Culture medium for culturing salivary gland tumor organoid, kit and application

Abstract

The invention discloses a culture medium for culturing salivary gland tumor organs, a kit and application, the culture medium comprises Advanced DMEM (Dulbecco's Modified Eagle Medium) / F12, and the following components based on the Advanced DMEM / F12: Antibiotic-Antimyotic, GlutaMAX, HEPES buffer solution, fibroblast growth factor FGF-10, EGF (Epidermal Growth Factor), TGF-beta I type receptor inhibitor, Wnt3a recombinant protein, Noggin recombinant protein, R-spondin-1 recombinant protein, N-acetylcysteine, nicotinamide, B27 supplement, dexamethasone, Primocin and Y27632. The kit comprises an N-1 supplement, an N-2 supplement, FGF2, ProstaglandinE2, FBS, 0CHIR 99021, Forskolin and BFGF (Brown Fibroblast Growth Factor).

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a culture medium, a kit and an application thereof for culturing salivary gland tumor organoids. Background Art

[0002] Research on major diseases with high incidence rates, such as lung cancer and breast cancer, has always been popular, and treatment methods are also diverse. However, for oral malignancies with relatively low incidence rates, especially salivary gland malignancies, there are fewer studies. Not only is diagnosis more difficult, but there is also a lack of treatment methods for them. In addition to conventional surgical treatment and radiotherapy and chemotherapy, there are few targeted drugs and other treatment methods, and the efficacy is poor, and patients often have a poor prognosis. Therefore, the key issue to be solved in salivary gland tumor research is to explore new targeted antibodies and effective therapeutic targeted drugs to improve diagnostic accuracy and patient survival. In-depth exploration of the pathogenesis of salivary gland tumors will help deepen the understanding of the mechanism of occurrence and development of salivary gland tumors. In vitro and in vivo tumor models that retain the genetic background and biobehavioral characteristics of patients' tumors and can simulate the heterogeneity of clinical patient tumors are effective tools for tumor research. Common preclinical tumor models are human-derived tumor cell lines (CDX), patient-derived tumor xenografts (PDX), and organoids. Compared with the first two, the latter can retain the genetic characteristics of the original tumor, has a short modeling time and a high success rate, and can well simulate the biological characteristics, occurrence and development process of the tumor.

[0003] In recent years, although there have been a few reports on PDO modeling of salivary gland tumors (such as adenoid cystic carcinoma) in domestic and foreign literature, there are still few reports on PDO modeling of other types of salivary gland tumors, especially research reports on PDO kits for salivary gland tumors. Summary of the invention

[0004] The present invention aims to provide a culture medium, a kit and an application for culturing salivary gland tumor organoids, which are used to solve the problem of PDO kits for other types of salivary gland tumors except adenoid cystic carcinoma in the field of salivary gland tumors.

[0005] The first invention of the present invention provides a culture medium for culturing salivary gland tumor organoids, the culture medium comprising: Advanced DMEM / F12, and the following components based on Advanced DMEM / F12: Antibiotic-Antimycotic, 0.5-2v%; GlutaMAX, 0.5-2v%; HEPES buffer, 0.5-2v%; fibroblast growth factor FGF-10, 50-300ng / ml; EGF, 10-300ng / ml; TGF-β type I receptor inhibitor, 10-1000nmol / L; Wnt3a recombinant protein, 300-800ng / ml; Noggin recombinant protein, 0.05-0.3μg / ml; R -spondin-1 recombinant protein, 0.05~0.3μg / ml; N-acetylcysteine, 0.75~2.5mmol / L; Nicotinamide, 5~30mmol / L; B27 supplement, 0.5~5v%; Dexamethasone, 0~5μmol / L; Primocin, 0~5mg / ml; Y27632, 0~30μmol / l; N-2 supplement 0-3%; FGF2, 0~10ng / ml; ProstaglandinE2, 0~3μmol / L; FBS, 0-5v / %; CHIR 99021, 0-0.4μmol / L; Forskolin, 0-2μmol / l; BFGF, 0~25μg / ml.

[0006] In one embodiment, the culture medium comprises: Advanced DMEM / F12, and the following components based on Advanced DMEM / F12: Antibiotic-Antimycotic, 1v%; GlutaMAX, 1v%; HEPES buffer, 1v%; fibroblast growth factor FGF-10, 100ng / ml; EGF, 50ng / ml; TGF-β type I receptor inhibitor, 100nmol / L; Wnt3a recombinant protein, 500ng / ml; Noggin recombinant protein, 0.1μg / ml; R-spondin-1 recombinant protein, 0.1μg / ml; N-acetylcysteine, 1mmol / L; nicotinamide, 10mmol / L; dexamethasone, 1μmol / L; Y27632, 10μmol / l; N-2 supplement 1%; fetal bovine serum, 1v%;

[0007] The salivary gland tumor organoids are salivary gland adenoid cystic carcinoma organoids (ACC).

[0008] In one embodiment, the culture medium comprises: Advanced DMEM / F12, and Advanced DMEM / F12 based on the following ingredients: Antibiotic-Antimycotic, 1v%; GlutaMAX, 1v%; HEPES buffer, 1v%; fibroblast growth factor FGF-10, 100ng / ml; EGF, 50ng / ml; TGF-β type I receptor inhibitor, 500nmol / L; Wnt3a recombinant protein, 500ng / ml; Noggin recombinant protein, 0.1μg / ml; R-spondin-1 recombinant protein, 0.1μg / ml; N-acetylcysteine, 1.25mmol / L; Nicotinamide, 10mmol / L; B27 supplement, 2v%; Dexamethasone, 1μmol / L; Primocin, 1mg / ml; Y27632, 10μmol / l; N-2 supplement 1%; FGF2, 5ng / ml; ProstaglandinE2, 1μmol / L; BFGF, 10μg / ml;

[0009] The salivary gland tumor organoids are salivary gland malignant pleomorphic adenoma organoids (MPA).

[0010] In one embodiment, the culture medium comprises: Advanced DMEM / F12, and Advanced The following ingredients are based on DMEM / F12: Antibiotic-Antimycotic, 1v%; GlutaMAX, 1v%; HEPES buffer, 1v%; fibroblast growth factor FGF-10, 100ng / ml; EGF, 50ng / ml; TGF-β type I receptor inhibitor, 500nmol / L; Wnt3a recombinant protein, 500ng / ml; Noggin recombinant protein, 0.1μg / ml; R-spondin-1 recombinant protein, 0.1μg / ml; N-acetylcysteine, 1.25mmol / L; Nicotinamide, 10mmol / L; B27 supplement, 2v%; Dexamethasone, 1μmol / L; Primocin, 1mg / ml; N-2 supplement 1%; FGF2, 5ng / ml; ProstaglandinE2, 1μmol / L; BFGF, 10μg / ml;

[0011] The salivary gland tumor organoid is a salivary gland pleomorphic adenoma organoid (PA).

[0012] In one embodiment, the culture medium comprises: Antibiotic-Antimycotic, 1v%; GlutaMAX, 1v%; HEPES buffer, 1v%; fibroblast growth factor FGF-10, 100ng / ml; EGF, 50ng / ml; TGF-β type I receptor inhibitor, 100nmol / L; Wnt3a recombinant protein, 500ng / ml; Noggin recombinant protein, 0.1μg / ml; R-spondin-1 recombinant protein, 0.1μg / ml; N-acetylcysteine, 1mmol / L; Nicotinamide, 10mmol / L; B27 supplement, 50x; Dexamethasone, 1μmol / L; Primocin, 1mg / ml; Y27632, 10μmol / l; FGF2, 5ng / ml; ProstaglandinE2, 2μmol / L; FBS, 2v / %; CHIR 99021, 0.2 μmol / L; Forskolin, 1 μmol / l; the x represents the multiple of the final dilution;

[0013] The salivary gland tumor organoids are salivary gland secretory carcinoma organoids (SSC).

[0014] In one embodiment, the culture medium comprises: Antibiotic-Antimycotic, 1v%; GlutaMAX, 1v%; HEPES buffer, 1v%; EGF, 50ng / ml; TGF-β type I receptor inhibitor, 100nmol / L; Wnt3a recombinant protein, 500ng / ml; Noggin recombinant protein, 0.1μg / ml; R-spondin-1 recombinant protein, 0.1μg / ml; N-acetylcysteine, 1mmol / L; Nicotinamide, 10mmol / L; B27 supplement, 50x; Y27632, 10μmol / l; wherein x represents the multiple of the final dilution;

[0015] The salivary gland tumor organoids are basal cell adenoma organoids (BCA).

[0016] In one embodiment, the culture medium comprises: Antibiotic-Antimycotic, 1v%; GlutaMAX, 1v%; HEPES buffer, 1v%; fibroblast growth factor FGF-10, 100ng / ml; EGF, 50ng / ml; TGF-β type I receptor inhibitor, 100nmol / L; Wnt3a recombinant protein, 500ng / ml; Noggin recombinant protein, 0.1μg / ml; R-spondin-1 recombinant protein, 0.1μg / ml; N-acetylcysteine, 1mmol / L; Nicotinamide, 10mmol / L; B27 supplement, 50x; Dexamethasone, 1μmol / L; Primocin, 1mg / ml; Y27632, 10μmol / l; FGF2, 5ng / ml; ProstaglandinE2, 1μmol / L; FBS, 2v / %; CHIR 99021, 0.24 μmol / L; Forskolin, 1 μmol / l;

[0017] The salivary gland tumor organoids are mucoepidermoid carcinoma organoids (MEC).

[0018] A second aspect of the present invention provides a kit comprising the above culture medium.

[0019] A third aspect of the present invention provides use of the above-mentioned culture medium or kit in culturing salivary gland tumor organoids.

[0020] A fourth aspect of the present invention provides a method for culturing salivary gland tumor organoids, the method comprising culturing the disrupted salivary gland tumor cells using the above-mentioned culture medium or kit to obtain the salivary gland tumor organoids.

[0021] A fifth aspect of the present invention provides salivary gland tumor organoids prepared by the above-mentioned culture method.

[0022] The sixth aspect of the present invention provides at least one of the following uses of the salivary gland tumor organoid:

[0023] Prepare or screen drugs for treating salivary gland tumor organoids;

[0024] Study the development mechanism of salivary gland tumor organoids;

[0025] Screening of relevant biomarkers in salivary gland tumor organoids.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The salivary gland tumor organoids provided by the present invention can be used to prepare or screen drugs for treating salivary gland tumor organoids, study the occurrence and development mechanism of salivary gland tumor organoids, screen relevant biomarkers of salivary gland tumor organoids, etc., which not only meets the needs of scientific research, but also provides a useful choice in clinical drug guidance.

[0028] The culture medium or kit of the present invention can enable salivary gland adenoid cystic carcinoma cells, malignant pleomorphic adenoma cells, pleomorphic adenoma cells, secretory cancer cells, basal cell adenoma cells, and mucoepidermoid carcinoma cells to be subcultured multiple times, and the morphology and properties of the primary salivary gland tumor 3D organoids can be maintained after subculture, thereby meeting the needs of large-scale replication of salivary gland tumor organoids.

[0029] The present invention targets the growth characteristics of salivary gland adenoid cystic carcinoma cells, malignant pleomorphic adenoma cells, pleomorphic adenoma cells, secretory cancer cells, basal cell adenoma cells, and mucoepidermoid carcinoma cells, respectively. The multiple cytokine components in the culture medium are compounded in specific proportions, and the components work synergistically to enable the salivary gland tumor cells to form 3D organoids that are close to the morphology and properties of real tumor tissue in a relatively short period of time.

[0030] The salivary gland tumor organoid model provided by the present invention can be co-cultured with immune cells or cancer-associated fibroblasts to form an organoid model that simulates the real microenvironment of the tumor, and is used to study the effects of immune cells and cancer-associated fibroblasts on the biological behavior of the tumor.

[0031] A large number of salivary gland tumor organoids obtained by culturing the culture medium provided by the present invention or a kit containing the culture medium can be used to establish an organoid biological sample library. The establishment of an organoid sample library can not only promote basic scientific research, but also be directly applied in clinical practice, providing important support for personalized medicine and new drug development.

[0032] Instruction Manual

[0033] Figure 1 This is the general picture of primary (P1) salivary gland ACC organoid cultured to day 8, 10×;

[0034] Figure 2 This is the general picture of P2 ACC organoid cultured to day 22, 10×;

[0035] Figure 3A HE image of P2 ACC organoid, 20×;

[0036] Figure 3B This is the HE staining result of ACC parental tumor tissue, 20×;

[0037] Figure 4A Immunohistochemical staining of ACC organoids at generation P2, 20×;

[0038] Figure 4B Immunohistochemical staining of ACC parental tumor tissue, 20×;

[0039] Figure 5 This is a general picture after MPA cell inoculation;

[0040] Figure 6 This is the general picture of primary (P1) MPA organoid culture on day 6, 10×;

[0041] Figure 7 This is the general picture of P2 MPA organoid cultured to day 6, 10×;

[0042] Figure 8 This is the general picture of MPA organoid cultured at P3 on day 6, 4×;

[0043] Fig. 9A HE staining of MPA organoid cultured at P2 on day 4, 40×;

[0044] Fig. 9B This is the HE staining result of MPA parent tumor tissue, 40×;

[0045] Fig. 10A Immunohistochemical staining of MPA organoids at generation P2, 40×;

[0046] Fig. 10B Immunohistochemical staining of MPA parental tumor tissue, 40×;

[0047] Fig.11 The results of the macroscopic and HE staining of the P3 generation PA organoid cultured to the 4th day and the HE staining of the PA parent tumor tissue, 10×;

[0048] Fig.12 Immunohistochemical staining of PA parental tumor tissue and P3 generation PA organoid, 10×;

[0049] Fig.13 This is a general picture of SSC organoids at generations P1, P2, and P3, 4×;

[0050] Fig.14 HE staining of SSC parental tumor tissue and P3 generation organoid, 40×;

[0051] Fig.15 Immunohistochemical staining of SSC parental tumor tissue and organoids, 40×;

[0052] Fig.16 Figure 1 shows the FISH staining results of SSC parental tumor tissue and organoids, 100×;

[0053] Fig.17 Results of SSC parental tumor and P2 generation organoids before and after 5 days of radiotherapy, ×;

[0054] Fig.18 This is the general picture of MEC organoids at P1, P2, and P3, 10×;

[0055] Fig.19 HE staining of MEC parental tumor tissue and organoid, 40×;

[0056] Fig. 20 Immunohistochemical staining of MEC parental tumor tissue and organoids, 40×;

[0057] Fig.21 Figure 1 shows the FISH staining results of MEC parental tumor tissue and organoids, 100×;

[0058] Fig. 22 This is a general picture of the P1 generation BCA organoid;

[0059] Fig.23 HE staining images of BCA parental tumor tissue and organoids;

[0060] Fig.24 This is an immunohistochemical staining image of BCA organoids and parental tumor tissues; Fig.24 A is the result of β-catenin staining of BCA organoids. Fig.24 B is the result of Calp staining of BCA organoids. Fig.24 C is the result of BCA organoid CK14 staining. Fig.24 D is the result of BCA organoid S100 staining. Fig.24 E is the result of BCA organoid P63 staining. Fig.24 F is the result of Ki67 staining of BCA organoids. Fig.24 a is the result of β-catenin staining of BCA parental tumor tissue, Fig.24 b is the Calp staining result of BCA parent tumor tissue, Fig.24 c is the CK14 staining result of BCA parent tumor tissue, Fig.24 d is the result of S100 staining of BCA parental tumor tissue. Fig.24 e is the result of P63 staining of BCA parent tumor tissue. Fig.24 f shows the Ki67 staining results of BCA parental tumor tissue. DETAILED DESCRIPTION

[0061] In this article, the range expressed by "a value to another value" is a summary expression method to avoid listing all the values ​​in the range one by one in the specification. Therefore, the description of a specific numerical range covers any value in the numerical range and a smaller numerical range defined by any value in the numerical range, just as if the arbitrary value and the smaller numerical range were explicitly written in the specification.

[0062] In this application, the use of "or" means "and / or" unless otherwise stated. In the case of multiple dependent claims, use of "or" refers to more than one of the preceding independent or dependent claims only in the alternative.

[0063] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0064] Example 1 Salivary gland adenoid cystic carcinoma (ACC) organoid culture

[0065] The specimen was obtained from the Department of Oral and Maxillofacial Head and Neck Oncology of the Ninth People's Hospital Affiliated to Shanghai Jiao Tong University. It was a 52-year-old female patient with salivary gland adenoid cystic carcinoma, with the primary lesion located in the parotid gland. Before the cryobiopsy, the patient and her guardian agreed and signed an informed consent form. The postoperative pathological diagnosis was: salivary gland adenoid cystic carcinoma.

[0066] (1) Specific training methods

[0067] (1.1) Tissue preservation

[0068] The biopsy samples were quickly placed in a centrifuge tube containing tissue preservation solution, the main component of which was DMEM (Shanghai Yuanpei Biotechnology Co., Ltd.), to which amphotericin B (at a concentration of 500 ng / ml in the preservation solution) (Suzhou Xinsaimei Biotechnology Co., Ltd.) and penicillin-streptomycin (Gibco) accounting for 5% of the preservation solution were added. The centrifuge tube was placed in an ice box and sent to the laboratory.

[0069] (1.2) Tissue fragmentation

[0070] In a biosafety cabinet, transfer the salivary gland adenoid cystic carcinoma tissue to a culture dish and soak the tissue in PBS-1 solution containing 5% penicillin-streptomycin for about 10 minutes. Transfer the tissue to a new culture dish containing PBS-1 solution and remove the capsule tissue. Cut the tissue into about 1 mm 3 Large and small tissue fragments.

[0071] (1.3) Tissue digestion

[0072] The tissue fragments were transferred to a 50 ml centrifuge tube containing digestive enzymes (2 mg / ml collagenase type II (Gibco) and 0.1 mg / ml DNase I (Applichem)) and digested at 37°C at 350 rpm for 2 h to obtain more cell pellets without cell adhesion. Then, serum-containing DMEM medium was added to terminate the digestion, and the tube was pipetted up and down 10 to 15 times to further promote tissue fragmentation.

[0073] (1.4) Filtering

[0074] The digested suspension was filtered with 100μm and 70μm filters in turn to obtain a cell suspension; centrifuged at 200g for 3min and the supernatant was discarded, the cell pellet was resuspended with PBS-1, and counted under a microscope. According to the counting results, an appropriate amount of preset culture medium was added to resuspend the cell pellet. The preset culture medium includes the following components:

[0075] 1v% Penicillin-Streptomycin Antibiotic-Antimycotic (Gibco), 1v% GlutaMAX (Gibco), 1v% HEPES buffer (Gibco), fibroblast growth factor (FGF-10) 100ng / ml (MCE), EGF 50ng / ml (MCE), A83-01 0.1μmol / l (MCE), Wnt3a recombinant protein 500ng / ml (MCE), Noggin recombinant protein 0.1μg / ml (MCE), R-spondin-1 recombinant protein 0.1μg / ml (MCE), N-Acetylcysteine ​​(NAC) 1mmol / L (MCE) , Nicotinamide (Nicotinamide) 10mmol / L (MCE), Dexamethasone (Dexamethasone) (MCE) 1μmol / L, 1v% N-2 supplement (MCE), Y-27632 (Rock inhibitor) 10μmol / l; wherein, in the culture medium, except Advanced DMEM / F12, the dosage of other components such as percentage or concentration are described based on 500mL Advanced DMEM / F12; N-2 supplement is 100XN-2 supplement.

[0076] (1.5) Vaccination

[0077] Take an appropriate amount of cell suspension and mix it with matrix gel (low growth factor type Matrigel, Corning Company), wherein each 40 μl matrix gel mixture contains about 10^4 cells, and inoculate it in a 24-well cell culture plate (NEST), and add 40 μl matrix gel mixture to each well. In the present invention, since the matrix gel is in a liquid state at 0-10°C and solidifies when the temperature rises, it is necessary to perform related operations of the matrix gel on ice, and it is best to use a pre-cooled gun tip to complete the mixing of the matrix gel and the cell suspension in the shortest time to prevent the matrix gel from solidifying.

[0078] (1.6) Primary salivary gland adenoid cystic carcinoma organoid culture

[0079] Place the inoculated 24-well plate upside down in a 37°C incubator (Thermo Fisher) for about 40 minutes. When the colloid solidifies, place the 24-well plate upright and add 450ul of the culture medium described in step (1.4) above to each well. Return to the 37°C incubator for culture. Thereafter, change the culture medium every 2 to 3 days. The above inversion operation is more conducive to the formation of a dome-shaped structure of the matrix gel and firm adhesion to the wall. During this period, observe the organoids under an inverted microscope. Generally, within 2 weeks, if the organoids do not grow larger, they are passaged. The passage ratio is 1:6 in a 24-well plate (that is, the primary salivary gland adenoid cystic carcinoma organoids in one well are passaged to 6 wells in a new 24-well plate).

[0080] (1.7) Passaging of salivary gland adenoid cystic carcinoma organoids

[0081] Discard the original culture medium, melt the matrix gel with pre-cooled PBS-1, gently blow down the mixture of matrix gel and organoids and transfer it to a centrifuge tube, centrifuge and discard the supernatant, resuspend with PBS-1 and centrifuge and discard the supernatant to remove all the matrix gel, add 2-3 ml of pancreatic enzyme (Suzhou Xinsaimei Biotechnology Co., Ltd.) to resuspend the precipitate and digest the spheres for 3 minutes (every 1 minute, you can gently blow a few times with the tip of the gun to further promote digestion), and terminate digestion. Centrifuge and discard the supernatant, then resuspend the precipitate with PBS-1. Count under the microscope, add an appropriate amount of culture medium to resuspend the cell precipitate according to the counting results; then inoculate and culture, the steps are the same as the primary salivary gland adenoid cystic carcinoma organoids.

[0082] (2) Morphological observation of salivary gland adenoid cystic carcinoma organoids

[0083] The morphology of salivary gland adenoid cystic carcinoma organoids at different growth generations was observed under an inverted microscope and photographed. Figure 1 As shown: The organoids are spherical in shape with continuous edges. After subculturing, the second generation (P2) salivary gland adenoid cystic carcinoma organoids were obtained. The microscopic appearance of the P2 generation was as follows: Figure 2 , relative to Figure 1, Figure 2 The tissue cell morphology of P2 organoids remained basically unchanged after medium passage.

[0084] (3) Identification of cell morphology and protein expression in salivary gland adenoid cystic carcinoma organoids

[0085] The P2 adenoid cystic carcinoma organoids were cultured for the 13th day of the second generation, and then the cultured adenoid cystic carcinoma organoids were harvested and stained with HE ( Figure 3A ), the staining results are similar to Figure 3B The HE staining results of clinical patient tumor tissues shown in the figure (data retrieved from the tissue sample library of Shanghai Ninth People's Hospital) were used for tissue morphology comparison. Figure 3A and 3B By comparison, it can be seen that the morphology of the organoids cultured by the method of the present invention is similar to that of clinical samples. The P2 generation adenoid cystic carcinoma organoids were cultured on the 13th day of the second generation, and then the cultured adenoid cystic carcinoma organoids were harvested and immunohistochemical staining of relevant molecular markers was performed ( Figure 4A ), histomorphology showed that the staining results of molecular markers (such as CK7, CK14, Ki67, MYB, S100, CK-PAN, P63) were similar to those of Figure 4B The tumor tissues of clinical patients shown in the figure (data retrieved from the tissue sample bank of Shanghai Ninth People's Hospital) were used for tissue morphology comparison. Figure 4A and 4B By comparison, it can be seen that the organoids cultured using the method of the present invention are similar to clinical samples.

[0086] Example 2 Salivary gland malignant pleomorphic adenoma organoid (MPA) culture

[0087] The specimen came from the Department of Oral and Maxillofacial Head and Neck Tumors of the Ninth People's Hospital Affiliated to Shanghai Jiao Tong University. It was a 62-year-old male patient with malignant pleomorphic adenoma, and the primary lesion was located in the parotid gland. Before the cryobiopsy, the patient and his guardian agreed and signed an informed consent form. The postoperative pathological diagnosis was: malignant pleomorphic adenoma of the parotid gland.

[0088] (1) Specific training methods

[0089] (1.1) Tissue preservation

[0090] The biopsy samples were quickly placed in a centrifuge tube containing tissue preservation solution, the main component of which was DMEM (Shanghai Yuanpei Biotechnology Co., Ltd.), to which amphotericin B (at a concentration of 500 ng / ml in the preservation solution) (Suzhou Xinsaimei Biotechnology Co., Ltd.) and penicillin-streptomycin (Gibco) accounting for 5% of the preservation solution were added. The centrifuge tube was placed in an ice box and sent to the laboratory.

[0091] (1.2) Tissue fragmentation

[0092] In a biosafety cabinet, transfer the malignant pleomorphic adenoma tissue to a culture dish and soak the tissue in PBS-1 solution containing 5% penicillin-streptomycin for about 10 minutes. Transfer the tissue to a new culture dish containing the above tissue washing solution and remove the capsule tissue. Cut the tissue into approximately 1 mm 3 Large tissue fragments.

[0093] (1.3) Tissue digestion

[0094] The tissue fragments were transferred to a 50 ml centrifuge tube containing digestive enzymes (2 mg / ml collagenase type II (Gibco) and 0.1 mg / ml DNaseI (Applichem)) and digested at 37°C at 300 rpm for 1.5 h to obtain more cell pellets without cell adhesion. Then, serum-containing DMEM medium was added to terminate the digestion, and the tube was pipetted up and down 10 to 20 times to further promote tissue fragmentation.

[0095] (1.4) Filtering

[0096] The digested suspension was filtered with 100 μm and 70 μm filters in turn to obtain a cell suspension; centrifuged at 200 g for 3 min, the supernatant was discarded, the cell pellet was resuspended with PBS-1, and counted under a microscope with a counting plate. According to the counting results, an appropriate amount of culture medium was added to resuspend the cell pellet.

[0097] The composition ratio of the culture medium is as follows: 500 ml Advanced DMEM / F12 (Gibco), 1 v% Antibiotic-Antimycotic (Gibco), 1 v% GlutaMAX (Gibco), 1 v% HEPES buffer (Gibco), 100 ng / ml fibroblast growth factor (FGF-10) (MCE), 100 ng / ml EGF (MCE), bFGF 10μg / ml (MCE), A83-01500nmol / L (MCE), Wnt3a recombinant protein 500ng / ml (MCE), Noggin recombinant protein 0.1μg / ml (MCE), Rspondin-1 recombinant protein 0.1μg / ml (MCE), N-acetylcysteine ​​(NAC) 1.25mmol / L (MCE), Nicotinamide (Nicotinamide) 10mmol / L (MCE), 2v% B27 supplement (MCE), Dexamethasone (MCE) 1μmol / L, Primocin (invivogen) 1mg / ml, 1v% N-2 supplement (MCE), FGF2 is 5ng / ml (MCE), Prostaglandin E21μmol / L (MCE), 1v% fetal bovine serum; wherein, in the culture medium, except for Advanced Except for DMEM / F12, the dosage of other components such as percentage or concentration is described based on 500mL Advanced DMEM / F12; B27 supplement is 50X B27 supplement; N-2 supplement is 100X N-2 supplement.

[0098] (1.5) Vaccination

[0099] Select an appropriate amount of cell suspension and mix it with matrix gel (low growth factor type Matrigel, Corning Company), wherein each 40 μl matrix gel mixture contains about 10 4 cells were seeded in a 24-well cell culture plate (NEST) and 40 μl of the matrix gel mixture was added to each well. Figure 5 shown.

[0100] Since the matrix gel is in liquid state at 0-10℃ and solidifies when the temperature rises, it is necessary to perform matrix gel-related operations on ice. At the same time, it is best to use a pre-cooled pipette tip to complete the mixing of the matrix gel and the cell suspension in the shortest time to prevent the matrix gel from solidifying.

[0101] (1.6) Primary Malignant Pleomorphic Adenoma Organoid Culture

[0102] Place the inoculated 24-well plate upside down in a 37°C incubator (Thermo Fisher) for about 45 minutes. After the colloid solidifies, place the 24-well plate upright and add 450ul of the culture medium described in step (4) above to each well. Return to the 37°C incubator for incubation. After that, change the culture medium every 2 to 3 days.

[0103] The above inversion operation is more conducive to the formation of a dome-shaped structure of the matrix gel and firm adhesion to the wall. During this period, the organoids are observed under an inverted microscope. Generally, within two weeks, if the organoids do not grow larger, they are passaged. The passage ratio is 1:6 in a 24-well plate (i.e., the primary malignant pleomorphic adenoma organoids in one well are passaged to 6 wells in a new 24-well plate).

[0104] (1.7) Passaging of Malignant Pleomorphic Adenoma Organoids

[0105] Discard the original culture medium, melt the matrix gel with pre-cooled PBS-1, gently blow down the mixture of matrix gel and organoids and transfer it to a centrifuge tube, centrifuge and discard the supernatant, resuspend with PBS-1 and centrifuge and discard the supernatant to remove all the matrix gel, add 1-2 ml of trypsin (Suzhou Xinsaimei Biotechnology Co., Ltd.) to resuspend the precipitate and digest the spheres for 3 minutes to terminate the digestion. Centrifuge and discard the supernatant, then resuspend the precipitate with PBS-1. Count under the microscope, add an appropriate amount of culture medium to resuspend the cell precipitate according to the counting results; then inoculate and culture, the steps are the same as those of primary malignant pleomorphic adenoma organoids.

[0106] (2) Observation of organoid morphology of malignant pleomorphic adenoma

[0107] The morphology of malignant pleomorphic adenoma organoids of different growth generations was observed under an inverted microscope and photographed. Figure 6 Shown: The organoid is spherical in shape with continuous edges.

[0108] After subculturing, the second generation (P2) malignant pleomorphic adenoma organoids were obtained. The microscopic appearance of P2 cultured to day 6 was as follows: Figure 7 After another subculture, the third generation (P3) malignant pleomorphic adenoma organoids were obtained. The microscopic appearance of P3 cultured to the sixth day was Figure 8 . Figure 7 , Figure 8 It shows that after subculture, the tissue cell morphology of P2 and P3 malignant pleomorphic adenoma organoids is similar to that of the primary malignant pleomorphic adenoma organoids and remains unchanged.

[0109] (3) Identification of cell morphology and protein expression in malignant pleomorphic adenoma organoids

[0110] P2 malignant pleomorphic adenoma organoids were cultured until day 4, and the organoids were harvested and stained with HE ( Fig. 9A) and compared the cell morphology with HE staining of the patient's tumor tissue ( Fig. 9B ) (The patient's tumor sample slice data were retrieved from the pathology archive of the Department of Oral Pathology, the Ninth People's Hospital affiliated to Shanghai Jiao Tong University). The comparison results showed that the cell morphology of the P2 generation malignant pleomorphic adenoma organoids was similar to that of the real tumor samples.

[0111] Immunohistochemical staining of protein expression (CK7, CK14, SMA) and cell proliferation index Ki67 expression was performed on P2 malignant pleomorphic adenoma organoids cultured to day 4. Fig. 10A ), and the staining results were compared with the immunohistochemical staining results of the above markers in the patient's tumor tissue ( Fig. 10B ) (The immunohistochemistry data of patient tumor samples were retrieved from the pathology archive of the tissue sample bank of the Department of Oral Pathology, the Ninth People's Hospital affiliated to Shanghai Jiao Tong University). The comparison results showed that the protein expression and proliferation index expression levels of the P2 generation organoids were similar to those of real tumor samples.

[0112] Example 3 Salivary gland pleomorphic adenoma organoid (PA) culture

[0113] PA organoid culture method:

[0114] 1) PA tissue samples

[0115] The sample was originally obtained from a 55-year-old male patient who underwent surgical resection in the Department of Oral and Maxillofacial Head and Neck Tumors of the Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. The patient was diagnosed with PA by postoperative pathology, and the primary lesion was located in the left parotid gland. The patient signed an informed consent form.

[0116] 2) Isolation of PA tissue

[0117] A typical area of ​​the tumor sample was selected and placed in a centrifuge tube containing tissue preservation solution within half an hour of ex vivo. The main component of the preservation solution was DMEM culture medium containing 5% penicillin-streptomycin double antibody. The centrifuge tube was stored at 4°C and transported to the laboratory within 1 hour.

[0118] 3) Pretreatment of PA tissue

[0119] Before the experiment, the surface of the biosafety cabinet was sprayed with 75% ethanol for disinfection, and the whole process was performed in the biosafety cabinet. The tumor sample tissue was transferred to a culture dish and soaked in PBS containing 5% penicillin-streptomycin for 10 minutes. After soaking, the tissue was transferred to a new culture dish containing the above tissue washing solution, and the tissue was clamped with sterile instruments (ophthalmic forceps, ophthalmic scissors, etc.), and then the capsule tissue was carefully cut off.

[0120] Use sterile surgical scissors to cut the tissue into pieces of about 1mm×1mm×1mm. Transfer the tissue fragments to a 50ml centrifuge tube, which contains 2mg / ml type II collagenase (Gibco) and 0.1mg / ml DNase I in DMEM high-glucose medium, and shake at 37℃ for 1h. Add DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin double antibody to terminate the digestion. Filter the digested suspension with 100μm and 70μm filters (biosharp) in turn to obtain a single cell suspension.

[0121] 4) Organoid culture

[0122] After washing the single cell suspension with PBS, count the cells on the counting plate. According to the counting results, add an appropriate amount of culture medium to resuspend the cell pellet, mix it with matrix gel at a ratio of 7:3, and inoculate it in a 48-well plate. Add 30 μL of gel droplets to each well. Place the inoculated 24-well plate upside down in the incubator at 37°C for about 20 minutes. After the gel solidifies, place the 24-well plate upright and add 500 μL of culture medium to each well. Then change the culture medium every 2-3 days. During this period, observe the number of organoids under the microscope. If the number of organoids meets the requirements, pass them on, including: discarding the culture medium, adding 4°C PBS to wash twice, aspirating PBS, and adding 500 μl 4°C ClutrexTM organoid harvesting solution to each well. Incubate at 4°C for 30 to 90 minutes, shake the 24-well plate appropriately, add matrix gel (to disaggregate) at the end of incubation, the dome-shaped structure disappears, and the organoids can be seen floating at the bottom of the well and transferred to a 15 ml centrifuge tube, centrifuged at 500×g for 3 minutes, and discard the supernatant. Wash twice with PBS, centrifuge, and discard the supernatant. Add TrypLETM Express enzyme, pipette evenly, and place in a 37°C incubator. Observe the cell separation under a microscope. When most of the organoids are dissociated into single cells, add DMEM culture medium containing 10% fetal bovine serum to quickly terminate the digestion and transfer to a 15 ml centrifuge tube.

[0123] Centrifuge at 500×g for 3 min, discard the supernatant, resuspend the pellet in organoid culture medium, mix with matrix gel, re-inoculate in a 24-well plate (30 μl / well), and let stand in a 37°C incubator for 20 min. After the matrix gel solidifies, add 500 μl of organoid culture medium to each well and culture in a 37°C incubator. The principle of taking one well and dividing it into 6 wells (1:6) was adopted for subculture.

[0124] 5) Observation of PA organoid morphology

[0125] The morphology of pleomorphic adenoma organoids of different growth generations was observed under an inverted microscope and photographed. (P3) The morphology of pleomorphic adenoma organoids on the 4th day of culture was as follows Fig.11 Shown: The organoid is spherical in shape with continuous edges.

[0126] 6) Identification of cell morphology and protein expression in pleomorphic adenoma organoids

[0127] The P3 malignant pleomorphic adenoma organoids were cultured until day 4, and the organoids were harvested and stained with HE ( Fig.11 ) and compared the cell morphology with HE staining of the patient's tumor tissue ( Fig.11 ) (The patient's tumor sample slice data were retrieved from the pathology archive of the Department of Oral Pathology, the Ninth People's Hospital affiliated to Shanghai Jiao Tong University). The comparison results showed that the cell morphology of the P23 generation pleomorphic adenoma organoids was similar to that of the real tumor samples.

[0128] P3 pleomorphic adenoma organoids cultured to day 4 were subjected to immunohistochemical staining of proteins (CK7, CK14, p63, ki-67, SMA and Calp) ( Fig.12 ), and the staining results were compared with the immunohistochemical staining results of the above markers in the patient's tumor tissue ( Fig.12 ) (The immunohistochemistry data of patient tumor samples were retrieved from the pathology archive of the tissue sample bank of the Department of Oral Pathology, the Ninth People's Hospital affiliated to Shanghai Jiao Tong University). The comparison results showed that the protein expression and proliferation index expression levels of the P3 generation organoids were similar to those of real tumor samples.

[0129] Example 4 Salivary gland secretory carcinoma organoid (SSC) culture

[0130] 1) SSC tissue samples

[0131] The sample was originally obtained from a 64-year-old male patient who underwent surgical resection in the Department of Oral and Maxillofacial Head and Neck Tumors of the Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. The patient was diagnosed with SSC by postoperative pathology, and the primary lesion was located in the left parotid gland. The patient signed an informed consent form.

[0132] 2) Isolation of SSC tissue

[0133] A typical area of ​​the tumor sample was selected and placed in a centrifuge tube containing tissue preservation solution within half an hour of ex vivo. The main component of the preservation solution was DMEM culture medium containing 5% penicillin-streptomycin double antibody. The centrifuge tube was stored at 4°C and transported to the laboratory within 1 hour.

[0134] 3) Pretreatment of SSC tissue

[0135] Before the experiment, the surface of the biosafety cabinet was sprayed with 75% ethanol for disinfection, and the whole process was performed in the biosafety cabinet. The tumor sample tissue was transferred to a culture dish and soaked in PBS containing 5% penicillin-streptomycin for 10 minutes. After soaking, the tissue was transferred to a new culture dish containing the above tissue washing solution, and the tissue was clamped with sterile instruments (ophthalmic forceps, ophthalmic scissors, etc.), and then the capsule tissue was carefully cut off.

[0136] Use sterile surgical scissors to cut the tissue into pieces of about 1mm×1mm×1mm. Transfer the tissue fragments to a 50ml centrifuge tube, which contains 2mg / ml type II collagenase (Gibco) and 0.1mg / ml DNaseI in DMEM high-glucose medium, and shake at 37°C for digestion for 1h. Add DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin double antibody to terminate the digestion. Filter the digested suspension with 100μm, 70μm and 40μm filters (biosharp) in turn to obtain a single cell suspension.

[0137] 6) Organoid Culture

[0138] After washing the single cell suspension with PBS, count the cells on the counting plate. According to the counting results, add an appropriate amount of culture medium to resuspend the cell pellet, mix it with matrix gel at a ratio of 1:1, and inoculate it in a 24-well plate. Place the inoculated 24-well plate upside down in an incubator at 37°C for about 30 minutes. After the gel solidifies, place the 24-well plate upright, add an appropriate amount of culture medium to each well, and then replace the culture medium every 2-3 days. During this period, observe the number of organoids under a microscope. Under the condition that the number of organoids meets the requirements, pass them on, including: discarding the culture medium, adding 4°C PBS to wash twice, aspirating PBS, and adding 500μl 4°C ClutrexTM organoid harvesting solution to each well. Incubate at 4°C for 30-90 minutes, shake the 24-well plate appropriately, add matrix gel (to disaggregate) at the end of incubation, the dome-shaped structure disappears, and the organoids can be seen floating at the bottom of the well and transferred to a 15ml centrifuge tube, centrifuged at 500×g for 3 minutes, and discard the supernatant. Wash twice with PBS, centrifuge, and discard the supernatant. Add TrypLETM Express enzyme, blow evenly, let it stand in a 37°C incubator, and observe the cell separation under a microscope. When most of the organoids are dissociated into single cells, add DMEM culture medium containing 10% fetal bovine serum to quickly terminate the digestion and transfer to a 15 ml centrifuge tube.

[0139] Centrifuge at 500×g for 3 min, discard the supernatant, resuspend the pellet in organoid culture medium, mix with matrix gel, re-inoculate in a 24-well plate (40 μl / well), and let stand in a 37°C incubator for 15-30 min. After the matrix gel solidifies, add 500 μl of organoid culture medium to each well and culture in a 37°C incubator. The principle of taking one well and dividing it into 6 wells (1:6) was adopted for subculture.

[0140] In this example, the culture medium consists of Advanced DMEM / F12, 1 wt % antimycin A (Gibco), 1 v / v % GlutaMAX (Gibco), 1 v / v % HEPES buffer (Gibco) and specific additive factors.

[0141] The specific growth factor is selected from: FGF-10 (e.g., 100 ng / ml), EGF (e.g., 50 ng / ml), FGF-2 (e.g., 5 ng / ml), A83-01 (0.1 μmol / ml), Wnt3a recombinant protein (e.g., 500 ng / ml), Noggin recombinant protein (e.g., 0.1 μg / ml), R-spondin-1 recombinant protein (e.g., 0.1 μg / ml), NAC (e.g., 1 mmol / ml), nicotinamide (e.g., 10 mmol / ml), 1×B27 supplement (1:50), antibiotic PrimocinTM (e.g., 1 mg / ml), ROCK inhibitor Y27632 (e.g., 10 umol / l), dexamethasone (e.g., 1 μmmol / l), fetal bovine serum (e.g., 2%), CHIR99021 (e.g., 0.2 μmol / l), prostaglandin E2 (e.g., 1 μmol / l), and forskolin (e.g., 1 μmol / l).

[0142] 7) Observation of the gross morphology of organoids

[0143] At each stage of different growth densities, the gross morphology of the subcultured organoids was observed using a stereo microscope and photographed. The organoid morphology was spherical with continuous edges. After passage to P2 and P3, the gross morphology remained unchanged, and the number of organoids increased with each passage, with the spheroids slowly increasing in size. Fig.13 .

[0144] In the laboratory, the organoids were cultured and subcultured to P3 and were deposited on May 14, 2024 in the China Center for Type Culture Collection (CCTCC), located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province (Postal Code: 430072), with the collection number CCTCCNO: C2024151 and the classification name Homo Sapiens.

[0145] 6) SSC organoid staining

[0146] The organoids obtained after culture were processed using a modified agar encapsulation method, including the following steps:

[0147] Fixation: Carefully pipette the organoid tissue into a 24-well cell culture plate, add 10% neutral buffered formaldehyde, and fix at room temperature for 2-4 hours. Pre-embedding: Preheat the frozen agar solution to a viscous state in advance. After the organoid tissue is fixed, carefully use a 3ml pipette to suck out the organoid tissue and place it in a disposable plastic embedding mold. Use a pipette to suck out an appropriate amount of agar solution and slowly add it around the organoid tissue until the agar wraps all the tissues. After the agar solidifies at room temperature, put it into 75% alcohol, 85% alcohol, 95% alcohol, and 100% alcohol for dehydration together with the mold, 5-10 minutes each time, to facilitate the agar block to be removed from the plastic embedding mold. Dehydration: Place the agar block containing the pre-embedded organoid tissue in the embedding box, and immerse it in 75% alcohol for 30 minutes, 85% alcohol for 30 minutes, 95% alcohol for 30 minutes, 100% alcohol for 1 hour, and 100% alcohol for 1 hour. Transparent and wax-immersed: xylene 30min, xylene 30min, paraffin 62℃ 30min, paraffin 62℃ 1h, paraffin 62℃ 1h. Gently pick up the agar block at the end of the treatment with tweezers, embed it with paraffin, slice it at 3μm, stain it with hematoxylin-eosin (HE), observe it with routine histology, and compare it with the primary tumor. By observing the HE sections of the passaged organoids and their corresponding primary tumor tissues, it was found that the passaged organoids retained the histopathological morphological characteristics of their corresponding primary tumors, such as Fig.14 .

[0148] 7) Immunohistochemical staining of SSC organoids

[0149] The salivary gland SSC organoids were stained with immunohistochemistry (IHC) of relevant protein markers using the Envision two-step method on the Roche fully automatic immunohistochemical staining machine BenchMark ULTRA. The results showed that the protein markers (Mammaglobin, CK7, Ki-67 and S-100) were consistent with the IHC results of the corresponding primary tumor tissues, see Fig.15 .

[0150] 8) Fluorescence in situ hybridization analysis of SSC organoids

[0151] Fluorescence in situ hybridization (FISH) was performed on the organoids of salivary gland SSCs: the paraffin-embedded organoids were sliced ​​into several 3μm sections and baked at 65℃ for 1-2h; dewaxed with xylene, hydrated with gradient alcohol, and the sections were placed in 90℃ distilled water for 30min. The tissue was digested in a water bath in 54℃ pepsin digestion solution for 20-30min, dehydrated with gradient alcohol, and air-dried. Under light-proof conditions, 5-10μL of probe (ETV6) was added, and the coverslip was covered with rubber cement to seal the slide. The hybridizer was denatured at 76℃ for 10min and hybridized at 42℃ for 18h. After the incubation, take out the slides, remove the rubber cement, and soak them in a 2×SSC / 0.3% NP-40 (pH7.0~7.2) solution preheated to 68°C for 2 minutes. Then soak the sections in a 2×SSC / 0.1% NP-40 solution at room temperature for 2 minutes, and finally place them in a 2×SSC buffer at room temperature for 2 minutes. Dehydrate with gradient alcohol, dry naturally in the dark, add 10μLDAPI counterstaining solution to the corresponding area of ​​probe hybridization, cover with a coverslip, stain at -20°C for 30 minutes, and observe the results under a fluorescence microscope. Look for tumor cells under three objective lenses: 10x, 40x, and 100x oil objective, and count the red and green signals in 20 cells under a 100x oil objective. FISH results showed that the ETV6 gene was broken in both P3 organoids and primary tumors ( Fig.16 ), the results remain consistent.

[0152] 9) Analysis of SSC organoids after radiotherapy

[0153] The results of radiotherapy sensitivity analysis of salivary gland SSC organoids were performed. The organoids were irradiated with different radiation doses of 0Gy, 4Gy, 8Gy and 16Gy before and after radiotherapy. Observation under an ordinary optical inverted microscope showed that there was no significant change in the P2 generation organoids 5 days after radiotherapy compared with before radiotherapy. The results suggest that SSC organoids are not sensitive to radiotherapy. For details, see Fig.17 .

[0154] Example 5 Salivary Gland Mucoepidermoid Carcinoma Organoid (MEC) Culture

[0155] 1) Culture of MEC organoids

[0156] Source of specimen: A 58-year-old male patient underwent surgical resection in the Department of Oral and Maxillofacial Head and Neck Tumors, the Ninth People's Hospital affiliated to Shanghai Jiao Tong University School of Medicine. Postoperative pathological diagnosis was mucoepidermoid carcinoma with the primary lesion located in the parotid gland. The patient signed an informed consent form.

[0157] Culture method: Select a typical area of ​​the tumor sample and place it in a centrifuge tube containing tissue preservation solution within half an hour of ex vivo. The main component of the preservation solution is DMEM + 5% penicillin-streptomycin double antibody. The centrifuge tube is placed in an ice box and stored at 4°C, and transported to the laboratory within 1 hour.

[0158] Before the experiment, the surface of the biosafety cabinet was sprayed with 75% ethanol for disinfection, and the entire operation was performed in the biosafety cabinet. The tumor sample tissue was transferred to a culture dish and soaked in PBS containing 5% penicillin-streptomycin double antibody for 10 minutes. After soaking, the tissue was transferred to a new culture dish containing the above tissue washing solution, and the tissue was clamped with sterile instruments (ophthalmic forceps, ophthalmic scissors, etc.), and then the capsule tissue was carefully cut off. Use sterile surgical scissors to cut the tissue into pieces of about 1mm×1mm×1mm. The tissue fragments were transferred to a 50ml centrifuge tube, which contained 2mg / ml type II collagenase (Gibco) and 0.1mg / ml DNase I in DMEM high-glucose medium, and digested at 37°C for 1h on a shaker. DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% double antibody was added to terminate the digestion. The digested suspension was filtered with 100μm, 70μm and 40μm filters (biosharp) in sequence to obtain a single-cell suspension. The single-cell suspension was washed with PBS and counted on a counting plate. According to the counting results, an appropriate amount of preset culture medium was added to resuspend the cell pellet, mixed with matrix gel in a 1:1 ratio, and inoculated into a 24-well plate.

[0159] The inoculated 24-well plate is placed upside down in an incubator at 37°C for about 30 minutes. After the gel solidifies, the 24-well plate is placed upright and an appropriate amount of a preset culture medium is added to each well. The preset culture medium may be composed of a culture medium Advanced DMEM / F12, 1% Antibiotic-Antimycotic, 2mmol / ml GlutaMAX, 10mmol / ml HEPES buffer, and specific additive factors. The specific growth factors may include fibroblast growth factor (FGF-10) 100ng / ml, EGF 50ng / ml, FGF-25ng / ml, A83-010.1umol / ml, Wnt3a recombinant protein 500ng / ml, Noggin recombinant protein 0.1μg / ml, R-spondin-1 recombinant protein 0.1μg / ml, N-acetylcysteine ​​(NAC) 1mmol / ml, Nicotinamide (Nicotinamide) 10mmol / ml, 1×B27 supplement (1:50), Primocin at a concentration of 1mg / ml, Rho-associated kinases (ROCK) inhibitor Y2763210umol / l, Dexamethasone 1ummol / l, 2% FBS, CHIR 990210.2umol / l, Prostaglandin E21umol / l, Forskolin 1umol / l. After that, the culture medium was replaced every 2-3 days.

[0160] 2) Gross morphological observation of MEC organoids

[0161] At each stage of different growth densities, the gross morphology of the subcultured organoids was observed using a stereo microscope and photographed. The organoid morphology was spherical with continuous edges. After passage to P2 and P3, the gross morphology remained unchanged, and the number of organoids increased with each passage, with the spheroids slowly increasing in size. Fig.18 .

[0162] 3) Morphological identification of MEC organoids

[0163] The organoids obtained after culture were processed using a modified agar encapsulation method:

[0164] Fixation: Carefully pipette the organoid tissue into a 24-well cell culture plate, add 10% neutral buffered formaldehyde, and fix at room temperature for 2-4 hours. Pre-embedding: Preheat the frozen agar solution to a viscous state in advance. After the organoid tissue is fixed, carefully use a 3ml pipette to suck out the organoid tissue and place it in a disposable plastic embedding mold. Use a pipette to suck out an appropriate amount of agar solution and slowly add it around the organoid tissue until the agar wraps all the tissues. After the agar solidifies at room temperature, put it into 75% alcohol, 85% alcohol, 95% alcohol, and 100% alcohol for dehydration together with the mold, 5-10 minutes each time, to facilitate the agar block to be removed from the plastic embedding mold. Dehydration: Place the agar block containing the pre-embedded organoid tissue in the embedding box, and immerse it in 75% alcohol for 30 minutes, 85% alcohol for 30 minutes, 95% alcohol for 30 minutes, 100% alcohol for 1 hour, and 100% alcohol for 1 hour. Transparent and wax-immersed: xylene 30min, xylene 30min, paraffin 62℃ 30min, paraffin 62℃ 1h, paraffin 62℃ 1h. Gently pick up the agar block at the end of the treatment with tweezers, embed it with paraffin, slice it at 3μm, stain it with hematoxylin-eosin (HE), observe it with routine histology, and compare it with the primary tumor. By observing the HE sections of the passaged organoids and their corresponding primary tumor tissues, it was found that the passaged organoids retained the histopathological morphological characteristics of their corresponding primary tumors, such as Fig.19 .

[0165] The salivary gland MEC organoids were stained with immunohistochemistry (IHC) of relevant protein markers using the Envision two-step method on the Roche fully automatic immunohistochemical staining machine BenchMark ULTRA. The results showed that the protein markers (AE1 / AE3, CK7, CK14, Ki-67, MUC-1, P63) were consistent with the IHC results of the corresponding primary tumor tissues, see Fig. 20 .

[0166] Fluorescence in situ hybridization (FISH) was performed on the organoids of salivary gland MECs: the paraffin-embedded organoids were sliced ​​into several 3μm sections and baked at 65℃ for 1-2h; dewaxed with xylene, hydrated with gradient alcohol, and the sections were placed in 90℃ distilled water for 30min. The tissue was digested in a water bath in 54℃ pepsin digestion solution for 20-30min, dehydrated with gradient alcohol and air-dried. Under light-proof conditions, 5-10μL of probe (MAML2) was added, and the coverslip was covered with rubber cement to seal the sections. The hybridizer was denatured at 76℃ for 10min and hybridized at 42℃ for 18h. After the incubation, the slides were removed, the rubber cement was removed, and the slides were immersed in a 2×SSC / 0.3% NP-40 (pH 7.0-7.2) solution preheated to 68°C for 2 minutes. The sections were then immersed in a 2×SSC / 0.1% NP-40 solution at room temperature for 2 minutes, and finally placed in a 2×SSC buffer at room temperature for 2 minutes. Dehydrate with gradient alcohol, dry naturally in the dark, add 10 μL of DAPI counterstaining solution to the corresponding area of ​​probe hybridization, cover with a coverslip, stain at 20°C for 30 minutes, and observe the results under a fluorescence microscope. Tumor cells were searched under three objective lenses: 10x, 40x, and 100x oil objective, and the red and green signals in 20 cells were counted under a 100x oil objective. FISH results showed that the MAML2 gene was broken in both P3 organoids and primary tumors ( Fig.21 ), the results remain consistent.

[0167] Example 6 Salivary Gland Basal Cell Adenoma Organoid (BCA) Culture

[0168] 1. Sample: A fresh tumor specimen was collected from BCA surgery at the Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine in October 2021. All relevant specimen acquisition and model establishment experimental procedures were approved by the Ethics Committee of the Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine.

[0169] 2. Main reagents and culture medium components: Advanced Dulbecco's modified Eagle's medium (DMEM): nutrient medium F12, 4-hydroxyethylpiperazineethanesulfonic acid [4-(2-hydroxyerhyl)piperazine-1-erhanesulfonicacid, HEPES] buffer, GlutaMAX additive, penicillin-streptomycin, B-27 additive, Australian fetal bovine serum, phosphate buffer solution (PBS), trypsin substitute (TrypLE Express) (all the above reagents were purchased from Gibco, the United States). DMEM (Shanghai Yuanpei Biotechnology Co., Ltd.); Matrigel (Corning, USA); serum-free cell cryopreservation medium (Suzhou Xinsaimei Biotechnology Co., Ltd.); deoxyribonuclease I (DNase I), collagenase (Sigma, USA); Noggin recombinant protein, transforming growth factor-β (TGF-β) type I receptor inhibitor (A83-01), recombinant human epidermal growth factor (EGF), Wnt3a recombinant protein, R-spondin-1 recombinant protein, N-acetyl-L-cysteine, Rho kinase inhibitor Y-27632, nicotinamide (all purchased from MCE, USA). CultrexTM organoid harvesting medium (R&D System, USA). Transformation related protein 63 (P63), cell proliferation associated antigen (Ki-67), cytokeratin 14.

[0170] BCA organoid culture medium: Advanced DMEM / F12, with the following ingredients added: GlutaMAX, 1v%; HEPES, 1v%; Antibiotic-Antimycotic, 100U / mL; N-Acetyl-L-cysteine, 1.25mmol / L; EGF, 50ng / mL; Noggin, 100ng / mL; A83-01, 0.1μmol / L; Wnt3a, 500ng / mL; R-spondin-1, 0.1μg / mL; Nicotinamide, 10mmol / L; Y-27632, 10μmol / L; B-27Supplement, used at 50× dilution.

[0171] 3. Cultivation methods:

[0172] (1) Isolation of human salivary gland BCA tumor cells and establishment and culture of in vitro organoids: Preheat the 24-well culture plate in a 37°C incubator in advance, and melt the matrix gel on ice 30 minutes in advance. Cut the tumor tissue into 3-5 mm diameter tissue blocks on a clean bench, rinse with serum-free DMEM culture medium, digest with collagenase and DNaseI enzymes at 37°C for 2 hours, and terminate the digestion with serum-containing DMEM culture medium. The digestion products were sieved through 100μm, 70μm, and 40μm filters in turn to form a single cell suspension. The cells were counted, and the cells were resuspended in organoid culture medium and mixed with matrix gel. 40μL / well was inoculated in a 24-well plate to form a dome-shaped matrix gel solidified droplet (matrixdome). The culture plate was inverted and incubated in a 37°C incubator for 15-30 minutes. After the matrix gel solidifies, add 500 μL of organoid culture medium to each well to completely cover the matrix gel droplets. Culture the organoids under 5% CO2 and 37°C conditions. Change the medium once every 2 to 3 days.

[0173] (2) Organoid passaging: discard the culture medium, add 4℃ PBS to wash twice, aspirate PBS, and add 500ul 4℃ Clutrex TM organoid harvesting solution to each well. Incubate at 4℃ for 30-90min, shake the 24-well plate appropriately. At the end of incubation, the matrix gel is depolymerized, the dome-shaped structure disappears, and the organoids can be seen floating at the bottom of the well and transferred to a 15mL centrifuge tube. Centrifuge at 500g for 3min and discard the supernatant. Wash twice with PBS, centrifuge, and discard the supernatant.

[0174] After adding TrypLE Express, blow and beat evenly, let it stand in a 37℃ incubator, and observe the cell separation under a microscope. When most of the organoids are dissociated into single cells, add DMEM culture medium containing 10% FBS to quickly terminate the digestion and transfer to a 15mL centrifuge tube. Centrifuge at 500g for 3min, discard the supernatant, resuspend the precipitate in the organoid culture medium, mix with the matrix gel, and re-inoculate it in a 24-well plate (40μL / well). Let it stand in a 37℃ incubator for 15-30min. When the matrix gel solidifies, add 500μL of organoid culture medium to each well and culture in a 37℃ incubator.

[0175] (3) Organoid cryopreservation: Discard the culture medium, add 4℃ Clutrex TM organoid harvesting solution to each well, and transfer to a 15mL centrifuge tube after the matrix gel is disaggregated. Centrifuge at 500g for 5 minutes and discard the supernatant. Pre-cool a 1mL pipette tip and resuspend the pellet in 4℃ serum-free cell cryopreservation solution. Aspirate the organoid cell suspension containing cryopreservation solution into a 2mL cryopreservation tube, place the cryopreservation tube in a gradient cooling cryopreservation box, transfer to a -80℃ refrigerator overnight, and place the cryopreservation tube in a liquid nitrogen tank for long-term storage the next day.

[0176] 4. Organoid morphological observation and immunohistochemical detection: The established BCA organoids were continuously cultured in vitro, and the organoid morphology was observed under a bright field microscope. After 14 days, the morphological characteristics of the organoids were obvious, and the organoids were fixed, pre-embedded in agar, embedded in paraffin, and stained with hematoxylin-eosin (HE). Specific steps: The agar block was melted in a 70°C water bath for 30 minutes. The BCA organoid culture medium was aspirated, the matrix gel was depolymerized with 4°C Clutrex TM organoid harvesting liquid, transferred to a centrifuge tube, and fixed with 10% formalin at room temperature for 2 hours. The organoid was transferred to the bottom of the embedding mold, and liquid agar was slowly added to the mold. After the agar solidified, the embedding block was removed and dehydrated in 70% alcohol, 85% alcohol, 95% alcohol, and 100% alcohol in turn, transparent with xylene, immersed in wax, embedded in paraffin, sliced, HE stained, and examined under a microscope.

[0177] The paraffin-embedded organoid blocks were cut into 3 μm white slices, dewaxed, and immunohistochemically detected using the EnVision two-step method according to the instructions. The primary antibodies were P63 (1:100, Maixin, China), Ki67 (1:100, Maixin, China), Cytokeratin14 (CK14, 1:100, Maixin, China), β-catenin (1:100, Maixin, China), S100 (1:100, Maixin, China), Calponin (Calp, 1:100, Maixin, China), CK14, Calp cytoplasmic brown staining, Ki67, P63, S100, β-catenin nuclear brown staining were positive expressions. PBS was used as a blank control instead of the primary antibody. The clinical paraffin-embedded samples of BCA were also subjected to immunohistochemical detection of the above primary antibodies.

[0178] result:

[0179] 1. In vitro culture: On the 1st to 3rd day of culture in the BCA organoid culture system, the tumor single cells wrapped in the matrix gel began to proliferate and divide, gradually growing to form cell clusters. The volume of the cell clusters continued to increase. On the 10th day, the organoid cell clusters were partially lobed, and on the 14th day, the organoids still maintained a good growth state ( Fig. 22 ).

[0180] 2. Morphological observation and immunohistochemical test results: HE staining results showed that the BCA tumor specimens under light microscopy showed tubular, beam-shaped and solid-shaped histology. The tumor cells were basal, cubic or short columnar, with eosinophilic cytoplasm and oval nuclei ( Fig.23 A). Tumor cells on the 14th day of the first generation of BCA organoid culture were arranged into solid cell clusters. The tumor cells were cubic or short columnar, with large, round or oval nuclei, eosinophilic cytoplasm, some vacuolated, unclear cell boundaries, and eosinophilic glassy material deposition around the cell nests ( Fig.23B). The number of organoids in the second and fourth generations decreased with each generation, but the HE histological morphology of the organoids grown was still similar to that of the first generation ( Fig.23 ). The results of immunohistochemistry showed that most of the BCA organoids were positive for β-catenin in the cytoplasm, a small number of cells were positive for nuclei, CK14 was strongly positive, some cells were positive for P63, Calp and S100 were not expressed, and some cells were positive for Ki67 ( Fig.24 AF). Some cells in the BCA original tumor were positive for β-catenin in the nucleus, expressed CK14 and P63, the cells in the solid mass area did not express Calp, S100 was positive for tumor stromal cells, and the Ki67 proliferation index was about 1-2% ( Fig.24 af). It can be seen that the in vitro BCA organoids basically retain the morphology and immunophenotypic characteristics of the original tumor cells.

[0181] The above disclosure is only the preferred embodiment of the present invention. The preferred embodiment does not describe all the details in detail, nor does it limit the invention to the specific implementation method described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can make good use of the present invention. The present invention is limited only by the claims and their full scope and equivalents.

[0182] Under the guidance of the present invention and the above-mentioned embodiments, it is easy for those skilled in the art to foresee that each raw material or its equivalent substitute, each processing method or its equivalent substitute listed or exemplified in the present invention can realize the present invention, and the upper and lower limit values ​​and interval values ​​of the parameters of each raw material and processing method can realize the present invention, and the embodiments are not listed one by one here.

Claims

1. A culture medium for culturing salivary gland tumor organoids, characterized in that: The culture medium comprises: Advanced DMEM / F12, and the following components based on Advanced DMEM / F12: Antibiotic-Antimycotic, 0.5-2v%; GlutaMAX, 0.5-2v%; HEPES buffer, 0.5-2v%; fibroblast growth factor FGF-10, 50-300ng / ml; EGF, 10-300ng / ml; TGF-β type I receptor inhibitor, 10-1000nmol / L; Wnt3a recombinant protein, 300-800ng / ml; Noggin recombinant protein, 0.05-0.3μg / ml; R -spondin-1 recombinant protein, 0.05~0.3μg / ml; N-acetylcysteine, 0.75~2.5mmol / L; Nicotinamide, 5~30mmol / L; B27 supplement, 0.5~5v%; Dexamethasone, 0~5μmol / L; Primocin, 0~5mg / ml; Y27632, 0~30μmol / l; N-2 supplement 0-3%; FGF2, 0~10ng / ml; ProstaglandinE2, 0~3μmol / L; FBS, 0-5v / %; CHIR 99021, 0-0.4μmol / L; Forskolin, 0-2μmol / l; BFGF, 0~25μg / ml.

2. The culture medium for culturing salivary gland tumor organoids according to claim 1, characterized in that: The culture medium comprises: Advanced DMEM / F12, and the following components based on Advanced DMEM / F12: Antibiotic-Antimycotic, 1v%; GlutaMAX, 1v%; HEPES buffer, 1v%; fibroblast growth factor FGF-10, 100ng / ml; EGF, 50ng / ml; TGF-β type I receptor inhibitor, 100nmol / L; Wnt3a recombinant protein, 500ng / ml; Noggin recombinant protein, 0.1μg / ml; R-spondin-1 recombinant protein, 0.1μg / ml; N-acetylcysteine, 1mmol / L; nicotinamide, 10mmol / L; dexamethasone, 1μmol / L; Y27632, 10μmol / l; N-2 supplement 1%; fetal bovine serum, 1v%; The salivary gland tumor organoid is a salivary gland adenoid cystic carcinoma organoid.

3. The culture medium for culturing salivary gland tumor organoids according to claim 1, characterized in that: The culture medium includes: Advanced DMEM / F12, and Advanced DMEM / F12 based on the following ingredients: Antibiotic-Antimycotic, 1v%; GlutaMAX, 1v%; HEPES buffer, 1v%; fibroblast growth factor FGF-10, 100ng / ml; EGF, 50ng / ml; TGF-β type I receptor inhibitor, 500nmol / L; Wnt3a recombinant protein, 500ng / ml; Noggin recombinant protein, 0.1μg / ml; R-spondin-1 recombinant protein, 0.1μg / ml; N-acetylcysteine, 1.25mmol / L; Nicotinamide, 10mmol / L; B27 supplement, 2v%; Dexamethasone, 1μmol / L; Primocin, 1mg / ml; Y27632, 10μmol / l; N-2 supplement 1%; FGF2, 5ng / ml; ProstaglandinE2, 1μmol / L; BFGF, 10μg / ml; The salivary gland tumor organoid is a salivary gland malignant pleomorphic adenoma organoid.

4. The culture medium for culturing salivary gland tumor organoids according to claim 1, characterized in that: The culture medium includes: Advanced DMEM / F12, and Advanced The following ingredients are based on DMEM / F12: Antibiotic-Antimycotic, 1v%; GlutaMAX, 1v%; HEPES buffer, 1v%; fibroblast growth factor FGF-10, 100ng / ml; EGF, 50ng / ml; TGF-β type I receptor inhibitor, 500nmol / L; Wnt3a recombinant protein, 500ng / ml; Noggin recombinant protein, 0.1μg / ml; R-spondin-1 recombinant protein, 0.1μg / ml; N-acetylcysteine, 1.25mmol / L; Nicotinamide, 10mmol / L; B27 supplement, 2v%; Dexamethasone, 1μmol / L; Primocin, 1mg / ml; N-2 supplement 1%; FGF2, 5ng / ml; ProstaglandinE2, 1μmol / L; BFGF, 10μg / ml; The salivary gland tumor organoid is a salivary gland pleomorphic adenoma organoid (PA).

5. The culture medium for culturing salivary gland tumor organoids according to claim 1, characterized in that: The culture medium includes: Antibiotic-Antimycotic, 1v%; GlutaMAX, 1v%; HEPES buffer, 1v%; fibroblast growth factor FGF-10, 100ng / ml; EGF, 50ng / ml; TGF-β type I receptor inhibitor, 100nmol / L; Wnt3a recombinant protein, 500ng / ml; Noggin recombinant protein, 0.1μg / ml; R-spondin-1 recombinant protein, 0.1μg / ml; N-acetylcysteine, 1mmol / L; Nicotinamide, 10mmol / L; B27 supplement, 50x; Dexamethasone, 1μmol / L; Primocin, 1mg / ml; Y27632, 10μmol / l; FGF2, 5ng / ml; ProstaglandinE2, 2μmol / L; FBS, 2v / %; CHIR 99021, 0.2 μmol / L; Forskolin, 1 μmol / l; the x represents the multiple of the final dilution; The salivary gland tumor organoids are salivary gland secretory cancer organoids.

6. The culture medium for culturing salivary gland tumor organoids according to claim 1, characterized in that: The culture medium includes: Antibiotic-Antimycotic, 1v%; GlutaMAX, 1v%; HEPES buffer, 1v%; EGF, 50ng / ml; TGF-β type I receptor inhibitor, 100nmol / L; Wnt3a recombinant protein, 500ng / ml; Noggin recombinant protein, 0.1μg / ml; R-spondin-1 recombinant protein, 0.1μg / ml; N-acetylcysteine, 1mmol / L; Nicotinamide, 10mmol / L; B27 supplement, 50x; Y27632, 10μmol / l; wherein x represents the multiple of the final dilution; The salivary gland tumor organoid is a salivary gland basal cell adenoma organoid (BCA).

7. The culture medium for culturing salivary gland tumor organoids according to claim 1, characterized in that: The culture medium includes: Antibiotic-Antimycotic, 1v%; GlutaMAX, 1v%; HEPES buffer, 1v%; fibroblast growth factor FGF-10, 100ng / ml; EGF, 50ng / ml; TGF-β type I receptor inhibitor, 100nmol / L; Wnt3a recombinant protein, 500ng / ml; Noggin recombinant protein, 0.1μg / ml; R-spondin-1 recombinant protein, 0.1μg / ml; N-acetylcysteine, 1mmol / L; Nicotinamide, 10mmol / L; B27 supplement, 50x; Dexamethasone, 1μmol / L; Primocin, 1mg / ml; Y27632, 10μmol / l; FGF2, 5ng / ml; ProstaglandinE2, 1μmol / L; FBS, 2v / %; CHIR 99021, 0.24 μmol / L; Forskolin, 1 μmol / l; The salivary gland tumor organoid is a salivary gland mucoepidermoid carcinoma organoid (MEC).

8. A kit comprising the culture medium according to any one of claims 1 to 7.

9. Use of the culture medium according to any one of claims 1 to 7 or the kit according to claim 8 in culturing salivary gland tumor organoids.

10. A method for culturing salivary gland tumor organoids, characterized in that: The culture method comprises culturing the disrupted salivary gland tumor cells using the culture medium described in any one of claims 1 to 7 or the kit described in claim 8 to obtain the salivary gland tumor organoids.

11. Salivary gland tumor organoids prepared by the culture method according to claim 10.

12. At least one of the following uses of the salivary gland tumor organoid according to claim 11: Prepare or screen drugs for treating salivary gland tumor organoids; Study the development mechanism of salivary gland tumor organoids; Screening of relevant biomarkers in salivary gland tumor organoids.

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