Cultivation method of enameloblastoma organoid and application of enameloblastoma organoid

Abstract

The invention discloses a culture method of an enameloblastoma organoid and application of the culture method, and belongs to the field of organoid culture. The method provided by the invention comprises the following steps: after cleaning an enameloblastoma tissue by using PBS (Phosphate Buffer Solution) containing double antibodies, cutting the enameloblastoma tissue into pieces; digesting the cut tissue with a tissue digestion solution to obtain a digested tissue suspension; carrying out cell filtration, centrifugation, red cracking, centrifugation, PBS washing and centrifugation on the digested tissue suspension; adding an organoid culture medium into the obtained cell precipitate, uniformly blowing the cells, and putting the cells into a low-adsorption plate for balling culture; when 2-5 single cells are gathered into a pellet, collecting a cell suspension, and centrifuging to obtain a preliminarily-formed enamel cell tumor organ pellet-like structure; and planting the pellet-like structure into matrigel, and adding an organoid culture medium for culturing to obtain the enameloblastoma organoid. According to the invention, the enameloblastoma-like organ which can be used for research and clinical application can be obtained with higher efficiency and better quality.

Description

Technical Field

[0001] The present invention belongs to the field of organoid culture, and specifically relates to an ameloblastoma organoid culture method and application thereof. Background Art

[0002] Ameloblastoma (AM) is a common benign odontogenic tumor of the maxillofacial region, which significantly affects the patient's quality of life and facial morphology. AM is highly locally invasive and has a high recurrence rate, which poses a challenge to treatment. Because ameloblastoma is prone to recurrence, patients may need to undergo multiple surgeries, which seriously affects their quality of life. The clinical classification of AM into cystic and solid types plays a decisive role in the choice of treatment options, but the cellular heterogeneity and pathogenesis between different clinical types are still unclear, and there is an urgent need to understand the key cellular subtypes that determine different clinical subtypes.

[0003] Organoid technology is a revolutionary in vitro cell culture method that enables people to construct three-dimensional cell models in the laboratory that simulate the structure and function of real tumors. These organoid tumor spheres are formed by guiding in a precisely controlled culture environment. They can not only replicate the microstructure of their corresponding tumors, but also exhibit corresponding physiological functions, providing a highly simulated platform for related disease research and drug testing. In the field of jaw tumor research, the application of organoid technology is particularly important. Ameloblastoma is a common jaw tumor with two clinical types: cystic and solid. Using organoid technology to construct a 3D environment that simulates these two types of ameloblastoma can deeply study their heterogeneity. This model can not only reflect the biological characteristics of tumor cells, but also reveal the differences between different clinical types, providing a theoretical basis for personalized treatment.

[0004] Compared with other tumor organoids, the culture of ameloblastoma organoids faces some special challenges. First, the source cells of ameloblastoma are relatively rare, and it is difficult to obtain sufficient high-quality cell samples. Secondly, the microenvironment of ameloblastoma is complex, and it is necessary to accurately simulate the cytokines and matrix conditions required for its growth. In addition, the invasive and recurrent characteristics of ameloblastoma also increase the complexity of organoid model construction. Finally, the oral cavity is a contaminated environment, and the risk of bacterial, mycoplasma and even fungal contamination in ameloblastoma organoids is high, which is one of the reasons for the low success rate.

[0005] The construction and application prospects of ameloblastoma organoid models are gradually being explored. For example, whole genome sequencing revealed the high frequency of BRAF gene mutations and SMO gene mutations in ameloblastoma, and these two mutations tend to be mutually exclusive. In addition, it has been reported that BRAF inhibitors are effective for patients carrying BRAFV600E mutations, but large-scale clinical studies are difficult to carry out, so the ameloblastoma organoid model provides a reliable platform for exploring targeted drug therapy. Through this ameloblastoma organoid model, drug screening can be performed to provide patients with personalized treatment plans, thereby bringing the possibility of treatment to more patients. Summary of the invention

[0006] The purpose of the present invention is to solve the problems of high contamination rate, low culture success rate and slow growth in the culture of ameloblastoma organoids in the prior art, and to provide a culture method and application of ameloblastoma organoids. The ameloblastoma organoids obtained by the present invention can simulate the structure and physiological function of ameloblastoma, which can not only study the heterogeneity mechanism of different clinical classifications of ameloblastoma, but also provide research objects for the treatment of ameloblastoma.

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

[0008] A method for culturing ameloblastoma organoids, comprising the following steps:

[0009] (1) The ameloblastoma tissue was washed with PBS containing double antibodies and then minced.

[0010] (2) Digesting the ameloblastoma tissue chopped in step (1) with a tissue digestion solution. When a large number of single cells are observed in the tissue digestion solution under a microscope, the digestion is stopped to obtain a digested tissue suspension.

[0011] The tissue digestion solution is preferably prepared by mixing solution A Basal Medium and solution B Supplement 20× in a tumor tissue digestion solution (BioGenous, K601003) in a volume ratio of 20:1.

[0012] (3) The digested tissue suspension obtained in step (2) is subjected to cell filtration, centrifugation, red cell lysis, centrifugation, PBS washing, and centrifugation; the obtained cell precipitate is added with organoid culture medium, the cells are blown evenly, and then placed in a low adsorption plate for spheroid culture. When 2-5 single cells are observed to aggregate into spheroids under a microscope, the cell suspension is collected and centrifuged, and the obtained precipitate is the initially formed ameloblastoma organoid spheroid-like structure.

[0013] (4) implanting the ameloblastoma organoid pellet-like structure obtained in step (3) into matrix gel, adding organoid culture medium for culturing to obtain ameloblastoma organoid.

[0014] Step (1) is preferably: in a sterile operating table, the ameloblastoma tissue is washed with precooled PBS containing double antibodies, and then washed with precooled PBS to remove the residual double antibodies, and the ameloblastoma tissue is cut into pieces using sterile instruments. The concentration of the double antibodies is preferably 5-10%; the ameloblastoma tissue is cut into pieces of about 1mm×1mm×1mm in size using sterile instruments.

[0015] In step (3), the centrifugation condition is preferably 1000-1400 rpm for 5-10 min.

[0016] Step (4) is preferably: the ameloblastoma organoid pellet-like structure is mixed by blowing with organoid culture medium, the cell suspension is added to the matrix gel, and after sufficient mixing, the cell pellet mixed with the matrix gel is dotted into the cell plate, with one droplet per well, and the volume is about 40-50 μL; after the matrix gel solidifies, the organoid culture medium is added for culturing, and the fresh organoid culture medium is replaced every 2-4 days until the ameloblastoma organoid is obtained.

[0017] In steps (3) and (4), the organoid culture medium preferably comprises the following components: basal culture medium, B27 supplement, N-acetyl-l-cysteine, nicotinamide, human epidermal growth factor, A83-01, human FGF10, human FGF2, prostaglandin E2, Laduviglusib, Y-27632, Forskolin, recombinant human R-spondin-1 protein, recombinant human Noggin protein; wherein the basal culture medium is preferably DMEM / F12 basal culture medium. Furthermore, the organoid culture medium contains the following components: basal culture medium, 1×B27 supplement, 1-1.5mmol / L N-acetyl-l-cysteine, 5-15mmol / L nicotinamide, 30-70ng / mL human epidermal growth factor, 300-700nmol / L A83-01, 5-15ng / mL human FGF10, 3-7ng / mL human FGF2, 0.5-1.5μmol / L prostaglandin E2, 0.1-0.5μmol / LLaduviglusib, 5-15nmol / L Y-27632, 0.5-1.5μmol / L Forskolin, 2-6% (w / v) R-spondin-1, 2-6% (w / v) Noggin. Furthermore, the organoid culture medium comprises the following components: basal culture medium, 1×B27 supplement, 1.25mmol / L N-acetyl-l-cysteine, 10mmol / L nicotinamide, 50ng / mL human epidermal growth factor, 500nmol / LA83-01, 10ng / mL human FGF10, 5ng / mL human FGF2, 1μmol / L prostaglandin E2, 0.3μmol / LLaduviglusib, 10nmol / L Y-27632, 1μmol / L Forskolin, 4% R-spondin-1, 4% Noggin. Furthermore, the organoid culture medium also comprises L-cysteine, and its content is preferably 5-15mmol / L.

[0018] The application of the ameloblastoma organoid culture method in preparing an ameloblastoma organoid model.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: the ameloblastoma organoid culture method of the present invention reduces contamination and promotes the rapid growth of ameloblastoma organoids by enhancing the tissue sample pretreatment step before culture, optimizing the tissue digestion method, and promoting single cell aggregation through low adsorption plates, significantly improving the efficiency and success rate of culture, enhancing the structural integrity and functional simulation ability of the organoids, and providing a high-quality model for biological research and drug screening of ameloblastoma. The present invention can obtain ameloblastoma organoids that can be used for research and clinical applications with higher efficiency and better quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The cell morphology of the cystic ameloblastoma organoid cultured in the present invention is observed under a microscope. The interior of the organoid is in a hollow cystic state.

[0021] Figure 2 This is a cell morphology observation diagram of the solid ameloblastoma organoid cultured in the present invention under a microscope. The organoid shows homogeneous epithelial cell agglomeration growth.

[0022] Figure 3 This is a typical picture of hematoxylin and eosin (HE) staining of the ameloblastoma organoid cultured in the present invention. HE staining shows that a cyst-like structure appears in the middle, surrounded by ameloblasts, which is consistent with the pathological manifestations of cystic ameloblastoma.

[0023] Figure 4 This is a typical picture of immunofluorescence staining of cystic ameloblastoma organoids cultured in the present invention. DAPI is a nuclear dye with blue fluorescence; ODAM, AMB and AMG are all labeled with red fluorescence, and MERGE is co-localization staining. It can be seen from the figure that the ameloblastoma organoids cultured in the present invention express three classic ameloblastoma markers.

[0024] Figure 5 The effect of different digestive solutions on solid ameloblastoma organoids obtained by culture. The left picture shows the cell morphology of ameloblastoma organoids cultured for 30 days after digestion with traditional digestive solution under a microscope, and the right picture shows the cell morphology of ameloblastoma organoids cultured for 30 days after digestion with A+B solution under a microscope. The red arrow points to the ameloblastoma organoids. It can be seen that the organoids obtained after digestion with A+B solution are more numerous, larger in size, and more spherical in shape than those obtained with traditional digestive solution.

[0025] Figure 6 Statistical chart of the number and volume of solid ameloblastoma organoids obtained after culture after digestion with different digestive fluids.

[0026] Figure 7The figure shows the effect of using a low-adsorption plate to promote single-cell aggregation on the solid ameloblastoma organoids obtained by culture. The left picture shows the cell morphology of ameloblastoma organoids under a microscope when the organoids were directly cultured for 30 days without using a low-adsorption plate to promote single-cell aggregation. The right picture shows the cell morphology of ameloblastoma organoids under a microscope when the organoids were first cultured for 30 days after promoting single-cell aggregation on a low-adsorption plate. The red arrow points to the ameloblastoma organoids. It can be seen that after promoting single-cell aggregation by low-adsorption, obvious ameloblastoma organoid formation can be observed in the early stage of organoid formation, and the number and volume of organoids are increased.

[0027] Figure 8 Statistical graph of the number and volume of solid ameloblastoma organoids obtained by culturing single cells without and with low-adhesion plates.

[0028] Fig. 9 The effect of adding L-cysteine ​​to the organoid culture medium on the solid ameloblastoma organoids obtained by culture. The left picture shows the cell morphology of ameloblastoma organoids under a microscope when the organoids were cultured in a medium without L-cysteine ​​for 30 days, and the right picture shows the cell morphology of ameloblastoma organoids under a microscope when the organoids were cultured in a medium with L-cysteine ​​for 30 days. The red arrow points to the ameloblastoma organoids, and it can be seen that the number and volume of ameloblastoma organoids in the culture medium with L-cysteine ​​added are increased compared with those without L-cysteine.

[0029] Fig.10 Statistical graph of the number and volume of solid ameloblastoma organoids obtained by culturing in medium without L-cysteine ​​(NC) and medium with L-cysteine. DETAILED DESCRIPTION

[0030] The present invention will be explained in detail below in conjunction with the following embodiments; it should be clear that the embodiments shown only represent several implementation methods of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should also be regarded as the scope of protection of the present invention.

[0031] Example 1

[0032] This embodiment provides a method for culturing ameloblastoma organoids, comprising the following steps:

[0033] (1) Obtaining ameloblastoma tissue samples clinically:

[0034] Tumor samples were obtained by sampling or surgical resection. At least 0.5 cm × 0.5 cm × 0.5 cm tissue was cut and rinsed three times with sterile saline. The tissue was placed in pre-cooled PBS phosphate buffer (HyClone, SH40007.01) containing 5% double antibody (Gibco, 15070063) and stored on ice during transportation.

[0035] (2) Preparation of tissue digestion fluid:

[0036] In a sterile operating table, use solution A Basal Medium and solution B Supplement 20× in tumor tissue digestion solution (BioGenous, K601003) to prepare tissue digestion solution in a volume ratio of solution A to solution B of 20:1.

[0037] (3) Pretreatment of ameloblastoma tissue:

[0038] In a sterile operating table, the sample obtained in step (1) was washed 4 times with precooled PBS phosphate buffer added with 5% double antibody, each time for 4 minutes, and then washed once with precooled PBS phosphate buffer without double antibody, for 4 minutes to wash away the residual double antibody. Then, the tissue sample block was cut into pieces of about 1 mm×1 mm×1 mm in size using sterile instruments (ophthalmic scissors, etc.).

[0039] (4) Tissue dissociation and digestion:

[0040] Transfer the chopped tissue sample to a 5 mL centrifuge tube or a 6-well plate or a 12-well plate, add 2-4 mL of the tissue digestion solution prepared in step (2), and digest in a 37°C constant temperature incubator. Digest for 5 minutes every half an hour with shaking. If a well plate is used, the digestion solution can be blown to promote digestion. The digestion time is about 1-2 hours, depending on the size of the tissue and the clinical classification of ameloblastoma (cystic and solid types). When a large number of single cells are observed in the tissue digestion solution under the microscope, the digestion can be stopped to obtain a digested tissue suspension.

[0041] (5) Low adsorption plates promote single cell aggregation and organoid culture:

[0042] Organoid culture medium was prepared according to the following composition: DMEM / F12 basal medium (HyClone, SH30023.01), 1×B27 supplement (Shanghai Yuanpei, S440J7), 1.25mmol / L N-acetyl-l-cysteine ​​(Sigma-Aldrich, A9165), 10mmol / L nicotinamide (Sigma-Aldrich, N0636), 50ng / mL human epidermal growth factor (PeproTech, AF-100-15), 500nmol / L A83-01 TGF-β type I receptor inhibitor (MCE, HY-10432), 10ng / mL human FGF10 (PeproTech, 100-26), 5ng / mL human FGF2 (PeproTech, 100-18B), 1μmol / L prostaglandin E2 (TocrisBioscience, 2296), 0.3μmol / L Laduviglusib (MCE, HY-10182), 10 nmol / L Y-27632 (MCE, HY-10071), 1 μmol / L Forskolin (MCE, HY-15371), 4% R-spondin-1 and 4% Noggin (both produced by the r-PEX protein expression platform of U-Protein Express BV).

[0043] The digested tissue suspension obtained in step (4) was subjected to cell filtration and centrifugation, and red blood cells were lysed by erythrocyte lysis solution (bioGenous, E238010) and centrifuged. The cell pellet was resuspended and washed with PBS buffer and then centrifuged. The above centrifugation speed and time were consistent, all of which were 1400rpm and 5min. The cell pellet was retained. 10μL of the above-mentioned organoid culture medium was first added to the cell pellet, the cells were blown evenly, and then the cell suspension was placed in a certain volume of organoid culture medium, and sphere culture was performed in a six-well low adsorption plate (Corning, 3471) (2mL culture medium per well of the six-well plate). After 24h, when 2-5 single cells aggregated into a spheroid under a microscope, the suspension containing the cells was collected and centrifuged at 1000rpm for 5min. The supernatant was discarded, and the obtained precipitate was a preliminarily formed ameloblastoma organoid spheroid-like structure. Add 10 μL of the above-mentioned organoid culture medium to the obtained cell sediment, blow and mix, then take 3 μL of cell suspension and add it to 300 μL Matrigel matrix gel (Corning, 356231), and gently blow the precipitated cell mass with a 200 μL gun tip to resuspend it; after sufficient mixing, the cell mass mixed with matrix gel is evenly dotted into a 24-well plate, 1 droplet per well, with a volume of about 40-50 μL. Put it in a cell culture incubator (37°C, 4% CO2) for 30 minutes until the matrix gel is fully solidified. After the matrix gel solidifies, add 500 μL of the above-mentioned organoid culture medium, the culture medium does not cover the matrix gel, and culture is carried out in a 37°C constant temperature incubator, and fresh organoid culture medium is replaced every 3 days. Organoid formation can be observed after 2-3 weeks of culture, and then culture is continued for 1 month. Microscopic measurement shows that most of the organoid diameters reach 100-200 μm or more. Figure 1 and Figure 2 This is a microscopic observation of a successfully cultured ameloblastoma-derived organoid, which exhibits spherical tumor characteristics. Figure 1 This is a picture of the cell morphology of cultured cystic ameloblastoma organoids observed under a microscope. Figure 2 This is a picture of the cell morphology of cultured solid ameloblastoma organoids observed under a microscope.

[0044] The more mature cystic ameloblastoma organoids were collected and cultured for about one month, and frozen sections were made and stained with hematoxylin and eosin (HE) to observe whether the organoid structure conformed to the histological morphology of ameloblastoma. The results are shown in Figure 3 Immunofluorescence staining was used to mark the expression of classic markers of cystic ameloblastoma, namely odontoblastoma-associated protein (ODAM), amelogenin (AMG) and amelogenin (AMB). Figure 4 HE staining and immunofluorescence staining results showed that ameloblastoma organoids were successfully constructed, which were consistent with the pathological manifestations of ameloblastoma and expressed the classic markers of ameloblastoma, ODAM, AMG, and AMB.

[0045] Example 2

[0046] (1) Obtaining ameloblastoma tissue samples clinically: Same as Example 1.

[0047] (2) Preparation of traditional tissue digestion solution: Prepare 10 mL of tissue digestion solution, including 9200 μL of serum-free DMEM / F12 medium, 250 μL of 40× Dispase II, 50 μL of deoxyribonuclease I (DNase I), and 500 μL of double antibody.

[0048] (3) Pretreatment of ameloblastoma tissue: Same as Example 1.

[0049] (4) Tissue dissociation and digestion: Move the chopped tissue sample blocks to a 5 mL centrifuge tube or a 6-well plate or a 12-well plate, add 2-4 mL of the tissue digestion solution prepared in step (2), and digest in a 37°C constant temperature incubator, shaking the centrifuge tube once every half an hour. The digestion time is 2-3 hours, depending on the size of the tissue and the clinical classification of ameloblastoma (cystic and solid types). When a large number of single cells are observed in the tissue digestion solution under the microscope, the digestion can be stopped to obtain a digested tissue suspension.

[0050] (5) Low adsorption plate promotes single cell aggregation and organoid culture: same as Example 1.

[0051] Compared with the tissue digestion solution prepared by using A solution BasalMedium and B solution Supplement 20× (A+B solution) in the present invention, the organoids obtained by culturing after digestion with A+B solution in the present invention are more numerous, larger in size, and closer in shape to a spherical shape ( Figure 5 , 6 ).

[0052] Example 3

[0053] (1) Obtaining ameloblastoma tissue samples clinically: Same as Example 1.

[0054] (2) Preparation of tissue digestion fluid: same as in Example 1.

[0055] (3) Pretreatment of ameloblastoma tissue: Same as Example 1.

[0056] (4) Tissue dissociation and digestion: Same as Example 1.

[0057] (5) Organoid culture: The digested tissue suspension obtained in step (4) was subjected to cell filtration and centrifugation, and red blood cells were lysed by erythrocyte lysis solution (bioGenous, E238010) and centrifuged. The cell pellet was resuspended and washed with PBS buffer and then centrifuged. The above centrifugation speed and time were consistent, both 1400rpm and 5min. The cell pellet was retained. 10μL of organoid culture medium was first added to the cell pellet, and the cells were blown evenly. Then 3μL of cell suspension was added to 300μL Matrigel matrix gel (Corning, 356231), and the precipitated cell clusters were gently blown with a 200μL gun tip to resuspend them; after sufficient mixing, the cell clusters mixed with matrix gel were evenly dotted into a 24-well plate, with 1 droplet per well, and the volume was about 40-50μL. Place the cells in a cell culture incubator for 30 minutes to allow the matrix gel to fully solidify. After the matrix gel solidifies, add 500 μL of organoid culture medium, with the culture medium covering the matrix gel. Culture the cells in a 37°C constant temperature incubator and replace with fresh organoid culture medium every 3 days.

[0058] The formation of organoids was observed under a microscope at 30 days of culture. Figure 7 , 8 ) showed that the present invention first promoted single-cell aggregation through a low-adsorption plate and then carried out organoid culture, which resulted in more ameloblastoma organoids and larger volumes than those formed by direct organoid culture.

[0059] Example 4

[0060] (1) Obtaining ameloblastoma tissue samples clinically: Same as Example 1.

[0061] (2) Preparation of tissue digestion fluid: same as in Example 1.

[0062] (3) Pretreatment of ameloblastoma tissue: Same as Example 1.

[0063] (4) Tissue dissociation and digestion: Same as Example 1.

[0064] (5) Low-adsorption plate promotes single-cell aggregation and organoid culture: Except for adding 10 mmol / L L-cysteine ​​to the organoid culture medium, the rest of the operation is the same as Example 1.

[0065] The formation of organoids was observed under a microscope at 30 days of culture. Fig. 9 , 10 ) showed that after adding 10mmol / L L-cysteine ​​to the culture medium, the number and size of ameloblastoma organoids were greater.

[0066] The above embodiments are only used to help illustrate the present invention. The implementation methods of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for culturing ameloblastoma organoids, characterized in that: The following steps are involved: (1) After washing the ameloblastoma tissue with PBS containing double antibodies, it was minced; (2) digesting the chopped tissue with a tissue digestion solution to obtain a digested tissue suspension; (3) filtering and centrifuging the digested tissue suspension obtained in step (2), splitting the red blood cells, centrifuging the cells, washing with PBS, and centrifuging the cells; adding organoid culture medium to the obtained cell precipitate, blowing the cells evenly, and placing them in a low-adsorption plate for spheroid culture; when 2-5 single cells aggregate into spheroids, collecting the cell suspension and centrifuging the cells to obtain a preliminarily formed ameloblastoma organoid spheroid-like structure; (4) implanting the spheroid structure obtained in step (3) into matrix gel, adding organoid culture medium for culturing to obtain ameloblastoma organoids.

2. The method for culturing ameloblastoma organoids according to claim 1, characterized in that: Step (1) is: in a sterile operating table, the ameloblastoma tissue is washed with pre-cooled PBS containing double antibodies by shaking, and then washed with pre-cooled PBS by shaking to wash away the residual double antibodies, and the ameloblastoma tissue is cut into pieces using sterile instruments.

3. The method for culturing ameloblastoma organoids according to claim 2, characterized in that: The concentration of the dual antibody is 5-10%.

4. The method for culturing ameloblastoma organoids according to claim 1, characterized in that: In step (2), the tissue digestion fluid is prepared by mixing solution A Basal Medium and solution B Supplement 20× in the tumor tissue digestion fluid.

5. The method for culturing ameloblastoma organoids according to claim 1, characterized in that: The centrifugal condition is 1000-1400 rpm for 5-10 min.

6. The method for culturing ameloblastoma organoids according to claim 1, characterized in that: Step (4) is as follows: the ameloblastoma organoid pellet-like structure is mixed by blowing with the organoid culture medium, the cell suspension is added to the matrix gel, and after sufficient mixing, the cell pellet mixed with the matrix gel is spotted into the cell plate, with one droplet per well, and the volume is 40-50 μL; after the matrix gel solidifies, the organoid culture medium is added for culturing, and the fresh organoid culture medium is replaced every 2-4 days until the ameloblastoma organoid is obtained.

7. The method for culturing ameloblastoma organoids according to claim 1, characterized in that: In steps (3) and (4), the organoid culture medium comprises the following components: basal culture medium, B27 supplement, N-acetyl-l-cysteine, nicotinamide, human epidermal growth factor, A83-01, human FGF10, human FGF2, prostaglandin E2, Laduviglusib, Y-27632, Forskolin, R-spondin-1, and Noggin.

8. The method for culturing ameloblastoma organoids according to claim 7, characterized in that: The organoid culture medium contains the following components: basal culture medium, 1×B27 supplement, 1-1.5mmol / L N-acetyl-l-cysteine, 5-15mmol / L nicotinamide, 30-70ng / mL human epidermal growth factor, 300-700nmol / L A83-01, 5-15ng / mL human FGF10, 3-7ng / mL human FGF2, 0.5-1.5μmol / L prostaglandin E2, 0.1-0.5μmol / L Laduviglusib, 5-15nmol / L Y-27632, 0.5-1.5μmol / L Forskolin, 2-6% R-spondin-1, 2-6% Noggin.

9. The method for culturing ameloblastoma organoids according to claim 7 or 8, characterized in that: The organoid culture medium also contains L-cysteine.

10. Use of the ameloblastoma organoid culture method according to any one of claims 1 to 9 in preparing an ameloblastoma organoid model.

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