Methods and applications of establishing mouse gastric squamous cell carcinoma cell lines and gastric cancer-related fibroblast cell lines.

Abstract

This application provides a method for establishing and applying mouse gastric squamous cell carcinoma cell lines and gastric cancer-related fibroblast cell lines. The culture medium for culturing these cell lines comprises L-WRN cell culture supernatant, and 100×N-2 additive (0.5 vol%–1.5 vol%), 50× vitamin A-free B-27 additive (1.5 vol%–2.5 vol%), N-acetylcysteine ​​amide (1 mM–1.5 mM), nicotinamide (8 mM–12 mM), FGF-10 (80 ng / mL–120 ng / mL), EGF (40 ng / mL–60 ng / mL), A83-01 (0.8 μM–1.2 μM), Y-27632 dihydrochloride (8 μM–12 μM), Glutamax additive (0.5 vol%–1.5 vol%), and Matrigel. The culture medium provided in this application contains 5 vol%–7 vol% Hepes buffer (8 mM–12 mM) and gastrin (0.8 nM–1.2 nM). This medium can significantly improve the success rate of in vitro culture of primary cell lines. The mouse gastric squamous cell carcinoma cell lines and gastric cancer-related fibroblast cell lines obtained from this medium are genetically stable and can be used to construct ectopic or orthotopic implantation models in immune-active mice.

Description

Technical Field

[0001] This application relates to the field of tumor cell line establishment technology, and in particular to a method and application for establishing a mouse gastric squamous cell carcinoma cell line and a gastric cancer-related fibroblast cell line. Background Technology

[0002] According to Global Cancer Statistics 2022, gastric cancer ranks fifth in incidence among malignant tumors worldwide. China accounts for 37% of new gastric cancer cases globally, ranking fifth in incidence and third in mortality among malignant tumors in China. Due to the lack of characteristic clinical symptoms in the early stages of gastric cancer, approximately 80%-90% of Chinese gastric cancer patients are diagnosed at an advanced or late stage. For patients with mid-to-late stage gastric cancer, surgery alone is often ineffective or even disqualifies them from surgery. Drug therapy, targeted therapy, and immunotherapy have become important adjuvant treatments for advanced gastric cancer. However, the efficacy of traditional chemotherapy has reached its limit, and the results of many large-scale phase III clinical trials of targeted therapies have been unsatisfactory, leading to a very poor overall prognosis for gastric cancer.

[0003] The emergence of immunotherapy, represented by immune checkpoint inhibitors, has transformed the treatment landscape for advanced solid tumors. In advanced gastric cancer, the publication of groundbreaking research data from studies such as CHECKMATE-649, KEYNOTE-811, and ORIENT-16 has shown promise for gastric cancer immunotherapy. However, immunotherapy is only effective in a subset of patients, and exploring biomarkers for immunocompetent populations remains a challenge in gastric cancer immunotherapy research. Furthermore, gastric cancer is a highly heterogeneous tumor, and precise analysis of its tumor microenvironment and the spatiotemporal interaction network between tumor cells and tumor cells during immunotherapy is crucial for elucidating the adaptive immune resistance mechanisms in gastric cancer patients. Mouse tumor implantation models are among the main models for exploring the tumor immune microenvironment, with mouse colorectal cancer-derived CT26 and MC38 cells and melanoma-derived B16 cells providing important tools for studying the in vivo tumor microenvironment.

[0004] To address the aforementioned key preclinical research challenges in gastric cancer, a suitable mouse-derived gastric cancer cell model is a crucial prerequisite for in vitro and in vivo studies of the tumor microenvironment. Currently, the only mouse-derived gastric squamous cell carcinoma cell line for gastric cancer is the MFC cell line, derived from the pre-gastric squamous cell carcinoma cells of the 615 mouse. This mouse lacks complete genome sequencing, making it unsuitable for gene modification studies. Furthermore, MFC cells inoculate into 615 mice, resulting in rapid tumor formation and progression, failing to reflect the chronic progression characteristics of clinical gastric cancer and leading to a short window for drug intervention, thus affecting the accuracy of drug treatment evaluation. In addition, compared to the C57BL / 6J mouse, the 615 mouse differs significantly from mainstream international models, resulting in fewer citations in international literature and relatively insufficient international comparability. Tumor-associated fibroblasts, as another major cellular component of the solid tumor microenvironment, can remodel the tumor extracellular matrix microenvironment and directly or indirectly participate in tumor growth, metastasis, and even induce tumor drug resistance through cell communication or bioactive molecules. However, mouse gastric cancer-associated fibroblast cell lines are currently lacking in gastric cancer research models, which limits comprehensive and in-depth research on the tumor microenvironment.

[0005] Therefore, obtaining genetically stable mouse gastric squamous cell carcinoma cell lines and their related fibroblast cell lines that can be used to construct preclinical research models with good immunological characteristics has become an urgent technical problem to be solved. Summary of the Invention

[0006] The purpose of this application is to provide a method for constructing a mouse gastric squamous cell carcinoma cell line and its related fibroblast cell line. The obtained mouse gastric squamous cell carcinoma cell line and gastric cancer-related fibroblast cell line are genetically stable and can be used to construct ectopic or orthotopic implantation models in immune-active mice. The specific technical solution is as follows:

[0007] The first aspect of this application provides a culture medium comprising L-WRN cell culture supernatant, and 100×N-2 additive 0.5 vol%-1.5 vol%, 50× vitamin A-free B-27 additive 1.5 vol%-2.5 vol%, N-acetylcysteine ​​amide 1 mM-1.5 mM, nicotinamide 8 mM-12 mM, FGF-10 80 ng / mL-120 ng / mL, EGF 40 ng / mL-60 ng / mL, A83-01 0.8 μM-1.2 μM, Y-27632 dihydrochloride 8 μM-12 μM, Glutamax additive 0.5 vol%-1.5 vol%, Matrigel 5 vol%-7 vol%, Hepes buffer 8 mM-12 mM, and gastrin 0.8 nM-1.2 nM.

[0008] In some embodiments of this application, the culture medium further includes: 0.08 vol%-0.12 vol% of primary cell antibiotics.

[0009] In some embodiments of this application, the method for preparing the L-WRN cell culture supernatant includes the following steps: L-WRN cells are cultured in DMEM complete medium containing FBS (fetal bovine serum), G418 and hygromycin B until the cell density reaches 85%-95%, then washed multiple times with PBS buffer, and then cultured in Advanced DMEM / F12 medium; the culture supernatant is collected at preset time intervals, the collected supernatants are combined and centrifuged, cell debris is removed and filtered, and the collected filtrate is mixed with Advanced DMEM / F12 medium to obtain the L-WRN cell culture supernatant.

[0010] In some embodiments of this application, in the method for preparing L-WRN cell culture supernatant, the DMEM complete culture medium containing FBS, G418 and hygromycin B has the following concentrations: FBS concentration is 8 vol%-12 vol%, G418 concentration is 0.4 mg / mL-0.6 mg / mL, and hygromycin B concentration is 0.4 mg / mL-0.6 mg / mL.

[0011] In some embodiments of this application, in the method for preparing L-WRN cell culture supernatant, the preset time interval is 20h-28h; and the number of times the culture supernatant is collected is 3-5 times.

[0012] In some embodiments of this application, in the method for preparing L-WRN cell culture supernatant, the centrifugation speed is 2500g-3500g and the centrifugation time is 12min-18min.

[0013] In some embodiments of this application, the filtration in the method for preparing L-WRN cell culture supernatant is performed using a 0.22-0.45 micrometer filter.

[0014] In some embodiments of this application, in the method for preparing L-WRN cell culture supernatant, the filtrate and the Advanced DMEM / F12 culture medium are mixed at a volume ratio of 1:(0.8-1.2).

[0015] The second aspect of this application provides the use of the culture medium described in the first aspect of this application in the establishment of mouse gastric squamous cell carcinoma cell lines and gastric cancer-associated fibroblast cell lines.

[0016] The third aspect of this application provides a method for establishing a mouse gastric squamous cell carcinoma cell line and a gastric cancer-related fibroblast cell line, comprising the following steps:

[0017] (1) The tumor tissue of spontaneous liver metastases in mice was washed multiple times with PBS buffer containing double antibodies, then cut into pieces and added to organoid lysis buffer for lysis to obtain tissue homogenate;

[0018] (2) The tissue homogenate is filtered using a sterile filter membrane. During filtration, the tissue remaining on the filter membrane is ground. The collected filtrate is then centrifuged to collect the lower layer of tissue cell mixture precipitate.

[0019] (3) Add red blood cell lysis buffer to the precipitate of the tissue cell mixture, let it stand, centrifuge, remove the supernatant, resuspend the precipitate using the culture medium described in the first aspect of this application, then add Matrigel, mix well and inoculate into a multi-well plate for culture; after a certain period of culture, add liquid and continue culture until some tissue fragments can be observed to adhere to the wall and primary cells sprout and grow from the tissue.

[0020] (4) The primary cells were digested with pancreatic enzyme digestion solution after growing for 7-10 days, and different types of cells were roughly separated by multiple differential digestion methods.

[0021] (5) The mouse gastric squamous cell carcinoma cell line and the gastric cancer-associated fibroblast cell line were obtained by flow cytometry.

[0022] In some embodiments of this application, in step (1), the volume ratio of the spontaneously metastatic liver metastasis tumor tissue in mice to the organoid lysis fluid is 1:(800-1200); the lysis is performed at 35℃-39℃ and 200rpm-300rpm for 8min-12min.

[0023] In some embodiments of this application, in step (2), the sterile filter membrane is a sterile filter membrane with a pore size of 70-100 micrometers, the centrifugation speed is 1200rpm-1800rpm, and the centrifugation time is 4min-6min.

[0024] In some embodiments of this application, in step (3), the volume ratio of the tissue-cell mixture precipitate to the red blood cell lysis buffer is 1:(2-4); the settling time is 2-4 min; the centrifugation speed is 1200 rpm-1800 rpm; the centrifugation time is 4-6 min; the volume ratio of the tissue-cell mixture precipitate to the culture medium described in the first aspect of this application is 1:50-100; the volume ratio of the tissue-cell mixture precipitate to the Matrigel is 1:1; and the replenishment time is 20-28 h of incubation.

[0025] The fourth aspect of this application provides mouse gastric squamous cell carcinoma cell lines and / or gastric cancer-associated fibroblast cell lines established by the methods described in the third aspect of this application.

[0026] The fifth aspect of this application provides the use of the mouse gastric squamous cell carcinoma cell lines and / or gastric cancer-associated fibroblast cell lines described in the fourth aspect of this application in constructing ectopic or orthotopic implantation models in immune-active mice.

[0027] The beneficial effects of this application are:

[0028] (1) This application is the first mouse gastric cancer cell line derived from a spontaneous tumor model of genetically engineered mouse gastric cancer established at home and abroad. It can be stably passaged and provides new experimental materials that are closer to clinical biological characteristics for exploring the microenvironment of human gastric cancer.

[0029] (2) The mouse gastric squamous cell carcinoma cell line mGC1955 provided in this application has high tumorigenicity and can successfully prepare gastric squamous cell carcinoma xenograft animal models. The prepared immune-active animal models can be used for basic research and screening of immunotherapy drugs, etc.

[0030] (3) The mouse gastric squamous cell carcinoma-related fibroblast cell line mCAF1955 provided in this application can be used to establish mouse gastric fibroblast-related research, providing favorable experimental materials for a comprehensive analysis of the tumor microenvironment;

[0031] (4) The cell line disclosed in this application can be used as an ideal cell line for preclinical research on the tumor microenvironment of human gastric cancer (including immunotherapy strategies, etc.);

[0032] (5) This application provides a "cocktail" culture medium formulation based on organoid culture medium, which can significantly improve the success rate of in vitro culture of primary cell lines;

[0033] In summary, this application, by obtaining the mouse gastric squamous cell carcinoma cell line mGC1955 and the gastric squamous cell carcinoma-associated fibroblast cell line mCAF1955 from spontaneous tumors in mice, can construct ectopic or in situ implantation models in immune-active mice. This can provide guidance for exploring the tumor microenvironment related to gastric cancer, including exploring biomarkers for immunotherapy efficacy, studying immune resistance mechanisms, developing new immunotherapy regimens, and conducting preclinical evaluations of new immunotherapies, and has high clinical application value.

[0034] Of course, implementing any product or method of this application does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other embodiments can be obtained based on these drawings.

[0036] Figure 1 This is an optical microscope image of the mGC1955 and mCAF1955 cell lines in the logarithmic growth phase in Example 2 of this application.

[0037] Figure 2 This is a growth curve diagram of the mGC1955 cell line and the mCAF1955 cell line in the logarithmic growth phase in Example 2 of this application;

[0038] Figure 3 This is an image showing the immunofluorescence detection results of the mGC1955 and mCAF1955 cell lines in the logarithmic growth phase in Example 2 of this application.

[0039] Figure 4 This image shows the tumor formation results 5 days after mGC1955 cells in the logarithmic growth phase of Example 3 of this application were inoculated into C57BL / 6J mice. Detailed Implementation

[0040] The technical solutions of this application will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on this application are within the scope of protection of this application.

[0041] The first aspect of this application provides a culture medium comprising L-WRN cell culture supernatant, and 100×N-2 additive 0.5 vol%-1.5 vol%, 50× vitamin A-free B-27 additive 1.5 vol%-2.5 vol%, N-acetylcysteine ​​amide 1 mM-1.5 mM, nicotinamide 8 mM-12 mM, FGF-10 80 ng / mL-120 ng / mL, EGF 40 ng / mL-60 ng / mL, A83-01 0.8 μM-1.2 μM, Y-27632 dihydrochloride 8 μM-12 μM, Glutamax additive 0.5 vol%-1.5 vol%, Matrigel 5 vol%-7 vol%, Hepes buffer 8 mM-12 mM, and gastrin 0.8 nM-1.2 nM.

[0042] In this application, 100×N-2 additive refers to N2 supplement (100×), which can be purchased from Gibco; 50× vitamin A-free B-27 additive refers to B27 supplement (50×), minus vitamin A, which can be purchased from Gibco; N-acetylcysteine ​​amide is N-Ace-L-Lys, which can be purchased from Sigma; Nicotinamide is Nicotinamide, which can be purchased from Sigma; FGF-10 refers to fibroblast growth factor 10, which can be purchased from Peprotech; EGF refers to epidermal growth factor, which can be purchased from Peprotech; A83-01 refers to TGF-β inhibitor (TGF inhibitor), which can be purchased from Selleck; the chemical formula of Y-27632 dihydrochloride is C 14 H 21 N3O·2HCl is available from Selleck; Glutamax additive refers to Gibco GlutaMAX™ additive, which is L-alanyl-glutamine (Ala-Glu), also known as alanyl-glutamine dipeptide, a modified L-glutamine alternative, and is available from Gibco; Matrigel is a mixture of extracellular matrix components containing laminin and various growth factors, and is available from Gibco; Hepes buffer is available from Gibco; Gastrin is available from Novoprotein; the primary cell antibiotic is Primocin.

[0043] The concentrations of each component in the culture medium provided in this application refer to the final concentrations in the culture medium.

[0044] The culture medium provided in this application is a "cocktail" culture medium formula based on organoid culture medium. This culture medium can significantly improve the success rate of in vitro culture of primary cell lines and can be used for primary cell line primary culture and passage culture.

[0045] In some embodiments of this application, the culture medium further includes: 0.08 vol%-0.12 vol% of primary cell antibiotics. The culture medium containing primary cell antibiotics in this application can be used for primary culture.

[0046] In some embodiments of this application, the method for preparing the L-WRN cell culture supernatant includes the following steps: L-WRN cells are cultured in DMEM complete medium containing FBS (fetal bovine serum), G418 and hygromycin B until the cell density reaches 85%-95%, then washed multiple times with PBS buffer, and then cultured in Advanced DMEM / F12 medium; the culture supernatant is collected at preset time intervals, the collected supernatants are combined and centrifuged, cell debris is removed and filtered, and the collected filtrate is mixed with Advanced DMEM / F12 medium to obtain the L-WRN cell culture supernatant.

[0047] In this application, the cell density can reach 85%, 87%, 89%, 91%, 93%, 95%, or any two values ​​in between.

[0048] The G418 (Geneticin) mentioned in this application is an aminoglycoside antibiotic; the hygromycin B mentioned in this application refers to hydromycin; the DMEM complete medium mentioned in this application refers to Dulbecco's Modified Eagle's Medium complete medium.

[0049] The L-WRN cells described in this application can be purchased from Meisen CTCC and are adherent mouse subcutaneous connective tissue cells. The L-WRN cells described in this application can be cultured in a 5% CO2 (volume fraction) incubator at 37°C.

[0050] The washing process described in this application, which involves multiple washes with PBS buffer, can be 2-3 washes using 1×PBS buffer.

[0051] The Advanced DMEM / F12 culture medium described in this application can be purchased from Gibco.

[0052] In some embodiments of this application, in the method for preparing L-WRN cell culture supernatant, the DMEM complete culture medium containing FBS, G418 and hygromycin B has the following concentrations: FBS concentration is 8 vol%-12 vol%, G418 concentration is 0.4 mg / mL-0.6 mg / mL, and hygromycin B concentration is 0.4 mg / mL-0.6 mg / mL.

[0053] In some embodiments of this application, in the method for preparing L-WRN cell culture supernatant, the preset interval is 20h-28h; and the culture supernatant is collected 3-5 times. In this application, the preset interval can be 20h, 22h, 24h, 26h, 28h, or a range of any two values ​​within this interval; and the culture supernatant can be collected 3, 4, or 5 times.

[0054] In some embodiments of this application, in the method for preparing L-WRN cell culture supernatant, the centrifugation speed is 2500g-3500g, and the centrifugation time is 12min-18min. In this application, the centrifugation speed can be 2500g, 2700g, 2900g, 3100g, 3300g, 3500g, or any combination of two values ​​therein; the centrifugation time can be 12min, 14min, 16min, 18min, or any combination of two values ​​therein.

[0055] In some embodiments of this application, the filtration in the method for preparing L-WRN cell culture supernatant is performed using a 0.22-0.45 micrometer filter.

[0056] In some embodiments of this application, in the method for preparing L-WRN cell culture supernatant, the collected filtrate and the Advanced DMEM / F12 culture medium are mixed at a volume ratio of 1:(0.8-1.2). In this application, the volume ratio of the collected filtrate to the Advanced DMEM / F12 culture medium can be 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, or any range of two values ​​within this range.

[0057] The second aspect of this application provides the use of the culture medium described in the first aspect of this application in the establishment of mouse gastric squamous cell carcinoma cell lines and gastric cancer-associated fibroblast cell lines.

[0058] The third aspect of this application provides a method for establishing a mouse gastric squamous cell carcinoma cell line and a gastric cancer-related fibroblast cell line, comprising the following steps:

[0059] (1) The tumor tissue of spontaneous liver metastases in mice was washed multiple times with PBS buffer containing double antibodies, then cut into pieces and added to organoid lysis buffer for lysis to obtain tissue homogenate;

[0060] (2) The tissue homogenate is filtered using a sterile filter membrane. During filtration, the tissue remaining on the filter membrane is ground. The collected filtrate is then centrifuged to collect the lower layer of tissue cell mixture precipitate.

[0061] (3) Add red blood cell lysis buffer to the precipitate of the tissue cell mixture, let it stand, centrifuge, remove the supernatant, resuspend the precipitate using the culture medium described in the first aspect of this application, then add Matrigel, mix well and inoculate into a multi-well plate for culture; after a certain period of culture, add liquid and continue culture until some tissue fragments can be observed to adhere to the wall and primary cells sprout and grow from the tissue.

[0062] (4) The primary cells were digested with pancreatic enzyme digestion solution after growing for 7-10 days, and different types of cells were roughly separated by multiple differential digestion methods.

[0063] (5) The mouse gastric squamous cell carcinoma cell line and the gastric cancer-associated fibroblast cell line were obtained by flow cytometry.

[0064] The mouse gastric squamous cell carcinoma cell line mGC1955 and the gastric cancer-associated fibroblast cell line mCAF1955 provided in this application are derived from a spontaneous gastric cancer model in p53 gene knockout mice. They are taken from spontaneous tumor liver metastases, and the tumor blocks are cut into tissue blocks and inoculated in a "cocktail" medium. After observing adherent cells sprouting and growing around the tissue blocks, they are digested and passaged. The "enzyme difference method" is used to roughly distinguish tumor cells from fibroblasts. Finally, flow cytometry is used to sort tumor cells and fibroblasts. The method provided in this application is highly operable, has low preparation cost, and is highly reproducible.

[0065] The spontaneous liver metastasis tumor tissue used in step (1) of this application is derived from the spontaneous gastric cancer model of P53 gene knockout mice. The construction method of the spontaneous gastric cancer model of P53 gene knockout mice includes the following steps: knocking out exons 2, 3, 4, 5 and 6 of the TP53 gene (GenBank Accession No. NC_000077.7 (Update Date 2024-02) nucleotide sequence, which includes 12 exons) in the genome of C57BL / 6 mice using the CRISPR-Cas9 system. After PCR identification, TP53 gene knockout C57BL / 6 mice (P53 gene knockout mice with systemic TP53 gene heterozygosity loss) were obtained. - / + ).

[0066] The organoid lysis fluid mentioned in step (1) of this application can be purchased commercially, for example, from Kelto. The term "shredding" in this application refers to using sterile scissors and forceps to shred the tissue block to 0.5 mm. 3 The procedure described in this application involves washing mouse spontaneous tumor liver metastases multiple times with PBS buffer containing bispecific antibodies, typically 2-3 times.

[0067] In step (1) of this application, a small amount of organoid lysis buffer can be added first, and the tissue block can be cut into pieces to 0.5 mm using sterile scissors and forceps. 3 Add the remaining organoid lysis buffer and mix well; alternatively, add all the organoid lysis buffer at once.

[0068] In step (2) of this application, grinding can be performed using a grinder; the filtrate can be collected using centrifuge tubes such as 10mL-15mL centrifuge tubes.

[0069] In step (3) of this application, the static temperature can be room temperature; the multi-well plate used can be a 6-well plate (Corning); the culture can be carried out in a 5% CO2, 37℃ constant temperature incubator; some tissue fragments can be observed to adhere to the wall, and primary cells can sprout and grow from the tissue, usually after 72 hours. Replenishment refers to supplementing the culture medium described in the first aspect of this application. This application does not have a particular limitation on the amount of replenishment, as long as it can achieve the inventive purpose of this application. For example, the volume of the supplemented culture medium described in the first aspect of this application is (2-5):1 of the original culture mixture.

[0070] In step (4) of this application, different types of cells can be roughly separated by multiple differential digestion methods according to the different digestion times of different types of cells.

[0071] In step (5) of this application, the two types of cells are separated by flow cytometry according to different markers of different types of cells, so as to obtain the mouse gastric squamous cell carcinoma cell line and the gastric cancer-related fibroblast cell line respectively.

[0072] In some embodiments of this application, in step (1), the volume ratio of the spontaneously generated mouse liver metastasis tumor tissue to the organoid lysis buffer is 1:(800-1200); the lysis is performed at 35℃-39℃ and 200rpm-300rpm for 8min-12min. The lysis described in this application can be performed in centrifuge tubes, such as 5mL-10mL tubes, placed in a shaker at 35℃-39℃ and 200rpm-300rpm. Depending on actual needs, a stop solution containing 8vol%-10vol% FBS complete culture medium can be used to terminate the lysis. In this application, the volume ratio of mouse spontaneous tumor liver metastasis tumor tissue to organoid lysate can be 1:800, 1:900, 1:1000, 1:1100, 1:1200, or any two values ​​thereof; the lysis rotation speed can be 200 rpm, 220 rpm, 240 rpm, 260 rpm, 280 rpm, 300 rpm, or any two values ​​thereof; and the lysis time can be 8 min, 9 min, 10 min, 11 min, 12 min, or any two values ​​thereof.

[0073] In some embodiments of this application, in step (2), the sterile filter membrane is a sterile filter membrane with a pore size of 70-100 micrometers, the centrifugation speed is 1200 rpm-1800 rpm, and the centrifugation time is 4 min-6 min. In this application, the centrifugation speed can be 1200 rpm, 1400 rpm, 1600 rpm, 1800 rpm, or any two values ​​in between; the centrifugation time can be 4 min, 5 min, 6 min, or any two values ​​in between.

[0074] In some embodiments of this application, in step (3), the volume ratio of the tissue-cell mixture precipitate to the red blood cell lysis buffer is 1:(2-4); the settling time is 2-4 min; the centrifugation speed is 1200 rpm-1800 rpm; the centrifugation time is 4-6 min; the volume ratio of the tissue-cell mixture precipitate to the culture medium described in the first aspect of this application is 1:(50-100); the volume ratio of the tissue-cell mixture precipitate to the Matrigel is 1:1; and the replenishment time is 20-28 h of incubation.

[0075] In this application, the volume ratio of the tissue cell mixture precipitate to the red blood cell lysis buffer can be 1:2, 1:3, 1:4, or any two values ​​in between; the settling time can be 2 min, 3 min, 4 min, or any two values ​​in between; the centrifugation speed can be 1200 rpm, 1400 rpm, 1600 rpm, 1800 rpm, or any two values ​​in between; and the centrifugation time can be 4 min, 5 min, 6 min, or any two values ​​in between.

[0076] In this application, the volume ratio of the tissue cell mixture precipitate to the culture medium described in the first aspect of this application can be 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, or any range of two values ​​therein.

[0077] The fourth aspect of this application provides mouse gastric squamous cell carcinoma cell lines and / or gastric cancer-associated fibroblast cell lines established by the methods described in the third aspect of this application.

[0078] The fifth aspect of this application provides the use of the mouse gastric squamous cell carcinoma cell lines and / or gastric cancer-associated fibroblast cell lines described in the fourth aspect of this application in constructing ectopic or orthotopic implantation models in immune-active mice.

[0079] In some embodiments of this application, the mouse is a C57BL / 6J mouse.

[0080] This application provides a mouse gastric squamous cell carcinoma cell line mGC1955 and a mouse tumor-associated fibroblast cell line mCAF1955, both of which can be stably passaged; and provides a method for culturing the above-mentioned mouse primary cells, namely, isolating them from tumor tissue that has metastasized from mouse primary gastric cancer to the liver, continuously culturing the primary cells using a specially prepared "cocktail" culture medium combined with an "enzyme differential method", and then isolating and purifying mouse gastric squamous cell carcinoma cells and tumor-associated fibroblasts that can be stably proliferated and passaged.

[0081] Example

[0082] The embodiments and comparative examples provided below illustrate the implementation of this application in more detail. Various tests and evaluations were conducted according to the methods described below. Furthermore, unless otherwise specified, percentage content in this application refers to mass percentage for solid-liquid mixtures and solid-phase-solid mixtures, and volume percentage for liquid-phase-liquid mixtures.

[0083] Example 1: Establishment of mouse gastric squamous cell carcinoma cell lines and tumor-associated fibroblast cell lines

[0084] 1. Preparation of "cocktail" culture medium

[0085] Freshly revived L-WRN cells (purchased from Meisen CTCC, adherent mouse subcutaneous connective tissue cells) were cultured in DMEM containing 10 vol% FBS, 0.5 mg / mL G418, and 0.5 mg / mL hygromycin B in a 5% CO2 incubator at 37°C. Once the cell density reached 90%, the cells were washed twice with 1×PBS buffer and then cultured in Advanced DMEM\F12 medium (purchased from Gibco). Culture supernatant was collected every 24 hours for 96 consecutive hours and centrifuged at 3000g for 15 min to remove cell debris. The supernatant was then filtered through a 0.22-micron filter. The culture supernatants collected over four consecutive days were mixed with an equal volume of Advanced DMEM\F12 medium to obtain the L-WRN cell culture supernatant.

[0086] Using L-WRN cell culture supernatant as the stock solution, prepare the "cocktail" culture medium according to Table 1 below:

[0087] Table 1 “Cocktail” culture medium

[0088] Note: When used as a primary culture medium, the "cocktail" medium also includes 0.1 vol% of Primocin (a primary cell antibiotic).

[0089] 2. Grinding treatment of tumor tissue

[0090] The obtained liver metastasis tumor tissue from the spontaneous gastric cancer model in P53 gene knockout mice was washed twice in 1×PBS buffer containing double antibodies (1% penicillin-streptomycin mixture), and a small amount of organoid lysis buffer (Ketoo Pharmaceuticals) was added. The tissue blocks were then cut into 0.5 mm pieces using sterile scissors and forceps. 3 Add organoid lysis buffer and mix well (the volume ratio of tumor tissue to organoid lysis buffer is 1:1000). Transfer to a 5 mL centrifuge tube and lyse and digest in a shaker at 37°C and 250 rpm for 10 min to obtain a tissue homogenate. Then filter the digested tissue homogenate through a 100 μm pore size sterile filter membrane, grind the tissue remaining on the filter membrane using a grinder, and then collect the filtrate into a 15 mL centrifuge tube. Centrifuge at 1500 rpm for 5 min and collect the lower layer of tissue cell mixture precipitate.

[0091] 3. Primary culture

[0092] Add 3 times the volume of erythrocyte lysis buffer to the lower layer of tissue cell mixture precipitate collected in step 2 above. After standing at room temperature for 3 min, centrifuge at 1500 rpm for 5 min, remove the supernatant, and resuspend the precipitate in the "cocktail" medium from step 1 above (including 0.1 vol% Primocin, with a volume ratio of tissue cell mixture precipitate to "cocktail" medium of 1:100). Add approximately 6 vol% Matrigel (with a volume ratio of tissue cell mixture precipitate to Matrigel of 1:1), mix well, and seed into 6-well plates. Incubate in a 5% CO2, 37°C incubator. After 24 hours, add supplemental medium. After 72 hours, some tissue fragments were observed to adhere to the plate, and primary cells (tumor cells and fibroblasts) sprouted and grew from the surrounding tissue.

[0093] 4. Passage breeding

[0094] After the primary cells have grown for 10 days, they are digested with pancreatic enzyme digestion solution. Based on the different digestion times of tumor-associated fibroblasts and tumor cells, a multiple differential digestion method is used to roughly separate the primary tumor cells and fibroblasts.

[0095] 5. Cell sorting

[0096] Further, based on different markers of epithelial cells and fibroblasts, the two cell types were separated by flow cytometry to obtain mouse gastric squamous cell carcinoma cell line mGC1955 and gastric cancer-associated fibroblast cell line mCAF1955, respectively.

[0097] Example 2: Identification of growth and biological characteristics of mouse gastric squamous cell carcinoma cell lines and tumor-associated fibroblast cell lines

[0098] Cell morphology observation: The mGC1955 and mCAF1955 cell lines in the logarithmic growth phase obtained in Example 1 were respectively placed under an inverted microscope to observe the morphology of live cells, such as... Figure 1 As shown, both cell lines exhibit adherent growth. The mGC1955 cell line is predominantly irregular polygonal, large and round, without contact inhibition, and can grow in overlapping patterns. The mCAF1955 cell line is predominantly spindle-shaped, thin and long.

[0099] Growth curve and doubling time determination: Cells in the logarithmic growth phase obtained in Example 1 were collected by centrifugation, and the cell concentration was adjusted to 2000 cells / 100 μL and seeded into 96-well plates. The plates were incubated for 2 days at 37°C, 5% CO2, and 70%-80% humidity, and monitored in real-time using the Incucyte live-cell imaging system. A cell growth curve was plotted with time on the x-axis and relative cell proliferation level on the y-axis, as shown below. Figure 2 As shown, the cell doubling time was calculated to be 11-15 hours.

[0100] Immunofluorescence assay: mGC1955 and mCAF1955 cell lines in logarithmic growth phase obtained in Example 1, positive control gastric squamous cell carcinoma MFC, and positive control mouse embryonic fibroblasts NIH / 3T3 were cultured to 70%–80% confluence. After washing with PBS, they were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and blocked with 5% BSA. Diluted primary antibody was added and incubated overnight at 4°C. After washing, fluorescent secondary antibody was added and incubated in the dark. After DAPI staining of the nuclei, the slides were mounted with anti-fluorescence quenching mounting medium. Fluorescence signals were observed under a fluorescence microscope, and expression levels were quantified using ImageJ software. The primary antibody was anti-α-SMA mouse monoclonal antibody (catalog number ab7817) purchased from Abcam (USA); the secondary antibody was anti-pan-CK mouse monoclonal antibody (catalog number #4545) purchased from Cell Signaling Technology (USA). Results are as follows: Figure 3 As shown, the mGC1955 cell line highly expresses pan-CK (red), consistent with the positive control gastric squamous cell carcinoma (MFC); the mCAF1955 cell line highly expresses α-SMA (green), consistent with the positive control mouse embryonic fibroblasts (NIH / 3T3).

[0101] Example 3: mGC1955 cell line used to construct C57BL / 6J mouse ectopic implantation model

[0102] The mGC1955 cells in logarithmic growth phase obtained in Example 1 were divided into 3×10⁻⁶ cells. 6 200 μL / mouse was injected subcutaneously into four female C57BL / 6J mice (5 weeks old, weighing 15g-19g); tumor formation at the injection site was observed 5 days after injection, and the results are as follows. Figure 4 As shown, a lump the size of a grain of rice can be felt under the skin.

[0103] This application utilizes mouse gastric squamous cell carcinoma cell line mGC1955 and gastric cancer-associated fibroblast cell line mCAF1955 obtained from spontaneous tumors in mice to construct ectopic or in situ implantation models in immune-active mice. This can provide guidance for exploring the tumor microenvironment related to gastric cancer, including exploring biomarkers for immunotherapy efficacy, studying immune resistance mechanisms, developing new immunotherapy regimens, and conducting preclinical evaluations of new immunotherapies, thus possessing high clinical application value.

[0104] The above description is only a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A culture medium comprising L-WRN cell culture supernatant, and 100× N-2 additive 0.5 vol%-1.5 vol%, 50× vitamin A-free B-27 additive 1.5 vol%-2.5 vol%, N-acetylcysteine ​​amide 1 mM-1.5 mM, nicotinamide 8 mM-12 mM, FGF-10 80 ng / mL-120 ng / mL, EGF 40 ng / mL-60 ng / mL, A83-01 0.8 μM-1.2 μM, Y-27632 dihydrochloride 8 μM-12 μM, Glutamax additive 0.5 vol%-1.5 vol%, Matrigel 5 vol%-7 vol%, Hepes buffer 8 mM-12 mM, and gastrin 0.8 nM-1.2 nM; Preferably, the culture medium further includes 0.08 vol% to 0.12 vol% of primary cell antibiotics.

2. The culture medium according to claim 1, wherein, The method for preparing the L-WRN cell culture supernatant includes the following steps: L-WRN cells were cultured in DMEM complete medium containing FBS, G418, and hygromycin B. When the cell density reached 85%-95%, the cells were washed multiple times with PBS buffer and then cultured in Advanced DMEM / F12 medium. The culture supernatant was collected at preset time intervals. The collected supernatants were combined, centrifuged, and filtered after removing cell debris. The collected filtrate was mixed with Advanced DMEM / F12 medium to obtain the L-WRN cell culture supernatant.

3. The culture medium according to claim 2, wherein, In the DMEM complete medium containing FBS, G418 and hygromycin B: the concentration of FBS is 8 vol%-12 vol%, the concentration of G418 is 0.4 mg / mL-0.6 mg / mL, and the concentration of hygromycin B is 0.4 mg / mL-0.6 mg / mL; The preset interval is 20h-28h; the culture supernatant is collected 3-5 times. The centrifugation speed is 2500g-3500g, and the centrifugation time is 12min-18min; The filtration is performed using a 0.22-0.45 micron filter; The filtrate and the Advanced DMEM\F12 culture medium were mixed at a volume ratio of 1:(0.8-1.2).

4. The use of the culture medium according to any one of claims 1-3 in the establishment of mouse gastric squamous cell carcinoma cell lines and gastric cancer-related fibroblast cell lines.

5. A method for establishing a mouse gastric squamous cell carcinoma cell line and a gastric cancer-related fibroblast cell line, comprising the following steps: (1) The tumor tissue of spontaneous liver metastases in mice was washed multiple times with PBS buffer containing double antibodies, then cut into pieces and added to organoid lysis buffer for lysis to obtain tissue homogenate; (2) The tissue homogenate is filtered using a sterile filter membrane. During filtration, the tissue remaining on the filter membrane is ground. The collected filtrate is then centrifuged to collect the lower layer of tissue cell mixture precipitate. (3) Add red blood cell lysis buffer to the precipitate of the tissue cell mixture, let it stand, centrifuge, remove the supernatant, resuspend the precipitate using the culture medium of any one of claims 1-3, then add Matrigel, mix well and inoculate into a multi-well plate for culture; after a certain period of culture, add liquid and continue culture until some tissue fragments can be observed to adhere to the wall and primary cells sprout and grow from the tissue. (4) The primary cells were digested with pancreatic enzyme digestion solution after growing for 7-10 days, and different types of cells were roughly separated by multiple differential digestion methods. (5) The mouse gastric squamous cell carcinoma cell line and the gastric cancer-associated fibroblast cell line were obtained by flow cytometry.

6. The method for establishing according to claim 5, wherein: In step (1), the volume ratio of the spontaneous liver metastasis tumor tissue in mice to the organoid lysis solution is 1:(800-1200); the lysis is performed at 35℃-39℃ and 200rpm-300rpm for 8min-12min.

7. The method for establishing according to claim 5, wherein: In step (2), the sterile filter membrane is a sterile filter membrane with a pore size of 70-100 micrometers, the centrifugation speed is 1200rpm-1800rpm, and the centrifugation time is 4min-6min.

8. The method for establishing according to any one of claims 5-7, wherein: In step (3), the volume ratio of the tissue cell mixture precipitate to the red blood cell lysis buffer is 1:(2-4); the settling time is 2 min-4 min; the centrifugation speed is 1200 rpm-1800 rpm; the centrifugation time is 4 min-6 min; the volume ratio of the tissue cell mixture precipitate to the culture medium according to any one of claims 1-3 is 1:(50-100); the volume ratio of the tissue cell mixture precipitate to the Matrigel is 1:1; and the replenishment time is 20 h-28 h of incubation.

9. The mouse gastric squamous cell carcinoma cell line and / or gastric cancer-associated fibroblast cell line established by the method according to any one of claims 5-8.

10. The application of the mouse gastric squamous cell carcinoma cell line and / or gastric cancer-associated fibroblast cell line according to claim 9 in constructing ectopic or orthotopic implantation models in immune-active mice; preferably, the mice are C57BL / 6J mice.

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