Application of a specific medium in constructing salivary gland organoids and method for constructing salivary gland organoids

Abstract

The present invention relates to the technical field of organoid culture, and specifically discloses the application of a specific culture medium in constructing salivary gland organoids and a method for constructing salivary gland organoids. The specific culture medium provided by the present invention has clear components and does not contain components of animal tissue origin, and is particularly suitable for the construction and culture of human salivary gland organoids, and can meet the needs of subsequent application transformation. The method provided by the present invention is simple and easy to operate, and has the advantages of high organoid construction efficiency, high success rate, high organoid quality, and fast amplification speed when constructing salivary gland organoids by using this method.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid culture, and specifically relates to the application of a specific culture medium in the construction of salivary gland organoids and a method for constructing salivary gland organoids. Background Art

[0002] The salivary gland is an important exocrine organ of the human body, playing important roles such as lubricating and cleaning the oral cavity, assisting digestion, and immune defense. However, salivary gland function is often damaged or reduced due to head and neck radiotherapy, salivary gland tumor resection, autoimmune diseases, aging, etc., resulting in xerostomia. Xerostomia is often accompanied by complications such as difficulty in chewing and swallowing, speech difficulties, loss of taste, oral infections, rampant caries, etc., seriously affecting the quality of life and physical health of patients. At present, the main treatment methods for xerostomia can only provide temporary relief, with poor curative effects. In recent years, researchers have found that the salivary gland organoid culture technology based on salivary gland adult stem cells is expected to become a new method for effectively treating salivary gland function damage and reduction.

[0003] However, currently, most of the methods for constructing salivary gland organoid models are based on hydrogel scaffolds made of proteins extracted from animal tissues. The hydrogel scaffolds used in this method include various components, such as collagen gels, fibrin gels, and Matrigel, etc. Moreover, the components of the culture medium currently used for constructing salivary gland organoids also mostly contain animal tissue extracts (such as bovine pituitary extract BPE, etc.). In this method, one of the main functions of the fiber scaffold is to support the development of salivary spheres, the aggregation, polarization, and differentiation of salivary gland cells. Using this method, salivary gland cells can be inoculated in the gel and divide to form spheroids. Through the regulation of growth factors in the culture medium, the spheroids further differentiate into acinar-like structures and express tight junction proteins (such as occludin, etc.) and aquaporins (such as AQP5, etc.). Since the protein components of animal tissue origin are unknown and cannot be used for future translational applications, it is necessary to develop a culture medium with clear components for the construction of human salivary gland organoids and establish a new culture method for salivary gland organoids to meet the needs of future translational applications. Summary of the Invention

[0004] The purpose of the present invention is to overcome the problems existing in the prior art that there are animal-derived substances with unclear composition in the culture medium currently used for constructing salivary gland organoids, making it difficult for this method to adapt to translational applications for humans, and to provide the application of a specific culture medium in the construction of salivary gland organoids and a method for constructing salivary gland organoids. The specific culture medium provided by the present invention has clear components, is particularly suitable for the construction and culture of human salivary gland organoids, and has the advantages of high organoid construction efficiency, high success rate, fast amplification speed, etc.

[0005] To achieve the above object, on the one hand, the present invention provides an application of a specific culture medium in constructing salivary gland organoids, wherein the specific culture medium comprises a basal medium and additives;

[0006] Among them, the basal medium comprises: DMEM / F12 medium, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and L-glutamine supplement;

[0007] The additives include: B-27 additive, nicotinamide, epidermal growth factor, fibroblast growth factor 10, A83-01, Wnt protein, noggin and R-spondin.

[0008] On the second aspect, the present invention provides the specific culture medium used in the application described in the first aspect.

[0009] On the third aspect, the present invention provides a method for constructing salivary gland organoids, the method comprising:

[0010] (A) Suspending and culturing salivary gland tissue cells with the specific culture medium described in the second aspect; or

[0011] (B) Embedding salivary gland tissue cells with Matrigel and then culturing them in the specific culture medium described in the second aspect.

[0012] On the fourth aspect, the present invention provides salivary gland tissue organoids constructed according to the method described in the third aspect.

[0013] Through the above technical solutions, the present invention can at least achieve the following beneficial effects:

[0014] (1) The components used in the specific culture medium provided by the present invention are clear and do not contain animal tissue-derived substances, laying a foundation for the future development of salivary gland organoids suitable for human use;

[0015] (2) The types and dosages of the additives in the specific culture medium provided by the present invention are optimized, and the organoid construction efficiency is relatively high when using this culture medium to construct human salivary gland organoids. Microscopic organoids can be observed after 24 hours of culture, and mature organoids with multi-cellular components can be obtained after 7 days of culture;

[0016] (3) The method for constructing salivary gland organoids provided by the present invention is simple to operate, can efficiently and stably obtain high-quality organoids, and is suitable for popularization and application. Description of the Drawings

[0017] Figure 1 It is an immunofluorescence staining diagram of salivary gland organoids obtained by the suspension culture method in Example 1.

[0018] Figure 2It is a morphological change diagram of salivary gland organoids during the culture process in Example 1 (the legend is 500 μm).

[0019] Figure 3 It is a comparison diagram of the HE staining, PAS staining, and AB-PAS staining results of salivary gland tissue and salivary gland organoids in Example 1.

[0020] Figure 4 It is an immunofluorescence staining diagram of salivary gland organoids obtained by the method of embedding and culturing with Matrigel in Example 2. Detailed implementation mode

[0021] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values. 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, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0022] The inventors of the present invention ingeniously discovered in the research that by adding specific additives to the culture medium commonly used for organoid construction and culture (such as DMEM / F12 medium, etc.), salivary gland organoids can be efficiently cultured. Through further research, the inventors also found that by the combination of different additives, it is possible to prepare a specific culture medium suitable for the construction and culture of salivary gland organoids without adding animal tissue-derived substances. Moreover, by optimizing the components in the culture medium and the addition amounts and ratios of each component, the construction efficiency and success rate of human salivary gland organoids and the quality of the obtained human salivary gland organoids can be further improved.

[0023] Based on the above discovery, on the one hand, the present invention provides the application of a specific culture medium in the construction of salivary gland organoids, and the specific culture medium includes a basal medium and additives;

[0024] Among them, the basal medium includes: DMEM / F12 medium, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and L-glutamine supplement;

[0025] The additives include: B-27 additive, nicotinamide, epidermal growth factor (EGF), fibroblast growth factor 10 (FGF-10), A83-01, Wnt protein, noggin, and R-spondin (RSPO).

[0026] The B-27 additive is a serum-free additive used in culture media. It can provide various nutrients and cytokines for the culture media and is usually available through commercial purchase. The purchased B-27 additive is usually provided in the form of a concentrated solution (such as 100× or 50×). Before use, it can be diluted with culture media or PBS first, or it can also be directly added to the culture media according to the required concentration.

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

[0028]

[0029] Wnt proteins are a highly conserved family of secreted glycoproteins and are important proteins in the Wnt signaling pathway. They can be added to the culture media as cytokines for organoid construction. The present invention places no particular limitation on the Wnt proteins used. Any Wnt protein that can be used for the construction of (human) salivary gland organoids is applicable to the present invention, and preferably Wnt-3a, etc.

[0030] R-spondin is a newly discovered secreted protein family in recent years. Currently, 4 members have been discovered (named RSPO-1, RSPO-2, RSPO-3, and RSPO-4 in sequence). It binds to LGR4 (Leucine-rich repeat-containing G protein-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 places no particular limitation on the R-spondin used. Any R-spondin that can be used for the construction of (human) salivary gland organoids is applicable to the present invention, and preferably RSPO-1, etc.

[0031] According to a preferred embodiment of the present invention, the basal culture medium further includes at least one of antibiotics, N-acetylcysteine (NAC), and dexamethasone. The present invention places no particular limitation on the selected antibiotics as long as they can be used for the construction of salivary gland organoids.

[0032] Preferably, the antibiotics are selected from antibiotics with antibacterial functions, and preferably penicillin and streptomycin (for example, for convenience of use, commercially available penicillin / streptomycin double-antibody products, etc. can be selected).

[0033] In the present invention, the dosage of the antibiotic only needs to achieve the effect of inhibiting microbial contamination. According to some preferred embodiments of the present invention, the dosage of the antibiotic is such that the specific medium contains 9000 - 11000 U / mL of penicillin and 9000 - 11000 μg / mL of streptomycin.

[0034] In the present invention, the L - glutamine supplement refers to a substance that can provide L - glutamine for the specific medium provided by the present invention. Any substance that can be used for organoid construction and can achieve this purpose is 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 has no particular limitation on the specific source of the selected L - glutamine supplement. It can be either a self - prepared related product containing the aforementioned components or a directly commercially available related product. For example, GlutaMAX produced by ThermoFisher can be selected. TM As the L - glutamine supplement in the present invention, its main components are L - glutamine and the dipeptide in the stable form of L - alanyl - L - glutamine.

[0035] Preferably, the dosage of the L - glutamine supplement is such that the content of L - glutamine provided by it in the specific medium is not less than 1 mM, preferably 1 - 4 mM.

[0036] Preferably, the dosage of HEPES is such that its concentration in the specific medium is not less than 5 mM, preferably 5 - 15 mM, more preferably 8 - 12 mM.

[0037] In the present invention, the protein components added to the specific medium, such as EGF, FGF - 10, Wnt protein, Noggin, RSPO, etc., can all be commercially available or self - prepared related products. The present invention has no particular limitation on the specific selection of the above - mentioned protein components, and those skilled in the art can select them according to actual needs.

[0038] In order to avoid the adverse effects of the introduction of animal - tissue - derived substances on the subsequent application of organoids in the human body, according to some preferred embodiments of the present invention, among the added components, EGF, FGF - 10, Wnt protein, Noggin, and RSPO are all recombinant human - derived proteins.

[0039] The inventors also found through a large number of experiments that by adjusting and optimizing the content of the added components in the specific medium provided by the present invention, the construction efficiency, success rate of salivary gland organoids, and the quality of the obtained salivary gland organoids can be further improved.

[0040] According to a preferred embodiment of the present invention, the dosage of the additive components is such that in the specific medium:

[0041] The final concentration of the B-27 additive is 0.5 - 1.5 mM, the final concentration of NAC is 1 - 1.5 mM, the final concentration of nicotinamide is 5 - 15 mM, the final concentration of EGF is 50 - 100 ng / mL, the final concentration of FGF-10 is 50 - 150 ng / mL, the final concentration of dexamethasone is 0.5 - 1.5 μM, the final concentration of A83-01 is 50 - 150 nM, the final concentration of Wnt protein is 300 - 700 ng / mL, the final concentration of Noggin is 50 - 150 ng / mL, and the final concentration of RSPO is 50 - 150 ng / mL.

[0042] Preferably, the final concentration of the B-27 additive is 0.8 - 1.2 mM, the final concentration of NAC is 1.2 - 1.3 mM, the final concentration of nicotinamide is 8 - 12 mM, the final concentration of EGF is 80 - 120 ng / mL, the final concentration of FGF-10 is 80 - 120 ng / mL, the final concentration of dexamethasone is 0.8 - 1.2 μM, the final concentration of A83-01 is 80 - 120 nM, the final concentration of Wnt protein is 400 - 600 ng / mL, the final concentration of Noggin is 80 - 120 ng / mL, and the final concentration of RSPO is 80 - 120 ng / mL.

[0043] The specific medium provided by the present invention can be applied to the construction and culture of any salivary gland organoids. For example, it can be used to culture human salivary gland organoids or salivary gland organoids of animal origin. According to a preferred embodiment of the present invention, the salivary gland organoids are human salivary gland organoids.

[0044] The second aspect of the present invention provides the specific medium used in the application described in the first aspect. The specific characteristics of this medium are as described above and will not be elaborated here.

[0045] Organoids are based on stem cells and self-assemble into tissues containing various tissue-specific cells with a spatial structure and function similar to normal tissues through three-dimensional culture and directed differentiation. [1] In 2009, the laboratory of Professor Hans Clevers successfully constructed mouse intestinal organoids with stable proliferation, self-renewal ability, and villous structure in Matrigel matrix gel for the first time. Subsequently, research related to organoids has developed vigorously in a short period of time, and the applications of organoids have become increasingly extensive. [2，3] Salivary gland organoids can be generated from various stem cells, including embryonic stem cells, induced pluripotent stem cells, and salivary gland-derived stem / progenitor cells. [4-10]。During the research process, the inventors found that the use of embryonic stem cells and induced pluripotent stem cells may bring serious risks, including gene mutations, teratogenicity, and immunogenic rejection reactions, making it difficult to apply them in translational research. Therefore, after extensive research, the inventors proposed a technical solution for establishing a salivary gland organoid model based on salivary gland-derived stem / progenitor cells in adult tissues. However, in previous studies on culturing organoids using human / animal salivary gland-derived stem / progenitor cells, animal-derived reagents (such as fetal bovine serum, type I rat tail collagen, etc.) were used, and the culture matrix needed to use Matrigel matrix glue or hyaluronic acid hydrogel containing bioactive basement membrane peptides [5-9] , the use of these components also makes the obtained organoids difficult to be used in translational applications. After extensive research, the inventors developed the specific culture medium provided by the present invention and found that when using this culture medium to construct salivary gland organoids, organoids can be obtained either by the commonly used method in the art of first embedding cells in matrix glue and then culturing, or by directly culturing salivary gland cells in suspension with this culture medium, breaking through the technical bottleneck that matrix glue must be used to achieve the construction of salivary gland organoids in the prior art, having high innovation and achieving great technological progress.

[0046] The third aspect of the present invention provides a method for constructing salivary gland organoids, and the method includes:

[0047] (A) Suspending and culturing salivary gland tissue cells with the specific culture medium described in the second aspect; or

[0048] (B) Embedding salivary gland tissue cells with matrix glue and then culturing them in the specific culture medium described in the second aspect.

[0049] The present invention has no particular limitation on the specific source of the salivary gland tissue cells used in the above method. According to a preferred embodiment of the present invention, wherein, the method further includes a step of digesting a salivary gland tissue sample with a digestive solution to obtain salivary gland tissue cells.

[0050] In the present invention, there is no particular limitation on the specific manner of digesting the salivary gland tissue sample and the specific components of the digestive solution used, and the conventional manner of digesting a tissue sample in the art to obtain tissue cells can be adopted. The inventors accidentally found in the research that by using specific digestive enzymes, the digestion efficiency of the salivary gland tissue sample can be effectively improved, and the cell yield and viability can be increased.

[0051] According to a preferred embodiment of the present invention, wherein, the digestive solution contains collagenase. Preferably, the collagenase is a mixture of collagenase I, collagenase II, and collagenase IV.

[0052] Preferably, in the digestive solution, the concentration ratio of collagenase I, collagenase II, and collagenase IV is 1∶0.5 - 1.5∶0.5 - 1.5. Preferably it is 1∶0.8 - 1.2∶0.8 - 1.2. More preferably, the concentration ratio of collagenase II and collagenase IV is 1∶0.5 - 1.5, preferably 1∶0.8 - 1.2.

[0053] More preferably, in the digestive solution, the concentration of collagenase I is 0.5 - 1.5 mg / mL. Preferably it is 0.8 - 1.2 mg / mL.

[0054] More preferably, in the digestive solution, the concentration of collagenase II is 0.5 - 1.5 mg / mL. Preferably it is 0.8 - 1.2 mg / mL.

[0055] More preferably, in the digestive solution, the concentration of collagenase IV is 0.5 - 1.5 mg / mL. Preferably it is 0.8 - 1.2 mg / mL.

[0056] According to a preferred embodiment of the present invention, wherein the digestion conditions include: digestion temperature 35 - 38°C, digestion time 30 - 60 min. To avoid incomplete digestion or over - digestion resulting in poor quality of the obtained salivary gland tissue cells, thereby affecting the construction efficiency and success rate of salivary gland organoids, it is preferred to observe the digestion situation every 10 - 20 min during digestion (for example, by visual observation and / or microscopic observation). In the present invention, there is no particularly strict limitation on the specific digestion time, as long as sufficient digestion effect is achieved, and the digestion time can be appropriately extended or shortened based on the actual digestion situation within the above - mentioned digestion time range. It is preferred to terminate digestion when it is visually observed that the salivary gland tissue is digested from light yellow tissue blocks to only white connective tissue remaining, and the digestive solution is relatively turbid, or when it is microscopically observed that the tissue is (basically) digested into cell clusters formed by 2 - 5 cells.

[0057] To ensure that a sufficient amount of cells can be obtained for the construction of salivary gland organoids, according to a preferred embodiment of the present invention, wherein the size of the salivary gland tissue sample is not less than 0.5 cm×0.5 cm×0 cm, preferably 0.5 - 1 cm×0.5 - 1 cm×0.5 - 1 cm. More preferably it is 0.6 - 0.8 cm×0.6 - 0.8 cm×0.6 - 0.8 cm.

[0058] To improve the digestion efficiency and effect, preferably, before digestion, the salivary gland tissue is crushed or sheared to form tissue fragments with a volume not exceeding 3 mm 3 , preferably 1 - 2 mm 3 of tissue fragments.

[0059] According to a preferred embodiment of the present invention, in method (A), the conditions for suspension culture include: a temperature of 35-38°C. Static culture is preferably used. For example, a culture container containing a culture medium and salivary gland tissue cells can be placed in a 5% CO2 cell culture incubator for culture.

[0060] Preferably, the initial concentration of the salivary gland tissue cells is 2.5×10 4 -5×10 4 cells / mL. Preferably, the addition amount of the culture medium does not exceed 70% of the volume of the culture container, and is preferably 40-60%. For example, when using a 96-well cell culture plate to construct salivary gland organoids, the addition amount of the culture medium per well does not exceed 280 μL, and preferably can be 200-250 μL.

[0061] In the method provided by the present invention, method (A) is simple and easy to operate, and does not rely on a hydrogel scaffold, avoiding the introduction of animal tissue-derived substances during the construction and culture of salivary gland organoids. At the same time, it can also reduce costs and simplify operations, thus being able to well meet the future application transformation needs of organoids.

[0062] According to a preferred embodiment of the present invention, in method (B), the embedding operation includes mixing a salivary gland tissue cell suspension and a matrix gel. Preferably, the volume ratio of the salivary gland tissue cell suspension to the matrix gel does not exceed 1:1, and is preferably 0.1-0.5:1.

[0063] In the method provided by the present invention, there are no special restrictions on the source and dosage of the matrix gel, as long as it can meet the needs of salivary gland organoid construction. For example, a matrix gel prepared according to the prior art can be selected, or a commercially available matrix gel (such as the matrix gel produced by Corning company, etc.) routinely selected in the field for organoid construction can also be directly used.

[0064] Preferably, the dosage (and concentration) of the salivary gland tissue cell suspension is such that the content of salivary gland tissue cells in the embedding system is 2.5×10 4 -5×10 4 cells / mL. Preferably, the inoculation amount of the matrix gel embedded with salivary gland tissue cells is 5-15% of the volume of the culture container. In the present invention, the matrix gel embedded with salivary gland tissue cells can be placed in the culture container at a dosage of 15-45 μL / drop, and then the temperature is raised to make the matrix gel solidify, and then the aforementioned specific culture medium is added and placed in an incubator for culture.

[0065] Preferably, the addition amount of the culture medium does not exceed 70% of the volume of the culture container, and is preferably 40-60%. For example, when constructing salivary gland organoids using a 96-well cell culture plate, the addition amount of the culture medium per well does not exceed 280 μL, and preferably can be 200-250 μL.

[0066] According to a preferred embodiment of the present invention, the method further includes the step of replacing the culture medium. In order to avoid the death or quality decline of organoids caused by the depletion of nutrients in the culture medium and the excessive accumulation of cell metabolic wastes due to not replacing 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 it is observed that the color of the culture medium changes. Preferably, the culture medium is replaced every 2-3 days.

[0067] According to a preferred embodiment of the present invention, the method further includes the passage of salivary gland organoids. The present invention has no particular limitation on the specific operation method during passage, and any method applicable to organoid passage in the art can be applicable to the present invention.

[0068] Preferably, passage is performed when the diameter of the organoids exceeds 100 μm, preferably reaching 150-250 μm.

[0069] The fourth aspect of the present invention provides salivary gland tissue organoids constructed by the method according to the third aspect.

[0070] The salivary gland tissue organoids constructed by the method provided by the present invention contain the main epithelial cell components of the salivary gland. For example, the salivary gland tissue organoids usually include cells of types such as acinar cells, duct cells, and myoepithelial cells. In addition, the salivary gland tissue organoids may also contain (a small amount of) stromal cells.

[0071] The present invention will be described in detail below through 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 used to limit the present invention.

[0072] In the following examples, GlutaMAX purchased from Gibco was used TMAs an L-glutamine supplement, when its concentration is 1×, the provided L-glutamine is approximately 200 mM. When the concentration of the double-antibody antibacterial solution (penicillin-streptomycin) is 1×, it contains 10,000 U / mL of penicillin and 10,000 μg / mL of streptomycin, purchased from Gibco, with the product number 15140122; the DMEM / F12 medium is purchased from Gibco, with the product number C11330500BT; the B-27 additive is purchased from Gibco, with the product number 17504-044; A83-01 is purchased from Sigma, with the product number SML0788; EGF, FGF-10, Wnt-3a, Noggin, and RSPO-1 are all recombinant human proteins, all purchased from Novoprotein.

[0073] In the following examples, unless otherwise specified, the "basic medium" refers to the basic medium prepared according to the ingredients listed in Table 1 of Preparation Example 1; the "specific medium" refers to the medium obtained by adding additive components to the basic medium prepared according to the ingredients listed in Table 1 of Preparation Example 1.

[0074] Preparation Example 1

[0075] (1) Preparation of the specific medium

[0076] Weigh the raw materials according to the formula in Table 1, and add the additive components to the basic medium and mix well to obtain the specific medium for constructing salivary gland organoids.

[0077] Table 1

[0078]

[0079] (2) Preparation of the digestive solution

[0080] According to the formula in Table 2, dilute and prepare collagenase I, collagenase II, and collagenase IV with PBS buffer to obtain the digestive solution for digesting salivary gland tissue samples.

[0081] Table 2

[0082] Component Content Collagenase I 1mg / mL Collagenase II 1mg / mL Collagenase IV 1mg / mL

[0083] Example 1

[0084] This example is used to illustrate the effect of constructing salivary gland organoids by suspension culture.

[0085] In this example, the salivary gland tissue samples used are samples obtained by surgery or biopsy, and the informed consent of relevant personnel has been obtained.

[0086] (1) Obtaining of tissue samples

[0087] A batch of salivary gland tissue samples with a size of approximately 0.5 cm × 0.5 cm × 0.5 cm were obtained through surgery or biopsy. After the samples were taken out of the body, they were placed in 15 mL sterile centrifuge tubes, with each tube containing 10 mL of pre-cooled basal medium. Immediately after collection, the samples were transported on ice.

[0088] (2) Digestion of tissue blocks

[0089] The obtained salivary gland tissue samples were rinsed 3 times with PBS containing 2× antifungal solution (pre-cooled at 4 °C before use) and then placed in a 6 cm sterile culture dish. The tissue sample blocks were cut into small pieces of 2 ± 1 mm 3 using sterile scissors, placed in a 15 mL centrifuge tube, and digestive solution was added (the composition is shown in Table 2 in detail, and the addition amount is about 10 times the volume of the tissue sample). The centrifuge tube was placed in a 37 °C constant temperature mixer for digestion for 60 min. During the digestion process, the digestion situation was observed every 15 min, and the tissue was gently pipetted with a 1 mL pipette tip with the tip cut off. When it was observed macroscopically that the light yellow tissue block was digested until only white connective tissue remained and the solution in the tube was relatively turbid, and under the microscope, the tissue block was digested into a cell mass mixture composed of 2 - 5 cells, 10 mL of basal medium was added to terminate the digestion.

[0090] Centrifuge at 200 G for 5 min at 4 °C, discard the supernatant, resuspend with 10 mL of basal medium, and filter the cell suspension through a 100 μm cell strainer. After filtration, centrifuge again at 200 G for 5 min at 4 °C.

[0091] (3) Organoid construction

[0092] Collect the centrifuged cell pellet, resuspend the cells with 1 mL of specific medium, and perform cell counting. The specific counting method includes: taking 10 μL of cell suspension and mixing it with 10 μL of trypan blue (0.4%), loading the mixture onto a cell counting chamber slide, and using an automatic cell counter to measure the cell density and viability. According to the test results, an appropriate amount of cell suspension was inoculated into a 96-well low-attachment cell culture plate, with about 0.75×10 4 ±0.25×10 4 cells inoculated in each well, and then a certain amount of specific medium was added to each well to make the final volume 200 μL / well.

[0093] The culture plate was placed in a humidified incubator at 37 °C and 5% CO2 for culture. The culture situation and organoid status were observed and recorded every day. The medium was changed every 2 - 3 days (when the color of the medium changed, usually when it became lighter and yellowish, it could be changed).

[0094] A total of 10 salivary gland tissue samples were used for organoid construction in this batch, and salivary gland tissue organoids were successfully obtained from all 10 samples, with a construction success rate of 100%. After observation, cell clusters with a diameter of approximately 50 μm could be observed in the wells about 24 h after inoculation. When cultured for 7 days, spherical or vesicular organoids with a diameter of approximately 200 μm were formed, and the diameter of the organoids was approximately 500 μm at 14 days.

[0095] Immunofluorescence staining was performed on the salivary gland organoids obtained at the 14th day of culture, and the results are as Figure 1 shown (blue staining in the figure is DAPI, representing cell nuclei). Figure 1 In (a), green fluorescence represents K5 (type II keratin, a basal cell stem progenitor cell marker), and red fluorescence represents CD133 (a functional marker of stem progenitor cells); Figure 1 In (b), green fluorescence represents K7 (cytokeratin, a duct cell marker), and red fluorescence represents αSMA (a myoepithelial cell marker); Figure 1 In (c), green fluorescence represents AQP5 (aquaporin, an acinar cell marker), and red fluorescence represents K19 (cytokeratin, a duct cell marker); Figure 1 In (d), green fluorescence represents Epcam (epithelial cell adhesion molecule, an epithelial cell marker), and red fluorescence represents Vim (vimentin, a mesenchymal cell marker). This indicates that the salivary gland organoids are composed of acinar cells, duct cells, myoepithelial cells, and a small amount of mesenchymal cells.

[0096] Figure 2 The morphological changes of salivary gland tissue organoids during the culture process under an optical microscope are shown in Figure 2 (a) to Figure 2 (e) are the observation results of the growth and morphological changes of salivary gland organoids in the same culture well on the 1st day (the day of cell inoculation into the culture plate is the 0th day), 4th day, 7th day, 11th day, and 14th day of culture, respectively.

[0097] The salivary gland tissue obtained by surgery or biopsy and the salivary gland organoids obtained by culturing using the above method to the 14th day were paraffin-embedded and sectioned, and then HE staining was performed. The results are as Figure 3 shown. Figure 3 The HE staining results of the salivary gland tissue are shown in (a), Figure 3 The HE staining results of the salivary gland tissue organoids are shown in (b); Figure 3 The periodic acid-Schiff (PAS) staining results of the salivary gland tissue are shown in (c); Figure 3 The PAS staining results of the salivary gland tissue organoids are shown in (d), indicating a positive reaction to salivary gland mucus; Figure 3(e) shows the Alcian blue - periodic acid Schiff (AB - PAS) staining results of salivary gland tissue; Figure 3 (f) shows the AB - PAS staining results of salivary gland tissue organoids, indicating a positive mucin reaction in the salivary gland. From the comparison in the figure, it can be seen that the salivary gland tissue organoids obtained by the above method are similar in cell composition to normal human tissues, have duct - like and acinar structures, and have a certain secretory function. This shows that the salivary gland tissue organoids have the potential for translational application.

[0098] (4) Organoid passage

[0099] When the diameter of all organoids reaches 100 - 200 μm (most of the organoids have a diameter of about 150 μm), passage is carried out. The specific method includes: centrifuging the culture plate and then discarding the supernatant, adding a small amount of specific medium to resuspend, putting a 10 μL pipette tip on the tip of a 1 μL pipette, and mechanically pipetting the resuspended organoids about 10 - 20 times to disperse them into smaller cell clusters (with a diameter of about 20 - 50 μm), then performing cell counting, and inoculating them in a new culture plate in the manner described in step (3) to complete the passage of the organoids.

[0100] After detection, it was found that after 3 passages, the salivary gland organoids could still maintain their basic morphological characteristics, and the results of immunofluorescence staining and HE staining also showed that the cell composition in the organoids was similar to that in Figure 1 as well.

[0101] Example 2

[0102] This example is used to illustrate the effect of constructing salivary gland organoids by embedding and culturing with Matrigel.

[0103] The process of constructing organoids is similar to that in Example 1. The difference is that in step (3), after cell counting, the cell suspension is pre - cooled on ice, and an appropriate amount of pre - cooled cell suspension is mixed with Matrigel (Matrigel produced by Corning) on ice. The volume ratio of the cell suspension to Matrigel does not exceed 0.5∶1. Then the mixture of the cell suspension and Matrigel is spread on the bottom of a 96 - well low - attachment culture plate pre - heated to 37 °C, 40 μL per well (the cell content is about 1×10 4 cells). The culture plate is placed in a 37 °C incubator for 20 - 30 min to allow Matrigel to solidify. Then 200 μL of specific medium (the composition is shown in Table 1 in detail) is added to each well, and the culture is carried out in a humidified incubator at 37 °C and 5% CO2. Observe and record the culture situation and the state of the organoids every day. Replace the medium every 2 - 3 days (when the color of the medium changes, usually when it becomes lighter and yellowish, it can be replaced).

[0104] A total of 10 salivary gland tissue samples were used in this batch for organoid construction. Among them, salivary gland tissue organoids were successfully obtained from all 10 samples, and the construction success rate was 100%. After observation, cell clusters with a diameter of approximately 50 μm could be observed in the wells about 24 h after inoculation. When cultured for 7 days, spherical or vesicular organoids with a diameter of approximately 200 μm were formed, and the diameter of the organoids was approximately 500 μm at 14 days.

[0105] The salivary gland organoids obtained at 14 days of culture were subjected to immunofluorescence staining, and the results are as Figure 4 shown (blue staining in the figure is DAPI, representing cell nuclei). Figure 4 (a), green fluorescence represents K5 (type II keratin, a basal cell stem progenitor cell marker), and red fluorescence represents CD133 (a functional marker of stem progenitor cells); Figure 4 (b), green fluorescence represents K7 (cytokeratin, a duct cell marker), and red fluorescence represents αSMA (a myoepithelial cell marker); Figure 4 (c), green fluorescence represents AQP5 (aquaporin, an acinar cell marker), and red fluorescence represents K19 (cytokeratin, a duct cell marker); Figure 4 (d), green fluorescence represents Epcam (epithelial cell adhesion molecule, an epithelial cell marker), and red fluorescence represents Vim (vimentin, a mesenchymal cell marker). This indicates that the salivary gland organoids are composed of acinar cells, duct cells, myoepithelial cells, and a small amount of mesenchymal cells.

[0106] Example 3

[0107] The method in Example 1 was used for the construction of salivary gland organoids, except that dexamethasone and NAC were not added when preparing the specific culture medium. The remaining operations and conditions were the same as those in Example 1.

[0108] A total of 3 salivary gland tissue samples were used in this batch for organoid construction. Among them, salivary gland tissue organoids were successfully obtained from all 3 samples, and the construction success rate was 100%. After observation, compared with Example 1, the culture speed of the salivary gland organoids in this example and the characteristics and quality of the obtained organoids were similar to those in Example 1. After immunofluorescence staining, HE staining, PAS staining, and AB-PAS staining of the obtained salivary gland organoids and observation, similar results to those in Figure 1 and Figure 3 were also shown.

[0109] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including the combination of each technical feature in any other suitable manner. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

[0110] References

[0111] 1. Lancaster, M.A. and J.A. Knoblich, Organogenesis in a dish: modeling development and disease using organoid technologies. Science, 2014. 345(6194): p. 1247 - 125.

[0112] 2. Sato, T., et al., Single Lgr5 stem cells build crypt - villus structures in vitro without a mesenchymal niche. Nature, 2009. 459(7244): p. 262 - 5.

[0113] 3. Spence, J.R., et al., Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature, 2011. 470(7332): p. 105 - 9.

[0114] 4. Tanaka, J., et al., Generation of orthotopically functional salivary gland from embryonic stem cells. Nat Commun, 2018. 9(1): p. 4216.

[0115] 5. Pringle, S., et al., Human Salivary Gland Stem Cells Functionally Restore Radiation Damaged Salivary Glands. Stem Cells, 2016. 34(3): p. 640 - 52. 6. Maimets, M., et al., Long - Term In Vitro Expansion of Salivary Gland Stem Cells Driven by Wnt Signals. Stem Cell Reports, 2016. 6(1): p. 150 - 62. 7. Sui, Y., et al., Generation of functional salivary gland tissue from human submandibular gland stem / progenitor cells. Stem Cell Res Ther, 2020. 11(1): p. 127.

[0116] 8. Yoon, Y.J., et a1., Salivary gland organoid culture maintains distinct glandular properties of murine and human major salivary glanlls. Nat Commun, 2022. 13(1): p. 3291.

[0117] 9. Srinivasan, P.P., et al., Primary Salivary Human Stem / Progenitor Cells Undergo Microenvironment - Driven Acinar - Like Differentiation in Hyaluronate Hydrogel Culture. Stem Cells Transl Med, 2017. 6(1): p. 110 - 120.

[0118] 10. Tanaka, J., et al., Human induced pluripotent stem cell-derived salivary gland organoids model SARS-CoV-2 infection and replication. Nat Cell Biol, 2022. 24(11): p. 1595-1605.

Claims

1. A method for constructing salivary gland organoids, characterized in that, The method includes: Suspending and culturing salivary gland tissue cells in a specific medium; The specific medium consists of a basal medium and additives; Among them, the basal medium consists of DMEM / F12 medium, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, L-glutamine supplement, and penicillin-streptomycin double antibody; The additives consist of: B-27 additive, nicotinamide, epidermal growth factor, fibroblast growth factor 10, A83-01, Wnt-3a protein, noggin, and R-spondin-1; The dosage of the L-glutamine supplement is such that the content of L-glutamine provided by it in the specific medium is 1-4 mM; The dosage of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid is such that its concentration in the specific medium is 5-15 mM; The dosage of the additives is such that in the specific medium: the final concentration of B-27 additive is 0.5-1.5 mM, the final concentration of nicotinamide is 5-15 mM, the final concentration of epidermal growth factor is 50-100 ng / mL, the final concentration of fibroblast growth factor 10 is 50-150 ng / mL, the final concentration of A83-01 is 50-150 nM, the final concentration of Wnt-3a protein is 300-700 ng / mL, the final concentration of noggin is 50-150 ng / mL, and the final concentration of R-spondin-1 is 50-150 ng / mL.

2. The method according to claim 1, wherein, The L-glutamine supplement is selected from at least one of L-glutamine, L-alanyl-L-glutamine, and oligopeptides formed thereby.

3. The method according to claim 1, wherein, The dosage of the antibiotic is such that the specific medium contains 9000-11000 U / mL of penicillin and 9000-11000 μg / mL of streptomycin.

4. The method according to claim 1, wherein, The dosage of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid is such that its concentration in the specific medium is 8-12 mM.

5. The method according to claim 1, wherein, Among the additives, epidermal growth factor, fibroblast growth factor 10, Wnt protein, noggin, and R-spondin are all recombinant human-derived proteins.

6. The method according to claim 1, wherein, The method further includes the step of digesting the salivary gland tissue sample with a digestive solution to obtain salivary gland tissue cells.

7. The method according to claim 6, wherein, The digestive solution contains collagenase.

8. The method according to claim 7, wherein, The collagenase is a mixture of collagenase I, collagenase II, and collagenase IV.

9. The method according to claim 8, wherein, In the digestive solution, the concentration ratio of collagenase I, collagenase II, and collagenase IV is 1:0.5-1.5:0.5-1.

5.

10. The method according to claim 8 or 9, wherein, In the digestive solution, the concentration of collagenase I is 0.5-1.5 mg / mL; And / or, in the digestive solution, the concentration of collagenase II is 0.5-1.5 mg / mL; And / or, in the digestive solution, the concentration of collagenase IV is 0.5-1.5 mg / mL.

11. The method according to claim 6, wherein, The digestion conditions include: digestion temperature 35-38 °C, digestion time 30-60 min; And / or, the size of the salivary gland tissue sample is not less than 0.5 cm × 0.5 cm × 0.5 cm.

12. The method according to claim 11, wherein, The size of the salivary gland tissue sample is (0.5-1) cm × (0.5-1) cm × (0.5-1) cm.

13. The method according to claim 11 or 12, wherein, Prior to digestion, the salivary gland tissue is crushed or sheared to form tissue fragments with a volume not exceeding 3 mm 3 .

14. The method according to claim 13, wherein, Before digestion, the salivary gland tissue is crushed or sheared to form tissue fragments with a volume of 1-2 mm 3 each.

15. The method according to claim 1, wherein, The conditions for the suspension culture include: a temperature of 35-38°C.

16. The method according to claim 1, wherein, The initial concentration of the salivary gland tissue cells is 2.5×10 4 -5×10 4 cells / mL.

17. The method according to claim 1, wherein The addition amount of the culture medium does not exceed 70% of the volume of the culture container.

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