Throat mucosa organoid culture medium, mouse throat mucosa organoid model and construction method and application thereof

Abstract

The invention discloses a throat mucosa organoid culture medium, a mouse throat mucosa organoid model and a construction method and application thereof, and relates to the technical field of organoid models. The method disclosed by the invention can be used for efficiently preparing the normal throat mucosa organ-like model of the mouse, and the model can reproduce histological and pathological characteristics of normal tissues; a reliable and stable research platform can be provided for research on normal throat tissue physiological functions and occurrence and development mechanisms and diagnosis and treatment methods of multiple benign and malignant diseases such as papilloma, oropharyngeal cancer and laryngeal cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid models, and in particular to a laryngeal mucosal organoid culture medium, a mouse laryngeal mucosal organoid model, and a construction method and application thereof. Background Art

[0002] As a key component of the upper respiratory tract mucosa, the laryngeal mucosa has many physiological functions, such as preventing aspiration, lubricating the vocal cords, warming and humidifying the air. Its lesions (such as infection, hyperplasia, and cancer) can seriously affect the patient's daily quality of life and even life safety. However, the difficulty in constructing models related to laryngeal diseases has always limited the ability to explore the mechanisms of related diseases and develop effective treatment strategies. Therefore, the in vitro culture of laryngeal mucosa has become increasingly indispensable in medical research and clinical practice.

[0003] At present, the in vitro models of laryngeal mucosa mainly include 2D primary epithelial cell models, 2D tumor cell lines, xenograft (PDX) models, and genetically engineered animal models [1,2]. Although 2D cell models are simple and easy to use, they cannot fully simulate the three-dimensional (3D) structure and complex cell-to-cell interactions of the laryngeal mucosa, which limits their accurate simulation of key physiological functions such as epithelial barrier function, cell polarity, and cell-to-cell communication. In addition, in the cultivation process of PDX models, they rely on immunodeficient mice or humanized mice, and genetic variation and unpredictable and uncontrollable environmental variables often become important factors affecting the reproducibility of their experimental results, resulting in relatively low reproducibility of the model. As a non-orthotopic primary tumor, it cannot simulate the in situ microenvironment of laryngeal tissue. Genetically engineered animal models achieve the purpose of gene mutation and related diseases by performing corresponding gene editing and other operations on mice during the embryonic period. However, this method is complicated to operate, has high cultivation costs, and a long breeding cycle, and is limited to some basic research applications. Therefore, the above models still have great limitations in the study of the physiological functions of laryngeal mucosal epithelium and related diseases.

[0004] As a research hotspot in the field of stem cells in the past decade, organoid research has a very broad development prospect [3-5]. In terms of drug screening, the three-dimensional structure of organoids is closer to the real state of the human body than ordinary two-dimensional cell culture, and is expected to be used as a method for precision medicine drug screening, which has positive significance for preclinical research on drug use; in terms of disease model establishment and mechanism research, organoids can reflect the interaction between cells-cells, cells-microenvironment, and cells-pathogens, and can establish individualized disease models and develop individualized treatments [6,7]. At present, some studies have developed tongue organoid models from patient-derived tissues [8,9]. However, since normal human pharyngeal mucosa is not easy to obtain, it is impossible to carry out large-scale organoid modeling and subsequent gene editing, drug screening, etc. In addition, clinical patient samples are very precious, the amount of tissue cells is small, and the success rate of model construction is also relatively low, which limits the application of the above models in the study of the physiological function of pharyngeal mucosal epithelium and related diseases.

[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 laryngeal mucosal organoid culture medium, a mouse laryngeal mucosal organoid model, and a construction method and application thereof to solve the above-mentioned technical problems.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides a laryngeal mucosal organoid culture medium, comprising: a basal culture medium DMEM / F12, and the basal culture medium comprises 5-15 w / v% Wnt3a, 5-15 w / v% R-spondin1, 40-60 ng / mL recombinant human EGF, 50-200 ng / mL recombinant human noggin, 1-2 mM N-acetylcysteine, 10-20 mM nicotinamide, 1-5 mM GlutaMAX, 1-5 w / v% penicillin and streptomycin, 0.5-1×B27, 1-5×N2, 5-20 μM Y-27632, 50-200 ng / mL FGF10 and 300-500 nM A8301.

[0009] In a second aspect, the present invention provides a method for constructing a mouse laryngeal mucosal organoid model, comprising the following steps:

[0010] S1: The chopped mouse throat tissue was digested with pancreatic enzyme to obtain the digested product;

[0011] S2: Eliminate the product after digestion, filter it, wash it to obtain a single cell suspension, and collect the cell precipitate;

[0012] S3: Resuspend the cell pellet in the extracellular matrix (ECM), mix every 10,000 to 20,000 cells with 25-30 μl of extracellular matrix, inoculate the cells into the cell culture wells, and place them in a 5% CO2, 37°C cell culture incubator for 15-30 min; add 150-200 μl of organoid culture medium to each well, place them in a cell culture incubator, and replace the culture medium every 2-3 days;

[0013] S4: Organoid passaging: After 7 to 10 days of culture, subculture is performed;

[0014] Preferably, the organoid culture medium comprises, in terms of final concentrations, a basal culture medium DMEM / F12, and the basal culture medium comprises 10 w / v% Wnt3a, 10 w / v% R-spondin1, 50 ng / mL recombinant human EGF, 100 ng / mL recombinant human noggin, 1 mM N-acetylcysteine, 10 mM nicotinamide, DMEM / F12, 2 mM GlutaMAX, 1 w / v% penicillin and streptomycin, 1x B27, 1x N2, 10 μM Y-27632, 200 ng / mL FGF10 and 500 nM A8301.

[0015] In a third aspect, the present invention provides mouse pharyngeal mucosal organoids obtained by the construction method.

[0016] In a fourth aspect, the present invention provides the use of mouse pharyngeal mucosal organoids in in vitro drug screening and / or detection.

[0017] In a fifth aspect, the present invention provides an in vitro detection platform, which comprises the above-mentioned mouse laryngeal mucosal organoids.

[0018] In a sixth aspect, the present invention provides a method for in vitro drug screening, the method comprising: applying the drug to be tested to the above-mentioned mouse throat mucosal organoids and / or the above-mentioned in vitro detection platform, and performing detection and analysis on the mouse throat mucosal organoids and / or the in vitro detection platform; the detection and analysis methods include image capture, histopathological analysis, DNA / RNA extraction and biomarker analysis.

[0019] The present invention has the following beneficial effects:

[0020] The present invention provides an organoid culture medium adapted to the pharyngeal mucosa of mice. The culture medium provided by the present invention improves the controllability of the survival rate, proliferation rate and differentiation direction of pharyngeal mucosal cells.

[0021] The present invention provides a method for constructing a mouse laryngeal mucosal organoid model, which is derived from the mouse itself, completely solving the problem of scarcity of clinical samples from patients, and making it easier to obtain experimental materials. Compared with 2D cell culture, the organoid model is superior in summarizing the 3D structure, heterogeneity and cell function of primary tissues. It can simulate the normal physiological functions of laryngeal tissues and the occurrence and development of related diseases, and is closer to the real situation in terms of genetic level, tissue microenvironment and pathophysiology.

[0022] The method for constructing a mouse pharyngeal mucosal organoid model provided by the present invention has the technical advantage of a shorter model building time. Obvious organoid formation can be observed 4-7 days after inoculation. The method provided by the present invention can significantly shorten the model building time, which is beneficial to the subsequent large-scale organoid construction and gene editing, drug screening and other applications.

[0023] The mouse laryngeal mucosa organoid model constructed by the mouse laryngeal mucosa organoid model construction method provided by the present invention has a modeling success rate of 100% and can be stably passaged and expanded.

[0024] Compared with the existing 2D cell models, the organoid model provided by the present invention can truly simulate the 3D structure and cell function of the primary tissue, while retaining the histological, pathophysiological characteristics and cell-to-cell interactions of the original tissue, and can more accurately simulate the tissue microenvironment of the laryngeal mucosa.

[0025] The organoid model efficiently prepared by the present invention is closer to the characteristics of normal pharyngeal mucosa, providing a useful tool for studying the physiological functions of pharyngeal tissues, the mechanisms of disease occurrence and development, and drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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.

[0027] Figure 1 This is a micrograph of the growth status of the normal pharyngeal mucosal organoids of mice at generation P0 in Example 1;

[0028] Figure 2 This is a micrograph of the growth status of the normal pharyngeal mucosal organoids of mice at generation P1 in Example 1;

[0029] Figure 3 This is the HE staining result of the normal pharyngeal mucosa organoid of mice in Example 1;

[0030] Figure 4This is the result of immunohistochemical staining of CK5 in normal mouse pharyngeal mucosa organoids in Example 1;

[0031] Figure 5 This is the result of immunohistochemical staining of CK14 in normal mouse pharyngeal mucosa organoids in Example 1;

[0032] Figure 6 This is the result of P63 immunohistochemical staining of normal mouse pharyngeal mucosa organoids in Example 1;

[0033] Figure 7 This is the result of Ki67 immunohistochemical staining of normal mouse pharyngeal mucosa organoids in Example 1;

[0034] Figure 8 It is a microscopic comparison of the growth status of normal pharyngeal mucosal organoids in mice of the embodiment and the comparison example. DETAILED DESCRIPTION

[0035] 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.

[0036] As a common experimental animal model, mice are not only similar to humans, but also have many advantages such as low cost, strong controllability, high throughput, and fast speed. Therefore, the present invention intends to use mouse pharyngeal mucosal tissue to achieve long-term in vitro culture of organoids, providing a new research idea for the study of pharyngeal related diseases.

[0037] In the first aspect, the present invention provides a laryngeal mucosal organoid culture medium, comprising: a basal culture medium DMEM / F12, and the basal culture medium contains 5-15w / v% Wnt3a, 5-15w / v% R-spondin1, 40-60ng / mL recombinant human EGF, 50-200ng / mL recombinant human noggin, 1-2mM N-acetylcysteine, 10-20mM nicotinamide, 1-5mMGlutaMAX, 1-5w / v% penicillin and streptomycin, 0.5-1×B27, 1-5×N2, 5-20μM Y-27632, 50-200ng / mL FGF10 and 300-500nM A8301.

[0038] In a preferred embodiment of the present invention, the organoid culture medium includes: a basal medium DMEM / F12, which contains 10 w / v% Wnt3a, 10 w / v% R-spondin1, 50 ng / mL recombinant human EGF, 100 ng / mL recombinant human noggin, 1 mM N-acetylcysteine, 10 mM nicotinamide, 2 mM GlutaMAX, 1 w / v% penicillin and streptomycin, 1xB27, 1x N2, 10 μM Y-27632, 200 ng / mL FGF10 and 500 nM A8301.

[0039] The culture medium adapted to mouse pharyngeal mucosal organoids provided by the present invention improves the controllability of the survival rate, proliferation rate and differentiation direction of pharyngeal mucosal cells.

[0040] In a second aspect, the present invention provides a method for constructing a mouse laryngeal mucosal organoid model, comprising the following steps:

[0041] S1: The chopped mouse throat tissue was digested with pancreatic enzyme to obtain the digested product;

[0042] S2: Eliminate the product after digestion, filter it, wash it to obtain a single cell suspension, and collect the cell precipitate;

[0043] S3: Resuspend the cell pellet in the extracellular matrix (ECM), mix every 10,000 to 20,000 cells with 25-30 μl of extracellular matrix, inoculate the cells into the cell culture wells, and place them in a 5% CO2, 37°C cell culture incubator for 15-30 min; add 150-200 μl of organoid culture medium to each well, place them in a cell culture incubator, and replace the culture medium every 2-3 days;

[0044] S4: Organoid passaging: After 7 to 10 days of culture, subculture is performed;

[0045] The organoid culture medium includes: a basic culture medium DMEM / F12, and the basic culture medium contains 5-15w / v% Wnt3a, 5-15w / v% R-spondin1, 40-60ng / mL recombinant human EGF, 50-200ng / mL recombinant human noggin, 1-2mM N-acetylcysteine, 10-20mM nicotinamide, 1-5mM GlutaMAX, 1-5w / v% penicillin and streptomycin, 0.5-1×B27, 1-5×N2, 5-20μM Y-27632, 50-200ng / mL FGF10 and 300-500nM A8301.

[0046] In a preferred embodiment of the present invention, the organoid culture medium includes: a basal medium DMEM / F12, which contains 10 w / v% Wnt3a, 10 w / v% R-spondin1, 50 ng / mL recombinant human EGF, 100 ng / mL recombinant human noggin, 1 mM N-acetylcysteine, 10 mM nicotinamide, 2 mM GlutaMAX, 1 w / v% penicillin and streptomycin, 1xB27, 1x N2, 10 μM Y-27632, 200 ng / mL FGF10 and 500 nM A8301.

[0047] The present invention selects low-cost and highly controllable mouse-derived pharyngeal tissue to construct a pharyngeal mucosal organoid model, optimizes the organoid culture medium, improves the survival rate, proliferation rate and controllability of the differentiation direction of pharyngeal mucosal cells, and has a short organoid formation time, a 100% modeling success rate, and can be stably passaged and expanded. Compared with the existing 2D cell model, the organoid model of the present invention can truly simulate the 3D structure and cell function of the primary tissue, while retaining the histological, pathophysiological characteristics and cell-to-cell interactions of the original tissue, and can more accurately simulate the tissue microenvironment of the pharyngeal mucosa; based on the organoid model, gene editing, drug screening, and in vivo transplantation operations can be carried out to deeply study the normal physiological function of the mucosa and the occurrence and development mechanism and treatment methods of related diseases. In addition, in view of the scarcity of patient-derived tissue samples collected based on biopsy or surgery and the small amount of tissue, the present invention uses mouse mucosal tissue for organoid modeling and culture, and experimental materials are easier to obtain, which is convenient for large-scale batch construction and application of organoid models. In summary, the present invention can efficiently prepare an organoid model that is closer to the characteristics of normal pharyngeal mucosa, providing a useful tool for studying the physiological functions of pharyngeal tissues, the mechanisms of disease occurrence and development, and drug development.

[0048] In order to facilitate rapid tissue digestion, the tissue needs to be minced in advance, for example, using small scissors to mince mouse tissue on ice or in an equally low temperature environment.

[0049] The protein at the junction between cells is degraded by trypsin digestion, and the cells become spherical due to the tension of their internal cytoskeleton, thereby obtaining cells separated from each other. The inventors found that trypsin digestion has better digestion efficiency than other collagenases.

[0050] The extracellular matrix (ECM) provides the necessary structural support and biochemical inducements for cell adhesion, growth and differentiation.

[0051] The use of the above-mentioned organoid culture medium can ensure that the cultured organoids have good proliferation ability. The organoids are composed of multiple cells, are hollow or solid, and are mostly keratinized epithelial cells in the center, with good activity characteristics. After removing Wnt3a, the growth rate, size and number of organoids will be affected; after removing R-spondin1, no obvious keratin differentiation was observed in the central cells of the organoids; after removing EGF, the growth state of the cultured organoids was poor, the size and number of the organoids were small, and no keratinocyte differentiation was observed. Therefore, the organoid culture medium of the present invention can efficiently prepare a mouse normal pharyngeal mucosa organoid model with good activity, and the model can reproduce the histological and pathological characteristics of normal tissues.

[0052] Wnt3a is involved in regulating cell development, proliferation, differentiation, adhesion, polarity, cell-cell communication, survival and self-renewal functions.

[0053] Epithelial growth factor (EGF) binds to its receptor and induces proliferative changes. EGF is a factor required for organoid culture.

[0054] R-spondin-1 plays a role in stem cell self-renewal and Wnt signaling activation. It is a key factor in the maintenance and proliferation of mouse organoid progenitor stem cells.

[0055] Noggin 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.

[0056] Y-27632 is a ROCK-I and ROCK-II inhibitor that inhibits apoptosis in mouse stem or progenitor cells.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] A8301 is an effective inhibitor of transforming growth factor β type I receptor ALK5, ALK4 and ALK7. It 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. It can be used in combination with Y-27632, CHIR99021, etc. to improve cell survival rate and proliferation ability.

[0061] In a preferred embodiment of the present invention, the extracellular matrix is ​​BME or Matrigel, and Matrigel is produced by Corning Company, with product number 356237.

[0062] In a preferred embodiment of the present invention, the digestion in step S1 includes: digesting the shredded mouse throat tissue with pancreatic enzyme for 30-45 minutes, blowing during the period; the mixing volume ratio of tissue to pancreatic enzyme is 1:5-10; too long digestion time will damage cell activity, and the subsequent biological properties of the obtained cells will be poor, and the digestion can be further terminated by PBS washing solution to terminate the effect of pancreatic enzyme on the cells.

[0063] The presence of free DNA in the cell suspension will reduce the activity of proteases, and free DNA will act as an adhesive to promote cell aggregation. To prevent naked DNA from entangled with living cells, DNase I should be added during pipetting, and the added amount accounts for 1% of the total digestion system volume. It can prevent cell aggregation by degrading free DNA released by dead cell lysis, and will not initiate the apoptotic pathway.

[0064] In a preferred embodiment of the present invention, in step S2, the product after tissue digestion is taken, filtered with a filter, washed with PBS to obtain a single cell suspension, and centrifuged at 400-450g for 3-5 minutes to collect the cell precipitate.

[0065] In a preferred embodiment of the present invention, the collected cell precipitate is washed with PBS, and the cell precipitate is collected again by centrifugation;

[0066] In a preferred embodiment of the present invention, the cell precipitate collected by centrifugation is subjected to red blood cell lysis, washed with PBS after lysis, and the cell precipitate is collected after centrifugation.

[0067] In a preferred embodiment of the present invention, the subculture of S4 comprises the following steps:

[0068] (1) adding serine protease to the cell culture wells from which the organoid culture medium has been removed for digestion until the organoids are in a single-cell state, and centrifuging to retain the cell pellet;

[0069] (2) Take the cell pellet obtained in step (1) and resuspend it in the extracellular matrix, with a subculture density of 1:3-5; inoculate the cells into the cell culture wells; let them stand in a 5% CO2, 37°C cell culture incubator for 15-30 minutes; add 150-200 μl of organoid culture medium to each well, place them in a cell culture incubator for culture, and replace the culture medium every 2-3 days.

[0070] The passaging density means that after the nth generation reaches the passaging standard, the cells are passed on according to the ratio of nth generation: n+1st generation = 1:3 to 5.

[0071] The serine protease is TrypLE digestive enzyme.

[0072] In a third aspect, the present invention provides mouse pharyngeal mucosal organoids obtained by the construction method.

[0073] The diameter of mouse throat mucosal organoids is 100-200μm. They are composed of multiple cells, hollow or solid, with keratinized epithelial cells in the center, and have good activity characteristics; the classic squamous epithelial markers CK5, CK14, and P63 are positive, and the proliferation marker Ki67 is positive.

[0074] In a fourth aspect, the present invention provides the use of mouse pharyngeal mucosal organoids in in vitro drug screening and / or detection.

[0075] In a preferred embodiment of the present invention, the in vitro drug screening is an in vitro high-throughput drug screening.

[0076] In a fifth aspect, the present invention provides an in vitro detection platform, which comprises the above-mentioned mouse pharyngeal mucosal organoids, thereby providing a stable and low-cost in vitro detection platform for drug screening that can simulate the in vivo environment, and is particularly useful for high-throughput drug screening.

[0077] In a sixth aspect, the present invention provides a method for in vitro drug screening, the method comprising: applying the drug to be tested to the above-mentioned mouse throat mucosal organoids and / or the above-mentioned in vitro detection platform, and performing detection and analysis on the mouse throat mucosal organoids and / or the in vitro detection platform; the detection and analysis methods include image capture, histopathological analysis, DNA / RNA extraction and biomarker analysis.

[0078] Histopathological analysis includes but is not limited to: HE staining and immunohistochemical staining.

[0079] 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.

[0080] 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.

[0081] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0082] Example 1

[0083] The present invention discloses a method for constructing a normal mouse pharyngeal mucosa organoid model.

[0084] The materials and reagents, experimental conditions and methods involved in the examples are as follows:

[0085] 1. Materials and Reagents

[0086] Equipment and materials: C57BL / 6 mice, 15ml / 50ml centrifuge tubes, 70μm / 100μm filter, 48-well cell culture dishes, refrigerated low-speed centrifuge, water bath.

[0087] Reagents: Trypsin (0.02% EDTA; GIBCO, Cat# R001100), DNase I (GIBCO, Cat# EN0521), PBS (GIBCO, Cat# C14190500BT), Matrigel (Corning, Cat# 356237), red blood cell lysis buffer (Solarbio, Cat# R1010)

[0088] Organoid culture medium: DMEM / F12 (GIBCO, Cat#11330500BT); 2mM GlutaMAX (GIBCO, Cat#35050-061); 1w / v% Penicillin and Streptomycin (GIBCO, Cat#15140-122); 1xB27 (GIBCO, Cat#A3582801); 1x N2 (GIBCO, Cat#17502048); 10μM Y-27632; 200ng / mL FGF10 (Peprotech, Cat#100-26-1000); 500nM A8301 (Peprotech, Cat#9094360); 50ng / mL human recombinant EGF (Peprotech, Cat#AF-100-15-1000); 100 ng / mL human recombinant noggin (Peprotech, Cat#120-10C-250); 10 mM Nicotinamide (Sigma, Cat#N0636); 1 mM N-acetylcysteie (Sigma, Cat#A9165); 10 w / v % Wnt3a condition medium (PeproTech, Cat#315-20-10); 10 w / v % R-spondin1 condition medium (PeproTech, Cat#120-38-20).

[0089] 2. Specific experimental steps

[0090] 1. Collection of mouse throat tissue: Dissect the mouse throat tissue, remove muscle tissue as much as possible, retain the mucosal epithelium, put it in PBS solution and store it on ice.

[0091] 2. Tissue digestion: Use small scissors to cut up mouse tissue on ice (or in an equivalent low temperature environment); collect the cut tissue, add 5 ml of trypsin to mix and transfer to a 50 ml centrifuge tube, then add 5 ml of trypsin and place in a shaker to shake and digest at 37°C, 120 rpm for 10 minutes; add DNase I (DNase I: digestion system = 1:100) to prevent naked DNA from entangled with living cells, continue shaking digestion for 25-35 minutes, use a pipette to blow about 50 times every 15 minutes to prevent tissue agglomeration, fully digest single cells, and observe the tissue digestion status in real time (digest until there is no obvious tissue to the naked eye, and the total tissue digestion time does not exceed 1 hour).

[0092] 3. Preparation of single cell suspension: After digestion, filter with a 70μm / 100μm filter and rinse with PBS to obtain a single cell suspension, transfer to a 15ml centrifuge tube, centrifuge at 400g for 5min, discard the supernatant, and collect the cell pellet; add 5ml PBS to wash to terminate digestion, centrifuge at 400g for 5min, discard the supernatant, and collect the cell pellet;

[0093] Since the mouse throat is rich in blood supply, this example takes into account that the collected cell pellet is red in color, so a red blood cell lysis operation is performed, 500 μl of red blood cell lysis buffer is added to resuspend the cell pellet, and after standing at room temperature for 2 minutes, PBS is added for washing, and the supernatant is discarded after centrifugation.

[0094] 4. Organoid inoculation and culture: Count the cells, resuspend the cell pellet in 25-30 μl Matrigel per well (about 20,000 cells per 30 μl), inoculate in the center of a 48-well plate to avoid bubbles and collapse of the matrix gel, and place in a 5% CO2, 37°C cell culture incubator for 15-30 min to solidify the Matrigel matrix gel; add 150-200 μl organoid culture medium to each well, add an equal amount of PBS solution to the surrounding wells to prevent the culture medium from evaporating, and culture in a 5% CO2, 37°C cell culture incubator. Replace the culture medium every 2-3 days, observe the growth of the organoids, and culture the normal mouse pharyngeal mucosal organoids. The growth status of the P0 generation is as follows: Figure 1 shown.

[0095] 5. Organoid passaging: After 7 to 10 days of culture, organoids can be subcultured when their diameter reaches 100 μm.

[0096] (1) Take out the 48-well plate for organoid culture, remove the organoid culture medium, add an appropriate amount of TrypLE digestion solution (about 500 μl per well), use the tip of a pipette to crush the matrix gel, pipette and mix well, and then transfer the cell suspension to a 15 ml centrifuge tube;

[0097] (2) Continue to pipette for more than 50 times and place in a 37°C water bath for digestion for 5 min;

[0098] (3) Add an appropriate amount of DNase I (reference ratio: 1:100) for treatment, use a pipette to gently blow about 50 times, and observe the cell digestion status under a microscope. You may observe a large number of cell clumps suspended in the digestion solution, and very few single cells. Place the centrifuge tube in a 37°C water bath for digestion for 5 minutes;

[0099] (4) Remove the centrifuge tube and continue to use the pipette to gently pipette several times. Observe under a microscope. If the organoids are digested to a single cell state, stop digestion. If there are still cell clumps, repeat the water bath digestion as above.

[0100] (5) After all organoids are in a single-cell state, place the centrifuge tube in a centrifuge at 400 g for 5 min, discard the supernatant, and retain the cell pellet;

[0101] (6) Add an appropriate amount of matrix gel to resuspend the cell pellet (passage density is 1:3-5), inoculate 30 μl of matrix gel per well in a new 48-well plate, place in a 5% CO2, 37°C incubator for 15-30 min to solidify the matrix gel, add 150-200 μl of organoid culture medium to each well, and culture in a 5% CO2, 37°C incubator. Replace the culture medium every 2-3 days and observe the growth of the organoids. Cultivate normal pharyngeal mucosal organoids of P1 mice, such as Figure 2 shown.

[0102] 3. Experimental Results Analysis

[0103] In this example, a total of 4 batches of normal mouse pharyngeal mucosa samples were collected and organoid models were established. Organoid formation was observed under a microscope and long-term subculture was possible, with a modeling success rate of 100%.

[0104] Example 2

[0105] In this example, pathological HE staining and immunohistochemical staining were performed on the pharyngeal mucosal organoids cultured in Example 1.

[0106] The specific steps of HE staining and immunohistochemical staining methods refer to the experimental operation methods described in the reference "A method for quickly preparing paraffin blocks of intestinal organoid samples", Journal of Clinical and Experimental Pathology, 2021, 37(09): 1131-1133. DOI: 10.13315 / j.cnki.cjcep.2021.09.026.

[0107] HE staining results of normal mouse pharyngeal mucosa organoids are as follows Figure 3 As shown, the results of CK5 immunohistochemical staining of normal mouse pharyngeal mucosa organoids are as follows Figure 4 As shown, the results of CK14 immunohistochemical staining of normal mouse pharyngeal mucosa organoids are as follows Figure 5 As shown, the results of P63 immunohistochemical staining of normal mouse pharyngeal mucosa organoids are as follows Figure 6 The results of Ki67 immunohistochemical staining of normal mouse pharyngeal mucosa organoids are shown in Figure 7 shown.

[0108] The results showed that the organoids were composed of multiple cells and were hollow or solid in shape, with mostly keratinized epithelial cells in the center, and had good activity characteristics; the classic squamous epithelial markers CK5, CK14, and P63 were positive, and the proliferation marker Ki67 was positive, which was consistent with the characteristics of normal mouse throat tissue and showed good proliferation ability.

[0109] Comparative Example 1 (Comparative Example 1)

[0110] Compared with Example 1, the only difference between this comparative example and Example 1 is that the organoid culture medium does not include cytokines Wnt3a and R-spondin1, and the other organoid culture medium compositions are the same as those in Example 1. Specifically: DMEM / F12 culture medium; 2mM GlutaMAX; 1% Penicillin and Streptomycin; 1x B27; 1x N2; 10μM Y-27632; 200ng / mL FGF10; 500nM A-8301; 100ng / mL human recombinant noggin; 10mM Nicotinamide; 1mM N-acetylcysteie; 50ng / mL human recombinant EGF.

[0111] The other organoid model construction methods are the same as in Example 1. The culture results are as follows Figure 8 As shown, compared with Example 1, the organoids cultured using this method grew slowly and were fewer in number, and the central keratinocytes were poorly differentiated.

[0112] Comparative Example 2 (Comparative Example 2)

[0113] Compared with Example 1, the only difference between this comparative example and Example 1 is that the organoid culture medium does not include cytokines EGF and R-spondin1, and the other organoid culture medium compositions are the same as those in Example 1. Specifically: DMEM / F12 culture medium; 2mMGlutaMAX; 1% Penicillin and Streptomycin; 1x B27; 1x N2; 10μM Y-27632; 200ng / mL FGF10; 500nM A-8301; 100ng / mL human recombinant noggin; 10mM Nicotinamide; 1mM N-acetylcysteie; 10% Wnt3a condition medium. The other organoid model construction methods are the same as in Example 1. The culture results are shown in Figure 1. Figure 8 As shown, the volume and number of organoids cultured by this method were significantly reduced, the growth was slow, and the organoid state was poor.

[0114] Comparative Example 3 (Comparative Example 3)

[0115] Compared with Example 1, the only difference between this comparative example and Example 1 is that the organoid culture medium does not include cytokines EGF and Wnt3a, and the other organoid culture medium compositions are the same as those in Example 1. Specifically, it is: DMEM / F12 culture medium; 2mM GlutaMAX; 1% Penicillin and Streptomycin; 1x B27; 1x N2; 10μM Y-27632; 200ng / mL FGF10; 500nM A-8301; 100ng / mL human recombinant noggin; 10mM Nicotinamide; 1mM N-acetylcysteie; 10% R-spondin1 condition medium.

[0116] The other organoid model construction methods are the same as in Example 1. The culture results are as follows Figure 8 As shown, the growth rate of organoids slowed down significantly after 5 days of culture, and the number of organoids formed was small.

[0117] Comparative Example 4 (Comparative Example 4)

[0118] The only difference between this comparative example and Example 1 is that the organoid culture medium does not include the cytokine R-spondin1, and specifically includes: DMEM / F12 culture medium; 2mM GlutaMAX; 1% Penicillin and Streptomycin; 1x B27; 1x N2; 10μM Y-27632; 200ng / mL FGF10; 500nM A-8301; 100ng / mL humanrecombinant noggin; 10mM Nicotinamide; 1mM N-acetylcysteie; 50ng / mL humanrecombinant EGF; and 10% Wnt3a condition medium.

[0119] The other organoid model construction methods are the same as in Example 1. The culture results are as follows Figure 8 As shown, the size of the organoids obtained by this method is similar to that of Example 1, but no obvious keratin differentiation is observed in the central cells of the organoids.

[0120] Comparative Example 5 (Comparative Example 5)

[0121] The only difference between this comparative example and Example 1 is that the organoid culture medium does not include the cytokine Wnt3a, and specifically includes: DMEM / F12 culture medium; 2mM GlutaMAX; 1% Penicillin and Streptomycin; 1x B27; 1xN2; 10μM Y-27632; 200ng / mL FGF10; 500nM A-8301; 100ng / mL human recombinant noggin; 10mM Nicotinamide; 1mM N-acetylcysteie; 50ng / mL human recombinant EGF; 10% R-spondin1 condition medium.

[0122] The other organoid model construction methods are the same as in Example 1. The culture results are as follows Figure 8 As shown, organoids grew more slowly, were smaller in size and formed in smaller numbers.

[0123] Comparative Example 6

[0124] The only difference between this comparative example and Example 1 is that the organoid culture medium does not include the cytokine EGF, and specifically includes: DMEM / F12 culture medium; 2mM GlutaMAX; 1% Penicillin and Streptomycin; 1x B27; 1x N2; 10μM Y-27632; 200ng / mL FGF10; 500nM A-8301; 100ng / mL human recombinant noggin; 10mM Nicotinamide; 1mM N-acetylcysteie; 10% Wnt3a condition medium; 10% R-spondin1 condition medium.

[0125] The other organoid model construction methods are the same as in Example 1. The culture results are as follows Figure 8 As shown, the organoids cultured by this method had poor growth status, small organoid size and number, and no keratinocyte differentiation was observed.

[0126] Comparative Example 7 (Comparative Example 7)

[0127] The only difference between this comparative example and Example 1 is that the components of the organoid culture medium do not include cytokines EGF, Wnt3a, and R-spondin1, and specifically include: DMEM / F12 culture medium; 2mM GlutaMAX; 1% Penicillin and Streptomycin; 1x B27; 1x N2; 10μM Y-27632; 200ng / mL FGF10; 500nM A-8301; 100ng / mL human recombinant noggin; 10mM Nicotinamide; 1mM N-acetylcysteie.

[0128] The other organoid model construction methods are the same as in Example 1. The culture results are as follows Figure 8 As shown, the organoids cultured by this method are in the form of vacuolated cell clusters with a small diameter and no keratinocyte differentiation.

[0129] In summary, the method of the present invention can efficiently prepare a normal mouse laryngeal mucosal organoid model, which can reproduce the histological and pathological characteristics of normal tissue, and can provide a reliable and stable research platform for the study of the physiological functions of normal laryngeal tissue and the occurrence and development mechanisms, diagnosis and treatment methods of various benign and malignant diseases such as papilloma, oropharyngeal cancer, and laryngeal cancer.

[0130] 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.

[0131] References

[0132] 1.Amelian,A.; Wasilewska,K.;Megias,D.;Winnicka,K.,Application ofstandard cell cultures and 3D in vitro tissue models as an effective tool indrug design and development.Pharmacol Rep 2017,69(5),861-870.

[0133] 2.Sacks,P.G.,Cell,tissue and organ culture as in vitro models tostudy the biology of squamous cell carcinomas of the head and neck.CancerMetastasis Rev 1996,15(1),27-51.

[0134] 3.Corrò,C.;Novellasdemunt,L.;Li,V.S.W.,A brief history oforganoids.Am J Physiol Cell Physiol 2020,319(1),C151-C165.

[0135] 4.Simian,M.;Bissell,M.J.,Organoids:A historical perspective ofthinking in three dimensions.J Cell Biol 2017,216(1),31-40.

[0136] 5.Kim,J.;Koo,B.-K.;Knoblich,J.A.,Human organoids:model systems forhuman biology and medicine.Nat Rev Mol Cell Biol 2020,21(10),571-584.6.Wang,F.;Weaver,V.M.;Petersen,O.W.;Larabell,C.A.;Dedhar,S.;Briand,P.;Lupu,R.;Bissell,M.J.,Reciprocal interactions between beta1-integrin and epidermalgrowth factor receptor in three-dimensional basement membrane breastcultures:a different perspective in epithelial biology.Proc Natl Acad Sci U SA 1998,95(25),14821-14826.

[0137] 7.Weaver,V.M.;Lelièvre,S.;Lakins,J.N.;Chrenek,M.A.;Jones,J.C.R.;Giancotti,F.;Werb,Z.;Bissell,M.J.,beta4 integrin-dependentformation ofpolarized three-dimensional architecture confers resistance toapoptosis innormal and malignant mammary epithelium.Cancer Cell 2002,2(3),205-216.

[0138] 8.Hisha,H.;Tanaka,T.;Ueno,H.,Lingual Epithelial Stem CellsandOrganoid Culture of Them.Int J Mol Sci 2016,17(2).

[0139] 9.Hisha,H.;Tanaka,T.;Kanno,S.;Tokuyama,Y.;Komai,Y.;Ohe,S.;Yanai,H.;Omachi,T.;Ueno,H.,Establishment of a novel lingualorganoid culture system:generation of organoids having mature keratinizedepithelium from adultepithelial stem cells.Sci Rep 2013,3,3224。

Claims

1. A laryngeal mucosal organoid culture medium, characterized in that: It includes: A basal medium DMEM / F12, wherein the basal medium comprises 5-15 w / v% Wnt3a, 5-15 w / v% R-spondin1, 40-60 ng / mL recombinant human EGF, 50-200 ng / mL recombinant human noggin, 1-2 mM N-acetylcysteine, 10-20 mM nicotinamide, 1-5 mM GlutaMAX, 1-5 w / v% penicillin and streptomycin, 0.5-1×B27, 1-5×N2, 5-20 μM Y-27632, 50-200 ng / mL FGF10 and 300-500 nM A8301; Preferably, the basal culture medium comprises 10 w / v% Wnt3a, 10 w / v% R-spondin1, 50 ng / mL recombinant human EGF, 100 ng / mL recombinant human noggin, 1 mM N-acetylcysteine, 10 mM nicotinamide, 2 mM GlutaMAX, 1 w / v% penicillin and streptomycin, 1×B27, 1×N2, 10 μM Y-27632, 200 ng / mL FGF10 and 500 nM A8301.

2. A method for constructing a mouse pharyngeal mucosal organoid model, characterized in that: It includes the following steps: S1: The chopped mouse throat tissue was digested with pancreatic enzyme to obtain the digested product; S2: Eliminate the product after digestion, filter it, wash it to obtain a single cell suspension, and collect the cell precipitate; S3: Resuspend the cell pellet in the extracellular matrix (ECM), mix every 10,000 to 20,000 cells with 25-30 μl of extracellular matrix, inoculate the cells into the cell culture wells, and place them in a 5% CO2, 37°C cell culture incubator for 15-30 min; add 150-200 μl of organoid culture medium to each well, place them in a cell culture incubator, and replace the culture medium every 2-3 days; S4: Organoid passaging: After 7 to 10 days of culture, subculture is performed; The organoid culture medium comprises: a basic culture medium DMEM / F12, and the basic culture medium comprises 5-15w / v% Wnt3a, 5-15w / v% R-spondin1, 40-60ng / mL recombinant human EGF, 50-200ng / mL recombinant human noggin, 1-2mM N-acetylcysteine, 10-20mM nicotinamide, 1-5mM GlutaMAX, 1-5w / v% penicillin and streptomycin, 0.5-1×B27, 1-5×N2, 5-20μM Y-27632, 50-200ng / mL FGF10 and 300-500nM A8301; Preferably, the basic culture medium comprises 10 w / v% Wnt3a, 10 w / v% R-spondin1, 50 ng / mL recombinant human EGF, 100 ng / mL recombinant human noggin, 1 mM N-acetylcysteine, 10 mM nicotinamide, DMEM / F12, 2 mM GlutaMAX, 1 w / v% penicillin and streptomycin, 1x B27, 1x N2, 10 μM Y-27632, 200 ng / mL FGF10 and 500 nM A8301.

3. The method for constructing a mouse pharyngeal mucosal organoid model according to claim 2, characterized in that: The extracellular matrix is ​​Matrigel or BME.

4. The method for constructing a mouse pharyngeal mucosal organoid model according to claim 2, characterized in that: The digestion in step S1 comprises: digesting the chopped mouse throat tissue with pancreatic enzyme for 30-45 minutes, during which the tissue and pancreatic enzyme are blown; the mixing volume ratio of the tissue to the pancreatic enzyme is 1:5-10; Preferably, DNase I is added during pipetting; Preferably, the added amount of DNase I accounts for 1% of the volume of the total digestion system.

5. The method for constructing a mouse pharyngeal mucosal organoid model according to claim 2, characterized in that: In the step S2, the product after the inactivation is filtered with a filter, washed with PBS to obtain a single cell suspension, and centrifuged at 400-450g for 3-5 minutes to collect the cell precipitate; Preferably, the collected cell pellet is washed with PBS, and the cell pellet is collected again by centrifugation; Preferably, the cell pellet collected by centrifugation again is subjected to erythrocyte lysis, washed with PBS after lysis, and the cell pellet is collected after centrifugation.

6. The method for constructing a mouse pharyngeal mucosal organoid model according to claim 2, characterized in that: The subculture comprises the following steps: (1) adding serine protease to the cell culture wells from which the organoid culture medium has been removed for digestion until the organoids are in a single-cell state, and centrifuging to retain the cell pellet; (2) Resuspending the cell pellet obtained in step (1) with an extracellular matrix at a cell culture density of 1:3-5; inoculating the cells into the cell culture wells; standing in a 5% CO2, 37°C cell culture incubator for 15-30 min; adding 150-200 μl of organoid culture medium to each well, placing in a cell culture incubator for culture, and replacing the culture medium every 2-3 days; Preferably, the serine protease is TrypLE digestive enzyme.

7. Mouse laryngeal mucosal organoids obtained by the construction method according to any one of claims 2 to 6.

8. Use of the mouse pharyngeal mucosal organoid according to claim 7 in in vitro drug screening and / or detection; Preferably, the in vitro drug screening is in vitro drug high-throughput screening.

9. An in vitro detection platform, characterized in that: The in vitro detection platform comprises the mouse pharyngeal mucosal organoid described in claim 7.

10. A method for in vitro drug screening, characterized in that: The method comprises: applying the drug to be tested to the mouse pharyngeal mucosal organoid of claim 7 and / or the in vitro detection platform of claim 9, and performing detection and analysis on the mouse pharyngeal mucosal organoid and / or the in vitro detection platform; Preferably, the detection and analysis method is selected from at least one of image capture, histopathological analysis, DNA / RNA extraction and biomarker analysis.

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