A culture medium and method for culturing digestive tract tumors using three-dimensional tumor slice technology

Abstract

The present invention discloses a culture medium and method for culturing digestive tract tumors using three-dimensional tumor slice technology. The culture medium is composed of the following components: FBS 5-10% v / v, Ham's F12 nutrient solution 20-30% v / v, recombinant human epidermal growth factor EGF 0.10-0.15 ng / mL, hydrocortisone 20-30 ng / mL, cholera toxin 0.05-0.15 nM, insulin 4-6µg / mL, Y-27632 dihydrochloride 9-11µM, cefoperazone sodium 10-30μg / mL, sulbactam sodium 5-15μg / mL, and DMEM as the remainder. The culture medium not only reduces the contamination rate, but also has high cell viability and an immune microenvironment similar to the original tissue, making it possible to evaluate the drug sensitivity of immunotherapy drugs for clinical digestive tract tumors by the in vitro 3D-TSCs culture method, and has high clinical application value and market potential.

Description

Technical Field

[0001] The invention relates to a culture medium, and in particular to a culture medium and a method for culturing digestive tract tumors using three-dimensional tumor slice technology. Background Art

[0002] Accurately predicting drug sensitivity is a prerequisite for the success of personalized cancer treatment, which requires tumor samples grown in a microenvironment that is most similar to its original conditions. Scientists have made many efforts to truly simulate the tumor microenvironment of cancer patients. For example, models such as 2D cells, 3D spheroids, organoids, zebrafish, and nude mouse PDXs have been used to evaluate the drug sensitivity response of anticancer drugs, but there are still challenges in creating an in vitro environment suitable for all types of tumors from different individuals. We previously established a new mouse tumor-derived three-dimensional tumor slice (3D-TSCs) drug sensitivity test platform for evaluating the drug sensitivity of targeted and immune checkpoint inhibitors (ICIs) (FQ Xing, et al. Accelerating precision anti-cancer therapy by time-lapse and label-free3D tumor slice cultureplatform, THERANOSTICS, 2021, 11(19): 9415-9430). The 3D-TSCs drug sensitivity testing platform has several major advantages: 1) It contains stromal cells, immune cells, and cancer cells; while primary patient-derived organoids (PDOs) lose the immune components of the original tumor due to digestion and passaging during the modeling process; 2) All resected tumor samples can be used for drug evaluation; 3) It is a fast and low-cost in vitro system. In fact, the evaluation process can be completed in 4-7 days, while PDOs take more than two weeks, and animal models and primary patient-derived tumor xenografts (PDXs) take more than two months to complete the evaluation due to modeling. These characteristics make 3D-TSCs technology a physiologically relevant model in the field of precision oncology for selecting the best and most effective drugs for each cancer patient. Whether it is antibody or cell immunotherapy, it is an effective technical model that provides a cost-saving, fast and easy-to-operate platform for anti-cancer drug discovery, which can accelerate precision anti-cancer treatment. In order to confirm that targeted drugs combined with ICIs have a synergistic effect, we tested the drug sensitivity of the combined drug based on 3D-TSCs constructed from mouse tumors and found that the combination of FGFR2 inhibitors and ICIs (PD-1 / PD-L1 antibodies) enhanced the drug sensitivity of tumors in mice. Currently, in clinical practice, tumor immunotherapy is based on the results of pathological immunohistochemistry, and there is no reliable model to test the drug sensitivity of immune drugs. Therefore, how to more accurately determine the sensitivity of patients to immunotherapy is of great clinical significance.

[0003] The 3D-TSCs we previously established mainly used mouse-derived tumors to conduct immune checkpoint drug sensitivity testing. However, there are huge differences between clinical samples and mouse tumors in terms of cells and environment. The in vitro culture of clinical samples is more susceptible to contamination and the modeling success rate is not high, which greatly affects the development of immune checkpoint drug sensitivity testing.

[0004] Moreover, compared with clinical tumor specimens of other organs, digestive tract tumors are very easy to be contaminated in vitro due to contact with food residues and digestive tract flora. In our experiments, we have tried to use disinfectants such as alcohol and povidone iodine to clean clinical tumor samples, increase the number of times PBS is used to clean specimens, sterilize operating tools such as scissors and blades with high-pressure steam, and add ultraviolet lamps to sterilize the vibrating slicer and operating environment, but none of them has a significant effect on the high contamination rate of digestive tract tumors cultured in 3D-TSCs. In addition, we added penicillin and streptomycin, an antibiotic commonly used in cell experiments, to the PBS used to clean specimens, and also added penicillin and streptomycin to the PBS used in the vibrating slicer slicing process, in order to achieve the best possible anti-contamination effect. The actual effect is not good, and the contamination rate remains above 90%, resulting in a large waste of specimens and experimental time, hindering the clinical application of 3D-TSCs technology. In practice, people often control contamination by increasing the content of penicillin and streptomycin during the culture of organoids. In the process of culturing three-dimensional tumor slices, we also borrowed the method of dealing with organoid contamination problems and increased the amount of penicillin and streptomycin. The addition of antibiotics may have little effect on the modeling of organoids, because the construction of organoids requires the extraction of cancer cells, and a small number of cells can be cultured and propagated without other tumor components, such as immune cells, so organoids themselves are not suitable for immune checkpoint drug sensitivity testing. In fact, antibiotics have anti-cancer effects while resisting pollution, which can cause great damage to the vitality of cancer cells. In particular, immune cells are particularly sensitive to antibiotics. During the experiment, we found that high concentrations of penicillin and streptomycin caused immune cells to stop growing or die under 3D-TSCs technology conditions, so immunotherapy efficacy evaluation often failed. Therefore, a culture medium for culturing digestive tract tumors using three-dimensional tumor slice technology that can reduce the contamination rate, maintain high cell vitality, and conduct immune checkpoint drug sensitivity testing is a key technical point of 3D-TSCs. Summary of the invention

[0005] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a culture medium for culturing digestive tract tumors using three-dimensional tumor slice technology.

[0006] Another object of the present invention is to provide a method for culturing digestive tract tumors using three-dimensional tumor slice technology. The method uses the above culture medium to overcome the contamination problem, maintain the high cell viability of the original tumor tissue, and obtain a tumor slice with an immune microenvironment similar to the original tumor microenvironment.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A culture medium for culturing digestive tract tumors using three-dimensional tumor slice technology, comprising the following components: FBS 5-10% v / v, Ham's F12 nutrient solution 20-30% v / v, recombinant human epidermal growth factor EGF 0.10-0.15 ng / mL, hydrocortisone 20-30 ng / mL, cholera toxin 0.05-0.15nM, insulin 4-6 µg / mL, Y-27632 dihydrochloride (Y-27632 2HCl) 9-11 µM, cefoperazone sodium 10-30 μg / mL, sulbactam sodium 5-15 μg / mL, and DMEM as the balance; preferably, the culture medium comprises the following components: FBS 7.5% v / v, Ham's F12 nutrient solution 25% v / v, recombinant human epidermal growth factor EGF 0.125 ng / mL, hydrocortisone 25 ng / mL, cholera toxin 0.1 nM, insulin 5 µg / mL, Y-27632 dihydrochloride (Y-27632 2HCl) 10µM, cefoperazone sodium 20μg / mL, sulbactam sodium 10μg / mL, and DMEM as the balance.

[0009] Application of the above culture medium in culturing digestive tract tumors with 3D-TSCs.

[0010] A method for culturing digestive tract tumors using three-dimensional tumor slice technology comprises the following steps:

[0011] (1) Prepare three-dimensional digestive tract tumor slices using three-dimensional tumor slice technology;

[0012] (2) Using the above culture medium to culture the three-dimensional digestive tract tumor slices obtained in step (1), a tumor slice that can be used for drug sensitivity testing is obtained.

[0013] The specific steps of step (1) are preferably as follows:

[0014] 1) Sterilize the fresh digestive tract tumor tissues collected from the clinic, as well as the operating instruments and operating environment, and divide them into tumor strips;

[0015] 2) Fix the tumor strips;

[0016] 3) Slice the fixed tumor strips using a microtome;

[0017] 4) The obtained slices are placed on the membrane of an insert culture dish and fixed with collagen gel to obtain three-dimensional digestive tract tumor slices.

[0018] The digestive tract tumors described in step 1) include gastric cancer and intestinal cancer.

[0019] The disinfectant used for the digestive tract tumor tissue in step 1) is preferably at least one of povidone-iodine and PBS containing antibiotics.

[0020] The composition of the antibiotics in the PBS containing antibiotics is preferably as follows: 400 U / mL penicillin, 400 μg / mL streptomycin, and 10 μg / mL amphotericin B.

[0021] The disinfection methods of the operating instruments described in step 1) include high temperature and high pressure sterilization, ozone sterilization and sterilization using disinfectant agents.

[0022] The disinfectant is preferably an ethanol solution with a concentration of 75%.

[0023] The disinfection methods of the operating environment include ozone sterilization and sterilization using disinfectant agents.

[0024] The disinfectant is preferably an ethanol solution with a concentration of 75%.

[0025] The specifications of the tumor strips described in step 1) are preferably as follows: a width of 0.3 to 0.6 cm and a length of no more than 1 cm.

[0026] The fixing agent in step 2) is low melting point agarose; more preferably, it is a low melting point agarose solution with a concentration of 5-9% w / v.

[0027] The thickness of the slices obtained by slicing in step 3) is preferably 200 to 300 μm.

[0028] The specific steps of step 4) are preferably: firstly spread collagen gel on the membrane of the insert culture dish, then place the slices, and then wrap the collagen gel on the slices.

[0029] The steps of spreading the collagen gel are as follows: placing the liquid containing the collagen gel on the membrane of the insert culture dish until the liquid containing the collagen is completely solidified.

[0030] The step of wrapping the slice with collagen gel is to add liquid containing collagen to the slice until the liquid containing collagen is completely solidified.

[0031] The liquid containing collagen gel is preferably prepared by the following steps: mixing the collagen gel pre-cooled on ice, 10× Ham's F-12 solution and sterile buffer in a volume ratio of 8:1:1.

[0032] The concentration of the collagen gel is preferably 5 mg / mL.

[0033] The collagen is preferably collagen with a molecular weight of 300 kDa.

[0034] The sterile buffer is preferably prepared by the following steps: dissolving 2.2 g NaHCO 3 in 100 mL of a solution containing 0.05 mol / L NaOH and 200 mM HEPES.

[0035] The coagulation environment temperature is preferably 30-40°C; more preferably 37°C.

[0036] The culture described in step (2) is to add the above culture medium to the periphery of the insert culture dish and replace the culture medium every 3 to 4 days.

[0037] Compared with the prior art, the present invention has the following advantages and effects:

[0038] The present invention provides a culture medium suitable for culturing digestive tract tumors with 3D-TSCs, which not only reduces the contamination rate, but also has high viability of cultured cells and an immune microenvironment similar to that of the original tissue, making it possible to evaluate the drug sensitivity of immunotherapy drugs for clinical digestive tract tumors by an in vitro 3D-TSCs culture method, and has high clinical application value and market potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a graph showing the contamination status of gastric cancer tumor slices obtained by culturing with six types of culture media using three-dimensional tumor slice technology.

[0040] Figure 2 These are photographs of cell viability detection of gastric cancer tumor sections cultured in three types of culture media.

[0041] Figure 3 The figure is a graph showing the cell viability test results of gastric cancer tumor sections cultured with three culture media; among them, the cell viability obtained with the implemented culture medium is 100%.

[0042] Figure 4 This is a graph showing the contamination status of colon cancer tumor slices obtained using 6 types of culture media using three-dimensional tumor slice technology.

[0043] Figure 5 These are photographs of cell viability detection of colorectal cancer tumor sections cultured in 4 types of culture media.

[0044] Figure 6 This is a graph showing the results of cell viability testing of colorectal cancer tumor sections cultured with three types of culture media; among them, the cell viability obtained with the implemented culture medium is 100%.

[0045] Figure 7 This is a graph showing the effect of culture medium on the immune microenvironment of colorectal cancer tumor slices.

[0046] Figure 8 This is a graph showing the contamination of colorectal cancer tumor sections by culture media with different concentrations of cefoperazone sodium and sulbactam sodium.

[0047] Fig. 9 This is a graph showing the results of cell viability testing of colorectal cancer tumor sections using culture media containing different concentrations of cefoperazone sodium and sulbactam sodium.

[0048] Fig.10 This is a graph showing the effects of culture media with different concentrations of cefoperazone sodium and sulbactam sodium on the immune microenvironment of colorectal cancer tumor sections. DETAILED DESCRIPTION

[0049] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0050] The main reagents and consumables used in the present invention are as follows:

[0051] DMEM, manufactured by GIBCO, catalog number 11966025;

[0052] Fetal bovine serum, manufactured by CellMax, catalog number SA301.02;

[0053] Ham's F-12 Nutrient Mix, manufactured by Gibco, catalog number 21700075;

[0054] Recombinant human epidermal growth factor, manufacturer is MedChemExpress (MCE), product number is HY-P7109;

[0055] Hydrocortisone, manufacturer is MedChemExpress (MCE), product number is HY-N0583;

[0056] Cholera toxin, manufacturer is MedChemExpress (MCE), product number is HY-P1446;

[0057] Insulin, manufacturer: Selleckchem, product number: S6955;

[0058] Y-27632 2HCl, manufacturer: MedChemExpress (MCE), product number: HY-10583;

[0059] Penicillin-streptomycin, manufactured by Gibco, catalog number 15140122;

[0060] Amphotericin B, manufacturer: Thermo Fisher Scientific, catalog number: 15290018;

[0061] Gentamycin, manufactured by Thermo Fisher Scientific, catalog number 15710064;

[0062] Cefoperazone sodium and sulbactam sodium, manufactured by Pfizer Pharmaceuticals Co., Ltd.;

[0063] PBS, manufacturer: Invitrogen, catalog number: 18912014;

[0064] Low melting point agarose, manufacturer is Macklin, product number is A800343-100g;

[0065] Insert culture dish, manufacturer is SPL Life Sciences, catalog number is 37524;

[0066] Cultrex 3-D Culture Matrix™ rat tail glue is manufactured by R&D, and the product number is 3447-020-01;

[0067] Anti -Ki67, brand is Zhongshan Jinqiao, item number is ZM-0167;

[0068] CD3 antibody, brand is Zhongshan Jinqiao, product number is ZA-0503;

[0069] CD4 antibody, brand is Zhongshan Jinqiao, product number is ZA-0519;

[0070] CD8 antibody, brand is Zhongshan Jinqiao, product number is ZA-0508;

[0071] CTLA-4 antibody, brand is Zhongshan Jinqiao, product number is ZM-0035;

[0072] PD-1 antibody, brand is Zhongshan Jinqiao, product number is ZM-0381;

[0073] DAB colorimetric kit, brand: Servicebio G1212-200T;

[0074] Hematoxylin Solution, Harris Modified, brand: Sigma, product number: HHS16-500mL;

[0075] Eosin Y Stain Solution, For HE, brand is Solarbio, product number is G1100-100mL.

[0076] Example 1

[0077] (1) Preparation: Wash the solid tumor of the digestive tract removed by surgery with normal saline for 2-3 times, then collect the tumor and transfer it in a 4°C pre-cooled culture medium (DMEM containing 10% v / v FBS and 1% penicillin / streptomycin, 1% penicillin / streptomycin refers to 100U / mL penicillin and 100μg / mL streptomycin). Wash the specimen 2-3 times with pre-cooled washing PBS in a biosafety cabinet, then use scissors and forceps to cut the tumor into tumor strips with a width of 0.3-0.6 cm and a length of no more than 1 cm. Prepare low-melting point agarose with a concentration of 5-9% (w / v), and fix the tumor strips when the agarose temperature drops to about 37°C. Place it on ice or in a 4°C environment for 20 minutes. The composition of the antibiotics in the washing PBS is as follows: 400U / mL penicillin, 400μg / mL streptomycin, and 10μg / mL amphotericin B.

[0078] (2) Tumor sectioning: Within 2-6 hours after surgery, the fixed tumor strips obtained in step (1) were sectioned under sterile conditions using a vibratome Leica VT1200 S (Leica Biosystems Nussloch GmbH, Germany) to obtain tumor tissue sections with a thickness of 200-300 μm.

[0079] (3) Slice culture: Pre-cooled A (rat collagen I), B solution (10 × Ham's F-12) and C solution (sterile buffer, obtained by dissolving 2.2g NaHCO3 in 100mL of a solution containing 0.05mol / L NaOH and 200mM HEPES) on ice were mixed in a volume ratio of 8:1:1 to obtain a liquid gel. 100 μL of the liquid gel solution was poured onto the membrane in a 12mm diameter culture dish insert (membrane pore size 0.4 μm). The culture dish insert was placed in a 37°C incubator for 20-30 minutes until the collagen liquid gel was completely solidified. Then the tumor slice prepared in step (2) was carefully placed on it and 100 μL of liquid gel was added to the top layer. It was solidified in a 37°C incubator for 20-30 minutes, and 400 μL of culture medium was added to the periphery of the culture dish insert. Thereafter, the culture medium was replaced every 3-4 days.

[0080] The culture media used in the optimization process are as follows:

[0081] Control 1 culture medium: Fetal bovine serum and gentamicin were added to Ham's F12 nutrient solution, wherein the concentration of the fetal bovine serum was 20% v / v, and the concentration of gentamicin was 50 μg / mL.

[0082] Control 2 culture medium: FBS 7.5% v / v, Ham's F12 nutrient solution 125 mL, recombinant human epidermal growth factor EGF 0.125 ng / mL, hydrocortisone 25 ng / mL, cholera toxin 0.1 nM, insulin 5 µg / mL, Y-27632 dihydrochloride (Y-27632 2HCl) 10 µM, penicillin 400 U / mL, streptomycin 400 μg / mL, DMEM was made up to 500 mL.

[0083] Control 3 culture medium: FBS 7.5% v / v, Ham's F12 nutrient solution 125 mL, recombinant human epidermal growth factor EGF 0.125 ng / mL, hydrocortisone 25 ng / mL, cholera toxin 0.1 nM, insulin 5 µg / mL, Y-27632 dihydrochloride (Y-27632 2HCl) 10 µM, penicillin 400 U / mL, streptomycin 400 μg / mL, amphotericin B 10 μg / mL, DMEM is diluted to 500 mL.

[0084] Control 4 culture medium: fetal bovine serum, cefoperazone sodium and sulbactam sodium were added to Ham's F12 nutrient solution, wherein the concentration of fetal bovine serum was 20% v / v, the concentration of cefoperazone sodium was 20 μg / mL, and the concentration of sulbactam sodium was 10 μg / mL.

[0085] Control 5 culture medium: FBS 7.5% v / v, Ham's F12 nutrient solution 125 mL, recombinant human epidermal growth factor EGF 0.125 ng / mL, hydrocortisone 25 ng / mL, cholera toxin 0.1 nM, insulin 5 µg / mL, Y-27632 dihydrochloride (Y-27632 2HCl) 10 µM, penicillin 400 U / mL, streptomycin 400 μg / mL, amphotericin B 10 μg / mL, cefoperazone sodium 20 μg / mL, sulbactam sodium 10 μg / mL, DMEM was diluted to 500 mL.

[0086] Implementation culture medium: FBS 7.5% v / v, Ham's F12 nutrient solution 125 mL, recombinant human epidermal growth factor EGF 0.125 ng / mL, hydrocortisone 25 ng / mL, cholera toxin 0.1 nM, insulin 5 µg / mL, Y-27632 dihydrochloride (Y-27632 2HCl) 10 µM, cefoperazone sodium 20 μg / mL, sulbactam sodium 10 μg / mL, DMEM is diluted to 500 mL.

[0087] (4) Test results:

[0088] 1) Collect gastric cancer specimens from 5 patients, and then use the above 6 culture media to culture the tumor slices for 4 days for each specimen according to steps (1) to (3), with 4 replicates for each group; add 0.5mL of 1mg / ml MTT staining solution and stain for 1 hour. If the culture medium is dark and turbid, it is considered to be contaminated. If the number of contaminated cells is ≥2, the group is classified as contaminated for statistics; if the number of contaminated cells is <2, the group is classified as non-contaminated. The contamination situation is counted, and the results are as follows: Figure 1 As shown in the figure: 5 specimens were cultured with the implementation medium, control medium 4, and control medium 5, and none of them was contaminated; the contamination rate was 60% when cultured with control medium 1 and control medium 2; and the contamination rate was 40% when cultured with control medium 3. It can be seen that the culture medium containing cefoperazone sodium and sulbactam sodium effectively reduced the contamination rate.

[0089] MTT is a classic staining reagent for detecting cell viability or proliferation. Deep purple staining indicates very high viability. Dead cells are generally difficult to stain, and light colors indicate low or no viability. Figure 2 This is a photograph of a specimen with clear culture medium after MTT staining. From the naked eye observation, the tumor obtained by culture in the culture medium is stained darker.

[0090] After MTT staining, the clear specimens were washed three times with PBS and then dissolved with isopropanol. The absorbance at OD570nm was measured using an ELISA reader to quantify cell viability. The results were as follows: Figure 3 As shown, among the 5 gastric cancer specimens, the cell viability of the tumor sections cultured with the implementation medium was generally higher than that with the control 4 medium and the control 5 medium.

[0091] 2) Collect colorectal cancer specimens from 20 patients, and then use the above 6 culture media to culture the tumor slices for 4 days for each specimen according to steps (1) to (3). Set up 4 replicates for each group. If the culture medium is dark and turbid after MTT staining, it is judged as contaminated. If the number of contaminated cells is ≥2, the group is classified as contaminated for statistics; if the number of contaminated cells is <2, the group is classified as non-contaminated. The contamination situation is counted. The results are as follows: Figure 4As shown, the contamination conditions are arranged in the order of control 5 medium < implementation medium < control 4 medium < control 3 medium < control 2 medium < control 1 medium.

[0092] Figure 5 This is a photograph of the specimen with clear culture fluid after MTT staining. From the naked eye observation, the tumor staining color obtained by culture in the implementation culture medium is generally darker than that of the control 5 culture medium, that is, the cell viability of the tumor slice obtained by culture in the implementation culture medium is high.

[0093] After MTT staining, the clear samples were washed with PBS three times and then dissolved with isopropanol. The OD was measured using an enzyme reader. 570nm Absorbance value, quantify cell viability, and the results are as follows Figure 6 As shown. It can be seen that among the 20 colorectal cancer specimens, the viability of 2 specimens could not be detected due to contamination, and the viability of tumor cells cultured in the implementation medium of the remaining 18 specimens was higher than that of the control medium 4, and the cell viability of the implementation medium of 15 specimens was higher than that of the control medium 5.

[0094] The above results show that the implementation of the culture medium is more conducive to maintaining the viability of colorectal cancer tumor slice cells while controlling contamination. Therefore, considering the anti-contamination situation and cell viability results, the implementation of the culture medium is more conducive to the three-dimensional tumor slice culture.

[0095] 3) The colorectal cancer tumor sections were cultured with the implementation medium for 4 days, and then fixed and immunohistochemically stained (IHC) (method reference Shu J, Dolman GE, Duan J, Qiu G, Ilyas M. Statistical colour models: an automated digital image analysis method for quantification of histological biomarkers. Biomed Eng Online. 2016; 15: 46.). The results are as follows Figure 7 As shown, it can be seen that tumor tissue cultured in culture medium can completely retain the immune components of solid tumors.

[0096] 4) The concentrations of cefoperazone sodium and sulbactam sodium were optimized. Different concentrations of cefoperazone sodium and sulbactam sodium in the culture medium were set. Ten colorectal cancer tumor sections were cultured for 4 days, with 4 replicates in each group. If the culture medium was dark and turbid after MTT staining, it was considered contaminated. If the number of contaminated cells was ≥2, the group was classified as contaminated for statistics. If the number of contaminated cells was <2, the group was classified as non-contaminated and the contamination was counted. The results are as follows Figure 8As shown, it can be seen that the culture medium containing 2 mg / mL cefoperazone sodium + 1 mg / mL sulbactam sodium has the best anti-contamination effect, followed by the culture medium containing 20 μg / mL cefoperazone sodium + 10 μg / mL sulbactam sodium.

[0097] The cell viability was quantified using MTT staining. Fig. 9 As shown, it can be seen that the tumor cell viability obtained by the culture medium containing 20 μg / mL cefoperazone sodium + 10 μg / mL sulbactam sodium is generally higher than that of the culture medium containing 2 mg / mL cefoperazone sodium + 1 mg / mL sulbactam sodium.

[0098] Immunohistochemical staining was used to detect the immune microenvironment of tumor sections. Fig.10 As shown, it can be seen that tumor tissues cultured in culture media containing different concentrations of cefoperazone sodium and sulbactam sodium can completely retain the immune components of solid tumors. However, the expression level of immune components in tumor sections cultured in the culture medium containing 20μg / mL cefoperazone sodium + 10μg / mL sulbactam sodium is higher than that in the culture medium containing 2mg / mL cefoperazone sodium + 1mg / mL sulbactam sodium.

[0099] In summary, 20 μg / mL cefoperazone sodium + 10 μg / mL sulbactam sodium is the best concentration for use. It maintains the best cell viability while effectively controlling specimen contamination and completely preserves the immune components of solid tumors, which is most conducive to drug sensitivity testing.

[0100] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A culture medium for culturing digestive tract tumors using three-dimensional tumor slice technology, characterized in that It is composed of the following ingredients: FBS 5-10% v / v, Ham's F12 nutrient solution 20-30% v / v, recombinant human epidermal growth factor EGF 0.10-0.15 ng / mL, hydrocortisone 20-30 ng / mL, cholera toxin 0.05-0.15 nM, insulin 4-6 µg / mL, Y-27632 dihydrochloride 9-11 µM, cefoperazone sodium 10-30 μg / mL, sulbactam sodium 5-15 μg / mL, and DMEM as the balance.

2. The culture medium for culturing digestive tract tumors using three-dimensional tumor slice technology according to claim 1, characterized in that The medium is composed of the following ingredients: FBS 7.5% v / v, Ham's F12 nutrient solution 25% v / v, recombinant human epidermal growth factor EGF 0.125 ng / mL, hydrocortisone 25 ng / mL, cholera toxin 0.1 nM, insulin 5 µg / mL, Y-27632 dihydrochloride 10 µM, cefoperazone sodium 20 μg / mL, sulbactam sodium 10 μg / mL, and DMEM as the balance.

3. Use of the culture medium according to claim 1 or 2 in culturing digestive tract tumors using three-dimensional tumor slice technology.

4. A method for culturing digestive tract tumors using three-dimensional tumor slice technology, characterized in that The steps include: (1) Prepare three-dimensional digestive tract tumor slices using three-dimensional tumor slice technology; (2) Using the culture medium described in claim 1 or 2 to culture the three-dimensional digestive tract tumor slices obtained in step (1) to obtain tumor slices that can be used for drug sensitivity testing.

5. The method for culturing digestive tract tumors using three-dimensional tumor slice technology according to claim 4, characterized in that: The specific steps of step (1) are as follows: 1) Sterilize the fresh digestive tract tumor tissues collected from the clinic, as well as the operating instruments and operating environment, and divide them into tumor strips; 2) Fix the tumor strips; 3) Slice the fixed tumor strips using a microtome; 4) The obtained slices are placed on the membrane of an insert culture dish and fixed with collagen gel to obtain three-dimensional digestive tract tumor slices.

6. The method for culturing digestive tract tumors using three-dimensional tumor slice technology according to claim 5, characterized in that: The disinfectant used for the digestive tract tumor tissue in step 1) is at least one of povidone-iodine and PBS containing antibiotics; The disinfection methods of the operating instruments described in step 1) include high temperature and high pressure sterilization, ozone sterilization and sterilization with disinfectant agents; The disinfection methods of the operating environment include ozone sterilization and sterilization using disinfectant agents.

7. The method for culturing digestive tract tumors using three-dimensional tumor slice technology according to claim 6, characterized in that: The composition of antibiotics in the antibiotic-containing PBS is as follows: 400 U / mL penicillin, 400 μg / mL streptomycin, and 10 μg / mL amphotericin B.

8. The method for culturing digestive tract tumors using three-dimensional tumor slice technology according to claim 5, characterized in that: The specifications of the tumor strips described in step 1) are as follows: 0.3 to 0.6 cm in width and no more than 1 cm in length; The fixing agent in step 2) is low melting point agarose; The slice thickness obtained by the slicing described in step 3) is 200 to 300 μm; The specific steps of step 4) are: firstly spread collagen gel on the membrane of the insert culture dish, then place the slice, and then wrap the collagen gel on the slice; The culture described in step (2) is to add the culture medium described in claim 1 or 2 to the periphery of the insert culture dish, and replace the culture medium every 3 to 4 days.

9. The method for culturing digestive tract tumors using three-dimensional tumor slice technology according to claim 8, characterized in that: The fixing agent is a low melting point agarose solution with a concentration of 5-9% w / v; The steps of spreading the collagen gel are as follows: placing a liquid containing the collagen gel on the membrane of the insert culture dish until the liquid containing the collagen gel is completely solidified; The step of wrapping the slice with collagen gel is to add liquid containing collagen gel to the slice until the liquid containing collagen gel is completely solidified.

10. The method for culturing digestive tract tumors using three-dimensional tumor slice technology according to claim 9, characterized in that: The collagen-containing liquid is prepared by the following steps: mixing the collagen pre-cooled on ice, 10×Ham's F-12 solution and sterile buffer in a volume ratio of 8:1:1; The sterile buffer solution is prepared by the following steps: dissolving 2.2 g NaHCO3 in 100 mL of a solution containing 0.05 mol / L NaOH and 200 mM HEPES; The solidification environment temperature is 30-40°C.

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