Method for constructing adult thyroid organoid by using novel thyroid organoid culture medium

Abstract

The invention provides a method for constructing an adult thyroid organ by using a novel thyroid organ culture medium. Specifically, the invention provides a thyroid organ culture medium, a kit and a culture method thereof, by adding special additives FGF2, Y27632 and BMP-4, cells with dryness are induced from tissues derived from adult thyroid, and thyroid organs closer to thyroid tissues are successfully obtained. Compared with the prior art, the thyroid organoid provided by the invention has the advantages that the number is obviously increased, the volume is larger, follicular structures closer to thyroid tissues in morphology can be observed, more thyroid functional proteins can be expressed, the passage frequency is more, the passage time is longer, and the application range is wider. The method has wide application prospects in the fields of establishment and passage of adult thyroid organs, research of thyroid or thyroid-related diseases and drug sensitivity screening of thyroid-related diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a method for constructing adult thyroid organoids using a novel thyroid organoid culture medium. Background Art

[0002] The thyroid is a butterfly-shaped organ that functions to synthesize, secrete, and store thyroid hormones. The thyroid is one of the important endocrine glands in the human body, and the thyroid hormones it secretes play a key role in promoting the body's metabolism and maintaining normal growth and development of the body. Epidemiological survey results show that currently in China, there are 10 million hyperthyroidism patients, 90 million hypothyroidism (hypothyroidism) patients, and more than 100 million patients with thyroid nodules and thyroid cancer. Conservatively estimated, there are currently more than 200 million thyroid patients in China.

[0003] Organoids are small clusters of cells that grow and form in a three-dimensional (3D) environment in vitro. They are derived from stem cells and self-organize and differentiate into functional cell types, thus generalizing the structure and function of organs in vivo. Stem cells that can form organoids include embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), and neonatal or adult stem cells (ASC). The self-organization process of organoids needs to be mediated by activating multiple signaling pathways of intrinsic cellular components or the external environment through the use of a culture medium (containing essential growth factors) and extracellular matrix (ECM).

[0004] Currently, there is relatively little research on normal adult thyroid organoids at home and abroad, and most of them remain in the preliminary culture stage. The main reason is that researchers have not found specific stem and progenitor cell populations in adult thyroid. Therefore, the establishment and passage of adult thyroid organoids pose great challenges, and there is an urgent need in this field to develop methods for constructing adult thyroid organoids. Summary of the Invention

[0005] To solve the above problems, the present invention discloses a method for constructing adult thyroid organoids using a novel thyroid organoid culture medium. The present invention constructs a culture medium with a new composition, induces cells with stemness from tissues derived from adult thyroid, successfully cultures thyroid organoids, and can be passaged for a long time, which is convenient for disease model construction and drug sensitivity screening.

[0006] In a first aspect of the present invention, there is provided a thyroid organoid culture medium, which comprises a basal medium and additives selected from the group consisting of fibroblast growth factor-2 (FGF2), Y27632, bone morphogenetic protein-4 (BMP-4), or a combination thereof.

[0007] In another preferred embodiment, the basal medium is selected from the group consisting of DMEM medium, Advanced DMEM / F12 medium, or a combination thereof; preferably, the basal medium is Advanced DMEM / F12 medium.

[0008] In another preferred embodiment, the additives include: FGF2, Y27632, and BMP-4.

[0009] In another preferred embodiment, the thyroid organoid culture medium further comprises one or more components selected from the group consisting of epidermal growth factor (EGF), R-spondin-1 (RSPO1), Wnt-3a, N-acetylcysteine, nicotinamide, B27, fibroblast growth factor-10 (FGF10), A83-01, forskolin.

[0010] In another preferred embodiment, the thyroid organoid culture medium comprises the following components:

[0011] (a) a basal medium (preferably Advanced DMEM / F12 medium);

[0012] (b) specific additives selected from the group consisting of fibroblast growth factor-2 (FGF2), Y27632, bone morphogenetic protein-4 (BMP-4), or a combination thereof; and

[0013] (c) basal additives selected from the group consisting of epidermal growth factor (EGF), R-spondin-1 (RSPO1), Wnt-3a, N-acetylcysteine, nicotinamide, B27, fibroblast growth factor-10 (FGF10), A83-01, forskolin, or a combination thereof.

[0014] In another preferred embodiment, the R-spondin-1 is a recombinant protein.

[0015] In another preferred embodiment, the Wnt-3a is a recombinant protein.

[0016] In another preferred example, based on the total volume of the culture medium, the concentration of FGF2 is 10 - 100 ng / ml; preferably 15 - 80 ng / ml; more preferably 20 - 50 ng / ml.

[0017] In another preferred example, the concentration of FGF2 is 20 ng / ml.

[0018] In another preferred example, the concentration of FGF2 is 50 ng / ml.

[0019] In another preferred example, based on the total volume of the culture medium, the concentration of Y27632 is 0.32 - 32 μg / ml; preferably 1 - 16 μg / ml; more preferably 2 - 8 μg / ml; most preferably 3.2 - 6.4 μg / ml.

[0020] In another preferred example, the concentration of Y27632 is 3.2 μg / ml.

[0021] In another preferred example, the concentration of Y27632 is 6.4 μg / ml.

[0022] In another preferred example, based on the total volume of the culture medium, the concentration of Y27632 is 10 - 50 μM.

[0023] In another preferred example, based on the total volume of the culture medium, the concentration of BMP-4 is 10 - 100 ng / ml; preferably 10 - 50 ng / ml; more preferably 10 - 30 ng / ml; most preferably 20 - 30 ng / ml.

[0024] In another preferred example, the concentration of BMP-4 is 20 ng / ml.

[0025] In another preferred example, the concentration of BMP-4 is 10 ng / ml.

[0026] In another preferred example, the concentration of BMP-4 is 30 ng / ml.

[0027] In another preferred example, based on the total volume of the culture medium, the concentration of EGF is 10 - 1000 ng / ml; preferably 50 - 500 ng / ml; more preferably 50 - 200 ng / ml; most preferably 50 ng / ml.

[0028] In another preferred example, based on the total volume of the culture medium, the concentration of R-spondin-1 is 50 - 500 ng / ml; preferably 100 - 400 ng / ml; more preferably 100 - 200 ng / ml; most preferably 200 ng / ml.

[0029] In another preferred embodiment, the concentration of Wnt-3a is 0 - 100 ng / ml based on the total volume of the medium; preferably 1 - 80 ng / ml; more preferably 10 - 40 ng / ml; most preferably 30 ng / ml.

[0030] In another preferred embodiment, the concentration of N-acetylcysteine is 1 - 10 mM based on the total volume of the medium; preferably 1 - 5 mM; more preferably 1 - 2 mM; most preferably 1.25 mM.

[0031] In another preferred embodiment, the concentration of nicotinamide is 1 - 100 mM based on the total volume of the medium; preferably 5 - 50 mM; more preferably 10 - 20 mM; most preferably 10 mM.

[0032] In another preferred embodiment, the concentration (volume percentage) of B27 is 0.1 - 5% based on the total volume of the medium; preferably 0.1 - 2%; more preferably 0.5 - 2%; most preferably 0.5%.

[0033] In another preferred embodiment, the concentration of FGF10 is 20 - 100 ng / ml based on the total volume of the medium; preferably 20 - 80 ng / ml; more preferably 20 - 40 ng / ml; most preferably 20 ng / ml.

[0034] In another preferred embodiment, the concentration of A83-01 is 0.1 - 10 μM based on the total volume of the medium; preferably 0.25 - 5 μM; more preferably 0.5 - 2 μM; most preferably 1 μM.

[0035] In another preferred embodiment, the concentration of forskolin is 1 - 20 μM based on the total volume of the medium; preferably 1 - 10 μM; more preferably 1 - 5 μM; most preferably 1 μM.

[0036] In a second aspect of the present invention, there is provided a culture medium kit for thyroid organoids, the culture medium kit comprising:

[0037] (Z1) A first container and a special additive placed in the first container, the special additive being selected from the group consisting of fibroblast growth factor-2 (FGF2), Y27632, bone morphogenetic protein-4 (BMP-4), or a combination thereof.

[0038] In another preferred embodiment, the culture medium kit further comprises:

[0039] (Z2) A second container and a basal additive placed within the second container, the basal additive being selected from the group consisting of epidermal growth factor (EGF), R-spondin-1 (RSPO1), Wnt-3a, N-acetylcysteine, nicotinamide, B27, fibroblast growth factor-10 (FGF10), A83-01, forskolin, or a combination thereof.

[0040] In another preferred embodiment, the culture medium kit further comprises:

[0041] (Z3) A third container and a basal culture medium placed within the third container.

[0042] In another preferred embodiment, the culture medium kit further comprises:

[0043] (Z4) A fourth container and a digestive solution or digestive enzyme placed within the fourth container.

[0044] In another preferred embodiment, the basal culture medium is selected from the group consisting of DMEM medium, Advanced DMEM / F12 medium, or a combination thereof; preferably, the basal culture medium is Advanced DMEM / F12 medium.

[0045] In another preferred embodiment, the main components of the digestive solution or digestive enzyme are selected from the group consisting of collagenase, DNase, Dispase, trypsin, or a combination thereof.

[0046] In another preferred embodiment, the collagenase comprises: type I collagenase.

[0047] In another preferred embodiment, the trypsin comprises: TrypLE.

[0048] In another preferred embodiment, the culture medium kit is used for preparing the thyroid organoid culture medium as described in the first aspect of the present invention.

[0049] In another preferred embodiment, the culture medium kit further comprises: an instruction manual, which records the concentrations of the components in the culture medium kit and the method for preparing the thyroid organoid culture medium as described in the first aspect of the present invention.

[0050] In another preferred embodiment, the instruction manual further records the guiding usage concentrations of the components in the culture medium kit when preparing the thyroid organoid culture medium as described in the first aspect of the present invention.

[0051] In another preferred embodiment, the first container, the second container, the third container, and / or the fourth container can be the same or different containers.

[0052] In a third aspect of the present invention, a method for culturing thyroid organoids is provided, and the method includes the steps of: culturing using the thyroid organoid culture medium as described in the first aspect of the present invention.

[0053] In another preferred example, the method is for non-disease diagnosis and non-disease treatment purposes.

[0054] In another preferred example, the method includes the steps of:

[0055] (S1) Provide a thyroid tissue sample to be cultured;

[0056] (S2) Digest and centrifuge the thyroid tissue sample to obtain a thyroid cell pellet;

[0057] (S3) Resuspend the thyroid cell pellet to obtain a thyroid cell suspension;

[0058] (S4) Mix the thyroid cell suspension with Matrigel and then perform cell seeding. After the Matrigel solidifies, add the thyroid organoid culture medium as described in the first aspect of the present invention for culturing; and

[0059] (S5) Under conditions suitable for culturing thyroid organoids, culture for 3 - 14 days to obtain the thyroid organoids.

[0060] In another preferred example, the thyroid tissue sample is a normal thyroid tissue sample or a pathological thyroid tissue sample (such as a thyroid cancer tissue sample).

[0061] In another preferred example, the step (S1) further includes a sub-step (S1a): removing adipose tissue.

[0062] In another preferred example, the step (S1) further includes a sub-step (S1b): washing the thyroid tissue sample with DPBS solution.

[0063] In another preferred example, the step (S1) further includes a sub-step (S1c): cutting the thyroid tissue sample into tissue blocks with a size of 1 - 3 mm 3 in size.

[0064] In another preferred example, in the step (S2), multiple digestions and centrifugations are performed.

[0065] In another preferred example, the step (S2) further includes a sub-step (S2a): removing the DPBS solution.

[0066] In another preferred example, in the step (S2), digestion is performed using a digestive solution or digestive enzyme, and the main components of the digestive solution or digestive enzyme are selected from the group consisting of: collagenase, DNase, Dispase, trypsin, or a combination thereof.

[0067] In another preferred example, the collagenase includes: type I collagenase.

[0068] In another preferred example, the trypsin includes: TrypLE.

[0069] In another preferred example, the centrifugation conditions in step (S2) are: centrifuging at 1000 - 2000 rpm (preferably 1500 rpm) or 200 - 400 g (preferably 350 g) for 5 - 10 min.

[0070] In another preferred example, in step (S3), the resuspension is carried out using a solution selected from the following group: basal medium, the thyroid organoid medium as described in the first aspect of the present invention, DPBS solution, or a combination thereof.

[0071] In another preferred example, step (S3) further includes sub-step (S3a): after resuspending the thyroid cell pellet, passing it through a 100 μM cell strainer, and collecting the thyroid cell solution after passing through the cell strainer.

[0072] In another preferred example, step (S3) further includes sub-step (S3b): centrifuging the thyroid cell solution after passing through the cell strainer, removing the supernatant, and resuspending the obtained thyroid cell pellet again to obtain a thyroid cell suspension.

[0073] In another preferred example, in step (S4), wait for the Matrigel to solidify under the condition of 37°C.

[0074] In another preferred example, step (S4) further includes the step: wait for the Matrigel to solidify in a cell culture incubator at 37°C and 5% CO2.

[0075] In another preferred example, step (S5) further includes the step: replacing the fresh thyroid organoid medium as described in the first aspect of the present invention every 2 - 3 days.

[0076] In another preferred example, the conditions suitable for culturing thyroid organoids in step (S5) include: culturing in a cell culture incubator at 37°C and 5% CO2.

[0077] In another preferred example, the method further includes the step:

[0078] (S6) Analyzing and / or identifying the thyroid organoids.

[0079] In another preferred example, the analysis and / or identification includes:

[0080] (1) Staining analysis (preferably, hematoxylin-eosin staining, for observing the physiological and pathological structure of the thyroid, such as the follicular structure unique to the thyroid);

[0081] (2) Immunohistochemistry (preferably, immunohistochemical detection of the expression of thyroid-related proteins, such as thyroglobulin and thyroid peroxidase);

[0082] (3) Immunohistocytofluorescence (preferably, immunohistochemical detection of the co-expression of thyroid-related proteins, such as the sodium-iodide symporter (an ion channel essential for thyroid hormone synthesis));

[0083] (4) Analysis or identification of the secretion and / or regulatory function of thyroid-related hormones (preferably, such as the secretion of thyroxine T4 and triiodothyronine T3; such as the regulation of T4 and T3 by thyroid-stimulating hormone (TSH));

[0084] (5) Transmission electron microscopy (such as observing the fine structure of the thyroid, including apical microvilli, pseudopods, and intracellular vesicles storing thyroglobulin); and / or

[0085] (6) Single-cell transcriptome sequencing (such as analyzing cell types, the developmental trajectory of thyroid organoids, and stem progenitor cells in thyroid organoids).

[0086] In the fourth aspect of the present invention, a thyroid organoid is provided, which is obtained by the method as described in the third aspect of the present invention.

[0087] In another preferred example, the thyroid organoid contains adult thyroid stem cells.

[0088] In the fifth aspect of the present invention, an adult thyroid stem cell is provided, which is obtained by the method as described in the third aspect of the present invention.

[0089] In another preferred example, the adult thyroid stem cell exists in the thyroid organoid.

[0090] In another preferred example, the adult thyroid stem cell can be identified by performing single-cell transcriptome sequencing on the thyroid organoid.

[0091] In the sixth aspect of the present invention, there is provided a use of the thyroid organoid culture medium as described in the first aspect of the present invention, or the culture medium kit as described in the second aspect of the present invention, and the use includes:

[0092] (a) being used as or for formulating a culture medium for culturing thyroid organoids;

[0093] (b) As an additive used in or for formulating a medium for culturing thyroid organoids;

[0094] (c) For culturing and / or forming adult thyroid stem cells;

[0095] (d) As a medium used in or for formulating a medium for culturing adult thyroid stem cells; and / or

[0096] (e) As an additive used in or for formulating a medium for adult thyroid stem cells.

[0097] In another preferred embodiment, the use is for non-disease diagnosis and non-disease treatment purposes.

[0098] In a seventh aspect of the present invention, there is provided a composition comprising: fibroblast growth factor-2 (FGF2), Y27632, and bone morphogenetic protein-4 (BMP-4).

[0099] In an eighth aspect of the present invention, there is provided a use of a composition as described in the seventh aspect of the present invention, and the use includes:

[0100] (a) For preparing a medium for culturing thyroid organoids and / or adult thyroid stem cells;

[0101] (b) For culturing and / or forming adult thyroid stem cells; and / or

[0102] (c) As an additive to a medium for thyroid organoids and / or adult thyroid stem cells.

[0103] In another preferred embodiment, the use is for non-disease diagnosis and non-disease treatment purposes.

[0104] In a ninth aspect of the present invention, there is provided a use of a thyroid organoid as described in the fourth aspect of the present invention, or an adult thyroid stem cell as described in the fifth aspect of the present invention, and the use includes:

[0105] (a) Studying the structure and / or function of the thyroid;

[0106] (b) Studying the structure and / or function of adult thyroid stem cells;

[0107] (c) Studying the structure and / or function of thyroid-related proteins;

[0108] (d) Studying the secretion and / or regulation of thyroid-related hormones;

[0109] (e) Studying the structure and / or function of thyroid organoids (such as studying cell types in thyroid organoids);

[0110] (f) Studying the developmental trajectory of the thyroid and / or thyroid organoids;

[0111] (g) Research on stem progenitor cells in the thyroid gland and / or thyroid organoids;

[0112] (h) Used as an in vitro model of the thyroid gland; and / or

[0113] (i) Used as an in vitro model of thyroid-related diseases.

[0114] In another preferred example, the use is for non-disease diagnosis and non-disease treatment purposes.

[0115] In another preferred example, the thyroid-related diseases include thyroid cancer.

[0116] It should be understood that within the scope of the present invention, each of the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings

[0117] Figure 1 Shows the number of thyroid organoids obtained by culturing with thyroid organoid media 1-5 in Examples 1-5 respectively. Among them, Example 1 is the experimental group using thyroid organoid media 1 in Example 1, Example 2 is the experimental group using thyroid organoid media 2 in Example 2, and so on.

[0118] Figure 2 Shows the bright-field images, hematoxylin-eosin staining images, and immunohistochemical images of marker proteins of thyroid organoids obtained by culturing with thyroid organoid media 1 or 5 in Examples 1 and 5. Among them, Example 1 is the experimental group using thyroid organoid media 1 in Example 1, and Example 5 is the experimental group using thyroid organoid media 5 in Example 5.

[0119] Figure 3 Shows the bright-field images, hematoxylin-eosin staining images, immunohistochemical images, and immunocytofluorescence images of marker proteins of thyroid organoids obtained by culturing with thyroid organoid media 5 or 6 in Examples 5 and 6. Among them, Example 5 is the experimental group using thyroid organoid media 5 in Example 5, and Example 6 is the experimental group using thyroid organoid media 6 in Example 6.

[0120] Figure 4 Shows the identification of the related hormone secretion and regulation functions of thyroid organoids obtained by culturing with thyroid organoid media 5 in Example 5. Among them, the control group is the concentrations of T3 and T4 detected in the culture medium supernatant when TSH stimulation is not added; the TSH group is the concentrations of T3 and T4 detected in the culture medium supernatant after TSH stimulation.

[0121] Figure 5 It shows the UMAP photo of cell types of thyroid organoids by single-cell sequencing, and a group of thyroid stem cells are found therein. Detailed implementation manners

[0122] After extensive and in-depth research and a large number of screenings, the present inventors unexpectedly discovered for the first time that a thyroid organoid culture medium formulated by specifically adding components of FGF2, Y27632, and BMP4 can induce stem cells with stemness from tissues derived from adult thyroid, and the cultured thyroid organoids have many advantages, such as a significantly increased number of formed thyroid organoids, the characteristics of the generated thyroid organoids being closer to those of thyroid tissues, enabling more passages and longer passage times, and the formed thyroid organoids having relatively complete functionality and being able to have functions basically consistent with those of the thyroid tissues of a normal organism, such as hormone secretion and regulation functions. In addition, by adjusting the proportions of the above-mentioned specifically added components, different effects on the morphology of thyroid organoids and the expression of marker proteins are obtained, which is beneficial to adjusting the corresponding culture protocol according to experimental or research purposes and culturing thyroid organoids that meet the requirements. The present invention has been completed on this basis.

[0123] Terms

[0124] To more easily understand the present disclosure, certain terms are first defined. As used in this application, unless otherwise clearly specified herein, each of the following terms shall have the meaning given below.

[0125] The term "about" may refer to a value or composition within an acceptable error range of a specific value or composition determined by a person of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0126] The terms "epidermal growth factor", "EGF (Epidermal Growth Factor)" are used interchangeably and refer to a heat-stable single-chain low-molecular polypeptide composed of 53 amino acid residues. After EGF specifically recognizes and binds to the EGF receptor on the target cell, a series of biochemical reactions occur, and ultimately it can promote the DNA synthesis and mitosis of the target cell. For example, EGF can promote the transport of small molecules such as potassium ions, deoxyglucose, and α-aminoisobutyric acid, increase the synthesis and secretion of extracellular matrix, and promote RNA, DNA, and protein synthesis, thereby providing energy for the proliferation and differentiation of human epidermal cells, effectively stimulating the growth of epidermal cells, and enhancing the cell metabolism ability.

[0127] The terms "R-spondin-1" and "RSPO1" are used interchangeably and refer to a member of the R-spondin family, which includes 4 members (RSPO1-4) and is a niche factor for maintaining adult stem cells in multiple organs. RSPO1 is a secreted activator protein with two cysteine-rich furin-like domains (FU-like CR) and one thrombospondin type 1 domain (TSR). As a classical Wnt signal enhancer, RSPO1 is currently widely used to promote the growth and survival of 3D organoids.

[0128] The term "Wnt-3a" refers to a member of the Wnt family of secreted proteins, which contains 19 human proteins. As the most representative signaling protein of the Wnt family, Wnt3a is widely distributed and plays a key role in regulating pleiotropic cell functions, including self-renewal, proliferation, differentiation, and motility. Wnt3a is also one of the most commonly used culture factors for organoid construction, and its high activity, batch-to-batch stability, contamination-free, and serum-free preparation are the keys to the success of organoid experiments.

[0129] The terms "N-acetylcysteine" and "N-acetylcysteine" are used interchangeably and refer to an organic compound containing a sulfhydryl group with the chemical formula C5H9NO3S. It can be added to cell culture media as a general amino acid source or as a general reducing agent to support the growth of T cells, neuronal progenitor / stem cells, or muscle progenitor / stem cells, respectively. As a reactive oxygen species (ROS) inhibitor, N-acetylcysteine can also effectively reduce the intracellular ROS level, which is beneficial to the improvement of cell viability.

[0130] The terms "nicotinamide" and "nicotinamide" are used interchangeably and are also known as niacinamide, which is an amide compound of nicotinic acid. Nicotinamide can be used as a nutrient component of the culture medium. In cells, it is incorporated into NADP+ and NAD+ and used as a coenzyme for various enzymatic redox reactions. It has been reported that nicotinamide can also inhibit sirtuin activity, thus being used to promote the formation and extend the lifespan of organoids.

[0131] The term "B27" refers to the B-27 supplement, which is a serum-free supplement widely used in tissue culture media for culturing cells and neurons and can effectively improve the growth and survival rate of cultured cells and neurons.

[0132] The terms "fibroblast growth factor-10" and "FGF10 (Fibroblast Growth Factor 10)" are used interchangeably and refer to a multifunctional mesenchymal-epithelial signaling growth factor, which is a member of the FGF family. It is mainly expressed in the epithelial cells and mesenchymal cells of embryos, fibroblasts, and adult tissues and organs such as the liver, lung, brain, and intestine, and plays diverse roles in different tissues and organs.

[0133] The term "A83-01" refers to a compound with the chemical formula C 18 H 12 N2O2, which is an effective inhibitor of TGF-β (transforming growth factor β) receptor kinase and can affect cell growth, proliferation, and transformation by inhibiting the TGF-β signaling pathway. Therefore, A83-01 is often used in the research on the regulatory mechanisms of cell proliferation and differentiation, cancer research, and stem cell culture and differentiation.

[0134] The terms "forskolin" and "Forskolin" are used interchangeably, also known as forskolin and maohousu, and refer to a hydrophobic activator that can stimulate the elevation of the activity of adenylate cyclase (all subtypes except type IX) in mammalian cells, thereby increasing the intracellular cAMP concentration. Therefore, Forskolin is usually used in various types of intact cells or tissue cultures to increase the CAMP level.

[0135] The term "TG (Thyroglobulin)" refers to thyroglobulin, which is a large molecular glycoprotein secreted by thyroid follicular epithelial cells. The vast majority is synthesized by thyroid cells and released into the residual cavity of thyroid follicles. Thyroglobulin is considered a special marker for the integrity of the thyroid gland. It is also a tumor marker for differentiated thyroid cancer (DTC) and can be an important reference index for the follow-up of patients with differentiated thyroid cancer after treatment. It can also be used to distinguish subacute thyroiditis and pseudo-thyrotoxicosis.

[0136] The term "TPO" is an abbreviation for thyroid peroxidase antibody (TPOAb, thyroid peroxidase antibody). TPO is an autoantibody that mainly acts on peroxidase in thyroid tissue. Peroxidase is one of the key enzymes for the synthesis of thyroid hormones in the thyroid gland. It catalyzes the iodination reaction of thyroglobulin and is an essential enzyme for thyroid hormone synthesis.

[0137] The term "sodium-iodide symporter" refers to the Na / I symporter (NIS), which is a glycoprotein expressed on the cell membrane of thyroid follicles. It is an ion channel essential for thyroid hormone synthesis and mediates the active uptake of iodine by thyroid cells in the presence of sodium ions. Its function is regulated by thyroid-stimulating hormone, iodine, various cytokines, and some other hormones.

[0138] The term "TSH" refers to thyroid-stimulating hormone, which is secreted by thyrotrophs in the pituitary gland and is mainly responsible for regulating the proliferation of thyroid cells, as well as the synthesis and secretion of thyroid hormones, playing an important regulatory role in maintaining normal thyroid function. When the TSH level increases, it stimulates the thyroid gland to secrete more thyroid hormones T3 and T4.

[0139] FGF2

[0140] Fibroblast Growth Factor-2 (FGF-2) is a member of the fibroblast growth factor family and has a high affinity for heparin. FGF-2 plays an important role in tendon-to-bone healing, cartilage repair, bone repair, and nerve regeneration. FGF-2 can specifically bind to tyrosine kinase receptors and activate the FGF / FGFR signaling pathway. Subsequently, FGF-2 affects cell proliferation, differentiation, apoptosis, and immune regulation by transducing other classical pathways. For example, FGF-2 regulates the JAK-STAT signaling pathway to regulate cartilage metabolism. FGF-2 can also act as a mitogen to accelerate cell proliferation.

[0141] Y27632

[0142] Y27632 is a selective inhibitor of Rho-associated serine-threonine protein kinase (ROCK) and belongs to small molecule compounds. Y-27632 can inhibit the activities of ROCK-1 and ROCK-2, thereby affecting processes such as cell proliferation, migration, adhesion, and cytokinesis. Y-27632 is widely used in research in the biological field, such as stem cell biology, tumor biology, neurobiology, etc. In stem cell research, Y-27632 can be used to induce the differentiation of stem cells and improve the survival rate of stem cells; in tumor research, Y-27632 can be used to inhibit the proliferation and metastasis of tumor cells; in neurobiology, Y-27632 can be used to study the growth and differentiation of nerve cells, etc.

[0143] BMP4

[0144] Bone morphogenetic protein-4, also known as bone morphogenetic protein (BMP)-4, is a member of the BMPs family. It has a general structural consistency with the overall structure of BMPs in terms of spatial structure, and only has its specificity in the sequence of the mature region. BMP-4 plays an important role in promoting the regeneration and repair of bone tissue. In addition, BMP4 is also closely related to inducing embryonic differentiation, guiding the differentiation of neural stem cells, regulating tumor growth and invasion, and some cardiovascular and cerebrovascular diseases.

[0145] The preproprotein of BMP4 consists of 400 - 500 amino acids and includes three parts: the N-terminal signal peptide, the preprotein folding region, and the C-terminal mature peptide. The carboxyl-terminal mature BMP4 protein can be cleaved from the preproprotein under the action of Furin, PC6, and PC7 to become a highly conserved BMP4 molecule composed of 116 amino acids. Its C-terminal contains 7 cysteine residues, 6 of which form 3 intramolecular disulfide bonds, called cysteine knots. The 7th cysteine can be glycosylated, thereby forming a covalent disulfide bond for dimerization with another monomer, thus forming a bioactive signaling molecule.

[0146] Thyroid hormone

[0147] The thyroid mainly secretes thyroxine T4 and triiodothyronine T3. Thyroxine T4 is a hormone synthesized and secreted by the thyroid gland and is a key hormone for maintaining normal human growth and development. It can promote human growth and development and also enhance human metabolism. Triiodothyronine T3 is a hormone synthesized and secreted by thyroid follicular cells, which can promote the synthesis of triiodothyronine in the body and promote human metabolism.

[0148] Thyroid organoid

[0149] Thyroid organoids are three-dimensional cell cultures cultured in vitro and have similar structures and functions to actual thyroid organs. Thyroid organoids can be used to simulate thyroid physiological functions and disease processes, which helps to study the pathogenesis and treatment methods of thyroid diseases.

[0150] Thyroid organoids have the ability of self-renewal and differentiation, and can continuously proliferate and differentiate during in vitro culture to form tissues with multi-layer cell structures. In addition, thyroid organoids can also be modified by gene editing technology to study the role of genes in thyroid development and disease occurrence.

[0151] By studying thyroid organoids, we can gain a deeper understanding of the physiological and pathological processes of the thyroid gland, providing new ideas and methods for future medical research, drug screening, etc.

[0152] Thyroid organoid culture medium

[0153] The present invention provides a thyroid organoid culture medium, which comprises a basal medium and additives selected from the following group: fibroblast growth factor-2 (FGF2), Y27632, bone morphogenetic protein-4 (BMP-4), or a combination thereof.

[0154] The above specially added components can promote the formation and development of thyroid organoids alone or in combination, and can significantly increase the number of formed thyroid organoids. It is also beneficial to make the generated thyroid organoids closer to the characteristics of thyroid tissue, and can enable more passages and longer passage times.

[0155] In addition, by adjusting the ratio of the above specially added components, it has different effects on the morphology and expression of marker proteins of thyroid organoids, which is beneficial to adjusting the corresponding culture protocol according to experimental or research purposes.

[0156] Preferably, the basal medium is selected from the following group: DMEM medium, Advanced DMEM / F12 medium, or a combination thereof; more preferably, the basal medium is Advanced DMEM / F12 medium.

[0157] Preferably, the thyroid organoid culture medium further comprises one or more components selected from the following group: epidermal growth factor (EGF), R-spondin-1 (RSPO1), Wnt-3a, N-acetylcysteine, nicotinamide, B27, fibroblast growth factor-10 (FGF10), A83-01, forskolin.

[0158] For example, the thyroid organoid culture medium comprises the following components:

[0159] (a) A basal medium (preferably Advanced DMEM / F12 medium);

[0160] (b) Specially added substances selected from the following group: fibroblast growth factor-2 (FGF2), Y27632, bone morphogenetic protein-4 (BMP-4), or a combination thereof; and

[0161] (c) Basic additives selected from the following group: epidermal growth factor (EGF), R-spondin-1 (RSPO1), Wnt-3a, N-acetylcysteine, nicotinamide, B27, fibroblast growth factor-10 (FGF10), A83-01, forskolin, or a combination thereof.

[0162] The preferred concentrations of the components in the thyroid organoid culture medium are as described above, and most preferably, they can be the concentrations of the components in Examples 5 and 6 of the present invention.

[0163] Using the thyroid organoid culture medium of the present invention, the formed thyroid organoids have relatively complete functionality and can have functions substantially consistent with those of the thyroid tissue of a normal organism, such as hormone secretion and regulation functions. It can also induce cells with stemness and can efficiently and stably establish a culture and formation system of thyroid organoids with normal physiological functions.

[0164] Therefore, the thyroid organoid culture medium of the present invention can be used for the following purposes:

[0165] (a) being used as or for formulating a culture medium for culturing thyroid organoids;

[0166] (b) being used as or for formulating an additive for a thyroid organoid culture medium;

[0167] (c) being used for culturing and / or forming adult thyroid stem cells;

[0168] (d) being used as or for formulating a culture medium for culturing adult thyroid stem cells; and / or

[0169] (e) being used as or for formulating an additive for an adult thyroid stem cell culture medium.

[0170] Thyroid organoid culture medium kit

[0171] The present invention also provides a thyroid organoid culture medium kit, and the culture medium kit includes:

[0172] (Z1) A first container and a special additive placed in the first container, and the special additive is selected from the group consisting of fibroblast growth factor-2 (FGF2), Y27632, bone morphogenetic protein-4 (BMP-4), or a combination thereof.

[0173] Preferably, the culture medium kit further includes:

[0174] (Z2) A second container and a basic additive placed in the second container, and the basic additive is selected from the group consisting of epidermal growth factor (EGF), R-spondin-1 (RSPO1), Wnt-3a, N-acetylcysteine, nicotinamide, B27, fibroblast growth factor-10 (FGF10), A83-01, forskolin, or a combination thereof;

[0175] (Z3) The third container and the basal medium placed therein; and / or

[0176] (Z4) The fourth container and the digestive fluid or digestive enzyme placed therein.

[0177] The main components of the digestive fluid or digestive enzyme can be selected from the following group: collagenase, DNase, Dispase, trypsin, or a combination thereof.

[0178] Additionally, the above-mentioned first container, second container, third container, and / or fourth container can be the same or different containers.

[0179] The medium kit can be used to prepare the thyroid organoid medium as described in the first aspect of the present invention. Based on this, the medium kit can further include: an instruction manual, which records the concentrations of the components in the medium kit and the method for preparing the thyroid organoid medium as described in the first aspect of the present invention. The instruction manual can also record the guiding usage concentrations of the components in the medium kit when preparing the thyroid organoid medium as described in the first aspect of the present invention.

[0180] Thyroid organoid culture method

[0181] The present invention also provides a method for culturing thyroid organoids, the method comprising the step of: culturing using the thyroid organoid medium as described in the first aspect of the present invention.

[0182] Preferably, the method comprises the steps of:

[0183] (S1) Provide a thyroid tissue sample to be cultured;

[0184] (S2) Digest and centrifuge the thyroid tissue sample to obtain a thyroid cell pellet;

[0185] (S3) Resuspend the thyroid cell pellet to obtain a thyroid cell suspension;

[0186] (S4) Mix the thyroid cell suspension with Matrigel and then perform cell seeding. After the Matrigel solidifies, add the thyroid organoid medium as described in the first aspect of the present invention for culture; and

[0187] (S5) Under conditions suitable for culturing thyroid organoids, culture for 3 - 14 days to obtain the thyroid organoids.

[0188] The thyroid tissue sample is a normal thyroid tissue sample or a pathological thyroid tissue sample (such as a thyroid cancer tissue sample). When using a normal thyroid tissue sample to culture thyroid organoids, it can be used as a model for in vitro study of normal thyroid; when using a pathological thyroid tissue sample to culture thyroid organoids, it can be used as a model for in vitro study of thyroid pathology.

[0189] Preferably, the method further includes the step of:

[0190] (S6) Analyzing and / or identifying the thyroid organoids.

[0191] The above analysis and / or identification may include:

[0192] (1) Staining analysis (preferably, hematoxylin-eosin staining, which can observe the physiological and pathological structure of the thyroid, such as the unique follicular structure of the thyroid);

[0193] (2) Immunohistochemistry (preferably, immunohistochemical detection of the expression of thyroid-related proteins, such as thyroglobulin, thyroid peroxidase);

[0194] (3) Immunohistocytofluorescence (preferably, immunohistochemical detection of the co-expression of thyroid-related proteins, such as sodium-iodide symporter (an ion channel essential for thyroid hormone synthesis));

[0195] (4) Analysis or identification of the secretion and / or regulatory function of thyroid-related hormones (preferably, such as the secretion of thyroxine T4 and triiodothyronine T3; such as the regulation of T4 and T3 by thyroid-stimulating hormone (TSH));

[0196] (5) Transmission electron microscopy (such as observing the fine structure of the thyroid, including apical microvilli, pseudopods, and intracellular vesicles storing thyroglobulin); and / or

[0197] (6) Single-cell transcriptome sequencing (such as analyzing cell types, the developmental trajectory of thyroid organoids, and stem progenitor cells in thyroid organoids).

[0198] The above analysis and / or identification can be used not only to identify the obtained thyroid organoids but also to verify or study their functional mechanisms.

[0199] The main advantages of the present invention

[0200] 1. Currently, domestic and foreign studies have not confirmed the existence of specific stem cell populations in adult thyroid. In the present invention, however, thyroid organoids are successfully obtained by using a culture medium specially supplemented with FGF2, Y27632, and BMP4 components, and stem cells existing in adult thyroid are first discovered by analyzing single-cell transcriptome sequencing.

[0201] 2. The culture medium formed by specifically adding components of FGF2, Y27632 and BMP4 in the present invention can effectively promote the formation and development of thyroid organoids. Moreover, the culture media formed by separately adding the BMP4 component, separately adding the FGF2 component, and separately adding the Y27632 component can also effectively promote the formation and development of thyroid organoids, significantly increasing the number of formed thyroid organoids.

[0202] 3. Specifically adding components of FGF2, Y27632 and BMP4 in the culture medium is not only beneficial to increasing the number of formed thyroid organoids, making the generated thyroid organoids closer to the characteristics of thyroid tissue, but also can adjust the corresponding culture protocol according to experimental or research purposes by regulating the proportions of the above specifically added components.

[0203] 4. Specifically adding components of FGF2, Y27632 and BMP4 in the culture medium enables more passages and longer passage times.

[0204] 5. By using the components of FGF2, Y27632 and BMP4 specifically added in the culture medium, the thyroid organoids formed in the present invention have relatively complete functionality and can have functions basically consistent with those of thyroid tissue in a normal organism, such as hormone secretion and regulation functions.

[0205] 6. By using the components of FGF2, Y27632 and BMP4 specifically added in the culture medium, the thyroid organoid culture medium formed in the present invention can induce cells with stemness and can efficiently and stably establish a culture and formation system of thyroid organoids with normal physiological functions.

[0206] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0207] Example 1: Thyroid organoid medium 1

[0208] In this embodiment, a thyroid organoid culture medium 1 is provided, which includes a basal medium (such as Advanced DMEM / F12 medium) and various components added according to the formula in Table 1 below (calculated based on the total volume of the culture medium).

[0209] Table 1

[0210]

[0211]

[0212] As shown in Table 1, the thyroid organoid medium 1 does not contain the components of FGF2, Y27632, and BMP4, and is used as a control medium in the present invention.

[0213] Example 2: Thyroid organoid medium 2

[0214] In this embodiment, a thyroid organoid medium 2 is provided, which comprises a basal medium (such as Advanced DMEM / F12 medium) and various components added according to the formulation in Table 2 below (calculated based on the total volume of the medium).

[0215] Table 2

[0216]

[0217] As shown in Table 2, the thyroid organoid medium 2 is specifically added with the component of BMP4, but does not contain the components of FGF2 and Y27632.

[0218] Example 3: Thyroid organoid medium 3

[0219] In this embodiment, a thyroid organoid medium 3 is provided, which comprises a basal medium (such as Advanced DMEM / F12 medium) and various components added according to the formulation in Table 3 below (calculated based on the total volume of the medium).

[0220] Table 3

[0221]

[0222]

[0223] As shown in Table 3, the thyroid organoid medium 3 is specifically added with the component of FGF2, but does not contain the components of Y27632 and BMP4.

[0224] Example 4: Thyroid organoid medium 4

[0225] In this embodiment, a thyroid organoid medium 4 is provided, which comprises a basal medium (such as Advanced DMEM / F12 medium) and various components added according to the formulation in Table 4 below (calculated based on the total volume of the medium).

[0226] Table 4

[0227]

[0228] As shown in Table 4, the thyroid organoid medium 4 is specially added with the component Y27632, but does not contain the components FGF2 and BMP4.

[0229] Example 5: Thyroid organoid medium 5

[0230] In this embodiment, a thyroid organoid medium 5 is provided, which includes a basal medium (such as Advanced DMEM / F12 medium) and various components added according to the formula in Table 5 below (based on the total volume of the medium).

[0231] Table 5

[0232]

[0233]

[0234] As shown in Table 5, the thyroid organoid medium 5 is specially added with the components FGF2 (20 ng / ml), Y27632 (3.2 μg / ml), and BMP4 (20 ng / ml).

[0235] Example 6: Thyroid organoid medium 6

[0236] In this embodiment, a thyroid organoid medium 6 is provided, which includes a basal medium (such as Advanced DMEM / F12 medium) and various components added according to the formula in Table 6 below (based on the total volume of the medium).

[0237] Table 6

[0238]

[0239] As shown in Table 6, the thyroid organoid medium 6 is specially added with the components FGF2 (50 ng / ml), Y27632 (6.4 μg / ml), and BMP4 (10 ng / ml).

[0240] Example 7: Thyroid organoid culture and detection of the number of thyroid organoids formed

[0241] In this embodiment, the thyroid organoid media 1 - 5 in Examples 1 - 5 are respectively used for culturing thyroid organoids.

[0242] The general culture method of thyroid organoids is as follows:

[0243] (S1) Provide a thyroid tissue sample to be cultured;

[0244] (S2) Digest and centrifuge the thyroid tissue sample to obtain a thyroid cell pellet;

[0245] (S3) Resuspend the thyroid cell pellet to obtain a thyroid cell suspension;

[0246] (S4) Mix the thyroid cell suspension with Matrigel and then perform cell seeding. After the Matrigel solidifies (preferably under the condition of 37°C), add the thyroid organoid culture medium as described in the first aspect of the present invention for culture; and

[0247] (S5) Under the conditions suitable for culturing thyroid organoids (preferably in a cell culture incubator at 37°C and 5% CO2), culture for 3 - 14 days to obtain the thyroid organoids.

[0248] Preferably, the step (S1) further includes: removing adipose tissue; washing the thyroid tissue sample with DPBS solution; and / or cutting the thyroid tissue sample into tissue blocks of 2 - 3 mm in size.

[0249] Preferably, the step (S2) further includes: removing the DPBS solution; adding a digestive solution or digestive enzyme (preferably its main components are: collagenase, DNase, Dispase, or a combination thereof) for digestion; and / or centrifuging under centrifugation conditions suitable for thyroid cells (for example, centrifuging at 1000 - 2000 rpm (such as 1500 rpm) or 200 - 400 g (such as 350 g) for 5 - 10 min);

[0250] Preferably, the step (S3) further includes: after resuspending the thyroid cell pellet, passing it through a 100 μM cell sieve, collecting the thyroid cell solution after passing through the cell sieve; centrifuging the thyroid cell solution after passing through the cell sieve, removing the supernatant, and then resuspending the obtained thyroid cell pellet again to obtain a thyroid cell suspension.

[0251] Preferably, the step (S4) further includes: waiting for the Matrigel to solidify in a cell culture incubator at 37°C and 5% CO2.

[0252] Preferably, the step (S5) further includes: replacing the fresh thyroid organoid culture medium as described in the first aspect of the present invention every 2 - 3 days.

[0253] After culturing to obtain thyroid organoids, detect the number of formed thyroid organoids.

[0254] As Figure 1 shown, it shows the number of thyroid organoids obtained by culturing with the thyroid organoid media 1 - 5 in Examples 1 - 5 respectively.

[0255] Compared with the thyroid organoid culture medium 1 without FGF2, Y27632, and BMP4 components, it was found that the number of thyroid organoids formed in the thyroid organoid culture medium 2 with only the BMP4 component added, the thyroid organoid culture medium 3 with only the FGF2 component added, and the thyroid organoid culture medium 4 with only the Y27632 component added was significantly increased, reaching about 1.5 - 2 times the corresponding number of thyroid organoids formed in the thyroid organoid culture medium 1.

[0256] In addition, the number of thyroid organoids formed using the thyroid organoid culture medium 5 in Example 5 was the largest, reaching about 2.7 - 2.8 times the corresponding number of thyroid organoids formed in the thyroid organoid culture medium 1.

[0257] The above results indicate that: (1) Adding the BMP4 component or the FGF2 component or the Y27632 component alone to the culture medium can effectively increase the number of thyroid organoids formed; (2) Adding the three components of FGF2, Y27632, and BMP4 simultaneously to the culture medium can further synergistically increase the number of thyroid organoids formed.

[0258] Example 8: Identification of the structure, morphology, and function of thyroid organoids

[0259] (1) In this example, the thyroid organoids cultured using the thyroid organoid culture medium 1 or 5 in Examples 1 and 5 were subjected to structure, morphology, and function identification.

[0260] As Figure 2 shown, the bright - field images, hematoxylin - eosin staining images, and immunohistochemical images of marker proteins of the thyroid organoids cultured using the thyroid organoid culture medium 1 or 5 in Examples 1 and 5 are respectively shown. Among them, Example 1 is the experimental group using the thyroid organoid culture medium 1 in Example 1, and Example 5 is the experimental group using the thyroid organoid culture medium 5 in Example 5.

[0261] The bright - field image results show that the thyroid organoids in Example 5 are larger in volume; the HE staining results show that the thyroid organoids in Example 5 are closer to the follicular structure of the tissue morphologically; the immunohistochemical results show that more thyroid function proteins such as TG, TPO, and T4 are expressed in the thyroid organoids in Example 5.

[0262] The above results indicate that: The thyroid organoids generated using the culture medium supplemented with FGF2, Y27632, and BMP4 components are closer to the characteristics of thyroid tissue.

[0263] (2) In this example, the thyroid organoids cultured using the thyroid organoid culture medium 5 or 6 in Examples 5 and 6 were subjected to structure, morphology, and function identification.

[0264] As Figure 3 shown, the bright-field images, hematoxylin-eosin staining images, immunohistochemical images of marker proteins, and immunocytofluorescence images of thyroid organoids obtained by culturing with the thyroid organoid culture medium 5 or 6 in Examples 5 and 6 are respectively shown. Among them, Example 5 is the experimental group using the thyroid organoid culture medium 5 in Example 5, and Example 6 is the experimental group using the thyroid organoid culture medium 6 in Example 6.

[0265] The results show that: the proportions of specially added FGF2, Y27632, and BMP4 in the culture medium have different effects on the morphology of thyroid organoids and the expression of marker proteins. Therefore, the special addition of FGF2, Y27632, and BMP4 in the culture medium not only helps to increase the number of formed thyroid organoids and makes the generated thyroid organoids closer to the characteristics of thyroid tissue, but also can adjust the corresponding culture protocol according to experimental or research purposes by regulating the proportions of the above-mentioned specially added components.

[0266] Example 9: Statistics of the minimum number of passages of thyroid organoids

[0267] In this example, the thyroid organoids were passaged using the thyroid organoid culture medium 1 or 5 in Examples 1 and 5 respectively, and the minimum number of passages was counted as shown in Table 7 below:

[0268] Table 7

[0269] Media used Number of passages Thyroid organoid medium 1 10 Thyroid organoid medium 5 15 (at least)

[0270] The results show that: compared with the thyroid organoid culture medium 1, the number of passages and the passage time are more when using the thyroid organoid culture medium 5 for passage.

[0271] Example 10: Identification of the hormone secretion and regulation functions of thyroid organoids

[0272] In this example, the thyroid organoids obtained by culturing with the thyroid organoid culture medium 5 in Example 5 were subjected to hormone secretion and regulation function identification.

[0273] As Figure 4 shown, when TSH stimulation was not added, T3 and T4 could already be detected in the culture medium supernatant, indicating that the formed thyroid organoids have normal hormone secretion physiological functions.

[0274] In addition, after adding TSH stimulation, the concentrations of T3 and T4 detected in the culture medium supernatant were significantly increased, indicating that TSH has a normal regulatory function on the hormone secretion of the formed thyroid organoids.

[0275] The above results show that: the thyroid organoids formed by the present invention have relatively complete functionality and can have functions basically consistent with those of the thyroid tissue of a normal organism.

[0276] Example 11: Single-cell transcriptome sequencing analysis of thyroid organoids

[0277] In this example, the thyroid organoids obtained by culturing with the thyroid organoid medium 5 in Example 5 were subjected to single-cell transcriptome sequencing analysis.

[0278] As Figure 5 shown, a group of thyroid stem cells was found in the formed thyroid organoids, which is the first discovery of stem cells in the adult thyroid.

[0279] The above results indicate that: the thyroid organoid medium of the present invention can induce cells with stemness, and can efficiently and stably establish a culture and formation system of thyroid organoids with normal physiological functions.

[0280] All documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. A thyroid organoid culture medium, characterized in that, The thyroid organoid culture medium includes a basal medium and additives selected from the group consisting of fibroblast growth factor-2 (FGF2), Y27632, bone morphogenetic protein-4 (BMP-4), or a combination thereof.

2. The thyroid organoid culture medium according to claim 1, characterized in that, The basal medium is selected from the group consisting of DMEM medium, Advanced DMEM / F12 medium, or a combination thereof; preferably, the basal medium is Advanced DMEM / F12 medium.

3. The thyroid organoid culture medium according to claim 1 or 2, characterized in that, The thyroid organoid culture medium further includes one or more components selected from the group consisting of epidermal growth factor (EGF), R-spondin-1 (RSPO1), Wnt-3a, N-acetylcysteine, nicotinamide, B27, fibroblast growth factor-10 (FGF10), A83-01, forskolin.

4. A culture medium kit for thyroid organoids, characterized in that, The culture medium kit includes: (Z1) A first container and a special additive placed in the first container, the special additive being selected from the group consisting of fibroblast growth factor-2 (FGF2), Y27632, bone morphogenetic protein-4 (BMP-4), or a combination thereof.

5. The culture medium kit according to claim 4, wherein, The culture medium kit further includes: (Z2) A second container and a basal additive placed in the second container, the basal additive being selected from the group consisting of epidermal growth factor (EGF), R-spondin-1 (RSPO1), Wnt-3a, N-acetylcysteine, nicotinamide, B27, fibroblast growth factor-10 (FGF10), A83-01, forskolin, or a combination thereof.

6. A method for culturing thyroid organoids, characterized in that, The method includes the step of culturing using the thyroid organoid culture medium according to any one of claims 1-3.

7. The method according to claim 6, wherein The method includes the steps of: (S1) Providing a thyroid tissue sample to be cultured; (S2) Digesting and centrifuging the thyroid tissue sample to obtain a thyroid cell pellet; (S3) Resuspending the thyroid cell pellet to obtain a thyroid cell suspension; (S4) Mixing the thyroid cell suspension with Matrigel and then performing cell seeding, and adding the thyroid organoid culture medium according to any one of claims 1-3 for culture after the Matrigel solidifies; and (S5) Culturing for 3-14 days under conditions suitable for culturing thyroid organoids to obtain the thyroid organoids.

8. The method according to claim 6 or 7, characterized in that, The method further includes the step of: (S6) Analyzing and / or identifying the thyroid organoids.

9. The method according to claim 8, wherein The analysis and / or identification includes: (1) Staining analysis (preferably, hematoxylin-eosin staining, which can observe the physiological and pathological structure of the thyroid, such as the unique follicular structure of the thyroid); (2) Immunohistochemistry (preferably, immunohistochemical detection of the expression of thyroid-related proteins, such as thyroglobulin, thyroid peroxidase); (3) Immunohistocytofluorescence (preferably, immunohistochemical detection of co-expression of thyroid-related proteins, such as sodium-iodide symporter (an ion channel essential for thyroid hormone synthesis)); (4) Analysis or identification of the secretion and / or regulatory function of thyroid-related hormones (preferably, such as the secretion of thyroxine T4 and triiodothyronine T3; such as the regulation of T4 and T3 by thyroid-stimulating hormone (TSH)); (5) Transmission electron microscopy (such as observing the fine structure of the thyroid, including apical microvilli, pseudopodia, and intracellular vesicles storing thyroglobulin); and / or (6) Single-cell transcriptome sequencing (such as analyzing cell types in thyroid organoids, the developmental trajectory of thyroid organoids, and stem progenitor cells in thyroid organoids, etc.).

10. Use of a thyroid organoid culture medium as described in any one of claims 1-3, or a culture medium kit as described in claim 4 or 5, said use comprising: (a) being used as or for formulating a culture medium for culturing thyroid organoids; (b) being used as or for formulating an additive for a thyroid organoid culture medium; (c) for culturing and / or forming adult thyroid stem cells; (d) being used as or for formulating a culture medium for culturing adult thyroid stem cells; and / or (e) being used as or for formulating an additive for an adult thyroid stem cell culture medium.

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