Medium-auricular-biliary-neoplasm organoid culture medium and construction method of medium-auricular-biliary-neoplasm organoid

Abstract

The invention discloses a medium-auricular-biliary-neoplasm organoid culture medium and a construction method of a medium-auricular-biliary-neoplasm organoid, and relates to the technical field of organoid models. By optimizing the formula of the culture medium, the survival rate, the proliferation rate and the controllability of the differentiation direction of the middle auricular and biliary neoplasm cells are remarkably improved, the organoid forming time is short, the modeling success rate is high, and stable passage amplification can be achieved. Compared with an existing in-vivo and in-vitro model, the middle ear and bile adipoma organ can truly reproduce the three-dimensional structure and the cell function of middle ear and bile adipoma. The organ with the middle ear and gallbladder neoplasm provides a more reliable and innovative platform for accurately simulating the tissue microenvironment of the middle ear and gallbladder neoplasm, can lay a foundation for subsequent gene editing and drug screening research, and is beneficial to deep exploration of the generation mechanism and treatment method of the middle ear and gallbladder neoplasm.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid models, and in particular to a middle ear cholesteatoma organoid culture medium and a method for constructing a middle ear cholesteatoma organoid. Background Art

[0002] Middle ear cholesteatoma is a cystic lesion of keratinized stratified squamous epithelium that occurs in the middle ear. Its pathogenesis is unknown, and it has clinical characteristics such as wide invasion and severe complications. Currently, surgery is the only effective radical treatment for middle ear cholesteatoma, but it still faces difficulties such as high invasiveness and high postoperative recurrence rate. Therefore, a reliable disease model is of great significance for in-depth exploration of the occurrence and development mechanism of middle ear cholesteatoma and the development of new treatment methods.

[0003] At present, the commonly used disease models for middle ear cholesteatoma are mainly: 1) in vitro models: including in vitro culture of middle ear cholesteatoma keratinocyte cell lines and middle ear cholesteatoma tissues; 2) in vivo models: including mouse external auditory canal keratinocyte growth factor gene transfection induction model, Mongolian gerbil spontaneous tumor model and mouse fibroblast and keratinocyte mixed injection tumor model. Keratinocyte cell lines are easy to culture and operate, can be used for large-scale experimental screening, and have low cost, but they are separated from the three-dimensional structure of the original tissue, lack the interaction between tissues and the influence of the microenvironment, and therefore cannot effectively simulate the complex biological behaviors of middle ear cholesteatoma such as invasive growth. In addition, cell lines will undergo gene mutations or phenotypic changes during long-term culture, and lack the genome stability in vivo. Tissue culture models can simulate the microenvironment and cell-cell interactions of in situ tissues, and better preserve the histological structure of cholesteatoma, but due to the limitations of culture conditions, tissue culture models can often only maintain short-term growth and differentiation, and cannot carry out long-term drug screening and functional studies. Although animal models can simulate the overall physiological process of middle ear cholesteatoma and have unique advantages in drug testing, animal models often have species differences and cannot fully reflect the pathological characteristics of human cholesteatoma. Therefore, existing disease models still have great limitations in the mechanism and translational research of middle ear cholesteatoma.

[0004] In recent years, organoid technology has shown great potential in disease model research, especially in simulating complex tissue microenvironments. Organoids self-organize into structures similar to in situ tissues through three-dimensional culture, which can more realistically reproduce the growth and invasion behavior of middle ear cholesteatoma and simulate multi-level disease microenvironments, including interactions between different cell types. This makes organoids an ideal drug screening platform with the advantages of high-throughput screening and personalized treatment, and can conduct precision medicine research based on patient samples. At the same time, compared with traditional animal models, organoids have significant advantages in reducing ethical disputes, reducing costs and improving experimental efficiency, and have become an important tool for promoting the research and treatment of cholesteatoma in the middle ear.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a middle ear cholesteatoma organoid culture medium and a method for constructing middle ear cholesteatoma organoids, thereby providing a more reliable and innovative technical platform for research in related fields.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides a middle ear cholesteatoma organoid culture medium, which includes a basal culture medium DMEM / F12, and the basal culture medium contains the following substances at final concentrations: 1-2w / v% penicillin-streptomycin, 2-8mM GlutaMAX, 1-5x B27, 1-5x N2, 10-20μM Y-27632, 50-200ng / mL human epidermal growth factor EGF, 100-200ng / mL recombinant human Noggin protein, 100-200ng / mL FGF10, 300-600nM A-8301, 10-20mM nicotinamide, 1-5mM N-acetylcysteine, 5-20w / v% R-spondin1 conditioned medium and 5-20w / v% Wnt3a conditioned medium.

[0009] In a second aspect, the present invention provides a method for constructing a middle ear cholesteatoma organoid, comprising the following steps:

[0010] S1: Take the middle ear cholesteatoma sample for tissue digestion and obtain the cell pellet after tissue digestion;

[0011] S2: Take the cell pellet and resuspend it with Matrigel gel, inoculate the cell suspension on the cell culture plate, and then add the above-mentioned middle ear cholesteatoma organoid culture medium to the culture wells where the cells were inoculated, and culture them in an incubator at 37℃±0.5℃, 5% CO2, and replace the culture medium every 2-3 days; obtain the middle ear cholesteatoma organoids after 7-10 days of culture.

[0012] In a third aspect, the present invention also provides a method for constructing a middle ear cholesteatoma organoid and the use of the constructed middle ear cholesteatoma organoid in in vitro drug screening and / or detection.

[0013] In a preferred embodiment of the present invention, the in vitro drug screening is an in vitro high-throughput drug screening.

[0014] In a fourth aspect, the present invention also provides an in vitro drug screening method, the method comprising: applying a drug to be tested to the middle ear cholesteatoma organoid constructed by the above-mentioned method for constructing the middle ear cholesteatoma organoid, and detecting and analyzing the middle ear cholesteatoma organoid.

[0015] The present invention has the following beneficial effects:

[0016] The present invention significantly improves the controllability of the survival rate, proliferation rate and differentiation direction of middle ear cholesteatoma cells by optimizing the culture medium formula, and the organoid formation time is short, the modeling success rate reaches 100%, and it can be stably passaged and expanded. Compared with the existing in vitro and in vivo models, the middle ear cholesteatoma organoid (model) of the present invention can truly reproduce the three-dimensional structure and cell function of middle ear cholesteatoma. The organoid contains both proliferative and keratinized epithelial cells, and the outer layer is formed by a small amount of collagen fibers wrapped, which is highly similar to human middle ear cholesteatoma tissue, and is highly consistent with the original tissue at the transcriptome, genome and protein levels. This model provides a more reliable and innovative platform for accurately simulating the tissue microenvironment of middle ear cholesteatoma, which can lay the foundation for subsequent gene editing and drug screening research, and help to explore the pathogenesis and treatment methods of middle ear cholesteatoma in depth. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 Figures 2 and 3 are the results of the growth status test of human middle ear cholesteatoma organoids; (A) Growth status of the P0 generation of human middle ear cholesteatoma organoids in Example 1; (B) Growth status of the P1 generation of human middle ear cholesteatoma organoids in Example 1; (C) Growth status of the P0 generation of human middle ear cholesteatoma organoids in Comparative Example 1; (D) HE staining microscopy of the human middle ear cholesteatoma organoids in Example 1; (E) Multicolor immunofluorescence staining microscopy of the human middle ear cholesteatoma organoids in Example 1. DETAILED DESCRIPTION

[0019] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0020] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0021] In a first aspect, the present invention provides a middle ear cholesteatoma organoid culture medium, which includes a basal culture medium DMEM / F12, and the basal culture medium contains the following substances at final concentrations: 1-2w / v% penicillin-streptomycin, 2-8mM GlutaMAX, 1-5x B27, 1-5x N2, 10-20μM Y-27632, 50-200ng / mL human epidermal growth factor EGF, 100-200ng / mL recombinant human Noggin protein, 100-200ng / mL FGF10, 300-600nM A-8301, 10-20mM nicotinamide, 1-5mM N-acetylcysteine, 5-20w / v% R-spondin1 conditioned medium and 5-20w / v% Wnt3a conditioned medium.

[0022] The present invention significantly improves the survival rate, proliferation rate and controllability of differentiation direction of middle ear cholesteatoma cells by optimizing the culture medium formula, and the formation time of middle ear cholesteatoma organoids is short, the modeling success rate reaches 100%, and they can be stably passaged and expanded. Compared with existing in vitro and in vivo models, the organoid model of the present invention can truly reproduce the three-dimensional structure and cell function of middle ear cholesteatoma. The organoid contains both proliferative and keratinized epithelial cells, and the outer layer is wrapped with a small amount of collagen fibers, which is highly similar to human middle ear cholesteatoma tissue, and is highly consistent with the original tissue at the transcriptome, genome and protein levels.

[0023] If the cytokines Wnt3a and R-spondin1 are removed, the organoids cultured in this culture medium will show obvious growth stagnation or degeneration within 2 to 3 days, and cell apoptosis or necrosis will occur within 5 days, and the modeling success rate will be 0%.

[0024] The cytokine Wnt3a mainly promotes the growth of organoids by activating the Wnt / β-catenin signaling pathway, maintaining the proliferation, self-renewal and undifferentiated state of stem cells; R-spondin1 mainly enhances the activity of the Wnt signaling pathway, further promoting the proliferation and self-renewal of stem cells in organoids and maintaining the integrity of tissue structure.

[0025] Epithelial growth factor (EGF) binds to its receptor and induces proliferative changes. EGF is a factor required for organoid culture.

[0026] Noggin protein is an endogenous inhibitor of bone morphogenetic protein, regulating cell differentiation, proliferation and apoptosis. It is one of the most basic factors for organoid culture.

[0027] Y-27632 is a ROCK-I and ROCK-II inhibitor that inhibits apoptosis of host stem or progenitor cells.

[0028] GlutaMAX is a glutamine supplement that can provide cells with a nitrogen source. During cell culture, it not only participates in the biosynthesis of cells, but also participates in the energy metabolism of cells, helping to maintain the growth and functionality of organoids.

[0029] B27 is a common culture medium supplement that can replace serum and is commonly used in neural stem cell culture and does not cause differentiation in organoid culture.

[0030] A8301 is an effective inhibitor of transforming growth factor β type I receptor ALK5, ALK4 and ALK7, which can block the growth inhibition induced by transforming growth factor β and inhibit Smad2 phosphorylation to maintain the self-renewal and proliferation of stem cells and inhibit cell apoptosis and senescence; combined with Y-27632, it can improve cell survival rate and proliferation ability.

[0031] N-acetylcysteine ​​is also known as N-acetylcysteie.

[0032] As a fibroblast factor, FGF10 plays a key role in morphogenesis by regulating cell proliferation, differentiation and migration. It binds to the high-affinity receptor FGFR on target cells, activates the signal transduction of kinases and transcription factors, and then leads to mitogenesis, differentiation, migration, angiogenesis and wound healing.

[0033] In a preferred embodiment of the present invention, the basal culture medium contains the following substances at final concentrations: 1 w / v% penicillin-streptomycin, 2 mM GlutaMAX, 1x B27, 1x N2, 10 μM Y-27632, 50 ng / mL human epidermal growth factor EGF, 100 ng / mL recombinant human Noggin protein, 200 ng / mL FGF10, 500 nM A-8301, 10 mM nicotinamide, 1 mM N-acetylcysteine, 10 w / v% R-spondin1 conditioned medium and 10 w / v% Wnt3a conditioned medium.

[0034] In a second aspect, the present invention provides a method for constructing a middle ear cholesteatoma organoid, comprising the following steps:

[0035] S1: Take the middle ear cholesteatoma sample for tissue digestion and obtain the cell pellet after tissue digestion;

[0036] S2: Take the cell pellet and resuspend it with Matrigel gel, inoculate the cell suspension on the cell culture plate, and then add the above-mentioned middle ear cholesteatoma organoid culture medium to the culture wells where the cells were inoculated, and culture them in an incubator at 37℃±0.5℃, 5% CO2, and replace the culture medium every 2-3 days; obtain the middle ear cholesteatoma organoids after 7-10 days of culture.

[0037] The method for constructing middle ear cholesteatoma organoids provided by the present invention can efficiently prepare an organoid model that is closer to the characteristics of middle ear cholesteatoma, and provides a favorable tool for studying the pathogenesis and treatment methods of middle ear cholesteatoma, drug screening, gene editing, etc.

[0038] In a preferred embodiment of the present invention, the construction method further comprises subculture: the primary middle ear cholesteatoma organoids obtained in S2 are subcultured.

[0039] In a preferred embodiment of the present invention, the subculture includes: taking organoid-matrigel mixed gel droplets for suspension treatment, centrifuging and discarding the supernatant, digesting with organoid digestion solution in a 37°C water bath for 2-10 minutes to obtain a cell pellet, resuspending the cell pellet, and counting the number of cell clusters; adding Matrigel gel to resuspend the cell pellet at a ratio of 200-400 cell clusters to 25ul Matrigel gel, and then inoculating the cell suspension into a cell culture plate; adding 150-200ul preheated organoid culture medium to each well, and culturing in a 37°C, 5% CO2 incubator; replacing the culture medium every 2-3 days.

[0040] In a preferred embodiment of the present invention, the middle ear cholesteatoma sample is from a primate mammal, mouse or rat; a primate mammal is such as a human, monkey or ape.

[0041] In a preferred embodiment of the present invention, the tissue digestion in step S1 includes: mixing the chopped human middle ear cholesteatoma sample with tissue digestion solution, and digesting at 37°C±0.5°C for 0.5-2h; then taking the supernatant for centrifugation to collect the cell precipitate.

[0042] In a third aspect, the present invention also provides a method for constructing a middle ear cholesteatoma organoid and the use of the constructed middle ear cholesteatoma organoid in in vitro drug screening and / or detection.

[0043] The middle ear cholesteatoma organoids provided by the present invention can truly reproduce the three-dimensional structure and cell function of the middle ear cholesteatoma, and are highly consistent with the original tissue at the transcriptome, genome and protein levels. Therefore, the middle ear cholesteatoma organoids can be used for drug screening for the treatment of middle ear cholesteatoma or for detection and analysis of middle ear cholesteatoma organoids, such as for evaluating the levels of various markers of the middle ear cholesteatoma organoids.

[0044] The detection and analysis method is selected from at least one of image capture, histopathological analysis, DNA and / or RNA extraction, and biomarker analysis.

[0045] In a preferred embodiment of the present invention, the in vitro drug screening is an in vitro high-throughput drug screening.

[0046] The application of drug screening includes at least one of the following implementation methods: if the drug to be tested can inhibit the invasive growth of the middle ear cholesteatoma of the middle ear cholesteatoma organoid, can cause the growth of the middle ear cholesteatoma organoid to stagnate or degenerate, and cause cell apoptosis or necrosis, it indicates that the drug has a good therapeutic effect.

[0047] In a fourth aspect, the present invention also provides an in vitro drug screening method, the method comprising: applying a drug to be tested to the middle ear cholesteatoma organoid constructed by the above-mentioned method for constructing the middle ear cholesteatoma organoid, and detecting and analyzing the middle ear cholesteatoma organoid.

[0048] In a preferred embodiment of the present invention, the detection and analysis method is selected from at least one of image capture, histopathological analysis, DNA and / or RNA extraction, and biomarker analysis.

[0049] Histopathological analysis includes but is not limited to: HE staining and immunohistochemical staining.

[0050] Immunohistochemical staining includes but is not limited to combining fluorescent or colorable chemicals on antibodies, and using the specific binding reaction between antigens and antibodies in immunological principles to detect the presence of target antigens in cells or tissues.

[0051] In the DNA / RNA extraction method, TRIzol is preferably used for lysis; in the image capture and biomarker analysis method, confocal Z-step imaging method is preferably used, so as to facilitate the capture of clearer mouse throat mucosal structure.

[0052] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0053] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0054] Example 1

[0055] This embodiment provides a method for constructing human middle ear cholesteatoma organoids.

[0056] 1. Materials and Reagents

[0057] Equipment and materials: human middle ear cholesteatoma tissue, centrifuge tubes (15ml, 50ml), 10cm culture dishes, 10ml pipettes, tips (200ul, 1000ul), 48-well cell culture plates, CO2 cell culture incubator, biological safety cabinet, optical microscope, low-speed refrigerated centrifuge, constant temperature water bath, pipette, ultra-low temperature storage refrigerator.

[0058] Reagents: tissue sample preservation solution (catalog number #01K05100), organoid culture medium, tissue digestion solution (catalog number #01K03100), organoid digestion solution (catalog number #01K02100), organoid freezing solution (catalog number #01K04100), tissue cleaning solution (catalog number #01K06500), rinse solution (catalog number #01K08100), digestion stop solution (catalog number #01K07100), Matrigel (Corning, Cat#356237), red blood cell lysis solution (Solarbio, Cat#R1010).

[0059] The formula of the organoid culture medium is as follows: DMEM / F12 (GIBCO, Cat#11330500BT); 1% Penicillin and Streptomycin (GIBCO, Cat#15140-122); 2mM GlutaMAX (GIBCO, Cat#35050-061); 1x B27 (GIBCO, Cat#A3582801); 1x N2 (GIBCO, Cat#17502048); 10μM Y-27632; 50ng / mL human recombinant EGF (Peprotech, Cat#AF-100-15-1000); 100ng / mL human recombinant noggin (Peprotech, Cat#120-10C-250); 200ng / mL FGF10 (Peprotech, Cat#100-26-1000); 500nM A-8301 (Peprotech, Cat#9094360); 10mM Nicotinamide (Sigma, Cat#N0636); 1mM N-acetylcysteie (Sigma, Cat#A9165); 10% R-spondin1 condition medium(PeproTech,Cat#120-38-20); 10% Wnt3a condition medium(PeproTech,Cat#315-20-10)

[0060] 2. Construction Method

[0061] (1) Tissue collection: Collect fresh human middle ear cholesteatoma samples. Aseptic operation should be performed during the collection process to avoid contamination. The tissue should be placed in tissue sample preservation solution (Cat. No. 01K05100) and transported at 2-8°C.

[0062] (2) Tissue sample cleaning: Use a 1ml pipette tip to thoroughly mix the tissue sample preservation solution, take 100ul into a 10cm culture dish, observe under a microscope, determine the cell shedding, cell activity, etc., and keep records. Use sterile tweezers to transfer the tissue sample to a new 50ml centrifuge tube, add 10ml tissue cleaning solution (Cat. No. 01K06500) to clean the tissue sample, and repeat 3-5 times.

[0063] (3) Tissue digestion: Use new sterile forceps to transfer the cleaned tissue to a new 10 cm culture dish, and use sterile scissors to cut the rinsed tissue into 1 mm pieces. 3 Depending on the size of the tissue, prepare 5-10 ml of tissue digestion solution (Cat. No. 01K03100) to collect the shredded tissue step by step, use a 10 ml pipette to draw half of the volume of tissue digestion solution in the culture dish to resuspend the tissue and transfer it to a 50 ml centrifuge tube, draw the other half of the tissue digestion solution to wash and collect the residual tissue in the culture dish and transfer it to the same centrifuge tube, and use a pipette to mix 10-20 times. Place the centrifuge tube in a 37°C water bath for 0.5-2h of digestion time. Take out the centrifuge tube every 10min and use a 10mL pipette to mix 10-20 times. Let it stand for 5-10s until most of the tissue blocks settle to the bottom of the centrifuge tube, then aspirate 50uL of supernatant and observe under a microscope; observe the release of tissue cells under an optical microscope. When cell clusters are released and most of the tissue is completely digested, add 2ml of digestion stop solution (catalog number #01K07100) to stop digestion. Let it stand for 5-10s until the remaining tissue blocks settle to the bottom of the centrifuge tube, collect the supernatant in a 15ml centrifuge tube, centrifuge at 300g for 5min, discard the supernatant, and collect the cell pellet.

[0064] (4) Organoid inoculation and culture: Preheat a 48-well cell culture plate in a 37°C incubator 1 hour in advance, prepare an ice box in advance, and perform all Matrigel-related operations on ice; according to the number of cell clusters, add an appropriate amount of Matrigel to resuspend the cell pellet, aspirate 25ul of the cell suspension and inoculate it into a 48-well cell culture plate, try to inoculate it in the center of the well plate to avoid collapsing of the gel. After inoculation, add an appropriate amount of cleaning solution to the surrounding wells, maintain the ambient humidity to avoid too fast evaporation of the culture medium, and add an appropriate amount of organoid culture medium to the surrounding clean wells for preheating (calculated as 200ul / well for inoculation wells); transfer the inoculated cell culture plate to a 37°C, 5% CO2 incubator and incubate for 30 minutes. After the matrix gel solidifies, remove the cell culture plate, add 200ul of preheated organoid culture medium to each well, and culture it in a 37°C, 5% CO2 incubator. Observe cell growth under a microscope every day and replace the culture medium every 2-3 days. The cultured P0 generation human middle ear cholesteatoma organoids are as follows Figure 1 As shown in Figure A.

[0065] (5) Organoid passaging: Select cells in the logarithmic growth phase for digestion and expansion, discard the culture medium in the cell culture plate, add 200ul of pre-cooled rinse solution (Cat. No. 01K08100) to each well, use a 1ml pipette to scrape the organoid-matrix gel mixture droplets, collect them in a 15ml centrifuge tube, use the pipette to blow 4-5 times to mix the suspension, centrifuge at 300-600g at 4℃ for 5min; discard the supernatant, add 1-2ml of organoid digestion solution (Cat. No. 01K02100), use the pipette to blow 4-5 times to mix the suspension, digest in a 37℃ water bath for 2-10min, take 5-10ul of cell suspension Observe the cell suspension under a microscope. If the diameter of most cell clusters is within the range of 50-80um, add 2 times the volume of cleaning solution to dilute and terminate digestion. Centrifuge at 300-600g for 5min at 4℃; discard the supernatant to obtain the cell pellet, add 3ml of rinse solution (Cat. No. 01K08100) to resuspend the pellet, mix the cell suspension thoroughly, take 20ul to count under the microscope, calculate the number of cell clusters (4 or more cells are counted as one cell cluster), centrifuge at 300g for 5min; according to the number of cell clusters, add appropriate amount of Matrigel to resuspend the cell pellet (200-400 cell clusters / 25ul Matrigel gel), aspirate 25ul of cell suspension and inoculate it into a 48-well cell culture plate. Try to inoculate it into the center of the well plate to avoid gel collapse. After inoculation, add an appropriate amount of cleaning solution to the surrounding wells, maintain environmental humidity, avoid too fast evaporation of the culture medium, and add an appropriate amount of preheated organoid culture medium to the clean wells around (calculated as 200ul / well for inoculation wells), incubate in a 37°C, 5% CO2 incubator for 30min. After the Matrigel gel solidifies, remove the cell culture plate, add 200ul of preheated organoid culture medium to each well, and culture in a 37°C, 5% CO2 incubator. Observe cell growth under a microscope every day, and replace the culture medium every 2-3 days. Culture the P1 generation of human middle ear cholesteatoma organoids, such as Figure 1 As shown in Figure B.

[0066] (6) Pathological verification of organoids: Fix the organoid samples with 4% neutral buffered formalin for 30 min; rinse twice with PBS, 1 min each time; add 75% ethanol, 85% ethanol, and 95% ethanol to a 24-well plate for dehydration for 10 min; after dehydration, gently remove the matrix gel wrapped in the organoid sample with a certain hardness with ophthalmic forceps, put it into a single-layer roll of paper of size 5 cm × 5 cm with the bottom of the matrix gel facing down, and draw a circle around the matrix gel with a pencil to locate it. After folding, put it into the embedding box with a printed number; put the embedding box into the fully automatic dehydrator for dehydration, transparentization, and wax dipping. The whole process should not exceed 2 hours. Open the roll of paper, find the intestinal organoid sample at the pencil circle, and use electric heating forceps to quickly place the sample into the center bottom of the hot embedding mold filled with wax liquid, transfer it to the small cold stage of the embedding machine and gently flatten it. After the bottom solidifies, quickly put it on the bottom box of the embedding box and move it to the large freezing stage for rapid cooling to make a tissue wax block; slice the wax block with a slice thickness of 4 μm. After routine dewaxing and hydration, paraffin sections were stained with hematoxylin for 2 min, differentiated with 0.5% hydrochloric acid ethanol for a few seconds, and stained with eosin for 20 s after returning to blue with warm water. They were dehydrated with gradient ethanol, made transparent with xylene, and sealed with an automatic sealing device. Multiple immunohistochemistry experiments were carried out using the Opal 6-Plex Manual Detection Kit-for Whole Slide Imaging (formerly Opal Polaris 7Color IHC Manual Detection Kit).

[0067] Comparative Example 1

[0068] In this comparative example, two batches of human middle ear cholesteatoma organoids were constructed. Compared with Example 1, the organoid culture medium used in this comparative example does not contain cytokines Wnt3a (mainly activating the Wnt / β-catenin signaling pathway to maintain the proliferation, self-renewal and undifferentiated state of stem cells, thereby promoting the growth of organoids) and R-spondin1 (mainly enhancing the activity of the Wnt signaling pathway to further promote the proliferation and self-renewal of stem cells in organoids and maintain the integrity of tissue structure), specifically: DMEM / F12 culture medium; 2mM GlutaMAX; 1x B27; 100ng / mL humanrecombinant noggin; 1x N2; 10μM Y-27632; 200ng / mL FGF10; 1w / v% Penicillin and Streptomycin; 500nM A-8301; 10mM Nicotinamide; 1mM N-acetylcysteie; 50ng / mL humanrecombinant EGF. Other operations are the same as in Example 1.

[0069] The culture results showed that compared with Example 1, the organoids cultured in this medium showed obvious growth stagnation or degeneration within 2 to 3 days, and cell apoptosis or necrosis occurred within 5 days, and the modeling success rate was 0% ( Figure 1 C).

[0070] Experimental Example 1

[0071] Two batches of human middle ear cholesteatoma organoids were constructed. Organoid formation was observed under a microscope and long-term subculture was possible. The modeling success rate was 100%. Pathological HE staining results showed that ( Figure 1 D in the figure shows that the organoid is composed of multiple cells and is hollow or solid. In the center, detached keratinized epithelial cells and keratin accumulation can be seen. At the same time, the results of multicolor immunofluorescence staining showed that ( Figure 1 E), the organoid contains both proliferative and keratinized epithelial cells, and the outer layer is wrapped with a small amount of collagen fibers, which is highly similar to human middle ear cholesteatoma tissue.

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A middle ear cholesteatoma organoid culture medium, characterized in that The invention comprises a basic culture medium DMEM / F12, and the basic culture medium comprises the following substances at final concentrations: 1-2 w / v% penicillin-streptomycin, 2-8 mM GlutaMAX, 1-5xB27, 1-5x N2, 10-20 μM Y-27632, 50-200 ng / mL human epidermal growth factor EGF, 100-200 ng / mL recombinant human Noggin protein, 100-200 ng / mL FGF10, 300-600 nM A-8301, 10-20 mM nicotinamide, 1-5 mM N-acetylcysteine, 5-20 w / v% R-spondin1 conditioned medium and 5-20 w / v% Wnt3a conditioned medium.

2. The middle ear cholesteatoma organoid culture medium according to claim 1, characterized in that The following substances are contained in the basic culture medium at final concentrations: 1 w / v% penicillin-streptomycin, 2 mM GlutaMAX, 1x B27, 1x N2, 10 μM Y-27632, 50 ng / mL human epidermal growth factor EGF, 100 ng / mL recombinant human Noggin protein, 200 ng / mL FGF10, 500 nM A-8301, 10 mM nicotinamide, 1 mM N-acetylcysteine, 10 w / v% R-spondin1 conditioned medium and 10 w / v% Wnt3a conditioned medium.

3. A method for constructing middle ear cholesteatoma organoids, characterized in that: It includes the following steps: S1: Take the middle ear cholesteatoma sample for tissue digestion and obtain the cell pellet after tissue digestion; S2: Take the cell pellet and resuspend it with Matrigel gel, inoculate the cell suspension on a cell culture plate, and then add the middle ear cholesteatoma organoid culture medium according to any one of claims 1-2 to the culture wells where the cells are inoculated, and culture in an incubator at 37°C ± 0.5°C and 5% CO2, replacing the culture medium every 2-3 days; and obtain the middle ear cholesteatoma organoid after 7-10 days of culture.

4. The method for constructing middle ear cholesteatoma organoids according to claim 3, characterized in that: The construction method also includes subculture: taking the primary middle ear cholesteatoma organoids obtained in S2 for subculture.

5. The method for constructing middle ear cholesteatoma organoids according to claim 4, characterized in that: The subculture includes: taking organoid-matrigel mixed gel droplets for suspension treatment, centrifuging and discarding the supernatant, digesting with organoid digestion solution in a 37°C water bath for 2-10 minutes to obtain cell pellets, resuspending the cell pellets, and counting the number of cell clusters; adding Matrigel gel to resuspend the cell pellets at a ratio of 200-400 cell clusters to 25ul Matrigel gel, and then inoculating the cell suspension into a cell culture plate; adding 150-200ul preheated organoid culture medium to each well, and culturing in a 37°C, 5% CO2 incubator; replacing the culture medium every 2-3 days.

6. The method for constructing middle ear cholesteatoma organoids according to claim 3, characterized in that: The middle ear cholesteatoma sample is from a primate mammal, a mouse or a rat; Preferably, the tissue digestion in step S1 comprises: mixing the chopped human middle ear cholesteatoma sample with a tissue digestion solution, and digesting at 37°C±0.5°C for 0.5-2h; then taking the supernatant and centrifuging it to collect the cell precipitate.

7. Use of the middle ear cholesteatoma organoid constructed by the method for constructing the middle ear cholesteatoma organoid as described in any one of claims 3 to 6 in in vitro drug screening and / or detection.

8. The use according to claim 7, characterized in that: The in vitro drug screening is an in vitro drug high-throughput screening.

9. A method for in vitro drug screening, characterized in that: The method comprises: applying the drug to be tested to the middle ear cholesteatoma organoid constructed by the method for constructing the middle ear cholesteatoma organoid according to any one of claims 3 to 6, and detecting and analyzing the middle ear cholesteatoma organoid.

10. The method for in vitro drug screening according to claim 9, characterized in that: The detection and analysis method is selected from at least one of image capture, histopathological analysis, DNA and / or RNA extraction, and biomarker analysis.

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