Method for constructing salivary gland mucus epidermoid carcinoma organoid

Abstract

The invention relates to the technical field of organoid, and particularly discloses a method for constructing a salivary gland mucus epidermoid carcinoma organoid. According to the method provided by the invention, the salivary gland mucoepidermoid cancer cells are cultured by adopting a specific culture medium with specific components, so that the high-quality organ of which the tissue cell morphology and the protein expression are highly consistent with those of the real salivary gland mucoepidermoid cancer cells is efficiently constructed; the problem that a salivary gland mucoepidermoid carcinoma in-vitro drug screening model capable of simulating in-vivo conditions of tumors is lacked is effectively solved, and an in-vitro model with high quality, high stability and high simulation degree is provided for scientific research and production work.

Description

Technical Field

[0001] The present invention relates to the technical field of organoids, and in particular to a method for constructing salivary gland mucoepidermoid carcinoma organoids. Background Art

[0002] Mucoepidermoid carcinoma (MEC) of salivary glands is one of the most common malignant tumors of salivary gland epithelial origin. According to the relative proportion of the three main cell types constituting the tumor, the degree of differentiation of tumor cells and the structure of the tumor, this tumor can be divided into three types: highly differentiated, poorly differentiated and moderately differentiated. Highly differentiated ones often present with symptoms such as dysphagia and paresthesia, and rarely metastasize to cervical lymph nodes and bloodstream, so the prognosis is good, with a high five-year survival rate of 90%-97%. Poorly differentiated ones, due to the characteristics of rapid tumor growth, invasion of surrounding tissues, high lymph node metastasis rate and bloodstream metastasis rate, are often accompanied by pain and facial paralysis, and the prognosis is poor, with a five-year survival rate of only 22.5%-37.5% and a ten-year survival rate of 0%. At present, the treatment of salivary mucoepidermoid carcinoma is still mainly based on traditional surgical treatment and postoperative radiotherapy, and there is no effective chemotherapy drug.

[0003] One of the main factors restricting the development and screening of chemotherapy drugs for salivary mucoepidermoid carcinoma is the lack of an in vitro screening model that can simulate the actual situation of tumors in vivo. At present, most studies on mucoepidermoid carcinoma are based on two-dimensional planar culture systems, and there is no application of 3D organoid culture technology. Tumor cells cultured in two dimensions cannot reflect the tissue morphology and cell function of real tumors, cannot reproduce the internal environment of tumors, and lose tumor heterogeneity, resulting in slow research progress on this type of cancer and difficulties in drug screening. Therefore, in order to further study the development mechanism and characteristics of salivary mucoepidermoid carcinoma and develop and screen targeted drugs, it is urgent to establish an in vitro model that can better simulate the environment and characteristics of tumors in vivo. Summary of the invention

[0004] The purpose of the present invention is to overcome the problems of the prior art such as the lack of an in vitro organoid culture system and model for salivary mucoepidermoid carcinoma, and to provide a method for constructing salivary mucoepidermoid carcinoma organoids. The method is stable, economical and practical, simple to operate, and can efficiently obtain high-quality salivary mucoepidermoid carcinoma organoids.

[0005] In order to achieve the above object, the present invention provides a method for constructing salivary gland mucoepidermoid carcinoma organoids, characterized in that the method comprises culturing salivary gland mucoepidermoid carcinoma cells using a salivary gland mucoepidermoid carcinoma organoid-specific culture medium;

[0006] The salivary gland mucoepidermoid carcinoma organoid-specific culture medium comprises a basal culture medium and supplementary components, wherein the basal culture medium is selected from Advanced DMEM / F12 culture medium, and the supplementary components are selected from L-glutamine supplement, 4-hydroxyethylpiperazineethanesulfonic acid, fibroblast growth factor, epidermal cell growth factor, WNT protein, R-spondin and B-27 additive.

[0007] The second aspect of the present invention provides a salivary gland mucoepidermoid carcinoma organoid obtained according to the method described in the first aspect.

[0008] Through the above technical solution, the present invention can at least achieve the following beneficial effects:

[0009] (1) In the method provided by the present invention, on the basis of fully screening and streamlining the additive components of the conditioned medium, by adopting a specific culture medium with a specific formula, the construction efficiency and success rate of salivary gland mucoepidermoid carcinoma organoids are improved, while the obtained organoids can better express their inherent activity characteristics, thereby achieving comprehensive characteristics that are highly similar to real salivary gland mucoepidermoid carcinoma tumors.

[0010] (2) The present invention uses a digestive solution with a specific formula to digest the salivary gland mucoepidermoid carcinoma tissue, thereby efficiently extracting salivary gland mucoepidermoid carcinoma cells and improving the cell yield and viability.

[0011] (3) The method provided by the present invention is simple, easy to use, economical and practical. In addition, the tissue cell morphology and protein expression of the salivary gland mucoepidermoid carcinoma organoids constructed by this method are highly consistent with those of real salivary gland mucoepidermoid carcinoma, providing a high-quality and high-fidelity in vitro model of salivary gland mucoepidermoid carcinoma for scientific research and production. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a diagram of the morphological changes of organoids during the process of constructing salivary gland mucoepidermoid carcinoma organoids using basal culture medium in Example 1.

[0013] Figure 2 This is a diagram of the morphological changes of organoids during the process of constructing salivary gland mucoepidermoid carcinoma organoids using specific culture medium in Example 1.

[0014] FIG. 3A to FIG. 3D This is a comparison of the immunofluorescence staining results of MEC tissue and organoid in Example 1 (the scale bars in the figures are all 100 μm).

[0015] Figure 4 This is a diagram showing the morphological changes of the passaged organoid P1 during the culture process in Example 1.

[0016] Figure 5This is a diagram showing the morphological changes of the passaged organoid P2 during the culture process in Example 1. DETAILED DESCRIPTION

[0017] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0018] In the present invention, "MEC" is an abbreviation of "salivary gland mucoepidermoid carcinoma", which have the same meaning and can be used interchangeably.

[0019] In the previous study, the inventors of the present invention found that it was difficult to successfully or stably culture salivary gland mucoepidermoid carcinoma organoids using only traditional two-dimensional culture medium or other salivary gland tumor conditioned medium. Therefore, based on the basal culture medium, the inventors increased or decreased different tumor growth and maintenance related additives according to the characteristics of the mucoepidermoid carcinoma tumor microenvironment, and established a relatively stable, practical and economical salivary gland adenoid cystic carcinoma organoid culture system. After a lot of research, the inventors cleverly found that by using a culture medium with specific ingredients to culture cells extracted from salivary gland mucoepidermoid carcinoma tissue, organoids with cell composition and characteristics highly similar to those of salivary gland mucoepidermoid carcinoma tissue in vivo can be obtained, thereby effectively solving the problem of the lack of in vitro organoid models of salivary gland mucoepidermoid carcinoma, and providing a powerful tool for the research of this type of cancer and related drug development.

[0020] Based on the above findings, the present invention provides a method for constructing salivary gland mucoepidermoid carcinoma organoids, the method comprising culturing salivary gland mucoepidermoid carcinoma cells using a salivary gland mucoepidermoid carcinoma organoid-specific culture medium;

[0021] The salivary gland mucoepidermoid carcinoma organoid-specific culture medium comprises a basal culture medium and supplementary components, wherein the basal culture medium is selected from Advanced DMEM / F12 culture medium, and the supplementary components are selected from L-glutamine supplement, 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), fibroblast growth factor (FGF-10), epidermal growth factor (EGF), Wnt protein, R-spondin (RSPO) and B-27 additive.

[0022] In the present invention, L-glutamine supplement refers to a substance that can provide L-glutamine for the salivary gland mucoepidermoid carcinoma organoid-specific culture medium used in the present invention. Any substance that can be used for organoid construction and can achieve this purpose can be applicable to the present invention. According to a preferred embodiment of the present invention, the L-glutamine supplement is selected from at least one of L-glutamine, L-alanyl-L-glutamine and oligopeptides formed therefrom. The present invention does not particularly limit the specific source of the selected L-glutamine supplement, which can be a related product prepared by itself containing the aforementioned ingredients, or a related product directly obtained through commercial purchase. For example, GlutaMAX produced by ThermoFisher can be used TM The L-glutamine supplement of the present invention has as its main components L-glutamine and L-alanyl-L-glutamine stable dipeptide.

[0023] Wnt protein is a highly conserved secretory glycoprotein family and an important protein in the Wnt signaling pathway. It can be added to the culture medium as a cytokine for organoid construction. The present invention has no particular limitation on the Wnt protein used. Any Wnt protein that can be used for the construction of salivary gland mucoepidermoid carcinoma organoids can be applied to the present invention, preferably Wnt-3a and the like.

[0024] R-spondin (RSPO) is a newly discovered secretory protein family in recent years. Currently, four members have been discovered (named RSPO-1, RSPO-2, RSPO-3, and RSPO-4, respectively). It binds to LGR4 (Leucine-rich repeat-containing Gprotein-coupled receptor 4, also known as GPR48) in the G protein-coupled receptor family and can activate the Wnt / β-catenin signaling pathway. The present invention has no particular restrictions on the R-spondin used, and any R-spondin that can be used for the construction of salivary gland mucoepidermoid carcinoma organoids can be applied to the present invention, preferably RSPO-1.

[0025] B-27 supplement is a serum-free supplement used in culture medium, which can provide a variety of nutrients and cytokines to the culture medium and can usually be obtained through commercial channels. The purchased B-27 supplement is usually provided in the form of a concentrated solution (e.g., 100× or 50×), which can be diluted with culture medium or PBS before use, or can be directly added to the culture medium at the desired concentration.

[0026] In order to further improve the construction efficiency of organoids and the quality level of the obtained organoids, according to some preferred embodiments of the present invention, the supplementary ingredients may also include at least one of Noggin, N-acetylcysteine ​​(NAC), nicotinamide, dexamethasone and A83-01.

[0027] A83-01 is an ALK5 inhibitor with CAS number 909910-43-6 and a structural formula as shown in the following formula (I).

[0028]

[0029] Preferably, the supplementary ingredients may also contain at least one of a TGF-β type I receptor inhibitor, Y-27632 (Rock inhibitor), and an N-2 additive.

[0030] According to a preferred embodiment of the present invention, the supplementary ingredients may also include antibiotics. The addition of antibiotics is to avoid microbial contamination during organoid construction and culture, which affects the success rate of organoid construction. The present invention has no particular restrictions on the selected antibiotics, as long as they can be used for salivary gland mucoepidermoid carcinoma organoid construction. Preferably, the antibiotics are selected from penicillin and / or streptomycin.

[0031] After repeated experiments, the inventors of the present invention found that when the aforementioned supplementary components are added to the basal culture medium in specific amounts, the construction efficiency of salivary gland mucoepidermoid carcinoma organoids and the quality of the obtained organoids can be further improved, thereby obtaining an in vitro organoid model that is more similar to the actual situation of the tumor in vivo.

[0032] According to a preferred embodiment of the present invention, the amount of the supplementary components is such that in the salivary gland mucoepidermoid carcinoma organoid-specific culture medium:

[0033] The final concentration of L-glutamine provided by the L-glutamine supplement is not less than 0.5 mM, preferably 0.5-3 mM;

[0034] The final concentration of 4-hydroxyethylpiperazineethanesulfonic acid is 5-15 mM;

[0035] The final concentration of fibroblast growth factor was 50-150 ng / mL;

[0036] The final concentration of epidermal growth factor was 10-100 ng / mL;

[0037] The final concentration of WNT protein was 300-800 ng / mL;

[0038] The final concentration of R-spondin was 50-150 ng / mL;

[0039] The final concentration of B-27 Supplement is 0.5×-3×.

[0040] Preferably, the amount of the supplementary components can also be such that in the salivary gland mucoepidermoid carcinoma organoid-specific culture medium:

[0041] The final concentration of noggin was 50-150 ng / mL;

[0042] The final concentration of N-acetylcysteine ​​was 0.1-2 mM;

[0043] The final concentration of nicotinamide was 5-15 mM;

[0044] The final concentration of dexamethasone was 0.1-2 μM;

[0045] The final concentration of A83-01 was 0.01-0.3 μM.

[0046] Preferably, the amount of antibiotics used is such that in the salivary gland mucoepidermoid carcinoma organoid-specific culture medium: the final concentration of penicillin is 50-200 U / mL, and / or the final concentration of streptomycin is 10-500 mg / mL.

[0047] More preferably, the amount of the supplement is such that the salivary gland mucoepidermoid carcinoma organoid-specific culture medium contains:

[0048] L-glutamine supplements provide L-glutamine at a final concentration of 1-3 mM;

[0049] The final concentration of 4-hydroxyethylpiperazineethanesulfonic acid is 8-12 mM;

[0050] The final concentration of fibroblast growth factor was 80-120 ng / mL;

[0051] The final concentration of epidermal growth factor was 40-60 ng / mL;

[0052] The final concentration of WNT protein was 400-600 ng / mL;

[0053] The final concentration of R-spondin was 80-120 ng / mL;

[0054] The final concentration of B-27 Supplement is 0.5×-1.5×.

[0055] Preferably, the amount of the supplementary components can also be such that in the salivary gland mucoepidermoid carcinoma organoid-specific culture medium:

[0056] The final concentration of noggin was 80-120 ng / mL;

[0057] The final concentration of N-acetylcysteine ​​is 0.5-1.5 mM;

[0058] The final concentration of nicotinamide was 8-12 mM;

[0059] The final concentration of dexamethasone was 0.5-1.5 μM;

[0060] The final concentration of A83-01 was 0.05-0.15 μM.

[0061] Preferably, the dosage of antibiotics is such that the final concentration of penicillin is 80-120 U / mL, and / or the final concentration of streptomycin is 50-150 μg / mL.

[0062] In the present invention, there is no particular limitation on the source of the salivary gland mucoepidermoid carcinoma cells used. In order to improve the efficiency of organoid construction, the present invention preferably extracts cells from relevant tissue samples for use before constructing salivary gland mucoepidermoid carcinoma organoids.

[0063] According to a preferred embodiment of the present invention, the method further comprises the step of digesting the salivary gland mucoepidermoid carcinoma tissue sample to obtain salivary gland mucoepidermoid carcinoma cells.

[0064] Preferably, the digestion treatment comprises digesting the salivary gland mucoepidermoid carcinoma tissue sample with a digestive solution.

[0065] The inventors of the present invention cleverly discovered in their research that a digestive solution prepared with specific digestive enzymes at specific concentrations can more efficiently digest salivary gland mucoepidermoid carcinoma tissue, increase cell yield and viability, and thereby obtain cells suitable for organoid construction.

[0066] According to a preferred embodiment of the present invention, the digestive fluid comprises hyaluronidase and collagenase, preferably hyaluronidase and collagenase II.

[0067] More preferably, the digestive solution comprises 5-15 mg / mL hyaluronidase and 5-15 mg / mL collagenase II. Preferably, in the digestive solution, the concentration ratio of hyaluronidase to collagenase II is 1:0.8-1.2.

[0068] In the present invention, there is no particular requirement for the reagents used to prepare the digestion solution. Generally, the digestion solution can be obtained by adding a specific concentration of digestive enzymes to a buffer suitable for tissue digestion and mixing. For example, the digestion solution can be prepared using a buffer such as PBS or a DMEM / F12 culture medium according to the concentrations of the hyaluronidase and collagenase II described above.

[0069] In the present invention, there is no particular limitation on the specific digestion method and conditions, as long as the cells can be efficiently extracted while ensuring that the cell yield and viability can reach a relatively satisfactory level.

[0070] According to a preferred embodiment of the present invention, the digestion treatment conditions include: a digestion temperature of 35-38°C and a digestion time of 30-90 min (preferably 50-70 min). In order to better disperse the cells and make the tissue blocks more fully contact with the digestion solution, the digestion is preferably carried out under an oscillation condition of 100-500 rpm. In order to avoid incomplete digestion or over-digestion resulting in insufficient number or quality of extracted cells, it is preferred to observe the digestion of the tissue every 10-15 min during the digestion process (for example, direct observation with the naked eye or observation under a microscope), and the digestion can be stopped when the tissue blocks are significantly reduced or even disappear.

[0071] In order to improve the digestion efficiency and avoid the problems of insufficient cell viability caused by too long digestion time of tissue blocks, preferably, the salivary gland mucoepidermoid carcinoma tissue sample is crushed into a volume not exceeding 3 mm 3 , preferably 0.5-1.5mm 3 The present invention has no particular limitation on the specific pulverization method, and any commonly used method in the art can be used, for example, a sterile scalpel or surgical scissors can be used to cut the tissue sample into the above size.

[0072] According to a preferred embodiment of the present invention, the method further comprises centrifuging and resuspending after terminating the digestion, and sieving the resuspension with a cell sieve (preferably with a pore size of 150 μm) to remove incompletely digested tissue and obtain a salivary gland mucoepidermoid carcinoma cell suspension.

[0073] In the present invention, there is no particular limitation on the specific method of constructing organoids using salivary gland mucoepidermoid carcinoma cells, and conventional organoid construction methods in the art can be used.

[0074] According to a preferred embodiment of the present invention, the method of culturing salivary gland mucoepidermoid cancer cells using a salivary gland mucoepidermoid cancer organoid-specific culture medium comprises: mixing the cultured salivary gland mucoepidermoid cancer cells with matrix gel to a concentration of 2×10 5 -5×10 5 The cells were suspended in matrix gel at a concentration of 100 cells / mL and inoculated into a culture container and cultured at 35-38°C for 10-25 days.

[0075] In the method provided by the present invention, there is no particular limitation on the source of the matrix gel, as long as it can meet the needs of constructing salivary gland mucoepidermoid carcinoma organoids. For example, a matrix gel prepared by oneself according to the prior art can be selected, or a commercially available matrix gel (such as the matrix gel produced by Corning) conventionally selected for organoid construction in the art can be directly used. Matrigel, etc.).

[0076] Preferably, before mixing the cultured salivary gland mucoepidermoid carcinoma cells with the matrigel, the cell suspension is centrifuged and the supernatant is discarded. The cell precipitate is then mixed with the matrigel to form a matrigel cell suspension. In order to prevent the matrigel from solidifying, the cell precipitate is preferably precooled on ice before mixing with the matrigel, and the mixing operation is completed on ice.

[0077] Preferably, the inoculation amount of the matrix gel cell suspension does not exceed 15% of the volume of the inoculation container, preferably 5-10%. For example, when a 96-well cell culture plate is used for organoid construction, the inoculation amount of the matrix gel cell suspension can be 20-50 μL / well, preferably 25-35 μL / well. In the present invention, the matrix gel cell suspension can be inoculated in the culture container and then heated to solidify the matrix gel, and then the aforementioned specific culture medium is added thereto and placed in an incubator for culture. Preferably, the amount of culture medium added does not exceed 70% of the volume of the culture container. Preferably, 40-60%. For example, when a 96-well cell culture plate is used for organoid construction, the amount of culture medium added can be 150-250 μL / well.

[0078] According to a preferred embodiment of the present invention, the method further comprises the step of replacing the culture medium. In order to avoid the death or quality decline of organoids caused by the exhaustion of nutrients in the culture medium, the accumulation of excessive metabolic waste of cells due to the failure to replace the culture medium for a long time, or the waste and increased culture cost caused by replacing the culture medium too frequently, the culture medium can be replaced when a significant change in the color of the culture medium (usually a lighter or yellower color) is observed. Preferably, the culture medium is replaced every 4-5 days.

[0079] According to a preferred embodiment of the present invention, the method further comprises the step of passaging the salivary gland mucoepidermoid carcinoma organoids obtained by culture.

[0080] Preferably, individual salivary gland mucoepidermoid carcinoma organoids are passaged when their diameter exceeds 500 μm, preferably (most of the organoids in the same culture vessel) reaches 500-800 μm.

[0081] The present invention has no particular limitation on the specific method of passaging, and the conventional operation method for passaging organoids in the art can be used. For example, the matrix gel solidified in the culture container can be taken out and melted, the organoid cell clusters can be dispersed, and then the supernatant can be discarded by centrifugation to obtain the organoid precipitate, and then the organoid precipitate can be resuspended with matrix gel, inoculated in the culture container, and the culture medium can be added.

[0082] The second aspect of the present invention provides a salivary gland mucoepidermoid carcinoma organoid obtained according to the method described in the first aspect.

[0083] The salivary gland mucoepidermoid carcinoma organoids obtained by the method provided by the present invention contain multiple types of cells, are closer to the composition of real tumor cells, and can well simulate the inherent activity characteristics of this cancer in vivo in vitro. The tissue cell morphology and protein expression are highly consistent with those of real salivary gland mucoepidermoid carcinoma, thereby highly similarly simulating the in vivo situation of the tumor.

[0084] According to a preferred embodiment of the present invention, the salivary gland mucoepidermoid carcinoma organoid contains at least one of epithelial cells, tumor stromal cells, neural cells, and immune cells.

[0085] The present invention will be described in detail below by way of examples. It should be understood that the following examples are only used to further explain and illustrate the content of the present invention by way of example, and are not intended to limit the present invention.

[0086] In the following examples, penicillin-streptomycin dual antibody (100× concentrated solution, in which the concentrations of penicillin and streptomycin are 10000 U / ml and 10000 μg / ml, respectively) was purchased from ThermoFisher, brand 10378016. (100× concentrated solution) was used as an L-glutamine supplement, and its L-glutamine provided at a concentration of 1× was approximately 2 mM. Matrigel was purchased from Corning Matrigel. Advanced DMEM / F12 medium was purchased from ThermoFisher, brand A4192001, B-27 supplement (50× concentrate) was purchased from ThermoFisher, brand A1486701, and A83-01 was purchased from MERCK, brand 909910-43-6. Protein components such as EGF, FGF-10, Wnt-3a, and RSPO-1 are all recombinant human proteins purchased from Nearshore Protein.

[0087] Preparation Example 1

[0088] (1) Preparation of culture medium

[0089] Weigh or measure the raw materials according to the formula in Table 1, add the supplementary components to the Advanced DMEM / F12 medium and mix well to obtain a specific medium for constructing salivary gland mucoepidermoid carcinoma organoids.

[0090] Table 1

[0091] Element Final concentration* Penicillin-Streptomycin 1 volume% HEPES 10mM <![CDATA[GlutaMAX TM ]]> 1 volume% B-27 Additive 2% by volume EGF 50ng / mL FGF-10 100ng / mL Wnt-3a 500ng / mL RSPO-1 100ng / mL

[0092] *In Table 1, volume % refers to the volume percentage of the component in Advanced DMEM / F12 medium when preparing the specific medium. For example, 1 volume % means adding 1 mL of the component to 100 mL of Advanced DMEM / F12 medium.

[0093] (2) Preparation of digestive juice

[0094] According to the formula in Table 2, collagenase II and hyaluronidase were diluted and prepared with PBS to obtain a digestion solution for digesting salivary gland mucoepidermoid carcinoma tissue samples.

[0095] Table 2

[0096] Element Final concentration Collagenase II 10mg / mL Hyaluronidase 10mg / mL

[0097] Example 1

[0098] This example is used to illustrate the effect of constructing salivary gland mucoepidermoid carcinoma organoids.

[0099] The salivary gland mucoepidermoid carcinoma tissue samples used in this example were samples obtained through surgery or biopsy, and informed consent was obtained from the relevant personnel.

[0100] (1) Tissue sample acquisition and digestion

[0101] A batch of salivary gland mucoepidermoid carcinoma tissue samples were obtained through surgery or biopsy, with an average size of 2 cm × 2 cm × 2 cm. Samples from different patients were numbered, and after removing the interstitial tissue as much as possible, they were cut into 1 mm pieces using sterile scissors. 3 Small pieces around.

[0102] Mix the sheared tissue blocks with digestion solution (see Table 2 for details), with the volume ratio of tissue blocks to digestion solution being approximately 1:10. Place the centrifuge tube containing the mixture of tissue blocks and digestion solution in a 37°C metal bath, set the speed to 300 rpm, and digest for 60 minutes. During the digestion period, observe the digestion every 10 minutes. When the tissue blocks are significantly reduced or disappear, add an appropriate amount of Advanced DMEM / F12 medium to terminate the digestion.

[0103] After terminating the digestion, the cell was filtered through a cell strainer (pore size 150 μm), and the filtrate was centrifuged at 350G for 5 min at 4°C, the supernatant was removed, and the cell pellet was resuspended in 1 mL of Advanced DMEM / F12 medium to obtain a cell suspension, which was then counted and the cell viability was calculated.

[0104] (2) Organoid Construction

[0105] The cell suspension was centrifuged at 350G for 5 min at 4°C, the supernatant was removed, and the cell pellet was precooled on ice. According to the cell counting results, an appropriate amount of melted matrix gel was added to obtain a cell concentration of approximately 3.3×10 5 / mL of matrix gel cell suspension.

[0106] The matrix gel cell suspension was inoculated in a 96-well low-adhesion cell culture plate at a volume of 30 μL / well (i.e., approximately 1×10 4 cells), and then 200 μL of the specific culture medium prepared in Preparation Example 1 was added to each well.

[0107] The culture plate was placed in a humidified incubator at 37°C and 5% CO2 for culture. The culture conditions and organoid status were observed and recorded every day, and the culture medium was replaced when it turned yellow (approximately once every 4-5 days).

[0108] The salivary gland mucoepidermoid carcinoma organoids were constructed according to the above method using the basic culture medium (DMEM / F12 culture medium, only antibiotics and double antibodies were added, without other supplementary components) as a control.

[0109] Figure 1 and Figure 2 Figure 2 shows the morphological changes of MEC organoids observed under an inverted microscope (4×) during culture. Figure 1 This is the case when the basal medium is used. Figure 2 This is the situation when using specific culture medium for construction. As can be seen from the figure, the MEC cell spheres cultured in the basic culture medium DMEM / F12 have poor morphology, small diameter, irregular edges, and scattered cells, indicating that the construction speed of the organoids is slow and the quality of the obtained MEC organoids is poor; when using specific culture medium, the formation of primary salivary gland mucoepidermoid carcinoma organoids can be observed at about 7 days of culture, the organoids grow fast, and the obtained MEC organoids have smooth edges and are surrounded by stromal cells. After 14-21 days of culture, the diameter of the organoids reaches 500μm and can be passaged.

[0110] Immunofluorescence staining was performed on MEC tissues and primary MEC organoids cultured for 21 days (denoted as P0), and the staining of tissues and organoids was observed using a fluorescence microscope (20×). The results are shown in FIG. 3A to FIG. 3D . FIG. 3A to FIG. 3D In the figure, “MERGE” is a fusion image of the staining results of three channels: red (Cy3.5 staining), green (FITC staining), and blue (DAPI staining). DAPI is used to stain the cell nucleus and represents the cell position. Figure 3A In, EPCAM is an epithelial cell adhesion molecule (epithelial cell marker), and αSMA is a smooth muscle actin (myofibroblast marker); Figure 3BIn, TUBB3 is tubulin β3 (neural marker), VIMENTIN is vimentin (mesenchymal cell marker); Figure 3C In, PANCK is a pan-spectrum keratin (tumor marker), and CD45 is a leukocyte common antigen (immune marker); Figure 3D In the study, KI67 is a nuclear protein (cell proliferation marker) and CD44 is a cell surface molecule (tumor stemness marker).

[0111] Depend on FIG. 3A to FIG. 3D Comparison of the staining results of tissues and organoids shows that the primary salivary gland mucoepidermoid carcinoma organoids obtained by the above method are highly similar to the cell composition of MEC tissues and can express specific cell markers in cancer tissues, indicating that the organoids can be used as an in vitro model that highly simulates the in vivo conditions of tumors.

[0112] (3) Organoid passaging

[0113] After discarding the culture medium, use Corning Cell Recovery Solution (about 150μL / well) to melt the matrix gel, gently blow down the mixture of matrix gel and organoids and transfer it to a centrifuge tube, and melt it at 4℃ for 1h. Use a pipette to blow the melted matrix gel and organoid mixture 10 times, and centrifuge it at 350G for 5min at 4℃. Discard the supernatant, add AdvancedDMEM / F12 to resuspend the precipitate, and wash the matrix gel attached to the organoid. To avoid the coagulation of the matrix gel, this operation is performed on ice. Centrifuge again at 350G for 5min at 4℃, discard the supernatant, and resuspend the cells with matrix gel. The amount of matrix gel used makes the subculture ratio about 1:3-4 (that is, the organoids in one culture well can be inoculated into 3-4 culture wells after subculture). Use a 1 mL pipette tip with a 10 μL pipette tip to blow the matrix gel cell suspension 20-25 times to disperse the organoid cell clusters, then inoculate the matrix gel cell suspension into a new 96-well low adhesion cell culture plate in the manner of step (2), and use the specific culture medium obtained in Preparation Example 1 for organoid subculture.

[0114] An inverted microscope (4×) was used to observe the morphological changes of the passaged organoids in the culture wells. The results are detailed in Figure 4 (first passage, denoted as P1) and Figure 5 (The second passage is recorded as P2). As can be seen from the figure, the MEC organoids after passage grow rapidly and have clear boundaries. As the culture time increases, the volume of the passaged organoids gradually increases. Immunofluorescence staining was used to further detect the passaged organoids of P1 and P2, and it was found that their cell composition and expression of cell markers were similar to those of P0, indicating that the characteristics of the MEC organoids constructed by the method provided by the present invention are stable and can be used as a stable in vitro model for drug screening and cancer mechanism research.

[0115] Example 2

[0116] This example is used to illustrate the effects of culture medium components on the construction of salivary gland mucoepidermoid carcinoma organoids.

[0117] (1) The method of Example 1 was followed, except that the specific culture medium used also contained 100 ng / mL Noggin.

[0118] The results showed that compared with the specific culture medium used in Example 1, the growth of organoids was slightly accelerated, the edges of the obtained MEC organoids were clearer, and the diameter was not significantly different from that in Example 1.

[0119] (2) The method of Example 1 was followed, except that NAC at a final concentration of 1 mM was further added to the specific culture medium.

[0120] The results showed that compared with Example 1, there was no significant difference in the growth rate, morphology and diameter of the organoids.

[0121] (3) The method of Example 1 was followed, except that nicotinamide was further added to the culture medium at a final concentration of 10 mM.

[0122] The results showed that compared with Example 1, there was no significant difference in the growth rate, morphology and diameter of the organoids.

[0123] (4) The method of Example 1 was followed, except that dexamethasone was further added to the culture medium at a final concentration of 1 μM.

[0124] The results showed that compared with Example 1, there was no significant difference in the growth rate, morphology and diameter of the organoids.

[0125] (5) The method of Example 1 was followed, except that A83-01 at a final concentration of 100 nM was further added to the culture medium.

[0126] The results showed that compared with Example 1, there was no significant difference in the growth rate, morphology and diameter of the organoids.

[0127] Example 3

[0128] This example is used to illustrate the effect of the treatment method in the digestion step on the construction of salivary gland mucoepidermoid carcinoma organoids.

[0129] (1) The method of Example 1 was followed, except that the digestion solution used contained only collagenase II (20 mg / mL).

[0130] The results showed that the tissue digestion speed slowed down and the number of cells obtained after digestion decreased. The obtained cells were used to construct MEC organoids according to the method in Example 1 (using specific culture medium), and it was found that the number of cultured cell spheres decreased and the diameter was reduced compared with Example 1.

[0131] (2) The method of Example 1 was followed, except that the digestive fluid used contained only hyaluronidase (20 mg / mL).

[0132] The results showed that the tissue digestion speed slowed down and the number of cells obtained after digestion decreased. The obtained cells were used to construct MEC organoids according to the method in Example 1 (using specific culture medium), and it was found that the number of cultured cell spheres decreased and the diameter was reduced compared with Example 1.

[0133] (3) The method of Example 1 was followed, except that the content of hyaluronidase in the digestive solution was adjusted to 5 mg / mL, and the content of collagenase II was adjusted to 15 mg / mL.

[0134] The results showed that under the same total volume of digestion solution, the number of cells obtained after digestion decreased. The obtained cells were used to construct MEC organoids according to the method in Example 1 (using specific culture medium), and it was found that the number of cultured cell spheres decreased compared with Example 1.

[0135] (4) The method of Example 1 was followed, except that the digestion solution was replaced with trypsin (prepared in PBS, with a concentration of about 20 mg / mL).

[0136] The results showed that the tissue digestion speed was accelerated, the cell viability and the number of live cells decreased after digestion. The obtained cells were used to construct MEC organoids according to the method in Example 1 (using a specific culture medium), and it was found that the number of cultured cell spheres was reduced compared with Example 1.

[0137] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A method for constructing salivary gland mucoepidermoid carcinoma organoids, characterized in that: The method comprises culturing salivary gland mucoepidermoid cancer cells using a salivary gland mucoepidermoid cancer organoid-specific culture medium; The salivary gland mucoepidermoid carcinoma organoid-specific culture medium comprises a basal culture medium and supplementary components, wherein the basal culture medium is selected from Advanced DMEM / F12 culture medium, and the supplementary components are selected from L-glutamine supplement, 4-hydroxyethylpiperazineethanesulfonic acid, fibroblast growth factor, epidermal cell growth factor, Wnt protein, R-spondin and B-27 additive.

2. The method according to claim 1, wherein: The supplement ingredients also include at least one of noggin, N-acetylcysteine, nicotinamide, dexamethasone, and A83-01; Preferably, the supplementary ingredients further comprise an antibiotic, preferably the antibiotic is selected from penicillin and / or streptomycin.

3. The method according to claim 1 or 2, wherein: The amounts of the supplements are such that the salivary gland mucoepidermoid carcinoma organoid-specific culture medium contains: The final concentration of L-glutamine provided by the L-glutamine supplement in the specific culture medium is not less than 0.5 mM, preferably 0.5-3 mM; The final concentration of 4-hydroxyethylpiperazineethanesulfonic acid is 5-15 mM; The final concentration of fibroblast growth factor was 50-150 ng / mL; The final concentration of epidermal growth factor was 10-100 ng / mL; The final concentration of WNT protein was 300-800 ng / mL; The final concentration of R-spondin was 50-150 ng / mL; The final concentration of B-27 additive was 0.5×-3×; Preferably, the amount of the supplementary components is such that the salivary gland mucoepidermoid carcinoma organoid-specific culture medium contains: The final concentration of noggin was 50-150 ng / mL; The final concentration of N-acetylcysteine ​​was 0.1-2 mM; The final concentration of nicotinamide was 5-15 mM; The final concentration of dexamethasone was 0.1-2 μM; The final concentration of A83-01 was 0.01-0.3 μM; Preferably, the dosage of the antibiotics is such that the final concentration of penicillin is 50-200 U / mL, and / or the final concentration of streptomycin is 10-500 μg / mL.

4. The method according to claim 1, wherein: The method further comprises the step of digesting the salivary gland mucoepidermoid carcinoma tissue sample to obtain salivary gland mucoepidermoid carcinoma cells.

5. The method according to claim 4, wherein: The digestion treatment comprises digesting the salivary gland mucoepidermoid carcinoma tissue sample with a digestive solution; Preferably, the digestive fluid comprises hyaluronidase and collagenase, preferably hyaluronidase and collagenase II; More preferably, the digestion solution comprises 5-15 mg / mL hyaluronidase and 5-15 mg / mL collagenase II.

6. The method according to claim 5, wherein: The digestion treatment conditions include: digestion temperature 35-38°C, digestion time 30-90min, preferably digestion under shaking conditions of 100-500rpm; Preferably, the salivary gland mucoepidermoid carcinoma tissue sample is crushed into a volume not exceeding 3 mm 3 The tissue pieces were then digested.

7. The method according to claim 1, wherein: The method of culturing salivary gland mucoepidermoid cancer cells using salivary gland mucoepidermoid cancer organoid-specific medium comprises: mixing the cultured salivary gland mucoepidermoid cancer cells with matrix gel to prepare a concentration of 2×10 5 -5×10 5 / mL of matrix gel cell suspension, and inoculate it into a culture container, and culture it at 35-38°C for 10-25 days; Preferably, the inoculation amount of the matrix gel cell suspension does not exceed 15% of the volume of the inoculation container, preferably 5-10%.

8. The method according to claim 1, wherein: The method further comprises the step of passaging the salivary gland mucoepidermoid carcinoma organoid obtained by culture; Preferably, individual salivary gland mucoepidermoid carcinoma organoids are passaged when their diameter exceeds 500 μm, preferably reaches 500-800 μm.

9. Salivary gland mucoepidermoid carcinoma organoid obtained according to the method of any one of claims 1 to 8.

10. The salivary gland mucoepidermoid carcinoma organoid according to claim 9, wherein: The salivary gland mucoepidermoid carcinoma organoid contains at least one of epithelial cells, tumor stromal cells, neural cells, and immune cells.

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