations, severely restricting the accuracy and clinical translational value of related research. These limitations are mainly reflected in the following aspects: 1. The vast majority of existing cell lines are "immortalized" cell lines established decades ago, undergoing long-term, countless generations of passage culture in the laboratory. This process inevitably leads to genetic drift and in vitro adaptive selection. Subclones with the fastest proliferation rate in the cell population gradually become dominant, causing significant differences in the genome and phenotype of the cell line compared to the primary tumor. Specifically, the expression levels of key oncogenes or tumor suppressor genes may change, failing to accurately reflect the molecular state of the tumor in vivo. Long-term in vitro culture may cause cells to lose their original hormone responsiveness (such as sensitivity to estrogen and progesterone), invasive and metastatic abilities, and other key malignant phenotypes, or exhibit non-physiological abnormalities. The same cell line may exhibit different biological behaviors due to genetic drift in different laboratories and under different culture conditions, making it difficult to replicate and verify research results across different teams and reducing the reliability of the research.

[0004] 2. Endometrial cancer is a highly heterogeneous disease, encompassing various molecular subtypes (such as POLE hypermutant, MSI-H, copy number high and low, etc.), with significant differences in treatment response and prognosis across different subtypes. Existing cell lines are not only limited in number, but more importantly, the few existing classic cell lines fall far short of representing the complex molecular landscape of endometrial cancer, resulting in a lack of suitable models for research on specific subtypes (especially rare subtypes). These cell lines have been purified into a single epithelial cell population through long-term passage, completely losing the complex tumor microenvironment of the primary tumor tissue, including cancer-associated fibroblasts, immune cells, and vascular endothelial cells, thus limiting their value in researching tumor-stromal interactions and immunotherapy.

[0005] 3. In the field of cell culture, cell cross-contamination and misidentification are long-neglected but extremely serious problems. Existing cell lines commonly suffer from the following: many early-established cell lines lack authoritative identification through short tandem repeat (STR) analysis, casting doubt on their true origin. For example, some so-called endometrial cancer cell lines may have been contaminated by rapidly growing cell lines such as HeLa, leading to studies based on flawed cell models and invalid conclusions. Besides STR analysis, many cell lines also lack systematic chromosomal karyotype analysis to demonstrate their genetic stability and lack pathogen detection reports (such as mycoplasma). These deficiencies in quality control significantly compromise the scientific rigor of experimental results.

[0006] 4. Due to the aforementioned genetic instability, model unrepresentativeness, and identity uncertainty, preclinical studies based on these traditional cell lines have performed poorly in predicting clinical efficacy. Drugs that show effectiveness in traditional cell lines often fail after entering clinical trials because they cannot simulate the heterogeneity and complexity of real patient tumors. The difficulty in using existing cell lines to establish personalized research models corresponding to specific patient gene mutations, treatment responses, and prognoses hinders the development of precision medicine.

[0007] 5. Although culturing primary cells directly from patient tumor tissue is an ideal way to solve the above problems, traditional primary culture methods have technical bottlenecks: the tissue digestion method is crude, resulting in a large loss of tumor stem cells or progenitor cells with proliferative potential; the operation process is not optimized enough, the risk of microbial contamination is high, leading to culture failure; primary cells have limited in vitro proliferation capacity and often enter a senescent state after a few generations, making it impossible to expand into a cell line that can be stably passaged, which is difficult to meet the needs of large-scale experiments.

[0008] In summary, existing endometrial cancer cell lines suffer from significant shortcomings in terms of genetic stability, model representativeness, rigorous identification, and clinical predictive value. Therefore, developing a method for efficiently and stably establishing novel endometrial cancer cell lines from primary tumor tissues and for systematically and standardizedly identifying them is crucial for advancing basic research and clinical translation in this field. Summary of the Invention

[0009] The purpose of this invention is: (1) to provide a method for successfully establishing and stably passaged (up to 100 generations or more) a cell line from primary endometrial cancer tissue, so as to overcome the genetic drift and changes in biological characteristics of existing cell lines caused by long-term passage.

[0010] (2) Provide the construction method and identification of the above-mentioned human endometrial cancer cell lines.

[0011] (3) Provide the methods for culturing, passage, and cryopreservation of the above-mentioned human endometrial cancer cell lines.

[0012] (4) Provide animal experimental applications of the above-mentioned human endometrial cancer cell lines.

[0013] The technical solution adopted by this invention to solve its technical problem is as follows: A primary human endometrial cancer cell line, named HEC2507, was derived from the primary lesion tissue of a patient with endometrioid adenocarcinoma and was not introduced with exogenous genes.

[0014] A method for establishing a primary cell culture system for endometrial cancer, comprising the following steps: S1. Obtain surgical specimens of endometrial cancer from patients. After fragmenting the specimens, infiltrate them into McCoy's 5A basal medium, wash with erythrocyte lysis buffer, allow the tissue blocks to settle, aspirate dry, repeat washing, rinse with Hank's buffer, aspirate clean, add collagenase type IV, and place in a constant temperature shaker for digestion. Collect the supernatant of the digestion solution, centrifuge, discard the supernatant, resuspend the cell clusters in McCoy's 5A complete medium, seed them in multi-well culture plates, and incubate in a static incubator. Change the McCoy's 5A complete medium periodically until the cell confluence reaches 90%. S2. Discard the culture medium from the primary cell culture plate and wash the plate with Hank's buffer. Add trypsin digestion solution containing EDTA and phenol red, and place the plate in an incubator for static culture. Add an equal volume of McCoy's 5A complete medium to neutralize, pipette the plate, and aspirate the cell suspension for centrifugation. Take the centrifuged cell suspension, discard the supernatant, add McCoy's 5A complete medium to resuspend the cells, transfer the cell suspension to a T25 culture flask, and continue culturing in an incubator until the cells have been passaged to the tenth generation or higher to obtain the human endometrial cancer cell line HEC2507.

[0015] Preferably, in step S1, the concentration of collagenase type IV is 1000 U / mL, and the digestion conditions are 37°C, 80 rpm, and digestion in a constant temperature shaker for 30 minutes.

[0016] Preferably, the McCoy's 5A complete culture medium in step S1 contains 1.5 mM L-alanyl-L-glutamine, 15% fetal bovine serum, and at least one antibiotic selected from penicillin, streptomycin, and gentamicin.

[0017] Further preferred formulations are: penicillin 100 U / mL, streptomycin 100 μg / mL, and gentamicin 50 μg / mL.

[0018] Preferably, the incubation conditions in both steps S1 and S2 are 5% CO2, 37°C, and relative humidity of not less than 95%.

[0019] Preferably, the specific operation of periodically replacing McCoy's 5A complete medium in step S1 includes: first aspirating the old medium, washing the culture plate with Hank's buffer, and then adding fresh McCoy's 5A complete medium.

[0020] Preferably, in step S1, the red blood cell lysis buffer is washed twice, and the settling time after each wash is 5 minutes.

[0021] Preferably, in step S2, the trypsin digestion solution contains 2.5 g / L trypsin, 0.2 g / L EDTA and 10 mg / L phenol red, and the digestion time is 3-5 min.

[0022] Preferably, the method for establishing a primary cell culture system for endometrial cancer proposed in this invention further includes cell cryopreservation and thawing steps: (1) Cryopreservation: After passage culture in step S2, cells were digested with trypsin digestion solution, resuspended in McCoy's 5A basal medium containing 10% DMSO and 20% fetal bovine serum, dispensed into cryovials, placed in a programmed cooling box at -80°C overnight, and then transferred to liquid nitrogen for long-term storage. (2) Thawing: Thaw the frozen cells quickly in a 37°C water bath, transfer them to centrifuge tubes containing McCoy's 5A complete medium, centrifuge at 1000 rpm for 4 minutes, discard the supernatant, resuspend the cells in fresh McCoy's 5A complete medium, and then inoculate them for culture.

[0023] In summary, compared with existing technologies, the method for establishing a primary cell culture system for endometrial cancer provided by this invention has the following beneficial effects: (1) The human endometrial cancer cell line of the present invention is obtained from human endometrial cancer tissue and can be stably passaged for more than 100 generations. By applying relevant experimental methods such as cell biology, tumor pathology and molecular oncology, the biological behavior of the cell line can be comprehensively evaluated, and it was found that the cell line has stable in vitro proliferation, unlimited passage ability, clonogenic ability and invasion and metastasis ability.

[0024] (2) The human endometrial cancer cell line of the present invention has stable characteristics and can be directly applied to important research related to cell biology, molecular biology, and anti-tumor drug screening. It is an ideal human endometrial cancer cell line for basic research and preclinical application.

[0025] (3) The present invention can efficiently separate active cells in endometrial cancer tissue through optimized tissue processing procedures and specific enzyme digestion methods, greatly improve the efficiency of obtaining primary cells and maintain good cell survival status.

[0026] (4) The present invention uses a combination of antibiotics with a specific ratio and a strict aseptic operation procedure, which significantly reduces the risk of microbial contamination during the culture process and ensures the purity of cell culture. Attached Figure Description

[0027] Other features, objects, and advantages of this application will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is a flowchart of the method for establishing a primary cell culture system for endometrial cancer according to the present invention; Figure 2 This is a morphological diagram of primary cultured HEC2507 cells; Figure 3 This is a morphological diagram of HEC2507 cells from the 42nd generation; Figure 4 This is a morphological diagram of HEC2507 cells from the 51st generation; Figure 5 This is a morphological diagram of HEC2507 cells from the 100th generation; Figure 6 This is a schematic diagram of the immunohistochemical identification results of the endometrial cancer cell line HEC2507. Detailed Implementation

[0028] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0029] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0030] Figure 1 This demonstrates a method for establishing a primary cell culture system for endometrial cancer, with reference to... Figure 1 The preparation method includes the following steps: S1. Obtain fresh surgical specimens of clinical endometrial cancer and perform primary cell isolation and culture using collagenase type IV digestion method.

[0031] S2. When the cell confluence reaches about 90%, perform cell passage culture to complete the establishment of HEC2507.

[0032] Step S1 specifically includes the following sub-steps: (1) Processing of specimen tissue: Collect samples during the operation, cut the samples into small pieces on the operating table, and then immerse the fresh excised tissue in McCoy's 5A basal medium; wash with red blood cell lysis buffer, let stand until the tissue block settles, aspirate the liquid, and repeat the washing once; then rinse the tissue specimen with Hank's buffer, aspirate the residual liquid, and add collagenase type IV.

[0033] (2) Digestion reaction: The tissue treated in step (1) was placed in a constant temperature shaker at 37°C and 80 rpm for 30 minutes for digestion.

[0034] (3) Centrifugation and culture: Collect the supernatant of the digestion solution obtained in step (2) and centrifuge it. Discard the supernatant, suspend the cell clusters in McCoy's 5A complete medium, and seed them into one well of a 6-well cell culture plate. Place the plate in a 5% CO2, 37°C incubator for static culture. When changing the medium, first remove the old medium, wash the culture plate with Hank's buffer, add fresh McCoy's 5A complete medium, and then place it back in a 5% CO2, 37°C incubator for static culture. When the cells in the culture plate grow to 90% confluence, transfer them to a T25 culture flask for culture.

[0035] The complete culture medium was McCoy's 5A medium containing 15% (v / v) fetal bovine serum and a mixture of antibiotics (including penicillin, streptomycin and gentamicin).

[0036] The specific method for step S2 is as follows: (1) Cell suspension and centrifugation: Discard the culture medium in the culture plate for primary culture, wash the culture plate with Hank's buffer; add trypsin digestion solution to make the digestion solution 100% cover the bottom of the cell culture plate; place in a 5% CO2, 37℃ incubator and incubate for 3-5 minutes; add an equal volume of McCoy's 5A complete culture medium to neutralize the cells, gently pipette the culture flask to detach the cells, aspirate the cell suspension, and centrifuge.

[0037] (2) Medium change and culture: Take the cell pellet from step (1) after centrifugation, discard the supernatant, add fresh cell culture medium (i.e., McCoy's 5A complete medium) to resuspend the cells, add the cell suspension to a new T25 culture flask, and place it in a 5% CO2, 37℃ incubator for static culture. After the cells are passaged to the tenth generation, the endometrial cancer cell line HEC2507 can be obtained. This cell line can be stably passaged to more than 100 generations.

[0038] In a specific embodiment, the human endometrial cancer cell line HEC2507 was established using postoperative tumor tissue from a 50-year-old patient with endometrioid adenocarcinoma (Chen xx). The details are as follows: The patient's postoperative pathological diagnosis was endometrioid adenocarcinoma, grade II, with partial mucinous differentiation, without involvement of the internal cervical os, and no definite intravascular tumor emboli or nerve bundle invasion was observed. Immunohistochemical results showed: ER (100%+), PR (100%+), PAX-8 (+), Vim (+), PTEN (+), WT-1 (-).

[0039] Immediately after tumor tissue was excised, it was placed in McCoy's 5A medium and transported to the laboratory within 30 minutes for primary cell isolation and culture.

[0040] The primary culture process is as follows: Fresh ex vivo tissue was infiltrated into 15 mL centrifuge tubes in complete McCoy's 5A medium and transported to the laboratory for processing. After centrifugation, the liquid in the sample tube was aspirated, and 5 mL of erythrocyte lysis buffer was added for washing. The tissue was allowed to settle, centrifuged again, aspirated, and then 5 mL of erythrocyte lysis buffer was added for washing once more. Then, 2 mL of Hank's buffer was added for washing once more. After aspirating all liquid, 2 mL of collagenase type IV solution was added, and the mixture was sealed and placed in a 37°C shaker at 80 rpm for 30 minutes for digestion. The supernatant was collected, centrifuged at 1000 rpm for 4 minutes, and discarded. The cell pellet was resuspended in 1 mL of complete medium and seeded into one well of a 6-well cell culture plate. The plate was then incubated in a 5% CO2 incubator at 37°C. Regularly observe the color of the culture medium and cell growth density. If the culture medium turns yellow, change the medium: aspirate the old medium, wash the wells with Hank's buffer, add fresh complete culture medium, and continue static culture at 37°C with 5% CO2. When the cells are in good growth condition and the confluence of the first-generation cells reaches approximately 90%, perform subculture. Figure 2 As shown.

[0041] The subculture procedure is as follows: Aspirate the culture medium from the primary culture plate, wash the plate with Hank's buffer, add 0.5 mL of trypsin digestion solution, gently shake to just cover the bottom of the culture plate, and incubate the plate in a 5% CO2, 37°C incubator for 3–5 minutes. Observe under a microscope. Once the cells are completely detached and suspended, add an equal volume of complete culture medium to neutralize the digestion reaction. Disperse the suspended cells in the culture plate by pipetting, and transfer the cell suspension to a centrifuge tube. Centrifuge at 1000 rpm for 4 minutes. Discard the supernatant, add 1 mL of cell culture medium (i.e., McCoy's 5A complete medium) to resuspend the cells, and transfer the cell suspension to a T25 culture flask containing 4 mL of cell culture medium. Continue static incubation in a 5% CO2, 37°C incubator.

[0042] The cell cryopreservation method is as follows: Prepare cryopreservation solution in advance, namely McCoy's 5A medium containing 10% (v / v) DMSO and 20% (v / v) fetal bovine serum. Discard the medium in the culture flask, wash the culture flask with Hank's buffer, add 1 mL of the above trypsin digestion solution (containing 2.5 g / L trypsin, 0.2 g / L EDTA and 10 mg / L phenol red), gently shake to cover the bottom of the cell culture flask with the cell digestion solution, and incubate at 37°C with 5% CO2 for 3-5 minutes. Observe the digestion status of the cells under a microscope. When all cells are digested to a suspension state, add 1 mL of complete medium to neutralize, pipette to suspend the cells in the culture flask, aspirate the cell suspension and transfer it to a centrifuge tube, centrifuge at 1000 rpm for 4 minutes. Discard the supernatant, add 1 mL of cryopreservation solution to resuspend the cells, transfer to cryovials, seal and label with cell name, passage number and cryopreservation date.

[0043] Primary cells were continuously passaged in vitro, and cell morphology was observed under a microscope at passages 42 and 51 (e.g., ...). Figure 3 , Figure 4 (As shown). Continue culturing to the 100th generation, and microscopic observation shows (as shown). Figure 5 HEC2507 cells lost contact inhibition and exhibited malignant growth with overlapping growth characteristics. The adherent cells grew in an irregular, cobblestone pattern, consistent with epithelial-like cells, indicating successful establishment of the cell line. Systematic identification and culture characteristic analysis of this cell line were performed, with the following results: The identification of the endometrial cancer cell line included: (1) Morphological identification: The cells were confirmed to be adherent, exhibiting a typical epithelial-like cell morphology, with tight connections between cells.

[0044] (2) Immunophenotypic identification: using immunohistochemistry or immunofluorescence techniques, epithelial markers: cytokeratin 7 (CK-7) showed positive expression, and PAX8 showed positive nuclear expression.

[0045] (3) Genetic identity identification: confirming that it is a human female-derived cell line with a unique genetic background and no cross-contamination.

[0046] (4) Chromosome karyotype analysis: The metaphase chromosomes of cells are analyzed by G-banding technique to assess the stability of their genetic material and whether there are any abnormalities.

[0047] The methods for culturing, passage, cryopreservation, and resuscitation of the endometrial cancer cell line are as follows: (1) Cell culture: Using McCoy's 5A complete medium, cells are placed in a cell culture incubator at 37°C with 5% CO2 for static culture. Depending on the color change of the medium (e.g., turning yellow) or the cell density, the medium is usually changed every 2-3 days.

[0048] (2) Passaging: When the cells grow to 80% to 90%, they need to be passaged for expansion. Discard the old culture medium in the culture flask. Add an appropriate amount of preheated Hank's buffer to wash the cell surface, and then discard it. Add an appropriate amount of trypsin digestion solution (containing 0.25% trypsin, 0.02% EDTA and 0.001% phenol red) to digest for 3-5 minutes, then add complete culture medium to stop digestion. After centrifugation, collect the cells, resuspend the cells, and seed the cell suspension into a new T25 culture flask at a passage ratio of 1:2 to 1:4, and add sufficient complete culture medium.

[0049] (3) Cryopreservation: Prepare the cryopreservation solution in advance, consisting of McCoy's 5A basal medium + 20% fetal bovine serum + 10% DMSO. After preparation, pre-cool at 4°C (Note: DMSO is toxic to cells and must be prepared and used immediately; operation should be rapid). Subsequently, follow the same procedure as in passage steps 1-3: centrifuge, discard the supernatant, resuspend the cells in the pre-cooled cryopreservation solution, and aliquot the cell suspension into cryovials. Finally, place the cryovials in a programmed cooling box and ultimately transfer them to liquid nitrogen for long-term storage.

[0050] (4) Thawing: Remove the cryovials from liquid nitrogen and immediately place them in a 37°C water bath, shaking rapidly to thaw them completely within 1-2 minutes. Thoroughly wipe the outer wall of the cryovials with 75% alcohol, open the cap, and transfer the cell suspension to a centrifuge tube containing 5-10 mL of complete culture medium. Centrifuge at 1000 rpm for 4 minutes and discard the supernatant (to remove the cryovial solution containing DMSO). Resuspend the cells in fresh complete culture medium, seed them into culture flasks, and incubate them in an incubator.

[0051] Systematic immunohistochemical identification of the established human endometrial cancer cell line HEC2507 confirmed that this cell line expresses multiple key protein markers. For example... Figure 6As shown, immunohistochemical staining revealed positive expression of CK-7 in HEC2507 cells, confirming their epithelial origin; positive expression of E-cadherin suggested the maintenance of intercellular connections between epithelial cells; strong positive expression of both estrogen and progesterone receptors, consistent with the hormone receptor expression characteristics of endometrial cancer cells; a positive reaction to the proliferation marker KI67, indicating active cell proliferation; specific expression patterns of p53 protein, providing a basis for further research on gene mutation status; positive nuclear expression of PAX8, supporting its Müllerian duct epithelial origin; and vimentin expression detection, providing a foundation for the study of cell epithelial-mesenchymal transition. These immunohistochemical results comprehensively validated the biological characteristics of the HEC2507 cell line at the protein expression level, providing important identification criteria for its application in endometrial cancer research.

[0052] The above description is merely a preferred embodiment of this application and an explanation of the technical principles employed. Those skilled in the art should understand that the scope of the invention involved in this application is not limited to technical solutions formed by specific combinations of the above-described technical features, but should also cover other technical solutions formed by arbitrary combinations of the above-described technical features or their equivalents without departing from the above-described inventive concept. For example, technical solutions formed by substituting the above features with (but not limited to) technical features with similar functions disclosed in this application.

Claims

1. A method for establishing a primary cell culture system for endometrial cancer, characterized in that, The method includes the following steps: S1. Obtain surgical specimens of endometrial cancer from patients. After fragmenting the specimens, infiltrate them in McCoy's 5A basal medium, wash with erythrocyte lysis buffer, allow the tissue blocks to settle, aspirate dry, repeat washing, rinse with Hank's buffer, aspirate clean, add collagenase type IV, and place in a constant temperature shaker for digestion. Collect the supernatant of the digestion solution, centrifuge, discard the supernatant, resuspend the cell clusters in McCoy's 5A complete medium, seed them in multi-well culture plates, and incubate in a static incubator. Change the McCoy's 5A complete medium periodically until the cell confluence reaches 90%. S2. Discard the culture medium from the culture plate used for primary cell culture, wash the culture plate with Hank's buffer; add trypsin digestion solution containing EDTA and phenol red, and place the culture plate in an incubator for static culture; add an equal volume of McCoy's 5A complete culture medium to neutralize, pipette the culture plate, aspirate the cell suspension and centrifuge; take the centrifuged cell solution, discard the supernatant, add McCoy's 5A complete culture medium to resuspend the cells, transfer the cell suspension to a T25 culture flask, and continue culturing in an incubator until the cells have been passaged to the tenth generation or higher to obtain the human endometrial cancer cell line HEC2507.

2. The method for establishing a primary cell culture system for endometrial cancer according to claim 1, characterized in that, The concentration of collagenase type IV used in step S1 is 1000 U / mL, and the digestion conditions are 37°C, 80 rpm, and digestion in a constant temperature shaker for 30 minutes.

3. The method for establishing a primary cell culture system for endometrial cancer according to claim 1, characterized in that, The McCoy's 5A complete culture medium described in step S1 contains 1.5 mM L-alanyl-L-glutamine, 15% fetal bovine serum, and at least one antibiotic selected from penicillin, streptomycin, and gentamicin.

4. The method for establishing a primary cell culture system for endometrial cancer according to claim 3, characterized in that, The working concentrations of the antibiotics are 100 U / mL for penicillin, 100 μg / mL for streptomycin, and 50 μg / mL for gentamicin.

5. The method for establishing a primary cell culture system for endometrial cancer according to claim 1, characterized in that, The incubation conditions in both steps S1 and S2 are 5% CO2, 37°C, and relative humidity of not less than 95%.

6. The method for establishing a primary cell culture system for endometrial cancer according to claim 1, characterized in that, The specific operation of periodically replacing McCoy's 5A complete medium in step S1 includes: first, removing the old medium, washing the culture plate with Hank's buffer, and then adding fresh McCoy's 5A complete medium.

7. The method for establishing a primary cell culture system for endometrial cancer according to claim 1, characterized in that, The red blood cell lysis buffer is washed twice in step S1, and the standing time is 5 minutes after each wash.

8. The method for establishing a primary cell culture system for endometrial cancer according to claim 1, characterized in that, The trypsin digestion solution in step S2 contains 2.5 g / L trypsin, 0.2 g / L EDTA and 10 mg / L phenol red, and the digestion time is 3-5 min.

9. The method for establishing a primary cell culture system for endometrial cancer according to claim 8, characterized in that, It also includes cell cryopreservation and thawing steps: (1) Cryopreservation: After passage culture in step S2, cells were digested with trypsin digestion solution, resuspended in McCoy's 5A basal medium containing 10% DMSO and 20% fetal bovine serum, dispensed into cryovials, placed in a programmed cooling box at -80°C overnight, and then transferred to liquid nitrogen for long-term storage. (2) Thawing: Thaw the frozen cells quickly in a 37°C water bath, transfer them to centrifuge tubes containing McCoy's 5A complete medium, centrifuge at 1000 rpm for 4 minutes, discard the supernatant, resuspend the cells in fresh McCoy's 5A complete medium, and then inoculate them for culture.

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