Mucous membrane melanoma organ and tumor-associated fibroblast co-culture model as well as culture medium, culture method and application thereof

Abstract

The invention provides a mucous membrane melanoma organoid and tumor-associated fibroblast co-culture model and a culture medium, a culture method and application thereof, the culture medium comprises a CAF culture medium and a PDO culture medium, and the CAF culture medium comprises a culture medium 1 and a culture medium 2; the culture medium 1 comprises DMEM (Dulbecco Modified Eagle Medium) high glucose, 20% fetal calf serum and 1% penicillin-streptomycin solution; the culture medium 2 comprises DMEM (Dulbecco Modified Eagle Medium) high glucose, 10% fetal calf serum, 5ng / ml bFGF and 1% penicillin-streptomycin solution; the PDO culture medium is composed of a PDO basic culture medium component and a PDO additive component. The co-culture model constructed by the culture medium provided by the invention can be continuously subcultured during in-vitro culture, and can still keep stable cellular morphology and proliferation speed after being subcultured for multiple times, so that the co-culture model can be applied to scientific research related to mucous membrane melanoma.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts, a culture medium, a culture method and an application thereof. Background Art

[0002] Mucosal melanoma (MM) is a subtype of malignant melanoma with high invasiveness, unknown pathogenesis, high incidence in East Asian populations, lack of therapeutic targets, and extremely poor prognosis, with a 5-year survival rate of less than 25%. Currently, there is no systematic clinical treatment recommendation for patients with recurrent and metastatic MM. The clinical effects of first-line / second-line chemotherapy regimens are not ideal, and the efficacy of immunotherapy is poor, far lower than that of skin melanoma. Most patients in the middle and late stages often have no drugs available.

[0003] In recent years, the development of immunotherapy has provided a promising treatment approach for patients with advanced melanoma, such as the promotion and application of immune checkpoint inhibitors (ICIs). Ipilimumab, Nivolumab, and Pembrolizumab have been approved for melanoma indications. However, the efficacy of immunotherapy in mucosal malignant melanoma is limited, and its efficacy is significantly lower than that of cutaneous melanoma. In the 2019 KEYNOTE-151 study data, the study evaluated the efficacy and safety of Pembrolizumab in the treatment of Chinese patients with advanced melanoma, with an ORR of 13.3% for mucosal melanoma. The POLARIS-01 study is a study of Toripalimab for Chinese patients with advanced refractory melanoma who have failed systemic treatment. A total of 128 patients were included, including 50 patients with acral subtype and 22 patients with mucosal subtype. The overall ORR was 17.3%, mPFS was 3.6 months, and mOS was expected to be 22.2 months. The ORR in the skin subtype can reach 34%, but in the evaluable mucosal subtype patients, no CR and PR were observed, and the ORR was 0. In general, immunotherapy has a certain relief effect on mucosal malignant melanoma, but its therapeutic effect, whether it is monotherapy or combination therapy, the effectiveness of MM on immunotherapy is far from that of cutaneous malignant melanoma. Therefore, it is an urgent problem to clarify the primary resistance mechanism of immunotherapy, explore potential combined treatment strategies, and enhance the anti-tumor immune efficacy of mucosal melanoma including OMM.

[0004] In recent years, although conventional treatment methods (such as surgery, radiotherapy, interferon, immunotherapy, etc.) and comprehensive treatment strategies have made certain progress in the clinical treatment of MM, a standardized clinical diagnosis and treatment plan or guideline has not yet been formed, which seriously affects the treatment and prognosis of MM patients. One of the main reasons is the lack of effective clinical trials and preclinical models for MM research, especially disease models that can reflect the MM tumor microenvironment.

[0005] A large number of different types of tumors have confirmed the presence of cancer-associated fibroblasts (CAFs) in the tumor microenvironment, and their extraction and passage methods have been reported. However, since the number of MM tumor samples is relatively small compared to common malignant tumors, and the success rate of CAF extraction and passage is low, it is difficult to establish a CAF cell line for MM. Therefore, there is no confirmation of the presence of CAFs in MM in reports, which has hindered the research progress of MM to a certain extent. Cancer-associated fibroblasts (CAFs) are an important component of the tumor microenvironment (TME) and often play a key role in tumor immune escape and immunotherapy resistance, but the specific mechanism is still unclear.

[0006] The definition of organoids is just like its name, which is an organ-like model. It is a type of microscopic three-dimensional structure that can self-assemble when various cells are cultured under certain conditions in vitro. The development of patient-derived organoids (PDO) technology has greatly promoted tumor research and achieved many exciting achievements. It also brings opportunities for the construction of preclinical models of mucosal melanoma and provides new possibilities for basic and clinical translational research on mucosal melanoma.

[0007] The tumor microenvironment is not only composed of tumor cells, but also includes endothelial cells, immune cells and fibroblasts. These cells together constitute a complex tumor microenvironment, and the interaction between cells has been shown to promote tumor growth, especially cancer-associated fibroblasts (CAF), which have been reported to affect the progression of cancer in many cancers. Therefore, it is particularly important to construct a co-culture system of CAF and PDO, because it can more realistically reflect and simulate the growth of tumors in the human body.

[0008] Currently, researchers have constructed a variety of organoids to simulate various tissue types and established a co-culture system with CAFs. However, there are certain difficulties in establishing a co-culture system of organoids and CAFs for mucosal melanoma, especially whether the mixed culture medium can enable CAFs to survive. Summary of the invention

[0009] In view of the defects of the prior art, the object of the present invention is to provide a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts and its culture medium, culture method and application.

[0010] The technical solution of the present invention is: In a first aspect, the present invention provides a culture medium for constructing a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts, wherein the culture medium of the co-culture model comprises a CAF culture medium and a PDO culture medium, wherein the CAF culture medium comprises a culture medium 1 and a culture medium 2; The culture medium 1 includes DMEM high glucose, 20wt.% fetal bovine serum, and 1wt.% penicillin-streptomycin solution; The culture medium 2 includes DMEM high glucose, 10wt.% fetal bovine serum, 5ng / ml bFGF, and 1wt.% penicillin-streptomycin solution; The PDO culture medium is composed of a PDO basic culture medium component and a PDO additive component. The PDO basic culture medium component includes Advanced DMEM / F12 and the following components based on Advanced DMEM / F12: 1% Antibiotic-Antimycotic, 1% GlutaMAX, and 1% HEPES buffer; the PDO additive component is composed of the following: 100 ng / ml fibroblast growth factor, 100 ng / ml EGF, 10 μg / ml bFGF, 500 nM / ml A83-01, 500 ng / ml Wnt3a recombinant protein, 0.1 μg / ml Noggin recombinant protein, 0.1 μg / ml R-spondin-1 recombinant protein, 1.25 mM N-acetylcysteine, 10 mM / ml nicotinamide, 1×B27 supplement, and 10 uM Rho-associated protein kinase inhibitor Y27632.

[0011] Further preferably, the CAF culture medium and the PDO culture medium are mixed in a weight ratio of 1:1.

[0012] A second aspect of the present invention provides a kit for constructing a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts, wherein the kit comprises the above-mentioned culture medium for constructing a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts.

[0013] A third aspect of the present invention provides a method for constructing a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts, wherein the construction method uses the above-mentioned culture medium or the above-mentioned kit to co-culture mucosal melanoma organoids and tumor-associated fibroblasts to obtain the co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts.

[0014] The method for constructing the co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts comprises the following steps: S1. Prepare a mucosal melanoma organoid suspension and a tumor-associated fibroblast suspension, wherein the cell number ratio of the mucosal melanoma organoid suspension and the tumor-associated fibroblast suspension is 1:(1-3); S2, mixing the mucosal melanoma organoid suspension and the tumor fibroblast suspension in S1 to obtain a mixed suspension; S3. Mix Matrigel and type I rat tail collagen in a volume ratio of 1:(1-2) to obtain a heavy suspension, resuspend the mixed suspension in S2 with the heavy suspension, inoculate into the well plate after gently blowing on ice, seed the gel droplets, add culture medium after the gel droplets solidify, and co-culture for 2-5 days to obtain a co-culture model of tumor-associated fibroblasts and mucosal melanoma organoids.

[0015] Further preferably, in step S3, Matrigel and type I rat tail collagen in the resuspension are mixed at a volume ratio of 1:2.

[0016] More preferably, in step S3, the concentration of type I rat tail collagen in the resuspension is 3.33 mg / ml.

[0017] Further preferably, in step S3, after the gel droplets are seeded, the well plate is placed in an incubator, and culture medium is added after the gel droplets are solidified.

[0018] A fourth aspect of the present invention provides a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts, wherein the co-culture model is prepared according to the above-mentioned method for constructing a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts.

[0019] The fourth aspect of the present invention provides the application of a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts in clarifying the primary resistance mechanism of immunotherapy, preparing or screening immune resistance; and exploring the immunotherapy of mucosal malignant melanoma.

[0020] Compared with the prior art, the present invention has the following beneficial effects: By using the culture medium for constructing a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts provided by the present invention, a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts with a stable growth state can be obtained, and after multiple passaging of the co-culture model, the relevant co-culture model still maintains a stable cell morphology and proliferation rate, so that it can be applied to scientific research related to mucosal malignant melanoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings: Figure 1 A schematic diagram of the growth status of cells in each group after culturing for 2 days in the three experimental groups provided in the third embodiment of the present invention (the three experimental groups are a single CAF group: CAF only, in which there are only CAF cells in the culture medium; a single PDO group: PDO only, in which there are only PDO cells in the culture medium; and a PDO+CAF group: PDO+CAF=1:3, in which PDO cells and CAF cells in the culture medium are co-cultured at a ratio of 1:3); Figure 2 A schematic diagram of the growth status of cells in each group after 5 days of culture in the three experimental groups provided in the third embodiment of the present invention (the three experimental groups are a single CAF group: CAF only, in which there are only CAF cells in the culture medium; a single PDO group: PDO only, in which there are only PDO cells in the culture medium; and a PDO+CAF group: PDO+CAF=1:3, in which PDO cells and CAF cells in the culture medium are co-cultured at a ratio of 1:3); Figure 3 A schematic diagram of the statistical results of the diameter of the cell spheres obtained by using statistical software to analyze the single PDO group and the PDO+CAF group provided in the third embodiment of the present invention (PDO only, only PDO cells in the culture medium, PDO+CAF group: PDO+CAF=1:3, PDO cells and CAF cells in the culture medium were co-cultured at a ratio of 1:3); Figure 4 Schematic diagram of HE staining results of mucosal melanoma organoids and parental tumors provided in the experimental examples of the present invention (P2 refers to the second-generation mucosal melanoma organoids, Figure 4 a: Schematic diagram of HE staining results of P2 generation HE×20; Figure 4 b: Schematic diagram of HE staining results of parental tumor samples); Figure 5 Schematic diagram of the immunohistochemical staining results of protein expression of mucosal melanoma organoids and parental tumors provided for the experimental examples of the present invention (P2 refers to the second-generation mucosal melanoma organoids; Figure 5 a: Schematic diagram of immunohistochemical staining results of P2 generation, IHC×20; Figure 5 b: Schematic diagram of immunohistochemical staining results of parental tumor samples). DETAILED DESCRIPTION

[0022] The present invention is described in detail below with reference to specific embodiments.

[0023] The following examples will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several changes and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.

[0024] First embodiment This embodiment provides a culture medium for constructing a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts.

[0025] The culture medium of the co-culture model includes CAF culture medium and PDO culture medium, wherein the CAF culture medium includes culture medium 1 and culture medium 2.

[0026] Culture medium 1: DMEM high glucose (Shanghai Yuanpei), 20% fetal bovine serum (Gibco), 1% penicillin-streptomycin solution (Gibco).

[0027] Culture medium 2: DMEM high glucose (Shanghai Yuanpei), 10% fetal bovine serum (Gibco), 5ng / ml bFGF (MCE), 1% penicillin-streptomycin solution (Gibco).

[0028] The PDO culture medium consists of a PDO basal culture medium component and a PDO additive component. The PDO basal culture medium component includes Advanced DMEM / F12 and the following components based on Advanced DMEM / F12: 1% Antibiotic-Antimycotic, 1% GlutaMAX, and 1% HEPES buffer; the PDO additive component consists of the following: fibroblast growth factor 100ng / ml, EGF 100ng / ml, bFGF 10μg / ml, A83-01500nM / ml, Wnt3a recombinant protein 500ng / ml, Noggin recombinant protein 0.1μg / ml, R-spondin-1 recombinant protein 0.1μg / ml, N-acetylcysteine ​​1.25mM, nicotinamide 10mM / ml, 1×B27 supplement, and Rho-associated protein kinase inhibitor Y27632 10uM.

[0029] When constructing a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts, CAF culture medium and PDO culture medium were mixed at a weight ratio of 1:1.

[0030] Compared with the culture medium currently used to culture the co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts, the culture medium provided in this embodiment, although the EGF reagent is not added to the culture medium 2, still achieves the normal growth of organoid cells, and achieves the optimal reduction of the amount of culture medium components, thereby effectively reducing the cost of the co-culture model. The formula of the culture medium provided in this embodiment is obtained through extensive experimental verification, which means that subsequent researchers can use it directly without the need for time-consuming exploratory experiments, which significantly improves the research efficiency and saves a lot of valuable time for the experimenters.

[0031] Second embodiment The present embodiment provides a kit for constructing a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts, and the kit includes the culture medium for constructing a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts in the first embodiment. The kit is based on the existing culture medium, which can effectively save the company's cost and time in the research and development process; based on the culture medium provided in the first embodiment, combined with a scientific research and development process, the successful development and market launch of the kit is ensured; the development of the kit not only reduces the company's cost, but also improves the research and development efficiency, and provides a powerful tool for the study of head mucosal malignant melanoma.

[0032] Third embodiment The present embodiment provides a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts and a construction method thereof. The construction method uses the culture medium provided in the first embodiment to co-culture mucosal melanoma organoids and tumor-associated fibroblasts to obtain a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts.

[0033] The method for constructing a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts includes the following steps: 1. Pretreatment: Cell counting 1. Take one well of the organoid in the 48-well plate for cell counting. Discard the original culture medium, add 1ml ice PBS to the well, pipette gently repeatedly, and transfer to a 1.5ml EP tube. Centrifuge and discard the supernatant, resuspend with ice PBS, centrifuge again and discard the supernatant to remove the residual Matrigel. (Generally, wash twice to remove the gel.) 2. Add 250 μl TripLE to the EP tube in step 1, pipette repeatedly, incubate in a 37°C incubator for 3-5 minutes, pipette repeatedly again, add PBS to a volume of 1 ml, and count the cells. The number of cells in 1 ml can be determined based on the counting results.

[0034] Principle: Use the PDO digestion and counting in one well to estimate the total cell number of organoids in other wells Prepare CAF cell suspension: 1) Digest adherent CAF cells with trypsin. Generally, digestion of CAF cells can be terminated after 2 minutes. Resuspend in 1 ml PBS and count the cells.

[0035] 2. Experimental steps for constructing a co-culture model In this example, three experimental groups were set up for mutual comparison to compare and analyze the growth conditions of cells in each group. The three experimental groups were a single CAF group (CAF only), a single PDO group (PDO only), and a PDO+CAF group (PDO+CAF=1:3). Three replicate wells were set up in each group. The specific co-culture steps were as follows: Take one well of the organoid from the 48-well plate for the experiment. Discard the original culture medium, add 1 ml of ice PBS to the well, pipette gently repeatedly, and transfer to a 1.5 ml EP tube. Centrifuge and discard the supernatant, resuspend with ice PBS, centrifuge again and discard the supernatant to remove the residual Matrigel, add 1 ml PBS to reselect, place on ice for later use, and count the cells.

[0036] Prepare mucosal melanoma organoid suspension and tumor-associated fibroblast suspension, wherein the cell number ratio of the salivary mucosal melanoma organoid suspension and the tumor-associated fibroblast suspension is 1:3.

[0037] According to the PDO single cell counting results: if the PDO suspension used for co-culture is required to have 20W cells (cell number), the number of CAF cells used for co-culture is 60W, that is, the mixture is based on the cell number of 1:3. Among them, the number of cells in the single PDO group is 20W, and the number of cells in the single CAF group is 20W.

[0038] Prepare CAF cell suspension: 1) Digest adherent CAF cells with trypsin. Generally, digestion of CAF cells can be terminated after 2 minutes. Resuspend in 1 ml PBS and count the cells.

[0039] 3. After the PDO suspension and CAF cell suspension are mixed, centrifuge at 1000 rpm and 4°C for 3-5 min. Carefully remove the supernatant (try not to leave too much liquid) and keep the precipitate on ice for later use.

[0040] 4. The above groups were resuspended with Matrigel and type I rat tail collagen mixture (volume ratio of 1:2, final collagen concentration of 3.33 mg / ml), and inoculated into 48-well plates after gently blowing on ice. Each well has 5 μl of one drop of gel, and each of the above three groups has 3 replicates. Note that before adding each drop of gel, gently blow the gel-cell mixture in the EP tube.

[0041] 5. Place the 48-well plate in a 37°C incubator for 20 minutes. After the gel droplets solidify, add culture medium.

[0042] 6. Mix CAF medium and PDO medium in a weight ratio of 1:1 and add to a 48-well plate, adding 250 μL to each well.

[0043] 7. After 5 days of co-culture, a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts was obtained, and the growth was observed under an inverted microscope (the indicators for quantifying proliferation were: sphere diameter or sphere area).

[0044] After 2 days of co-culture, the growth status was observed under an inverted microscope. Figure 1 As shown by Figure 1 It can be seen that using the culture medium of the co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts provided by the present invention, the cells in the single CAF group, the single PDO group and the CAF+PDO group can survive and grow normally, indicating that the culture medium provided by the present invention has good applicability and safety.

[0045] After 5 days of co-culture, the growth status was observed under an inverted microscope. Figure 2 As shown by Figure 2 It can be seen that in the CAF+PDO group, the number and volume of PDO were greater than those in the single CAF group and the single PDO group, indicating that the presence of CAF makes PDO grow and proliferate faster.

[0046] After 3 days of co-culture, the diameter of the cell spheres was statistically analyzed as follows: Figure 3 As shown, through the use of statistical software analysis, it was concluded that in the CAF+PDO group, the diameter of the PDO spheres was significantly larger than that in the single PDO group, which indicated that when CAF and PDO were co-cultured, the growth state of PDO was better and the growth rate was faster.

[0047] Application Examples This application example provides the application of the co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts in clarifying the primary resistance mechanism of immunotherapy, preparing or screening immune resistance; and exploring the application of immunotherapy for mucosal malignant melanoma.

[0048] Experimental example The following experimental example identified the cell morphology and protein expression of mucosal melanoma organoids, and verified again that the PDO obtained by using the culture medium provided in the first embodiment can maintain a stable cell morphology after passage.

[0049] The patient tumor sample slice data were retrieved from the pathology archive of the Department of Oral Pathology, the Ninth People's Hospital Affiliated to Shanghai Jiao Tong University.

[0050] PDO passage experiment operation process: Discard the original culture medium, melt the matrix gel with pre-cooled PBS, gently blow down the mixture of matrix gel and organoids and transfer it to a centrifuge tube, centrifuge and discard the supernatant, resuspend with PBS and centrifuge and discard the supernatant to remove all the matrix gel, add 1-2 ml of trypsin (Suzhou Xinsaimei Biotechnology Co., Ltd.) to resuspend the precipitate and digest for 3 minutes to terminate the digestion. Centrifuge and discard the supernatant, then resuspend the precipitate with PBS. Count the cells under the microscope, and resuspend the cells with culture medium according to the counting results; then inoculate and culture.

[0051] The inoculated 24-well plate was placed upside down in a 37°C incubator (Thermo Fisher) for about 45 minutes. After the colloid solidified, the 24-well plate was placed upright, 450 ul of the culture medium provided in the first embodiment was added to each well, and the plate was returned to the 37°C incubator for culture. The culture medium was then replaced every 2-3 days.

[0052] The above inversion operation is more conducive to the formation of a dome-shaped structure of the matrix gel and firm adhesion to the wall. During this period, the organoids are observed under an inverted microscope. Generally, within two weeks, if the organoids do not grow larger, they are passaged. The passage ratio is 1:6 in a 24-well plate (i.e., the organoids in one well are passaged to 6 wells in a new 24-well plate).

[0053] HE staining experimental operation process: The organoids obtained after culture were processed using a modified agar encapsulation method: Fixation: Carefully pipette organoid tissue into a 24-well cell culture plate, add 10% neutral buffered formaldehyde, and fix at room temperature for 1-3 hours; Pre-embedding: Preheat the frozen agar solution to a viscous state in advance. After the organoid tissue is fixed, use a 3ml pipette to suck out the organoid tissue and place it in a disposable plastic embedding mold. Use a pipette to suck out an appropriate amount of agar solution and slowly add it around the organoid tissue until the agar wraps all the organoid tissue. After the agar solidifies at room temperature, put it into 75% alcohol, 85% alcohol, 95% alcohol, and 100% alcohol for dehydration together with the mold, each time for 5-20 minutes, to facilitate the agar block to be removed from the plastic embedding mold; Dehydration: Place the agar block containing pre-embedded organoid tissue in the embedding box and immerse it in 75% alcohol for 45 minutes, 85% alcohol for 45 minutes, 95% alcohol for 45 minutes, 100% alcohol for 1 hour, and 100% alcohol for 1 hour. Transparent and wax-immersed: xylene 30 min, xylene 30 min, paraffin 62°C 1 h, paraffin 62°C 1 h, paraffin 62°C 1 h; Sectioning: Gently pick up the agar block after treatment with tweezers, embed it in paraffin, and slice it into 3μm sections; Staining: Hematoxylin & Eosin (HE) staining, routine histological observation, and comparison with MM parental tumor Protein expression staining experimental operation process: Immunohistochemistry (IHC) analysis: Paraffin-embedded tissues were sliced ​​several times at 3 μm and baked at 65°C for 1-3 hours; after dewaxing and hydration, antigen retrieval was performed in EDTA buffer at pH 8.0 for 20 minutes; the slices were blocked with 3% H2O2 solution for 10 minutes to eliminate the activity of endogenous catalase in the tissues. After rinsing with PBS three times, primary antibodies (HMB45, MelanA, TYRO, SOX10 and S100) were added and incubated at room temperature for 1 hour. After rinsing with PBS three times, secondary antibodies were added and incubated at room temperature for 30 minutes. After rinsing with PBS three times, DAB was used for color development for 2 minutes and hematoxylin was used for counterstaining for 1 minute, and the slices were sealed with neutral gum and observed.

[0054] The P2 generation mucosal melanoma organoids were cultured to the 4th day, and the organoids were harvested and stained with HE. The cell morphology of the organoid staining results was compared with the HE staining results of the patient's tumor tissue. The comparison results are as follows: Figure 4 As shown, P2 mucosal melanoma organoid cells ( Figure 4 a) Morphology and parental tumor samples ( Figure 4 b) Similar.

[0055] Immunohistochemical staining of protein expression (HMB45, MelanA, TYRO, SOX10 and S100) was performed on P2 mucosal melanoma organoids cultured to day 4. The staining results of mucosal melanoma organoids were compared with those of patient tumor tissues. Figure 5 As shown, P2 organoids ( Figure 5 a) Protein expression levels compared with parental tumor samples ( Figure 5 b) similar.

[0056] The above experiments show that the culture medium provided by the present invention not only has good applicability and safety, but also the PDO obtained by using the culture medium provided by the present invention can maintain a stable morphology.

[0057] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. A culture medium for constructing a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts, characterized in that: The culture medium of the co-culture model includes CAF culture medium and PDO culture medium, wherein the CAF culture medium includes culture medium 1 and culture medium 2; The culture medium 1 includes DMEM high glucose, 20% fetal bovine serum, and 1% penicillin-streptomycin solution; The culture medium 2 includes DMEM high glucose, 10% fetal bovine serum, 5ng / ml bFGF, and 1% penicillin-streptomycin solution; The PDO culture medium is composed of a PDO basic culture medium component and a PDO additive component. The PDO basic culture medium component includes Advanced DMEM / F12 and the following components based on Advanced DMEM / F12: 1% Antibiotic-Antimycotic, 1% GlutaMAX, and 1% HEPES buffer; the PDO additive component is composed of the following: 100 ng / ml fibroblast growth factor, 100 ng / ml EGF, 10 μg / ml bFGF, 500 nM / ml A83-01, 500 ng / ml Wnt3a recombinant protein, 0.1 μg / ml Noggin recombinant protein, 0.1 μg / ml R-spondin-1 recombinant protein, 1.25 mM N-acetylcysteine, 10 mM / ml nicotinamide, 1×B27 supplement, and 10 uM Rho-associated protein kinase inhibitor Y27632.

2. The culture medium for constructing a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts according to claim 1, characterized in that: The CAF culture medium and the PDO culture medium were mixed at a weight ratio of 1:

1.

3. A kit for constructing a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts, characterized in that: A culture medium for constructing a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts as described in any one of claims 1-2.

4. A method for constructing a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts, characterized in that: The mucosal melanoma organoids and tumor-associated fibroblasts are co-cultured using the culture medium described in any one of claims 1-2 or the kit described in claim 3 to obtain the co-culture model of the mucosal melanoma organoids and tumor-associated fibroblasts.

5. The method for constructing a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts according to claim 4, characterized in that: The following steps are involved: S1. Prepare a mucosal melanoma organoid suspension and a tumor-associated fibroblast suspension, wherein the cell number ratio of the mucosal melanoma organoid suspension and the tumor-associated fibroblast suspension is 1:(1-3); S2, mixing the mucosal melanoma organoid suspension and the tumor fibroblast suspension in S1 to obtain a mixed suspension; S3. Mix Matrigel and type I rat tail collagen in a volume ratio of 1:(1-5) to obtain a heavy suspension, resuspend the mixed suspension in S2 with the heavy suspension, inoculate into the well plate after gently blowing on ice, seed the gel droplets, add culture medium after the gel droplets solidify, and co-culture for 2-5 days to obtain a co-culture model of tumor-associated fibroblasts and mucosal melanoma organoids.

6. The method for constructing a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts according to claim 4, characterized in that: In step S3, Matrigel and type I rat tail collagen in the resuspension are mixed at a volume ratio of 1:

2.

7. The method for constructing a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts according to claim 4, characterized in that: In step S3, the concentration of type I rat tail collagen in the resuspension is 3.33 mg / ml.

8. The method for constructing a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts according to claim 4, characterized in that: In step S3, after the gel droplets are planted, the well plate is placed in an incubator, and after the gel droplets solidify, culture medium is added.

9. A co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts, characterized in that: The co-culture model is prepared according to the method for constructing a co-culture model of mucosal melanoma organoids and tumor-associated fibroblasts according to any one of claims 4 to 8.

10. The mucosal melanoma organoid and tumor-associated fibroblast co-culture model according to claim 9, characterized in that: The co-culture model has at least one of the following applications: a. Application in clarifying the primary resistance mechanism of immunotherapy and preparing or screening immune resistance; b. Application in exploring immunotherapy for mucosal malignant melanoma.

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