Thyroid cancer organoid culture medium, thyroid cancer organoid culture method and drug sensitivity detection method

Abstract

The invention discloses a thyroid cancer organoid culture medium, a thyroid cancer organoid culture method and a drug sensitivity detection method, and belongs to the technical field of organoid models. The thyroid cancer organoid culture medium provided by the invention comprises a basic culture medium and the following components: 0.5 to 2 percent (V / V) of N2, 0.5 to 2 percent (V / V) of penicillin-streptomycin-amphotericin, 0.5 to 5 mM of N-acetylcysteine, 5 to 30 mM of Nicotinamide, 1 to 10 mu M of CHIR99021, 5 to 50 ng / mL of hEGF, 1 to 20 mu M of a TGF-beta receptor inhibitor and 1 to 50 M of a p38 MAPK inhibitor. The thyroid cancer organoid culture medium is utilized in the thyroid cancer organoid culture process, the improvement of a culture method and a drug sensitivity detection method is combined, the organoid construction efficiency reaches up to 90% or above, the thyroid cancer organoid culture and drug sensitivity period can be greatly shortened, and the thyroid cancer organoid culture medium is low in cost and easy to popularize. Good popularization and application prospects are realized.

Description

Technical Field

[0001] This invention belongs to the field of organoid model technology, specifically relating to a culture medium for thyroid cancer organoids, a method for culturing thyroid organoids, and a method for drug sensitivity detection. Background Technology

[0002] Thyroid cancer is the most common malignant tumor of the endocrine system, and its global incidence has been rising steadily in recent years. Its pathological subtypes are complex, and treatment responses and prognoses exhibit significant heterogeneity. Current clinical decisions rely on postoperative pathological risk stratification, with major interventions including lobectomy, central lymph node dissection, selective adjuvant radioactive iodine therapy, and thyroid-stimulating hormone (TSH) suppression therapy. However, in the face of aggressive subtypes (such as poorly differentiated and undifferentiated thyroid carcinoma) or advanced radioactive iodine-refractory lesions, the response rate of existing treatment strategies declines rapidly, and the cumulative drug toxicity problem is prominent, leading to impaired patient quality of life and a bottleneck in prolonging survival. Especially for patients with rare mutations (such as NTRK fusions and BRAF non-V600E mutations), the selection of targeted therapy lacks high-level evidence, resulting in significant blind spots and uncertainties in clinical drug use.

[0003] The aforementioned challenges highlight the urgency of developing precise in vitro drug sensitivity models. Patient-derived organoid models faithfully preserve the tissue structure, cell population heterogeneity, and key signaling pathway activity of the primary tumor, providing an ideal platform for preclinical drug sensitivity profiling analysis. Utilizing organoids from thyroid cancer for drug sensitivity testing (Organoid-based Drug Sensitivity Test, ODST) not only provides clinicians with intuitive in vitro evidence before treatment, predicting patients' actual sensitivity to specific targeted drugs or chemotherapy regimens, thus enabling personalized medicine, but also allows for in-depth exploration of effective therapeutic targets in rare mutation populations, promoting the rapid implementation and optimization of personalized treatment plans. This is of great significance for improving the level of precision treatment for thyroid cancer.

[0004] Despite the immense potential of thyroid cancer organoid technology, its translational application remains limited by two core obstacles: First, the high cost of the culture system. Current protocols heavily rely on various recombinant growth factors and small molecule inhibitors (such as R-spondin1, Noggin, and A83-01), with reagent costs accounting for over 70% of the total cost, severely restricting the technology's accessibility in low- and middle-income healthcare institutions. Second, the high time cost. Existing technologies typically require 3-4 weeks or even longer from sample collection to obtaining stable drug sensitivity results. This long waiting time is particularly problematic for rapidly progressing late-stage patients, potentially causing them to miss the optimal treatment window and significantly diminishing the technology's clinical timeliness and practicality. It is noteworthy that current patents (such as CN114891748A) primarily focus on optimizing culture medium components or improving culture methods, failing to fundamentally resolve the "time-cost" paradox. Therefore, developing a thyroid cancer organoid construction and drug sensitivity technology that is economical, rapid, and stable is of great practical significance for shortening the clinical decision-making waiting period, lowering the technological application threshold, and ultimately benefiting a wider range of patients. This invention is proposed based on this urgent need. Summary of the Invention

[0005] In view of this, one objective of the present invention is to provide a low-cost culture medium for thyroid cancer organoids; another objective of the present invention is to provide an economical and rapid method for culturing thyroid cancer organoids and a method for drug sensitivity detection.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A first aspect of the present invention provides a culture medium for culturing thyroid cancer organoids, comprising a basal culture medium and the following components: 0.5-2% (V / V) N2, 0.5-2% (V / V) penicillin-streptomycin-amphotericidal, 0.5-5 mM N-acetylcysteine, 5-30 mM Nicotinamide, 1-10 μM CHIR99021, 5-50 ng / mL hEGF, 1-20 μM TGF-β receptor inhibitor, 1-50 µM p38MAPK inhibitor. Dissolve the above components in Advanced DMEM / F12 medium to obtain the final product.

[0007] illustrate: N2 is a serum-free cell culture additive that can replace B27 to provide essential hormones and siderophores, maintaining basic cell functions and proliferation.

[0008] N-acetylcysteine ​​acts as an antioxidant, scavenging reactive oxygen species, protecting stem cells from oxidative damage, and promoting cell survival in the early stages of organoid formation.

[0009] Nicotinamide maintains stem cell pluripotency by inhibiting histone deacetylase (HDAC), supporting the self-renewal of thyroid cancer organoids.

[0010] In thyroid cancer organoid culture, N-acetylcysteine, Nicotinamide, N2 and penicillin-streptomycin-amphotericidal constitute an antioxidant-metabolic homeostasis axis, which synergistically inhibits ferroptosis and maintains stem cell characteristics. CHIR99021 is a small molecule Wnt pathway agonist that replaces R-spondin1 to activate β-catenin signaling, driving stem cell proliferation and organoid growth.

[0011] hEGF stimulates the epithelial cell receptor (EGFR) to activate the downstream MAPK pathway, promoting the division of thyroid cancer cells and the expansion of organoids.

[0012] The TGF-β receptor inhibitor selected is A83-01, which works by blocking epithelial-mesenchymal transition (EMT) and preventing excessive proliferation of fibroblasts and organoid differentiation.

[0013] The p38 MAPK inhibitor selected is SB202190, which reduces stress-induced apoptosis and maintains the quiescent state and long-term culture stability of stem cells.

[0014] In thyroid cancer organoid culture, CHIR99021, hEGF, A83-01 and SB202190 constitute a proliferation-survival signaling axis, synergistically activating the Wnt and EGFR pathways and releasing the proliferation brakes induced by TGF-β and stress. The two axes work in parallel to achieve efficient establishment and long-term expansion of organoids.

[0015] In some preferred embodiments of the present invention, the culture medium further comprises 5-20 µM Y-27632.

[0016] The second aspect of this invention provides a method for culturing thyroid cancer organoids, which involves constructing and culturing thyroid cancer organoids using surgical samples from thyroid cancer. The specific technical solution is as follows: (1) Take tissue samples obtained from thyroid cancer surgery, place them in tissue preservation solution, and transport them to the laboratory at 0-8℃; the tissue preservation solution contains the following components: 1-5 mM Glutamax, 1-2% (V / V) penicillin-streptomycin-amphoteric B, 1-3% (V / V) HEPES, 5-10% (V / V) fetal bovine serum, and the remainder is the basic culture medium AdvancedDMEM / F12.

[0017] (2) Immerse the sample in tissue washing solution and incubate at 25°C and 200 rpm for 30 min with shaking. Replace the washing solution every 5 min during this period. The tissue washing solution contains the following components: 1-2% (V / V) penicillin-streptomycin-amphoteric acid B, 0.1-0.5 mg / mL hyaluronidase, and 0.05-0.5 mg / mL DNase I, dissolved in 1×DPBS. Hyaluronic acid is a negatively charged high molecular weight polysaccharide and an important component of the extracellular matrix (ECM). It is widely present in connective tissue, epithelial tissue, and nerve tissue and participates in many biological processes. Studies have found that hyaluronic acid is widely present in the tumor microenvironment. The presence of hyaluronic acid affects subsequent sample processing. The sample washing solution contains 0.1-0.5 mg / mL hyaluronidase, which reduces the large number of bubbles generated by hyaluronic acid during the washing process and makes the ECM framework looser, thereby improving the sample recovery rate and organoid construction efficiency.

[0018] (3) Cut the cleaned sample into small pieces of mushy tissue with a side length of less than 0.3 mm, resuspend it in thyroid cancer organoid culture medium containing Y-27632, and filter it together with the culture medium through a 40-mesh cell sieve. (4) Collect the filtrate and centrifuge to obtain cell pellet. After suspending the cell pellet in thyroid cancer organoid culture medium containing Y-27632, add it to a 6-well plate with ultra-low adsorption and culture. Add 3 ml of thyroid cancer organoid culture medium containing Y-27632 to each well. (5) Incubate overnight at 37°C with shaking at 120 rpm, and replace with the culture medium without Y-27632 after 2 days; (6) Cultured continuously in a shaking environment of 120 rpm and at 37°C, with the culture medium changed every 2 days; cultured for 5-7 days until organoids mature.

[0019] The third aspect of this invention provides a method for detecting drug sensitivity in thyroid cancer organoids, which is performed using thyroid cancer organoids obtained by the culture method described in the second aspect. The specific steps are as follows: Calculate the required number of organoids based on the amount of drug to be tested. Use a wide-mouth pipette tip to draw up the culture medium suspension containing thyroid cancer organoids obtained in the second aspect above, add the drug solution, and then perform drug sensitivity analysis.

[0020] Note: After processing, P0 generation thyroid cancer organoids were first cultured in a medium containing Y-27632. This is because Y-27632 can prevent apoptosis of cells after mechanical dissociation from the tissue matrix by inhibiting the Rho / ROCK signaling pathway, thereby significantly improving the survival rate and colony formation efficiency of P0 generation cells. In subsequent culture, Y-27632 needs to be removed in a timely manner to avoid its long-term inhibition of cytoskeleton remodeling, which would prevent organoids from forming normal lumen or polar structures.

[0021] The beneficial effects of this invention are as follows: This invention provides a method for culturing and susceptibility testing of thyroid cancer organoids. Firstly, based on the pathogenesis of thyroid cancer, a suitable culture medium for the growth of thyroid cancer organoids can be prepared and continuously cultured. The obtained tumor organoids maintain consistency with the original tissue in terms of histopathological structure and tumor heterogeneity. Secondly, the method is low-cost and highly efficient: the cost of 100ml of thyroid cancer organoid culture medium is less than 500 yuan; the organoid construction efficiency is over 90%. Thirdly, it significantly shortens the thyroid cancer organoid culture and susceptibility testing cycle, providing stable susceptibility results in just 7-10 days. This invention, which develops an economical, rapid, and stable thyroid cancer organoid construction and susceptibility testing technology, aims to shorten the clinical decision-making waiting period, lower the technical application threshold, and ultimately benefit a wider range of patients. Attached Figure Description

[0022] Figure 1 The images show the morphological images of thyroid carcinoma organoids cultured in Example 2, Comparative Example 2, and Comparative Example 4, respectively. Figure 2 This is a morphological image of the thyroid carcinoma organoid cultured in Comparative Example 3; Figure 3 HE and immunohistochemical staining images of primary thyroid cancer tissue, from left to right: HE, TTF1, CK19, and Ki67; Figure 4 HE and immunohistochemical staining images of thyroid carcinoma organoids obtained in Example 2, from left to right: HE, TTF1, CK19, and Ki67; Figure 5 The following are statistical charts showing the drug sensitivity results of Example 3, Comparative Example 5, and Comparative Example 6, respectively. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0025] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0026] All reagents and raw materials used in the examples can be purchased from the market or prepared by known methods.

[0027] Example 1: A tissue preservation solution for thyroid cancer, comprising the following components: 2 mM Glutamax, 1% (v / v) penicillin-streptomycin-amphoteric B, 1% (v / v) HEPES, 10% (v / v) fetal bovine serum, with the remainder being the basal medium Advanced DMEM / F12.

[0028] A tissue washing solution for thyroid cancer, comprising the following components: 2% (V / V) penicillin-streptomycin-amphoteric B, 0.1 mg / mL hyaluronidase, 0.05 mg / mL DNase I, dissolved in 1×DPBS.

[0029] A culture medium for thyroid cancer organoids, comprising the following components: 1% (v / v) N2, 1% (v / v) penicillin-streptomycin-amphotericidal, 1.25 mM N-acetylcysteine, 10 mM Nicotinamide, 3 μM CHIR99021, 50 ng / mL hEGF, 5 μM A83-01, 10 µM SB202190. Dissolve the above components in Advanced DMEM / F12 medium to obtain the final product.

[0030] A culture medium for thyroid cancer organoids containing Y-27632 comprises the following components: 1% (v / v) N2, 1% (v / v) penicillin-streptomycin-amphotericidal, 1.25 mM N-acetylcysteine, 10 mM Nicotinamide, 3 μM CHIR99021, 50 ng / mL hEGF, 5 μM A83-01, and 10 µM SB202190. The above components are dissolved in Advanced DMEM / F12 medium to obtain the culture medium.

[0031] Example 2: The thyroid cancer organoids were constructed using the reagents and culture medium prepared in Example 1. The specific steps are as follows: (1) Take tissue samples obtained from thyroid cancer surgery, place them in tissue preservation solution, and transport them to the laboratory at 0-8℃; (2) Immerse the sample in tissue cleaning solution and incubate at 25°C and 200 rpm for 30 min with shaking. Change the cleaning solution every 5 min during this period. (3) Cut the cleaned sample into small pieces of mushy tissue with a side length of less than 0.3 mm, resuspend it in thyroid cancer organoid culture medium containing Y-27632, and filter it together with the culture medium through a 40-mesh cell sieve. (4) Collect the filtrate and centrifuge to obtain cell pellet. After suspending the cell pellet in thyroid cancer organoid culture medium containing Y-27632, add it to a 6-well plate with ultra-low adsorption and culture. Add 3 ml of thyroid cancer organoid culture medium containing Y-27632 to each well. (5) Incubate overnight at 120 rpm and 37°C, and replace with the culture medium without Y-27632 after 2 days; (6) Culture continuously at 120 rpm and 37°C, and change the culture medium every 2 days; culture for 5-7 days until organoids mature.

[0032] Example 3: The thyroid carcinoma organoids obtained in Example 2 were subjected to drug sensitivity testing, and the specific steps are as follows: (1) Count the number of organoids in the culture medium under a microscope and calculate the density. Calculate the required number of organoids based on the required amount of drug to be detected, and take the corresponding culture medium suspension. (2) Under stereomicroscopy, carefully aspirate thyroid cancer organoids into each well of a 96-well plate using a 200μl wide-mouth pipette tip. The number and size of thyroid cancer organoids in each well should be kept as consistent as possible. (3) Carefully aspirate the culture medium from the 96-well plate while observing (avoid organoid loss during this process). (4) Add the pre-prepared drug solution and continue to incubate in a 37°C, 5% CO2 incubator for 48-72 hours; (5) Add CellTiter-Glo 3D according to the manufacturer's instructions and read the value using an ELISA reader; (6) Summarize the drug sensitivity data and output the drug sensitivity results.

[0033] Example 4: HE and immunohistochemical staining identification were performed as follows: After culturing the thyroid cancer organoids obtained in Example 2 for 25 days, the organoids cultured in 6-well plates were removed and fixed with an appropriate amount of 4% paraformaldehyde for 12 hours. After undergoing gradient dehydration with alcohol and clearing with xylene, they were embedded in paraffin and sectioned transversely and longitudinally. At the same time, the original thyroid cancer tissue of this case was also fixed, dehydrated, cleared, and embedded in paraffin. After sectioning, HE and immunohistochemical staining (markers: Ki67, TTF1, CK19) were performed on the organoids and the original tissue, respectively, to identify their morphology and structure.

[0034] Comparative Example 1: A tissue preservation solution for thyroid cancer, comprising the following components: 2 mM Glutamax, 1% (v / v) penicillin-streptomycin-amphoteric B, 1% (v / v) HEPES, 10% (v / v) fetal bovine serum, with the remainder being the basal medium Advanced DMEM / F12.

[0035] A tissue washing solution for thyroid cancer, comprising the following components: 2% (V / V) penicillin-streptomycin-amphoteric B, 0.1 mg / mL hyaluronidase, 0.05 mg / mL DNase I, dissolved in 1×DPBS.

[0036] A sample digestion solution for thyroid cancer contains the following components: 1 mg / mL collagenase IV, 1 U / mL elastase, 0.1 mg / mL DNase I, and 10 µM Y-27632. The solution is prepared by dissolving the above components in Advanced DMEM / F12 medium and should be used immediately. A culture medium for thyroid cancer organoids, comprising the following components: 1% (V / V) N2, 1% (V / V) penicillin-streptomycin-amphotericidal, 1.25 mM N-acetylcysteine, 3 μM CHIR99021, 5 μM A83-01, 10 µM SB202190. Dissolve the above components in Advanced DMEM / F12 medium to obtain the final product.

[0037] A culture medium for thyroid cancer organoids containing Y-27632, comprising the following components: 1% (v / v) N2, 1% (v / v) penicillin-streptomycin-amphotericidal, 1.25 mM N-acetylcysteine, 3 μM CHIR99021, 5 μM A83-01, and 10 µM SB202190. The above components are dissolved in Advanced DMEM / F12 medium to obtain the culture medium.

[0038] Comparative Example 2 The thyroid cancer organoids were constructed using the reagents (except for the sample digestion solution) and culture medium prepared in Comparative Example 1. The specific steps are as follows: (1) Take tissue samples obtained from thyroid cancer surgery, place them in tissue preservation solution, and transport them to the laboratory at 0-8℃; (2) Immerse the sample in tissue cleaning solution and incubate at 25°C and 200 rpm for 30 min with shaking. Change the cleaning solution every 5 min during this period. (3) Cut the cleaned sample into small pieces of mushy tissue with a side length of less than 0.3 mm, resuspend it in thyroid cancer organoid culture medium containing Y-27632, and filter it together with the culture medium through a 40-mesh cell sieve. (4) Collect the filtrate and centrifuge to obtain cell pellet. After suspending the cell pellet in thyroid cancer organoid culture medium containing Y-27632, add it to a 6-well plate with ultra-low adsorption and culture. Add 3 ml of thyroid cancer organoid culture medium containing Y-27632 to each well. (5) Incubate overnight at 120 rpm and 37°C, and replace with the culture medium without Y-27632 after 2 days; (6) Culture continuously at 120 rpm and 37°C, and change the culture medium every 2 days; culture for 5-7 days until organoids mature.

[0039] Comparative Example 3: The thyroid cancer organoids were constructed using the reagents and culture medium prepared in Example 1. The specific steps are as follows: (1) Take tissue samples obtained from thyroid cancer surgery, place them in tissue preservation solution, and transport them to the laboratory at 0-8℃; (2) Immerse the sample in tissue cleaning solution and incubate at 25°C and 200 rpm for 30 min with shaking. Change the cleaning solution every 5 min during this period. (3) Collect the cleaned sample, use a sterile scalpel and surgical scissors to cut the sample into small pieces of mushy tissue with a side length of less than 0.3 mm, transfer the sample to 5 times the sample volume of sample digestion solution, digest at 200 rpm and 37°C on a constant temperature shaker for 3-5 min, and terminate digestion with 10 times the sample digestion solution volume of Advaced DMEM / F12. (4) Collect the digested cell suspension and filter it through a 100 μm filter screen; (5) Centrifuge at 500×g, 4℃ for 8 min, discard the supernatant, collect the sample precipitate, add thyroid cancer culture medium, and after forming a cell suspension, mix thoroughly with Matrigel and drop evenly into the well plate at 30-40 μL / drop. (6) After incubating in a 37℃ incubator for 30 min to allow the matrix gel to fully solidify, remove the well plate and add thyroid cancer organoid culture medium containing Y-27632. Place it in an incubator for continuous culture. After 2 days, replace it with thyroid cancer organoid culture medium without Y-27632. (7) Culture continuously in a 37℃ incubator, change the medium every 2 days, and culture for 7-14 days until the organoids mature.

[0040] Comparative Example 4: The thyroid cancer organoids were constructed using the reagents and culture medium prepared in Example 1. The specific steps are as follows: (1) Take tissue samples obtained from thyroid cancer surgery, place them in tissue preservation solution, and transport them to the laboratory at 0-8℃; (2) Immerse the sample in tissue cleaning solution and incubate at 25°C and 200 rpm for 30 min with shaking. Change the cleaning solution every 5 min during this period. (3) Cut the cleaned sample into small pieces of mushy tissue with a side length of less than 0.3 mm, resuspend it in thyroid cancer organoid culture medium containing Y-27632, and filter it together with the culture medium through a 40-mesh cell sieve. (4) Collect the filtrate and centrifuge to obtain cell pellet. After suspending the cell pellet in thyroid cancer organoid culture medium containing Y-27632, add it to a 6-well plate with ultra-low adsorption and culture. Add 3 ml of thyroid cancer organoid culture medium containing Y-27632 to each well. (5) Incubate at 37°C overnight, and replace with the culture medium without Y-27632 after 2 days; (6) Incubate continuously at 37℃ under static conditions, and change the culture medium every 2 days; incubate for 8-12 days.

[0041] Comparative Example 5: The thyroid carcinoma organoids obtained from Comparative Example 3 were subjected to drug sensitivity testing. The specific steps are as follows: (1) The culture medium in the culture plate was blown up and down to break the Matrigel, and the organoid suspension was transferred to a 15 mL centrifuge tube; (2) To help remove Matrigel, add 10 mL of pre-cooled DMEM medium. Centrifuge at 1300 rpm for 5 min at 4°C. (3) Remove the supernatant, add organoid digestion solution to the precipitate and mix well. Incubate at 37°C for 5-10 min until the organoid cell clusters are digested into single cells. Centrifuge at 1300 rpm for 5 min at 4°C. (4) Resuspend in 5 mL of DMEM medium (depending on the amount of precipitate), and take 10 µL of the resuspended solution for counting; (5) Calculate the required number of organoids based on the required number of drugs to be tested, and centrifuge the corresponding culture medium suspension at 1300 rpm for 5 min at 4℃. (6) Add the calculated thyroid cancer organoid culture medium and Matrigel, inoculate the suspension into 96-well plates / 384-well plates, and then place the 96-well plates / 384-well plates inoculated with organoids in a 37°C incubator for 5-7 days until organoids are formed. (7) After organoid formation, add the pre-prepared drug solution and continue to incubate in a 37℃ 5% CO2 incubator for 48-72 hours; (8) Add CelltiterGlo 2.0 according to the manufacturer's instructions and read the value using an enzyme-linked immunosorbent assay (ELISA) reader; (9) Summarize the drug sensitivity data and output the drug sensitivity results.

[0042] Note: Due to limitations in the growth environment of organoids and the need to maintain the uniformity of organoid morphology and size during drug sensitivity testing, a new round of gelation, passage, and re-culture is required for organoid samples before drug sensitivity testing. Drug sensitivity testing can only be performed after the organoids have grown to a certain extent.

[0043] Comparative Example 6: The thyroid carcinoma organoids obtained in Example 2 were subjected to drug sensitivity testing, and the specific steps are as follows: (1) Count the number of organoids in the culture medium under a microscope and calculate the density. Calculate the required number of organoids based on the required amount of drug to be detected, and take the corresponding culture medium suspension. (2) Under stereomicroscopy, carefully aspirate thyroid cancer organoids into each well of a 96-well plate using a 200 μl ordinary pointed pipette tip. The number and size of thyroid cancer organoids in each well should be kept as consistent as possible. (3) Carefully aspirate the culture medium from the 96-well plate while observing (avoid organoid loss during this process). (4) Add the pre-prepared drug solution and continue to incubate in a 37°C, 5% CO2 incubator for 48-72 hours; (5) Add CellTiter-Glo 3D according to the manufacturer's instructions and read the value using an ELISA reader; (6) Summarize the drug sensitivity data and output the drug sensitivity results.

[0044] Figure 1 From left to right, these are morphological images of the cultures obtained in Example 2, Comparative Example 2, and Comparative Example 4, respectively. Figure 2 This is a morphological diagram of the culture obtained in Comparative Example 3. Figure 1 As shown, compared with Comparative Examples 2 and 4, the organoids obtained in Example 1 are plump, have smooth edges, and are numerous, while the cultures obtained in Comparative Examples 2 and 4 have noticeably less smooth edges, indicating poor self-assembly. In particular, the central region of the culture in Comparative Example 2 shows obvious metabolic stagnation or necrosis. Therefore, the culture effect of Example 2 is significantly better than that of Comparative Examples 2 and 4, indicating that changing the culture medium formula or altering the culture conditions in steps (5) and (6) during organoid construction directly affects the culture effect of thyroid cancer organoids. Compared with the static culture in Comparative Example 4, Example 2 of this invention uses a oscillating environment at a certain rotation speed for culture. This can effectively prevent cell sedimentation and necrosis by providing gentle and uniform fluid shear force, simulating the physical environment of tissue fluid flow in vivo, while also helping to maintain internal nutrient exchange and metabolic waste excretion. Although the organoids obtained in Example 2 and Comparative Example 3 are not completely identical in morphology, both are plump, numerous, and can be cultured to a large size, indicating that the organoid culture effects of both are superior.

[0045] Figure 3 , Figure 4 HE and immunohistochemical staining results were shown for the original tissue and the organoids obtained in Example 2, respectively. HE staining results showed that the cells in the original thyroid cancer tissue and the organoids cultured in Example 2 were similar in terms of nucleus, cytoplasm, and nucleocytoplasmic ratio. Immunohistochemical results showed that the expression of three markers in the organoids obtained in Example 2 was consistent with that in the original tissue: TTF1 and CK19 were positive, while Ki67 was negative. These results indicate that the thyroid cancer organoids constructed in Example 2 can better retain the genetic characteristics and biological behavior of the original tissue. This is because the fluid shear force generated by the 120 rpm oscillation condition effectively promotes the maintenance of the organoid's dense spherical shape and phenotype. Figure 5 From left to right, the charts show the drug sensitivity results for Example 3, Comparative Example 5, and Comparative Example 6. In the charts, drug 1 is canvatinib, drug 2 is toremifene, and drug 3 is exemestane. Figure 5As shown, the drug sensitivity results of Example 3 and Comparative Example 5 show a consistent trend and good data parallelism between replicates; conversely, the drug sensitivity results of Comparative Example 6 are inconsistent with those of Example 3 and Comparative Example 5, and the data parallelism between replicates is poor. This indicates that compared with Example 3, replacing the "wide-mouth pipette tip" with a "normal pointed pipette tip" in step (2) of Comparative Example 6 directly affects the accuracy of the drug sensitivity results. This is because the shearing force of the normal pointed pipette tip during the aspiration process will cause mechanical damage to the organoids, thereby directly affecting the overall state and cell viability of the organoids. On the other hand, the time required for Example 3 and Comparative Example 5 from primary tissue treatment to drug sensitivity result output is approximately 9 days and 20 days, respectively. (Both are averages). Therefore, although the drug sensitivity results of the two are consistent, in terms of time efficiency, Example 3 is significantly better than Comparative Example 5. The main reason is that, apart from the slightly shorter organoid formation time, the organoids cultured in Example 2 can be directly subjected to drug sensitivity testing in Example 3 without the need for time-consuming organoid digestion, passage, and other steps. On the other hand, in Comparative Example 5, due to the limitations of the organoid growth environment and the need to maintain the uniformity of organoid morphology and size during drug sensitivity testing, a new round of gelation, passage, and re-culture of organoid samples is required before drug sensitivity testing. Drug sensitivity testing can only be performed after the organoids have grown to a certain extent, which greatly prolongs the time cycle.

[0046] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A culture medium for thyroid cancer organoids, characterized in that, It includes the basal culture medium and the following components: 0.5-2% (V / V) N2, 0.5-2% (V / V) penicillin-streptomycin-amphotericidal, 0.5-5 mM N-acetylcysteine, 5-30 mM Nicotinamide, 1-10 μM CHIR99021, 5-50 ng / mL hEGF, 1-20 μM TGF-β receptor inhibitor, 1-50 µM p38MAPK inhibitor.

2. The thyroid cancer organoid culture medium according to claim 1, characterized in that, The basal culture medium is Advanced DMEM / F12 medium.

3. The thyroid cancer organoid culture medium according to claim 1, characterized in that, The TGF-β receptor inhibitor is A83-01.

4. The thyroid cancer organoid culture medium according to claim 1, characterized in that, The p38MAPK inhibitor is SB202190.

5. The thyroid cancer organoid culture medium according to any one of claims 1 to 4, characterized in that, It also includes Y-27632, the content of which is 5-20 µM.

6. A method for culturing thyroid cancer organoids, characterized in that, Includes the following steps: Obtain thyroid tissue samples to be cultured and preserve them in tissue preservation solution; After washing, the thyroid tissue sample was minced into small, meat-like pieces, resuspended in the thyroid cancer organoid culture medium as described in claim 5, then filtered through a sieve, centrifuged, and the cell pellet was collected. The cell pellet was then resuspended in the thyroid cancer organoid culture medium as described in claim 5 and cultured. The culture method is as follows: The organoids were cultured overnight at 37°C in a shaking environment; after 2 days, the culture medium was replaced with the thyroid cancer organoid culture medium as described in any one of claims 1 to 4 and the culture medium was replaced every 1 to 3 days. The organoids were cultured for 5 to 7 days until they matured, and thyroid cancer organoids were obtained.

7. The cultivation method according to claim 6, characterized in that, The tissue preservation solution contains the following components in the following amounts: 1-5 mM Glutamax, 1-2% (V / V) penicillin-streptomycin-amphoteric B, 1-3% (V / V) HEPES, 5-10% (V / V) fetal bovine serum, and the balance being the basal medium Advanced DMEM / F12.

8. The cultivation method according to claim 6, characterized in that, The thyroid tissue sample was cleaned using a tissue cleaning solution containing the following components: 1-2% (V / V) penicillin-streptomycin-amphoteric B, 0.1-0.5 mg / mL hyaluronidase, and 0.05-0.5 mg / mL DNase I, dissolved in 1×DPBS.

9. A method for detecting drug sensitivity in thyroid cancer organoids, characterized in that, It was accomplished using thyroid cancer organoids obtained by the culture method described in any one of claims 6-8.

10. The method for detecting drug sensitivity in thyroid cancer organoids according to claim 9, characterized in that, The procedure includes the following steps: calculating the required number of organoids based on the required amount of drug to be detected; using a wide-mouth pipette tip to draw up a culture medium suspension containing thyroid cancer organoids as described in any of claims 6-8; adding the drug solution for culturing; and then performing drug sensitivity analysis.

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