Salvary gland neuroendocrine cancer organoid and culture method, culture medium and application thereof

Abstract

The invention discloses a salivary gland neuroendocrine cancer organoid and a culture method, a culture medium and application thereof. The culture medium is prepared from Advanced DMEM (Dulbecco's Modified Eagle Medium) / F12 and the following components based on the Advanced DMEM / F12: 1wt.% of Antibiotic, 1wt.% of GlutaMAX, 1wt.% of an HEPES buffer solution and a specific addition factor. Wherein the specific growth factor comprises 100 ng / ml of fibroblast growth factor FGF 10, 50 ng / ml of EGF, 2 nM of bFGF, 300 ng / ml of Wnt3a recombinant protein, 300 ng / ml of Noggin recombinant protein, 0.3 [mu] g / ml of R-spondin-1 recombinant protein, 1.25 mM of N-acetylcysteine, 10 mM / ml of nicotinamide and a 1x B27 supplement. The culture medium and the culture method disclosed by the invention are suitable for culturing the salivary gland neuroendocrine cancer organoid, and the obtained organoid not only can predict the therapeutic response with reliable curative effect and verify the effectiveness of clinical drug treatment, but also can assist in researching the pathogenesis, development process, diagnosis, treatment and the like of diseases.

Description

Technical Field

[0001] The present invention relates to the field of medicine and biology, and in particular to a salivary gland neuroendocrine carcinoma organoid, a culture method thereof, a culture medium and an application thereof. Background Art

[0002] Neuroendocrine carcinoma is an epithelial tumor derived from neuroendocrine cells. It is common in the pancreas, gastrointestinal tract, and lungs. It is rare for it to occur in the salivary glands of the head and neck, accounting for less than 0.3% of head and neck malignant tumors. Due to its high malignancy, atypical clinical manifestations, and high rate of local metastasis, most patients have already developed local and distant metastases when they are diagnosed, so the prognosis is often poor. Literature reports that the median survival time is 16 months, and the average median survival time of patients with distant metastases is only 5 months. The current main treatment method is a combination of surgical resection and adjuvant radiotherapy and chemotherapy. However, it is relatively rare and there is a lack of experience in treatment, so the effect is often poor.

[0003] Patient-derived organoids (PDO) are three-dimensional (3D) tissue cultures that can not only predict reliable therapeutic responses through modeling of salivary neuroendocrine carcinoma, but also verify the effectiveness of clinical drug treatments. They can also assist in the study of the pathogenesis, development, diagnosis and treatment of the disease. There have been reports of the establishment of organoid models of neuroendocrine carcinomas in other parts of the body, but there is a lack of means to establish organoid models of oral and maxillofacial neuroendocrine carcinomas. This is not only because the culture requirements of PDO are high and the success rate is low, but also because of the lack of high-quality samples, which has had a certain impact on the research of salivary neuroendocrine carcinoma. Summary of the invention

[0004] In view of the defects of the prior art, the object of the present invention is to provide a salivary gland neuroendocrine carcinoma organoid and a culture method, culture medium and application thereof.

[0005] The technical solution of the present invention is: The first aspect of the present invention provides a culture medium for culturing salivary gland neuroendocrine carcinoma organoids, the culture medium comprising Advanced DMEM / F12, and the following components based on Advanced DMEM / F12: 1wt.% Antibiotic Antimycotic, 1wt.% GlutaMAX, 1wt.% HEPES buffer and specific added factors; wherein the specific growth factors include fibroblast growth factor FGF 10 100ng / ml, EGF 50ng / ml, bFGF 2nM, Wnt3a recombinant protein 300ng / ml, Noggin recombinant protein 300ng / ml, R-spondin-1 recombinant protein 0.3μg / ml, N-acetylcysteine ​​1.25mM, nicotinamide 10mM / ml, and 1x B27 supplement.

[0006] A second aspect of the present invention provides a kit for culturing salivary gland neuroendocrine carcinoma organoids, the kit comprising the above-mentioned culture medium for salivary gland neuroendocrine carcinoma organoids.

[0007] A third aspect of the present invention provides a method for culturing salivary gland neuroendocrine carcinoma organoids, which requires using the above-mentioned salivary gland neuroendocrine carcinoma organoid culture medium or salivary gland neuroendocrine carcinoma organoid kit for culturing to obtain the salivary gland neuroendocrine carcinoma organoids.

[0008] The specific steps of the salivary gland neuroendocrine carcinoma organoid culture method are as follows: (a) Tissue fragmentation The salivary gland neuroendocrine carcinoma tumor tissue is crushed to obtain tissue fragments; (b) Tissue digestion digesting the tissue fragments with digestive enzymes to obtain a suspension of salivary gland neuroendocrine carcinoma; (c) Filtration The salivary gland neuroendocrine carcinoma suspension is filtered, the supernatant is discarded by centrifugation, the precipitate is resuspended to obtain a single cell suspension of salivary gland neuroendocrine carcinoma, and the single cell suspension is inoculated and cultured using the above-mentioned salivary gland neuroendocrine carcinoma organoid culture medium or salivary gland neuroendocrine carcinoma organoid kit to obtain salivary gland neuroendocrine carcinoma organoids.

[0009] More preferably, the digestive enzymes include 2 mg / ml type II collagenase, 0.1 mg / ml DNase I, and 0.5 mg / ml hyaluronidase.

[0010] Further preferably, in step (c), culturing refers to culturing in an environment of 37° C. and a CO 2 concentration of 5%.

[0011] Further preferably, in step (c), during the culture period, the medium is replaced every 2-3 days.

[0012] Further preferably, in step (c), the suspension of salivary neuroendocrine carcinoma is filtered through 100 μm, 70 μm and 40 μm cell filters in sequence.

[0013] A fourth aspect of the present invention provides a salivary gland neuroendocrine carcinoma organoid, wherein the organoid is cultured by the above-mentioned salivary gland neuroendocrine carcinoma organoid culture method.

[0014] The fifth aspect of the present invention provides the use of salivary neuroendocrine carcinoma organoids in studying the pathogenesis of salivary neuroendocrine carcinoma, preparing or screening drugs for treating salivary neuroendocrine carcinoma; and the use of salivary neuroendocrine carcinoma organoids in preparing drug evaluation or screening models.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The salivary gland neuroendocrine carcinoma organoids obtained using the culture medium and culture method provided by the present invention can be subcultured for multiple times, and the organoids obtained after subculture maintain the same morphology and properties as the primary salivary gland neuroendocrine carcinoma organoids, meeting the needs of large-scale replication of salivary gland neuroendocrine carcinoma organoids.

[0016] 2. The salivary gland neuroendocrine carcinoma organoids obtained using the culture medium and culture method provided by the present invention have cell tissue morphology and expression of protein molecular markers that are highly consistent with clinical samples, and can effectively maintain cell tissue specificity.

[0017] 3. The salivary gland neuroendocrine carcinoma organoids obtained using the culture medium and culture method provided by the present invention can be used to screen therapeutic drugs for salivary gland neuroendocrine carcinoma, study the occurrence and development mechanism of salivary gland neuroendocrine carcinoma, screen salivary gland neuroendocrine carcinoma-related biomarkers, etc., which can not only meet the needs of scientific research, but also provide useful options in clinical drug guidance. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings: Figure 1 A schematic diagram of the passaging of salivary gland neuroendocrine carcinoma organoids provided for the third embodiment of the present invention (P1 refers to the first generation of salivary gland neuroendocrine carcinoma organoids, P2 refers to the second generation of salivary gland neuroendocrine carcinoma organoids, and P5 refers to the fifth generation of salivary gland neuroendocrine carcinoma organoids); Figure 2Schematic diagram of HE staining results of P2 and P5 salivary gland neuroendocrine carcinoma organoids and parental tumors provided for the experimental examples of the present invention (HE, 20×; PDO refers to salivary gland neuroendocrine carcinoma organoids; P2 refers to the second generation of salivary gland neuroendocrine carcinoma organoids; P5 refers to the fifth generation of salivary gland neuroendocrine carcinoma organoids; Figure 2 a: The left picture is a schematic diagram of the HE staining results of P2 salivary gland neuroendocrine carcinoma organoids, and the right picture is a schematic diagram of the HE staining results of the parental tumor; Figure 2 b: From left to right, the schematic diagram of HE staining results of the second-generation salivary gland neuroendocrine carcinoma organoids, the schematic diagram of HE staining results of the fifth-generation salivary gland neuroendocrine carcinoma organoids, and the schematic diagram of HE staining results of the parental tumor sample); Figure 3 Schematic diagram of the IHC staining results of the P2 salivary gland neuroendocrine carcinoma organoids and the parental tumor provided for the experimental examples of the present invention (IHC, 20×; PDO refers to salivary gland neuroendocrine carcinoma organoids; Figure 3 a: Schematic diagram of IHC staining results of the second-generation salivary gland neuroendocrine carcinoma organoids; Figure 3 b: Schematic diagram of IHC staining results of parental tumor samples). DETAILED DESCRIPTION

[0019] After a large number of experiments and in-depth research, the present invention provides an in vitro construction method of salivary gland neuroendocrine carcinoma organoids, extracts human salivary gland neuroendocrine cancer cells, and induces the construction of organoids, observes their morphology under a microscope, and observes and verifies the tissue and cell morphology and protein expression of salivary gland neuroendocrine carcinoma organoids through HE staining and immunohistochemistry, so as to obtain organoids that are highly consistent with the tissue and cell morphology, protein expression, etc. of the patient's tumor sample.

[0020] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0021] The present invention is described in detail below with reference to specific embodiments.

[0022] First embodiment The present embodiment provides a culture medium for culturing salivary gland neuroendocrine carcinoma organoids, the culture medium comprising Advanced DMEM / F12, and the following components based on Advanced DMEM / F12: 1wt.% AntibioticAntimycotic, 1wt.% GlutaMAX, 1wt.% HEPES buffer, and specific added factors; wherein the specific growth factors include fibroblast growth factor FGF 10 100ng / ml, EGF 50ng / ml, bFGF 2nM, Wnt3a recombinant protein 300ng / ml, Noggin recombinant protein 300ng / ml, R-spondin-1 recombinant protein 0.3μg / ml, N-acetylcysteine ​​1.25mM, nicotinamide 10mM / ml, and 1x B27 supplement.

[0023] The culture medium provided in this embodiment can obtain morphologically stable salivary gland neuroendocrine carcinoma organoids, and can successfully construct organoids in batches, which means that subsequent researchers can use them directly without having to conduct time-consuming exploratory experiments, significantly improving research efficiency and saving a lot of valuable time for experimenters.

[0024] Second embodiment This embodiment provides a kit for culturing salivary gland neuroendocrine carcinoma organoids. The kit in this embodiment includes the culture medium of the salivary gland neuroendocrine carcinoma organoids in the first embodiment.

[0025] The kit is based on existing culture medium, which can effectively save enterprises' costs and time in the research and development process; based on the culture medium for culturing salivary neuroendocrine carcinoma organoids provided in the first embodiment, combined with scientific research and development processes, the successful development and market launch of the salivary neuroendocrine carcinoma organoid culture kit is ensured; the development of the kit not only reduces the cost of the enterprise, but also improves the research and development efficiency, providing a powerful tool for the research of salivary neuroendocrine carcinoma.

[0026] Third embodiment The present embodiment provides a salivary gland neuroendocrine carcinoma organoid and a culture method thereof. The present embodiment uses the culture medium provided in the first embodiment to culture salivary gland neuroendocrine carcinoma cells to obtain the salivary gland neuroendocrine carcinoma organoid.

[0027] Sample source: A 76-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 was diagnosed with neuroendocrine carcinoma by postoperative pathology, with the primary lesion located in the left submandibular area. The patient signed an informed consent form.

[0028] (1) The culture method of salivary gland neuroendocrine carcinoma organoids, the specific steps are as follows: The biopsy sample was quickly placed in a centrifuge tube containing tissue preservation solution, the main components of which were DMEM + amphotericin B (500 ng / ml) + 5% penicillin-streptomycin. The centrifuge tube was placed in an ice box and transported to the laboratory.

[0029] Operate in a biosafety cabinet, transfer the tissue to a culture dish, soak the tissue in PBS containing 5% penicillin-streptomycin for about 10 minutes, then transfer to another PBS culture dish also containing 5% penicillin-streptomycin, and remove the capsule tissue in the culture dish; (a) Tissue fragmentation The salivary gland neuroendocrine carcinoma tumor tissue was broken into pieces of about 1 mm 3 The tissue fragments are of different sizes, and the tissue fragments are obtained; (b) Tissue digestion The tissue fragments were transferred to a 50 ml centrifuge tube containing digestive enzymes and digested at 37°C for 1 h. Afterwards, serum-containing DMEM medium was added to terminate the digestion and obtain a suspension of salivary gland neuroendocrine carcinoma; Digestive enzymes include 2mg / ml type II collagenase, 0.1mg / ml DNase I, and 0.5mg / ml hyaluronidase.

[0030] (c) Filtration The salivary gland neuroendocrine carcinoma suspension is filtered using 100 μm, 70 μm, and 40 μm cell filters in sequence, the supernatant is discarded after centrifugation, and the precipitate is resuspended to obtain a single cell suspension of salivary gland neuroendocrine carcinoma.

[0031] After washing the single cell suspension of salivary gland neuroendocrine carcinoma with PBS, count the cells on a counting plate, add an appropriate amount of culture medium according to the counting results to resuspend the cell pellet, mix it with matrix gel, and inoculate it in a 24-well plate. The inoculated 24-well plate was placed upside down in a 37°C incubator for about 40 minutes. After the gel solidified, the 24-well plate was placed upright, and an appropriate amount of culture medium was added to each well. The cells were cultured at 37°C with a CO2 concentration of 5%.

[0032] During the culture period, the medium was changed every 2-3 days.

[0033] (2) Subculture of salivary gland neuroendocrine carcinoma organoids During the culture of salivary gland neuroendocrine carcinoma organoids, observe the number of salivary gland neuroendocrine carcinoma organoids under a microscope as needed. When the number of salivary gland neuroendocrine carcinoma organoids is sufficient, perform subculturing. The subculturing steps are as follows: Discard the original culture medium, melt the matrix gel with pre-cooled PBS, gently blow the mixture of matrix gel and salivary gland neuroendocrine carcinoma organoids and transfer it to a centrifuge tube. After centrifugation, add PBS to wash, remove all the matrix gel, add trypsin to digest for 3 minutes, and add culture medium to terminate the digestion. Count the cells under the microscope, centrifuge and discard the supernatant, resuspend the precipitate with an appropriate amount of culture medium, and mix it with matrix gel (add 40μl matrix gel to each well of the 24-well plate, and about 10 4 After inoculation, the 24-well plate was inverted and placed in a 37°C incubator for about 40 minutes. After the colloid solidified, the 24-well plate was placed upright and an appropriate amount of culture medium was added to each well for culture.

[0034] During the culture period, the culture medium was replaced every 2-3 days.

[0035] (3) Morphological observation of salivary gland neuroendocrine carcinoma organoids At each stage of different growth densities, a stereo microscope was used to observe the gross morphology of the subcultured salivary gland neuroendocrine carcinoma organoids and take photos to record them. The organoid morphology is spherical with continuous edges. After being passed from P1 (first-generation salivary gland neuroendocrine carcinoma organoids) to P2 (second-generation salivary gland neuroendocrine carcinoma organoids) and P5 (fifth-generation salivary gland neuroendocrine carcinoma organoids), the gross morphology remained unchanged, and the number of organoids increased after each passaging, with the spheroids showing a slow enlargement trend. P1, P2, and P5 generations of salivary gland neuroendocrine carcinoma organoids are shown in Figure 2. Figure 1 As shown, the gross morphology remains unchanged, and the number of organoids increases after each passage, and the spheroids show a slow enlargement trend, indicating that the salivary gland neuroendocrine carcinoma organoids obtained by using the culture medium provided in the first embodiment can maintain a stable cell morphology after passage.

[0036] (4) Morphological identification of salivary gland neuroendocrine carcinoma organoids 4.1) HE staining Salivary gland neuroendocrine carcinoma organoids were processed using a modified agar-encapsulation method as follows: Fixation: Carefully pipette salivary gland neuroendocrine carcinoma 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 salivary gland neuroendocrine carcinoma organoid tissue is fixed, use a 3ml pipette to suck out the salivary gland neuroendocrine carcinoma 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 salivary gland neuroendocrine carcinoma organoid tissue until the agar wraps all the salivary gland neuroendocrine carcinoma organoid tissue; wait for the agar to solidify at room temperature, and put it into 75% alcohol, 85% alcohol, 95% alcohol, and 100% alcohol for dehydration together with the mold, each time for 5-10 minutes, to facilitate the agar block to be removed from the plastic embedding mold; Dehydration: Place the agar block containing pre-embedded salivary gland neuroendocrine carcinoma 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 30 min, xylene 30 min, paraffin 62°C 30 min, paraffin 62°C 1 h, paraffin 62°C 1 h; Sectioning: Gently pick up the agar block after treatment with tweezers, embed it in paraffin, and slice it into 3μm sections; Staining: Hematoxylin & Eosin (HE) staining, routine histological observation. Figure 2 As shown, P2 salivary gland neuroendocrine carcinoma organoid cells ( Figure 2 a) Morphology similar to parental tumor of salivary gland neuroendocrine carcinoma ( Figure 2 b) Similar.

[0037] 4.2) Immunohistochemistry (IHC) The second-generation salivary gland neuroendocrine carcinoma organoids embedded in paraffin were sliced ​​into several 3μm continuous slices and baked at 65℃ for 2-4h; after dewaxing and hydration, antigen retrieval was performed in EDTA buffer with a pH of 8.0 for 20min; the slices were blocked with 3% H2O2 solution for 20min to eliminate the activity of endogenous catalase in the tissue. After rinsing with PBS three times, the primary antibody (CHGA, AE1 / AE3, EMA, P63, FLI-1, KI-67, NSE and SYN) was added and incubated at room temperature for 1 hour. After rinsing with PBS three times, the secondary antibody was added and incubated at room temperature for 30min. After rinsing with PBS three times, DAB was used for color development for 5min and hematoxylin was used for counterstaining for 1min, and the slices were sealed with neutral gum. The results are shown in Figure 3 As shown, P2 salivary gland neuroendocrine carcinoma organoids ( Figure 3 a) Protein expression levels and their correlation with parental tumors of salivary gland neuroendocrine carcinoma ( Figure 3 b) Similar.

[0038] The salivary gland neuroendocrine carcinoma organoids were subjected to HE staining and immunohistochemical staining and compared with the salivary gland neuroendocrine carcinoma parent tumor. The results showed that the subcultured salivary gland neuroendocrine carcinoma organoids maintained the histopathological morphological characteristics of their corresponding parent tumors, and the culture medium provided by the present invention is suitable for salivary gland neuroendocrine carcinoma organoids.

[0039] Application Examples This application example provides the salivary gland neuroendocrine carcinoma organoids described in the third embodiment, and their application in studying the pathogenesis of salivary gland neuroendocrine carcinoma, preparing or screening drugs for treating salivary gland neuroendocrine carcinoma; and their application in preparing a drug evaluation or screening model using salivary gland neuroendocrine carcinoma organoids.

[0040] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. A culture medium for culturing salivary gland neuroendocrine carcinoma organoids, characterized in that: The culture medium includes Advanced DMEM / F12, and the following components based on Advanced DMEM / F12: 1wt.% AntibioticAntimycotic, 1wt.% GlutaMAX, 1wt.% HEPES buffer and specific added factors; the specific growth factors include fibroblast growth factor FGF 10 100ng / ml, EGF 50ng / ml, bFGF 2nM, Wnt3a recombinant protein 300ng / ml, Noggin recombinant protein 300ng / ml, R-spondin-1 recombinant protein 0.3μg / ml, N-acetylcysteine ​​1.25mM, nicotinamide 10mM / ml, and 1x B27 supplement.

2. A kit for culturing salivary gland neuroendocrine carcinoma organoids, characterized in that: A culture medium comprising the salivary gland neuroendocrine carcinoma organoid according to claim 1.

3. A method for culturing salivary gland neuroendocrine carcinoma organoids, characterized in that: The salivary gland neuroendocrine carcinoma organoids are cultured using the culture medium for salivary gland neuroendocrine carcinoma organoids according to claim 1 or the kit for salivary gland neuroendocrine carcinoma organoids according to claim 2 to obtain the salivary gland neuroendocrine carcinoma organoids.

4. The method for culturing salivary gland neuroendocrine carcinoma organoids according to claim 3, characterized in that: The specific steps are as follows: (a) Tissue fragmentation The salivary gland neuroendocrine carcinoma tumor tissue is crushed to obtain tissue fragments; (b) Tissue digestion digesting the tissue fragments with digestive enzymes to obtain a suspension of salivary gland neuroendocrine carcinoma; (c) Filtration The salivary gland neuroendocrine carcinoma suspension is filtered, the supernatant is discarded by centrifugation, the precipitate is resuspended to obtain a single cell suspension of salivary gland neuroendocrine carcinoma, and the single cell suspension is inoculated and cultured using the salivary gland neuroendocrine carcinoma organoid culture medium described in claim 1 or the salivary gland neuroendocrine carcinoma organoid kit described in claim 2 to obtain salivary gland neuroendocrine carcinoma organoids.

5. The method for culturing salivary gland neuroendocrine carcinoma organoids according to claim 4, characterized in that: The digestive enzymes include 2 mg / ml type II collagenase, 0.1 mg / ml DNase I, and 0.5 mg / ml hyaluronidase.

6. The method for culturing salivary gland neuroendocrine carcinoma organoids according to claim 4, characterized in that: In step (c), culturing refers to culturing at 37°C and a CO2 concentration of 5%.

7. The method for culturing salivary gland neuroendocrine carcinoma organoids according to claim 4, characterized in that: In step (c), during the culture period, the medium is replaced every 2-3 days.

8. The method for culturing salivary gland neuroendocrine carcinoma organoids according to claim 4, characterized in that: In step (c), the suspension of salivary gland neuroendocrine carcinoma is filtered through 100 μm, 70 μm and 40 μm cell filters in sequence.

9. A salivary gland neuroendocrine carcinoma organoid, characterized in that: The salivary gland neuroendocrine carcinoma organoid is obtained by culturing the salivary gland neuroendocrine carcinoma organoid according to any one of claims 3 to 8.

10. The salivary gland neuroendocrine carcinoma organoid according to claim 9, characterized in that The organoid has at least the following applications; 1) Study the pathogenesis of salivary gland neuroendocrine carcinoma and prepare or screen drugs for the treatment of salivary gland neuroendocrine carcinoma; 2) Use salivary gland neuroendocrine carcinoma organoids to prepare drug evaluation or screening models.

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