A lung squamous carcinoma organoid culture medium and culture method

Abstract

The application provides a lung squamous carcinoma organoid culture medium and a culture method, the lung squamous carcinoma organoid culture medium is composed of R-spondin-1, Noggin, EGF, FGF10, FGF7, A83-01, Y-27632, Nicotinamide, Insulin, B27 Supplement, N-Acetylcysteine, Alpelisib, GlutaMAX, primary cell antibiotic and the like. The lung squamous carcinoma organoid culture medium can efficiently culture lung squamous carcinoma tissues and successfully obtain organoids, the formed organoids are highly similar to primary lung squamous carcinoma tissues in cell type, polarity and spatial hierarchical structure, and truly reproduce the microscopic pathological characteristics of tumors. Moreover, the organoids can be stably subcultured for a long time, and provide an ideal and reliable preclinical model for the pathogenesis research, personalized drug screening and new therapy development of lung squamous carcinoma.

Description

Technical Field

[0001] This invention relates to the field of organoid culture technology, specifically to a culture medium and method for lung squamous cell carcinoma organoids. Background Technology

[0002] Squamous cell carcinoma of the lung is one of the major subtypes of lung cancer, accounting for approximately 25%-30% of non-small cell lung cancers. Its incidence is closely related to smoking. Originating from squamous metaplasia of the bronchial epithelium, squamous cell carcinoma is highly heterogeneous and aggressive, with limited clinical treatment options and a poor prognosis. Currently, treatment for squamous cell carcinoma mainly relies on surgery, chemotherapy, and radiotherapy, but drug resistance and recurrence are prominent issues, and there is a lack of effective individualized treatment models.

[0003] Organoids are tissue analogs with a certain spatial structure formed by in vitro three-dimensional (3D) culture of adult stem cells or pluripotent stem cells. They are highly similar to the corresponding organs in terms of cellular components and tissue structure, and have become an important tool for disease research, drug screening and precision medicine.

[0004] Lung squamous cell carcinoma and lung adenocarcinoma have different origins and pathological characteristics, resulting in significant differences in their biological characteristics, including growth characteristics, invasiveness, and metastasis rate. Therefore, the composition of the culture medium during organoid culture has a significant impact on different pathological types of lung cancer. However, there is currently a lack of publicly available culture medium formulations specifically for lung squamous cell carcinoma.

[0005] Therefore, it is of great significance to provide an organoid culture medium with good stability that is suitable for long-term passage culture of lung squamous cell carcinoma and its application method. Summary of the Invention

[0006] In view of this, and to address the gaps in existing lung squamous cell carcinoma organoid culture technology, this invention provides a lung squamous cell carcinoma organoid culture medium and culture method.

[0007] To achieve the above technical objectives, the technical solution adopted in this application is as follows: In a first aspect, the present invention provides a lung squamous cell carcinoma organoid culture medium, wherein the lung squamous cell carcinoma organoid culture medium comprises, in a final concentration: 50-500 ng / mL R-spondin-1, 10-500 ng / mL Noggin, 10-300 ng / mL EGF, 1-50 μg / mL FGF10, 5-200 ng / mL FGF7, 0.5-10 μM A83-01, 1-20 μM Y-27632, 1-30 mM Nicotinamide, 1-50 μg / mL Insulin, 1-2× B27 Supplement, 0.5-20 mM N-Acetylcysteine, 0.1-1 μM Alpelisib, 1× GlutaMAX, and 20-500 μg / mL primary cell antibiotic; all of the above components are dissolved in DMEM / F12 culture medium.

[0008] Preferably, the lung squamous cell carcinoma organoid culture medium comprises, in final concentration: 100-300 ng / mL R-spondin-1, 50-300 ng / mL Noggin, 50-200 ng / mL EGF, 5-20 μg / mL FGF10, 20-100 ng / mL FGF7, 0.5-8 μM A83-01, 1-15 μM Y-27632, 1-25 mM Nicotinamide, 5-30 μg / mL Insulin, 1-2× B27 Supplement, 1-15 mM N-Acetylcysteine, 0.1-0.8 μM Alpelisib, 1× GlutaMAX, and 50-300 μg / mL primary cell antibiotic; all of the above components are dissolved in DMEM / F12 culture medium.

[0009] More preferably, the lung squamous cell carcinoma organoid culture medium comprises, in final concentration: 200-300 ng / mL R-spondin-1, 80-150 ng / mL Noggin, 50-100 ng / mL EGF, 5-10 μg / mL FGF10, 25-60 ng / mL FGF7, 1-5 μM A83-01, 5-15 μM Y-27632, 5-20 mM Nicotinamide, 5-20 μg / mL Insulin, 1-1.5× B27 Supplement, 1-10 mM N-Acetylcysteine, 0.1-0.3 μM Alpelisib, 1× GlutaMAX, and 100-200 μg / mL primary cell antibiotic; all of the above components are dissolved in DMEM / F12 culture medium.

[0010] Secondly, the present invention provides the application of the lung squamous cell carcinoma organoid culture medium described in the first aspect in the preparation of lung squamous cell carcinoma organoids.

[0011] Thirdly, the present invention provides a method for culturing lung squamous cell carcinoma organoids, the method comprising: processing the obtained lung squamous cell carcinoma tissue into cell clusters, and then resuspending the cell clusters in the culture medium described in the first aspect; further mixing the cell resuspension with matrix gel, and after solidification, adding the culture medium described in the first aspect for constant temperature culture to obtain lung squamous cell carcinoma organoids.

[0012] Preferably, the cell concentration in the cell resuspension is (0.5~5.0)×10⁻⁶. 3 The concentration of cells / μl is preferably (2.0~5.0)×10⁻⁶. 3 per μl.

[0013] Preferably, the volume ratio of the cell resuspension to the matrix gel is 1:1~2, more preferably 1:1.3~1.5.

[0014] Preferably, the control parameters for the isothermal culture include: temperature 37±1℃, 5% CO2, and culture time of 3~7 days.

[0015] Optionally, the culture method further includes: passage culture of the obtained lung squamous cell carcinoma organoids.

[0016] Fourthly, the present invention provides the application of lung squamous cell carcinoma organoids obtained by the culture method described in the third aspect in preparing preclinical models for studying the pathogenesis of lung squamous cell carcinoma and / or screening drugs for the treatment of lung squamous cell carcinoma and / or developing therapies for lung squamous cell carcinoma.

[0017] Compared with the prior art, the present invention has the following beneficial effects: Firstly, the lung squamous cell carcinoma organoid culture medium provided by this invention contains specific components required for the culture of lung squamous cell carcinoma tissue organoids, enabling efficient culture of lung squamous cell carcinoma tissue. During the culture process, the formed organoids are highly similar to primary lung squamous cell carcinoma tissue in cell type, polarity, and spatial hierarchical structure, realistically reproducing the microscopic pathological features of the tumor. Furthermore, the organoids can be stably passaged for a long period, maintaining the same gene lineage, tissue structure, and drug sensitivity characteristics as the primary tissue, providing an ideal and reliable preclinical model for the study of the pathogenesis of lung squamous cell carcinoma, personalized drug screening, and the development of novel therapies.

[0018] Secondly, the lung squamous cell carcinoma organoid culture medium of the present invention does not require the addition of bovine serum albumin (FBS), the most common component in existing organoid cultures, thus completely avoiding batch-to-batch variations, cytotoxicity, and interference from unknown inhibitors caused by the use of FBS. Furthermore, the composition is well-defined and simple, resulting in lower costs. The various components work synergistically to create a microenvironment conducive to the self-renewal and growth of lung squamous cell carcinoma stem cells. Attached Figure Description

[0019] The invention will be further illustrated with reference to the accompanying drawings, but the embodiments in the drawings do not constitute any limitation on the invention. For those skilled in the art, other drawings can be obtained based on the following drawings without any creative effort.

[0020] Figure 1 This is an optical microscope image of lung squamous cell carcinoma organoids obtained by culturing them at low cell concentrations using the lung squamous cell carcinoma organoid culture medium from Example 1 in Example 5 of the present invention.

[0021] Figure 2 This is an optical microscope image of lung squamous cell carcinoma organoids obtained by culturing them at high cell concentrations using the lung squamous cell carcinoma organoid culture medium from Example 1 in Example 5 of the present invention.

[0022] Figure 3 The images shown are optical microscope images of lung squamous cell carcinoma organoids cultured using the culture medium of Example 1 in Example 6 of the present invention, where A and B are images at different magnifications.

[0023] Figure 4 The images shown are optical microscope images of lung squamous cell carcinoma organoids cultured using the culture medium from Example 2 in Example 7 of this invention, where A and B are images at different magnifications.

[0024] Figure 5 This is an optical microscope image of a lung squamous cell carcinoma organoid cultured using the culture medium from Example 3 in Example 8 of the present invention.

[0025] Figure 6 This is an optical microscope image of a lung squamous cell carcinoma organoid cultured using the culture medium from Example 4 in Example 9 of the present invention.

[0026] Figure 7 HE staining image of lung squamous cell carcinoma organoids obtained in Example 5 of this invention.

[0027] Figure 8 This is an IHC staining image of lung squamous cell carcinoma organoids obtained in Example 5 of the present invention.

[0028] Figure 9 This is an optical microscope image of lung squamous cell carcinoma organoid cultured and passaged to the third generation in Example 11 of the present invention.

[0029] Figure 10 This is an optical microscope image of lung squamous cell carcinoma organoid cultured to the 5th generation in Example 12 of the present invention.

[0030] Figure 11 This is an optical microscope image of lung adenocarcinoma tissue cultured using the lung squamous cell carcinoma organoid culture medium from Example 1 in Example 13 of the present invention.

[0031] Figure 12 This is an optical microscope image of lung squamous cell carcinoma tissue cultured for 5 days in Comparative Example 1 of this invention.

[0032] Figure 13 This is an optical microscope image of lung squamous cell carcinoma tissue cultured for 5 days in Comparative Example 2 of this invention.

[0033] Figure 14 This is an optical microscope image of lung squamous cell carcinoma tissue cultured for 5 days in Comparative Example 3 of this invention. Detailed Implementation

[0034] A specific embodiment of the present invention provides a lung squamous cell carcinoma organoid culture medium, which comprises, in a final concentration: 50-500 ng / mL R-spondin-1, 10-500 ng / mL Noggin, 10-300 ng / mL EGF, 1-50 μg / mL FGF10, 5-200 ng / mL FGF7, 0.5-10 μM A83-01, 1-20 μM Y-27632, 1-30 mM Nicotinamide, 1-50 μg / mL Insulin, 1-2× B27 Supplement, 0.5-20 mM N-Acetylcysteine, 0.1-1 μM Alpelisib, 1× GlutaMAX, and 20-500 μg / mL primary cell antibiotic; all of the above components are dissolved in DMEM / F12 culture medium. Within the range of the composition of the culture medium, the present invention can successfully culture and obtain spherical lung squamous cell carcinoma organoids, which can be passaged at least three times.

[0035] The lung squamous cell carcinoma organoid culture medium of some preferred embodiments of the present invention comprises, in final concentration: 100-300 ng / mL R-spondin-1, 50-300 ng / mL Noggin, 50-200 ng / mL EGF, 5-20 μg / mL FGF10, 20-100 ng / mL FGF7, 0.5-8 μM A83-01, 1-15 μM Y-27632, 1-25 mM Nicotinamide, 5-30 μg / mL Insulin, 1-2× B27 Supplement, 1-15 mM N-Acetylcysteine, 0.1-0.8 μM Alpelisib, 1× GlutaMAX, and 50-300 μg / mL primary cell antibiotic; all of the above components are dissolved in DMEM / F12 culture medium. With further optimization of the composition, lung squamous cell carcinoma organoids can be cultured to achieve faster and greater cell proliferation and a more stable passage process.

[0036] The lung squamous cell carcinoma organoid culture medium of the further preferred embodiment of the present invention comprises, in final concentration: 200-300 ng / mL R-spondin-1, 80-150 ng / mL Noggin, 50-100 ng / mL EGF, 5-10 μg / mL FGF10, 25-60 ng / mL FGF7, 1-5 μM A83-01, 5-15 μM Y-27632, 5-20 mM Nicotinamide, 5-20 μg / mL Insulin, 1-1.5× B27 Supplement, 1-10 mM N-Acetylcysteine, 0.1-0.3 μM Alpelisib, 1× GlutaMAX, and 100-200 μg / mL primary cell antibiotic; all of the above components are dissolved in DMEM / F12 culture medium. After further optimization of the composition, the spherical structure of lung squamous cell carcinoma organoids obtained by culture is more uniform, with smooth and clear edges, and it proliferates rapidly. It can be passaged at least 5 times.

[0037] Furthermore, in this invention, different cell densities in the cell resuspension are correlated with the organoid formation rate and activity; excessively high or low densities may lead to low organoid formation rates, structural abnormalities, or central necrosis. When using the aforementioned lung squamous cell carcinoma organoid culture medium for cell culture, the cell concentration is controlled at (0.5~5.0) × 10⁻⁶. 3 The concentration of [number] cells / μl is preferably controlled within (2.0~5.0)×10⁻⁶. 3 A ratio of 1 organoid per μl ensures that the resulting organoids are structurally stable, uniform in size, and have suitable proliferation potential.

[0038] Furthermore, for the culture of lung squamous cell carcinoma organoids, the volume ratio of cell resuspension to matrix gel is 1:1~2, preferably 1:1.3~1.5, to ensure that the gel solidifies rapidly and fully encapsulates the cells, providing appropriate mechanical support, establishing cell polarity, and promoting intercellular interactions. This can better maintain the heterogeneity of lung squamous cell carcinoma cells, squamous differentiation characteristics (such as keratinization), and the hypoxic necrotic core inside the tumor.

[0039] In the description of this invention, it should be noted that the lung squamous cell carcinoma organoid is a "miniature lung squamous cell carcinoma tumor" with a three-dimensional spherical structure that is cultured in vitro. It retains the complex structural features and typical pathological features of the original lung squamous cell carcinoma tumor and can provide a powerful tool for biological research, disease modeling and drug testing of lung squamous cell carcinoma.

[0040] Furthermore, unless specific conditions are specified in the examples, they should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0041] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0042] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0043] Example 1 This embodiment provides a lung squamous cell carcinoma organoid culture medium, the composition of which, according to the final concentration, is as follows: 200 ng / mL R-spondin-1, 100 ng / mL Noggin, 80 ng / mL EGF, 10 μg / mL FGF10, 50 ng / mL FGF7, 5 μM A83-01, 10 μM Y-27632, 10 mM Nicotinamide, 10 μg / mL Insulin, 1× B27 Supplement, 2 mM N-Acetylcysteine, 0.1 μM Alpelisib, 1× GlutaMAX, and 200 μg / mL primary cell antibiotics were all dissolved in DMEM / F12 culture medium.

[0044] Example 2 This embodiment provides a lung squamous cell carcinoma organoid culture medium, the composition of which, according to the final concentration, is as follows: 300 ng / mL R-spondin-1, 150 ng / mL Noggin, 50 ng / mL EGF, 5 μg / mL FGF10, 30 ng / mL FGF7, 2 μM A83-01, 5 μM Y-27632, 20 mM Nicotinamide, 20 μg / mL Insulin, 1.5× B27 Supplement, 5 mM N-Acetylcysteine, 0.3 μM Alpelisib, 1× GlutaMAX, and 100 μg / mL primary cell antibiotics were all dissolved in DMEM / F12 culture medium.

[0045] Example 3 This embodiment provides a lung squamous cell carcinoma organoid culture medium, the composition of which, according to the final concentration, is as follows: 100 ng / mL R-spondin-1, 50 ng / mL Noggin, 150 ng / mL EGF, 20 μg / mL FGF10, 100 ng / mL FGF7, 8 μM A83-01, 3 μM Y-27632, 10 mM Nicotinamide, 30 μg / mL Insulin, 2× B27 Supplement, 1 mM N-Acetylcysteine, 0.5 μM Alpelisib, 1× GlutaMAX, and 50 μg / mL primary cell antibiotics were all dissolved in DMEM / F12 culture medium.

[0046] Example 4 This embodiment provides a lung squamous cell carcinoma organoid culture medium, the composition of which, according to the final concentration, is as follows: 50 ng / mL R-spondin-1, 20 ng / mL Noggin, 30 ng / mL EGF, 50 μg / mL FGF10, 100 ng / mL FGF7, 0.5 μM A83-01, 10 μM Y-27632, 30 mM Nicotinamide, 50 μg / mL Insulin, 2× B27 Supplement, 20 mM N-Acetylcysteine, 0.8 μM Alpelisib, 1× GlutaMAX, and 300 μg / mL primary cell antibiotics were all dissolved in DMEM / F12 culture medium.

[0047] Example 5 This embodiment provides a method for culturing lung squamous cell carcinoma organoids, which uses the lung squamous cell carcinoma organoid culture medium of Example 1. The culture method includes the following steps: The lung squamous cell carcinoma specimen in this embodiment was obtained from a patient with advanced lung squamous cell carcinoma. The patient was a male, diagnosed at age 74, with a Ki-67 positivity rate of approximately 30%, and the tumor was moderately differentiated. He had a history of smoking. His initial clinical stage was stage III, and his TNM stage was T4N2M0. The diagnosis of advanced lung squamous cell carcinoma is based on a comprehensive assessment of clinical imaging and pathological examination results. The pathological diagnostic criteria for lung squamous cell carcinoma include the presence of squamous differentiation features in the tumor tissue and the expression of squamous cell carcinoma-related markers, including but not limited to p40, p63, and CK5 / 6, while simultaneously showing no or low expression of adenocarcinoma-related markers.

[0048] With the patient's informed consent obtained, a biopsy tissue was taken from the primary lesion in the patient's lung via percutaneous lung puncture. The fresh tumor tissue, approximately 0.2 × 0.2 × 2 cm in size, was used as the starting sample for organoid culture of squamous cell carcinoma of the lung in this embodiment.

[0049] S1. Transfer the obtained lung squamous cell carcinoma specimen into a centrifuge tube, then wash with sterile saline solution for 30 seconds, remove the supernatant, and wash again with sterile saline solution. Repeat the washing process three times to remove impurities from the tissue surface. In a biosafety cabinet, transfer the sample to a 6 cm culture dish and use sterilized surgical scissors on ice to mince the tissue to a size of 1-3 mm. 3 Add 5 mL of type III collagenase and digest at 37°C with shaking for 30 min. After digestion, add 5 mL of DMEM / F12 medium to terminate digestion. Centrifuge at 1000 rpm for 5 min, carefully remove the supernatant to obtain cell clusters with 3-50 cells each. Divide the cell clusters into two parts and resuspend the cells in the lung squamous cell carcinoma organoid culture medium of Example 1 until the cell concentration is 2*10⁻⁶ cells / mL. 4 / 20μl and 5*10 4 / 20μl; S2. Mix the two portions of cell resuspension with Matrigel separately and then plate them. The volume ratio of each portion of cell resuspension to Matrigel is 1:1.5, and each droplet is 20 μL for seeding. S3. After the two portions of the mixed gel from step S2 have solidified for 5 minutes, add the lung squamous cell carcinoma organoid culture medium from Example 1 to each portion. Then, incubate them in a constant temperature incubator at 37°C and 5% CO2 concentration for 5 days, changing the culture medium every 2 days to obtain the lung squamous cell carcinoma organoids. 2*10 4 The culture results at a cell concentration of 20 μl are as follows: Figure 1 As shown, 5*10 4 The culture results at a cell concentration of 20 μl are as follows: Figure 2 As shown, both cell concentrations yielded lung squamous cell carcinoma organoids with intact spherical structures and smooth edges. However, compared to lower cell concentrations, higher cell concentrations resulted in organoids with relatively larger volumes and smaller inter-organoid distances. Appropriately decreasing or increasing the cell culture concentration had some impact on the size and spacing of the resulting organoids, but did not affect the final organoid formation. Therefore, this invention preferably controls the cell concentration in the cell resuspension at (2.0~5.0) × 10⁻⁶. 3 The concentration of cells / μl can be appropriately increased to (0.5~5)×10⁻⁶. 3 per μl.

[0050] Example 6 This embodiment provides a method for culturing lung squamous cell carcinoma organoids, which uses the lung squamous cell carcinoma organoid culture medium of Example 1. The culture method includes the following steps: The lung squamous cell carcinoma specimen in this embodiment was obtained from a patient with advanced lung squamous cell carcinoma. The patient was a male, diagnosed at age 76, with a Ki-67 positivity rate of approximately 50%, poorly differentiated tumor, and a history of smoking. His initial clinical stage was IV, and his TNM stage was T3N2M1. The diagnostic methods and criteria for advanced lung squamous cell carcinoma are as described in Example 5. With the patient's informed consent, a percutaneous lung biopsy was performed on the primary lesion in the patient's lung. A fresh tumor tissue of approximately 0.2 × 0.2 × 2 cm was used as the starting sample for organoid culture of lung squamous cell carcinoma in this embodiment.

[0051] S1. Transfer the obtained lung squamous cell carcinoma specimen into a centrifuge tube, then wash with sterile saline solution for 30 seconds, remove the supernatant, and wash again with sterile saline solution. Repeat the washing process three times to remove impurities from the tissue surface. In a biosafety cabinet, transfer the sample to a 6 cm culture dish and use sterilized surgical scissors on ice to mince the tissue to a size of 1-3 mm. 3 Add 5 mL of type III collagenase and digest at 37°C with shaking for 30 min. After digestion, add 5 mL of DMEM / F12 medium to terminate digestion. Centrifuge at 1000 rpm for 5 min, carefully remove the supernatant to obtain cell clusters with 3-50 cells per cluster. Resuspend the cells in the lung squamous cell carcinoma organoid culture medium from Example 1 to a cell concentration of 5*10⁻⁶ cells / mL. 4 / 20μl; S2. After mixing the cell resuspension with Matrigel, plate the cells. The volume ratio of cell resuspension to Matrigel is 1:1.5, and each droplet contains 20 μL. S3. After the mixed gel from step S2 has solidified for 5 minutes, add the lung squamous cell carcinoma organoid culture medium from Example 1, and then incubate in a constant temperature incubator at 37°C and 5% CO2 concentration for 5 days. The culture medium is changed every 2 days during this period to obtain the lung squamous cell carcinoma organoids. The culture results are as follows... Figure 3 As shown, lung squamous cell carcinoma organoids with intact spherical structures and smooth edges were obtained.

[0052] Example 7 This embodiment provides a method for culturing lung squamous cell carcinoma organoids, using the lung squamous cell carcinoma organoid culture medium from Example 2. This culture method is identical in operation to that of Example 6 (and the sample source is also the same). The culture results are as follows: Figure 4 As shown, lung squamous cell carcinoma organoids with intact spherical structures and smooth edges were obtained.

[0053] Example 8 This embodiment provides a method for culturing lung squamous cell carcinoma organoids, using the lung squamous cell carcinoma organoid culture medium of Example 3. This culture method is identical in operation to that of Example 6 (and the sample source is also the same). The culture results are as follows: Figure 5 As shown, lung squamous cell carcinoma organoids with intact spherical structures and smooth edges were obtained, but the cell proliferation was not as high as in Examples 6 and 7.

[0054] Example 9 This embodiment provides a method for culturing lung squamous cell carcinoma organoids, using the lung squamous cell carcinoma organoid culture medium from Example 4. This culture method is identical in operation to that of Example 6 (and the sample source is also the same). The culture results are as follows: Figure 6 As shown, lung squamous cell carcinoma organoids with intact spherical structures and smooth edges were obtained, but the cell proliferation was significantly lower than that in Examples 6 and 7.

[0055] Example 10 In Example 5, based on high cell concentration (5*10) 4 Lung squamous cell carcinoma organoids obtained by culture (20 μl) were further subjected to morphological identification and functional verification: I. The obtained lung squamous cell carcinoma organoids were stained with hematoxylin and eosin (HE) using the following method: 1) Embedding and sectioning: The organoid is embedded in an embedding mold, and then cut into 4-6μm sections by a microtome and attached to a glass slide to prevent detachment.

[0056] 2) Baking the slides: Place the slides on a slide holder and put them in an oven at 65°C for 30 minutes to dry the moisture on the slides and melt the paraffin wax.

[0057] 3) Dewaxing: Dewax with xylene three times, 10 minutes each time; then rinse with 100% alcohol three times, 1 minute each time; finally rinse with running water for 1 minute.

[0058] 4) H&E staining: First stain with hematoxylin for 8 min, then wash with water for 1 min, then differentiate with 1% hydrochloric acid alcohol for 1-2 seconds, then rinse with running water for 30 min, then stain with 1% eosin for 1-2 min, and finally rinse with running water for 1 min.

[0059] 5) Fixation after staining: Immerse in 95% alcohol and 100% alcohol in sequence, twice for each reagent, for 2 minutes each time.

[0060] 6) Clearing and sealing: Use xylene to clear for 2 minutes, remove and let dry, then seal with neutral resin.

[0061] Observing tissue morphology and structure under a regular optical microscope, such as Figure 7 As shown in the figure, the organoids exhibit significant nuclear atypia, abundant cytoplasm, and tumor cells grow in nests or sheets, displaying characteristics of lung squamous cell carcinoma.

[0062] II. The obtained lung squamous cell carcinoma organoids were subjected to immunohistochemical (IHC) staining as follows: 1) Embedding and sectioning: The organoid is embedded in an embedding mold, and then cut into 4-6μm sections by a microtome and attached to a glass slide to prevent detachment.

[0063] 2) Baking the slides: Place the slides on a slide holder and put them in an oven at 65°C for 30 minutes to dry the moisture on the slides and melt the paraffin wax.

[0064] 3) Dewaxing: Dewax with xylene three times, 10 minutes each time; then rinse with 100% alcohol three times, 1 minute each time; finally rinse with running water for 1 minute.

[0065] 4) Antigen retrieval: Immerse the slides in citrate antigen retrieval solution (pH 6.0) and place them in a microwave oven for high-temperature antigen retrieval. After naturally cooling to room temperature, rinse three times with PBS.

[0066] 5) Blocking and incubation with primary antibody: Add 3% hydrogen peroxide solution to the tissue and incubate at room temperature in the dark for 15 minutes. After rinsing with PBS, add 5% bovine serum albumin (BSA) and block at room temperature for 30 minutes. Discard the blocking solution, do not wash, and directly add an appropriate amount of diluted primary antibody working solution (TTF1, p40, p63 antibody), and incubate overnight in a humidified chamber at 4°C.

[0067] 6) Secondary antibody incubation and staining: The next day, remove the sections from 4℃ and allow them to warm to room temperature for 45 minutes. After rinsing thoroughly with PBS three times, add the corresponding HRP-labeled secondary antibody working solution and incubate at room temperature for 60 minutes. After rinsing again with PBS, add freshly prepared DAB staining solution and control the staining time under a microscope. Immediately stop the staining process with running water after the staining is complete.

[0068] 7) Counterstaining and mounting: Counterstain the cell nuclei with hematoxylin solution. After about 3-5 minutes, rinse with running water to restore blue color. Then, dehydrate with a series of alcohols, clear with xylene, and finally mount with neutral resin.

[0069] Observing tissue morphology and structure under a regular optical microscope, such as Figure 8 As shown in the figure, this organ is TTF-1 negative, P40 positive, and P63 positive, which is consistent with the IHC results of clinical puncture tissue, confirming that this organ successfully reproduces the typical pathological features of lung squamous cell carcinoma.

[0070] Example 11 In Example 5, based on high cell concentration (5*10) 4 Lung squamous cell carcinoma organoids obtained from culture ( / 20 μl) were subjected to multiple passage cultures, with the specific procedures as follows: 1. Use a pipette to remove the culture medium from the culture dish, take 1-2 ml of TrypLE TME×press to digest the gel drop containing lung squamous cell carcinoma organoids, place it in an incubator, and digest at 37℃ for 15 min; 2. Add 10 ml of sterile Hank's balanced salt solution, centrifuge at 1000 rpm for 3 min; 3. Discard the supernatant, resuspend the organoids in 200 μl of the culture medium from Example 1, add 250 μl of Matrigel and mix well. Drop the mixture onto a new 35 mm culture dish, let it stand for 5 min, transfer it to an incubator, invert it, and after 40 min, add 2-4 ml of culture medium. Incubate at 37°C with 5% CO2. After 6 days of subculturing, the second generation of lung squamous cell carcinoma organoids was obtained. Subculturing was continued using this method, and after 21 days of culture, the third generation of lung squamous cell carcinoma organoids was obtained. The tissue morphology and structure observed under a regular light microscope are as follows: Figure 9 As shown, lung squamous cell carcinoma organoids have good structural morphology, and even after three generations, lung squamous cell carcinoma organoids can still maintain the original tissue morphology and structure.

[0071] Example 12 Referring to the high cell concentration (5*10) in Example 5 4 Lung squamous cell carcinoma organoids obtained by culturing 20 μl of the culture were passaged multiple times. The passage culture procedure was the same as in Example 10, with passage every 7 days. After 35 days of culture, the 5th generation lung squamous cell carcinoma organoids were obtained. The tissue morphology and structure of the organoids were observed under a regular light microscope as follows. Figure 10 As shown, the lung squamous cell carcinoma organoids have a good structural morphology, and even after 5 generations, the lung squamous cell carcinoma organoids can still maintain the original tissue morphology and structure.

[0072] Example 13 This embodiment provides a method for culturing lung adenocarcinoma organoids, which uses the lung squamous cell carcinoma organoid culture medium of Example 1. The culture method includes the following steps: The lung adenocarcinoma specimen in this embodiment was obtained from a 63-year-old male patient with advanced lung adenocarcinoma. The Ki-67 positivity rate was approximately 50%, the tumor was poorly differentiated, and the patient had a history of smoking. The initial clinical stage was IV, and the TNM stage was T2N1M1. The diagnosis of lung adenocarcinoma was determined based on a combination of clinical imaging and pathological examination results. The pathological diagnostic criteria for lung adenocarcinoma included the presence of glandular structures or glandular differentiation features in the tumor tissue, and the expression of lung adenocarcinoma-related markers, including but not limited to TTF-1 and Napsin A, while showing no or low expression of squamous cell carcinoma-related markers. With the patient's informed consent, a percutaneous lung biopsy was performed on the primary lesion in the patient's lung. A fresh tumor tissue sample of approximately 0.2 × 0.2 × 2 cm was used as the starting sample for lung adenocarcinoma organoid culture in this embodiment.

[0073] S1. Transfer the obtained lung adenocarcinoma specimen into a centrifuge tube, then wash with sterile saline solution for 30 seconds, remove the supernatant, and wash again with sterile saline solution. Repeat the washing process three times to remove impurities from the tissue surface. In a biosafety cabinet, transfer the sample to a 6 cm culture dish and use sterilized surgical scissors on ice to mince the tissue to a size of 1-3 mm. 3 Add 5 mL of type III collagenase and digest at 37°C with shaking for 30 min. After digestion, add 5 mL of DMEM / F12 medium to terminate digestion. Centrifuge at 1000 rpm for 5 min, carefully remove the supernatant to obtain cell clusters with 3-50 cells per cluster. Resuspend the cells in the lung squamous cell carcinoma organoid culture medium from Example 1 to a cell concentration of 5*10⁻⁶ cells / mL. 4 / 20μl; S2. After mixing the cell resuspension with Matrigel, plate the cells. The volume ratio of cell resuspension to Matrigel is 1:1.5, and each droplet contains 20 μL. S3. After the mixed gel from step S2 has solidified for 5 minutes, add the lung squamous cell carcinoma organoid culture medium from Example 1, and then incubate in a constant temperature incubator at 37°C and 5% CO2 concentration for 5 days, changing the culture medium every 2 days during this period. The culture results are as follows. Figure 11 As shown, although some lung adenocarcinoma organoids with intact spherical structures can be obtained, the overall cell condition is poor. Some cell boundaries are blurred, and there appear to be unseparated or unformed cells adhering to the surrounding tissues. Few well-formed organoids are formed, their morphology is irregular, their cavity-like structures are atypical, and their growth rate is slow. Overall, the culture efficiency is low, cell proliferation is minimal, and the results are unsatisfactory. Therefore, this culture medium is unsuitable for culturing lung adenocarcinoma organoids.

[0074] Comparative Example 1 This comparative example provides a lung squamous cell carcinoma organoid culture medium, which differs from the culture medium in Example 1 in that it does not contain Alpelisib; otherwise, it is the same as in Example 1. Using the culture medium of this comparative example, the high cell concentration (5*10⁻⁶) was referenced in Example 5. 4 Lung squamous cell carcinoma tissue (tissue source same as in Example 5) was cultured using a 20 μl culture process. The results after 5 days are as follows: Figure 12 As shown, under culture conditions without Alpelisib, after 5 days of culture, the resulting three-dimensional cells exhibited heterogeneous morphology, irregular edges, and significant size differences. Furthermore, there appeared to be unformed cells adhering to the cell edges, suggesting that in the absence of Alpelisib, it is impossible to form organoids with uniform structure, regular shape, stability, and high proliferation.

[0075] Comparative Example 2 This comparative example provides a lung squamous cell carcinoma organoid culture medium, which differs from the culture medium in Example 1 in that it lacks FGF10; otherwise, it is the same as Example 1. Using the culture medium of this comparative example, the high cell concentration (5*10⁻⁶) was referenced in Example 5. 4 Lung squamous cell carcinoma tissue (tissue source same as in Example 5) was cultured using a 20 μl culture process. The results after 5 days are as follows: Figure 13 As shown, the cell spheroidization rate was significantly reduced, the overall diameter of the formed organoids was smaller, and the growth rate was slower, suggesting that the organoid expansion capacity was limited in the absence of FGF10.

[0076] Comparative Example 3 This comparative example provides a lung squamous cell carcinoma organoid culture medium. The difference between this medium and the medium in Example 1 is that the final concentration of Alpelisib is 1.5 μM; otherwise, it is the same as in Example 1. Using the culture medium of this comparative example, the high cell concentration (5*10⁻⁶) was referenced in Example 5. 4 Lung squamous cell carcinoma tissue (tissue source same as in Example 5) was cultured using a 20 μl culture process. The results after 5 days are as follows: Figure 14 As shown, when using higher concentrations of Alpelisib, the organoid formation rate in the culture system decreases significantly, with only a small number of small organoids observed, and most forming only loose cell clusters or incomplete spherical structures. Therefore, the final concentration of Alpelisib in the system should not exceed 1.0 μM. If the amount of Alpelisib added is too small, for example, below 0.05 μM, the observed results of this invention are that the number of organoids is generally too small, and batch-to-batch culture is unstable and has poor reproducibility. Therefore, it is more suitable to control the amount of Alpelisib added at 0.1-1 μM, preferably at 0.1-0.8 μM, and most preferably at 0.1-0.3 μM.

[0077] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A culture medium for lung squamous cell carcinoma organoids, characterized in that, The lung squamous cell carcinoma organoid culture medium, in its final concentration, comprises: 50-500 ng / mL R-spondin-1, 10-500 ng / mL Noggin, 10-300 ng / mL EGF, 1-50 μg / mL FGF10, 5-200 ng / mL FGF7, 0.5-10 μM A83-01, 1-20 μM Y-27632, 1-30 mM Nicotinamide, 1-50 μg / mL Insulin, 1-2× B27 Supplement, 0.5-20 mM N-Acetylcysteine, 0.1-1 μM Alpelisib, 1× GlutaMAX, and 20-500 μg / mL primary cell antibiotics; all of the above components are dissolved in DMEM / F12 culture medium.

2. The lung squamous cell carcinoma organoid culture medium according to claim 1, characterized in that, The lung squamous cell carcinoma organoid culture medium, in its final concentration, comprises: 100-300 ng / mL R-spondin-1, 50-300 ng / mL Noggin, 50-200 ng / mL EGF, 5-20 μg / mL FGF10, 20-100 ng / mL FGF7, 0.5-8 μM A83-01, 1-15 μM Y-27632, 1-25 mM Nicotinamide, 5-30 μg / mL Insulin, 1-2× B27 Supplement, 1-15 mM N-Acetylcysteine, 0.1-0.8 μM Alpelisib, 1× GlutaMAX, and 50-300 μg / mL primary cell antibiotics; all of the above components are dissolved in DMEM / F12 culture medium.

3. The lung squamous cell carcinoma organoid culture medium according to claim 1 or 2, characterized in that, The lung squamous cell carcinoma organoid culture medium, in its final concentration, comprises: 200-300 ng / mL R-spondin-1, 80-150 ng / mL Noggin, 50-100 ng / mL EGF, 5-10 μg / mL FGF10, 25-60 ng / mL FGF7, 1-5 μM A83-01, 5-15 μM Y-27632, 5-20 mM Nicotinamide, 5-20 μg / mL Insulin, 1-1.5× B27 Supplement, 1-10 mM N-Acetylcysteine, 0.1-0.3 μM Alpelisib, 1× GlutaMAX, and 100-200 μg / mL primary cell antibiotics; all components are dissolved in DMEM / F12 culture medium.

4. The use of the lung squamous cell carcinoma organoid culture medium according to any one of claims 1 to 3 in the preparation of lung squamous cell carcinoma organoids.

5. A method for culturing lung squamous cell carcinoma organoids, characterized in that, The culture method includes: processing the obtained lung squamous cell carcinoma tissue into cell clusters, and then resuspending the cell clusters in the culture medium according to any one of claims 1 to 3; further mixing the cell resuspension with matrix gel, and after solidification, adding the culture medium according to any one of claims 1 to 3 for isothermal culture to obtain lung squamous cell carcinoma organoids.

6. The cultivation method according to claim 5, characterized in that, The cell concentration in the cell resuspension was (0.5~5.0)×10⁻⁶. 3 per μl.

7. The cultivation method according to claim 5, characterized in that, The cell concentration in the cell resuspension was (2.0~5.0)×10⁻¹⁰. 3 per μl.

8. The cultivation method according to any one of claims 5 to 7, characterized in that, The volume ratio of the cell resuspension to the matrix gel is 1:1~2.

9. The cultivation method according to any one of claims 5 to 7, characterized in that, The volume ratio of the cell resuspension to the matrix gel is 1:1.3~1.

5.

10. The cultivation method according to claim 5, characterized in that, The control parameters for the isothermal culture include: temperature 37±1℃, 5% CO2, and culture time of 3~7 days.

11. The cultivation method according to claim 5, characterized in that, The culture method also includes further passage culture of the obtained lung squamous cell carcinoma organoids.

12. The use of lung squamous cell carcinoma organoids obtained by the culture method according to any one of claims 5 to 11 in preparing preclinical models for studying the pathogenesis of lung squamous cell carcinoma and / or screening drugs for the treatment of lung squamous cell carcinoma and / or developing therapies for lung squamous cell carcinoma.

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